DISRUPTION OF CELLULAR CALCIUM HOMEOSTASIS AND MITOCHONDRIAL
FUNCTION FOLLOWING CHRONIC METHYLMERCURY TREATMENT
By
Sara Michelle Ciotti

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Pharmacology and Toxicology – Environmental Toxicology - Doctor of Philosophy
2013

ABSTRACT
DISRUPTION OF CELLULAR CALCIUM HOMEOSTASIS AND MITOCHONDRIAL
FUNCTION FOLLOWING CHRONIC METHYLMERCURY TREATMENT
By
Sara Michelle Ciotti
Methylmercury (MeHg) is a potent environmental neurotoxicant that impairs several
neurological functions upon short and long-term exposure. MeHg causes cell-type specific
damage in the cerebellum and this effect seems to be due to a disruption of intracellular calcium
2+

2+

([Ca ]i) homeostasis and voltage-gated Ca

channel (VGCC) function. The striatum may be a

sensitive target due to its high expression of Cav1.3, an L-type VGCC. Exposure to low levels of
MeHg throughout ones lifetime is of contemporary concern. In viro and in vivo studies were
designed to mimic a lifetime exposure to low-levels of MeHg. Following prolonged exposure to
low-nanomolar MeHg in vitro we found that MeHg causes a time- and subtype-dependent block
2+

of VGCCs, as well as increased resting [Ca ]i. Chronic MeHg exposure in vivo suggests that
MeHg interacts with AMPA receptors altering the post-synaptic response to glutamate at striatal
medium spiny neurons. Chronic MeHg treatment also altered striatal synaptosomal
mitochondrial function. This work contributes to our understanding of the consequences of longterm exposure to MeHg on neuronal function.

ACKNOWLEDGEMENTS

First and foremost I would like to thank my mentor Dr. William D. Atchison for his
invauluable guidance throughout my graduate career. I also want to thank my thesis committee
members- Dr. Susan Barman, Dr. John Goudreau, and Dr. Steven Heidemann- for their
suggestions and careful consideration of my experiments. I would also like to thank the
Department of Pharmacology and Toxicology as well as the Center for Integrative Toxicology.
They have provided me with the opportunity to come to MSU and train in the company of
excellent scientists.
I would also like to acknowledge Dr. Ravindra Hajela for his continued support and
guidance throughout my time in the lab. To my current and former lab mates- Dawn Autio, Dr.
April Neal, Dr. Wen-hsin Ku, Dr. Erin Wakeling, Dr. Elizabeth Molina-Campos, Dr. Brenda
Marrero-Rosado, Dr. Aaron Bradford, Alexandra Colón-Rodríguez, Duanghathai Wiwatrana,
Kaitlyn Sherer, Heidi Hannon, Rosa Jaiman, and Mónica Ríos-Cabanillas- for making the lab an
enjoyable place to work. To Ivelisse Cruz-Torres and Kaye Long-Roldan helping me run the in
vitro experiments. I would not have been able to finish all of my experiments without their
assistance.
Finally, I am forever grateful for my my family. They have always believed in me and
have been a constant source of love and support. I would not have been able to finish graduate
school without them. To my husband James, thank you for your patience, love, and support. He
stayed by my side throught this journey and I couldn’t have done this without him.
iii

TABLE OF CONTENTS

LIST OF TABLES

vii

LIST OF FIGURES

viii

KEY TO ABBREVIATIONS

xiv

CHAPTER ONE: INTRODUCTION
A)
Background
a.
Methylmercury
i.
Methylmercury in the environment
ii.
Methylmercury and human health
b.
Neuronal calcium regulation
i.
Calcium regulation
ii.
Calcium and aging
iii.
Methylmercury and calcium regulation
c.
Striatal and nigrostriatal dopamine system
i.
Neuronal organization
ii.
Methylmercury and the striatum and the NSDA system
B)
Objectives and Rationale
a.
Hypothesis and aims
b.
Model systems
i.
SH-SY5Y cell model
ii.
Synaptosomes
iii.
Striatal slice
c.
Techniques
i.
Calcium imaging
ii.
Mitochondrial function

1
2
2
2
3
8
8
13
14
17
17
21
22
22
23
23
25
26
27
27
29

CHAPTER TWO: PERTURBATION OF CALCIUM REGULATION FOLLOWING
ACUTE AND CHRONIC METHYLMERCURY TREATMENT IN DIFFERENTIATED
SH-SY5Y CELLS
31
A)
Abstract
32
B)
Introduction
34
C)
Materials and methods
37
Chemicals and solutions
37
Cell culture
39
Single-cell microfluorimetry
39
MeHg treatments
41
2+
Determination of [Ca ]i
42
Statistics
44
D)
Results
45
iv

E)

a.
Acute methylmercury treatment
b.
Chronic methylmercury treatment
Discussion

45
48
54

CHAPTER 3: STRIATAL SLICES FROM AGED ANIMALS ARE MORE
SUSCEPTIBLE TO METHYLMERCURY-INDUCED CALCIUM INCREASE THAN
YOUNG ANIMALS
57
A)
Abstract
58
B)
Introduction
59
C)
Materials and methods
62
Chemicals and solutions
62
Slice preparation
62
Methylmercury treatment
62
Confocal imaging
63
Statistics
64
D)
Results
66
a.
Confocal imaging
66
b.
Comparative susceptibility of striatal and cerebellar slices
66
E)
Discussion
70
CHAPTER 4: CHRONIC METHYLMERCURY TREATMENT ALTERS STRIATAL
CALCIUM REGULATION AND SYNAPTOSOME MITOCHONDRIAL FUNCTION
73
A)
Abstract
74
B)
Introduction
75
C)
Materials and methods
78
Chemicals and solutions
78
Animal care and treatments
78
Tissue collection
79
Striatal synaptosome isolation
79
Mitochondrial function analysis
80
Striatal slice preparation
81
High speed calcium imaging
82
Statistics
83
D)
Results
89
a.
Animal health and behavior
89
b.
Trunk blood Hg concentration
89
c.
Mitochondrial function
91
2+
98
d.
High speed [Ca ]i imaging
E)
Discussion
106
CHAPTER 5: SUMMARY AND CONCLUSIONS
Summary and conclusions
v

111
112

REFERENCES

127

vi

LIST OF TABLES

2+

Table 1.1

Characteristics of HVA and LVA Ca channels. Summary of the properties
2+
of HVA and LVA Ca channels. Table from: Marrero et al., (2013). …………9

Table 3.1.

No difference in time-to-peak fluorescence between cerebellar granule cells
and striatal medium spiny neurons. The time-to-peak fluoresce was determined
for cerebellar granule cells and striatal medium spiny neurons in response to
20μM and 100μM MeHg. No differences were detected between the time-to-peak
fluorescence (min) in the cerebellum and striatum at any age. Data are expressed
at means ± SEM. (n ≥4).…………………………………………………………69

vii

LIST OF FIGURES

Figure 1.1

2+

Acute MeHg exposure causes a biphasic increase in [Ca ]i. Diagram
2+
representing the MeHg-induced biphasic increase in [Ca ]i. Acute MeHg
2+

exposure causes the release of Ca
2+

from the SER and mitochondria followed by

extracellular Ca entry. For interpretation of the references to color in this and all
other figures the reader is referred to the electronic version of this dissertation. 15
Figure 1.2.

Organization of the basal ganglia. The basal ganglia is a highly organized
circuit composed of multiple nuclei. GABAergic (inhibitory) projections are
represented in red and glutamatergic (excitatory) projections are represented in
green. This figure was modified from Obeso et al. (2008). ……………………..18

Figure 1.3

A graphic representation of the location of SNc neurons in the human brain.
SNc cell bodies are located in the midbrain and their axons project to the striatum
where they release DA.…………………………………………………………..19

Figure 1.4

A depiction of an XF Analyzer experiment measuring mitochondrial OCR.
Perturbation of mitochondrial oxidative phosphorylation allows for the
determination of mitochondrial basal respiration, ATP production, maximal
respiration, spare capacity, non-mitochondrial respiration, and proton leak.…....30

Figure 2.1

Acute MeHg treatment in differentiated SH-SY5Y cells caused a biphasic
2+
increase in [Ca ]i. (A-B) Representative 340nm, 380nm, and ratio of
(F340nm/F380nm) fura-2 tracings from a single differentiated SH-SY5Y cell
treated acutely with 5μM MeHg. (A) Representative 340nm tracing is shown in
2+
black. 340nm fluorescence corresponds to changes in Ca -bound fura-2. The
onset of phase 1 was the time at which the 340nm tracing increases from baseline.
Representative 380nm tracing is shown in grey. 380nm fluorescence corresponds
to changes in unbound fura-2. The onset of phase 2 was the time at which the
380nm tracing had a sustained decrease from baseline. (B) Representative ratio of
(F340nm/F380nm). An increase in fluorescence of the ratio corresponds to an
2+

increase in [Ca ]i. Acute 5μM MeHg treatment caused a biphasic increase in
2+
[Ca ]i.…………………………………………………………………………...38
Figure 2.2

Measurement of VGCC function. (A) Schematic diagram of how VGCC
function was measured. VGCCs were activated by depolarizing the cell
2+
membrane with 40mM KCl which activated VGCCs and allowed Ca influx
through L- and N-type channels. (B) Representative fura-2 tracing from a single
viii

differentiated SH-SY5Y cell treated with 0nM MeHg for 48h. Application of
2+
40mM KCl is indicated by the arrows. The amplitude of KCl-induced Ca influx
was measured for each depolarization and averaged to determine a mean
amplitude for each experiment (n=1).……………………………………………40
Figure 2.3

Determination of VGCC subtype sensitivity to chronic MeHg treatment. (A)
Schematic diagram of how VGCC function was measured. SH-SY5Y cells were
pretreated with either 1μM GVIA or 5μM nifedipine (as shown in diagram) and
2+
then depolarized with 40mM KCl. Subsequent Ca influx was due solely to flow
2+

through the unblocked channel resulting in decreased Ca influx. (B)
Representative fura-2 tracing from a single differentiated SH-SY5Y cell treated
with 0nM MeHg for 48h. Application of 40mM KCl is indicated by the arrows.
The first depolarization was considered the control and occurred with no
antagonist present. The following 3 depolarizations occurred in the presence of
antagonist. The amplitude of KCl-induced Ca2+ influx was quantified for each
depolarization. The mean amplitude of the depolarizations in the presence of
antagonist was divided by the amplitude of the control depolarization to
determine the percent of control.………………………………………………...43
Figure 2.4

Acute MeHg caused a concentration-dependent decrease in the time-to-onset
of Phase 1 and Phase 2 in differentiated SH-SY5Y cells. Differentiated SH2+
SY5Y cells were superfused with 1, 2, 5, or 10μM MeHg and changes in [Ca ]i
were continuously monitored. MeHg caused a concentration dependent decrease
in the time-to-onset of phase 1 and phase 2. Acute treatment with 5 and 10μM
MeHg resulted in a significant decrease in the onset of phase 1 and 2 compared to
1μM MeHg. Data are expressed as mean ± SEM. * = significant difference
compared to 1μM MeHg (One-Way ANOVA, p<0.05, n≥4).…………………...46

Figure 2.5

N- or L-type VGCC antagonists delay the onset of phase 2. Differentiated SHSY5Y cells were superfused with 2μM MeHg alone, 2μM MeHg + 1μM GVIA,
2+
or 2μM MeHg + 5μM Nif and changes in [Ca ]i were continuously monitored.
VGCC antagonists did not delay the onset of phase 1. Co-treatment with GVIA or
Nif significantly delayed the onset of phase 2. Data are expressed as mean ±
SEM. * = significant difference compared to 2μM MeHg alone (One-Way
ANOVA, p<0.05, n≥3). …………………………………………………………47

Figure 2.6

Chronic MeHg caused decreased KCl-induced Ca influx. Differentiated SHSY5Y cells were treated with 0 or 20nM MeHg for 48 or 72h. Following MeHg
2+
treatment cells were loaded with fura-2AM and KCl-induced Ca influx was
measured. (A) Forty-eight hour 20nM MeHg treatment caused a slight reduction
in the maximum amplitude of KCl-induced Ca2+ influx. (B) Seventy-two hour
20nM MeHg caused a significant decrease in the maximum amplitude of KCl2+
induced Ca influx (Unpaired t-test, p = 0.018, n ≥4).…………………………49

