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This is to certify that the
dissertation entitled

THE NEURODEVELOPMENTAL EFFECTS OF CHRONIC
MODERATE PRENATAL CAFFEINE EXPOSURE IN RATS

presented by

DEBORAH ELIZABETH SOELLNER

has been accepted towards fulﬁllment
of the requirements for the

Ph.D. degree in Neuroscience

 

 

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THE NEURODEVELOPMENTAL EFFECTS OF CHRONIC
MODERATE PRENATAL CAF F EINE EXPOSURE IN RATS

By

Deborah Elizabeth Soellner

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Neuroscience

2009

ABSTRACT

THE NEURODEVELOPMENTAL EFFECTS OF CHRONIC MODERATE
PRENATAL CAF F EINE EXPOSURE IN RATS

By

Deborah Elizabeth Soellner

Caffeine is the most widely used psychostimulant in the world. Although women
are currently advised to reduce caffeine intake throughout pregnancy, it is estimated that
70-95% of pregnant women continue to consume the drug. The widespread use of
caffeine is of concern because it crosses both the placental and blood brain barriers to
accumulate in the developing brain. Furthermore, the half-life of caffeine is greatly
prolonged in the developing fetus, being twenty fold that of an adult. Caffeine is an
adenosine receptor antagonist and adenosine is a well-known critical neuromodulator
during brain development. Adenosine functions to reduce neuronal activity thus
providing protection against excitotoxicity. When adenosine receptors are inhibited by
caffeine, there is an overall increase in glutamate neurotransmission particularly in brain
regions with an abundance of Al adenosine receptors such as the hippocampus.
Glutamate receptors play a role in numerous developmental processes including activity-
dependent migration, synapse formation, and synaptic plasticity. Therefore, increased
glutamate neurotransmission in the developing brain as a result of prenatal caffeine
exposure could have numerous neurodevelopmental consequences. In order to elucidate
the effects of prenatal caffeine exposure, pregnant rats were exposed to chronic moderate

doses of caffeine (75, 150, or 300 mg/L) ad Iibitum in their drinking water throughout

gestation. Moderate doses and oral administration were chosen to maintain human
relevance of the studies.

The objectives of this dissertation were to determine whether caffeine and its
primary metabolites accumulate in the fetal brain and the effects of prenatal exposure to
these drugs on hippocampal glutamate receptor expression, glutamate-mediated calcium
transients, learning and memory behaviors, and anatomy. The ﬁndings presented in this
dissertation show that chronic prenatal exposure to moderate doses of caffeine results in
an accumulation of caffeine and its primary metabolites in the fetal brain. Prenatal
caffeine exposure also alters protein expression of the NR1, NR2A, and NRZB subunits
of the NMDA receptor and reduces glutamate-induced peak calcium transients in
caffeine-exposed hippocampal neurons. Further, caffeine-exposed offspring show
impaired learning and memory behavior in both the novel object recognition task and
radial arm maze, but not in the Morris water maze. Lastly, prenatal caffeine exposure
results in a signiﬁcant increase in total hippocampal volume in caffeine-exposed juvenile
rats, however this effect does not persist into adulthood. In summary, the studies in this
dissertation present novel ﬁndings of cellular, behavioral, and anatomical

neurodevelopmental effects following moderate exposure to prenatal caffeine.

ACKNOWLEDGEMENTS

I would like to thank many people who supported and encouraged me during my
dissertation studies. I would like to thank my committee members, all of whom made
this dissertation possible. First, Dr. Cheryl Sisk who provided immeasurable advice and
support during my graduate studies for which I am extremely thankful. Dr. Keith
Lookingland who read each chapter of my dissertation providing invaluable feedback
during the writing process, took me under his wing as one of his own students, and
allowed me to make his lab a second home. Dr. Hongbing Wang who also opened up his
lab to me during my studies and provided critical insight to many of the studies I
completed. Dr. Peter Cobbett who has always been a student advocate and who never
hesitated to take a moment of his time to discuss my research or listen. Dr. Marc
Breedlove who helped me attain my predoctoral NRSA and was always supportive of my
research pursuits. And Dr. Joseph Nunez with whom I began the studies completed in
this dissertation and who supported my research ideas and pursuit of my own scientiﬁc
interests.

I would also like to thank numerous other faculty and staff who mentored and
supported me: Dr. James Galligan and Dr. Cindy Jordan for allowing me to gain teaching
experience in their classes, Dr. Stephanie Watts and the Program Project Grant for
allowing me to use the HPLC equipment in the core facility, Dr. John Goudreau for
allowing me to use his laboratory space, Dr. Nigel Paneth and Dr. Cheryl Sisk for
allowing me to rotate in their laboratories during my ﬁrst year, and Dr. Barbara Clancy

who allowed me to gain neuroscience laboratory experience as an undergraduate and who

iv

encouraged me to pursue graduate studies. I would also like to thank Robert Burnett who
helped tremendously with the setup and completion of the HPLC experiments.

I had the pleasure of working in numerous laboratories throughout my graduate
studies. I would like to thank all of the undergraduates, graduate students, and postdocs
who enriched each of these experiences. In the Nunez lab, I would like to thank Theresa
Grandys, Jacob McClean, Ilana Mark, Lianna Greene, Sarah Rothstein, Michael Mazur,
and Chris Gertiser. In the Goudreau/Lookingland lab, I would like to thank Bahareh
Behrouz, Kelly Janis, Sam Pappas, Tyrell Simkins, Chelsea Tieman, Matt Bensky, Dr.
Yu, and Dr. Baek. In the Wang lab, I would like to thank Dr. Xianju Zhou, Fei Zheng,
Yan Wang, and Dr. Ming Zhang. In the Sisk lab, I would like to thank Margaret Bell,
Kayla De Lorrne, Dr. Heather Molenda—Figueira, Ray Figueira, Dr. Eman Ahmed, and
Jane Venier.

I am grateful for funding from the National Institutes of Health and the
Neuroscience Program which supported my doctoral studies.

Lastly, I would like to thank my family and friends who have unconditionally
supported me during my studies. I cannot put in words how much I appreciate the love
and encouragement of my family. I also cannot thank my husband Josh enough for
always being by my side. I also want to thank the friends I have made during my
graduate studies and know that I will take more than a degree from this experience.
There are far too many friends to list but I would especially like to thank Lace Svec,
Margaret Bell, Stephanie Miller, Bahareh Behrouz, and Kelly Janis for always being
available to listen and for being the greatest friends a person could ask for. Without each

of these people, these dissertation studies would not have been possible.

TABLE OF CONTENTS

LIST OF TABLES ......................................................................................................... ix

LIST OF FIGURES ........................................................................................................ x
CHAPTER 1

INTRODUCTION AND SPECIFIC AIMS .................................................................... 1

A. Caffeine Consumption, Metabolism, and Distribution ................................. l

B. Actions of Caffeine and its Metabolites ........................................................ 3

C. Adenosine and its Receptors ......................................................................... 6

D. Developmental Expression of A1 and A2A receptors .................................. 7

E. Al and A2A Adenosine Receptors ................................................................ 8

F. A1 and A2A Receptor Knockout and Transgenic Studies .......................... 15

G. Pharmacological Manipulation of A1 and A2A Receptor Function ........... 17

H. Perinatal Caffeine Exposure and Adenosine Receptor Regulation ............. 19

I. The Hippocampus and Learning and Memory ............................................. 20

J. Perinatal Development of the Hippocampus ................................................ 24

K. Glutamate Receptors and Development ...................................................... 26

L. Prenatal Caffeine and Development ............................................................ 30

M. Summary ..................................................................................................... 34

N. Hypotheses ................................................................................................... 35

0. Speciﬁc Aims ............................................................................................... 35
CHAPTER 2

CAFFEINE AND ITS ACTIVE METABOLITES ARE ELEVATED IN THE FETAL

BRAIN AFTER CHRONIC GESTATIONAL EXPOSURE ....................................... 38

A. Introduction ................................................................................................. 38

B. Materials and Methods ................................................................................ 41

Animals ....................................................................................................... 41

Chemicals .................................................................................................... 41

Plasma Extraction ....................................................................................... 42

Brain Preparation ........................................................................................ 42

Protein Determination ................................................................................. 43

Extraction Recovery .................................................................................... 43

HPLC Analyses of Caffeine and its Major Metabolites ............................. 44

Statistics ...................................................................................................... 45

C. Results ......................................................................................................... 46

Chromatograms and Standard Curve .......................................................... 46

Extraction Efﬁciency .................................................................................. 47

Gestational Caffeine Consumption ............................................................. 51

Methylxanthine Concentration in Male and Female Offspring .................. 51

Methylxanthine Concentrations in Fetal and Maternal Plasma and Brain ..52

D. Discussion .................................................................................................... 60

vi

CHAPTER 3
CHRONIC GESTATIONAL CAFFEINE EXPOSURE ALTERS GLUTAMATE
RECEPTOR EXPRESSION AND CALCIUM IN FLUX IN THE DEVELOPING

HIPPOCAMPUS ........................................................................................................... 66

A. Introduction ................................................................................................. 66

B. Materials and Methods ................................................................................ 69

Animals ....................................................................................................... 69

Primary Hippocampal Cell Cultures ........................................................... 69

Calcium Imaging ......................................................................................... 70

Western Blot Analysis ................................................................................ 73

High Performance Liquid Chromatography ............................................... 74

Statistics ...................................................................................................... 76

C. Results ......................................................................................................... 78

Peak Calcium Response to Glutamate ........................................................ 78

Total NMDA Receptor Expression in the Developing Hippocampus ........ 81

Activation of Calcium Dependent Cascades ............................................... 85

High Performance Liquid Chromatography ............................................... 85

D. Discussion .................................................................................................... 88
CHAPTER 4

CHRONIC PRENATAL CAFFEINE EXPOSURE IMPAIRS NOVEL OBJECT
RECOGNITION AND RADIAL ARM MAZE BEHAVIORS IN ADULT RATS ....96

A. Introduction ................................................................................................. 96
B. Materials and Methods .............................................................................. 100
Prenatal Caffeine Treatment ..................................................................... 100
Behavioral Tests ........................................................................................ 1 01
Novel Object Recognition Test ................................................................. 102
Radial Arm Maze ...................................................................................... 103
Standard Morris Water Maze .................................................................... 104
Advanced Morris Water Maze .................................................................. 105
Statistics .................................................................................................... 106
C. Results ....................................................................................................... 107
Water Intake .............................................................................................. 107
Novel Object Recognition Test ................................................................. 107
Radial Arm Maze ...................................................................................... 108
Standard Morris Water Maze .................................................................... 115
Advanced Morris Water Maze .................................................................. 116
D. Discussion ................................................................................................. 123
CHAPTER 5
CHRONIC PRENATAL CAFFEINE EXPOSURE INCREASES HIPPOCAMPAL
VOLUME IN JUVENILE RATS ............................................................................... 130
A. Introduction ............................................................................................... 130
B. Materials and Methods .............................................................................. 133
Animals ..................................................................................................... 133

vii

Cavalieri Estimation of Volume ............................................................... 134

Statistics .................................................................................................... 135

C. Results ....................................................................................................... 138

D. Discussion .................................................................................................. 143
CHAPTER 6

DISCUSSION ............................................................................................................. 149

REFERENCES ........................................................................................................... 1 61

viii

LIST OF TABLES

Table 2-1. The plasma extraction efﬁciency of acetone and methanol were compared for
each methylxanthine. Data represent [(peak height of the sample/peak height of
standard) x 100] i SEM ..................................................................................................... 50

Table 4-1. Mean (:t SEM) values of radial arm maze pre-training time (3), training path
lengths (cm), time to retrieve all cereal rewards (s), and body weights before and after
food deprivation ............................................................................................................... 114

Table 4-2. Mean (:t SEM) values for advanced Morris water maze path length (cm) in
the place learning, reversal, and working memory tasks ................................................. 122

Table 5-1. Volumes (mm3) for the whole hippocampal formation, Ammon’s horn, and

dentate gyrus for control and caffeine-treated juveniles and adults. Data represent the
average volume (:h SEM) ................................................................................................. 142

ix

LIST OF FIGURES

Figure 1-1. Caffeine elicits differential concentration-dependent effects within the brain.
After consumption of a single cup of coffee, the concentrations of caffeine attained are
likely acting exclusively at the A1 and A2A adenosine receptors. Supraphysiological
concentrations of caffeine, which approach toxic levels, are needed to induce inhibition
of phosphodiesterase activity, blockade of GABA(A) receptors, and activation of
intracellular calcium release. Reproduced from F redholm et al., 1999 .............................. 5

Figure 1-2. The actions of (A) adenosine and (B) caffeine at the Al receptor. (A) When
adenosine binds the A1 receptor (1) it decreases adenylyl cyclase activity (AC) and
cAMP formation (2). Adenosine also reduces calcium entry via voltage-gated calcium
channels (3) and increases potassium efﬂux via potassium channels, which results in
hyperpolarization of neuron and decreased neurotransmitter release (5). (B) When
caffeine antagonizes the A1 receptor (1), adenosine can no longer inhibit adenylyl
cyclase activity resulting in an increase in both adenylyl cyclase activity and CAMP
formation (2). Further, voltage-gated calcium channels are open which allows calcium
entry (3) and potassium channels are blocked (4). The accumulation of positive calcium
and potassium ions within the neuron allows the cell to be more easily depolarized,
thereby increasing neurotransmitter release (5) ................................................................. 13

Figure 1-3. The actions of adenosine and caffeine at the A2A receptor located
postsynaptically on striatal GABAergic intemeurons. Adenosine normally binds to the
A2A receptor to stimulate adenylyl cyclase (AC) activity, which increases GABAergic
output and results in decreased motor activity. When caffeine blocks adenosine from
binding to the A2A receptor, adenylyl cylcase activity is decreased resulting in decreased
GABAergic output to the basal ganglia which increases overall motor activity. SN=
substantia nigra .................................................................................................................. ‘14

Figure 2-1. HPLC-UV detection of caffeine and its major metabolites in standards.
Shown is a chromatogram of 625 ng/ml standard solution. Standards of 0-10 ug/ml were
prepared by serial dilution in 0.1% acetic acid prior to each analysis. The retention times
for each methylxanthine were approximately 1) Theobromine — 5.61, 2) Paraxanthine —
8.49, 3) Theophylline - 9.57, B-Hydroxyethyltheophylline — 10.72, and 5) Caffeine —
16.10. AU = Absorbance Unit .......................................................................................... 48

Figure 2-2. Standard curves for caffeine, its metabolites, and the internal standard [3-
hydroxyethyltheophylline. Standards were prepared in 0.1% acetic acid across
concentrations of 78.1— 5000 ng/ml. Linear correlation coefﬁcients (R2) above 0.9998
were found for each standard by comparing the peak height across each concentration.
The lowest limit of detection was 39.1 ng/ml .................................................................... 49

Figure 2-3. Maternal intake of either untreated tap water (control) or 75, 150, or 300
mg/L caffeine-treated tap water across each gestational week. Data represent the mean

daily intake :t SEM for each week. Water intake increased throughout gestation in all
treatment groups, however dams consuming 300 mg/L caffeine-treated water drank
signiﬁcantly less than dams treated with either no or low caffeine doses ......................... 55

Figure 2-4. Concentrations of caffeine in fetal and maternal (A) brain and (B) plasma
following prenatal caffeine exposure. Data represent the mean caffeine concentration (d:
SEM) reached following exposure to each dose. Chronic prenatal exposure to caffeine
results in a signiﬁcant accumulation of caffeine within the fetal brain when compared to
the maternal brain, however there is no difference in caffeine concentrations in fetal and
maternal plasma ................................................................................................................. 56

Figure 2-5. Concentrations of theobromine in fetal and maternal (A) brain and (B)
plasma following prenatal caffeine exposure. Data represent the mean theobromine
concentration (d: SEM) reached following exposure to each dose. Chronic prenatal
exposure to caffeine results in a signiﬁcant accumulation of theobromine within the fetal
brain when compared to the maternal brain that increased by dose. No differences
between fetal and maternal plasma concentrations of theobromine were found ............... 57

Figure 2-6. Concentrations of paraxanthine in fetal and maternal (A) brain and (B)
plasma following prenatal caffeine exposure. Data represent the mean paraxanthine
concentration (:5 SEM) reached following exposure to each dose. Chronic prenatal
exposure to caffeine results in a signiﬁcant accumulation of paraxanthine within the fetal
brain when compared to the maternal brain that increased by dose. No differences
between fetal and maternal plasma concentrations of paraxanthine were found ............... 58

Figure 2-7. Concentrations of theophylline in fetal and maternal (A) brain and (B)
plasma following prenatal caffeine exposure. Data represent the mean theophylline
concentration (:h SEM) reached following exposure to each dose. Chronic prenatal
exposure to caffeine results in a signiﬁcant accumulation of theophylline within the fetal
brain when compared to the maternal brain that increased by dose. No differences
between fetal and maternal plasma concentrations of theophylline were found ............... 59

Figure 3-1. The change in ratio between resting intracellular calcium and peak responses
following glutamate administration are shown in both the hippocampal cell cultures (A)
and a corresponding trace recording (B) during live cell imaging. Both the images and
trace represent the ratio of bound to unbound intracellular Fura-2 .................................. 72

Figure 3-2. Baseline resting intracellular Ca2+ ratio in cultured hippocampal neurons
from control and prenatal caffeine-treated neurons. There were no signiﬁcant differences
between resting intracellular Ca2+ ratios between treatments. Data represent the average
resting Fura-2 ratio :1: SEM ................................................................................................ 79

Figure 3-3. Peak calcium transients following brief application of 2, 5, 10, or 50uM
glutamate in hippocampal neurons cultured from control or caffeine-exposed pups. A
calcium transient less than 20% over baseline was classiﬁed as a low response (A), 20-
100% change over baseline as a medium response (B), and higher than 100% change

xi

from baseline as a high response (C). In high responding neurons, caffeine treatment
resulted in a reduced calcium transient. Data represent the mean calcium peak
:1: SEM ................................................................................................................................ 80

Figure 3-4. Total NR1 protein expression in the developing hippocampus on postnatal
day 1 (A), postnatal day 5 (B), postnatal day 10 (C), and postnatal day 20 (D) in control
and caffeine-exposed (75, 150, or 300 mg/L) offspring. Data represent the mean optical
density :1: SEM. (E) Representative immunoblots from each treatment group (n=3) at
each postnatal time period. A main effect of treatment was found on postnatal days 5, 10,
and 20. * indicates signiﬁcant difference from control (Tukey; p<0.05) ......................... 82

Figure 3-5. Total NR2A protein expression in the developing hippocampus on postnatal
day 1 (A), postnatal day 5 (B), postnatal day 10 (C), and postnatal day 20 (D) in control
and caffeine-exposed (75, 150, or 300 mg/L) offspring. Data represent the mean optical
density :t SEM. (E) Representative immunoblots from each treatment group (n=3) at
each postnatal time period. A main effect of treatment was found on postnatal days 10
and 20. * indicates signiﬁcant difference from control (Tukey; p<0.05) ......................... 83

Figure 3-6. Total NR2B protein expression in the developing hippocampus on postnatal
day l (A), postnatal day 5 (B), postnatal day 10 (C), and postnatal day 20 (D) in control
and caffeine-exposed (75, 150, or 300 mg/L) offspring. Data represent the mean optical
density i SEM. (E) Representative immunoblots from each treatment group (n=3) at
each postnatal time period. A main effect of treatment was found on postnatal day 1.

* indicates signiﬁcant difference from control (Tukey; p<0.05) ....................................... 84

Figure 3-7. Phosphorylated-CREB (CAMP response element binding protein) expression
in the developing hippocampus on postnatal day 10 (A) and postnatal day 20 (B)
following acute caffeine treatment in control and caffeine-exposed (75, 150, or 300
mg/L) offspring. Acute caffeine treatment consisted of a single dose caffeine (10 mg/kg)
administered orally by pipette followed by a 1.5 hour delay prior to tissue collection.
Data represent the mean optical density 1 SEM. (C) Representative immunoblots from
each treatment group (n=3) at both postnatal time periods. No signiﬁcant differences in
pCREB activation were found between control and caffeine-exposed offspring .............. 86

Figure 3-8. Phosphorylated-ERK1/2 (extracellular signal related kinase 1/2) expression
in the developing hippocampus on postnatal day 10 (A) and postnatal day 20 (B)
following acute caffeine treatment in control and caffeine-exposed (75, 150, or 300
mg/L) offspring. Acute caffeine treatment consisted of a single dose caffeine (10 mg/kg)
administered orally by pipette followed by a 1.5 hour delay prior to tissue collection.
Data represent the mean optical density i SEM. (C) Representative immunoblots from
each treatment group (n=3) at both postnatal time periods. No signiﬁcant differences in
pERK activation were found between control and caffeine-exposed offspring ................ 87

Figure 4-1. Maternal intake of either water or caffeinated tap water across each

gestational week. Data represent the mean daily intake :t SEM for each week. Water
intake increased throughout gestation but did not signiﬁcantly differ by treatment ....... 111

xii

Figure 4-2. (A) Effects of prenatal caffeine on the total time spent exploring both objects
in the novel object recognition test during each 3 min trial. Data represent the mean
exploration time in seconds :1: SEM for pre-training, 3 hour recognition memory, and 24
hour recognition memory. There was a signiﬁcant effect of treatment on total exploration
time during pre-training, with caffeine-treated animals exploring the objects less. (B)
Effects of prenatal caffeine exposure on memory retention of the novel object at 3 and 24
hours after pre-training. Data represent the percent of time i SEM spent exploring the
novel object. Prenatal exposure to caffeine signiﬁcantly reduced memory retention of the
novel object at 24 hours after pre-training. Controls n=l3; Caffeine n=16 .................... 112

Figure 4-3. Effects of prenatal caffeine exposure on the number of (A) working memory
errors and (B) reference memory errors performed during Radial arm maze testing, with
caffeine-treated animals performing signiﬁcantly more memory errors. Data represent
the average number of errors at SEM performed during each block (Trials 1-2, 3-4, 5-6, 7-
8, 9-10). Control n=l3; Caffeine=n=16 ........................................................................... 113

Figure 4-4. (A) Effects of prenatal caffeine on latency to ﬁnd the hidden platform during
pre-training and across the three trial blocks (block 1=trials 1-4, block 2 = trials 5-8,
block 3 = trials 9-12) in the Morris water maze. Data represent the average latency d:
SEM needed to ﬁnd the platform during each 60 5 trial. (B) Effects of prenatal caffeine
on path lengths in the Morris water maze. Path length was measured as the total distance
in centimeters :t SEM swam during each trial averaged across the 3 trial blocks. Control
n=15; Caffeine n=16 ........................................................................................................ 119

Figure 4-5. Effects of prenatal caffeine on latency to ﬁnd the hidden platform during (A)
place learning and (B) reversal in the Morris water maze across the four trials conducted
each day over the ﬁrst 6 days of testing. Data represent the average latency i SEM
needed to ﬁnd the platform during each 60 3 trial. Control n=15; Caffeine n=16 ........... 120

Figure 4-6. Effects of prenatal caffeine on latency to ﬁnd the hidden platform during
working memory tasks in the Morris water maze across the four trials conducted each day
over the last 3 days of the total 9 days of testing. Data represent the average latency :1:
SEM needed to ﬁnd the platform during each 60 5 trial. Control n=15;

Caffeine n=16 .................................................................................................................. 121

Figure 5-1. Representative coronal sections highlighting the rat hippocampus to show
the delineation of the Ammon’s horn and dentate gyrus used for the bilateral volume
measurements (Swanson, 1998). The entire area within the medium gray area was used
to determine the area of Ammon’s horn, while the entire dark gray area area was used to
determine area of the dentate gyrus. Both areas combined were used to determine the
area of the hippocampal formation .................................................................................. 137

Figure 5-2. Maternal intake of water (n=4) or caffeinated tap water (n=4) from
gestational day 4 until the day of birth (gestational day 23 i 1 day). Data represent the
mean daily intake i SEM for each gestational week. Water intake increased throughout
gestation, but there was no main effect of treatment on intake ....................................... 140

xiii

Figure 5-3. Effect of prenatal caffeine on total hippocampal volume in (A) juveniles (n-
16) and (B) adults (n=27). Data represent the average volume i SEM. Juvenile rats
show a signiﬁcant main effect of treatment (p<0.05) whereas adult rats show no main
effect of treatment but a main effect of sex (p<0.00) ...................................................... 14]

xiv

Chapter 1. Introduction and Speciﬁc Aims

A. Caffeine Consumption, Metabolism, and Distribution

Caffeine is the most widely consumed psychoactive drug in the world due to its
presence in coffee, tea, chocolate, soft drinks and numerous medications. The caffeine
content in food and beverages varies considerably, depending on the food or beverage
and its method of preparation. For instance, an 8 oz. cup of coffee contains
approximately 40-160 mg caffeine depending on whether it is prepared by brew or drip.
A single cup of tea contains approximately 30-70 mg depending on the type of tea leave
used. Soft drinks contain between 30-45 mg caffeine per 8 oz. The daily intake of
caffeine in the United States is estimated to be 4.0 mg/kg in a 70 kg person
(approximately 280 mg/day) (Barone & Roberts, 1996).

Once consumed, caffeine is rapidly absorbed by the stomach and intestines to
reach the blood (Fredholm er al., 1999). Caffeine’s hydrophobic chemical composition
allows it to cross all biological membranes, including the blood brain barrier, to be
distributed throughout the body (McCall et al., 1982). Peak plasma concentrations are
reached within 15-120 minutes of intake, with consumption of a single cup of coffee
resulting in plasma concentrations of 1-10 uM caffeine (or 0.25-2 mg/L) (Fredholm et
al., 1999). Excessive consumption of caffeine can produce toxic effects. In rats, caffeine
is toxic at doses greater than 200 mg/kg, resulting in seizures, which may lead to death
(Bonati et al., 1984-1985). In humans and animals, the effects of caffeine are biphasic.
At low doses, such as those commonly consumed in a few cups of coffee, caffeine

produces stimulatory effects such as increased locomotor activity, enhanced attention,

and reduced fatigue. However, at high doses, caffeine leads to reduced locomotor
activity, inability to concentrate, increased anxiety, and other negative side effects.
Caffeine is a methylxanthine that is metabolized by the cytochrome P-450
enzyme CYP1A2 in the liver to three primary dimethylxanthines: theophylline,
theobromine, and paraxanthine. These metabolites, like caffeine, are pharmacologically
active within the body and brain. The metabolism of caffeine is similar in humans and
rodents, however the primary metabolites differ. For instance, in humans the primary
metabolite of caffeine is paraxanthine (Nehlig & Derby, 1994), whereas in rats, the
primary metabolite of caffeine is theobromine although the levels of each metabolite have
been found to be comparable (Eteng et al., 1997). The half-life of caffeine is
approximately 2-4 hours in the adult human and 1-2 hours in the adult rat (Bonati et al. ,
1984-1985). In the adult rat, the half-life of each metabolite is approximately 5-6 hours
for theobromine, 4-5 hours for theophylline, and 1-2 hours forparaxanthine. For
humans, the half-life of each metabolite is approximately double the half-life in rats. The
half-life of caffeine and its metabolites can be prolonged by various factors including
pregnancy and the use of oral contraceptives. During the third trimester of pregnancy, the
half-life of caffeine is increased to 10-20 hours (Aldridge et al., 1981). The use of
caffeine during pregnancy is also of concern because it is estimated that the half-life of
caffeine in a newborn ranges from 50 to 100 hours with a more prolonged half-life in the
fetus (Amaud, 1993). The slower clearance of caffeine in the newborn and fetus is due to
a deﬁciency in cytochrome P-450 activity (Aranda er a1. , 1979a). Cytochrome P-450
enzymes develop during early postnatal life and clearance of caffeine does not attain

adult rates until 6 months of age (Aranda er al. , 1979a; Parsons & Neims, 1981).

The psychostimulatory effects of caffeine are a result of its actions within the
central nervous system. Caffeine enters the brain by both passive diffusion as well as a
carrier-mediated adenine transport system (McCall etal., 1982). Caffeine enters the
brain more rapidly than its metabolites due to its increased lipid solubility and decreased
plasma protein binding. In adult rats, a single oral dose of 5 or 25 mg/kg results in
caffeine levels that exceed metabolite levels in both the brain and plasma (Wilkinson &
Pollard, 1993). However, each metabolite attained measurable levels within the plasma
after 5 min and within the brain by 30-60 minutes. In mice, brain levels of caffeine
exceeded levels of each metabolite as quickly as 15 minutes following exposure (Shi &
Daly, 1999). Therefore, caffeine enters the brain rapidly to produce its effects, but is
more quickly metabolized allowing its active metabolites to then exert their effects within

the nervous system.