2+

ix

Figure 2.7

Chronic MeHg treatment caused a time- and subtype specific block of
2+
VGCCs. The maximum amplitude of KCl-induced Ca influx was measured in
cells treated with 0 or 20nM MeHg for 48 or 72h. Cells were depolarized 4 times
using 40mM KCl. The first depolarization was performed with KCl alone (control
depolarization) the following 3 were performed in the presence of a VGCC
2+
antagonist. The amplitude of KCl-induced Ca influx was quantified for each
depolarization. The mean amplitude of depolarizations in the presence of
antagonist was divided by the amplitude of control depolarization to determine
percent (%) of control. Data are represented as percent (%) of control
depolarization. (A) Forty-eight hour 20nM MeHg treatment, caused a significant
2+
decrease in Ca influx through L-type VGCCs and no change in KCl-induced
2+

Ca influx through N-type channels (Unpaired t-test, p = 0.0122, n=3). (B)
Seventy-two hour 20nM MeHg treatment caused a significant decrease in KCl2+
induced Ca influx through N - and L-type VGCCs (Unpaired t-test, p<0.05,
n≥3). * = significantly different than the 0nM MeHg group. Data are expressed as
mean ± SEM.…………………………………………………………………….51
Figure 2.8

2+

Chronic MeHg treatment caused a significant increase in resting [Ca ]i.
Differentiated SH-SY5Y cells were treated with 0 or 20nM MeHg for 72h and the
2+
resting [Ca ]i was calculated. Cells treated with 20nM MeHg had a significant
2+

increase in mean resting [Ca ]i compared to 0nM MeHg treated cells.* =
significant difference compared to 0nM MeHg group (Unpaired t-test, p<0.05, n =
3).………………………………………………………………………………...53
Figure 3.1

Striatal slices from aged animals are more susceptible to 20μM MeHg than
slices from 1mo animals. Striatal slices from 1mo, 9mo, and 21mo mice were
loaded with Fluo4 and superfused with 20μM MeHg for 45min. (A) Relative
Flou4 fluorescence (F/F0) of striatal slices from 1mo, 9mo, and 21mo mice to
20μM MeHg treatment. (B) The time-to-peak fluorescence was quantified for
each group. Striatal slices from 9 and 21mo mice had a significantly decreased
time-to-peak compared to slices from 1mo animals. Data are means ± SEM (n≥4)
* = significantly different than 1mo animals.(p<0.05, One-Way ANOVA,
Tukey’s post-test).………………………………………………………………..65

Figure 3.2

Age had no effect on the response of striatal slices to 100μM MeHg
treatment. Striatal slices from 1 and 9mo animals were loaded with Fluo4 and
superfused with 100μM MeHg for 21min. (A) Relative Fluo4 fluorescence (F/F0)
response of striatal slices from 1 and 9mo mice to 100μM MeHg treatment. (B)
There was no difference in the time-to-peak effect between 1 and 9mo striatal
slices to 100μM MeHg treatment. Data are means ± SEM (n≥4).……………….67

x

Figure 4.1

Six or twelve month MeHg or Isr/MeHg treatments did not alter mouse
weight. Male balb/c mice were treated with 0ppm Isr/0ppm MeHg (Con/Con),
0ppm Isr/6.25ppm MeHg (Con/MeHg), 2ppm Isr/0ppm MeHg (Isr/Con), 2ppm
Isr/6.25ppm MeHg (Isr/MeHg) for 6 or 12mo. Throughout the treatment period
mice were weighed three times a week and their mean weight per week was
determined. (A) Six or (B) twelve month treatments did not alter mouse weight.
Treatment and time had a significant effect on mouse weight following 6 and
12mo treatments (Two-Way ANOVA, Bonferroni post-test, p<0.05) (n≥12). Data
are expressed as mean ± SEM.…………………………………………………..84

Figure 4.2

Six or twelve month MeHg or Isr/MeHg treatments did not alter mouse
performance on the rotarod. Male balb/c mice were treated with 0ppm Isr/0ppm
MeHg (Con/Con), 0ppm Isr/6.25ppm MeHg (Con/MeHg), 2ppm Isr/0ppm MeHg
(Isr/Con), 2ppm Isr/6.25ppm MeHg (Isr/MeHg) for 6 or 12mo. During the first
6mo of treatment mouse ability to remain on an accelerating rotarod was tested
every 4wks and then every 2wks for the duration of the treatment. (A) Six or (B)
twelve month treatments did not alter mouse ability to remain on an accelerating
rotarod. Treatment and time had a significant effect on mouse ability to remain on
the accelerating rotorod following 6 and 12mo treatments (Two-Way ANOVA,
Bonferroni post-test, p<0.05) (n≥12). Data are expressed as mean ± SEM.…….85

Figure 4.3

Six or twelve month MeHg or Isr/MeHg treatments did cause a consistent
pattern of altered mouse limb cross. Male balb/c mice were treated with 0ppm
Isr/0ppm MeHg (Con/Con), 0ppm Isr/6.25ppm MeHg (Con/MeHg), 2ppm
Isr/0ppm MeHg (Isr/Con), 2ppm Isr/6.25ppm MeHg (Isr/MeHg) for 6 or 12mo.
Throughout the treatment period the extent of mouse hind limb cross was scored
three times a week and their mean score per week was determined. The extent of
hind limb cross was assigned a score based on severity. A score of 0, 1, or 2 were
assigned to no cross, partial cross, and complete cross, respectively. (A) Mean
limb cross test scores for six month treatment. Con/Con hind limb score is
significantly different than Con/MeHg hind limb score on week 25. Con/Con hind
limb score is significantly different than Isr/MeHg hind limb score during weeks 8
and 9. (B) Mean limb cross test scores for twelve month treatment. Con/Con hind
limb score is significantly different than Isr/MeHg hind limb score during weeks
39 and 48. Treatment and time had a significant effect on mouse hind limb score
(Two-Way ANOVA, Bonferonni post-test, p<0.05). * = Con/Con is significantly
different than Con/MeHg. # = Con/Con is significantly different than Isr/MeHg.
Data are expressed as mean ± SEM (n≥12).……………………………………..87

Figure 4.4

Six and twelve month MeHg treatment caused blood Hg accumulation. Male
balb/c mice were treated with 0ppm Isr/0ppm MeHg (Con/Con), 0ppm
Isr/6.25ppm MeHg (Con/MeHg), 2ppm Isr/0ppm MeHg (Isr/Con), 2ppm
Isr/6.25ppm MeHg (Isr/MeHg) for 6 or 12mo. Following anaesthesia with
isofluorane truck blood was collected and later analyzed for Hg content. MeHg
xi

and Isr/MeHg caused significant Hg accumulation following (A) 6mo and (B)
12mo treatment (One-Way ANOVA, Tukey’s post-test, p<0.05) (n=4).………90
Figure 4.5

MeHg or Isr/MeHg treatment did not alter mitochondrial bioenergetics.
Mitochondrial bioenergetics of striatal synaptosomes was determined following
treatment period. Oxygen consumption rate (OCR, pMoles/min) before and after
the addition of oligomycin (1µM), Carbonyl cyanide 4(trifluoromethoxy)phenylhydrazone (FCCP) (2µM), antimycin A (1µM), and
rotenone (1µM). No differences were detected in OCR between treatment groups
following (A) 6mo or (B) 12mo treatment period. Data are means ± SEM (n≥4).
……………………………………………………………………………………92

Figure 4.6

Mitochondrial function was unaltered following 6mo MeHg or Isr/MeHg cotreatment. Quantification of mitochondrial basal respiration, ATP production,
maximum respiration and spare capacity striatal synaptosomes. MeHg or
Isr/MeHg co-treatment caused no significant differences in (A) basal respiration
(B) ATP production (C) maximal respiration or (D) spare capacity. Data are
means ± SEM (n≥5).……………………………………………………………..93

Figure 4.7

Mitochondrial function was unaltered following 12mo MeHg or Isr/MeHg
co-treatment. Quantification of striatal synaptosome mitochondrial basal
respiration, maximum respiration, and spare capacity. MeHg or Isr/MeHg cotreatment caused no significant differences in (A) basal respiration (B) maximal
respiration or (C) spare capacity. Data are means ± SEM (n≥5).………………..95

Figure 4.8

Twelve month Isr/MeHg treatment caused a significant increase in ATP
production in both striatal and cortical synaptosome mitochondria.
Quantification of (A) striatal and (B) cortical synaptosome ATP production. 12mo
Isr/MeHg treatment caused a significant increase in ATP production compared to
both Isr/Con and Con/MeHg treatment groups. * = significant difference (OneWay ANOVA, Tukey’s post-test, p<0.05) (n≥5). Data are means ± SEM.……..97

Figure 4.9

Twelve month MeHg or Isr/MeHg did not alter striatal response to KCl2+
induced [Ca ]i increase. Striatal slices were loaded with fura-2AM and then
exposed to 40mM KCl for 2min followed by 8min of ACSF. Fura-2 fluorescence
was continuously monitored. Data are presented as relative fura-2 fluorescence
(Fluorescence at time 0/fluorescence at time x). An increase in fura-2
2+
fluorescence corresponds to an increase in [Ca ]i. Time-to-peak fluorescence
2+

was determine for each treatment group. (A) Striatal [Ca ]i response to 40mM
KCl.(B) Time-to-peak and (C) amplitude of response was quantified. No
Differences were detected between treatment groups. Data are means ± SEM
(n≥3).……………………………………………………………………………100

xii

Figure 4.10

Twelve month MeHg or Isr/MeHg did not alter striatal response to NMDA2+
induced [Ca ]i increase. Striatal slices were loaded with fura-2AM and then
exposed to 50μM NMDA and 10μM glycine for 2min followed by 8min of
ACSF. Fura-2 fluorescence was continuously monitored. Data are presented as
relative fura-2 fluorescence (Fluorescence at time 0/fluorescence at time x). An
2+
increase in fura-2 fluorescence corresponds to an increase in [Ca ]i. Time-topeak fluorescence was determine for each treatment group. (A) Striatal [Ca2+]i
response to 50μM NMDA.(B) Time-to-peak and (C) amplitude of response was
quantified. Many slices did not respond to NMDA application resulting in an n
value less than 3.……………………………… ………………………………102

Figure 4.11

Twelve month MeHg treatment caused a significanltly decreased time-topeak fluorescence following application of AMPA. Striatal slices were loaded
with fura-2AM and then exposed to 25μM AMPA for 2min followed by 8min of
ACSF. Fura-2 fluorescence was continuously monitored. Data are presented as
relative fura-2 fluorescence (Fluorescence at time 0/fluorescence at time x). An
2+
increase in fura-2 fluorescence corresponds to an increase in [Ca ]i. Time-topeak fluorescence was determine for each treatment group. (A) Striatal [Ca2+]i
response to AMPA (B) 12mo MeHg treatment caused a significantly faster timeto-peak fluorescence in response to AMPA (p<0.05, unpaired student t-test, n≥3).
(C) Amplitude of response was not affected by treatment. Data are means ± SEM
(n≥3).……………………………………………………………………………104

Figure 5.1

A schematic representation of the effects of prolonged exposure to MeHg in
differentiated SH-SY5Y cells. Exposure of differentiated SH-SY5Y cells with
20nM MeHg resulted in a time- and VGCC subtype-dependent block of
2+
2+
depolarization-dependent [Ca ]i increase. MeHg blocked KCl-induced Ca
increase through L-type VGCCs after a 48h exposure and through both L- and N2+
type VGCCs after 72h exposure. The resting [Ca ]i was also significantly
increased following 72h MeHg exposure.……………………………………...113

Figure 5.2

Schematic diagram of the effects of MeHg on the striatal circuit. MeHg acts at
2+
striatal MSNs to causes increases in [Ca ]i as well as alter mitochondrial ATP
production. It has also been shown to cause increase in both DA and glutamate
release. These actions along with potential direct interactions with AMPA
2+
receptors results in altered post-synaptic Ca response to AMPA receptor
activation.……………………………………………………………………….114

xiii

KEY TO ABBREVIATIONS

ACSF - Artificial cerebrospinal fluid
AM - Acetoxymethyl ester
AMPA - 2-Amino-3-hydroxy-5-methyl-5-isoxazole propionic acid
ANOVA - Analysis of variance
ATP - Adenosine 5'-triphosphate
BBB - Blood brain barrier
2+

Ca

- Calcium

2+

Ca e - Extracellular calcium
2+

Ca i - Intracellular calcium
2+

[Ca ]i - Intracellular calcium concentration
2+

Ca m – Mitochondrial calcium
2+

Ca m – Mitochondrial calcium concentration
Con - Control
CNS - Central nervous system
xiv