B. Actions of Caffeine and its Metabolites

Caffeine is known to have multiple dose-dependent biological actions. The four
best characterized effects of caffeine are 1) inhibition of cyclic nucleotide
phosphodiesterase activity leading to reduced cyclic AMP degradation, 2) ryanodine
receptor mediated stimulation of intracellular calcium release, 3) antagonism of
GABA(A) receptors, and 4) antagonism of adenosine receptors (Daly, 2007). At the
concentrations attained following normal consumption of caffeine, it is unlikely that
caffeine has any effects through the ﬁrst three mechanisms (Figure 1-1). For instance, it
has been shown that caffeine and theophylline are relatively weak phosphodiesterase

inhibitors and levels of these compounds must reach concentrations of 500 uM and 100

pM, respectively, in order to exert inhibitory effects (Daly, 2000). Caffeine increases the
sensitivity of ryanodine receptors to intracellular calcium causing opening of calcium
release channels (Daly, 2000), however millimolar concentrations of caffeine must be
used in order to elicit ryanodine receptor mediated intracellular calcium release
(McPherson et al. , 1991). Similar to the ryanodine receptor, caffeine inﬂuences
GABA(A) receptors only at extremely high doses. Caffeine inhibits the binding of both
GABA and the benzodiazepine diazepam to GABA(A) receptors (Marangos et al., 1979).
However, the afﬁnity of caffeine for GABA receptors is low and it is estimated that
concentrations needed to effectively block these receptors would have to be
approximately 40 times greater than concentrations acquired following normal coffee
consumption (Fredholm et al., 1999). Further, caffeine may act primarily on glycine
receptors. Caffeine signiﬁcantly inhibits glycine receptors at concentrations of 500 pM,

‘whereas concentrations of 4mM are needed to produce similar inhibition of GABA
receptors (Daly, 2007). Although not well studied, millimolar concentrations of caffeine
may also have direct effects on numerous ion channels, including voltage sensitive L-
type calcium channels, potassium channels, and sodium channels (Daly, 2000). Given
the millimolar concentrations required for caffeine activate these mechanisms, the

primary effects of caffeine within the nervous system are likely mediated through

antagonism of adenosine receptors.

1 cup of coffee

Toxic doses

 

 

 

 

 

100 V
1,. A O x
/ ' [I
if A e v
/ ,‘f 1’ l
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80 A 97‘ .1 o i
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:55 A1 receptor J... .1..- . -- receptors
‘5 i 3 ’ '
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"x ‘ " Phos hodiesterase
A e ,1! 9
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0.001 0.01 0.1 1 10

Concentration of caffeine (mM)

Figure 1-1. Caffeine elicits differential concentration-dependent effects within the brain.
After consumption of a single cup of coffee, the concentrations of caffeine attained are
likely acting exclusively at the A1 and A2A adenosine receptors. Supraphysiological
concentrations of caffeine, which approach toxic levels, are needed to induce inhibition

of phosphodiesterase activity, blockade of GABA(A) receptors, and activation of
intracellular calcium release. Reproduced from F redholm et al., 1999.

 

C. Adenosine and its Receptors

The main target of caffeine and its metabolites is antagonism of adenosine
receptors. Four heptahelical G-protein-coupled adenosine receptors have been classiﬁed
and named the A1 , A2A, A28, and A3 receptors (F redholm et al., 2000). Although
adenosine is not classiﬁed as a neurotransmitter, it has considerable neuromodulatory
effects, including general inhibition of neuronal activity (Fisone et al., 2004). Adenosine
is found ubiquitously throughout the brain and is synthesized both intracellularly and
extracellularly. Adenosine can be formed via two main pathways. The ﬁrst pathway
involves the breakdown of adenine nucleotides, such as ATP. When this occurs, ATP is
dephosphorylated to produce AMP, which then forms adenosine (F redholm et al., 1999;
Fisone et al., 2004). Thus, adenosine levels produced via this pathway are highly
dependent on the utilization of ATP. A second pathway involves the hydrolysis of S-
adenosyl-homocysteine, although this pathway is not thought to be the primary pathway
involved in adenosine formation (Fisone et al., 2004; Fredholm et al., 2005). Levels of
intracellular adenosine are in the 25-250 nM range and increase with cellular activity,
oxygen utilization, and neurotransmitter release (Fredholm, 2007). Adenosine is broken
down by adenosine kinase and adenosine deaminase (F redholm, 2007). Adenosine
kinase functions to regulate physiological levels of adenosine and is the primary enzyme
responsible for maintaining low levels of intracellular adenosine (Fredholm et al., 2005).
Adenosine deaminase functions to reduce supraphysiological levels of adenosine
produced during pathological conditions such as hypoxia or ischemia. During these
conditions, adenosine levels rise rapidly due to the massive release of ATP, which is

rapidly converted to adenosine (Fredholm, 2007). Because adenosine generally functions

within the brain to reduce the level of neuronal activity, it plays a prominent role in
protecting the brain from excitotoxic cell death caused by hypoxia, seizures, and
inﬂammation (Fredholm, 2007).

Of the four adenosine receptors, the A1 and A2A adenosine receptors bind
caffeine with the highest affinity. These receptors are primary receptors antagonized by
caffeine and therefore are responsible for caffeine’s effects within the nervous system
(F redholm et al., 1999). The A28 receptor, although blocked by caffeine, is only
functionally active when endogenous adenosine reaches extremely high levels. These
levels are generally only attained during pathological conditions, such as hypoxia-
ischemia, and thus caffeine has little effect at these receptors during normal
circumstances (F redholm, 2007). Further, caffeine has been shown to have little action at
A3 receptors (Fredholm et al., 1999). Theophylline, paraxanthine, and theobromine also
target the A1 and A2A adenosine receptors. Theophylline and paraxanthine are more
potent than caffeine at non-selectively inhibiting both the A1 and A2A adenosine
receptors (Shi & Daly, 1999). Theobromine has a lower afﬁnity than caffeine at both A1
and A2A receptors, but it is selective for the A1 receptor (Shi & Daly, 1999). Therefore,
to understand the effects of caffeine in the brain, the combined actions of caffeine and its
metabolites must be considered since they all exert effects on adenosine receptor

function.

D. Developmental Expression of Al and A2A Receptors
The A1 and A2A receptors are both expressed during gestational development. In

the developing rat brain, the Al receptor is one of the earliest receptors expressed

(Rivkees et al., 2001) with mRN A detectable by gestational day 14 and adult patterns of
distribution established by gestational day 20 (Weaver, 1996). By gestational day 14,
functional binding of the A1 receptor can be detected in low levels throughout the brain
using radioligand binding with the A1 receptor antagonist DPCPX (Rivkees, 1995). A2A
receptors are also present prenatally (Svenningsson et al., 1999) with mRNA expression
detectable in the striatum by gestational day 14 in rats (Weaver, 1993). In humans,
adenosine receptors are believed to be the earliest receptor system developed (Herlenius
& Lagercrantz, 2001) and as a result may be susceptible to the effects of prenatal caffeine
exposure. Developmentally, adenosine plays a key role in the inhibition of neuronal
activity (Fisone et al., 2004), formation of axons (Turner et al., 2002), and

neuroprotection (Rivkees et al. , 2001 ).

E. A1 and A2A Adenosine Receptors

The A1 and A2A adenosine receptors are G-protein-coupled and are characterized
by their differential effects on adenylyl cyclase, with A1 receptors being inhibitory and
A2A receptors being stimulatory to adenylyl cyclase (Londos & Wolff, 1977). The Al

receptors are linked to both Gi and Go proteins, whereas A2A receptors are linked to Gs

and Golf proteins. A] receptor expression is highest in the cortex, hippocampus and

cerebellum, although the receptor is ubiquitously expressed throughout the brain. In each
region, A1 receptors are primarily located on presynaptic nerve terminals (F redholm &
Dunwiddie, 1988). Activation of Gi protein-coupled A1 receptors leads to inhibition of
adenylyl cyclase activity, decreases in CAMP concentrations, and consequently inhibition

of N, P, and Q-type voltage gated calcium channels (Fredholm er al. , 2000). Activation

of Go-protein-coupled Al receptors leads to an activation of G-protein-dependent inward
rectifying potassium channels causing neuronal hyperpolarization (Figure 1-2; Schubert
et al., 1997). These two actions 1) activation of potassium channels and 2) inhibition of
calcium channels, allows adenosine via the A1 receptor to inhibit neuronal activity and
diminish transmitter release, providing protection against excitotoxicity (Schubert et al. ,
1997). In the hippocampus, adenosine acting at the A1 receptor can decrease presynaptic
calcium inﬂux via the N, P, and Q-type voltage gated calcium channels. This decrease in
calcium inﬂux leads to an inhibition of synaptic transmission at mossy ﬁbre synapses
(Gundlﬁnger et al. , 2007).

A2A receptors play a stimulatory rather than inhibitory role. Thus, activation of
Gs-protein-coupled A2A receptors leads to stimulation of adenylyl cyclase and activation
of L-type calcium channels (F redholm et al., 2003). A2A receptor expression is highest
in dopamine-containing regions of the brain, such as the striatum, nucleus accumbens,
and olfactory tubercle (Fredholm et al., 2007). In the striatum, A2A receptors are located
postsynaptically on GABAergic medium spiny neurons. Adenosine increases adenylyl
cyclase activity, which results in increased activity of GABAergic medium spiny
neurons, which project to the basal ganglia. Increased activity of the GABAergic
neurons results in greater inhibition of cells in the basal ganglia, which reduces overall
motor activity (Figure 1-3). Recently, it has been found that Al and A2A receptors are
sometimes co-expressed. For instance, these receptors have been shown to form
heteromers in glutamatergic nerve terminals in the striatum (Ciruela er al. , 2006).

Current understanding of the neuromodulatory actions of adenosine may be broadened if

it is found that the A1 and A2A receptors produce opposing actions within the same
regions of the brain.

A1 receptor mediated inhibition of calcium channels and activation of potassium
channels results in decreased transmitter release. A1 receptor activation decreases
transmission of almost every major neurotransmitter within the brain, including
glutamate, GABA, dopamine, and serotonin (Dunwiddie & Masino, 2001).
Glutamatergic transmission is the most effected in that adenosine is able to almost
completely abolish release of glutamate. For instance, in hypothalamic cultures, in the
absence of glutamate transmission, adenosine has little effect on intracellular calcium,
however when glutamate transmission is not blocked, adenosine can decrease glutamate
transmission resulting in decreased postsynaptic calcium inﬂux (Obrietan er al. , 1995).
These results suggest that the effects of adenosine at the A1 receptor are glutamate
dependent.

Understanding the actions of adenosine at the A1 and A2A receptors makes it
possible to understand how antagonism of these receptors by caffeine produces its
stimulatory effects. Antagonism of A1 receptors by caffeine reduces the inhibition of
adenylyl cyclase, which leads to an increase in neurotransmitter release and an overall
increase in neuronal excitability (Figure 1-2). Increased activity of neurons in the cortex
and hippocampus is thought to mediate the ability of caffeine to increase attention and
information processing. Antagonism of A2A receptors leads to a reduction in adenylyl
cyclase activity, which in turn reduces the activity of the GABAergic medium spiny

neurons projecting to the basal ganglia. This allows neurons in the basal ganglia to

10

become more active and is believed to account for the ability of caffeine to increase
locomotor activity (Figure 1-3).

Recently, it has been shown that caffeine acts via A1 receptors on cortical nerve
terminals to enhance glutamate transmission (Wang, 2007). Application of caffeine (3
uM) or the A1 receptor antagonist DPCPX facilitates glutamate release. However, when
both antagonists were applied at the same time, there was no additional facilitation of
glutamate release suggesting a common mechanism of actibn (Wang, 2007). Further, Al
receptors play an important role in limiting excitotoxicity following seizures or hypoxic
conditions, with antagonism of these receptors exacerbating damage (Fredholm, 2007).

Although both the Al and A2A receptors are present and affected by caffeine, the
primary focus of the studies presented in this dissertation will be on the A] receptor due
to its widespread distribution throughout the brain and its interactions with glutamate
neurotransmission. The A1 receptor is the most abundant of the four adenosine receptors
within the brain and is distributed throughout the brain and spinal cord, with the highest
levels of expression in the hippocampus, cerebellum, and cerebral cortex (Fastbom er al.,
1986; Jarvis et al., 1987; Weaver, 1996; Svenningsson et al., 1997; Dunwiddie &
Masino, 2001). Although A2A receptors are also expressed in the hippocampus, their
most prominent levels of expression are in dopamine rich brain regions such as the
striatum (Svenningsson et al. , 1999). The role of the A2A receptors in the hippocampus
has begun to be understood through the use of knockout animals. Currently, it is
hypothesized that the actions of the A2A receptor in the hippocampus are the result of its
ability to modulate the Al receptor (Johansson et al., 2001). Thus, the role of the A2A

receptors in the hippocampus will be discussed further with regards to its effects on A1

11

receptor function. Currently, the cellular consequences of blocking the regulatory
functions of either adenosine receptor during development are unknown, however

receptor knockout studies have provided some insight.

12

A Presynaptically

   

Actions of
Adenosine

    
    

a
e
o
E] c... . E
K+
B Presynaptically
Actions of
Caffeine
o
e
e
e
. ‘ 5:
[Z] 032* e" ' El @

..O. K+

Figure 1-2. The actions of (A) adenosine and (B) caffeine at the A1 receptor. (A) When
adenosine binds the A1 receptor (1) it decreases adenylyl cyclase activity (AC) and
cAMP formation (2). Adenosine also reduces calcium entry via voltage-gated calcium
channels (3) and increases potassium efﬂux via potassium channels, which results in
hyperpolarization of neuron and decreased neurotransmitter release (5). (B) When
caffeine antagonizes the Al receptor (1), adenosine can no longer inhibit adenylyl
cyclase activity resulting in an increase in both adenylyl cyclase activity and CAMP
formation (2). Further, voltage-gated calcium channels are open which allows calcium
entry (3) and potassium charmels are blocked (4). The accumulation of positive calcium
and potassium ions within the neuron allows the cell to be more easily depolarized,
thereby increasing neurotransmitter release (5).

l3

Adenosine alone Caffeine SN

SN Dopaminergic Dopaminergic
Excitation input Excitation
from cortex from cortex

   
  

  
  
 

inhibits AC Striatal

   

   

Striatal Stimulates AC .
GABAergic GABAergic
lMemeuron Increased CAMP Decreased cAMP Inter neuron

      

. Decreased GABAergic
Increased GABAergic .
output to basal ganglia output to basal ganglia

Decreased motor activity Increased motor activity

Figure 1-3. The actions of adenosine and caffeine at the A2A receptor located
postsynaptically on striatal GABAergic intemeurons. Adenosine normally binds to the
A2A receptor to stimulate adenylyl cyclase (AC) activity, which increases GABAergic
output and results in decreased motor activity. When caffeine blocks adenosine from
binding to the A2A receptor, adenylyl cylcase activity is decreased resulting in decreased
GABAergic output to the basal ganglia which increases overall motor activity. SN=
substantia nigra.

F. Al and A2A Receptor Knockout and Transgenic Studies

9 The developmental effects of chronic antagonism of adenosine receptors by
caffeine may be similar to the lack of functional A1 and A2A receptors in knockout
studies. Knockout studies have provided valuable insight into the neuromodulatory roles
of adenosine and have been instrumental in understanding how caffeine is acting at each
receptor to produce its effects. Importantly, these studies have shown that adenosine
receptors play a signiﬁcant role in numerous behaviors, including learning and memory.

A1 receptor knockout mice (AlR-/-) do not exhibit birth defects or decreased
birth weights, but do differ at the cellular level when compared to wildtype mice (AIR
+/+) (Johansson et al., 2001). In hippocampal slices from A1R-/- mice, application of
adenosine does not inhibit glutamate transmission or induce potassium mediated
postsynaptic hyperpolarization as it does in wildtype mice (Masino et al., 2002). Fast
excitatory postsynaptic potentials (fEPSPs) are increased in hippocampal slices in AIR
wildtype mice after the application of AIR antagonists, however this effect is abolished
in AIR knockout mice (Johansson etal., 2001). Together, these results show that
adenosine does indeed act at the A] receptor to modulate glutamatergic
neurotransmission. Further, AIR knockout mice lack paired pulse facilitation in
hippocampal slices but do not show any differences in long-term potentiation (LTP) or
long-term depression (LTD) when compared to wildtype mice (Gimenez-Llort et al.,
2005). ‘

Behaviorally, A1 receptor knockout animals have similar performance to wildtype
animals in sensorimotor reﬂex tests, including tests of grasping ability, visual placing,

and locomotor activity (Johansson et al., 2001). However, AIR knockout mice show

15

impairments in anxiety and habituation tests. AIR knockout and AI R heterozygous mice
show increased measures of anxiety, with knockout mice entering less and spending less
total time in the lighted area during dark-light testing (J ohansson et al. , 2001). Similar
results were found using an elevated plus maze to test anxiety (Gimenez-Llort et al. ,
2002). In a six-arm radial tunnel maze, AIR knockout mice habituated signiﬁcantly less
to the maze, and exhibited greater total activity and arm explorations than wildtype mice
(Gimenez-Llort et al. , 2005). However, in open-ﬁeld testing AIR knockout mice show
decreased exploratory behavior (Gimenez-Llort et al., 2002). Strikingly, AIR knockout
mice do not show any deﬁcits in various paradigms of the water maze, indicating no
differences in spatial or working memory in these mice (Gimenez-Llort et al. , 2005).

Genetic manipulation of the A2A receptor also results in changes in learning and
memory behaviors. A2A receptor knockout mice show improved spatial recognition
memory in a Y-maze task, which is a spatial two-trial recognition task (Wang et al.,
2006). A2A knockout mice also exhibit higher'anxiety and decreased exploratory
behavior (Ledent et al., 1997). Interestingly, transgenic rats with an over-expression of
A2A receptors in the brain exhibit working memory deﬁcits. Speciﬁcally, these rats
show impaired working memory in a novel object recognition task, a repeated acquisition
Morris water maze task, and a six-arm radial tunnel maze task when compared to
wildtype rats (Gimenez-Llort et al., 2007). These ﬁndings suggest that activation of
adenosine receptors decreases memory abilities, while reduced receptor function or a lack
of receptors results in memory improvements.

Mice over-expressing adenosine kinase, a key enzyme involved in adenosine

degradation, also exhibit alterations in learning and memory behaviors (Yee et al. , 2007).

16

An increase in adenosine kinase activity functions to reduce the overall levels of
adenosine available within the brain, thereby reducing adenosine receptor activity.
Transgenic mice over-expressing adenosine kinase exhibit deﬁcits in both working and
reference memory Morris Water maze tasks, as well as deﬁcits in acquisition of a
conditioned response to a conditioned stimulus (Yee et al., 2007). These ﬁndings are in
contrast to studies using knockout mice and suggest that involvement of adenosine in
learning and memory behaviors may not be as simple as originally believed, i.e. reduced
receptor activity enhances memory, while increased receptor activity impairs memory.
In knockout and transgenic studies, compensatory mechanisms must be taken into
account when considering the behavioral changes that occur in the brain due to the lack
or altered expression of a functional receptor. In A2A heterozygous and homozygous
mice A1 receptor expression is altered such that in A2A heterozygous mice there is a
decrease in A1 receptor radioligand binding in the hippocampus, whereas in A2A
knockout mice there is an increase in A1 receptor binding (Snell et al., 2000). Up- or
down-regulation of the A1 receptors in the hippocampus may be a factor in the learning
and memory related changes observed in A2A receptor knockout mice. Unfortunately,
there are not many studies available discussing the compensatory mechanisms that may

occur following loss of the A1 or A2A adenosine receptors in these animals.

G. Pharmacological Manipulation of Al and A2A Receptor Function
Pharmacological manipulation of A1 and A2A receptors with selective agonists
and antagonists has also provided insight into the function of these receptors and their

role in learning and memory. A1 receptor agonists disrupt learning and memory in rats

17

(Zanindast & Shafaghi, 1994; Ohno & Watanabe, 1996). Administration of the Al
receptor agonist N6-cyclohexyladenosine (CHA) into the rat dorsal hippocampus
signiﬁcantly impairs working memory, whereas an A2A agonist fails to induce working
memory deﬁcits (Ohno & Watanabe, 1996). Infusion of an adenosine agonist to the
posterior cingulate cortex also impairs memory function in an inhibitory avoidance task
(Pereira et al., 2005). The posterior cingulate cortex is an area known to contain
projections to the hippocampus and is currently believed to modulate hippocampal
ﬁrnction. In summary, increased A1 receptor activity in the hippocampus and related
cortical areas impairs learning and memory.

On the other hand, blockade of A1 or A2A receptors with speciﬁc antagonists
facilitates learning and memory behaviors (Hauber & Bareiss, 2001). In adult rats,
caffeine facilitates learning and memory behaviors in paradigms such as the Morris water
maze (Angelucci et al., 2002). The facilitative effects of caffeine on learning and
memory are thought to result from its non-selective antagonism of A1 and A2A
receptors. Similarly, antagonism of A1 receptors by DPCPX (8-cyclopentyl-1,3-
dipropylxanthine) infusion to the posterior cingulate cortex facilitates memory retention
in a inhibitory avoidance task (Pereira et al. , 2002). Therefore, blockade of adenosine
receptors and the concurrent reduction of receptor activity in the hippocampus and related
cortical areas results in enhanced learning and memory behaviors.

The behavioral changes that result from altering adenosine receptor function align
well to the effects of adenosine agonists and antagonists on long-term potentiation (LTP)
and long-term depression (LTD). Al receptor agonists impair LTP in the hippocampus

while A1 receptor antagonists enhance LTP (de Mendonca & Ribeiro, 1990). The A1

18

receptor antagonist DPCPX greatly enhances the effects of LTP, likely due to an increase
. in excitatory neurotransmitter release which occurs after adenosine antagonism in the
hippocampus (de Mendonca & Ribeiro, 2000). A link between adenosine receptor
function and learning memory is beginning to be established, however, it is unknown
whether chronic prenatal caffeine exposure can cause long-term changes in adenosine

receptor function that may alter learning and memory.

H. Perinatal Caffeine Exposure and Adenosine Receptor Regulation

Determination of the effects of perinatal caffeine exposure on adenosine receptor
expression has proved difﬁcult due the effects of dose and timing of caffeine exposure.
For instance, Al receptor expression is up-regulated following neonatal caffeine
exposure (Guillet & Kellogg, 1991) but down-regulated following fetal caffeine exposure
(Leon et al., 2002). In animals treated with caffeine during postnatal days 2-6, a
signiﬁcant increase in A1 receptor binding is detectable in the cortex, hippocampus, and
cerebellum, indicating an increase in receptor expression that persists into adulthood
(Guillet & Kellogg, 1991).

In the fetal brain, A1 receptors are signiﬁcantly decreased following chronic
exposure to caffeine (by 49%) or theophylline (by 41%) (Leon et al. , 2002). However,
the same prenatal treatment results in increased A] receptor mRNA (Leon et al. , 2002).
Levels of the Gi proteins, to which A1 receptors are coupled, are also found to decrease
in the fetal brain following caffeine (by 15.2 %) or theophylline (by 18.4 %) treatment,
along with an increase in Gi mRNA (Leon et al., 2005a; Leon et‘a1., 2005b). The levels

of mGluRs are also decreased with caffeine (by 27 %) or theophylline (by 15 %)

l9

treatment in fetal brain tissue (Leon er a1. , 2005a). It is hypothesized that the decrease in
mGluRs may be due to increased glutamate release resulting in over-stimulation of
glutamate receptors and consequently receptor down-regulation.

Some studies show no changes in A1 and A2A receptor expression following
developmental caffeine exposure. Aden et al. (2000) found that a moderate dose of
caffeine administered throughout gestation does not signiﬁcantly alter A1 or A2A
receptor levels in fetal and newborn brains.

If Caffeine exposure during brain development alters adenosine receptor
expression in the hippocampus and cortical areas related to learning and memory, the
changes in adenosine receptor expression may alter learning and memory behaviors.
Accordingly, it is reasonable to hypothesize that if developmental caffeine exposure
increases adenosine receptor expression in the brain this may result in impairments in
learning and memory, whereas decreases in adenosine receptor expression may result in

enhanced learning and memory.

I. The Hippocampus and Learning and Memory

The role of the hippocampus in learning and memory behaviors is well known
and the integrity of hippocampal circuits is essential for spatial and temporal memory
paradigms (Eichenbaum, 2000). Spatial learning, assessed by tasks such as the Morris
water maze, is disrupted in rats with hippocampal lesions (Morris et al. , 1982).
Similarly, both recognition memory and working memory tasks are disrupted following
lesions of the temporal lobe (Clark et al., 2000; Laroche et al., 2000). In humans,

hippocampal damage is correlated to the extent of a patient’s retrograde and anterograde

20

amnesia, such that damage to only the CA1 regions produces mild memory loss, while
widespread damage throughout the hippocampus produces severe memory loss (Squire &
Zola, 1996). Administration of selective antagonists directly into the hippocampus prior
to analysis of hippocampal-dependent behavior has indicated speciﬁc receptors involved
in learning. Antagonism of NMDA receptors with AP5 results in impaired spatial
memory in the water maze, but does not impair visual discrimination behavior (Morris et
al., 1986). This was one of the ﬁrst studies to identify a role for NMDA receptors in
speciﬁc learning behaviors. Furthermore, as the dose of APS is increased in the
hippocampus and presumably more NMDA receptors are blocked, performance in the
water maze is increasingly impaired (Davis et al., 1992). Hippocampal infusion of
NMDA antagonists also impairs behavior in step-down inhibitory avoidance tasks
(Roesler et al., 1998), delayed matching-to-place tasks (Steele & Monis, 1999), and
numerous working memory tasks. I

Hippocampal neurons are well known for demonstrating LTP. Although a direct
link between learning and memory and LTP has yet to be elucidated, there is robust
evidence that these processes are integrated. Many studies found that in addition to
impairments of learning and memory behavior, NMDA receptor blockade impairs LTP
(Davis et al. , 1992; Tsien et al., 1996). Following increasing doses of APS administered
directly into the hippocampus, both spatial learning and LTP are impaired in a correlated,
dose-dependent fashion. Thus, doses that do not impair LTP in vivo are not associated
with spatial learning deﬁcits, while doses that do impair LTP are associated with
impaired spatial learning (Davis et al., 1992). Mice lacking NMDA receptors in the CA1

region of the hippocampus exhibit both spatial memory deﬁcits and impaired LTP (Tsien

21

et al., 1996). Conversely, over-expression of the NR28 subunit of the NMDA receptor in
the mouse hippocampus facilitates learning and memory behaviors in a novel object
recognition task, a spatial water maze task, and a contextual fear-conditioning task.
Hippocampal slices from NR2B subunit over-expressing mice also exhibit enhanced LTP
(Tang et al. , 1999). The link between NMDA receptor function, LTP, and learning and
memory behaviors is widely studied and supported, although the direct relationship
between these processes is still not fully elucidated.

Calcium inﬂux through postsynaptic NMDA receptors is an integral step in the
signaling cascades that mediate many forms of hippocampal neuroplasticity (Miyamoto,
2006). LTP is the most studied form of synaptic plasticity in the hippocampus. The
molecular mechanisms underlying LTP are complex and not completely understood, but
a few key components have been elucidated. In a simpliﬁed version of this molecular
cascade, glutamate is released in the hippocampus, which activates postsynaptic NMDA
receptors. Activation of NMDA receptors allows calcium entry into the postsynaptic cell,
where calcium activates numerous calcium-dependent kinases. Activation of
calcium/calmodulin-dependent protein kinase II (CaMKII) is believed to be necessary for
LTP in the hippocampus (Lynch, 2004; Malenka & Bear, 2004). Phosphorylation of
CaMKII has been associated with both increased AMPA receptor conductance and
insertion of AMPA receptors into the postsynaptic density following LTP (Derkach et al. ,
1999; Liao et al., 2001). Activation of CaMKIV and mitogen-activated protein
kinase/extracellular signal-related kinase pathway (MAPK/ERK) modulates
phosphorylation of CAMP response element binding protein (CREB), which is involved

in gene transcription (Lynch, 2004; Malenka & Bear, 2004). Importantly, inhibitors of

22

ERK have been shown to block both LTP and CREB activation (Impey et al., 1998).
Furthermore, CREB activation has been shown to be an important transcription factor for
long-term protein synthesis, an essential step for maintenance of synaptic plasticity
(Lynch, 2004; Malenka & Bear, 2004). CREB-dependent protein synthesis plays a role
in development of new spines, 3 morphological change that occurs following LTP and is
believed to account for increased synaptic efﬁcacy. Disruption of ERK or CREB
activation completely abolishes LTP, making these signaling molecules an important
measure of activation of this signaling cascade (Lynch, 2004).

As discussed previously, the high levels of adenosine receptor expression in the
hippocampus play a prominent role in modulating learning and memory as well as LTP,
likely through glutamate mediated mechanisms. In the hippocampus, adenosine receptor
activation reduces glutamatergic synaptic transmission by decreasing presynaptic calcium
inﬂux and thereby reducing vesicle release (Gundlﬁnger et al. , 2007). The inhibition of
adenosine by antagonists such as caffeine increases glutamate release thereby activating
postsynaptic glutamatergic receptors (Wang, 2007). Because glutamate receptors play an
important role in hippocampal plasticity as well as learning and memory, an increase in
glutamate receptor activity may alter NMDA receptor activity as well as activation of
ERK and CREB. These changes could lead an overall change in LTP as well as learning
and memory.

Induction of LTP in the neonatal hippocampus has a developmental proﬁle that
varies by age and distinct signaling pathways. It has been demonstrated that LTP cannot
be induced in the rat hippocampus prior to postnatal day 8, with LTD being the

predominant form of synaptic plasticity during this stage of neonatal development

23

(Dudek & Bear, 1993). However, other studies show induction of LTP as early as
postnatal day 2 in the rat hippocampus if postsynaptic depolarization and presynaptic
stimulation occur simultaneously (Durand et al., 1996). Postsynaptic depolarization is
likely necessary because during early postnatal development NMDA receptor activity
predominates whereas AMPA receptors are functionally silent (Liao et a1. , 1999).

In the neonatal hippocampus, distinct signaling pathways are associated with
induction of LTP. The CaMKII pathway, which is essential during adult LTP, is not
needed during neonatal LTP induction (Yasuda et al., 2003). Rather, the cAMP-
dependent PKA pathway seems to play a major role in induction of LTP prior to
postnatal day 8 (Yasuda et al., 2003). The CAMP-dependent PKA pathway may be
directly affected by caffeine in neonates. As discussed previously, blockade of the A1.
and A2A adenosine receptors by caffeine results in an increase or decrease, respectively,
of adenylyl cyclase and cAMP activity. An alteration in cAMP activity is coupled to
activation of the PKA pathway since PKA activation is dependent on cAMP activity.
Although the regulation and timing of the developmental switch between the neonatal
CAMP-dependent PKA pathway and the CaMKII dependent pathway is unknown, it is
possible that developmental caffeine exposure is modulating this switch and interfering

with LTP induction in the developing brain.

J. Perinatal Development of the Hippocampus
The development of the hippocampus and dentate gyrus occurs during gestation
and early neonatal periods, making the hippocampus especially susceptible to

developmental drug exposure. In the rodent hippocampus, all of the pyramidal cells and

24

neurons in Ammon’s horn are formed prior to birth (Bayer, 1980a, b). Dentate gyrus
development takes place between gestational day 14 and the second postnatal week of
life, with a peak of activity around postnatal day 5-7 (Schlessinger et al., 1975).
Neurogenesis of dentate gyrus granule cells occurs postnatally and continues at a reduced
rate throughout life. Granule cells increase their dendritic trees until PN14 after which
the branches are sculpted to form adult-like morphological features (Rahimi & Claiborne,
2007 ).

In humans, hippocampal development occurs over a prolonged period of
gestation. By gestational week 15, neurogenesis of pyramidal neurons is visible in
Ammon’s horn and adult-like morphological features of the CAI-CA3 ﬁelds are
established by the 23-25th gestational week (Arnold & Trojanowski, 1996a, b). Similar
to the ﬁndings in rats, neurogenesis in the granule cell layer of the dentate gyrus in
humans begins around gestational week 14 (Humphrey, 1967) and continues postnatally
(Seress et al., 2001). Prenatal exposure to drugs during these critical stages of
hippocampus and dentate gyrus formation can alter the overall morphology of both
structures in treated offspring.

Adenosine, acting at the Al receptors, depresses glutamatergic activity and
regulates giant depolarizing potentials within the hippocampus during the ﬁrst week of
postnatal life in rodents (Saﬁulina et al., 2005). Giant depolarizing potentials are a trait
of the developing hippocampus and are characterized by recurrent spontaneous
membrane depolarizations (Ben-Ari et al. , 1989). Giant depolarizing potentials are
instrumental in shaping the synaptic connections of the hippocampal circuitry and are

mediated by the actions of GABA and glutamate in the developing brain (Saﬁulina et al. ,

25

2006). Alterations in the frequency of giant depolarizing potentials may effect the
development of hippocampal neural networks (Dzhala et al. , 1999). If caffeine
antagonizes the A1 adenosine receptors in the hippocampus, such that adenosine can no
longer regulate or depress the frequency of the giant depolarizing potentials, the
development of the hippocampus may be negatively affected. Further, glutamatergic
NMDA receptors play a fundamental role in activity-dependent migration, synapse
formation, and functional connectivity of the developing hippocampus (Ben-Ari &
Holmes, 2006). Caffeine may increase glutamate release in the developing hippocampus

thereby disrupting these processes.

K. Glutamate Receptors and Development

Glutamate receptors play a critical role in numerous developmental processes
including axon migration, synaptogenesis, and synaptic plasticity. Alterations of
glutamatergic activity during development may interrupt or postpone critical
developmental processes. Thus, it is necessary to understand the normal ontogeny and
regulation of glutamate receptors during development in order to determine if the
regulation of these critical receptors is altered by prenatal caffeine exposure.

lonotropic glutamate receptors are categorized into three subclasses based on the
ligand speciﬁcity of each receptor. The three subclasses include the N-methyl-D-
aspartate (NMDA), a-amino-3-hydoxy-5-methyl-4-isoxazole propionic acid (AMPA),
and kainate receptors, and are speciﬁc for ligands of the same name. The NMDA
receptors are comprised of tetrarneric assemblies of the obligate NR1 subunit along with

NR2 subunits (NR2A-NR2D) and NR3 subunits (NR3A-NR3B) (Dingledine et al.,

26

1999). The majority of NMDA receptors in the hippocampus are NR1/NR2A or
NR1/NR28 subunit assemblies. AMPA receptor subunits are classiﬁed as GluRl-GluR4
and normally form tetrameric complexes of GluR2 with either GlurRl or GluR3
(Dingledine et al., 1999). Kainate receptor subunits are classiﬁed as GluRS-GluR7 or
KAI-KA2 and form either homomeric or heteromeric four unit assemblies. NMDA and
AMPA receptors play a predominant role in development and thus the expression and
ﬁrnction of these receptors is of primary importance.

During prenatal and postnatal development, the ionotropic glutamate receptors are
expressed in both temporally and regionally speciﬁc patterns. Glutamate receptor
expression is elevated during development when compared to expression in the mature
brain, with peak levels of expression corresponding to neonatal periods of synaptogenesis
(Ritter et al., 2002). The NR1 subunits are found prenatally throughout the brain with
expression detectable in the rat cortex as early as embryonic day 14 (Babb et al., 2005).
Peak levels of NR1 expression occur in the neonatal rat hippocampus between postnatal
days 3-14 (Ritter etal., 2002). The NR2A and NR2B subunits are differentially
expressed, allowing distinct pharmacological properties mediated by each receptor to.
predominate during different periods of brain development. By embryonic day 14, NR28
expression is detectable in the rat cortex with almost double the level of expression of the
NR1 subunit (Babb et al., 2005). High levels of NR2B expression are maintained until
postnatal day 7 after which levels begin to decline (Ritter et al., 2002). In contrast,
NR2A expression is very low prenatally, but begins to increase around postnatal day 10
in the rat hippocampus and reaches peak levels of expression around postnatal day 21

(Ritter et al., 2002). The NR2A subunit is characterized by fast channel kinetics and

27

increased channel open probability, whereas the NR2B subunit is characterized by slower
channel kinetics and decreased channel open probability (Lau & Zukin, 2007). The
developmental switch from NRZB to NR2A receptor expression and the distinct
pharmacological properties of each subunit is believed to play an important role in the
activation of different intracellular cascades and synaptic plasticity (Lau & Zukin, 2007).
However, the exact role and mechanisms behind the switch in subunit expression have
remained elusive.

AMPA receptor subunits are also detectable in the embryonic brain. GluRl
subunit expression is detectable in the rat cortex as early as embryonic day 15 (Martin et
al., 1998). Unlike the developmental proﬁles of other subunits, GluRl expression
remains fairly constant in the hippocampus during prenatal and postnatal development
(Ritter et al., 2002). Expression of GluR2/3 subunits is also detectable during embryonic
development. In the rat hippocampus, GluR2 subunits show a rapid increase in
expression immediately following birth, which peaks at postnatal day 10 and then rapidly
declines to prenatal levels of expression by postnatal day 18 (Ritter et al., 2002). GluR3
subunit expression in the rat hippocampus is very low around birth and increases to peak
levels around postnatal day 14 after which it remains elevated into adulthood (Ritter et
al., 2002).

Given the complex and dynamic patterns of NMDA and AMPA receptor
expression in the developing brain, it is not surprising that developmental drug exposure
during these time periods can interrupt the delicate timing of receptor expression. For
instance, gestational and neonatal ethanol exposure signiﬁcantly increases NR2A subunit

expression on postnatal day 10 in both the hippocampus and cortex (Nixon et al. , 2002,

28

2004). However, perinatal alcohol exposure reduces radioligand binding of the NMDA
antagonist MK-801 in the hippocampus and cortex of treated rats (Diaz-Granados et al. ,
1997) and decreases NMDA-receptor mediated calcium transients in cultured neurons
(Lee et al., 1994; Gruol et al., 1998) suggesting a decrease in expression or function of
NMDA receptors. Neonatal nicotine exposure also alters the expression of NMDA
receptor subunits in the developing brain. Exposure to nicotine on postnatal days 8-12
increases NR2A mRN A in the rat forebrain and enhances the duration of NMDA-
mediated EPSPs (Hsieh et al. , 2002). Similarly, prenatal exposure to morphine alters the
kinetic properties of NMDA receptors in neonatal hippocampal pyramidal neurons (Yang
et al., 2000). It is currently unknown whether perinatal exposure to caffeine induces
alterations in glutamate receptor expression or function. However, as the previous
studies elucidate, changes in NMDA receptor expression can lead to changes in
intracellular calcium signaling which may disrupt calcium-dependent signaling cascades.

Glutamate receptors also play a critical role in modulating excitotoxic and
apoptotic cell death. Increased NMDA receptor activation promotes excitotoxic cell
death (Choi, 1987; Chen et al., 1997), whereas prolonged inhibition of activity induces
apoptosis (Olney, 2002). Neonatal caffeine exposure (50 mg/kg administered 3 times per
day) induces cell death in the rat dentate gyrus, thalamus, hypothalamus, and caudate
putamen as early as one day following treatment (Kang et al., 2002). Similarly, cortical
(Kang et al., 2002) and cerebellar (Gepdiremen et al., 1998) cultures exposed to 250uM
caffeine exhibit signs of apoptosis following one day of continual exposure. Attenuation
of cell death occurs after administration of cycloheximide, an anti-apoptotic drug that

inhibits protein synthesis, to cortical cultures (Kang et al., 2002) and nimodipine, a

29

calcium channel blocker, to cerebellar cultures prior to caffeine exposure (Gepdiremen et
al., 1998). These ﬁndings suggest that developmental caffeine exposure may induce cell

death via glutamate and calcium dependent mechanisms.

L. Prenatal Caffeine and Development

Given the numerous neuromodulatory roles of adenosine receptors during
development and the widespread use of caffeine during pregnancy, it is surprising that
there is not an abundance of research on the effects of prenatal exposure to caffeine. It is
estimated that 70-95% of women consume caffeine during pregnancy (Fredholm et al.,
1999). Caffeine can readily cross the placental and blood-brain barriers to reach the
developing fetus and its brain (Amaud, 1993). The altered half-life of caffeine in the
fetus and pregnant women may result in prolonged effects of caffeine in the fetal brain.
In non-pregnant adults, the half-life of caffeine varies between 2-6 hours while in
pregnant women the half-life is increased to 10-20 hours (Aldridge et al., 1981). The
effect of caffeine is further prolonged in the fetirs and newborn infants due to a lack of
cytochrome P-450 activity (Aranda et al., 1979b) resulting in a half-life of caffeine
ranging from 50 to 100 hours (Amaud, 1993). Thus, once an expectant mother has
cleared caffeine from her system, the drug or its metabolites may accumulate and remain
in the fetus for days.

The prolonged exposure of the fetus to caffeine can result in an accumulation of
caffeine and its metabolites in the developing brain. After a single dose of caffeine (5 or

25 mg/kg) to pregnant rats, the levels of the primary metabolites of caffeine,

theophylline, theobromine and paraxanthine, are three times greater in the fetal brain than

30

in the plasma (Wilkinson & Pollard, 1993). In the brain, the main target of caffeine is
antagonism of adenosine receptors. The expression of Al receptor mRNA is detectable
using in situ hybridization by gestational day 14 (GD14) in the developing rat brain
(Weaver, 1996), and by GD20 the distribution of Al receptor mRNA resembles that of an
adult rat. Functional binding of the Al receptor can be detected in low levels throughout
the brain by gestational day 14 using radioligand binding with the antagonist DPCPX
(Rivkees, 1995). Although adenosine is not classiﬁed as a neurotransmitter, it has
considerable neuromodulatory effects, including general inhibition of neuronal activity
(Fisone et al., 2004). As discussed previously, the A1 and A2A adenosine receptors bind
caffeine with high afﬁnity and are considered to be crucial for the stimulatory effects of
caffeine (Fredholm et al., 1999). It is currently unknown whether caffeine acts in the
developing brain via the same mechanisms known to mediate the actions of caffeine in
the adult brain. Further, the effects of chronic moderate prenatal caffeine exposure are
not well understood. This area needs further examination given the numerous studies that
have indicated that high dose prenatal caffeine exposure is harmful to developing
offspring. I

High-dose prenatal exposure to caffeine has detrimental effects on development.
Mothers who consumed greater than 300 mg/day of caffeine during the third trimester of
pregnancy have an increased risk of delivering a small for gestational age infant (Vik et
al., 2003). In rodents, decreased birth weight and delayed eye opening results from in
utero caffeine exposure to as little as 25 mg/kg/day (West et al., 1986). In humans,
intake of caffeine decreases birth weight by approximately 28 g per 100 mg of daily

caffeine consumption (Bracken et al., 2003). At doses equivalent to one to two cups of

31

coffee, prenatal caffeine exposure does not alter brain weight, although caffeine at higher
doses of between two and twelve cups of coffee does decrease fetal brain weight (Tanaka
et al., 1983, 1987; Yazdani et al., 1990). Congenital malformations occur only at supra-
pharrnacological doses (consumption of 10-14 cups of coffee) and thus, caffeine exposure
in humans in not considered teratogenic (N ehlig & Derby, 1994). Overall, the scientiﬁc
literature has mainly focused on the effects of caffeine on birth weight, brain weight,
timing of birth, rates of miscarriage, and congenital malformations and most reports
indicate that moderate prenatal caffeine exposure has only mild effects on these gross
measures of health.

The developmental effects of low to moderate caffeine exposure are beginning to
be studied and understood. Recently it has been shown that daily neonatal exposure to
caffeine (50 mg/kg) results in a persistent increase of dendritic length and arborization of
neurons located in the prefrontal cortex (Juarez-Mendez er al., 2006). Intact neonatal
hippocampal cultures exposed to acute caffeine (50 uM) exhibit seizure-like activity
following a brief anoxic episode, whereas in control hippocampi, anoxia only induces a
depression of neuronal activity (Dzhala et al., 1999). This epileptogenic activity resulted
from blockade of A1 receptors. Furthermore, caffeine withdrawal in newborns
chronically exposed to prenatal caffeine has become of interest. Newborns of mothers
who chronically consumed high levels of caffeine throughout their pregnancies exhibit
increased irritability, jitteriness, and vomiting during the ﬁrst week after birth (Martin et
al., 2007). Prenatal caffeine exposure is also a risk factor for respiratory problems in
newborns, with exposed infants exhibiting episodes of apnea (suspended breathing) and

tachypnea (abnormally fast breathing) (Saadani-Makki er al. , 2004). Lastly, neonatal

32

exposure to low doses of caffeine signiﬁcantly decreases the protein and cholesterol
concentrations in brains of treated rat pups (N akamoto er al. , 1988). Interestingly, this
effect occurs following low dose, but not high dose treatment, suggesting that low dose
exposure may have distinct effects from high dose exposure. Further studies are needed
to fully understand the long-term effects of chronic developmental caffeine exposure.
Learning and memory behaviors are also altered following perinatal caffeine
exposure. Prenatal administration of 150 mg/L caffeine throughout gestation induces
hyperactivity in open ﬁeld testing in adult offspring (Grimm & Frieder, 1988). Higher
doses of 300 or 450 mg/L induce learning disabilities. Similarly, prenatal doses of 28
mg/kg, but not 36 mg/kg, induce increased locomotor and rearing activity in open-ﬁeld
testing in adult offspring (Hughes & Beveridge, 1990). Interestingly, young rats exposed
prenatally to caffeine have altered locomotor responses to the NMDA receptor antagonist
MK-801 (da Silva et al., 2005). This suggests that prenatal caffeine exposure leads to a
persistent change in NMDA receptors, perhaps due to increased glutamate release after
antagonism of A1 receptors. Neonatal exposure to caffeine also signiﬁcantly decreases
passive avoidance learning and retention. Caffeine exposure to 15-20 mg/kg/day on
postnatal days 2-6 results in slowed avoidance learning in juvenile rats (Fisher & Guillet,
1997). In adult rats, retention but not learning is altered following developmental
caffeine exposure to 15-20 mg/kg/day on postnatal days 2-6. Adult female rats have
signiﬁcantly improved retention of passive avoidance learning whereas male rats have
signiﬁcantly reduced retention when tested 72 hours after training (Fisher & Guillet,

1997). Recently, it has been shown that caffeine exposure the same administration on

33

postnatal days 2-6 signiﬁcantly decreases 24 hour memory retention of passive avoidance

in both males and females when tested on postnatal days 35-37 (Pan & Chen, 2007).

M. Summary

Caffeine is the most widely used psychostimulant in the world. The use of this drug
during pregnancy is common but controversial. Although women are currently advised to
reduce caffeine intake throughout pregnancy, it is estimated that 70-95% of pregnant women
continue to consume the drug. The widespread use of caffeine is of concern because it
crosses both the placental and blood brain barriers to accumulate in the developing brain.
Furthermore, the half-life of caffeine is greatly prolonged in the developing fetus, being
twenty fold that of an adult. Caffeine primarily acts as an adenosine receptor antagonist and
therefore may increase glutamate neurotransmission in the developing brain, speciﬁcally in
the developing hippocampus due to an abundance of A1 adenosine receptors in this brain
region. Increased glutamate neurotransmission during development may alter the expression
and function of NMDA receptors, which may induce long-term changes in calcium-
dependent intracellular cascades, learning and memory behaviors, and hippocampal anatomy.
In order to elucidate the effects of gestational caffeine exposure, pregnant rats were exposed
to chronic moderate doses of caffeine. Rats are an appropriate model given the similar
pharmacokinetics of caffeine metabolism between rats and humans. Metabolic body weight
(=body weightA3/4) calculations were used based on ﬁndings that 20 mg/kg in a rat is

equivalent to 4-6 cups of coffee in a 70 kg human (Nehlig & Derby, 1994).

34

N. Hypotheses
l. Caffeine and its metabolites accumulate in the developing brain during chronic

prenatal exposure.

2. Accumulation of caffeine and its metabolites in the fetal brain antagonizes A1
adenosine receptors, which increases glutamate neurotransmission in the developing
hippocampus and alters the expression of hippocampal NMDA receptors and calcium-

dependent signaling cascades.

3. Alterations in glutamate-mediated hippocampal signaling results in long-term changes

in hippocampal-dependent learning and memory behaviors.

4. Alterations in glutamate-mediated hippocampal signaling results in long-term changes

in hippocampal anatomy.

0. Specific Aims
Specific Aim 1: Does chronic prenatal caffeine exposure result in an accumulation of
caffeine and its primary metabolites in the fetal brain?

Chapter 2 presents the results from high-performance liquid chromatography (HPLC)
studies that determined the concentrations of caffeine and its primary metabolites in maternal
and fetal samples of brain and plasma following chronic administration of caffeine. Maternal
rats were provided 75, 150, or 300 mg/L ad libitum caffeinated water throughout gestation.

On gestational day 21-23, fetal and maternal samples of plasma and brain were collected for

35

HPLC determination of caffeine, theobromine, theophylline, and paraxanthine
concentrations. The results reveal that caffeine and its metabolites signiﬁcantly accumulate

in the fetal brain following chronic maternal caffeine consumption.

Speciﬁc Aim 2: Does chronic prenatal caffeine exposure alter the expression of
hippocampal NMDA receptors and calcium-dependent signaling?

Chapter 3 presents the results of Western blot and calcium imaging studies in which
the expression of NMDA receptors and calcium-dependent signaling in the hippocampus
were studied. For the Western blot analyses, the protein expression of the NR1, NR2A, and
NR2B NMDA receptors were measured in hippocampal tissue collected from control and
prenatal caffeine-treated offspring on postnatal days 1, 5, 10, and 20. Western blot analyses
were also performed on hippocampal tissue from control and prenatal-caffeine treated
offspring acutely treated with caffeine on postnatal days 10 and 20 to determine whether
there were any long-term changes in calcium-dependent cascades following prenatal caffeine
exposure. For this study, levels of phospho-CREB and phospho-ERK proteins were
determined. Lastly, calcium imaging was performed on hippocampal cultures from control
and caffeine-treated pups. Glutamate was brieﬂy applied during the live cell recordings to
determine the resulting peak calcium transient. The results reveal that total protein
expression of the NR1, NR2A, and NR2B subunits are altered by prenatal caffeine at
different postnatal ages. Further, peak calcium transients were reduced in caffeine-treated
neurons, however pCREB and pERK activation were not found to be signiﬁcantly different

in control and treated animals.

36

Speciﬁc Aim 3: Does chronic prenatal caffeine exposure alter hippocampal-
dependent learning and memory behaviors in adult rats?

Chapter 4 presents the results of behavioral studies in which the novel object
recognition task, radial arm maze, and Morris water maze were used to determine if
chronic prenatal caffeine exposure alters learning and memory behaviors in adult rats.
Pregnant rats were provided 75 mg/L caffeine throughout gestation and the offspring
underwent behavior testing on postnatal days 110-150. The results reveal that novel
object recognition and radial arm maze behaviors are impaired in caffeine-treated

offspring while Morris water maze behaviors are unimpaired.

Speciﬁc Aim 4: Does chronic prenatal caffeine exposure alter the volume of the
hippocampus in juvenile and adult rats?

Chapter 5 presents the results of stereological studies using the Cavalieri
estimator method to determine whether hippocampal volume is altered in caffeine-
exposed offspring. Pregnant rats were provided 75 mg/L caffeine throughout gestation
and tissue from offspring was collected on postnatal day 30 for juvenile analysis and
postnatal day 150 for adult analysis. The results reveal a signiﬁcant increase in total
hippocampal volume in caffeine-treated juvenile rats, which does not persist into

adulthood.

37

Chapter 2. Caffeine and its Active Metabolites are Elevated in the Fetal Brain after

Chronic Gestational Exposure

A. Introduction

Caffeine, a methylxanthine, is the most widely used psychoactive drug in the
world due to its presence in coffee, tea, colas, and chocolate (Fredholm et al., 1999). It is
estimated that 70-80% of pregnant women regularly consume caffeine during pregnancy
despite ongoing disagreement about the developmental consequences of fetal exposure
(Pastore & Savitz, 1995). Human and animals studies have shown that moderate prenatal
caffeine consumption does not induce gross malformations of the fetus (Linn er al., 1982;
Kurppa et al., 1983). However, in humans and non-human primates, maternal caffeine
consumption has been positively correlated with increased risk of fetal death late in
gestation (Gilbert et al., 1988; Bech et al., 2005; Weng et al., 2008).

Methylxanthines cross both the placental and blood brain barriers to reach the
developing fetal brain (Ikeda et al., 1982; Tanaka et al., 1984; Amaud, 1993). Within the
fetal brain, these substances have a prolonged duration of action due to an increased fetal
half-life (Aranda et al., 1979a). The increased half-life of caffeine is due to both a lack of
cytochrome P-450 1A2 (CYP1A2) metabolism in the fetus and slowed maternal
metabolism of caffeine during the second and third trimesters of pregnancy (Aldridge et
al., 1981; Knutti et al., 1981; Brazier et al., 1983). Slowed fetal elimination of caffeine
and its metabolites may result in accumulation of these substances to detrimental levels

during chronic prenatal exposure.

38

In humans and rodents, caffeine is demethylated to three primary metabolites:
paraxanthine, theobromine, and the0phylline. These metabolites are all biologically
active, and like caffeine, function as adenosine receptor antagonists at physiological
doses (Fredholm et al., 1999). There are four known adenosine receptors, which include
the A1, A2A, A28, and A3 receptors (Fredholm et al., 2000). Of the adenosine
receptors, A1 receptors have the highest levels of expression, with mRNA detectable by
gestational day 14 in the rat hippocampus and cerebellum (Rivkees, 1995; Weaver,
1996). Adenosine is a critical neuromodulator during brain development playing a major
role in axon guidance (Turner et al., 2002), white matter development (Rivkees et al. ,
2001), and neuronal inhibition (F isone et al., 2004). Chronic prenatal caffeine exposure
has been shown to alter fetal brain development by reducing adenosine receptor
expression (Aden et al. , 2000; Leon et al., 2002), altering DNA and protein content
(Tanaka et al. , 1983), and delaying developmental outcomes such as locomotor reﬂexes
(Tchekalarova et al., 2005).

Previous reports in the literature have shown that as little as a single maternal dose
of caffeine can lead to elevated levels of methylxanthines in the fetal brain. For example,
a single maternal dose of 5 or 25 mg/kg caffeine by gavage on gestational day 20 results
in metabolite levels three times higher in the fetal brain than in fetal plasma (Wilkinson
& Pollard, 1993). Similarly, an intramuscular injection of radiolabeled caffeine causes
signiﬁcantly higher accumulation of caffeine in the fetal brain as compared to the
maternal brain after 8 hours (Galli et al. , 1975). Elevated levels of caffeine and its
metabolites in the fetal brain following maternal intake are of concern due to the varied

actions of high concentrations of caffeine which inhibit phosphodiesterase activity and

39

induce intracellular calcium release (Fredholm et al., 1999). The use of injections for
caffeine administration may result in bypassing ﬁrst-pass metabolism inithe liver,
resulting in fetal levels that are not comparable to those reached after oral maternal
intake.

The purpose of the present study was to determine the concentrations of caffeine
and its metabolites in maternal and fetal samples of brain and plasma following chronic
oral ad libitum ingestion of caffeine. Maternal rats consumed approximately 30—60
ml/day of 75, 150, or 300 mg/L caffeinated water. On gestational day 21-23, fetal and
maternal samples of plasma and brain were collected for high performance liquid
chromatographic (HPLC) determination of caffeine and metabolite concentrations.
Individual male and female fetal samples were collected to determine if there is a sex
difference in prenatal caffeine metabolism. The results reveal that caffeine and its

metabolites are elevated in the fetal brain of both male and female rats following chronic

maternal caffeine consumption.

40

B. Materials and Methods

Animals

Fourteen pregnant Sprague-Dawley rats (Charles River Laboratories, Wilmington,
MA, USA) were provided either caffeinated tap water (75, 150, 300 mg/L; n=4 per
treatment) or tap water alone (n=2) from gestational day 4 until gestational day 21-23 and
intake was recorded daily. On gestational day 21-23, the dams were anesthetized and
removal of embryonic pups was performed. Plasma and whole brain samples were
collected for four pups from each litter (2 males and 2 females) for a total of n=16 pups
per dose. Plasma and whole brain samples were also collected from each dam.

Trunk blood from pups and dams was collected in heparinized glass capillary
tubes, immediately transferred to centrifuge tubes, and centrifuged at 1000 g for 10 min
at 4°C. Plasma was removed and stored at -80°C. Whole brains from pups and dams
were removed and immediately ﬂash frozen in 2-methylbutane. Brains were stored at -

80°C.

Chemicals

Caffeine, theophylline, 7-(B-hydroxyethyl)theophylline, theobromine, and 1,7-
dimethylxanthine (paraxanthine) were obtained from Sigma, St. Louis, MO. Methanol
and glacial acetic acid (99% HPLC grade) were obtained from Fisher Scientiﬁc.
Stock solutions of caffeine (1 mg/ml), theobromine (1 mg/ml), theophylline (1 mg/ml),
paraxanthine (1 mg/ml), and 7-(B-hydroxyethyl)theophylline (1 mg/ml) were prepared in

0.1% acetic acid. All stock solutions were stored at -80°C and have been previously

41

shown to be stable for at least 6 months (Hartley et al., 1985). Standards (0-10 pg/ml)
were prepared by serial dilution in 0.1% acetic acid from the stock solutions prior to each

HPLC run.

Plasma Extraction

Waters C18 solid-phase extraction preparation columns (1.5 ml capacity) were
used for rapid sample extraction. The columns were pre-conditioned by drawing 2 x 2 ml
methanol followed by 2 x 2 ml water through the column. A single plasma sample (100
pl) and the internal standard 7-(B-hydroxyethyl)theophylline (133 ng/ 100 pl) were pulled
into the column and allowed to equilibrate for 2 min. The column was washed with 2 x 2
ml water followed by 3 x 1 ml acetone. The eluent was evaporated to dryness using a
speed-vac (Savant, SVC100) and reconstituted in 150 ul of 1% glacial acetic acid-
methanol (83:17) mobile phase by vortexing. Injections of 20 111 were made for each

plasma sample.