DA – Dopamine
DAT – Dopamine transporter
DHP - Dihydropyridine
DMSO - Dimethyl sulfoxide
ECAR – Extracellular acidification rate
EGTA - Ethylene glycol tetraacetic acid
F - Fluorescence
FBS - Fetal bovine serum
FCCP - Cyanide 4-(trifluoromethoxy) phenylhydrazone
Fluo-4 NW - Fluo 4 no wash
Fura-2 AM - Fura-2 acetoxymethylester
GABA – Gamma-aminobutyric acid
GPe – External globus pallidus
GPi – Internal globus pallidus
GVIA - ω - Conotoxin GVIA
h - Hour(s)
HBS - HEPES buffered saline
xv

HEPES - 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid
HVA – High voltage activated
IP3 - Inositol-1,4,5-triphosphate
Isr - Isradipine
LVA – Low voltage activated
M - Molar
MEM - Minimum essential medium
MeHg - Methylmercury
min - Minute(s)
mo - Month
MSN – Medium spiny neuron
μ – Micro
MIB – Mitochondrial isolation buffer
mo - Month
mPTP - Mitochondrial transition pore
Nif - Nifedipine
nm - Nanometer

xvi

nM - Nanomolar
NMDA - N-methyl-D-aspartate
NSDA - Nigrostriatal dopamine
NT - Neurotransmitter
OCR - Oxygen consumption rate
PCB – Polychlorinated biphenyl
PD – Parkinson’s disease
PND - Postnatal day
ppm - Parts per million
RA - Retinoic acid
ROS - Reactive oxygen species
RT-PCR – Real time polymerase chain reaction
RyR - Ryanodine receptor
SEM - Standard error of the mean
SER - Smooth endoplasmic reticulum
SERCA - Smooth endoplasmic reticulum Ca2+ ATPase
SNc – Substantia nigra pars compacta
xvii

TPEN - N,N,N’,N’,-tertrakis-(2-pyridylmethyl)ethylediamine
VGCC - Voltage-gated calcium channel
yr - Year
Zn

2+

- Zinc

xviii

CHAPTER ONE: INTRODUCTION

1

A) Background
a. Methylmercury
i. Methylmercury in the environment
0

+

Mercury (Hg) is a persistent heavy metal that can be found in three forms: Hg , Hg and
2+

Hg . Hg and its compounds are neurotoxic environmental contaminants of contemporary
concern. Hg enters the environment from both natural and anthropogenic sources. Natural
sources of Hg include degassing of the earth’s crust, volcanic emissions, and soil erosion.
Anthropogenic sources include coal burning, waste combustion, and gold mining (Clarkson and
Magos, 2006; Wade, 2013). Mercury forms an amalgam with gold, creating an easy and
affordable method for its extraction from lower-grade ore. Its heavy use with artisan gold mining
has made it the worlds leading source of mercury pollution, releasing more mercury into the
environment than all of the world’s coal plants combined (Wade, 2013). Once released into the
+

atmosphere Hg is oxidized to Hg and then returns to the earth’s surface in rainwater. The
+

majority of Hg is reduced back to the vapor state and then returns to the atmosphere. However a
portion is methylated by methanogenic bacteria to form MeHg. MeHg then bioaccumulates up
the food chain, with the highest sources being piscivorous fish or marine mammals such as tuna
and pilot whale, respectively. Consequently, humans are mainly exposed to MeHg through the
consumption of fish and marine mammals. In an attempt to reduce Hg pollution many nations
around the world came together to form the Minamata Convention. Their goal is to reduce
anthropogenic Hg pollution up to 15% by the year 2020 (Lubick and Malakoff, 2013). Although
2

anthropogenic sources of Hg pollution can be reduced, Hg persists in the environment; it cannot
be destroyed and it does not degrade.
ii. Mercury and human health
Hg and Hg compounds have been used in a number of different applications throughout
history. The first known use dates back thousands of years. The Chinese used Hg to prepare red
ink (Clarkson and Magos, 2006). Hg has been used as an important constituent of drugs for
centuries. It has been used as an ingredient in many diuretics, antibacterials, ointments, and
laxatives. Hg was also used as an antifungal agent on seed grain. The fluidity of Hg has led to its
being used in thermometers, barometers, and on dynamic electrodes. As previously mentioned,
Hg is commonly used in artisan gold mining, which is currently the largest source of
anthropogenic pollution (Wade, 2013). Although it has been extensively used throughout history
for a variety of applications it current usage is limited due to the known adverse human health
effects.
Hg and Hg compounds are handled differently by the body, and clinical signs and
0

symptoms associated with their poisoning differ. Exposure to Hg is most commonly associated
0

with occupational exposures. Signs and symptoms associated with Hg poisoning include
tremors, polyneuropathy, loss of memory, insomnia, and neurocognitive disorders. Exposure to
2+

Hg

2+

was associated with its use in medical products. Hg

does not cross the blood brain

barrier (BBB). However it is extremely toxic to the kidneys causing severe renal dysfunction
(Ashner et al., 2013).

3

The primary target of MeHg is the central nervous system. When ingested MeHg is
efficiently (90%) absorbed in the gastrointestical tract. From there it enters the bloodstream
where it primarily binds to hemoglobin in the red blood cells. MeHg then distributes throughout
the body (Brunton et al., 2008). MeHg is highly reactive via oxidatition with the amino acid
cysteine. The MeHg-cysteine complex mimics methionine and is a substrate for the neutral
amino acid transporter allowing it to enter into the cell (Yin et al., 2008). MeHg is also
lipophilic, allowing it some ability to cross lipid membranes. MeHg is mainly eliminated through
the liver via excretion to the bile and gut. However, it undergoes extensive enterohepatic
circulation allowing reabsorption into the bloodstream (Brunton et al., 2008).
The neurotoxic impact of exposure to MeHg had not been understood until a seminal
paper by Hunter and Russell (1954). In this paper they described a case study of a 23 yr old man
exposed to MeHg dust for 4mo while working. Six mo after the exposure he presented to the
hospital with paresthesia of his hands and forearms, ataxia, and constriction of the visual fields.
Fifteen years after the exposure he died and an autopsy was performed. The most striking finding
of the autopsy was gross atrophy of the cerebellum. Upon microscopic examination a loss of the
cerebellar granule cells was evident while the neighboring purkinje cells were generally spared.
Hunter and Russell’s case report highlighted the latency between exposure and presentation of
symptoms and the cell type-specific damage in the cerebellum associated with MeHg poisoning.
There have been two very well known episodes of mass MeHg poisonings; the first
occurred in Minamata, Japan and the second occurred in Iraq. Although very unfortunate, these
exposures allowed scientists to understand the patholophysiology of MeHg poisonings in
humans. In the 1950’s the Chisso factory in Mimamata, Japan was producing acetylaldehyle. Hg
4

was used as a catalyst in the production and discharged into Minamata Bay, a fertile fish
spawning ground. Consequently people who lived near and around Minamata Bay were poisoned
by the daily consumption of heavily contaminated fish from the bay. In Iraq, in 1971, wheat seed
coated with a MeHg-containing fungicide that was meant to be used for planting was instead
used to make bread. People who consumed the bread made with the MeHg-contaminated seed
had MeHg poisoning (Bakir, 1973). Interestingly, victims of both chronic (Minamata) and acute
(Iraq) MeHg poisonings presented with the same signs and symptoms. Following a characteristic
latent period which can range from weeks to months, depending on the extent of exposure, signs
and symptoms begin to appear. Characteristic signs and symptoms include paresthesia (loss of
sensation in the extremities), ataxia (loss of coordination in gait), dysarthia (slurred speech), loss
of hearing, and constriction of vision (Harada, 1968; Bakir, 1973). Several of the neurological
signs presented by patients have plausible links to alterations in voltage-gated calcium channel
(VGCC) function. The severity of symptoms depends of the exposure dose, the duration, and the
stage of development that exposure takes place (for review see Castoldi, 2008). An example of
this is the severe damage that occurs to the developing fetus when exposed to MeHg. MeHg
crosses the BBB and placenta. The developing fetus can be adversely affected even when the
mother has no symptoms of MeHg poisoning. Children exposed in utero during the mass
poisoning in Minamata presented with severe cognitive and motor deficits (Harada et al., 1968).
Two unique populations which rely heavily on the daily consumption of fish or sea
mammals as their main food source are inhabitants of the Faroe and Seychelles Islands. The
Faroe Islands are located in the Arctic Ocean between Norway and Iceland. Inhabitants in the
Faroe Islands consume both fish and marine mammals which may also accumulate other
5

environmental contaminants such as polychlorinated biphenyls (PCBs) (Weihe et al., 1996). The
Seychelles Islands are located in the Indian Ocean off the eastern coast of Africa. Inhabitants of
the Seychelles Islands consume only fish and not marine mammals. Scientists have studied these
populations and focused on the impact of prenatal MeHg exposure and its association with
adverse neurodevelopmental outcomes (Davidson et al., 2006, 2010, 2011; Debes et al., 2006;
Stokes-Riner et al., 2011; Yorifuji et al., 2011; Grandjean et al., 2012; Strain et al., 2012;
Karagas et al., 2012). Studies following inhabitants of the Faroe Islands found significant
neurological deficits in children exposed prenatally to MeHg (Grandjean et al., 2012). In
contrast, studies of inhabitants of the Seychelles, found no adverse outcomes due to long-term
fish consumption (Davidson et al., 2011). The disparity between neurobehavioral outcomes may
be due to the genetic differences between the two populations as well as the presence of other
environmental contaminants such as PCBs present in marine mammals which exacerbate
neuronal damage in the Faroe Island population.
The contemporary concern is for MeHg exposure which occurs over a lifetime. Long
term exposure (lifetime or exposure during critical windows of development) could enhance
processes associated with normal aging, thereby facilitating the onset of senescence-related
neurodegeneration. This may ultimately lead to neuropathological changes associated with
degenerative diseases. The role of environmental toxicants in neurodegenerative disease has long
been speculated (Johnson and Atchison, 2009; Vance et al., 2010; Williams et al., 2010; Cannon
and Greenamyre, 2011; Madison et al., 2012). The causative agent in the development of the
sporadic form of many neurodegenerative diseases is unknown and reseachers have speculated
that environmental influences could play a role by altering neuronal function.
6

One such neurodegenerative disorder in which the environment is thought to play a role
is Parkinson’s disease (PD) (Landrigan et al., 2005). PD is strongly associated with aging and
afflicts over one million people in the United States; that number is on the rise with an aging
population (de Lau et al., 2004, Dorsey et al., 2007). During the development of PD the most
severely afflicted neurons are the neurons of the nisgrostriatal dopamine (NSDA) system (Braak
and Braak, 2000). NSDA neuron cell bodies are located in the substantia nigra pars compacta
(SNc) and project their axons to the striatum where they release dopamine (DA), which is
involved in the regulation of the movement and coordination (Bergman, 1998). The severity of
symptoms in PD is associated with the extent of SNc neuronal loss (Dauer and Prezedborski,
2003; Fahn, 2003). PD has two forms - familial and sporadic. Increased risk for the development
of the familial form of PD has been linked to mutations in a number of genes including, DJ-1,
PINK1 and Parkin (Schapira, 2008). The sporadic form of PD is the most common and the
cause is unknown. As such, initiating or predisposing factors such as environmental exposure or
lifestyle are thought to play a major role in sporadic PD. Post mortem autopsy reports from
humans with PD present evidence of mitochondrial dysfunction and an increased production of
reactive oxygen species (ROS) in the SNc of PD patients (Dexter et al., 1989). It has been
postulated that exposure to an environmental toxicant which targets the mitochondria could be a
factor in the development of the disease however the results are ambiguous. Development of the
sporadic form has been associated with a high body burden of Hg (Ngim and Devathasan, 1989),
however this was not the case in other studies (Ohlson and Hogstedt, 1981; Semchuk et al.,
1993; Gorell et al., 1999). Epidemiological studies of the Faroe Island Inuits, a population with
chronic environmental exposure to MeHg, report an increased (approximately 2-fold) prevalence

7

of PD compared to the general population (Wermuth et al., 1997; Wermuth et al., 2000;
Wermuth et al., 2007).Studies of the association of PD and exposure to MeHg in these people
have found that prenatal exposure to MeHg has no affect on the development of PD (Petersen et
al., 2008b). However, long-term consumption of whale meat and blubber in adulthood increases
is associated with the development of PD (Petersen et al., 2008a). The increased risk for the
development of PD may be influenced by the presence of PCBs and other environmental
contaminants in whale meat and blubber (Weihe et al., 1996; Petersen et al., 2008a). Although
these studies have found an association with MeHg exposure and PD, it is not thought that MeHg
exposure causes PD.
b. Neuronal calcium regulation
i. Calcium regulaion
2+

Ca

is a key regulator of a variety of neuronal processes such as neurotransmitter
2+

release, growth cone elongation, and gene transcription. Extracellular Ca
2+

2+

(Ca e)

2+

concentration is 10,000 higher than [Ca ]i. Thus Ca e entry is a tightly regulated process. A
2+

neuron can utilize many different Ca
2+

receptors, store-operated Ca

entry pathways, some of which are: VGCCs, glutamate

channels, and transient receptor potential channels. VGCCs are

intimately involved in the regulation of MeHg-induced toxicity and will be discussed in detail
below.
2+

2+

VGCCs are the main entry portals for Ca e. They mediate Ca

influx as a result of

depolarization of the plasma membrane, an energy-independent process. There are two classes of

8

Type

Channel
Subtype

Antagonist

Phenylalkylamines
, dihydropyridines,
benzothiazapines,
and ω-agatoxin
IIIA

L

HVA

ω-agatoxin IVA
(P-type),
ω-conotoxin
MVIIC (Q-type)

P/Q

α1Coding
Gene
α1S
(CaV1.1)
α1C
(CaV1.2)

α1D
(CaV1.3)

Cell/TissueSpecific
Expression
Skeletal muscle

Sensory transduction

Striatal medium
spiny neurons

Neurotransmitter
release

Substantia nigra
pars compacta
Retina

Purkinje neurons
and cerebellar
granule cells
Thalamus

Only expressed in
neurons
α1B
ω-conotoxin GVIA
Nociceptive
(CaV2.2)
dorsal root
ganglion neurons
Presynaptic
α1E
terminals
SNX 482
(CaV2.3)
Hippocampus
Kurtoxin,
α1G-I
Dendrites and cell
mibefradil,
(CaV3.1bodies of some
amiloride
3.3)
CNS neurons

N

R
LVA

T

2+

Table 1.1 Characteristics of HVA and LVA Ca
2+

HVA and LVA Ca

Plasticity/cardiac
muscle contraction

Pacemaker activity
Sensory transduction
Neurotransmitter
release in
neuromuscular
junctions and CNS
Neurotransmitter
release
Involved in
depolarization of
thalamic neurons
Neurotransmitter
release
Depolarization
Neurotransmitter
release
Depolarization
Rhythmic burst firing
and pacemaker
activity

channels. Summary of the properties of

channels. Table from: Marrero et al., (2013).

9

Excitationcontraction coupling

CNS (dendrites
and cell
bodies)/cardiac
muscle
Cochlea

Presynaptic
terminals
α1A
(CaV2.1)

Function

VGCCs which are grouped according to the degree of depolarization from resting membrane
potential that is needed for their activation: high-voltage activated (HVA) and low-voltage
activated (LVA). Their properties are summarized in Table 1.1. HVA channels include L-, N-,
P/Q-, and R-type VGCCs. They are separated based on their pharmacological and
electrophysiological properties. HVA channels are multimeric proteins composed of one α1, β,
α2δ, and sometimes γ subunits. The pore-forming α1 subunit confers the ion selectivity and
pharmacological properties of the channels. The accessory subunits β, α2δ, and γ modulate the
voltage-dependence of activation and inactivation kinetics of the channel. Of particular
importance to the work in this dissertation are L- (Cav1.1-4) and N-(Cav2.2) VGCCs. L-type
VGCCs were named “L” because of their long lasting current (for review see: Catterall et al.,
2005). L-type channels are antagonized by dihydropyridines (DHPs), such as nifedipine (Nif)
and isradipine (Isr) (for review see: Kochegarov, 2003). Both of these antagonists were utilized
in experiments descriped in this dissertation. N-type VGCCs were named “N” because they were
first identified in neurons (for review see: Catterall et al., 2005). N-type channels are irreversibly
antagonized by ω-conotoxin GVIA (GVIA), which is a snail toxin isolated from the Conus
geographus snail (Kerr and Yoshikami, 1984; Olivera et al., 1984). The function of HVA
VGCCs in response to MeHg is a primary focus of this dissertation.
2+

The main sites of Ca

2+

buffering are the Ca

buffering proteins, mitochondria, and
2+

smooth endoplasmic reticulum (SER). Maintenance of Ca
2+

and loss of Ca

homeostasis is critical for neuronal

buffering ability of either the mitochondria or SER could result in neuronal
2+

death (Bernardi et al., 1999). The role of the mitochondria in the maintenance of Ca
10

homeostasis and adenosine 5'-triphosphate (ATP) production is central to this dissertation and is
discussed in detail below.
2+

Mitochondria have a very important role in the dynamic process of Ca

homeostasis.

2+

The mitochondria have a low affinity but high capacity for Ca , thus they require a strong
2+

stimulus to induce Ca

2+

not serve as large Ca

uptake (Somlyo et al., 1985). Under basal conditions mitochondria do
2+

stores however, mitochondrial Ca

2+

([Ca ]m) levels do fluctuate in

response to external stimuli such as neurotransmitter release (Denton and McCormac, 1980;
2+

Hansford and Castro, 1981). Ca

entry into the mitochondria is an energetically favorable

process because the inner mitochondrial membrane has a negative membrane potential of
approximately 140-180mV (Brand and Murphy, 1986; Bygrave, 1977; Hansford 1985; Reid et
2+

al., 1966; Senior, 1988). Ca
2+

allows diffusion of Ca

2+

enters the mitochondria via the Ca

uniporter. The uniporter

down its electrochemical gradient from the cytosol, across the inner

mitochondrial membrane, to be stored in the mitochondrial matrix (Gunter and Gunter, 1994).
2+

Small increases in [Ca ]m stimulate ATP production (Tarasov et al., 2012). However, when
2+

large amounts of Ca

enter the mitochondria it can result in mitochondrial membrane

depolarization and an uncoupling of ATP synthesis (Sparagna et al., 1995; Wingrove and Gunter,
2+

1986) demonstrating that there is a balance between Ca

entry and maintenance of the
2+

mitochondrial membrane potential. The main neuronal Ca

11

+

2+

efflux pathway is the Na /Ca

+

+

exchanger (Crompton et al., 1976). The mitochondria contain a Na /H exchanger, which allows
+

the efflux of Na (Gunter and Gunter, 1994). Under conditions of extreme oxidative stress or
2+

[Ca ]m overload it can cause the opening of the mitochondrial permeability transition pore
(mPTP). The mPTP is a non-selective pore that forms across the inner mitochondrial membrane
which allows the passage of any molecule less than 1500 Da (Szabo and Zoratti, 1992; Chernyak
and Bernardi, 1996; Pastorino et al., 1999; Szalai et al., 1999; He et al., 2000; Petronilli et al.,
2001) and is linked to the induction of cell death (Fang et al., 2008; Gandhi et al., 2009). Acute
MeHg exposure causes the opening of the mPTP (Polunas et al., 2011; Limke et al., 2001) and
inhibition of its opening protects neurons against MeHg-induced cytotoxicity (Limke et al.,
2001; Qu et al., 2013).
The mitochondria are known as the powerhouse of the cell because of their essential role
in oxidative phosphorylation and production of ATP (for review see: Tarasov et al., 2012). They
contain all of the necessary machinery for oxidative phosphorylation (ATP synthase and
complexes I - IV) in their inner membrane. The mitochondrial intermembrane proton gradient
drives ATP synthesis (Nelson and Cox, 2005). The rate of mitochondrial oxidative
phosphoylation is a tightly regulated process adapting to the neuronal demand in order to
maintain neuronal function. Exposure to MeHg places an increased ATP demand on the neuron.
2+

MeHg causes an increase in [Ca ]i which is buffered by the mitochondria in an energy2+

independent manner or by the SER via the smooth endoplasmic reticulum Ca

ATPase
2+

(SERCA). It could also be removed from the cytosol by the plasma membrane Ca

12

ATPase.

However, in aging, mitochondria are deplolarized (Nicholls, 2002; Toescu, 2005) which causes
2+

them to buffer Ca

and produce ATP less efficiently. This could make them more vulnerable to

MeHg.
ii. Calcium and aging
In trying to understand the cellular mechanisms that regulate the aging process
2+

Khachaturian (1987) hypothesized that neuronal aging was due to Ca
2+

cytosol. This came to be known as the ‘Ca

changes within the

aging hypothesis’. The hypothesis has evolved with

increased understanding of changes in the cellular processes during aging but the main premise
has remained the same. It is now thought that the breakdown of cellular processes involved in
2+

Ca

homeostasis contributes to cellular dysfunction and ‘aging’ (Toescu and Vreugdenhil,

2010). Changes that occur in the mitochondria may have the most impact on neuronal aging.
2+

2+

Mitochondria are instrumental in regulating [Ca ]i and are also large sources of Ca . In aged
2+

neurons, the mitochondria are depolarized (Toescu, 2005). This results in a decreased Ca

gradient across the mitochondrial membranes resulting in a decreased ability to buffer changes in
2+

cytosolic Ca

and decreased capacity for ATP production. It has also been reported that aged

neurons have a smaller number of larger mitochondrial that are less efficient in the production of
ATP (Toescu, 2005). In aged neurons the ER has decreased expression of SERCA and releases
2+

larger amounts of Ca

through inositol-1,4,5-triphosphate receptors (IP3) and ryanodine
2+

receptors (RyR) which contribute to the cytosolic Ca

13

load (Toescu, 2007; Toescu and

2+

Vreugdenhil, 2010). Aged neurons also have decreased Ca -ATPase expression at the plasma
membrane (Toescu, 2007). All of these factors contribute to an inability of aged neurons to
2+

efficiently buffer changes in [Ca ]i as well as a decreased ability to respond to an increased
demand for ATP. These changes influence an aged neuron’s ability to respond to changes in
2+

[Ca ]i, leaving them vulnerable to stressors and more susceptible to environmental toxicants
2+

that alter [Ca ]i such as MeHg.
iii. Methylmercury and calcium regulation
MeHg is a very promiscuous molecule which inhibits many different cellular processes
simultaneously causing cellular dysfunction and damage. However, what is so intriguing about
MeHg is that despite its promiscuity, it causes cell type-specific damage. The cell type specific
damage is not due to the fact that MeHg only accumulates in those cells which have been found
to die, such as the cerebellar granule cells. On the contrary, MeHg has been found to accumulate
to a greater extent in the cerebellar purkinje cells than the cerebellar granule cells in the rat
(Møller-Madson, 1991). Therefore, cells sensitive to MeHg must have unique targets or
phenotypes which make them susceptible to MeHg-induced damage. Much of the work done in
our and other labs has been aimed at understanding what makes certain cell types uniquely
sensitive to MeHg.
Membrane proteins are the first to come in contact with exogenous compounds, so it is
not surprising that MeHg alters the function of membrane ion channels. As an initial screen of
potential ion channel targets of MeHg, the effects of acute application of MeHg on various ion
channels on cerebellar granule cells was tested. It was found that 1μM MeHg significantly
14

2+

Figure 1.1. Acute MeHg exposure causes a biphasic increase in [Ca ]i. (A) Diagram
representing the MeHg-induced biphasic increase in [Ca2+]i. Acute MeHg exposure causes the
2+

2+

release of Ca from the SER and mitochondria followed by extracellular Ca entry. For
interpretation of the references to color in this and all other figures the reader is referred to the
electronic version of this dissertation.

15

reduced current through GABAA receptors and VGCCs but not inwardly rectifying or voltagedependent potassium channels (Yuan et al., 2005). This study demonstrated that MeHg
specifically targets certain ion channels. Acute application of MeHg in a number of different
neuronal preparations has been shown to alter the function of VGCCs. MeHg inhibits the influx
of

45

2+

Ca

into synaptosomes (Atchison et al., 1986), and blocks dihydropyridine- (DHP)
2+

sensitive (L-type) and DHP-insensitive (N-type) Ca

channels in PC12 cells (Shafer et al.,

1990). These effects on VGCCs are irreversible and occur irrespective of channel configuration
(Sirois and Atchison, 1996). These studies have demonstrated that VGCCs are unique targets of
MeHg.
2+

MeHg also disrupts the dynamic Ca

homeostasis process (Komulainen and Bondy,

1987; Denny and Atchison, 1994; Marty and Atchison, 1997). Acute application of MeHg to
cerebellar granule cells results in a concentration-dependent biphasic increase in [Ca2+]i (Marty
and Atchison, 1997). The first phase is due to release of Ca2+i stores, of which a large component
is due to release from the mitochondria (Limke, 2001). The second phase is due to Ca2+e entry
(Marty and Atchison, 1997). This is shown in Figure 1.1. Co-treatment with Nif or GVIA results
in a significant delay in the time-to-onset of both phase 1 and phase 2, demonstrating that
VGCCs are involved in MeHg-induced Ca2+ dysregulation (Marty and Atchison, 1997). VGCC
antagonists have also been shown to delay the onset of behavior impairments and mortality in
mice (Bailey et al, 2013; Sakamoto et al., 1996). These studies support the role of VGCCs in
MeHg-induced neurotoxicity.