Brain Preparation

Extraction of caffeine and its metabolites in fetal brain was performed by
homogenizing each brain in 400 pl tissue buffer (0.1 M phosphate/citrate containing 15%
methanol) for 30 sec. Homogenized tissue was sonicated brieﬂy (5 x 1 sec) and
centrifuged at 18,000g for 10 min. The supernatant was immediately removed and
brought to a ﬁnal volume of 550 pl using tissue buffer. Injections of 50 pl were made for

each brain sample extract.

42

Extraction of caffeine and its metabolites in maternal brain was performed by
homogenizing each brain in 4 m1 tissue buffer for l min. Homogenized tissue was
sonicated brieﬂy (10 x 1 sec) and centrifuged. The supernatant was removed and brought
to a ﬁnal volume of 5.5 ml using tissue buffer to maintain the same ratio used for the fetal

brain extraction. Injections of 50 pl were made for each brain sample.

Protein Determination

One ml of 1.0N NaOH was added to the tissue pellet for determination of protein
levels using a standard Lowry assay (Lowry et al., 1951). Each sample was sonicated
and vortexed until dissolved. Five pl of each protein sample was added to 95 pl NaOH.
A single ml of Reagent A (50 ml sodium carbonate, 500 pl cupric sulfate, and 500 pl
KNA tartrate) was added to each protein sample for 10 min followed by 100p] of Folin-
Phenol Reagent for 30 min. Protein standards (12.5, 25, 50 pg/10pl) were run in
duplicate. Protein levels were assayed using a spectrophotometer set to a wavelength of

700nm.

Extraction Recovery

Normal rat plasma used to determine extraction efﬁciency was spiked with 100
11ng each of internal standard 7-(B-hydroxyethyl)theophylline, caffeine, theophylline,
theobromine, and paraxanthine. For extraction, 100 pl of the mix solution was passed
through the preparation column using the same procedure described above. The
extraction efﬁciencies of both acetone and methanol were tested. Extraction efﬁciency

was determined as [(peak height of the sample)/(peak height of standard) x 100].

43

HPLC Analyses of Caffeine and its Major Metabolites

A reverse-phase C18 column was used with a Waters 510 HPLC system, Waters
717 autosarnpler, and Waters 996 Photodiode array for analyses of caffeine and its
metabolites. A mobile phase of 1% glacial acetic acid-methanol (83:17) was ﬁltered and
degassed prior to use (Hartley et al., 1985). A ﬂow-rate of 1.0 ml/min was used with UV
detection wavelength set at 270nm. Waters Millennium32 Version 4.0 software was used
to process the data.

Levels of caffeine and its primary metabolites were determined in plasma using
20 pl injections of plasma extracts for each sample. Assays were performed on
individual plasma samples from control and caffeine exposed male and female pups.
High (1,250 ng/ml) and low (156.3 ng/ml) standards were injected between every four
consecutive samples to ensure retention times were maintained. The peak height for
caffeine and its metabolites was used and calculated as [(sample peak height/standard
peak height)*(dilution factor)*(l/extraction efﬁciency)* 10] to determine the
concentration of each peak of interest (ng/ml).

Levels of caffeine and its primary metabolites were determined in brain using 50
pl injections of brain extracts for each sample. Assays were performed on each
individual fetal and maternal brain. High (625 ng/ml) and low (312.5 ng/ml) standards
were injected between every four consecutive samples. The peak height was used and
calculated as [(sarnple peak height/standard peak height)*(dilution factor)*1000]/[sample

protein content].

44

Statistics

Statistical assessment of weekly water intake was performed using two-way
repeated measures analysis of variance (week, treatment). Linear regression was used to
determine R2 values for standard curves. One-way analysis of variance for sex was used
to compare concentrations of each metabolite in male and female offspring. Two-way
analysis of variance (dose, age) was used to compare the concentrations of caffeine and
each metabolite in maternal and fetal brain and plasma across the three doses. A level of
p<0.05 was required to obtain statistical signiﬁcance. All analyses were performed using

the SYSTAT program (version 11.0).

45

C. Results

Chromatograms and Standard curve

A representative chromatogram resulting from a 50 pl injection of a 625 ng/ml
standard mixture containing theobromine, paraxanthine, theophylline, B-
hydroxyethyltheophylline and caffeine, respectively, is shown in Figure 2-1A. Standards
of 19.5-10,000 11ng concentration were prepared by serial dilution in 0.1% acetic acid
prior to each analysis. No methylxanthine peaks were observed in control
Chromatograms. Further, there were no discemable differences between Chromatograms
of males and females. There was excellent resolution between peaks with approximate
retention times of: 5.8 min for theobromine, 8.9 min for paraxanthine, 10.1 min for
theophylline, 11.4 min for the internal standard B-hydroxyethyltheophylline, and 17.4
min for caffeine. 7

As shown in Figure 2-2, standard curves for caffeine and its metabolites were
made by comparing the peak height across concentrations ranging from 78.1—5000 ng/ml.
The standard curves for caffeine and each metabolite were found to be linear with
correlation coefﬁcient (R2) values above 0.9998 for each standard (Figure 2-2). The

threshold of detection for all metabolites was 39.1 ng/ml.

46

Extraction Eﬁciency

Using the extraction methods described for plasma, acetone was determined to
have higher extraction efﬁciency with less variance (99-101% :t 0.8; Table 2-1) for
caffeine, its metabolites, and the internal standard as compared to methanol (88-91% i

5.4). Thus, acetone was used as our ﬁnal eluent.

47

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a s3 1 aezaaooﬁ a .3.» r 8335.5 a .66 r 05:858.; : benign“
20>» 058.55%:on some Sm moﬁt mouse? 2:. .mibmce. some 9 Sta Eon 058m .XL .0 E

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.30 o
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48

Standard Curve

DTheobromine

25000 ‘ _ a
OParaxanthrne

E 20000 - ATheophylline .
.g’ X Internal Standard ,
I 15000 . OCaffeine ,
it *
«a 10000 - : ,
n- v

 

5000 ‘ , ;
-.Asgt

 

0 I . ‘ I W, ' r I r r l
0 1000 2000 3000 4000 5000
Concentration (nglml)

Figure 2-2. Standard curves for caffeine, its metabolites, and the internal standard [3-
hydroxyethyltheophylline. Standards were prepared in 0.1% acetic acid across
concentrations of 78. l— 5000 ng/ml. Linear correlation coefﬁcients (R2) above 0.9998
were found for each standard by comparing the peak height across each concentration.
The lowest limit of detection was 39.1 ng/ml.

49

Acetone Methanol

Theobromine 101.3 i 0.7 91.1 :1: 5.3
Paraxanthine 101.1 :E 0.7 91.1 i 5.1
Theophylline 101.0 i 0.6 91.0 :1: 5.3
Internal Standard 101.5 :1: 0.8 91.0 :t 5.4
Caffeine 99.2 :1: 0.3 88.3 :t 5.4

Table 2-1. The plasma extraction efﬁciency of acetone and methanol were compared for
each methylxanthine. Data represent [(peak height of the sample/peak height of
standard) x 100] :1: SEM.

50

Gestational Caffeine Consumption

Pregnant dams were administered ad libitum tap water (n=2) or caffeinated water
(n=12) from gestational day (GD) 4 through GD 21-23 with intake recorded daily. Daily
intake ranged from approximately 40-60 ml/day. The average water intake in control and
caffeine-treated dams signiﬁcantly increased each week (Figure 2-3; within-group
analyses — [F(2,124)=27.425, p<0.01]) and showed a signiﬁcant main effect of treatment
[F(l ,62)=4.136, p<0.05], but did not show a week by treatment interaction. To determine
whether the main effect of treatment was a result of the different doses of caffeine
administered, a two-way repeated measures analysis of variance for week by dose was
performed. This analysis also revealed a signiﬁcant within-group effect of week
[F(2,120)=46.012, p<0.01] as well as a signiﬁcant main effect of dose [F (3,60)=10.262,
p<0.01] but no week by dose interaction. Interestingly, the dams administered 300 mg/L
caffeine had signiﬁcantly reduced intake each week when compared to the control and
lower dose administrations (Figure 2-3). Reduced intake may be a result of the

increasingly bitter taste of caffeine in the drinking water at higher doses.

Methylxanthine Concentrations in Male and Female Offspring
One-way analysis of variance for sex revealed no differences in brain
concentrations between male and female offspring for caffeine, theobromine,
paraxanthine, or theophylline levels. Similarly, there were no sex differences in plasma
concentrations between male and female offspring for caffeine or any of its metabolites.
Based on these ﬁndings, data from male and female offspring were combined for all

other analyses.

51

Methylxanthine Concentrations in Fetal and Maternal Brain and Plasma

Two-way analysis of varian5ce (dose, age) revealed a signiﬁcant difference
between fetal and maternal brain concentrations for caffeine and each metabolite.
Interestingly, two-way analysis of variance (dose, age) for plasma concentrations did not
reveal any differences between maternal and fetal levels for caffeine or any metabolite.

Concentrations of caffeine were found to be signiﬁcantly greater in the fetal brain
than the maternal brain [F(1,54)=4.406, p<0.05] however there was no signiﬁcant effect
of dose or dose by age interaction (Figure 2-4A). Caffeine concentrations were
approximately 3 times higher in the fetal brain than the maternal brain with mean fetal
concentrations of 6.2, 21.8, and 35.6 rig/mg and mean maternal concentrations of 1.9, 7.9,
and 10.3 rig/mg across the three doses of 75, 150, and 300 mg/L caffeine, respectively.

Plasma concentrations of caffeine were found to signiﬁcantly differ across dose
[F(2,54)=4.623, p<0.02] however there was no difference between maternal and fetal
levels nor was there a dose by age interaction (Figure 2-4B). Plasma caffeine levels
increased with dose in both dams and their offspring such that mean maternal and fetal
levels were 199.9 and 185.1 ng/ml at the 75 mg/L dose, 740.6 and 644.4 ng/ml at the
150mg/L dose, and 1013.4 and 1366.6 ng/ml at the 300 mg/L dose.

As shown in Figure 2-5A, concentrations of theobromine were also found to be
signiﬁcantly greater in the fetal brain than the maternal brain [F (1,54)=47.332, p<0.001].
Furthermore, there was a signiﬁcant effect of dose [F(2,54)=l6.023, p<0.001] as well as
a signiﬁcant dose by age interaction [F(2,54)=7.3 l 7, p<0.002]. Theobromine

concentrations were the highest of any methylxanthine in the fetal brain with mean fetal

concentrations of 11.7, 22.6, and 51.7 ng/mg and mean maternal concentrations of 1.1,

52

2.6, and 8.7 ng/mg across the three doses of 75, 150, and 300 mg/L caffeine, respectively.
Concentrations of theobromine are 6-11 times higher in the fetal brain than the maternal
brain at the doses used.

Plasma concentrations of theobromine were found to signiﬁcantly differ across
dose [F (2,54)=40.227, p<0.001], however there was no difference between maternal and
fetal levels nor was there a dose by age interaction (Figure 2-SB). Plasma theobromine
levels increased by dose in both dams and their offspring such that mean maternal and
fetal levels were 525.5 and 491.5 ng/ml at the 75 mg/L dose, 892.1 and 802.3 ng/ml at the
150mg/L dose, and 2616.0 and 2212.3 ng/ml at the 300 mg/L dose. Similar to brain
concentrations, plasma concentrations of theobromine were higher than caffeine and the
other metabolites.

Paraxanthine concentrations were also found to be signiﬁcantly higher in the fetal
brain when compared with the maternal brain [F(1,54)=26.243, p<0.001] and there was
also a signiﬁcant main effect of dose [F (2,54)=7.293, p<0.002] as well as a signiﬁcant
dose by age interaction [F (2,54)=5.035, p<0.01] (Figure 2-6A). Across the three
incremental doses of caffeine, mean fetal brain concentrations of paraxanthine were 5.6,
6.2, and 19.9 ng/mg, whereas mean maternal brain concentrations were 0.3, 0.6, and 1.7
ng/mg. Thus, concentrations of paraxanthine are approximately 11-18 times higher in the
fetal brain when compared to the maternal brain depending on the dose despite overall
concentrations of paraxanthine being the lowest in the brain compared to the other
methylxanthines.

Plasma concentrations for paraxanthine did not differ between maternal and fetal

samples, however there was a main effect of dose [F (2,54)=23. 1 9, p<0.001] but no dose

53

by age interaction (Figure 2-6B). Mean maternal and fetal levels of plasma paraxanthine
were 172.5 and 142.6 ng/ml at the 75 mg/L dose, 338.6 and 234.9 ng/ml at the 150 mg/L
dose, and 1016.3 and 868.6 ng/ml at the 300 mg/L dose.

Similar to caffeine and the other metabolites, theophylline levels were also found
to be signiﬁcantly elevated in the fetal brain when compared to the maternal brain
[F(l,54)=28.488, p<0.001] (Figure 2-7A). A signiﬁcant main effect of dose
[F (2,54)=16.124, p<0.001] and dose by age interaction [F(2,54)=6.299, p<0.004] were
also found. Theophylline concentrations were lower than caffeine and theobromine
_ levels in the fetal brain with mean fetal concentrations of 7.2, 10.8, and 28.0 ng/mg and
mean maternal concentrations of 2. 1 , 4.7, and 7.6 rig/mg across the three doses of 75,
150, and 300 mg/L caffeine, respectively. Overall, fetal brain concentrations of
theophylline are approximately 3 times higher than maternal brain concentrations at each
dose.

Plasma concentrations of theophylline did not differ between dams and their
offspring however there was a signiﬁcant effect of dose [F(2,54)=31.977, p<0.001] but
no dose by age interaction (Figure 2-7B). Mean maternal and fetal levels of plasma
theophylline were 249.5 and 208.4 ng/ml at the 75 mg/L dose, 453.1 and 373.8 ng/ml at

the 150 mg/L dose, and 1392.0 and 1071.0 11ng at the 300 mg/L dose.

54

F H d U r

Water Consumption

807

\l
C?

I)
(no:
00

pr.

take (m

 

40-i

In

Daily
00
f

I

m
‘1’

~......- Control —9— 150 mg/L

1

—\
C.’

- O- 75 mg/L - ¥-- 300 mg/L

41

 

O

 

r l |

Week 1 Week 2 Week 3

Figure 2-3. Maternal intake of either untreated tap water (control) or 75, 150, or 300
mg/L caffeine-treated tap water across each gestational week. Data represent the mean
daily intake :t SEM for each week. Water intake increased throughout gestation in all
treatment groups, however dams consuming 300 mg/L caffeine-treated water drank
signiﬁcantly less than dams treated with either no or low caffeine doses.

55

50-
45-
40s
35-
30-i

204

Concentration (ng/mg)

 

Caffeine Brain

 

 

M,/l~“” r
ﬂ’/-/l l l

 

Concentration (ng/ml)
3
o
C?

75 mglL 150 mg/L 300 mgIL

Caffeine Plasma

 

 

 

 

 

75 mgIL 150 mgIL 300 mglL

 

' Fetal ,.....-..-.- Maternal

Figure 2-4. Concentrations of caffeine in fetal and maternal (A) brain and (B) plasma
following prenatal caffeine exposure. Data represent the mean caffeine concentration (:1:
SEM) reached following exposure to each dose. Chronic prenatal exposure to caffeine
results in a signiﬁcant accumulation of caffeine within the fetal brain when compared to
the maternal brain, however there is no difference in caffeine concentrations in fetal and
maternal plasma.

56

60— Theobromine Brain

Concentration (ng/mg)

 

0 ﬂ” Mr # Mm r I
75 mglL 150 mglL 300 mglL

 

 

4000— Theobromine Plasma

Concentration (ng/ml)
N
o
o
O
J

 

 

 

75 mglL 150 mglL 300 mglL

+ Fetal Maternal

 

Figure 2-5. Concentrations of theobromine in fetal and maternal (A) brain and (B)
plasma following prenatal caffeine exposure. Data represent the mean theobromine
concentration (i SEM) reached following exposure to each dose. Chronic prenatal
exposure to caffeine results in a signiﬁcant accumulation of theobromine within the fetal
brain when compared to the maternal brain that increased by dose. No differences
between fetal and maternal plasma concentrations of theobromine were found.

57

30- Paraxanthine Brain
25-
20-
15—

10—
5- I——

Concentration (ng/mg)

 

“ ..vrvr“ I
K M a
q U3 1‘...” v'”

',‘ __,.. W ..— m.‘ g.
”V

l l l

75 mglL 150 mglL 300 mglL

 

2000— Paraxanthine Plasma
1750-

..x ..L
N UT
01 O
O O
l I

1000 a
750 —
500—
250 —

Concentration (ng/ml)

 

 

 

75 mglL 150 mglL 300 mglL

I Fetal .. Maternal

 

Figure 2-6. Concentrations of paraxanthine in fetal and maternal (A) brain and (B)
plasma following prenatal caffeine exposure. Data represent the mean paraxanthine
concentration (:1: SEM) reached following exposure to each dose. Chronic prenatal
exposure to caffeine results in a signiﬁcant accumulation of paraxanthine within the fetal
brain when compared to the maternal brain that increased by dose. No differences
between fetal and maternal plasma concentrations of paraxanthine were found.

58

Concentration (ng/ml)

30-

25~

20-

‘15-

Concentration (ng/mg)

Theophylline Brain

 

 

 

 

75 mglL 150 mglL 300 mglL

Theophylline Plasma

,.
/

 

 

75 mglL 150 mglL 300 mglL

{ Fetal 5:;- Maternal

 

Figure 2-7. Concentrations of theophylline in fetal and maternal (A) brain and (B)
plasma following prenatal caffeine exposure. Data represent the mean theophylline
concentration (:1: SEM) reached following exposure to each dose. Chronic prenatal
exposure to caffeine results in a signiﬁcant accumulation of theophylline within the fetal
brain when compared to the maternal brain that increased by dose. No differences
between fetal and maternal plasma concentrations of theophylline were found.

59

D. Discussion

This report demonstrates that chronic maternal intake of caffeine results in 3-fold
or higher concentrations of caffeine and its metabolites in the fetal brain than in the
maternal brain. In the fetal brain, theobromine had the highest concentration followed by
lower concentrations of caffeine, paraxanthine, and theophylline, respectively. All of the
metabolites showed higher concentrations in the fetal brain as compared to the maternal
brain. Thus, the total methylxanthine level in the fetal brain was greatly elevated in
comparison to the methylxanthine levels in the maternal brain. In contrast, there were no
differences between caffeine and metabolite levels in the fetal and maternal plasma.
Importantly, these ﬁndings indicate that measurements of plasma levels in newborns
underestimate elevated methylxanthine levels in the brain.

Increased doses of caffeine administered to dams resulted in a dose dependent
increase in caffeine and metabolite levels in the fetal brain while there were only slight
increases in methylxanthine levels in the maternal brain across the three doses. Thus, the
increase of caffeine and metabolite levels in the maternal brain appears linear, whereas in
the fetal brain, the increase in methylxanthine concentration following exposure to the
300 mg/L dose appears to be non-linear. This suggests that repeated exposure to
caffeine, especially to high doses, may lead to an accumulation of caffeine and its
metabolites in the fetal brain due to decreased clearance of these drugs during
development.

The elevated levels of caffeine in the developing fetal brain are likely due to

caffeine’s prolonged half-life in the fetus. Once ingested, caffeine is rapidly absorbed

60

from the gastrointestinal tract and distributed throughout the body within 45 min (Marks
& Kelly, 1973; Bonati et al., 1982; Arnaud, 1993). Caffeine is a hydrophobic compound
and as such freely crosses all biological membranes including the placental and blood-
brain barriers (Ikeda et al. , 1982; Kimmel et al. , 1984; Tanaka et al. , 1984;. Abdi et al. ,
1993). In non-pregnant adults, the half-life of caffeine is estimated to be 2-4 hours
(Amaud, 1987). During the second and third trimesters of pregnancy, the half-life of
caffeine is estimated to be 10-15 hours in expectant mothers (Aldridge et al., 1981;
Knutti et al., 1981; Brazier et al., 1983). In the developing fetus, the immature
cytochrome P-450 1A2 metabolic pathway greatly prolongs the half-life of caffeine and
its metabolites (Aranda et al., 19793). It is estimated that the half-life of caffeine in
newborn infants can range from 50-100 hours, with higher estimates for the half-life of
caffeine in the fetus (Aranda et al., 1977; Parsons & Neims, 1981; Amaud, 1993). Thus,
once an expectant mother clears caffeine from her system, the drug could maintain
substantial levels in the fetus for prolonged periods of time.

The results of the present study demonstrate that methylxanthine levels are higher
in the fetal brain than in maternal brain despite the fact that there are no differences
between matemal and fetal plasma. The cause of sequestration of methylxanthines in the
fetal brain is not well understood. It is known that caffeine freely crosses the placental
barrier to reach the fetus. Bidirectional transport of caffeine across the placental barrier
would allow equal concentrations of methylxanthines in maternal and fetal plasma
(McCall et al., 1982; Kimmel et al., 1984). Elevated levels of caffeine and its
metabolites in the fetal brain suggest an inability of caffeine to readily cross from the

fetal brain back into circulation. This could be due to various factors including saturation

61

of carrier-mediated facilitated diffusion systems, protein binding of caffeine in the brain,
or caffeine sequestration in relatively lypophillic brain tissue. However, further research
is needed to understand the cause of caffeine sequestration in the fetal brain.

Although a direct comparison between the brain and plasma levels in the current
data may not be warranted, it does appear that levels of caffeine and its metabolites are
higher in plasma than in brain in both the dam and her offspring. Therefore, it is possible
that there may be a mechanism to keep caffeine from entering the brain that is better
functioning in the adult compared to the fetus, and thus fetal levels of methylxanthines
are elevated compared to maternal levels. However, such a mechanism blocking caffeine
from entering the brain has not been found.

It is also important to consider the effects of cytochrome P-450 metabolism in the
liver and brain on the elevated levels of methylxanthines in the fetus. As mentioned
previously, in the developing fetus, the immature cytochrome P-450 1A2 metabolic
pathway greatly prolongs the half-life of caffeine and its metabolites (Aranda et al. ,
1979a). Cytochrome P-450 enzymes are found in brain tissue and therefore may
contribute to the elevated levels of methylxanthines seen in the fetal brain. However, the
levels of these enzymes in the brain are only 0.5-2% of that found in the liver (Hedlund et
a1, 2001). Thus, slowed metabolism of P-450 enzymes in the brain is likely making a
very minor contribution to the elevated levels of caffeine in the fetal brain.

Chronic maternal intake of caffeine resulted in elevated levels of caffeine as well
as elevated levels of theobromine, theophylline, and paraxanthine in the fetal brain.
Because these metabolites are biologically active, the levels of each metabolite must be

considered to determine the cumulative effects of xanthines on the developing brain. In

62

rats, theobromine is the major metabolite of caffeine whereas in humans paraxanthine is
the major metabolite (Eteng et al., 1997; Fredholm et al., 1999). Thus, it is not
surprising that concentrations of theobromine were found to be higher than
concentrations of caffeine and the other metabolites in dams and their offspring.
Furthermore, caffeine and theobromine are poorly bound by plasma proteins leaving 70-
85% of these drugs free to cross the blood-brain barrier (Bonati et al., 1984; Wilkinson &
Pollard, 1993). This is unlike theophylline which readily binds plasma proteins and thus
does not enter the brain as rapidly (Birkett et al., 1985; Ararnayona et al., 1991; Stahle et
al., 1991). Within the brain, paraxanthine is equally as potent as caffeine at antagonizing
adenosine receptors, whereas theophylline is thought to be 3-5 times more potent than
caffeine (Benowitz et al. , 1995; Daly, 2000). Theobromine is a weaker adenosine
antagonist than caffeine, however has considerable effects as a vasodilator (Shi & Daly,
1999; Daly, 2000). Thus, the availability of these substances to enter the fetal brain and
potency of these substances within the brain are important determinants of the total
effects of xanthines on brain development.

Caffeine and its major metabolites function as adenosine receptor antagonists
(Fredholm, 1980; Daly et al., 1981; Daly & Fredholm, 1998). Elevated levels of these
methylxanthines within the fetal brain may cause persistent antagonism of adenosine
receptors during brain development. Chronic antagonism of these receptors may alter
normal brain development since adenosine is one of the earliest neuromodulators in the
developing brain (Rivkees et al. , 2001). Current reports in the literature have shown that
disruption of adenosine receptor ﬁrnction during development alters neonatal respiratory

patterns (Montandon et al., 2008), hypoxic response (Back at al., 2006), seizure activity

63

(Tchekalarova et al., 2006, 2007), and white matter development (Turner et al., 2002). In
humans, maternal consumption of greater than 200 mg caffeine per day has been shown
to double the risk of miscarriage (Weng et al., 2008). It has also been found that elevated
levels of paraxanthine in fetal plasma are positively correlated with intrauterine growth
restriction and small for gestational age newborns (Grosso et al., 2006). Thus, elevated
levels of these adenosine receptor antagonists in the fetal brain throughout gestation may
have detrimental effects on fetal development.

Caffeine metabolism in rats and humans is very similar, making rats an excellent
animal model for studying the effects of prenatal caffeine exposure (Amaud, 1993). To
maintain relevance to humans, doses of 0.075 to 0.3 g/L were utilized in the present
study. These doses are well within the range of human caffeine intake and low compared
to most caffeine studies, which use a range of 0.2-1 g/L (Aden et al., 2000; Leon et al. ,
2002; Picard et al., 2008). One drawback to using a prenatal rat model is the short length
of gestation (21-24 days). Newborn rats are altricial, or underdeveloped, in comparison
to newborn humans, and it is common to consider postnatal days 1-7 in the rat parallel to
the third trimester of development in humans (Clancy et al., 2007). In the present study,
embryonic pups were used to avoid experimental issues with timing of birth and lack of
control over neonatal exposure when suckling. This resulted in a much shorter period of
exposure than would be seen in the human fetus if the mother drank caffeine throughout
pregnancy. It is likely that the longer duration of exposure in humans could lead to even
higher fold accrunulation in the fetal brain.

In summary, moderate maternal caffeine intake leads to accumulation of caffeine

and its bioactive metabolites in the fetal brain. Elevated levels of these methylxanthines

64

may disrupt normal adenosine receptor function during development. High maternal
caffeine consumption may lead to toxic levels of caffeine and its metabolites in the fetal
brain increasing the risk of miscarriage and fetal growth restriction. Assays of newborn
plasma may not be indicative of levels of caffeine in the newborn brain. Thus, expectant
women should be cautioned to limit or discontinue the use of caffeine during pregnancy
due to the prolonged half-life of the drug in the fetus and the potential for accumulation

in the fetal brain.

65

Chapter 3. Chronic Gestational Caffeine Exposure Alters Glutamate Receptor

Expression and Calcium Inﬂux in the Developing Hippocampus

A. Introduction

Glutamate receptors play a role in numerous developmental processes including
activity-dependent migration, synapse formation, and synaptic plasticity. lonotropic
glutamate receptors include the N-methyl-D-aspartate (N MDA), or-amino-3-hydoxy-5-
methyl-4-isoxazole propionic acid (AMPA), and kainate receptor subtypes. Of these,
NMDA receptors exhibit increased calcium permeability making their activation critical
to the induction of numerous calcium-dependent cascades that are integral to processes
such at long-term potentiation (Dingledine et al. , 1999). NMDA receptors are tetrameric
complexes comprised of the NR1, NR2A-D, and NR3A-B subunits. The hippocampus
predominantly contains NR1/NR2A and NR1/NRZB oligomers (Dingledine et al., 1999).
During development, the expression of these subunits is temporally and spatially
regulated. The obligate NR1 subunit is expressed during both embryonic and postnatal
periods (Lujan et al. , 2005). The NRZB and NR2A subunits undergo a developmental
switch in expression such that NRZB subunits are highly expressed during pre- and early
postnatal periods after which NR2A subunit expression predominates (Lujan et al. ,
2005). Each of these subunits confers distinct kinetic properties allowing the function of
NMDA receptors to be modulated during development.

The dynamic timing of NMDA receptor expression during critical periods of

brain development makes these receptors susceptible to the effects of perinatal drug

66

exposure. Ethanol exposure during prenatal or postnatal development alters many
aspects of NMDA receptor expression and function. In the neonatal hippocampus and
cortex, NR2A receptor expression is increased in ethanol exposed animals (Nixon et al. ,
2002, 2004). NMDA receptor-mediated calcium transients are reduced in cerebellar
granule neurons after four consecutive days of postnatal ethanol exposure (Gruol et al. ,
1998). Similarly, there is reduced binding of the NMDA receptor antagonist MK-801 in
the hippocampus of ethanol-treated animals (Diaz-Granados et al., 1997). Like ethanol,
nicotine and morphine also induce alterations in the expression and function of NMDA
receptors in the developing brain. Exposure to nicotine increases NR2A mRN A in the rat
forebrain and enhances the duration of NMDA-mediated excitatory postsynaptic
potentials (EPSPs) (Hsieh et al., 2002). Similarly, prenatal morphine exposure alters the
kinetic properties of NMDA receptors in neonatal hippocampal pyramidal neurons (Yang
etal,2000)

Given the effects of numerous commonly used drugs on NMDA receptor function
and expression, it is surprising that prenatal exposure to caffeine and its potential effects
on the glutamatergic system have not been previously investigated. Caffeine is the most
commonly consumed psychostimulant drug in the world and an estimated 70-95% of
women consume this drug during pregnancy (Fredholm et al., 1999). Caffeine readily
crosses both the placental and blood brain barriers to reach the fetus (Amaud, 1993) and
accumulates in the developing brain (Chapter 2; Wilkinson & Pollard, 1993). Caffeine
acts as an adenosine receptor antagonist, and blockade of adenosine receptors by caffeine
results in an increase in neurotransmitter release and an overall increase in neuronal

excitability (Fredholm et al., 1999). Speciﬁcally, caffeine facilitates glutamate

67

transmission, particularly in brain regions with the high levels of A1 adenosine receptor
expression such as the cortex and hippocampus (Wang, 2007). Therefore, if prenatal
exposure is increasing glutamate neurotransmission in the developing brain, caffeine may
alter the development and function of critical glutamatergic systems.