16

2+

MeHg specifically targets mitochondrial regulation by causing a decrease in Ca

uptake

and inducing its release (Levesque and Atchison, 1991). Activation of the mPTP (Limke and
2+

Atchison, 2002) leads to further Ca -mediated damage, and is associated with MeHg-induced
delayed cell death in cerebellar granule cells. Prevention of mPTP activation by cyclosporin
reduces the extent of cerebellar granule cell cytotoxicity. MeHg also causes generation of
reactive oxygen species (ROS) (LeBel et al., 1990; Sarafian and Verity, 1991; Yee and Choi,
1996) and induces a delayed-onset cell death (Sarafian et al., 1989; Marty and Atchison, 1998).
2+

Therefore any deficiency in Ca

buffering or extrusion pathways in a neuron may increase its

sensitivity to MeHg-induced damage.
c. Striatal and nigrostriatal dopamine system
i. Neuronal organization
The neuronal circuit of the basal ganglia includes a highly organized group of subcortical nuclei
involved in motor, associative, cognitive, and mnemonic functions (Bolam et al., 2000). The
nuclei include the striatum, globus pallidus, subthalamic nucleus (STN), and SNc. The
predominant input into the basal ganglia is from the cortex, and all of the output from the basal
ganglia is directed through the thalamus back to the cortex. Diseases associated with the basal
ganglia include both Huntington’s and PD as well as obsessive-compulsive disorder (Kreitzer
and Malenka, 2008). Thus understanding the structure and function of the basal ganglia has
become important in understanding the pathology of these diseases. The research described in
this thesis is focused on the effects of MeHg on neurons of the striatum and SNc.

17

Cortex

(Glutamatergic)

Thalamus

Striatum

(Glutamatergic)

(GABAergic)

GPe

(GABAergic)

GPi

(GABAergic)

STN

(Glutamatergic)

SNpc

Inhibition

(DAergic)

Excitation

Figure 1.2. Organization of the basal ganglia. The basal ganglia is a highly organized circuit
composed of multiple nuclei. GABAergic (inhibitory) projections are represented in red and
glutamatergic (excitatory) projections are represented in green. This figure was modified from
Obeso et al., (2008).

18

SNc

Figure 1.3. A graphic representation of the location of SNc neurons in the human brain.
SNc cell bodies are located in the midbrain and their axons project to the striatum where they
release DA.

19

The striatum plays a very important role in motor control, habit formation and motivated
behavior. Its function is also altered in a number of neuropsychiatric disorders such as PD
(Chuhma, et al., 2011). The striatum has a small population of interneurons: slow-firing large
cholinergic neurons, fast-spiking parvalbumin expressing GABAergic neurons, and burst-firing
somatostatin/nitric oxide expressing neurons (Bolam et al., 2000). The principal neurons of the
striatum are the GABAergic medium spiny neurons (MSN) which make up 90-95% of all
neurons in the striatum. MSNs receive glutamatergic input from the cortex and thalamus and
dopaminergic input from SNc (Bolam et al., 2000). There are two classes of MSNs, which differ
based on the type of DA receptor they express (Surmeier et al., 2007). The striatonigral MSNs
express the DA D1 receptor and make up the direct-pathway. These neurons project to the
internal globus pallidus (GPi) and substantia nigra pars reticula (Kreitzer and Malenka, 2008).
The striatopallidal MSNs express the DA D2 receptor and make up the indirect-pathway. These
neurons project to the external globus pallidus (GPe) neurons and which in turn project to the
STN (Kreitzer and Malenka, 2008). A representation of this circuit is shown in Figure 1.2. DA
plays an essential role in the modulation of the direct- and indirect-pathway. Activation of the D1
receptors activates MSNs while activation of D2 receptors inhibits MSN activity (Kreitzer and
Malenka, 2008). Environmental toxicants such as MeHg, which alter neurotransmission or
VGCC function, would alter MSN function, fundamentally altering the function of the basal
ganglia curcuit.
NSDA neuronal cell bodies are located in the SNc and project their axons to the striatum
where they release DA, which is involved in the regulation of the movement and coordination
(Bergman, 1998). This is shown in Figure 1.3. SNc neurons have a unique physiological

20

property, in that they autonomously generate action potentials in the absence of synaptic input
(Grace and Bunney, 1983). These spontaneously occurring action potentials rely on the influx of
2+

Ca

2+

through Cav1.3 channels, an L-type voltage-gated Ca

channel (Bonci et al, 1998; Ping

and Shepard, 1996; Puopolo et al., 2007). As a result, during neuronal activity these neurons
2+

have much greater cytosolic Ca

2+

fluctuations. This places a much higher demand on Ca
2+

buffering organelles to maintain [Ca ]i at non-toxic levels (Wilson and Callaway, 2000; Chan et
2+

2+

al., 2007). The elevated [Ca ]i and increased demand on Ca

buffering organelles leaves SNc
2+

neurons at an increased risk for damage by environmental toxicants that disrupt Ca

regulation.

ii. Methylmercury and the striatum and the NSDA system
Several studies have demonstrated that neurons of the basal ganglia are susceptible to the
neurotoxic effects of MeHg. Lesions occur in the striatum following acute application of MeHg
in rats (O’Kusky et al., 1988; O’Kusky and McGeer 1989; Wakabayashi et al., 1995; Sakamoto
et al., 1998). Alterations in neuronal function of dopaminergic neurons has also been shown in
cells in culture (Gotz et al., 2002; Tiernan et al., 2013), striatal synaptosomes (Komulainen and
Tuomisto, 1982, 1985), striatal slices (Kalisch and Racz, 1996), and in c. elegans (Vanduyn et al.
2010; Vanduyn and Nass, 2013). A number of the earliest studies demonstrated that exposure to
MeHg resulted in a concentration-dependent increase in DA release (Tsuzuki et al., 1982;
Komulainen and Tuomisto, 1982; McKay et al., 1986; O’Kusky et al., 1988; Kalisch and Racz,
1996). Researchers went on to find that MeHg-induced increase in DA occured independently of
2+

Ca e (Minnema et al., 1989; Kalisch and Racz, 1996; Faro et al., 2002). This suggests that
21

2+

MeHg may act intracellularly to induce release of Ca i stores, which causes neurotransmitter
2+

release or it blocks DA uptake via the DA transporter (DAT). MeHg induces release of Ca i
(Levesque and Atchison, 1991; Marty and Atchison, 1997) which may contribute to the
increased DA release however, researchers also found that MeHg inhibits DA uptake (Bartolome
et al., 1982; Rajanna et al., 1990; Faro et al., 2002). The extent of MeHg-induced DA release can
be reduced though the addition of glutathione and cysteine which have free –SH groups which
will bind MeHg and reduce its availability (Faro et al., 2005). NMDA receptor antagonists also
reduce the extent of MeHg-induced DA release (Faro et al., 2002). Exposure of primary DA
neuronal cultures to MeHg resulted in decreased neurite number and cell shrinkage (Gotz et al.,
2002). Work in rat striatal synaptosomes suggest that MeHg induces mitochondrial dysfunction
2+

due to increased [Ca ]i (Dreiem and Seegal, 2007). This work supports the hypothesis that SNc
neurons are susceptible to MeHg-induced toxicity. Increased release and reduced DA uptake in
the striatum would result in prolonged exposure and activation of D1/D2 receptors on MSNs,
2+

this in combination with MeHg-induced [Ca ]i may result in increased susceptibility to MeHginduced damage in MSNs.
B) Objectives and Rationale
a. Hypothesis and aims
We hypothesize that exposure to low-doses of MeHg throughout one’s lifetime will
reduce mitochondrial ATP production and alter Ca

2+

regulation resulting in neuronal

dysfunction; these effects will be exacerbated with aging. Previous studies have characterized the

22

2+

effects of acute MeHg on mitochondrial function and [Ca ]i homeostasis (for review see:
Limke and Atchison, 2004). However, the response of an aged animal to MeHg or the
2+

consequences of long-term exposure to MeHg on mitochondrial function or Ca

regulation have

not been investigated. Three projects were designed to address these gaps in our knowledge. The
first, presented in Chapter 2, uses differentiated SH-SY5Y cells as a model for dopaminergic
2+

neurons. Chapter 2 addresses the effects of acute and chronic MeHg treatment on [Ca ]i and
2+

VGCC function. The second project, presented in Chapter 3, uses Ca

imaging of striatal slices

from young and aged animals to address the effects of aging in response to acute MeHg
treatment. Finally, the effects of chronic exposure to a low-dose of MeHg on mitochondrial
2+

function and Ca

regulation is investigated in Chapter 4. Together these aims characterize the

effects of aging in response to acute MeHg as well as the effects of chronic MeHg treatment on
2+

Ca

regulation.

b. Model systems
i. SH-SY5Y cell model
Although an in vivo system may better mimic what occurs in a human, they also
introduce a number of factors that sometimes complicate your experiment making it difficult to
interpret the data. A useful alternative is a human-derived cell line. In vitro studies allow for the
investigation of mechanisms in an isolated system without the confounding factors present in
animal studies. SH-SY5Y cells are an extensively characterized immortalized human
neuroblastoma cell line that have been utilized as a model in toxicology, neuronal differentiation,

23

and to study different aspects of neurodegenerative diseases (Biedler et al., 1978; Pahlman et al.,
1990; Wang et al., 2011; Noelker et al., 2012). SH-SY5Y cell line was sub-cloned from the SKN-SH cell line, which was isolated from a bone marrow biopsy of a 4yr old female with a
neuroblastoma (Bielder et al., 1973; Bielder et al., 1978; Ross et al., 1983). In the
undifferentiated state these cells are proliferative and express low levels of neuronal markers.
However, with the addition of all-trans-retinoic acid (RA) SH-SY5Y cells acquire a neuron-like
phenotype they produce an extensive neuronal network and express neuronal markers such as
tyrosine hydroxylase, neuron-specific enolase and neuronal nuclei protein (Nordin-Andersson et
al., 1998; Lopes et al., 2010; Pahlman et al., 1990). In the differentiated state SH-SY5Y cells
also express functional VGCCs primarily, N- and L-type Ca2+ channels (Morton et al., 1992;
Reuveny and Narahashi, 1993).
SH-SY5Y cells are most commonly utilized as an in vitro model for neurodegenerative
diseases (Agholme et al., 2013; Qualis et al., 2013; Yu et al., 2013), however they have also been
used to study the toxic effects of MeHg. Some of the first studies demonstrated that SH-SY5Y
cells are susceptible to MeHg-induced cytotoxicity following acute exposure (Nordin-Andersson
et al., 1998; Sanfeliu et al., 2001; Toimela and Tahti, 2004). MeHg-induced cytotoxicity induces
oxidative stress and cells can be protected with the addition of antioxidants (Kim et al., 2005;
Toyama et al., 2007; Franco et al, 2009). MeHg also disrupts SH-SY5Y cell differentiation (Kim
et al., 2007). Petroni et al., (2012, 2013) found that exposure to 100nM MeHg for 48h induced
oxidative stress leading to cytotoxicity and this can be prevented by co-treatment with an NMDA
receptor antagonist. Differentiated SH-SY5Y cells were used in Chapter 2 to study the effects of
2+

chronic MeHg treatment on VGCC function and Ca

24

regulation.

ii. Synaptosomes
Synaptosomes are isolated nerve endings prepared from the rodent brain. They are
isolated using density gradient centrifugation of brain homogenate. Synaptosomes were first
isolated in 1958 to identify the subcellular compartment of acetylcholine (Hebb and Whittaker,
1958). They have since been fully characterized and used extensively in neuroscience research
(for review see: Whittaker, 1992). Synaptosomes are essentially nerve endings that are pinched
off and then re-form their plasma membrane. They maintain a resting membrane potential, can
2+

regulate Ca , produce ATP, and produce, store and release neurotransmitter (Whittaker, 1992;
Whittaker et al., 1964). The main advantage of using synaptosomes is the ability to study nerve
terminal function in isolation.
Synaptosomes have been extensively used to investigate alterations in presynaptic
neuronal function following MeHg exposure. Many of the early studies utilized synaptosomes to
characterize changes in neurotransmitter uptake and release due to MeHg exposure (Braken et
al., 1981; Komulainen and Tuomisto, 1982; Komulainen and Tuomisto, 1985; Minnema et al.,
1989; Rajanna et al., 1990). These studies demonstrated that MeHg induced DA release and
inhibited its uptake in rat brain synaptosomes. Synaptosomes have been used in our lab to study
2+

the effects of MeHg on intracellular Ca

homeostasis, mitochondrial function and VGCC

function (Atchison et al., 1986; Shafer and Atchison 1989; Shafer et al., 1990; Hare and
Atchison, 1992; Levesque et al., 1992; Denny et al., 1993). Based on these studies we learned
2+

that acute MeHg treatment blocks Ca

influx through VGCCs, inhibits the binding of DHP-

antagonists, interacts with nerve-terminal mitochondria causing a depolarization of the

25

2+

membrane and Ca

2+

release, and induces extracellular Ca

entry. These studies have been
2+

instrumental in furthering out understanding on the role of MeHg-induced Ca

dysregulation.