The purpose of the present study was to determine the effects of prenatal caffeine on
NMDA receptor expression and calcium-dependent signaling in the hippocampus. Total
protein expression of the NR1, NR2A, and NRZB NMDA subunits was measured in
hippocampal tissue from control and prenatal caffeine-exposed offspring. Phospho-CREB
and phospho-ERK proteins were also analyzed following acute caffeine treatment on
postnatal days 10 or 20 in control and prenatal caffeine-exposed offspring to determine
whether there are long-term changes in calcium-dependent cascades due to prenatal caffeine
exposure. Additionally, calcium imaging was performed on hippocampal cultures from
control and caffeine-exposed pups. Glutamate was brieﬂy applied during live cell recordings
to determine the resulting peak calcium transients. The results reveal that protein expression
of the NR1, NR2A, and NR2B subunits is altered by prenatal caffeine, with each subunit
exhibiting increased or decreased expression during a speciﬁc time period. Further, peak
calcium transients were reduced in caffeine-treated neurons, whereas phospho-CREB and
phospho-ERK activation were not signiﬁcantly different between control and treated

animals.

68

B. Materials and Methods

Animals

Pregnant Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA,
USA) were provided either caffeinated tap water (75, 150, or 300 mg/L) or tap water
alone from gestational day 4 until birth and intake was recorded daily. For Western blot
analysis of total receptor expression, offspring were housed with each dam until postnatal
day 1, 5, 10, or 20 when hippocampal tissue was collected. Six pups were collected at
each time point for each treatment group. For Western blot analysis of phosphorylated
receptor expression, offspring were housed with each dam until postnatal days 10 or 20.
Pups were administered caffeine (10 mg/kg) orally by pipette and kept on 37°C heating
pads for 1.5 hours after which hippocampal tissue was immediately collected. Six pups
were collected at each time point for each treatment group. For calcium imaging studies,
dams were provided with caffeinated tap water (75mg/L) or tap water alone throughout
gestation, and male and female pups were collected on postnatal day 0 for hippocampal

cell cultures.

Primary Hippocampal Cell Cultures

Within 24 hours of birth, the bilateral hippocampi from male and female rat pups
were collected and separately cultured by prenatal treatment. Tissue was dissected into
HBSS+ (88ml sterile dHZO, 10ml 10X Hank’s Balanced Salt Solution (Gibco,
Invitrogen, Carlsbad, CA), lml 1.0M pH 7.3 HEPES buffer (Gibco), lml penicillin

(10,000 units) plus streptomycin (10,000 pg; Gibco)) and incubated in HBSS+ with 2.5%

69

trypsin for 15 min at 37°C. Supernatant was removed and tissue was rinsed twice with
HBSS+ followed by treatment with DNAase and dissociation by trituration. Dissociated
cells were seeded on 25mm poly-L-lysine coated coverslips which were stored in 100mm
petri dishes with 10ml plating medium (86ml MEM (Gibco), 10ml normal horse serum
(Gibco), 3m120% ﬁlter sterilized glucose, lml 100mM sodium pyruvate) and incubated
for 4 hours at 37°C, 5% CO2. Coverslips were then transferred to individual 35mm petri
dishes, which contained 3ml Neurobasal+ (lml 8-27 supplement (Gibco), lml penicillin
(10,000 units) plus streptomycin (10,000 pg; Gibco), 125ul L-glutamine (Gibco), and
48ml phenol red free Neurobasal (Gibco)). Cells were allowed to plate until day in vitro

3 (DIV3) when they were used for calcium imaging.

Calcium Imaging

Cultured hippocampal cells were used for calcium imaging on DIV3 as described
(Nunez & McCarthy, 2007). The cell permeable ﬂuorescent indicator fura-2
acetoxymethyl ester (fura-2-AM) was used for all imaging experiments. Cells were
loaded with fura-2-AM (3 pM) in DMSO (<0.5%) for 30 min at 37°C. Coverslips were
then placed on a perfusion chamber mounted to a Nikon Eclipse TE-2000U inverted
microscope and rinsed for 30 min with a physiological saline solution (PSS; 137ml NaCl,
20ml KCl, 4ml MgC12, 12ml CaC12, 40ml HEPES (Gibco), and 90ml 20% glucose
brought to a 4L with ultrapure dHZO) to remove extracellular dye. Illumination was
provided by a high-speed Sutter DG-5 wavelength switcher while ﬂuorescent images
were obtained using a Roper Coolsnap Cascade 5128 cooled CCD camera with on-chip

multiplication gain. Universal Imaging Metamorph/Metaﬂuor Imaging software (version

70

6.0) was used for all image acquisition and analysis. Baseline intracellular calcium
measurements were collected over a 5 min period during which cells were superfused
with PSS for each new ﬁeld of view. This was followed by a 1 min exposure to 2, 5, 10,
or 50 pM glutamate followed by a 5 min rinse with PSS (Figure 3-1). The maximal
intracellular peak calcium response, baseline calcium, and percentage of cells responding
to each treatment were analyzed. The intracellular calcium concentrations were not
directly measured and thus all references to peak levels are referring to the percent
change over baseline resting Fura-2 ratios. The relationship between the calcium ratio
and the overall change in the intracellular calcium concentration cannot be assumed to be
equal or directly proportional (i.e. a doubling of the ratio is not equivalent to a doubling

of the intracellular calcium concentration).

71

PSS rinse

Baseline Glutamate Agonist

 

A

       

 

 

 

 

Time (min)

Figure 3-1. The change in ratio between resting intracellular calcium and peak responses
following glutamate administration are shown in both the hippocampal cell cultures (A)
and a corresponding trace recording (8) during live cell imaging. Both the images and
trace represent the ratio of bound to unbound intracellular Fura-2.

72

Western Blot Analysis

On postnatal days 1, 5, 10, or 20, hippocampi were microdissected from both
hemispheres and immediately ﬂash frozen in 2-methylbutane. Tissue was weighed and
sonicated brieﬂy in 10pI/mg tissue buffer (50mM B-glycerophosphate, 1.5mM EGTA,
0.1mM sodium orthovanadate, lmM DTT, Complete mini protease inhibitor cocktail
(Roche)). Protein concentrations were normalized using a BCA protein assay kit (Pierce,
Rockford, IL). 100p] homogenate was added to an equal volume of SDS loading buffer
(lOmM Tris-HCl buffer pH 6.8, 10% glycerol, 2% sodium dodecyl sulfate, 0.01%
bromophenol blue, 5% B-mercaptoethanol). Samples were boiled for 10min,
electrophoresed on 10% Tris-Glycine gels (Invitrogen, Novex, Carlsbad, CA), and
transferred to PVDF membranes. For each primary antibody, overnight incubation at
4°C in 5% nonfat milk (Carnation, USA) made with PBS with 0.1% Triton-X was
performed. For analysis of total receptor expression, the following primary antibodies
were used: NR1 (1:2000, Cell Signaling), NR2A (1:1000, Cell Signaling), NR28
(1:1000, Cell Signaling). For analysis of receptor activity, phospho-CREB (1:2000, Cell
Signaling) and phospho-ERK1/2 (1:1000, Cell Signaling) were used. Membranes were
washed in PBS with 0.01% Triton-X before being incubated with HRP-conj ugated
secondary antibodies (1:5000, Pierce) at room temperature for 1 hour. Beta-Actin
(125000, Sigma) was used as a loading control. Proteins were visualized with enhanced
chemiluminescence (SuperSignal West Pico, Pierce, Rockford, IL) and exposed to ﬁlm
for several periods to obtain optimal signal. Membranes were probed multiple times by

rinsing with stripping buffer (1% SDS, 0.2% glycine, pH 2.0) before incubating with

73

another primary antibody. Quantiﬁcation of bands was performed using Scion Image

(NIH, Bethesda, Maryland, USA).

High Performance Liquid Chromatography

On PN5 and PNIO, trunk blood and whole brains from 4 pups in each treatment
group were collected for HPLC determination of caffeine and its metabolites. Blood was
collected in heparinized glass capillary tubes, immediately transferred to centrifuge tubes,
and centrifuged at 1000 g for 10min at 4°C. Plasma was removed and stored at -80°C.
Whole brains from pups and dams were removed and immediately ﬂash frozen in 2-
methylbutane. Brains were stored at -80°C.

Caffeine, theophylline, 7-(B-hydroxyethyl)theophylline, theobromine, and 1,7-
dimethylxanthine (paraxanthine) were obtained from Sigma, St. Louis, MO. Methanol
and glacial acetic acid (99% HPLC grade) were obtained from Fisher Scientiﬁc. Stock
solutions of caffeine (1 mg/ml), theobromine (1 mg/ml), theophylline (1 mg/ml),
paraxanthine (1 mg/ml), and 7-(B-hydroxyethyl)theophylline(1 mg/ml) were prepared in
0.1% acetic acid. All stock solutions were stored at -80°C and have been previously
shown to be stable for at least 6 months (Hartley et al., 1985). Standards (0-10 pg/ml)
were prepared by serial dilution in 0.1% acetic acid from the stock solutions prior to each
HPLC run.

Waters C18 solid-phase extraction preparation columns (1.5 ml capacity) were
used for rapid sample extraction. The columns were pre-conditioned by drawing 2 x 2 ml
methanol followed by 2 x 2 ml water through the column. A single plasma sample (100

pl) and the internal standard 7-(B-hydroxyethyl)theophylline (133 ng/ 100 pl) were pulled

74

into the column and allowed to equilibrate for 2 min. The column was washed with 2 x 2
ml water followed by 3 x 1 ml acetone. The eluent was evaporated to dryness using a
speed-vac (Savant, SVCIOO) and reconstituted in 150 ul of 1% glacial acetic acid-
methanol (83:17) mobile phase by vortexing. Injections of 20 111 were made for each
plasma sample.

Extraction of caffeine and its metabolites in fetal brain was performed by
homogenizing each PN5 brain in 1 ml tissue buffer (0.1 M phosphate/citrate containing
15% methanol) and each PNIO brain in 2 ml tissue buffer for 30 sec. Homogenized
tissue was sonicated brieﬂy (5 x 1 sec) and centrifuged at 18,000g for 10 min. The
supernatant was immediately removed and brought to a ﬁnal volume of 1.375 ml for PN5
samples and 2.75 ml for PNIO samples using tissue buffer. Injections of 50 pl were made
for each brain sample extract.

Two ml of 1.0N NaOH was added to the tissue pellet for determination of protein
levels using a standard Lowry assay (Lowry et al. , 1951). Each sample was sonicated
and vortexed until dissolved. Five pl of each protein sample was added to 95 pl NaOH.
A single ml of Reagent A (50ml sodium carbonate, 500 pl cupric sulfate, and 500 pl
KNA tartrate) was added to each protein sample for 10 min followed by lOOpl of F 01in-
Phenol Reagent for 30 min. Protein standards (12.5, 25, 50 pg/ 10pl) were run in
duplicate. Protein levels were assayed using a spectrophotometer set to a wavelength of
700nm.

A reverse-phase C18 column was used with a Waters 510 HPLC system, Waters
717 autosampler, and Waters 996 Photodiode array for analyses of caffeine and its

metabolites. A mobile phase of 1% glacial acetic acid-methanol (83:17) was ﬁltered and

75

degassed prior to use (Hartley et al., 1985). A ﬂow-rate of 1.0 ml/min was used with UV
detection wavelength set at 270nm. Waters Millennium32 Version 4.0 software was used
to process the data.

Levels of caffeine and its primary metabolites were determined in plasma using
20 pl injections of plasma extracts for each sample. Assays were performed on
individual plasma samples from control and caffeine exposed male and female pups.
High (1,250 ng/ml) and low (156.3 ng/ml) standards were injected between every four
consecutive samples to ensure retention times were maintained. The peak height for
caffeine and its metabolites was used and calculated as [(sample peak height/standard
peak height)*(dilution factor)*(1/extraction efﬁciency)* 10] to determine the
concentration of each peak of interest (ng/ml).

Levels of caffeine and its primary metabolites were determined in brain using 50
pl injections of brain extracts for each sample. Assays were performed on each
individual fetal and maternal brain. High (625 ng/ml) and low (312.5 ng/ml) standards
were injected between every four consecutive samples. The peak height was used and
calculated as [(sample peak height/standard peak height)*(dilution factor)*1000]/[sample

protein content].

Statistics

Statistical analyses of the calcium imaging data were performed using one-way
analysis of baseline and two-way analysis of variance (treatment, concentration) for the
peak calcium transient. The Western blot data were analyzed by one-way analysis of

variance by treatment for each individual blot. For all main effects, post-hoe (Tukey)

76

tests were performed. A level of p<0.05 was required to obtain statistical signiﬁcance.

All analyses were performed using the SYSTAT program (version 11.0).

77

C. Results

Peak Calcium Response to Glutamate

One-way analysis of resting intracellular calcium revealed no differences between
baseline calcium ratios in control and caffeine-exposed neurons (Figure 3-2). Two-way
analysis (treatment, concentration) of the peak calcium transient following brief
application of 2, 5, 10, or 50 pM glutamate revealed a signiﬁcant main effect of treatment
[F(1,1423)=7.129, p<0.01], a main effect of concentration [F(3,1439)=169.445, p<0.01],
and a treatment by concentration interaction [F(3,1439)=3.506, p<0.02]. To further
investigate these ﬁndings, the peak calcium response of each cell was classiﬁed as a low
responder (less than 20% change over baseline), medium responder (20—100% change
over baseline), or high responder (over 100% change from baseline). Two-way analysis
of variance (treatment, concentration) revealed no signiﬁcant effects of treatment or
concentration in low responding neurons (Figure 3-3A). In medium responding neurons,
there was a signiﬁcant main effect of glutamate concentration [F(3, 662)=25.605,
p<0.001], but no effect of treatment and no treatment by concentration interaction
(Figure 3-3B). In high responding neurons, there was a signiﬁcant main effect of
treatment [F (1 ,472)=4.902, p<0.003] as well as a signiﬁcant main effect of glutamate
concentration [F(3, 472)=4.954, p<0.003] (Figure 3-3C). These ﬁndings indicate that
caffeine-exposed neurons exhibit a signiﬁcantly reduced peak calcium transient when
compared to control neurons. Further, in both treatment groups, application of higher

concentrations of glutamate induces a larger calcium transient.

78

Baseline

 

 

 

 

 

 

 

Control Caffeine

Figure 3-2. Baseline resting intracellular Ca2+ ratio in cultured hippocampal neurons
from control and prenatal caffeine-treated neurons. There were no signiﬁcant differences
between resting intracellular Ca2+ ratios between treatments. Data represent the average
resting Fura-2 ratio 3: SEM.

79

20' Low
A

 

% change over baseline

 

 

2uM 5uM 10uM 50uM

100- Medium

 

% change over baseline
c»
‘7"

 

 

2uM 5uM 10uM 50uM

High

.5 .a. ..s .5. N
N ﬁr 09 G O
O O O O O
l I l l J

% change over baseline

 

 

100 r 1 r r
2uM 5uM 10uM 50uM

 

-I— Control ~e- Caffeine

Figure 3-3. Peak calcium transients following brief application of 2, 5, 10, or 50 pM
glutamate in hippocampal neurons cultured from control or caffeine-exposed pups. A
calcium transient less than 20% over baseline was classiﬁed as a low response (A), 20-
100% change over baseline as a medium response (8), and higher than 100% change
from baseline as a high response (C). In high responding neurons, caffeine treatment
resulted in a reduced calcium transient. Data represent the mean calcium peak :1: SEM.

80

Total NMDA Receptor Expression in the Developing Hippocampus

Analysis of the total protein expression of the NR1 subunit in the developing
hippocampus revealed no statistical difference between control and caffeine-exposed
animals on postnatal day 1 (Figure 3-4A). However, a statistically signiﬁcant main
effect of NR1 receptor expression was found on postnatal day 5 [F(3,6)=7.046, p<0.03]
(Figure 3-48), postnatal day 10 [F (3,7)=5 .543, p<0.03] (Figure 3-4C), and postnatal day
20 [F(3,7)=8.229, p<0.02] (Figures 3-4D), with caffeine exposure increasing NR1
expression. Figure 3-4E shows representative immunoblots of total NR1 protein
expression at each postnatal time point.

Analysis of the total protein expression of the NR2A subunit in the developing
hippocampus revealed no statistical differences between control and caffeine-exposed
animals on postnatal days 1 or 5 (Figure 3-5A, 8). However, a main effect of treatment
was found on both postnatal day 10 [F(3,8)=32.068, p<0.01]and postnatal day 20
[F (3,8)=10.159, p<0.02] (Figure 3-SC, D). Representative immunoblots of total NR2A
protein expression at each postnatal time point are shown in Figure 3-5E.

Analysis of the total protein expression of the NR28 subunit in the developing
hippocampus revealed a main effect of treatment on postnatal day 1 [F(3,7)=6.554,
p<0.02] with caffeine-exposed animals having reduced NR28 protein expression as
compared to controls (Figure 3-6A). However, there was no signiﬁcant main effect
between groups on postnatal days 5, 10, or 20. Representative immunoblots of total

NR28 protein expression at each postnatal time point are shown in Figure 3-6E.

81

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Figure 3-4. Total NR1 protein expression in the developing hippocampus on postnatal
day 1 (A), postnatal day 5 (B), postnatal day 10 (C), and postnatal day 20 (D) in control
and caffeine-exposed (75, 150, or 300 mg/L) offspring. Data represent the mean optical
density i SEM. (E) Representative immunoblots from each treatment group (n=3) at
each postnatal time period. A main effect of treatment was found on postnatal days 5, 10,
and 20. * indicates signiﬁcant difference from control (Tukey; p<0.05).

 

 

 

 

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Figure 3-5. Total NR2A protein expression in the developing hippocampus on postnatal
day 1 (A), postnatal day 5 (B), postnatal day 10 (C), and postnatal day 20 (D) in control
and caffeine-exposed (75, 150, or 300 mg/L) offspring. Data represent the mean optical
density 1: SEM. (E) Representative immunoblots from each treatment group (n=3) at
each postnatal time period. A main effect of treatment was found on postnatal days 10
and 20. * indicates signiﬁcant difference from control (Tukey; p<0.05).

83

Optical Density >
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Optical Density 0
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N

Con

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PN

PN5

PN20 PN10

 

75 150 300

PN10

75 150 300
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NR23 anal 1mm

PN5

0.4

Optical Dans
P
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Con 75 150 300

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Figure 3-6. Total NR2B protein expression in the developing hippocampus on postnatal
day l (A), postnatal day 5 (B), postnatal day 10 (C), and postnatal day 20 (D) in control
and caffeine-exposed (75, 150, or 300 mg/L) offspring. Data represent the mean optical
density i SEM. (E) Representative immunoblots from each treatment group (n=3) at
each postnatal time period. A main effect of treatment was found on postnatal day 1.

* indicates signiﬁcant difference from control (Tukey; p<0.05).

84

Activation of Calcium Dependent Cascades

Analysis of the protein expression of phospho-CREB following acute exposure to
caffeine revealed no signiﬁcant differences between prenatal control and prenatal
caffeine-exposed groups on postnatal day 10 or postnatal day 20 (Figure 3-7A, B).
Representative immunoblots of phosho-CREB protein expression for both time periods
are shown in Figure 3-7C.

Similarly, analysis of the protein expression of phospho-ERKl/Z following acute
caffeine exposure revealed no signiﬁcant differences between control and caffeine-
exposed animals on postnatal day 10 or postnatal day 20 (Figure 3-8A, B).
Representative immunoblots of phosph-ERK1/2 protein expression are shown in Figure

3-8C.

High Performance Liquid Chromatography

Analysis of brain and plasma samples in PN5 and PN10 offspring exposed to
prenatal caffeine revealed no remaining caffeine, theobromine, theophylline, or
paraxanthine at either postnatal period. Thus, caffeine and its primary metabolites are

metabolized from the brain and circulation within 5 days of birth in rats.

85

 

 

A 1 PN10 B 1 PN20

3.0.8a 50.8~

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Con 75 150 300 Con 75 150 300

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EpCREB~._m- u- u;
0- Actln ” a w a
gunman- an a. O-
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Figure 3-7. Phosphorylated-CREB (CAMP response element binding protein) expression
in the developing hippocampus on postnatal day 10 (A) and postnatal day 20 (B)
following acute caffeine treatment in control and caffeine-exposed (75, 150, or 300
mg/L) offspring. Acute caffeine treatment consisted of a single dose caffeine (10 mg/kg)
administered orally by pipette followed by a 1.5 hour delay prior to tissue collection.
Data represent the mean optical density :t SEM. (C) Representative immunoblots ﬁ'om
each treatment group (n=3) at both postnatal time periods. No signiﬁcant differences in
pCREB activation were found between control and caffeine-exposed offspring.

pERK

 

 

PN10 PN20
A 1— B1

30.8“ 308—
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PN20 PN10

 

Figure 3-8. Phosphorylated-ERK1/2 (extracellular signal related kinase 1/2) expression
in the developing hippocampus on postnatal day 10 (A) and postnatal day 20 (B)
following acute caffeine treatment in control and caffeine-exposed (75, 150, or 300
mg/L) offspring. Acute caffeine treatment consisted of a single dose caffeine (10 mg/kg)
administered orally by pipette followed by a 1.5 hour delay prior to tissue collection.
Data represent the mean optical density i SEM. (C) Representative immunoblots from
each treatment group (n=3) at both postnatal time periods. No signiﬁcant differences in
pERK activation were found between control and caffeine-exposed offspring.

D. Discussion

Results from the present study demonstrate that chronic prenatal exposure to
caffeine alters the expression of NMDA receptors in the neonatal hippocampus and
reduces glutamate-induced calcium transients in caffeine-exposed primary hippocampal
neurons. The NR1, NR2A, and NR2B subunits all exhibited slight differences in protein
expression at various postnatal periods. The NR1 subunit showed increased expression
on postnatal days 5, 10, and 20 in caffeine-exposed offspring. In contrast, the NRZB
subunit showed decreased expression in caffeine-exposed animals on postnatal day 1, but
this effect was no longer observed on postnatal day 5. On postnatal day 10, the NR2A
subunit had increased expression only in offspring prenatally exposed to the highest dose
of caffeine. Offspring exposed to the two lower doses of caffeine showed reduced NR2A
expression on postnatal day 10, but by postnatal day 20 the NR2A subunit showed
elevated expression in the caffeine-exposed groups. The changes in subunit expression at
PN5 and later are not likely due to a direct action of caffeine and its metabolites in the
brain since HPLC analysis showed no detectable levels of these metabolites at these time
points. Interestingly, prenatal caffeine exposure also reduced glutamate-induced calcium
transients in hippocampal cultures from treated offspring. Speciﬁcally, caffeine-treated
hippocampal cells showed diminished maximal calcium transients aﬁer glutamate
administration when compared to the maximal calcium transients seen in control cultures.
Despite this reduction in total calcium inﬂux in caffeine-treated cells, there were no

treatment differences detected between the levels of phosphorylated ERK and CREB

88

proteins, suggesting that sufﬁcient levels of intracellular calcium are still being attained
to induce these calcium-dependent cascades.

The altered expression of NMDA receptors in the neonatal hippocampus
following prenatal caffeine treatment corresponds to the period of peak expression for
each subunit. It is well known that the NMDA receptors are spatially and temporally
regulated during brain deve10pment (Lujan et al., 2005). In general, NMDA receptor
expression is elevated during development and expression levels decrease as the brain
matures. Periods of peak receptor expression have been found to correlate with neonatal
periods of synaptogenesis during the brain growth spurt, which occurs during the ﬁrst
three postnatal weeks in rats (Ritter et al., 2002). The NR1 subunits are expressed during
both prenatal and postnatal development, with peak levels of expression occurring around
postnatal days 3-14 in the rat (Ritter et al., 2002). The NR2A and NR2B subunits
undergo a developmental switch in expression, allowing the diverse pharmacological
properties of each subunit to predominate during distinct periods of brain development.
The NR2B subunit is characterized by slower channels kinetics and decreased channel
open probability, whereas the NR2A subunit is characterized by fast channel kinetics and
increased channel open probability (Lau & Zukin, 2007). The NRZB subunit is
expressed prenatally and maintains high levels of expression until postnatal day 7 aﬁer
which levels decline (Ritter et al., 2002). In contrast, NR2A expression levels are very
low during early prenatal and postnatal development. NR2A expression increases around
postnatal day 10 and peaks around postnatal day 21 (Ritter et al., 2002). The current
ﬁndings suggest that prenatal caffeine treatment may alter this delicate timing of peak

receptor expression. For instance, caffeine-exposed offspring showed increases in NR1

89

subunit expression only at the time points of postnatal days 5, 10, and 20 which
correspond to the highest levels of expression for this subunit. Similarly, altered NRZB
subunit expression was found only on postnatal day 1 when expression of these subunits
is highest. NR2A subunit expression was altered on postnatal days 10 and 20 when
elevated expression of this subunit becomes apparent. Thus, prenatal caffeine exposure
may selectively increase or decrease subunit expression during neonatal periods normally
associated with high levels of subunit expression. This may disrupt the delicate timing of
subunit expression, which mediates many important developmental processes.

NMDA receptor expression is dynamically regulated during development and is
inﬂuenced by neuronal activity. It has been found that chronic activation of NMDA
receptors results in receptor internalization and degradation via the ubiquitin-proteasome
system (Lau & Zukin, 2007). Further, it is believed that immature synapses are
especially susceptible to activity-dependent remodeling with NR1/NRZB receptor
complexes undergoing rapid internalization during development. Therefore, if prenatal
caffeine exposure is increasing glutamate neurotransmission and thereby increasing
glutamate receptor activity, it is possible that the increase in activity results in receptor
internalization and degradation resulting in less overall NMDA receptor expression. This
may be a factor mediating the decreased maximal calcium response seen following
prenatal caffeine exposure.

It is also possible that prenatal caffeine exposure is altering the expression of
AMPA receptors in the developing hippocampus, which may affect glutamate-mediated
signaling. AMPA receptors are expressed during prenatal and postnatal development

although many of these receptors are thought to be quiescent during early development.

90

GluRl subunit expression is detectable in the rat cortex as early as embryonic day 15
(Martin et al., 1998). Unlike the developmental proﬁles of other subunits, GluRl
expression remains fairly constant in the hippocampus during prenatal and postnatal
development (Ritter et al., 2002). Expression of GluR2/3 subunits is also detectable
during embryonic development. In the rat hippocampus, GluR2 subunits show a rapid
increase in expression immediately following birth, which peaks at postnatal day 10 and
then rapidly declines to prenatal levels of expression by postnatal day 18 (Ritter et al.,
2002). The effects of prenatal caffeine exposure on AMPA receptor expression is
currently unknown yet it is possible that increased glutamate neurotransmission is
increasing activation of AMPA receptors during development and altering their
expression in the hippocampus. It is also possible that changes in AMPA receptor
expression may alter NMDA receptor function due to the AMPA receptor-mediated
depolarization needed to fully activate NMDA receptors. Further research is needed to
fully elucidate the effects of prenatal caffeine exposure on AMPA receptor expression.
Another factor that requires consideration is subunit-dependent NMDA receptor
desensitization. NMDA receptor desensitization occurs after prolonged exposure to
glutamate and is likely an important neuroprotective mechanism (Dingledine et al.,
1999). Changes in subunit expression between control and caffeine-exposed offspring
could alter subunit-mediated receptor kinetics and thereby alter Ca2+ permeability. For
instance, receptors containing NR1/NR2A assemblies exhibit faster rise and decay
currents when compared to receptors comprised of NR1/NR2B assemblies (Chen et al. ,
1999). As discussed previously, the NR2B subunit is characterized by slower channels

kinetics and decreased channel open probability, whereas the NR2A subunit has fast

91

channel kinetics and increased channel open probability. Thus, in response to glutamate
NR2A-containing receptors open more quickly and desensitize more rapidly than do
NR2B-containing receptors (Yashiro & Philpot, 2008). Since NR2B subunit-containing
receptors have slow deactivation they likely carry more overall charge and allow more
Ca2+ entry during a single synaptic event (Sobczyk et al., 2005). Therefore, it is possible
that altered expression of the NR2A and NR2B subunits following prenatal caffeine
exposure may account for the decreased maximal calcium transient seen in caffeine-
exposed neurons. Unfortunately, the expression proﬁle of these subunits in day in vitro 3
hippocampal cultures was not assessed making the kinetic proﬁles of these neurons
unable to be determined. Electrophysiological studies of caffeine-exposed neurons
would provide invaluable information of how receptor kinetics may be altered.

Several other factors could be reducing the maximal calcium inﬂux in caffeine-
exposed neurons. lrnportantly, baseline resting calcium levels did not differ between
treatments as this could have many consequences on overall cell function and calcium
signaling. However, because calcium entry through NMDA receptors can induce
intracellular calcium release as well as membrane depolarization sufﬁcient to open
voltage-gated calcium channels, it cannot be ruled out that any alteration in calcium
inﬂux via these mechanisms may contribute to the reduced peak calcium amplitude seen
in caffeine-exposed cultures. Future studies utilizing speciﬁc antagonists to block
calcium inﬂux via these mechanisms are needed.

Interestingly, the images obtained during the live calcium imaging recordings
suggest that peak calcium levels increase most in the nuclear portion of the cell rather

than being contained in the somatic cytosol. It has recently been shown that there is no

92

nuclear barrier to calcium in hippocampal neurons, such that inﬂux of calcium at the cell
membrane readily propagates calcium towards and into the nuclear compartment (Eder &
Bading, 2007). Thus, the ﬁndings obtained in the current study may suggest that
signaling of nuclear calcium is altered. However, it is unknown whether the appearance
of increased calcium in the nuclear compartment is merely an artifact of the calcium
imaging protocol used for these studies. For instance, the shape of the cells in culture
may inﬂuence the appearance of the recording such that a spherical shape may result in
the highest light exposure in the center, or nuclear portion, of the cell. To fully elucidate
the cellular compartments involved in the calcium signal, much higher resolution would
be needed. Thus, the location of the calcium signal within the soma cannot be described
in precise detail.

Calcium inﬂux through NMDA receptors is known to activate numerous
intracellular cascades including the CAMP and MAPK/ERKv(mitogen-activated protein
kinase/extracellular signal-related kinase) pathways (Poser & Storm, 2001). Activation
of the MAPK/ERK pathway modulates phosphorylation of CREB (cAMP response
element binding protein) (Lynch, 2004; Malenka & Bear, 2004). CREB activation is an
important transcription factor for long-term protein synthesis and plays an essential role
in hippocampal synaptic plasticity (Lynch, 2004; Malenka & Bear, 2004). Because ERK
and CREB activation occur as downstream events following NMDA-receptor mediated
calcium inﬂux, the present study sought to determine whether altered calcium inﬂux
following prenatal caffeine exposure could alter activation of these signaling factors.
Previously, caffeine has been shown to activate ERK in the hippocampus and cortex

(Okano et al., 2008). Similarly, glutamate administration in vitro has been shown to

93

induce phosphorylation of CREB in primary hippocampal cultures (Mabuchi et al. ,
2001). Interestingly, removal of calcium from the culture medium abolished glutamate-
induced CREB phosphorylation, suggesting that CREB activation is dependent upon
calcium inﬂux (Mabuchi et al. , 2001). Moreover, neither the AMPA/kainate receptor
antagonist CNQX nor the voltage-gated calcium channel antagonist nifedipine blocked
glutamate-induced CREB phosphorylation. However, CREB phosphorylation was
blocked by application of the NMDA receptor antagonist MK-801, suggesting that CREB
activation is dependent on calcium inﬂux via NMDA receptors (Mabuchi et a1. , 2001).
The ﬁndings presented in this study suggest that CREB and ERK activation in the
hippocampus are not altered in offspring exposed to caffeine prenatally. Thus, it is likely
that there is still sufﬁcient calcium inﬂux to activate these cascades or that calcium inﬂux
is no longer altered by postnatal days 10 and 20. Nevertheless, these ﬁndings suggest
that prenatal caffeine exposure does not alter these critical signaling cascades.

Interestingly, the alterations seen in NMDA receptor expression and ﬁinction after
prenatal caffeine exposure are similar to the ﬁndings from prenatal ethanol exposure
studies. Prenatal ethanol exposure results in diminished glutamate-induced calcium
transients in primary cultures from treated rats (Lee et al., 1994; Spuhler-Phillips et al. ,
1997). Furthermore, NR2A receptor expression in the neonatal hippocampus and cortex
is increased in ethanol exposed animals (Nixon et al., 2002, 2004). During prenatal
development, ethanol acts as a neuronal stimulant rather than a depressant due to the
reverse in the chloride gradient in GABAergic neurons. Speciﬁcally, the intracellular
chloride concentration is elevated in immature neurons leading to depolarization

following activation of GABA receptors (Ben-Ari, 2002). Thus, the actions of ethanol

94

and caffeine may be functionally similar in the developing brain, with both drugs leading
to an increase in overall neuronal excitation and a consequent disruption of normal
NMDA-receptor expression and function.

In summary, prenatal caffeine exposure alters the peak developmental expression
of NMDA subunits and diminishes the maximal glutamate-induced calcium transients in
treated neurons. Caffeine is known to antagonize adenosine receptors, which normally
function to reduce neuronal activity in the brain (F redholm, 2007). When adenosine
receptors are inhibited, there is an overall increase in glutamate neurotransmission
particularly in brain regions with an abundance of adenosine receptors such as the cortex
and hippocampus (Wang, 2007). The ability of caffeine to increase neuronal stimulation
via blockade of adenosine receptors may lead to these alterations in NMDA receptor
function due to chronic increased stimulation of these glutamatergic systems during
development. Alterations in NMDA receptor expression or function may have long-term
consequences due to the importance of these receptors in synapse formation, synaptic

plasticity, and numerous other critical developmental processes.

95

Chapter 4. Chronic prenatal caffeine exposure impairs novel object recognition and

radial arm maze behaviors in adult rats.

A. Introduction

Caffeine, a methylxanthine, is one of the most widely consumed psychoactive
drugs in the world due to its presence in coffee, tea, chocolate, soft drinks and numerous
medications (Fredholm et al., 1999). Caffeine is rapidly absorbed throughout the body
and brain. At moderate doses, caffeine exerts its stimulatory effects on behavior directly
via blockade of adenosine receptors. Of the four characterized adenosine receptors (A1,
A2A, A28, and A3), caffeine has its primary actions at the A] and A2A receptors
(F redholm, 1980). A1 receptors are the most abundant of the adenosine receptors and are
highly expressed throughout the cerebral cortex, hippocampus, and cerebellum (Fastbom
et al., 1987; Jarvis et al., 1987; Svenningsson et al., 1997; Dunwiddie & Masino, 2001).
In the developing rat brain, the A1 receptor is one of the earliest receptors expressed
(Rivkees et al. , 2001) with mRN A detectable by gestational day 14 and adult patterns of
distribution established by gestational day 20 (Weaver, 1996). A2A receptors are
restricted in expression to dopaminergic regions of the brain (Svenningsson et al., 1999),
with mRNA expression detectable in the striatum by gestational day 14 in rats (Weaver,
1993). Developmentally, adenosine plays a key role in the inhibition of neuronal activity
(F isone etal., 2004), formation of axons (Turner et al., 2002), and neuroprotection
(Rivkees et al. , 2001). Gestational exposure to caffeine may inhibit the actions of

adenosine within the fetal brain, thereby altering normal brain development.

96

Maternal consumption of caffeine during pregnancy is of concern due to the
extended half-life of the drug in the fetus. Caffeine rapidly crosses both the placental and
blood-brain barriers to reach the fetal brain (Ikeda et al., 1982; Tanaka et al., 1984;
Arnaud, 1993). In the fetus, the metabolism and elimination of caffeine are slowed due
to a lack of cytochrome P-450 activity, resulting in a prolonged half-life (Aranda et al.,
1979a). The half-life of caffeine is estimated to be 50-100 hours at the end of gestation,
which may lead to accumulation of caffeine in the fetal brain (Aranda et al., 1977;
Parsons & Neims, 1981; Amaud, 1993). In the fetus, the half-life of caffeine is further
protracted due to slowed maternal metabolism of caffeine during pregnancy. During the
last trimester of pregnancy, the half-life of caffeine is estimated to be 10-15 hours in the
expectant mother (Aldridge et al., 1981; Knutti et al. , 1981; Brazier et al., 1983). Thus,
the altered half-life of caffeine in the fetus and expectant mother may greatly prolong the
actions of caffeine within the fetal brain.

Perinatal exposure to caffeine causes persistent alterations in brain development.
In the neonatal rat brain, concentrations of protein and cholesterol are decreased
following low dose (1 or 2 mg/l 00 g body weight) caffeine exposure (N akamoto et al.,
1988). Caffeine injections (50 mg/kg/day) on postnatal days 1-12 cause an enduring
increase in the dendritic length of pyramidal neurons within the rat prefrontal cortex
(Juarez-Mendez et al., 2006). In juvenile rats, caffeine exposure throughout gestation
and lactation diminishes the stimulatory locomotor response to MK-801, an NMDA
receptor antagonist, suggesting a persistent change in NMDA receptor function (da Silva
et al., 2005). Thus, developmental exposure to caffeine may cause long-term alterations

in the brain.

97

Perinatal exposure to caffeine also alters juvenile and adult locomotor behavior
and emotional reactivity. Numerous studies have shown decreased levels of activity in
both juvenile and adult rat offspring following developmental caffeine exposure (Hughes
& Beveridge, 1991; Zimmerberg et al., 1991). Caffeine exposed offspring exhibit
heightened emotional reactivity such that male offspring have increased defecation and
decreased exploration in the open ﬁeld and longer latencies to emerge from a light-dark
box (Hughes & Beveridge, 1986, 1991). However, increased activity and reduced
anxiety following postnatal caffeine exposure have also been shown. Juvenile rats show
increased pain sensitivity and reduced anxiety following caffeine exposure (15-20
mg/kg/day) across postnatal days 26 (Pan & Chen, 2007). Further, juvenile rats exhibit
hyperactivity, which persists into adulthood in an open ﬁeld task after exposure to
caffeine (20 mg/kg/day) across postnatal days 7-11 (Tchekalarova et al., 2005).

The long-term behavioral effects of chronic prenatal exposure to caffeine on
measures of learning and memory have not extensively investigated. Rat pups exposed to
1 or 9 mg/kg caffeine during the ﬁrst week of life exhibit hypoactivity at two weeks of
age and impairment in a spatial operant learning task during adulthood (Zimmerberg et
al., 1991). Following neonatal caffeine exposure, passive avoidance learning is
signiﬁcantly impaired in male and female juvenile rats (Pan & Chen, 2007). In adult
animals, neonatal caffeine (15-20 mg/kg/day on PN2-6) treated female rats have
signiﬁcantly improved retention of passive avoidance learning at 24 and 72 hours after
training, while male rats have signiﬁcantly reduced retention (Fisher & Guillet, 1997).
Thus, prenatal caffeine exposure may alter adult behavior in a sexually dimorphic

fashion, although these sex differences may be undetectable in pre- or peri-pubertal

98

animals. Importantly, sex differences in response to perinatal caffeine exposure may help
determine whether males or females are more susceptible to caffeine induced
developmental effects.

The aim of the present study was to document the effect of chronic prenatal
exposure to caffeine on cognitive behavior in adult male and female rats. The study
investigated whether daily oral administration of caffeine (approximately lOmg/kg) to
pregnant rat dams could alter long-term learning and memory in offspring. Speciﬁcally,
prenatal caffeine-treated and control offspring underwent testing in adulthood in three
well-established learning and memory paradigms, including the novel object recognition

task, radial arm maze, and Morris water maze.

99

B. Materials and Methods

Prenatal Caffeine Treatment

Female Sprague-Dawley rats 60-70 days of age were mated within our colony at
Michigan State University. Males were housed with females for 4 days to ensure that one
estrous cycle was completed. Males were removed after 4 days and this day was counted
as gestational day-4 to approximate the day of birth. From gestational day 4 until the day
of birth, pregnant dams were administered ad libitum tap water (n=4) or 75 mg/L
caffeinated tap water (n-4) with intake measured daily. Caffeine administration was
started early in gestation to determine the effects of chronic exposure since it has been
shown previously that caffeine can accumulate in the fetal brain (Wilkinson & Pollard,
1993). The dose was chosen aﬁer calculating metabolic body weight (=body weight"3/4)
(Nehlig & Derby, 1994). Oral administration was used to maintain the human relevance
of the study.

On postnatal day 2 (day of birth is postnatal day 0) the sex of each offspring was
determined and litters were culled to 4 males and 4 females. No observational
differences in litter size, sex ratio, or maternal nest building, retrieval, and nursing
behavior were noted following birth. Each litter contained approximately 12-14 pups and
there was no difference in mean body weights between control (6.57 :t 0.104 g) and
caffeine-treated (6.75 at 0.243 g) offspring. The pups were weaned on postnatal day 25
(PN25) to 2-3 animals per cage such that animals of the same litter and sex were housed
together. Offspring were housed in a temperature and humidity controlled environment

with a 12 h light/dark cycle (lights off at 0500 hours) through adulthood. Ad libitum

100

standard chow and tap water were available except during radial arm maze training. The
experiments were approved by the Michigan State University Institutional Animal Use

and Care Committee and followed NIH guidelines for animal use.

Behavioral Tests

To minimize the stress associated with behavioral testing, animals were handled
for two minutes daily for two weeks prior to the start of testing. In the ﬁrst set of
behavior testing, twenty-nine male and female offspring from four litters (control male,
n=5; control female, n=8; prenatal caffeine-treated male, n=8; and prenatal caffeine-
treated female, n=8) underwent three neurobehavioral tests chosen to measure
hippocampal-dependent and -independent learning and memory. Object recognition
testing, radial arm maze, and Morris water maze were assessed between PN 110-150,
sequentially. The testing order was chosen so that less stressful behavior tasks were
performed prior to more stressﬁil tasks. The stress induced by each task was previously
determined by the defecation and grooming behavior of animals during each task, with
almost no defecation and grooming behaviors in the object recognition test and very high
levels of defecation and grooming in the Morris water maze. In the second set of
behavior tests, thirty-two male and female offspring from four litters (control male, n=8;
control female, n=8; prenatal caffeine-treated male, n=8; and prenatal caffeine-treated
female, n=8) underwent 10 consecutive days of testing in the advanced Morris water
maze. Prior to each behavior test, the animals were placed in the testing room 30 min
before testing to allow habituation to the environment. All testing was performed

between noon and 4 pm. During each test, the experimenter remained outside the testing

101

room except between trials. The behavior room has numerous extra-maze cues such as
shelving, counters, and signs. Testing was always performed under dim illumination.
Each test was recorded and tracked using Smart tracking system (San Diego

Instruments). Behavior was scored by at least two observers blind to the treatment

groups.

Novel Object Recognition Test

The novel object recognition task was used to assess non-spatial memory. The
testing arena was a 40 cm x 60 cm open area to which rats were habituated for 10 min
over two days prior to pre-training and training. During pre-training, the animals were
allowed to explore two identical objects (A, A) for 3 min. Memory retention testing was
performed at both 3 and 24 hours after pre-training. To determine memory retention at 3
hours, animals were allowed to investigate a familiar object (A) and a novel object (B)
for 3 min. Memory retention was again tested at 24 hours following pre-training with the
familiar object (A) and a novel object (C). The objects used were small plastic toys of
varying color and shape. Objects were placed 12 cm from each wall and 18 cm from
each other in the maze. Objects were counterbalanced within the arena during each trial
and rinsed with 70% alcohol between trials. Time exploring each object was assessed,
which included approaching within 2 cm of the object with the face, sniffing, biting, and
pawing the object. Sitting or standing on the object was not counted as exploration. The
training exploration time was calculated as exploration of objects A1 + A2. The
exploration time during the 3 and 24 hour retention tasks were calculated as A + B and A

+ C respectively. The discrimination index was calculated as the (exploration of novel

102

object —- exploration of familiar object) / (total exploration time). A discrimination index
above zero indicates the animal explored the novel object more than the familiar object

and retained a memory of the familiar object.

Radial Arm Maze

The radial arm maze was used to assess hippocampal-dependent working and
reference memory. The radial arm maze is a spatial navigation memory task involving
primarily the hippocampus and prefrontal cortex (Olton, 1987). Two weeks prior to
behavior testing, animals were individually housed and began food deprivation until 90%
of their starting body weight was reached. During this time, animals were handled,
weighed, and habituated to a cereal reward daily. The eight-arm maze consisted of a 25
cm diameter round center platform from which 65 cm walled arms emerge. To reduce
the stress of the novel environment, rats were pre-trained to the radial arm maze for one
session per day over 7 consecutive days. During each pre-training session, a cereal
reward was moved closer to the end of each arm to ensure the rats learned to search the
entire length of the arm for the reward. Each animal was allowed 10 min to consume the
rewards in all 8 arms.

Radial arm maze training occurred over 10 consecutive days, one trial per day.
During training, food cups were placed at the end of each arm to ensure that the rats
could not see the cereal reward from the center platform. Four of the eight food cups
were baited with a reward and the sequence of baiting (arms 2, 3, 5, and 7) remained
constant. Animals were placed onto the center platform at the beginning of each trial and

were allowed 10 min to retrieve all cereal rewards. After eating all 4 cereal rewards or

103

once 10 min had elapsed, animals were placed in their home cage. Each day animals
were given food pellets (gram amount given equaled 12-16% of their body weight) after
the completion of the behavior session. The maze was cleaned with ethanol between
each trial. Upon completion of radial arm maze testing, standard chow was again freely
available to the rats. Memory acquisition was assessed as the decrease in the number of
errors between each trial. Reference memory errors were scored as entries into an arm
that was not baited such that animals would need to recall from previous days which arms
were not baited to avoid error. Working memory errors were scored as reentries into an
arm from which food had already been eaten such that animals would have to recall

within a given trial which arms they had already visited.

Standard Morris Water Maze

The standard Morris water maze (Morris, 1984) was used as a measure of
hippocampal-dependent spatial learning and memory. The maze was a 2 m diameter, 1 m
tall circular pool in which the water was kept at 26 d: 1 °C. During pre-training, rats
underwent three consecutive trials from separate starting points. Rats were allowed 60
sec per trial to ﬁnd the non-submerged platform. Between each of the three trials, rats
were allowed to sit upon the platform for 20 see. If an animal did not ﬁnd the platform
within the allowed time, an experimenter would guide the animal to the platform.

Morris water maze training began two days after pre-training. Rats underwent 12

non-consecutive 60 sec trials starting at random from one of four cardinal directions. At
the start of each trial, rats were placed into the water facing the wall of the maze. The

platform (8 inch diameter) was submerged beneath water made opaque with non-toxic

104

I /_'

white paint and always remained in the same location within the maze. Rats were
removed from the maze after ﬁnding the platform or once the allowed time had elapsed
and were dried and returned to a holding cage. The inter-trial interval was approximately
30 min. Latency to ﬁnd the platform and path length were recorded using the Smart
tracking system.

A probe test was done on the same day after the completion of the 12 trials.
During the 30 second probe test, the platform was completely removed from the water.
The number of times the animals crossed the location where the platform was previously

located was assessed.

Advanced Morris water maze

Three paradigms were tested in the Morris water maze across nine consecutive
days; Days 1-3 examined place learning, days 4-6 examined reversal, and days 7-9
examined working memory. The water maze and recording software used was the same
as described previously.

For the place learning task, rats underwent 4 non-consecutive trials per day
starting once from each cardinal direction. At the start of each trial, rats were placed into
the water facing the wall of the maze. The platform was submerged beneath the opaque
water and remained in the same location all three days. Rats were given 60 sec to ﬁnd
the hidden platform and if unsuccessful were guided to the platform location by an
experimenter. All animals were allowed to rest for 20 sec on the platform before being

returned to their home cage. No pre-training occurred prior to day l of testing. A 30 sec

105

probe test in which the platform was completely removed from the water was performed
on day 3 after the completion of the place learning trials.

For the reversal task, rats underwent 4 non-consecutive trials per day starting once
from each cardinal direction and were again allowed 60 sec per trial. However, the
platform was located opposite to its position during the place learning task. The platform
remained in the new location all three days.

During the working memory task, rats underwent 4 consecutive trials per day
starting from the same location. The platform was moved to a new location each day and
the starting location was also varied each day. The rats were allowed 60 sec per trial to
ﬁnd the submerged platform and then were allowed a 20 sec rest on the platform before

beginning the next trial.

Statistics

Statistical assessment of weekly water intake was performed using a two-way
repeated measures analysis of variance (week, treatment). For assessment of the object
recognition test, two-way analysis of variance (sex, treatment) was performed on the pre-
training, 3 hr memory, and 24 hr memory trials. Radial arm maze and Morris water maze
were assessed by two-way repeated measures analysis of variance (sex, treatment). All

analyses were performed using the SYSTAT program (version 11.0).

106

C. Results

Water Intake

During the time of caffeine exposure (gestational day 4 through birth), the
average water intake in control and caffeine-treated dams signiﬁcantly increased each
week (Figure 4-1; within-group analyses — F(2,12)=23.054, p<0.001) and showed a
signiﬁcant week by treatment interaction [F(2,12)=6.885, p<0.01] with caffeine-treated
animals having greater intake than controls during week 3. However, there was no

signiﬁcant main effect of treatment on water intake.

Novel Object Recognition Test
Total exploration time

During pre-training, two-way analysis of variance revealed a signiﬁcant effect of
treatment [F (1 ,25)=l 8.94, p<0.001] on the total amount of time spent exploring the novel
objects (Figure 4-2A). Caffeine-treated animals spent less total time exploring the novel
objects during pre-training. However, the total time spent exploring the novel and
familiar objects was not signiﬁcantly different between treatments during the 3 hour and
24 hour recognition memory tests. At 3 hours, there was a signiﬁcant effect of sex
[F (l ,25)=5.455, p<0.05] on total exploration time, with females spending more time
(54.1 :1: 5.3 sec) exploring than males (37.8 i 3.8 sec) yet this effect was not seen at 24

hours.

107

Memory Retention Testing

At 3 hours following pre-training, there was no signiﬁcant effect of treatment, '
sex, or treatment by sex interaction on the novelty discrimination index (Figure 4-23).
At 24 hours following pre-training, two-way analysis of variance showed a signiﬁcant
effect of treatment [F(l,25)=7.652, p<0.02] but no signiﬁcant effect of sex or treatment
by sex interaction on the discrimination index of novel object exploration (Figure 4-2B).
These ﬁndings indicate that at 24 hours following pre-training, the caffeine-treated
animals do not distinguish between the novel and familiar objects as well as control

animals.

Radial Arm Maze
Pre-training
Across the 7 consecutive days of pre-training in the radial arm maze, a two-way
repeated measures AN OVA revealed no signiﬁcant between-group effect of treatment,
sex, or treatment by sex interaction on the total time required to retrieve all food rewards.
However, there was a signiﬁcant within-group effect of time [F (6,150)=49.879, P<0.001]

indicating that animals learned the task (Table 4-1).

Training
There was a signiﬁcant between-group effect of treatment on the number of
working memory errors [F (1, 25)=4.357, P<0.05] across the ﬁve trial blocks (trials 1-2,
3-4, 5-6, 7-8, 9-10) of the radial arm maze. Working memory errors were assessed as the

number of reentries into arms already entered during the same trial. Caffeine-treated

108

animals made more errors than controls (Figure 4-3A). A signiﬁcant within-group
difference by trial block was observed [F(4,100)=2.988, p<0.03] indicating that animals
learned the task across the ﬁve blocks.

Between-group analyses also revealed a signiﬁcant main effect of treatment on
the number of reference memory errors [F(l, 25)=6.503; P<0.02] with caffeine-treated
animals making more errors than controls (Figure 4-3B). Reference memory errors were
assessed as entries into an arm that never contained a reward. A signiﬁcant within-group
difference was also found across the 5 trial blocks [F(4,100)=3.201, P<0.02] representing
a decrease in reference memory errors in both treatment groups as the task was acquired.

There was no signiﬁcant between-group effect of treatment, sex, or treatment by
sex interaction on path length. Path length was measured as the total distance in cm
covered across each of the 5 trial blocks. There was a signiﬁcant within—group effect of
path length [F(4,100)=8.601, P<0.001] indicating that as the rats learned the task they
decreased their traveling distance to obtain the rewards (Table 4-1).

Across the 10 consecutive days of testing, there was no signiﬁcant between-group
effect of treatment, sex, or treatment by sex in the total time required for animals to
retrieve all 4 food rewards. As in pre-training, there was a signiﬁcant within-group effect
of time [F(9,225)=3.672, P<0.003] across the trial blocks indicating acquisition of the
task (Table 4-1).

Two-way ANOVA revealed no signiﬁcant effect of treatment or treatment by sex
interaction on body weights at the start of food deprivation and at the conclusion of food
deprivation (Table 4-1). However, a signiﬁcant effect of sex was observed for body

weights prior to food deprivation [F(l ,25)=335.97, p<0.001] and at the conclusion of

109

food deprivation [F (1,25)=3 74.42, p<0.001]. Thus, both male and female rats began food
deprivation at a healthy weight and lost weight to the same extent during radial arm maze

testing.

110

Water Consumption

 

 

 

 

80-
70— T.
.L
E 60- "'
E: 50— i
if:
g 40~
>5
7;,- 30—
o
20-
10-
0 I l 1
Week 1 Week 2 Week 3
—I— Control ‘ Caffeine

Figure 4-1. Maternal intake of either water or caffeinated tap water across each
gestational week. Data represent the mean daily intake 3: SEM for each week. Water
intake increased throughout gestation but did not signiﬁcantly differ by treatment.

111

Exploration Time (s)

Discrimination Index

0
p
_l

l

(A)

O)
O
I

01
O
l

A
O
l

 

O
1

Total Exploration Time

 

- Control
[:1 Caffeine

 

 

 

 

 

 

.5
"1’

 

Pre-training

 

 

   

 

 

3 Hour

_,
3 hour 24 hour
Novel Object Recognition
*
r —"l

 

   

24 Hour

Figure 4-2. (A) Effects of prenatal caffeine on the total time spent exploring both objects
in the novel object recognition test during each 3 min trial. Data represent the mean
exploration time in seconds :t SEM for pre-training, 3 hour recognition memory, and 24
hour recognition memory. There was a signiﬁcant effect of treatment on total exploration
time during pre-training, with caffeine-treated animals exploring the objects less. (B)
Effects of prenatal caffeine exposure on memory retention of the novel object at 3 and 24
hours after pre-training. Data represent the percent of time :t SEM spent exploring the
novel object. Prenatal exposure to caffeine signiﬁcantly reduced memory retention of the
novel object at 24 hours aﬁer pre-training. Controls n=1 3; Caffeine n=16.

112

(A) RAM Working Memory

- Control
6— [:1 Caffeine

 

Working Memory Errors

 

 

 

 

 

 

 

I 1
Block 1 Block 2 Block 3 Block 4 Block 5

7, RAM Reference Memory

Reference Memory Errors

 

 

 

 

 

 

 

 

 

Block 1 Block 2 Block 3 Block 4 Block 5

Figure 4-3. Effects of prenatal caffeine exposure on the number of (A) working memory
errors and (B) reference memory errors performed during Radial arm maze testing, with
caffeine-treated animals performing signiﬁcantly more memory errors. Data represent
the average number of errors i SEM performed during each block (Trials l-2, 3-4, 5-6, 7-
8, 9-10). Control n=l3; Caffeine=n=16.

113

 

 

 

 

 

Control Control Caffeine Caffeine
Male Female Male Female
(n=5) (n=8) (n=8) (n=8)
PreTmirﬁjnifime (s)
Day 1 482 :1: 68 569 d: 31 361:1: 36 551 :t 38
Day 2 285 :t 56 385 :t 53 255 i 54 315 i 32
Day3 215i19 266i64 212i39 l80:t29
Day4 230141 253:64 185i3l 194i41
Days 151:1:15 127524 124i12 243i62
Day6 1713:33 l89:l:3l 136i22 139i16
Day7 228:1:42 208i19 137i8 181i28
RAM Training— Path Length Em)
Block 1 3862 d: 658 5042 i 795 4129 i 325 5857 i 785
Block 2 3546 i 309 4272 :1: 310 5354 :1: 589 4705 :t 389
Block 3 4244 i 720 3724 i 369 4434 :1: 567 4555 :t 440
Block 4 3598 i 264 3626 i 569 3523 d: 450 3065 :t 222
Block 5 3278 i 167 2793 i 445 2908 i 239 3270 d: 253
RAM Trainirg— Time to Retrieve Rewards (3)
Block] 116ill 1643:40 115i12 171i12
Blockz 9953 119i10 147122 1153:15
Block3 133131 112.8:t10 104:1:6 112i9
Block4 115i5 116.1i16 93:1:12 841:6
Blocks 113i8 106.5ill 933:16 96i9
Body Weights (gL
Start Weight 435 :1: 8 418 :h 12 247 :t 6 238 :1: 10
End Weight 393 i 8 378 i 10 227 :t 4 226 i 7

Table 4-1. Mean (i SEM) values of radial arm maze pre-training time (5), training path

lengths (cm), time to retrieve all cereal rewards (s), and body weights before and after

food deprivation.