As such, this model system was used to study the presynaptic effects of chronic MeHg treatment
on mitochondrial function in striatal synaptosomes in chapter 4.
iii. Striatal Slice
One particularly useful model system is the acute live brain slice. This technique was
developed to study the function of neurons in an environment similar to that in vivo, as some of
the integrity of the neuronal network is preserved and neurons maintain some contacts present in
the brain (Andersen et al., 1977; Schwartzkroin and Andersen, 1975; Skrede and Westgaard,
1971; Yamamoto and McIlwain, 1966). Thin slices (200μm) of neuronal tissue are taken from
the brain and are able to maintain viability for many hours when kept in an oxygenated artificial
cerebrospinal fluid (ACSF) buffer. The effects of acute MeHg exposure on synaptic function
have been studied in hippocampus and cerebellar brain slices in our laboratory (Yuan and
Atchison, 1993, 1994, 1995, 1997, 2003). Cerebellar and brainstem slices have also been used to
2+

study the effects of [Ca ]i regulation using imaging techniques (Yuan and Atchison, 2007;
2+

Johnson et al., 2011). Live brain slices were utilized to study the [Ca ]i response of striatal
MSNs from different age animals to acute MeHg treatment in Chapter 3 and to study the effects
2+

of chronic MeHg treatment on MSN Ca

regulation in Chapter 4.

26

c. Techniques
i. Calcium imaging
2+

From the previous described background it is clear that MeHg alters neuronal Ca
2+

regulation. There are a number of tools currently available to study changes in [Ca ]i (for
review see: Paredes et al., 2008). We decided to utilize the fluorescent fluorophores fura-2
acetoxymethyl ester (fura-2AM) and fluo-4 no wash (Fluo-4NW). Both of these fluorophores
2+

have been extensively used to monitor MeHg-induced [Ca ]i changes (Hare et al., 1993; Marty
and Atchison, 1997; Limke et al., 2002; Yuan and Atchison, 2007; Johnson et al., 2011).
2+

The Ca

2+

fluorophore fura-2AM was utilized for ratiometric [Ca ]i measurements in

SH-SY5Y cells in Chapter 2 and in striatal slices in Chapter 4. Fura-2 has a high affinity for
2+

2+

Ca , however it will also will bind other divalent cations such as zinc (Zn ). The use of a
ratiometric fluoriphore compensates for variability in fluorophore loading between cells,
particularly when loading fluorophore in thick tissue slices. The -AM group confers fura-2 lipid
solubility, allowing it to cross the cell membrane. During incubation the -AM group is cleaved
by intracellular esterases. Subsequently fura-2 is trapped inside the cell where it preferentially
2+

binds to Ca . Fura-2 is excited using 340nm and 380nm (corresponding to bound vs. unbound
fura-2) and emits at 520nm. An increase in 340nm/380nm ratio corresponds to an increase in
2+

[Ca ]i.

27

2+

In addition to its use as continuously monitoring changes in [Ca ]i, fura-2 can be used to
2+

determine the resting [Ca ]i of cells. Fura-2 fluorescence is continuously monitored under
conditions of maximal fluorescence when fura-2 is saturated and under minimum fluoresnce
2+

when there is zero Ca

present. Based on the fluorescence values under resting, maximum, and
2+

minimum fluorescence, the resting [Ca ]i can be determined. The mathematical relationship
described by Grynkeiwicz et al. (1985), states:
2+

[Ca ]i = Kd * Sf2/Sb2 * (R-Rmin)/(R-Rmax)
2+

Where Kd (222nM) is equal to the dissociation constant for fura-2-Ca
2+

binding. The values of
2+

Sf2 and Sb2 are equal to the value of 380nm fluorescence in Ca -free and Ca -saturating
conditions, respectively. The values are R, Rmin, and Rmax are equal to the ratio value under
2+

2+

resting, Ca -free, and Ca -saturating conditions. It has been previously determined that MeHg
2+

induces Zn

release (Denny and Atchison, 1994), which can then be bound by fura-2 altering
2+

2+

estimations of [Ca ]i. Therefore the Zn

chelator TPEN was present during all of the

2+

experiments when [Ca ]i was determined.
2+

Fluo-4NW was utilized in chapter 3 to monitor spatcial changes in Ca i in striatal slices
2+

in response to acute MeHg treatment. Fluo-4 is a Ca
2+

sensitive fluorophore commonly used to

monitor changes in Ca i. Unlike fura-2, fluo4 is not a ratiometric fluorophore and cannot be
28

2+

used to determine [Ca ]i. Fluo-4 is excited at 485nm and emits at 520nm. An increase in fluo-4
2+

fluorescence corresponds to an increase in Ca i.
ii. Mitochondrial function
The Extracellular Flux Analyzer (XF Analyzer, Seahorse Biosciences) was used to
measure mitochondrial function of striatal synaptosomes described in chapter 4. The XF
Analyzer provides real-time measurments of the extracellular flux of oxygen and protons in
medium surrounding live cells, synaptosomes, or mitochondria. Addition of 4 different
chemicals- oligomyocin, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP),
rotenone, and antimycin A- perturbs mitochondrial function and allows for the determination of
the bioenergetic capacity of mitochondria. The use of each chemical provides information about
different aspects of the bioenergetic capacity. Oligomycin inhibits ATP synthesis and allows for
the determination of the oxygen consumption devoted to ATP synthesis. FCCP is an uncoupling
agent and causes a collapse of the inner-mitochondrial membrane potential, its addition allows
for the determination of the maximal respiration capacity. Finally, addition of a combination of
antimycin A and rotenone which are inhibitors of complex I and III respectively, inhibit
mitochondrial respiration and allows for the determination of the oxygen devoted to nonmitochondrial respiration. This is demonstrated in Figure 1.4. The XF Analyzer has been utilized
to study changes in cellular metabolism in response to toxicants or disease states as well as a
number of different physiologic changes (for review see: Brand and Nicholls, 2011).

29

600

OCR(pMol/min)

500
400

Spare
Capacity

300
ATP
200
100
0

Basal
Respiration

Maximum
Respiration

Proton
Leak
Non-mitochondrial Respiration

0

9

18

27

36

42
54
63
Time (min)

72

81

90

98

Figure 1.4. A depiction of an XF Analyzer experiment measuring mitochondrial OCR.
Perturbation of mitochondrial oxidative phosphorylation allows for the determination of
mitochondrial basal respiration, ATP production, maximal respiration, spare capacity, nonmitochondrial respiration, and proton leak.

30

CHAPTER TWO
PERTURBATION OF CALCIUM REGULATION FOLLOWING ACUTE AND
CHRONIC METHYLMERCURY TREATMENT IN DIFFERENTIATED SH-SY5Y
CELLS

31

A) Abstract
2+

MeHg causes a loss of the cerebellar granule cells in the cerebellum in a Ca

dependent

manner. In vitro studies have shown that acute MeHg treatment caused a biphasic increase in
2+

[Ca ]i which can be delayed by VGCC antagonists, demonstrating the role of VGCCs in
2+

MeHg-induced Ca

dysregulation. Others have shown effects of MeHg on DA release;

numerous studies have addressed the acute effects of MeHg on cholinergic and dopaminergic
cell lines and primary neurons. However, exposure to low levels of MeHg during lifetime are
lacking. As such, our aim was to study effects of chronic MeHg treatment on VGCC function in
differentiated SH-SY5Y cells, a dopaminergic cell line. These cells have not been extensively
2+

used to study MeHg neurotoxicity, so we first characterized the acute effects of MeHg on [Ca ]i
2+

in SH-SY5Y cells. Changes in [Ca ]i were measured using Fura-2; cells were perfused
continuously with 1, 2, 5, or 10μM MeHg. MeHg caused a biphasic, concentration-dependent
increase in fura-2 fluorescence; the times to onset of both phases were inversely proportional to
the [MeHg]. At 1μM MeHg phase 1 and phase 2 occurred at ~11 and 49min respectively,
whereas at 10μM phase 1 occurred within 2 min and phase 2 within 16 min. Nif (5μM) and
GVIA (1μM), L- and N-type antagonists respectively, delayed the onset of phase 2 but not phase
1. Effects of chronic MeHg treatment on VGCC function were subsequently characterized. Cells
2+

were treated with 0 or 20nM MeHg for 48 or 72h. Changes in [Ca ]i were monitored using
2+

fura-2, KCl-induced (40mM) Ca2+ influx was monitored as an index of VGCC function. Ca

influx through L- and N-type VGCCs was isolated using GVIA and Nif respectively. The 48h
32

2+

treatment significantly decreased KCl-induced Ca

2+

treatment significantly decreased KCl-induced Ca

influx through L-type VGCCs; the 72h
influx through both L- and N-type VGCCs.
2+

The 72h treatment also caused a significant increase in resting [Ca ]i. Thus chronic MeHg
2+

2+

treatment significantly increases resting [Ca ]i and decreased KCl-induced Ca

33

increase.

B) Introduction
MeHg is an environmental neurotoxicant that causes cell-type specific damage in the
central nervous system. Humans are most commonly exposed to MeHg through the consumption
of contaminated seafood. The effects of both acute and long-term exposure to MeHg on humans
results in cerebellar ataxia, paresthesia, and constriction of the visual field (Bakir, 1973; Harada,
1995). Development of these symptoms may be related to MeHg-induced alterations in VGCC
2+

2+

function and [Ca ]i homeostasis. The effects of acute MeHg treatment on Ca i regulation and
VGCC function have been characterized. However the effects of chronic MeHg are largely
unknown.
Granule cells of the cerebellum are particularly sensitive to the effects of MeHg (Hunter
and Russell, 1954). Acute MeHg treatment of primary cerebellar granule cells results in a
2+

2+

biphasic increase in [Ca ]i. The first phase is due to release of Ca i stores from the
mitochondria and SER (Hare and Atchison, 1995; Limke and Atchison, 2001). The second phase
is due to extracellular Ca

2+

2+

(Ca e) entering the cell (Marty and Atchison, 1997). Both phase 1

and phase 2 can be delayed through the use of the L- or N-type VGCC antagonists, Nif and
GVIA respectively (Marty and Atchison, 1997). This evidence demonstrates the role of VGCCs
2+

in MeHg-induced Ca -dysregulation. Acute application of MeHg also alters function of VGCCs
in a number of different neuronal preparations. MeHg inhibits the influx of

45

2+

Ca

into neuronal

synaptosomes (Atchison et al., 1986), and blocks DHP sensitive (L-type) and DHP-insensitive

34

2+

(N-type) Ca

channels in PC12 cells (Shafer et al., 1990). These effects on VGCCs are

irreversible and occur irrespective of channel configuration (Sirois and Atchison, 1996).
Changes in neurotransmission have also been associated with MeHg exposure. Acute
MeHg treatment causes increased DA release. This has been demonstrated in cells in culture
(Tiernan et al., 2013), striatal synaptosomes (Dreiem et al., 2009), striatal slices (Kalisch and
Racz, 1996), and in vivo (Faro et al., 2000, 2003, 2007). Acute MeHg exposure causes an
2+

increase in [Ca ]i so it is not surprising that it also causes increased neurotransmitter release in
DA neurons. Taken together, the data support the hypothesis that DA neurons are susceptible to
2+

MeHg-induced Ca

dysregulation.

Long-term exposure to low levels of MeHg throughout ones lifetime are of contemporary
concern. The goal of this study was to determine the effects of chronic MeHg exposure on
2+

VGCC function in differentiated SH-SY5Y cells, KCl-induced Ca

influx was used as an index

of VGCC function and membrane integrity. Chronic treatment could result in no change,
2+

increased Ca

2+

influx, or decreased Ca
2+

treatment with MeHg blocks Ca

influx upon KCl-induced VGCC activation. Acute

current through VGCCs (Shafer et al., 1990; Sirois and
2+

Atchison, 2000). However, MeHg also mediates a biphasic increase in [Ca ]i, which can be
2+

delayed with an L- or N-type Ca

channel antagonist (Marty and Atchison, 1997). These data

seem to be contradictory. However one explanation may be that MeHg uses VGCCs for entry
into the cell, which competitively blocks the channel. Once inside the cell, MeHg interacts with

35

2+

the channel to enhance extracellular Ca
2+

block VGCCs causing decreased Ca

influx. We expect that at least initially MeHg will

influx. These changes will be time-dependent.

SH-SY5Y cells are a well characterized immortalized human neuroblastoma cell line,
which can be differentiated with the addition of RA. Upon differentiation they produce an
extensive neural network, express tyrosine-hydroxylase, and functional L- and N-type VGCCs
(Nordin-Andersson et al., 1998; Lopes et al., 2010; Pahlman et al., 1990; Morton et al., 1992;
Reuveny and Narahashi, 1993). Very limited work has been done to characterize the effects of
acute MeHg treatment in SH-SY5Y cells. It has been shown that they are susceptible to MeHg2+

induced ROS production and cytotoxicity (Petroni et al., 2010 and 2011). Changes in [Ca ]i
following acute MeHg treatment have not been characterized in differentiated SH-SY5Y cells.
2+

The goals of this study were two fold. The first was to characterize the [Ca ]i response
to acute MeHg treatment and determined the contribution of VGCCs in the SH-SY5Y cell
response. The second goal was to determine the effects of chronic MeHg treatment on VGCC
function in differentiated SH-SY5Y cells. The results of this study demonstrate that acute MeHg
2+

treatment causes the characteristic biphasic increase in [Ca ]i in differentiated SH-SY5Y cells.
The time-to-onset of phase 2 can be delayed with Nif or GVIA. Chronic MeHg treatment causes
a time- and subtype specific block of VGCCs. These data demonstrate that acute and chronic
MeHg treatment alters VGCC function in a similar manner.

36

C) Materials and methods
Chemicals and solutions
Methylmercuric (II) chloride was obtained from Aldrich Chemical (Milwaukee, WI).
Fura-2AM, minimum essential medium (MEM), F12, antibiotic/antimycotic, collagen I- rat tail,
ionomycin, and tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) were obtained from Life
Technologies (Carlsbad, CA). RA and Nif were obtained from Sigma-Aldrich (St. Louis, MO).
Fetal bovine serum (FBS) was obtained from Atlanta Biologicals (Flowery Branch, GA). GVIA
was obtained from Alamone Labs (Jerusalem, Israel).
HEPES buffered saline (HBS) contained (mM) 135 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 20
d-glucose and, 20 HEPES, pH 7.3. The 40mM KCl solution contained the same components as
HBS except that the NaCl was lowered to 115.4 mM. Rmin solutions contained (mM) 130 NaCl,
5.4 KCl, 0.8 MgSO4, 20 d-glucose, 20 HEPES and, 10 EGTA. Rmax solutions contained (mM)
150 NaCl, 5.4 KCl, 8 CaCl2, 0.8 MgSO4, 20 d-glucose and, 20 HEPES. Both Rmin and Rmax
2+

solutions contained 10μM ionomycin and 35μM TPEN. Ionomycin is a Ca
2+

added to the solutions to facilitate Ca

ionophore. It was

transport across the plasma membrane. MeHg treatment

2+

2+

alters intracellular [Zn ] (Denny and Atchison, 1994) which can bind to fura-2 and skew Ca
2+

measurements. TPEN was added to the loading solution to chelate Zn
recording.

37

2+

prior to initiating Ca

A.

P2

50

340nm
380nm

105

44

85

41

65

380nm

340nm

47

125

38
35

45

P1
0

9.00 18.0 27.0 36.0 45.0

25

Time(min)

Ratio(F340/F380)

B.

0.80
0.68
0.56
0.44
0.32
0.20

0

9.00 18.0 27.0 36.0 45.0
Time(min)

Figure 2.1. Acute MeHg treatment in differentiated SH-SY5Y cells caused a biphasic
2+
increase in [Ca ]i. (A-B) Representative 340nm, 380nm, and ratio of (F340nm/F380nm) fura-2
tracings from a single differentiated SH-SY5Y cell treated acutely with 5μM MeHg. (A)
Representative 340nm tracing is shown in black. 340nm fluorescence corresponds to changes in
2+
Ca -bound fura-2. The onset of phase 1 was the time at which the 340nm tracing increases from
baseline. Representative 380nm tracing is shown in grey. 380nm fluorescence corresponds to
changes in unbound fura-2. The onset of phase 2 was the time at which the 380nm tracing had a
sustained decrease from baseline. (B) Representative ratio of (F340nm/F380nm). An increase in
2+

fluorescence of the ratio corresponds to an increase in [Ca ]i. Acute 5μM MeHg treatment
2+
caused a biphasic increase in [Ca ]i.
38

Cell Culture
SH-SY5Y cells (ATCC #CRL-2266) used in these studies were from passages 3-10 from
our receipt. Cells were grown in 1:1 MEM/F12 (v/v) medium plus 10% (v/v) FBS and
antibiotic/antimycotic (SH-SY5Y medium). For all experiments, 2.5x105 cells/mL were seeded
on collagen (250µg/ml)- coated glass coverslips. Twenty-four hours after plating, the medium
was replaced with SH-SY5Y medium containing 10μM RA (differentiation medium). Cells were
grown for 5 days in differentiation medium with a medium exchange every 48h.
Single-cell microfluorimetry
Microfluorimetry experiments were performed using a IX-70 microscope (Olympus,
Tokyo, Japan) coupled to an IonOptix (Milton, MA) system with a heated perfusion system
2+

2+

(37°C, 3ml/min). Alterations in [Ca ]i were monitored through the use of the Ca -sensitive
ratiometric fluorophore fura-2. SH-SY5Y cells were loaded with 3μM fura-2AM in HBS for
45m at 37°C. During incubation the -AM group of fura-2 is cleaved by esterases in the cell.
2+

Subsequently, ionized fura-2 is trapped inside the cell where it binds preferentially to Ca . Prior
to initiating recording, cells were washed with HBS for 10m to remove excess unbound fura-2
and allow the cells to reach homeostasis. Cells were then depolarized with 40mM KCl to check
2+

cell viability and assure they are able to buffer increases in [Ca ]i. If cells did not respond to
KCl, they were not used for data analysis. Fura-2 was excited at 340 and 380nm (bound and
unbound fura-2, respectively). Changes in fura-2 fluorescence ratio were recorded at 510nm. An
2+

increase in the 340nm/380nm ratio corresponds to an increase in [Ca ]i.

39

Ratio(F340/F380)

B.

1.00
0.80
0.60

40mM KCl

0.40
0.20
0.00
0.00

6.00

12.0

18.0

24.0

30.2

Time (min)

Figure 2.2. Measurement of VGCC function. (A) Schematic diagram of how VGCC function
was measured. VGCCs were activated by depolarizing the cell membrane with 40mM KCl
2+
which activated VGCCs and allowed Ca influx through L- and N-type channels. (B)
Representative fura-2 tracing from a single differentiated SH-SY5Y cell treated with 0nM MeHg
for 48h. Application of 40mM KCl is indicated by the arrows. The amplitude of KCl-induced
2+
Ca influx was measured for each depolarization and averaged to determine a mean amplitude
for each experiment (n=1).

40

MeHg treatments
Cells were loaded with fura-2 and then superfused with 1, 2, 5, or 10μM MeHg. All MeHg
solutions were prepared on the day of experiment from a 10mM stock solution. The contribution
2+

of N- or L-type VGCC’s in MeHg-induced [Ca ]i increases were determined through the use of
VGCC antagonists. The contribution of N-type VGCC’s was determined by pre-treating cells
with 1μM GVIA. The contribution of L-type VGCC’s was determined by the co-treatment with
5μM nifedipine. The onset of the first phase was measured manually by determining the time at
which the 340nm tracing rose irreversibly from baseline (Figure 2.1a). The onset of the second
phase was determined as the time at which the 380 tracing had an irreversible decrease from
baseline (Figure 2.1a). The ratio of these tracing demonstrates that acute MeHg treatment results
2+

in a biphasic increase in [Ca ]i (Figure 2.1b).
To determine the effects of prolonged MeHg treatment on VGCC function, differentiated
SH-SY5Y cells were treated with 0 or 20nM MeHg for 48 or 72h. MeHg treatment medium
contained no FBS or antibiotic/antimycotic. Medium was exchanged every 24h. Upon
completion of the treatment period, cells were loaded with fura-2AM using the method that was
described previously. VGCC function was measured by depolarizing the cell membrane using
2+

40mM KCl, which activated VGCCs and allowed Ca

influx (Figure 2.2a,b). Using the
2+

340nm/380nm tracing the maximum amplitude of KCl-induced Ca

influx was measured for

each depolarization and averaged to determine a mean amplitude for each plate (n=1). The
function of L- and N-type VGCCs was determined through the use of Nif and GVIA. Cells were
depolarized 4 times with 40mM KCl. The first depolarization was considered the control and

41

occurred with no antagonist present. The following 3 depolarizations occurred in the presence of
2+

an antagonist (Figure 2.3a,b). The amplitude of KCl-induced Ca

influx was quantified for each

depolarization. The mean amplitude of the depolarizations in the presence of antagonist was
divided by the amplitude of the control depolarization to determine the percent of control. KCl2+

induced Ca

influx through L-type VGCCs was isolated by blocking N-type VGCCs. Cells
2+

were incubated with 1μM GVIA for 5m prior to the application of KCl. KCl-induced Ca

influx

through N-type VGCC’s was isolated by blocking L-type VGCCs with 5μM Nif. Nif was present
in HBS and KCl solutions throughout the experiment.
2+

Determination of [Ca ]i
2+

Fura-2 was used to determine the [Ca ]i of treated and untreated cells. The mathematical
2+

relationship of fura-2 fluorescence and [Ca ]i was described by Grynkiewicz et al. (1985), it
states:
2+

[Ca ]i = Kd * Sf2/Sb2 * (R - Rmin)/(Rmax - R)
2+

2+

Calculating the resting [Ca ]i is possible by controlling the [Ca ]i of cells. This is done by
2+

removing essentially all Ca

(Rmin) and saturating fura-2 (Rmax). The value used for the
2+

dissociation constant for fura-2-Ca

binding (Kd) was 225nM (Ionoptix, Tools for

Cardiovascular Research). R is equal to the resting ratio value. The ratio of Sf2 and Sb2 is equal
2+

to the value of the denominator (380nm) under zero Ca

42

2+

conditions (Sf2) and saturating Ca

Ratio (F340/F380)

B.

1.00

5M Nifedipine

0.80
0.60

40mM KCl

0.40
0.20
0.00
0.00

6.00

12.0

18.0

24.0

30.0

Time (min)
Figure 2.3. Determination of VGCC subtype sensitivity to chronic MeHg treatment. (A)
Schematic diagram of how VGCC function was measured. SH-SY5Y cells were pretreated with
either 1μM GVIA or 5μM nifedipine (as shown in diagram) and then depolarized with 40mM
2+
KCl. Subsequent Ca influx was due solely to flow through the unblocked channel resulting in
2+

decreased Ca influx. (B) Representative fura-2 tracing from a single differentiated SH-SY5Y
cell treated with 0nM MeHg for 48h. Application of 40mM KCl is indicated by the arrows. The
(Figure 2.3 cont’d) first depolarization was considered the control and occurred with no
antagonist present. The following 3 depolarizations occurred in the presence of antagonist. The
2+
amplitude of KCl-induced Ca influx was quantified for each depolarization. The mean
amplitude of the depolarizations in the presence of antagonist was divided by the amplitude of
the control depolarization to determine the percent of control.
43

conditions (Sb2). SH-SY5Y cells were treated with 0 or 20nM MeHg for 72h and then loaded
with fura-2 as previously described. Cells were washed for 5min with normal HBS to allow
establishment of a baseline. Next cells were perfused with Rmin solution for 5min. The Rmin
2+

solution contains no Ca
2+

all Ca

2+

and EGTA, a Ca

chelator. Perfusion with this solution will remove

present and the 340nm/380nm will reach its minimum value. The Rmin solution is
2+

followed by perfusion with the Rmax solution for 5min. The Rmax solution contains 8mM Ca
which will saturate fura-2 and the 340nm/380nm will reach its maximum value.
Statistics

Comparisons of mean time-to-onset were made using One-Way ANOVA followed by
Tukey-Kramer multiple comparisons post hoc test, p<0.05. Comparisons of 2 means were made
using an unpaired two-tailed student t-test, p<0.05.