114

Standard Morris Water Maze
Pre-training
Two-way analysis of variance revealed no signiﬁcant effect of treatment, sex, or
treatment by sex interaction on the pre-training component of the Morris water maze
(Figure 4-4A). This indicates that the animals did not exhibit motor or sensory deﬁcits

that would interfere with the procedural learning of the task.

Training

Two-way repeated measures ANOVA revealed no signiﬁcant effect of treatment,
sex, or treatment by sex interaction across the 3 trial blocks (trials 1-4, trials 5-8, trial 9-
12; Figure 4-4A) on water maze latency. There was a signiﬁcant within—group
difference across the three trial blocks [F(2,50)=32.65, P<0.001] indicating that all
animals were able to decrease their latency to ﬁnd the platform across the trial blocks.
Additionally, no signiﬁcant effect of treatment, sex, or treatment by sex interaction was
observed in the probe trial, indicating that all animals were successfully able to spatially
locate the region of the platform by the ﬁnal trial.

Between-group analyses revealed a signiﬁcant main effect of treatment on path
length [F(l,25)=6.992; P<0.02] across the 3 trial blocks, with caffeine-treated animals
swimming less distance than controls (Figure 4-4B). Path length was measured as the
total distance (cm) swam in each trial averaged across the 3 trial blocks. A signiﬁcant
within- group difference was also found across the 3 trial blocks [F(2,50)=3 7.3 72,

P<0.001] along with a signiﬁcant within-group block by treatment effect [F(2,50)= 3.466,

115

P<0.04] due to the increased path length of both groups in the ﬁrst trial block when

compared to path length in the second and third trial blocks.

Advanced Morris Water Maze
Place Learning Task

Two-way repeated measures ANOVA revealed no signiﬁcant effect of treatment,
sex, or treatment by sex interaction across the 3 days of place learning (Day 1: trials 1-4,
Day 2: trials 5-8, Day 3: trial 9-12; Figure 4-5A) on latency to ﬁnd the platform. There
was a signiﬁcant within-group difference across the three days [F(2,54)=88.05, P<0.001]
indicating that all animals were able to decrease their latency to ﬁnd the platform as they
learned the task. Additionally, no signiﬁcant effect of treatment, sex, or treatment by sex
interaction was observed in the probe trial conducted on Day 3, indicating that all animals
were successfully able to spatially locate the region of the platform by the ﬁnal trial.

Between-group analyses revealed no signiﬁcant difference of treatment, sex, or
treatment by sex interaction on place learning path length. Path length was measured as
the total distance (cm) swam averaged across the 4 trials swam each day. A signiﬁcant
within-group difference was also found across the 3 days [F(2,54)=62.54, P<0.001]
indicating that the path length decreased as animals learned to ﬁnd the platform (Table 4-

2).

Reversal Task
Two-way repeated measures ANOVA revealed no signiﬁcant effect of treatment,

sex, or treatment by sex interaction across the 3 days of reversal testing (Day4: trials 1-4,

116

Day 5: trials 5-8, Day6: trial 9-12; Figure 4-5B) on latency to ﬁnd the hidden platform.
There was a signiﬁcant within-group difference across the three days [F (2,54)=16. 14, ,
P<0.001] indicating that all animals were able to decrease their latency to ﬁnd the
platform as they learned the task. Additionally, there was a signiﬁcant within-group day
by treatment by sex.interaction [F(2,54)=3.5 78, P<0.035].

Between-group analyses revealed no signiﬁcant difference of treatment, sex, or
treatment by sex interaction on reversal path length. A signiﬁcant within-group
difference was also found across the 3 days of reversal testing [F(2,54)=15.297, P<0.001]
along with a signiﬁcant within-group day by treatment by sex effect [F(2,54)= 3.432,

P<0.04] (Table 4-2).

Working Memory Task

Two-way repeated measures ANOVA revealed no signiﬁcant effect of treatment,
sex, or treatment by sex interaction on latency to ﬁnd the platform across the 4 trials of
each of the three days of repeated acquisition working memory testing. There was a
signiﬁcant within-group difference across the four trials each day (Day 7: [F(3,81)=8.65,
P<0.001], Day 8: [F(3,8l)=21 .41, P<0.001], Day 9: [F(3,8l)=13.694, P<0.001] indicating
that animals were able to ﬁnd the platform more rapidly each day during the consecutive
trials.

Two-way repeated measures ANOVA revealed no signiﬁcant effect of treatment,
sex, or treatment by sex interaction on path length across the 4 trials of each of the three
days of repeated acquisition working memory testing. There was a signiﬁcant within-

group difference across the four trials each day (Day 7: [F(3,81)=6.82, P<0.001], Day 8:

117

[F(3,81)=17.15, P<0.001], Day 9: [F (3,81)=13.16, P<0.001] indicating that the path
length shortened during each consecutive trial as the animals learned the location of the

hidden platform (Table 4-2).

118

(A) Morris Water Maze
50— '
45~ + Control

40— '3“ . Caffeine

(00)
CM

Latency (s)
N
‘f‘

20-4

33:
11

 

U!
l

 

 

O

Pretraining Block 1 Block 2 Block 3

(B) Path Length
30004
—I— Control

- -. as, __... Caffeine

2500-—

—I N
01 O
O O
O O
l l

 

10004 i

Path Length (cm)

500—

 

 

 

Block 1 Block 2 Block 3

Figure 4-4. (A) Effects of prenatal caffeine on latency to ﬁnd the hidden platform during
pre-training and across the three trial blocks (block l=trials 1-4, block 2 = trials 5-8,
block 3 = trials 9-12) in the Morris water maze. Data represent the average latency i
SEM needed to ﬁnd the platform during each 60 3 trial. (B) Effects of prenatal caffeine
on path lengths in the Morris water maze. Path length was measured as the total distance

in centimeters i SEM swam during each trial averaged across the 3 trial blocks. Control
n=15; Caffeine n=16.

119

 

(A)
60 -

)
A 01
e C:

Latency (s
(.0
‘7

Place Learning Testing

 

 

 

20 —
1 O _, -.— Control
~~ $ Caffeine
0 l 1 I
Day 1 Day 2 Day 3

(B)

60-—
50—
A 40..

30-

Latency (s

20—

10—

 

Reversal Testing

 

-.- Control
4 Caffeine

 

Day 4 Day 5 Day 6

Figure 4-5. Effects of prenatal caffeine on latency to ﬁnd the hidden platform during (A)
place learning and (B) reversal in the Morris water maze across the four trials conducted
each day over the ﬁrst 6 days of testing. Data represent the average latency a: SEM
needed to ﬁnd the platform during each 60 5 trial. Control n=15; Caffeine n=16.

120

Working Memory Testing

”W

.—

—.— Control
50" I '

mil; Caffeine

     

 

 

 

 

‘15
O l l l l l l l l T l
1- N to V 1- N M Q 1- N 1") Q
i: t l- t t l- r- l: t l— l- I-
Day 7 Day 8 Day 9

Figure 4-6. Effects of prenatal caffeine on latency to ﬁnd the hidden platform during
working memory tasks in the Morris water maze across the four trials conducted each day
over the last 3 days of the total 9 days of testing. Data represent the average latency :1:
SEM needed to ﬁnd the platform during each 60 3 trial. Control n=15; Caffeine n=16.

121

Pl:

Re‘

111

131
the

 

 

 

Control Control Caffeine Caffeine
Male Female Male Female
Place Learning Path (n=7) (n=8) (n=8) (n=8)
Length (cm) -

Day] 2182i 182 2571:5257 2732i 16] 2125:: 186
Day2 1524:: 181 1252i 189 1360::247 1320i 167

Day 3 770 d: 156 1232 i 268 1171 :t 205 890 i 224

Reversal Path Length (cm)
Day4 l449i249 2249i408 1911 21:255 1613 :1:325
Day 5 1264 :t 227 1884 i 245 1927 :1: 279 1327 :1: 326
Day6 968i237 1188i198 737i91 1148i 175
Workigg Memrﬂ Path Length (cm)

Day 7 - Trial 1 2753 i 973 2318 :t 636 2026 i 744 1658 i 403
Day 7 - Trial 2 930 :t 372 1722 :l: 603 1269 d: 296 1023 i 395
Day 7 - Trial 3 854 d: 219 788 i 300 548 i 129 1233 i 475
Day 7 - Trial 4 591:1: 290 583 i193 473 :1: 130 713 d: 303
Day 8 - Trial 1 3192 i 597 2905 a: 975 3802 :t 762 3639 :1: 812
Day 8 - Trial 2 1247 :1: 202 1549 :1: 443 2886 :1: 809 729 :1: 125
Day8-Trial3 1310i318 1102i266 1839il96 1114i308
Day8-Trial4 1647::401 944:1:221 841 :1:75 814:1: 191
Day 9 - Trial 1 1684 i 273 3650 i 734 2301 :1: 604 2115 :1: 537
Day 9 — Trial 2 814 :t 180 1069 :1: 349 1553 i 399 1338 i 525
Day 9 - Trial 3 1083 :t 457 1352 :t 369 777 :1: 297 1029 :1: 199
Day9-Trial4 1117i391 798i137 869i358 544i 100

Table 4-2. Mean (i SEM) values for advanced Morris water maze path length (cm) in
the place learning, reversal, and working memory tasks.

122

D. Discussion

In this report, it was demonstrated that chronic prenatal exposure to a moderate
dose of caffeine disrupts learning and memory behaviors in adult male and female rats.
Speciﬁcally, it was shown that prenatal caffeine exposure impairs 24 hour memory
retention in the novel object recognition task and impairs spatial learning in the radial
arm maze. Previous studies in rats have shown that prenatal caffeine exposure alters the
concentration of DNA, protein, and cholesterol in the newborn brain (N akamoto et al. ,
1988), decreases fetal brain weight (Tanaka et al. , 1987), delays developmental
milestones (Tchekalarova et al., 2005), and alters juvenile behavior (Pan & Chen, 2007).
However, the long-term effects of prenatal caffeine exposure have not been extensively
studied. Unlike many previous studies that used a high dose of caffeine and an injection
or gavage administration paradigm, a low dose of approximately 10 mg/kg/day was used
along with self-administration in the drinking water. This dose and method of
administration was chosen to maintain human relevance and to avoid the stress associated
with injections or gavage. No main effects of treatment on water intake were found
between control and caffeine-treated dams indicating that caffeine-treated dams were not
averse to the taste of the caffeinated water. These ﬁndings demonstrate that chronic oral
intake of caffeine throughout gestation can lead to subtle alterations in adult cognitive
behavior in rats.

The novel object recognition test, which measures the natural propensity of a rat
to explore a novel versus familiar object (Ennaceur & Delacour, 1988), revealed that after

a 24 hour inter-trial interval, exploration of the novel object is reduced in animals

123

exposed to prenatal caffeine. This task has both an exploratory behavior component as
well as a memory retention component such that an animal must have sufﬁciently
explored the familiar object during the pretest phase in order to distinguish between it and
a novel object later during the test phase (Ennaceur & Delacour, 1988; Ennaceur &
Meliani, 1992). Caffeine-treated animals exhibited less total exploration time during pre-
training than controls. This is similar to previous ﬁndings in other behavioral paradigms
showing prenatal caffeine-treated adult animals exhibit decreased exploratory behavior,
which may be due to altered emotional reactivity in caffeine treated animals (Hughes &
Beveridge, 1986). However, the total exploration time of control and caffeine-treated
animals during the 3 and 24 hour tests were not signiﬁcantly different. At 3 hours, both
groups had a discrimination index greater than zero indicating preference for the novel
over the familiar object. After a 24 hour delay, caffeine-treated animals had a
discrimination index less than zero indicating that they could no longer distinguish
between the familiar and novel objects indicating a deﬁcit in memory, exploratory
behavior, or attentional processes.

A deﬁcit in the performance of caffeine-treated animals in the radial arm maze
was also found, with caffeine-treated animals performing signiﬁcantly more reference
and working memory errors than control animals. Increased errors performed by
caffeine-treated animals indicate that prenatal caffeine exposure may alter the developing
hippocampus and prefrontal cortex affecting both working and reference memory in
adults.

Unlike the ﬁndings in the radial arm maze, no deﬁcits were found in the Morris

water maze. In the standard version of the Morris water maze employed, there was

124

approximately a 30-minute delay between trials, with all 12 trials and the probe trial run
on the same day. Thus, the memory retention demands of this version of the water maze
task were greatly diminished. However, in the advanced Morris water maze task, the
memory demands were increased in both the reversal and working memory paradigms
and still there were no differences between control and caffeine-treated animals. This
may be due to many differences between the Morris water maze and radial arm maze
tasks, including level of difﬁculty, delay between trials, appetitive vs. aversive
motivation used, and the type of memory tested.

In the simple version of the water maze, the caffeine-treated animals had a
signiﬁcantly shorter path length when compared to controls, indicating that caffeine-
treated animals performed as well as, or better, than controls in directly ﬁnding the
platform. The decrease in path length in caffeine-treated animals may be due to the
aversive motivation used in the water maze. If prenatal caffeine exposure increases
anxiety in juveniles and adults, as has been shown previously (Hughes & Beveridge,
1986, 1991), these animals may utilize a more direct escape route to avoid the stressful
conditions inherent to the water maze. Previous studies have shown that animals with
prior exposure to stressful experiences have enhanced learning and memory in the water
maze when compared to unstressed animals (Yang et al., 2003).

It is also possible that prenatal caffeine exposure alters motivational or
exploratory aspects of behavior. It has been shown previously that perinatal caffeine
exposure alters locomotor activity, increases emotional reactivity, and increases adrenal
weight, particularly in males (Hughes & Beveridge, 1986, 1991). Each of the tasks

presented in this study has a motivational aspect such that caffeine-treated animals may

125

have differences in motivation to explore a novel object or a novel arena. As previously
discussed, in the simple version of the Morris water maze, there is a facilitating effect of
caffeine treatment on path length. The caffeine-treated animals appear to be more
motivated to ﬁnd the hidden platform than control animals, with the largest difference in
path length occurring during the ﬁrst trial. Perhaps this difference may be attributed to
decreased exploratory behavior such that caffeine-treated animals are less motivated to
explore the maze in their initial exposure to the novel area. However, no differences
were found in path length or latency to ﬁnd the platform on any of the advanced Morris
water maze tasks. Nevertheless, it is possible that prenatal caffeine exposure may be
altering motivational and exploratory aspects of behavior making it difﬁcult to know
whether and how these changes may alter learning and memory testing.

Another concern is that gestational caffeine intake may alter maternal behavior
and the intrauterine environment of dams, either of which may alter behavior of the
offspring. Recently, it has been shown that wildtype mice born to dams heterozygous for
the Al adenosine receptor exhibit hyperactivity as adults, similar to wildtype mice
exposed to caffeine (0.3g/L) from gestational day 1 to postnatal day 21 (Bjorklund et al.,
2008). Dams heterozygous for the A1 receptor express approximately half the number of
Al receptors in the cortex and hippocampus. Similarly, chronic caffeine exposure leads
to a blockade of approximately half the Al adenosine receptors in the brain. These
studies suggest that alterations in maternal A1 receptor ﬁrnction may have long-lasting
changes on offspring behavior. Bjorklund et al. (2008) hypothesize that the alterations in
offspring behavior are likely due to changes in the prenatal intrauterine environment

rather than maternal behavior, since they observed no differences in maternal behavior.

126

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Others have shown no changes in maternal behavior, as assessed by nesting activity as
well as licking and grooming behaviors, in dams exposed to caffeine (Picard et al., 2008)
and no effects of cross-fostering on reduced effects of caffeine on the offspring (Ajarem
& Brain, 1993). Therefore, it seems unlikely that the behavioral changes reported are due
to alterations in maternal behavior, however, it is not possible to completely rule out the
effects of the intrauterine environment and maternal behavior as important factors.

The mechanisms by which prenatal caffeine exposure may alter the brain are not

well understood. It is known that caffeine targets the adenosine receptor in both the

 

developing and adult brain (F redholm et al. , 1999), however it is unknown whether
caffeine exerts the same action at these receptors in the fetus and adult. Prenatal
exposure to caffeine decreases A1 adenosine receptor expression in the fetal rat brain
(Aden et al., 2000; Leon et al., 2002). In the brain of young adult rats, A1 receptor levels
are up-regulated in the cortex, hippocampus, and cerebellum following postnatal caffeine
treatment (Guillet & Kellogg, 1991). Thus, exposure to caffeine during development can
have long-term effects on adenosine receptor expression. Further, the levels of mGluRs
are decreased in the fetal brain following caffeine and theophylline treatment (Leon et al. ,
2005a). Enduring changes in adenosine or glutamate receptor expression following
perinatal caffeine exposure may contribute to changes in learning and memory.

Recent studies in transgenic animals have shown that altered adenosine receptor
expression impairs learning and memory behavior. Similar to the current ﬁndings,
transgenic rats over-expressing the adenosine A2A receptor were found to have impaired
novel object recognition and increased errors in a 6-arm radial tunnel maze while

showing no impairments in place learning and reversal tasks in the Morris water maze

127

(Gimenez-Llort et al., 2007). A1 receptor knockout animals also show no impairments in
the Morris water maze, while showing slowed habitutation to the 6-arm tunnel maze and
impaired paired pulse facilitation of long-term potentiation (Gimenez-Llort et al., 2005).
Thus, if chronic prenatal caffeine is altering adenosine receptor expression in adults, it
seems likely that animals'may have impaired learning and memory which is not detected
using the Morris water maze alone.

In the current study, no sex differences were found in learning and memory
behaviors as a result of prenatal caffeine exposure. This ﬁnding was surprising given the
previous literature on sex dependent effects of developmental caffeine exposure. In adult
rats, it has been shown that neonatal caffeine exposure (15-20 mg/kg/day on PN2-6) has
differential sex effects on passive avoidance learning with females exhibiting enhanced
memory retention at 24 and 72 hours after training, while males exhibit signiﬁcantly
reduced retention at both time periods (Fisher & Guillet, 1997). Interestingly, the same
neonatal caffeine treatment does not cause gender speciﬁc effects in juvenile passive
avoidance memory tests (Fisher & Guillet, 1997). Prenatal caffeine exposure has also
been shown to alter passive avoidance learning in a sex dependent fashion. Adult female
offspring of caffeine treated dams (60 mg/kg/day on GDl3-l9) showed signiﬁcantly
enhanced retention at 25 days after training when compared to placebo treated controls
(Swenson et al., 1990). Sexually dimorphic effects of prenatal caffeine exposure have
also been shown on measures of locomotion and anxiety in the open ﬁeld, with male
animals exhibiting decreased activity and increased defecation (Hughes & Beveridge,
1991). In humans, mean intake of approximately 300 mg/day caffeine during the third

trimester of pregnancy was associated with an increased risk of lowered birth weight in

128

males, but not in females (Vik et al., 2003). Thus, the current literature suggests that
males may be more susceptible to the detrimental effects of developmental caffeine
exposure. However, the current study and others (Zimmerberg et al. , 1991) suggest that
both males and females may be affected in speciﬁc learning and memory paradigms.
Future research is needed to determine why developmental caffeine exposure renders
males more susceptible to some detrimental effects of caffeine, while affecting other
behaviors equally in both sexes.

In summary, prenatal caffeine exposure alters some learning and memory
behaviors in adult male and female rats. Caffeine’s ability to cross both the placental and
blood-brain barrier allows it to enter the fetal brain where it has actions at adenosine
receptors. Developmental changes in the expression or function of adenosine receptors
within the hippocampus and cortex following prenatal exposure to caffeine may
contribute to the long-term alterations in learning and memory reported here. Thus,
pregnant women should consume caffeine with caution as it may have long-term

developmental consequences on multiple facets of behavior including cognition.

129

Chapter 5: Chronic prenatal caffeine exposure increases hippocampal volume in

juvenile rats

A. Introduction

Caffeine is the most widely consumed psychoactive stimulant in the world
(Fredholm et al., 1999). Caffeine is commonly consumed during pregnancy despite
uncertainty about the neurodevelopmental effects of prenatal caffeine exposure.
Consumption during pregnancy is of concern because caffeine and its metabolites cross
both the placental and blood brain barriers where they accumulate in the fetal brain
(Ikeda et al., 1982; Tanaka et al., 1984; Amaud, 1993). As shown previously in Chapter
2, caffeine and its metabolites are each higher in the fetal brain when compared to the
maternal brain regardless of dose.

Caffeine and its metabolites are methylxanthines that function as adenosine
receptor antagonists. Of the four known adenosine receptors expressed within the brain,
the A1 and A2A receptors mediate the stimulatory effects of caffeine (F redholm, 1980).
The A1 receptors are located throughout the cortex, hippocampus, and cerebellum
(Fastbom et al. , 1987) whereas the A2A receptors are located in regions of the brain
containing axon terminals of dopaminergic neurons, primarily the striatum (Svenningsson
et al. , 1999). The Al and A2A receptors are expressed in the fetal brain with mRNA
detectable for both receptors by gestational day 14 and thus may be antagonized by

prenatal caffeine (Weaver, 1993; Weaver, 1996).

130

The effects of antagonism of A1 and A2A adenosine receptors during
development are not well understood, however, caffeine could act at these receptors to
produce neurotoxic effects in the developing brain. In neonatal rats, apoptotic cell death
is signiﬁcantly increased in the cortex, hippocampus, thalamus, and hypothalamus 12
hours after a single injection of caffeine (100 mg/kg on postnatal day 3; Black et al. ,
2008) and 24 hours following three caffeine injections (50 mg/kg on postnatal day 7;
(Kang et al., 2002). At lower doses (2.5, 5, or 10 mg/kg/day on postnatal days 4-10),
caffeine does not signiﬁcantly increase cell death (Desfrere et al., 2007). However,
exposure to low dose caffeine during development results in decreased cell proliferation
in the dentate gyrus and subventricular zone and also causes a transient and dose-
dependent decrease in astrocyte expression in the hippocampus and striatum of neonatal
rats (Desfrere et al. , 2007). Caffeine (lg/L) administered throughout gestation and
lactation increases acetylcholinesterase activity in the hippocampus of neonatal rats,
supporting the hypothesis that caffeine acts at the A1 and A2A receptors during
development to increase neuronal activity (da Silva et al., 2008). Therefore, caffeine
could act at the A1 and A2A receptors to dose-dependently alter cell proliferation and
cell death in the developing brain.

Caffeine induced alterations in cell proliferation and cell death during
development could result in gross anatomical changes within the brain. Prenatal caffeine
exposure signiﬁcantly decreases brain weight in newborn rats (Tanaka et al., 1987).
Neonatal caffeine administration (50 mg/kg/day on postnatal days 1-12) increases the
dendritic length of pyramidal neurons in the prefrontal cortex in both juvenile and adult

rats (Juarez-Mendez et al., 2006). The increase in dendritic length of the pyramidal

131

neurons of the prefrontal cortex may be a direct result of adenosine receptor antagonism
in the cortex. It may also be an indirect effect of increased activity in other regions of the
brain. For instance, the prefrontal cortex receives glutamatergic inputs from the
hippocampus suggesting that Al receptor antagonism in the hippocampus may increase
activity in the prefrontal cortex. However, the effects of developmental caffeine
exposure on the hippocampal anatomy are unknown. The aim of the present study was to
document the effect of moderate chronic prenatal exposure to caffeine (10 mg/kg/day) on
hippocampal volume in juvenile and adult male and female rats. The ﬁndings suggest
that daily oral administration of caffeine to pregnant rat dams increases the volume of the
hippocampus and dentate gyrus in juvenile offspring but that this volume difference does

not persist into adulthood.

132

B. Materials and Methods

Animals

Female Sprague-Dawley rats 60-70 days of age were mated within our colony at
Michigan State University. From gestational day 4 until the day of birth, pregnant dams
were administered ad libitum tap water or 75 mg/L caffeinated tap water. Daily water
intake was recorded. Oral administration was used to avoid the stress associated with
injections or gavage administration.

On postnatal day 2 (PN2), litters were culled to 4 males and 4 females. On PN25,
pups were weaned to 2-3 animals per cage according to litter and sex. All animals were
housed in a temperature and humidity controlled environment with a 12 h light/dark cycle
(lights on at 0500 hours) with standard chow available ad libitum. The experiments were
approved by the Michigan State University Institutional Animal Care and Use Committee
and followed NIH guidelines for animal use.

Juvenile (n=16) and adult (n=27) animals were perfusion ﬁxed on either PN3O or
PN150, respectively, with 4% paraforrnaldehyde in 0.1M phosphate buffer. Brains were
kept in ﬁxative overnight and then transferred to a para-sucrose (4:30%) solution and
stored until sectioned. Coronal 50pm sections were collected through the entire brain
with every third section mounted on gelatin-coated slides and stained with methylene-

blue azure II.

133

Cavalieri Estimation of Volume

The volume of the anterior to posterior bilateral extent of the hippocampus and
dentate gyrus was measured using the Cavalieri method (Gundersen et al., 1999). For
analysis, the volume of the hippocampal formation was subdivided into two regions of
interest: 1) Ammon’s horn which is comprised of the CAI-CA3 ﬁelds and 2) the dentate
gyrus (Figure 5-1). The delineation of each region of interest was made using a rat brain
atlas for each section (Paxinos and Watson, 2005). The anterior-most tissue section used
for volume analysis was a randomly chosen section within 150 um of the start of the
hippocampus and dentate gyrus. Each subsequent tissue section used for volume analysis
was approximately 300 um (or six tissue sections) from the previous section. The entire
extent of the hippocampus and dentate gyrus were analyzed with the posterior-most tissue
section being the last section in the series to contain Ammon’s horn or dentate gyrus in
either hemisphere.

A total of 12-14 sections were analyzed for each juvenile animal and a total of 18-
20 sections were analyzed for each adult animal. Analysis was performed using a 4X
objective and 75 um grid spacing with the Stereolnvestigator software (MicroBrightField
version 8.03, Williston, Vermont), which was coupled to a Nikon Eclipse 80i microscope
with a motor-driven LEP stage and Optronics Microﬁre cooled CCD camera. The
Cavalieri method was used to determine the surface area of each region, which was
multiplied by the tissue thickness to determine the volume of each plane, using the
following equation:

Volume = 2p >< (a/p) X t X s

134

where 2p is the total number of grid points overlying a region of interest, (a/p) is the area
represented by each point on the grid, t is the tissue thickness per section, and s is the
section sampling interval. The total volume of a brain region was determined by
summing the area across all the tissue planes (12-20 total) through the entire depth of the
tissue samples (between 3600-6000um).

Measurements of a 2400um length cross-section of the brain were taken to ensure
that the volume of the brain did not alter hippocampal volume measurements. Because
tissue can shrink during the staining process it is important to determine that volumetric
differences are not merely the result of a total brain volume difference. Differences in
brain volume were determined by drawing a contour around each brain section to ﬁnd the
area of that section. The anterior most section sampled was the ﬁrst section to contain
hippocampus and the last section was that located 2400um posterior of the ﬁrst section
sampled. The volume of this section of brain was determined using the equation
previously described.

The extent of the hippocampus was determined by counting the number of
sections that contained hippocampus. This number was then multiplied by the thickness
of each section to determine the approximate anterior to posterior extent of the

hippocampus.
Statistics

Statistical assessment of weekly water intake was performed using two-way

repeated measures analysis of variance (week, treatment). For all other analyses, two-

135

way analysis of variance (sex, treatment) was performed. All statistics were performed

using the Systat program (version 11.0).

136

 

 

l I Ammon's Horn

1 Dentate Gyrus

 

Figure 5-1. Representative coronal sections highlighting the rat hippocampus to show
the delineation of the Ammon’s horn and dentate gyrus used for the bilateral volume
measurements (Swanson, 1998). The entire area within the medium gray area was used
to determine the area of Ammon’s horn, while the entire dark gray area area was used to
determine area of the dentate gyrus. Both areas combined were used to determine the
area of the hippocampal formation.

137

C. Results

During the time of caffeine exposure (gestational day 4 through birth), the
average water intake in control and caffeine-treated dams signiﬁcantly increased each
week (Figure 5-2; within-group analyses, p=0.00). However, there was no signiﬁcant
main effect of treatment on water intake.

In juvenile male and female rats, two-way analysis of variance revealed a
signiﬁcant effect of treatment on total hippocampal formation volume [F(l ,12)=7.324,
p<0.020] with caffeine treated juveniles having a 20.6% greater hippocampal volume
when compared with controls (Figure 5-3A). Also, there was a signiﬁcant effect of
treatment on Ammon’s horn volume [F (1,12)=5.124, p<0.05] and dentate gyrus volume
[F(l,12)=12.603, p<0.01], with caffeine treated juveniles having a 19.6% larger volume
of Ammon’s horn and a 20.0% larger dentate gyrus volume when compared to controls
(Table 5-1). There were no signiﬁcant effects of sex or treatment by sex interactions
across any of the volume measurements.

In adult male and female rats, there were no signiﬁcant effects of treatment or
treatment by sex interactions across any of the volume measurements. However, two-
way analysis of variance showed a signiﬁcant effect of sex on hippocampal volume
[F(l,23)=21.853, p<0.000] with females having a 9.6% decrease in volume when
compared to males (Figure 5-3B). There was also a signiﬁcant effect of sex on
Ammon’s horn volume [F (1,23)=15.344, p<0.01] and dentate gyrus volume
[F(l,23)=21.646, p<0.01] with an 8.1% and 12.2% increase in male volumes,

respectively, when compared to females (Table 5-1).

138

There was no signiﬁcant effect of treatment on the volume of the 2400um extent
of whole brain analyzed for juveniles or adults. However, there was a signiﬁcant effect
of treatment [F(l,12)= 16.124, p<0.01] on the anterior to posterior length of the
hippocampus in juveniles such that caffeine treated animals had a mean extent of 4406 :1:

49pm whereas control animals had an extent of 3675 :1: 165 um.

139

Water Consumption

air—”i
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0 O O
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01
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00
O
1

Daily Intake (ml)
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Week 1 Week 2 Week 3
+Control “-0- Caffeine

Figure 5-2. Maternal intake of water (n=4) or caffeinated tap water (n=4) from
gestational day 4 until the day of birth (gestational day 23 :1: 1 day). Data represent the
mean daily intake i SEM for each gestational week. Water intake increased throughout
gestation, but there was no main effect of treatment on intake.

140

A Juvenile Hippocampal Volume

*
B Male

 

I Female

 

 

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0

Volume (mm?)
M N
0 0|

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Control Caffeine

B Adult Hippocampal Volume

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60 -
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40 t

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Volume MW)

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104

 

 

 

 

Control Caffeine

Figure 5-3. Effect of prenatal caffeine on total hippocampal volume in (A) juveniles (n-
16) and (B) adults (n=27). Data represent the average volume d: SEM. Juvenile rats
show a signiﬁcant main effect of treatment (p<0.05) whereas adult rats show no main
effect of treatment but a main effect of sex (p<0.00).

141

 

Hippocampal Ammon's Dentate
Formation Horn Gyrus

Juvenile

Control Male (n=4) 33.8 (4.0) 23.7 (3.2) 10.0 (0.9)
Control Female (n=4) 34.1 (1.7) 24.1 (1.6) 10.1 (0.5)
Caffeine Male (n=4) 42.4 (2.6) 29.7 (2.1) 12.8 (0.6)
Caffeine Female (n=4) 39.5 (0.8) 27.6 (0.6) 11.9 (0.3)
Adult

Control Male (n=5) 62.3 (1.8) 39.4 (1.2) 22.9 (0.7)
Control Female (n=7) 55.4 (1.1) 35.8 (0.7) 19.6 (0.6)
Caffeine Male (n=7) 59.3 (1.6) 37.9 (1.0) 21.4 (0.7)
Caffeine Female (n=8) 54.2 (0.8) 35.1 (0.5) 19.1 (0.4)

Table 5-1. Volumes (mm3) for the whole hippocampal formation, Ammon’s horn, and
dentate gyrus for control and caffeine-treated juveniles and adults. Data represent the

average volume (:1: SEM).

142

D. Discussion

Results for the present study demonstrate that chronic prenatal caffeine exposure
to a moderate dose of caffeine increases total hippocampal volume in juvenile rats but
that this volume increase does not persist into adulthood. Speciﬁcally, chronic prenatal
caffeine exposure increases the volume of the hippocampus in juveniles by
approximately 20% when compared with controls. This volume increase occurs in both
caffeine-treated males and females, however males exhibit the greatest change.
Interestingly, this treatment effect does not persist into adulthood and it is unknown what
changes may occur during puberty to eliminate the caffeine-induced alterations in
hippocampal volume.

These data suggest that changes in volume that result from prenatal caffeine
exposure are dependent on the overall anterior to posterior extent of the hippocampus.
Analysis of the total number of sections containing hippocampus or dentate gyrus
revealed that caffeine-treated animals have an approximate 700 um greater length of the
hippocampus when compared to controls. Because most studies do not report the anterior
to posterior extent of the hippocampus, it is not known whether this difference accounts
for the volume differences seen after other prenatal drug exposures.

The sex difference found in adult animals was not surprising given that this
difference has been widely reported. Adult male rats have increased volume (Diamond et
al., 1983), increased cell number (Juraska et al., 1989), and increased dendritic
arborization of the hippocampus when compared adult females. Although the caffeine-

treated juvenile animals appear to have a sex difference, there was no main effect of sex

143

between caffeine-treated males and females. Therefore, the sex-dependent effects on
hippocampal volume are not observed prior to puberty.

After a thorough review of the literature, the present study appears to be the ﬁrst
to document the effects of prenatal caffeine exposure on hippocampal volume, making it
difﬁcult to compare these ﬁndings to the previous reports. However, numerous
developmental drug exposure studies using other drugs of abuse have shown alterations
in hippocampal volume and neuronal number in pre-pubertal and adult rats. Postnatal
exposure to alcohol during PN days 4-9 has been shown to decrease the volume of CA1
and CA3 in PN10 rats, with an associated decrease in CA1 pyramidal cell number (Livy
et al., 2003). In PN3O rats exposed to ethanol perinatally, CA1 volumes were larger
while the number of pyramidal neurons in the CA1 was lower than controls (Gonzalez-
Burgos et al. , 2006). Similar to the present ﬁndings, perinatal cocaine exposure resulted
in a greater volume of the dentate granule cell layer in juveniles but this effect was
reversed in adults (Ismail & Bedi, 2007). In the same animals, juveniles showed a
decrease in the total number of pyramidal cells whereas adults showed a decrease in both
pyramidal and granule cells when compared to controls. Therefore, it is not uncommon
for drug exposure during perinatal development to alter hippocampal volume and neuron
number. However, it is unknown why alterations in volume and neuron number are oﬂen
transient and do not persist into adulthood.

The relationship between hippocampal volume and behavior has just recently
begun to be elucidated. For instance, hippocampal volume measurements taken using
MRI technology are important predictors of disease states. Diseases such as depression,

schizophrenia, bipolar disorder, and post-traumatic brain disorder have been associated

144

 

with shrinkage of the hippocampus (Czeh & Lucassen, 2007). Each of these diseases is
associated with speciﬁc behaviors, which may or may not be related to the associated
changes in hippocampal volume, but further research will likely determine whether there
is a direct structure-function relationship. Interestingly, the use of MRI techniques to
study these diseases in humans has provided some evidence that the hippocampal volume
can change throughout the lifespan. In some depressed patients who receive anti-
depressant treatment, the volume of the hippocampus is normalized (Czeh & Lucassen,
2007).

Many MRI studies in humans have also correlated decreased hippocampal volume
to decreased memory, although this sentiment is widely debated (Van Petten, 2004).
Interestingly, meta-analysis of the relationship between memory abilities and
hippocampal volume has shown a positive relationship between smaller hippocampal
volumes and increased memory abilities in healthy adolescents. However, hippocampal
volume varies widely in normal healthy adults (Van Petten, 2004). In rats, approximately
26% of dentate gyrus granule cells labeled during peak granule cell proliferation in
neonates die, whereas granule cells developed in adulthood are much more stable (Dayer
et al. , 2003). Therefore, it is possible that the hippocampus is reorganizing and changing
volumes throughout the lifespan and that alterations in volume in juveniles are lost during
hippocampal reoganization in adulthood. Further, it is likely that hippocampal volume
may be directly related to learning and memory behaviors (Van Petten, 2004), however
more research into this structure-function relationship is needed.

The development of the hippocampus and dentate gyrus occurs during gestation

and early neonatal periods. In the rodent hippocampus, all of the pyramidal cells and

145

neurons in Ammon’s horn are formed prior to birth (Bayer, 1980a, b). Unlike neurons
that form Ammon’s horn, neurogenesis of dentate granule cells occurs postnatally and
continues at a reduced rate throughout life. In the rat, dentate gyrus development takes
place between gestational day 14 and the second postnatal week of life, with a peak of
activity around postnatal day 5-7 (Schlessinger et al. , 1975). Granule cells increase their
dendritic trees until PN14 after which the branches are sculpted to form adult-like
morphological features (Rahimi & Claiborne, 2007). In humans, hippocampal
development occurs over a prolonged period of gestation. By gestational week 15,
neurogenesis of pyramidal neurons is visible in Ammon’s horn with adult-like
morphological features of the CA1-CA3 ﬁelds established by the 23-25th gestational
week (Arnold & Trojanowski, 1996a, b). Neurogenesis in the granule cell layer of the
dentate gyrus begins around gestational week 14 (Humphrey, 1967) and continues
postnatally (Seress et al., 2001). Therefore, it is not surprising that prenatal exposure to
drugs during these critical stages of hippocampus and dentate gyrus formation can alter
the overall morphology of both structures in treated offspring.

Caffeine’s actions within the developing brain and how these actions may affect
hippocampal development are not well understood. The A1 and A2A receptors, which
mediate caffeine’s stimulatory actions, are expressed in the fetal hippocampus of both
rodents and humans (Weaver, 1993; Weaver, 1996). It is known that adenosine, acting at
the Al receptors, depresses glutamatergic activity and regulates giant depolarizing
potentials within the hippocampus during the ﬁrst week of postnatal life in rodents
(Saﬁulina et al., 2005). Giant depolarizing potentials are a trait of the developing

hippocampus and are characterized by recurrent spontaneous membrane depolarizations

146

(Ben-Ari et al., 1989). Giant depolarizing potentials are instrumental in shaping the
synaptic connections of the hippocampal circuitry and are mediated by the actions of
GABA and glutamate in the developing brain (Saﬁulina et al., 2006). Alterations in the
frequency of giant depolarizing potentials may alter the development of hippocampal
neural networks (Dzhala et al. , 1999). If caffeine antagonizes the adenosine receptors in
the hippocampus, primarily the A1 receptors, such that adenosine can no longer regulate
or depress the frequency of the giant depolarizing potentials, the development of the
hippocampus may be negatively affected. Further, glutamatergic NMDA receptors play a
fundamental role in activity-dependent migration, synapse formation, and functional
connectivity of the developing hippocampus (Ben-Ari & Holmes, 2006). Caffeine may
increase glutamate release in the developing hippocampus thereby disrupting these
processes, which may account for the increase in volume observed in the present study.

Caffeine also has numerous indirect effects on hippocampal development.
Acute administration of caffeine has been shown to increase hippocampal cell death in
the neonatal brain (Kang et al., 2002; Black et al., 2008), decrease cell proliferation in the
dentate gyrus and astrocyte expression in the hippocampus (Desfrere et al., 2007), and
increase acetylcholinesterase activity in the hippocampus (da Silva et al. , 2008). It is
possible that caffeine-induced alterations in cell death, neurogenesis, glial cell number, or
activity dependent synaptogenesis may account for changes in hippocampal volume.

In summary, exposure to a moderate dose of caffeine throughout gestation alters
the volume of the hippocampus in juveniles, but these differences are no longer present in
adult offspring. The use of caffeine during pregnancy is of concern because it crosses

both the placental and blood-brain barriers to reach the developing fetal brain. The

147

actions of caffeine within the fetal brain are not well understood but caffeine may act as
an antagonist of adenosine receptors within the developing fetal brain, which may alter
pyramidal and granule cell neurogenesis, migration, and synaptic connectivity in the
hippocampus and dentate gyrus. As the hippocampus plays a critical role in learning and
memory, care should be taken to reduce caffeine consumption during pregnancy until a

more thorough understanding of its effects on neurodevelopment becomes available.

148

Chapter 6: Discussion

Caffeine is the most widely used psychoactive drug in the world. Although it is
estimated that 70-95% of women continue to consume caffeine during pregnancy (F redholm
et al., 1999), the effects of prenatal caffeine exposure have not been well investigated. The
use of caffeine during pregnancy is of concern because it crosses both the placental and
blood brain barriers to reach the developing fetus (Amaud, 1993) where the half-life of
caffeine and its primary metabolites are greatly prolonged due to an immaturity of
cytochrome P-450 metabolism (Aranda et al., 1979a). The slowed metabolism of caffeine
and its metabolites in the fetus results in methylxanthine accumulation in the developing
brain (Chapter 2; Wilkinson & Pollard, 1993) and poses a potential risk to neural
development if unsafe levels are reached. Caffeine is an adenosine receptor antagonist and
adenosine is a well-known critical neuromodulator during brain development (F isone et al. ,
2004). Adenosine functions to reduce neuronal activity thus providing protection against
excitotoxicity (F redholm, 2007). When adenosine receptors are inhibited, there is an overall
increase in glutamate neurotransmission particularly in brain regions with an abundance of
adenosine receptors such as the cortex and hippocampus (Wang, 2007). Given the
widespread effects of caffeine and the frequency of prenatal exposure, it is imperative to
better understand the effects of the drug on the developing brain. The objectives of this
dissertation were to determine whether caffeine and its primary metabolites accumulate in the
fetal brain and what effects these drugs have on glutamate receptor expression, glutamate-
mediated calcium transients, hippocampal-dependent learning and memory behaviors, and

hippocampal anatomy.

149

In order to elucidate the effects of caffeine exposure, pregnant rats were exposed to
chronic moderate doses of caffeine throughout gestation. The model of chronic exposure to
moderate doses of caffeine in the drinking water was chosen to ensure that ﬁndings from this
dissertation are as relevant to the human situation as possible. Self-reports of pregnant
women who consume caffeine estimate their daily intake to range between 100-300 mg
(Christian & Brent, 2001). This range is likely due to current medical recommendations,
which suggest that pregnant women reduce intake to less than 300 mg/day. Metabolism of
caffeine and its primary metabolites is known to occur at a faster rate in rats than in humans.
For instance, the half-life of caffeine in adult humans is approximately 2-4 hours whereas the
half-life in adult rats is approximately 1-2 hours (Bonati et al., 1984-1985). Therefore, much
of the previous literature suggests use of a metabolic body weight conversion to determine
approximately equivalent doses between rats and humans. Metabolic body weight (=body
weightA3/4) calculations used were based on ﬁndings that 20 mg/kg in a rat is equivalent to
4-6 cups of coffee in a 70 kg human (Nehlig & Derby, 1994). The dose most commonly
used in this dissertation was 75 mg/L of which pregnant rats consumed approximately 40-60
ml/day, or an intake of approximately 10 mg/kg/day in the rat, which is approximately
equivalent to consumption of 2-3 cups of coffee per day in humans. This dosing regimen
aligns well to self-reports of human consumption. The additional higher doses of 150 and
300 mg/L described in Chapters 2 and 3 were used to allow comparisons to the previous
literature, in which doses of 300-1000 mg/L are most common. Thus, the studies in this
dissertation not only align well to the human condition they also present novel ﬁndings of

neurodevelopmental effects following low to moderate exposure to prenatal caffeine.

150

Rats are an excellent animal model of caffeine exposure due to the similar
pharmacokinetics of the drug between rats and humans. In both rats and humans,
caffeine is metabolized into the three primary metabolites paraxanthine, theobromine, and
theophylline with nearly superimposable plasma curves (Fredholm et al., 1999).
However, one drawback to using a prenatal rat model is the short length of gestation (21 -
24 days) in rats. Newborn rats are underdeveloped in comparison to newborn humans
' and it is common to consider postnatal days 1-7 in the rat parallel to the third trimester of
development in humans (Clancy et al., 2007). This shortened period of gestation in rats
results in a much shorter period of prenatal exposure than would be seen in the human
fetus if a mother drank caffeine throughout pregnancy. Furthermore, in humans
extensive brain development occurs during gestation whereas in rats the brain growth
spurt occurs primarily during postnatal development (Bayer, 1980a, b). For the studies
presented in this dissertation, rats were exposed to caffeine only during prenatal
development. This period of exposure was chosen to avoid differences between placental
and lactational transfer, dosing, and metabolism of caffeine. However, the differences
between gestational length and prenatal brain development between rats and humans
should not be overlooked. It is possible that the ﬁndings following prenatal exposure in
rats under reﬂect the neurodevelopmental consequences of prenatal caffeine exposure in
humans.

The studies in this dissertation focused primarily on the effects of prenatal
caffeine exposure on the developing hippocampus. The hippocampus was chosen
because it has an abundance of A1 adenosine receptors, which are likely to be

antagonized during caffeine exposure. The hippocampus is also well known to be a brain

151

region particularly susceptible to prenatal insult. For instance, hippocampal development
is impaired following prenatal exposure to alcohol, hypoxia-ischemia, seizures, anesthetic
medications, and stress. The hippocampus is also well characterized both anatomically
and behaviorally, which lends to enhanced experimentation of this region. Prior to this
dissertation work, the effects of prenatal caffeine on the developing hippocampus had not
been well investigated, making this a valuable region for analysis.

The ﬁrst objective of this dissertation (Chapter 2) was to determine whether
chronic moderate exposure to prenatal caffeine resulted in an accumulation of caffeine
and its primary metabolites in the fetal brain. A previous study showed that caffeine and
its metabolites accumulate in the fetal brain, however this study used a single maternal
dose of 5 or 25 mg/kg administered by gavage (Wilkinson & Pollard, 1993). Because the
pharmacokinetics of caffeine may differ following bolus and intermittent intake, it was
important to reevaluate these ﬁndings with an ad libitum administration paradigm.
Furthermore, it was important to assess whether repeated exposures to caffeine result in
higher-fold accumulation due to the prolonged half-life and lack of metabolism of
caffeine in the fetus. Results from the ﬁrst objective show that caffeine and its
metabolites do accumulate in the fetal brain when compared with the maternal brain.
Speciﬁcally, it was found that maternal intake of caffeine results in at least 3-fold higher
concentrations of caffeine and its metabolites in the fetal brain than in the maternal brain.
If the levels of all four bioactive methylxanthines are considered in combination, the
levels in the fetal brain are greatly elevated in comparison to the levels in the maternal
brain. In contrast, no differences were found between caffeine and metabolite levels in

the fetal and maternal plasma. Importantly, these ﬁndings indicate that measurements of

152

plasma levels in newborns may underestimate actual bioactive methylxanthine levels in
the brain. Therefore, studies of methylxanthine levels in humans, which are performed in
plasma, may greatly underestimate the actual risk posed to the developing brain of
offspring whose mothers consume caffeine.

The results from this study have numerous implications for caffeine use during
pregnancy. As discussed previously, caffeine and its metabolites are greatly elevated in
the fetal brain when compared to the maternal brain, even following exposure to the
lowest dose. It is known that at high concentrations caffeine can elicit actions beyond
antagonism of adenosine receptors. These actions include inhibition of phosphodiesterase
activity, blockade of GABA(A) receptors, and direct effects at numerous ion channels,
including sodium and potassium channels (Daly, 2000). In animal studies, caffeine
administration of doses greater than 20 mg/kg produces depressant rather than stimulant
effects on motor behavior (Fredholm et al. , 1999). Although the exact mechanisms
behind this biphasic response are unknown, it is believed that the behavioral changes
likely result from inhibition of phosphodiesterase activity or changes in GABA(A)
receptor activity. With regards to elevated levels of methylxanthines in the developing
brain, it is currently unknown whether the levels reached can elicit any of the actions
described above, however it is worth considering that there may be effects beyond
antagonism of adenosine receptors. The current ﬁndings also show that methylxanthine
levels reached in the fetal brain increase in a dose-dependent manner with increasing
maternal intake. Therefore, mothers who consume large amounts of caffeinated
beverages each day during pregnancy will likely be exposing the brains of their

developing offspring to extremely high methylxanthine concentrations.

153

Numerous follow-up studies would provide additional invaluable information
regarding the elevated levels of methylxanthines within the developing brain. In the
current study, analysis was performed on whole brains. It would be highly informative to
determine whether caffeine is accumulating at different rates in various regions of the
developing brain. This analysis was not performed in the current study because it proved
very difficult to dissect the hippocampus, cortex, and other brain regions of interest from
the same fetal brain. However, the use of brain slicing and Palkovits punch technique
could allow for this type of analysis. Further, it would be interesting to determine
whether prenatal caffeine exposure is altering neurotransmitter levels within the
developing brain. High performance liquid chromatography with electrochemical
detection could be used to determine the levels of GABA and glutamate following
prenatal caffeine exposure. Similar techniques could also be used to determine the levels
of adenosine within the brain, and it is unlikely that sensitivity would be an issue as
demonstrated in Chapter 2. Such studies would. provide a better understanding of how
prenatal caffeine exposure is altering neurotransmission and which receptors may
consequently be most affected. Although it is hypothesized that glutamate
neurotransmission is increased in the developing hippocampus following caffeine
exposure, such a study would provide more direct evidence for this hypothesis.

The second objective of this dissertation (Chapter 3) was to determine the effects
of prenatal caffeine exposure on NMDA receptor expression and glutamate-mediated
calcium signaling in the neonatal hippocampus. The results showed that the NR1, NR2A,
and NR2B subunits all exhibit slight differences in protein expression at various postnatal

periods, which correspond to peak periods of expression for each subunit. Interestingly,

154

prenatal caffeine exposure also reduced glutamate-induced calcium transients in
hippocampal cultures from treated offspring. Caffeine-treated hippocampal cells showed
diminished maximal calcium transients after glutamate administration when compared to
the maximal calcium transients seen in control cultures. However, no treatment
differences were detected between the levels of phosphorylated ERK and CREB proteins,
suggesting that these calcium-dependent cascades are not affected by prenatal caffeine
exposure. The results from these studies indicate that prenatal caffeine exposure is likely
altering glutamate-mediated neurotransmission and consequently altering expression of
the NR1, NR2A, and NR2B subunits. However, the changes found in subunit expression
appear to be subtle and transient. Therefore, it is unknown whether prenatal caffeine
exposure is causing long-term alterations in glutamate-receptor expression and whether
these changes are of enough magnitude to alter glutamate-receptor dependent processes
such as learning and memory or LTP. It does appear from these studies that early
alterations in receptor expression may affect glutamate-mediated calcium transients.
However, it is unknown whether the reduced maximal peak response seen in caffeine-
exposed hippocampal neurons results from a reduction in overall receptor number or
from a shift in subunit composition that could lead to altered receptor kinetics. Further, it
is possible that changes in AMPA receptor expression may be altered following prenatal
caffeine exposure. Increased glutamate neurotransmission is likely also acting on AMPA
receptors, which are expressed during prenatal and early postnatal development.
Therefore, there may be changes in AMPA receptor expression, which may reduce the
AMPA-mediated depolarization needed to fully activate NMDA receptors. It is also

possible that the reduced calcium transients in caffeine-exposed cultures are mediated by

155

changes in intracellular calcium-induced calcium release or voltage-gated calcium
channels. Thus, it cannot be ruled out that any alteration in calcium inﬂux via these
mechanisms may contribute to the reduced peak calcium amplitude until speciﬁc
antagonists are used to block these sources of calcium.

Although subtle changes in glutamate receptor expression could have numerous
developmental consequences, ﬁirther research is needed to ﬁrlly understand the extent
and magnitude of these changes following prenatal caffeine exposure. One obvious
follow-up study would be to repeat the calcium imaging with the use of speciﬁc voltage-
gated calcium channel antagonists as well as blockers of intracellular calcium channels,
such as the ryanodine receptors. A preliminary study of this type was conducted during
the course of this dissertation, yet it was very difﬁcult to determine whether the
antagonists were working correctly as the responses following glutamate-administration
did not appear to be different regardless of whether antagonists were used in the medium.
For this reason, the use of antagonists was not further pursued for these dissertation
studies. However, if this study could be repeated while performing both
electrophysiology as well as calcium imaging, one could test whether the antagonists
were working correctly prior to conducting the live cell imaging. This setup would also
provide invaluable information of changes in receptor kinetics that may occur following
prenatal caffeine exposure. Another intriguing study would be to perform ﬂuorescent
triple-label immunohistochemistry for the NR1, NR2A, and NR2B subunits in tissue
sections. Like Western blots analyses, such a study would allow for determination of
changes in subunit expression, although it might be difﬁcult to detect subtle changes.

More importantly, it would allow for region speciﬁc determinations of subunit expression

156

within the CA1-3 ﬁelds and the dentate gyrus as well as localization of speciﬁc subunits
to speciﬁc cells within the subdivisions of the hippocampus. Such a study would further
our understanding of both receptor trafﬁcking and region speciﬁc expression following
caffeine exposure.

The third objective of this dissertation (Chapter 4) was to determine the long-term
effects of prenatal caffeine exposure on learning and memory behaviors. For this study,
behavioral assessments in the novel object recognition task, radial arm maze, and Morris
water maze were conducted. The results reveal impaired behavior in caffeine-exposed
offspring in both the novel object recognition task and radial arm maze, but not in the
Morris water maze. Importantly, these results indicate that prenatal caffeine exposure has
long-term neurodevelopmental effects on behavior, however these effects are subtle.
Although the caffeine-exposed animals had more performance errors than control
animals, they learned each task after repeated trials. In the Morris water maze, the
caffeine-exposed animals actually performed as well as or slightly better than control
animals. Thus, the behavioral effects appear to be subtle yet persistent and do not affect
all hippocampal-dependent behaviors. Importantly, these ﬁndings have potential
implications for children exposed to caffeine prenatally and support previous ﬁndings
showing deﬁcits in learning following caffeine-exposure in animals. Many learning
deﬁcits are not detected in children until they are in elementary or middle school.
Because learning deﬁcits are often subtle and not easily detectable early in development,
there is a lack of longitudinal research investigating whether prenatal caffeine exposure

results in long-term alterations of learning in children. It does not seem likely that

157

prenatal caffeine exposure results in large learning and memory deﬁcits, however even
subtle changes could hinder educational success.

An intriguing follow-up to the behavioral studies performed in this dissertation
would be to determine whether there are long-term alterations in LTP following prenatal
caffeine exposure. If changes in LTP were found, this would be the ﬁrst study reported
in the literature with regards to prenatal caffeine. Further, such a study would provide a
more direct link between the hypothesis that glutamate receptor expression or function is
altered following prenatal caffeine and that these changes affect learning and memory
behaviors. Another intriguing follow-up study would be to utilize adenosine receptor
knockout mice to repeat many of the experiments presented in this dissertation. The use
of adenosine receptor knockout animals would further elucidate whether the effects of
prenatal caffeine exposure on glutamate receptor expression, calcium transients, and
learning and behavior are mediated through antagonism of the A1 receptor.

The ﬁnal objective of this dissertation (Chapter 5) was to determine whether there
are anatomical alterations in the hippocampus following prenatal caffeine exposure.
Speciﬁcally, stereological analysis with the Cavalieri estimator method was used to
determine whether there are hippocampal volume differences between control and
caffeine-exposed offspring. The results reveal a signiﬁcant increase in total hippocampal
volume in caffeine-treated juvenile rats, which does not persist into adulthood. These
results are similar to previous ﬁndings showing that neonatal caffeine administration (50
mg/kg/day on postnatal days 1-12) increases dendritic length of pyramidal neurons in the
preﬁ‘ontal cortex in both juvenile and adult rats (Juarez—Mendez et al. , 2006). If the

hypotheses presented in this dissertation are correct, the increase in hippocampal volume

158

 

in juveniles may result from altered glutamatergic activity in the developing brain since
glutamate receptor activity is known to play a key role in cell proliferation and activity-
dependent cell survival. However, because glutamate receptor activity was not directly
tested in the current studies, this remains speculative. Since measures of hippocampal
volume provide limited information, follow-up studies utilizing more in-depth anatomical
measurements are necessary to fully understand the effects of prenatal caffeine exposure
on hippocampal anatomy. One intriguing follow-up would be a study of Golgi-
impregnated neurons to determine if (similar to neurons in the prefrontal cortex)
hippocampal neurons exhibit increased dendritic length and arborization. If this were the
case, this may help explain why overall volume of the hippocampus is increased at
speciﬁc ages. Further, it would be interesting to increase the number of time periods
sampled to better understand how hippocampal size is being regulated between puberty
and adulthood.

Overall, this dissertation has added substantial novel ﬁndings to the area of
prenatal caffeine research, an area that has been vastly understudied in the past few
decades. Like most research, the ﬁndings presented here have raised far more questions
about the consequences of prenatal caffeine exposure than they have answered. Future
research is needed to further elucidate the subtle molecular and cellular consequences of
prenatal caffeine exposure. It would be extremely interesting and clinically important to
utilize a perinatal model for such research to more effectively recreate gestational
exposure in humans. The ﬁndings using such a model are likely to be more robust and
substantially different from the results of prenatal exposure alone. Although this

dissertation highlighted many changes in the hippocampus, numerous brain regions may

159

be affected by prenatal caffeine exposure. Therefore, molecular and cellular analysis is
needed in other brain regions. A few recent studies have highlighted changes in the
prefrontal cortex indicating that perhaps more research into this area is currently being
pursued. Lastly, a better understanding of the actions of caffeine within the developing
brain is needed. In this dissertation, it was hypothesized that caffeine is increasing
glutamate neurotransmission via adenosine receptor blockade. Although this hypothesis
is reasonable based on the known actions of adenosine, the developing brain does not
always respond to drugs in the same fashion as the adult brain. Therefore, additional
experiments must be done to fully understanding the relationship between caffeine,

adenosine, and glutamate neurotransmission in the developing brain.

160

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