44

D) Results
a. Acute methylmercury treatment
Events that occur over a prolonged period of time, such as chronic treatment, can result in
direct as well as secondary or tertiary results that may be far-removed consequences of
prolonged exposure. As such, the first crucial step in determining the chronic effects of MeHg on
differentiated SH-SY5Y cells is to parse out the initiating events. This was achieved by
2+

characterizing the Ca

response to acute MeHg treatment. Differentiated SH-SY5Y cells were

loaded with fura-2AM and then superfused with 1, 2, 5, or 10μM MeHg until P2 was reached
and the 340nm/380nm tracing plateaued (there was no further increase in intracellular calcium)
2+

(Figure 2.1A,B). Acute MeHg treatment caused a biphasic increase in [Ca ]i (Figure 2.1B). The
time-to-onset of phase 1 was the time at which the 340nm fluorescence tracing increased and the
time-to-onset of phase 2 was the time that the 380nm fluorescence tracing irreversibly decreased
from baseline (Figure 2.1A). The time-to-onset of phase 1 and phase 2 was inversely related to
[MeHg] with a decrease in the time-to-onset with increasing [MeHg] (Figure 2.4). At 1μM
MeHg phase 1 and phase 2 occurred in approximately 11min and 49min respectively. When cells
were treated with 5 and 10μM MeHg there was a statistically significant hastening in the timesto-onset of phase 2 compared to 1μM MeHg. These results are consistent with work done in rat
cerebellar granule cells (Marty and Atchison, 1997).
The next step was to determine if L-or N-type VGCCs contribute to the biphasic increase
2+

in [Ca ]i in differentiated SH-SY5Y cells. This was investigated by antagonizing L- or N-type

45

T ime (min)

60

1µ M

48

2µ M

36

5µ M

24
12
0

10µM

*

*

* *

Phas e 1

Phas e 2

Figure 2.4. Acute MeHg caused a concentration-dependent decrease in the time-to-onset of
Phase 1 and Phase 2 in differentiated SH-SY5Y cells. Differentiated SH-SY5Y cells were
2+
superfused with 1, 2, 5, or 10μM MeHg and changes in [Ca ]i were continuously monitored.
MeHg caused a concentration dependent decrease in the time-to-onset of phase 1 and phase 2.
Acute treatment with 5 and 10μM MeHg resulted in a significant decrease in the onset of phase 1
and 2 compared to 1μM MeHg. Data are expressed as mean ± SEM. * = significant difference
compared to 1μM MeHg (One-Way ANOVA, p<0.05, n≥4).

46

45

T ime (min)

36
27
18

* *

MeHg
MeHg+GVIA
MeHg+Nif

9
0

Phas e 1

Phas e 2

Figure 2.5. N- or L-type VGCC antagonists delay the onset of phase 2. Differentiated SHSY5Y cells were superfused with 2μM MeHg alone, 2μM MeHg + 1μM GVIA, or 2μM MeHg +
2+
5μM Nif and changes in [Ca ]i were continuously monitored. VGCC antagonists did not delay
the onset of phase 1. Co-treatment with GVIA or Nif significantly delayed the onset of phase 2.
Data are expressed as mean ± SEM. * = significant difference compared to 2μM MeHg alone
(One-Way ANOVA, p<0.05, n≥3).

47

VGCC with nifedipine (5μM) or GVIA (1μM) respectively prior to the application of 2μM
MeHg. Cells were loaded with fura-2AM and pretreated with Nif or GVIA and then superfused
with 2μM MeHg. Treatment with 2μM MeHg alone resulted in the onset of phase 1 and phase 2
at approximately 4min and 30min respectively. Application of Nif or GVIA statistically
significantly delayed the time-to-onset of phase 2 to approximately 36min (Figure 2.5). These
data are similar to work completed in rat cerebellar granule cells (Marty and Atchison, 1998).
However in granule cells VGCC antagonists delay the onset of phase 1 and phase 2 indicating
that these two cell types may differ in the intracellular events that initiate the onset of phase 1.
b. Chronic methylmercury treatment
2+

Acute MeHg exposure clearly alters intracellular Ca

homeostasis in differentiated SH-

SY5Y cells as well as primary neuronal cultures and immortalized cell lines (Denny and
Atchison, 1996; Marty and Atchison, 1997; Limke and Atchison, 2001). Yet the effects of
2+

prolonged exposure to low-level MeHg on Ca

regulation have been largely ignored. To begin

to fill this gap in our knowledge, experiments were designed to assess the effects of chronic
MeHg treatment on VGCC function. Previous studies have utilized a long-term treatment
paradigm in undifferentiated SH-SY5Y cells (Petroni et al., 2011) and PC12 cells (Shafer et al.,
2002). Shafer et al., (2002) utilized low (10-50) nM MeHg to study MeHg-induced cell death
+

2+

and MeHg effects on voltage-sensitive sodium (Na ) and Ca

currents. Because cells were

treated for a prolonged period of time (48 or 72h) we chose to utilize 20nM MeHg, which did not
induce a significant level of cell detachment, neurite retraction, or cell death. Differentiated SHSY5Y cells were treated with 0 or 20nM MeHg for 48 or 72h and then loaded with fura-2AM,
2+

KCl- (40mM) induced Ca

influx was monitored as a measure of VGCC function (Figure 2.2A,
48

0nM MeHg
0.80

0.48
0.32
0.16
0.00

B.

0.80
72h

0.64

(E -1)

Amplitude (arbitrary units)

48h

0.64

(E -1)

Amplitude (arbitrary units)

A.

20nM MeHg

0.48

*

0.32
0.16
0.00

2+

Figure 2.6. Chronic MeHg caused decreased KCl-induced Ca influx. Differentiated SHSY5Y cells were treated with 0 or 20nM MeHg for 48 or 72h. Following MeHg treatment cells
2+
were loaded with fura-2AM and KCl-induced Ca influx was measured. (A) Forty-eight hour
20nM MeHg treatment caused a slight reduction in the maximum amplitude of KCl-induced
2+
Ca influx. (B) Seventy-two hour 20nM MeHg caused a significant decrease in the maximum
2+

amplitude of KCl-induced Ca

influx (Unpaired t-test, p = 0.018, n ≥4).

49

B). Cells were depolarized 4 times with KCl; the maximum amplitude of each depolarization
was measured and then averaged for cell. A mean amplitude for each plate was determined
2+

(n=1). The maximum amplitude of KCl-induced Ca

influx was decreased in cells treated with

20nM MeHg compared to cells treated with 0nM MeHg (Figure 2.6A). Seventy-two hour
treatment with 20nM MeHg caused a significant decrease in the maximum amplitude of KCl2+

induced Ca

influx compared to cells treated with 0nM MeHg (Figure 2.6B). These experiments

demonstrate that long-term MeHg treatment disrupts VGCC function in a time-dependent
manner.
2+

We next determined if decreased Ca

influx was due to decreased influx through L- or

N-type channels, or both. A similar experimental design was used as described in the previous
experiments except the L- and N-type VGCC antagonists Nif and GVIA were utilized to isolate
current through specific subtypes of VGCC. Cells were treated for 48 or 72h and then loaded
2+

with fura-2AM and KCl-induced Ca

influx was used as an indirect measure of VGCC

function. Cells were depolarized with 40mM KCl 4 separate times. The first depolarization was
considered the control and occurred with no antagonist present. The following 3 depolarizations
2+

occurred in the presence of antagonist. Ca

influx through N-type VGCCs were isolated by
2+

antagonizing L-type VGCCs with Nif and Ca

influx through L-type VGCCs was isolated by
2+

antagonizing N-type VGCCs with GVIA. The amplitude of KCl-induced Ca

influx was

quantified for each depolarization. The mean amplitude of depolarizations in the presence of
antagonist was divided by the amplitude of the control depolarization to determine the percent of
control. Forty-eight hour 20nM MeHg treatment caused a statistically significant decrease in
50

% of Control

A.

0nM MeHg
100

20nM MeHg
48h

80

*

60
40
20
0

N-Type

L-Type
VGCC

% of Control

B.

100
72h

80

*

60
40

*

20
0

N-Type

L-Type
VGCC

Figure 2.7. Chronic MeHg treatment caused a time- and subtype specific block of VGCCs.
2+
The maximum amplitude of KCl-induced Ca influx was measured in cells treated with 0 or
20nM MeHg for 48 or 72h. Cells were depolarized 4 times using 40mM KCl. The first
depolarization was performed with KCl alone (control depolarization) the following 3 were
2+
performed in the presence of a VGCC antagonist. The amplitude of KCl-induced Ca influx
was quantified for each depolarization. The mean amplitude of depolarizations in the presence of
antagonist was divided by the amplitude of control depolarization to determine percent (%) of
control. Data are represented as percent (%) of control depolarization. (A) Forty-eight hour
2+
20nM MeHg treatment, caused a significant decrease in Ca influx through L-type VGCCs and
2+

no change in KCl-induced Ca influx through N-type channels (Unpaired t-test, p = 0.0122,
n=3). (B) Seventy-two hour 20nM MeHg treatment caused a significant decrease in KCl-induced
2+
Ca influx through N - and L-type VGCCs (Unpaired t-test, p<0.05, n≥3). * = significantly
different than the 0nM MeHg group. Data are expressed as mean ± SEM.

51

2+

KCl-induced Ca

2+

influx through L-type VGCCs and had no effect on Ca

influx through N-

type VGCCs (Figure 2.7A). Seventy-two hour 20nM MeHg treatment caused a statistically
2+

significantly decrease in KCl-induced Ca

influx through both N and L-type VGCCs compared

to cells treated with 0nM MeHg (Figure 2.7B). These data demonstrate that prolonged MeHg
treatment alters L- and N-type VGCC function in a subtype- and time-dependent manner. Based
our previous experiments which demonstrated that acute MeHg exposure caused a biphasic
2+

2+

increase in [Ca ]i we hypothesized that chronic MeHg treatment may increase resting [Ca ]i.
To test this hypothesis, differentiated SH-SY5Y cells were treated with 0 or 20nM MeHg for 72h
2+

and the resting [Ca ]i was measured using fura-2. Seventy-two hour 20nM MeHg treatment
2+

resulted in a statistically significant increase in resting [Ca ]i (Figure 2.8).

52

0nM

20nM

[Ca 2+ ]i (nM)

400
*

300
200
100
0

2+

Figure 2.8. Chronic MeHg treatment caused a significant increase in resting [Ca ]i.
Differentiated SH-SY5Y cells were treated with 0 or 20nM MeHg for 72h and the resting
2+
[Ca ]i was calculated. Cells treated with 20nM MeHg had a significant increase in mean resting
2+

[Ca ]i compared to 0nM MeHg treated cells.* = significant difference compared to 0nM MeHg
group (Unpaired t-test, p<0.05, n = 3).

53

E) Discussion
2+

Disruption of Ca

homeostasis appears to be a central event in MeHg-induced toxicity.

Both primary cells in culture and acute live brain slices from rodents respond to acute MeHg
2+

treatment with an increase in [Ca ]i (Hare and Atchison, 1994; Marty and Atchison, 1997;
Edwards et al., 2005; Yuan and Atchison, 2007; Ramanathan and Atchison, 2011) for cells in
culture this can be differentiated kinetically into two distinct phases. Acute MeHg in
2+

differentiated SH-SY5Y cells causes a biphasic increase in [Ca ]i and the time-to-onset is
inversely related to [MeHg]. Nif or GVIA delay the onset of phase 2. Our results are consistent
with previous reports (Marty and Atchison, 1997). However, the characteristics of the biphasic
response appear to differ between cell types. Cerebellar granule cells are a known target of
2+

MeHg and the occurrence of phase 1 is highly dependent on release of mitochondrial Ca

(Limke and Atchison, 2001), it can be delayed by VGCC antagonists. In contrast, the rat
neuroblastoma cell line NG108-15 responds to acute MeHg with 3 distinct phases. Phase 1 is
2+

2+

primarily due to Ca
and finally the 2

nd

release from the SER this is followed by a non-Ca
2+

Ca

2+

phase due to Ca

phase due to Zn

2+

entry. The SH-SY5Y cell response is similar to that
2+

seen in the NG108-15 cell line. This insinuates that phase 1 in SH-SY5Y cells is due to Ca

release from the SER which cannot be delayed with the addition of Nif or GVIA. Therefore the
initiation of phase 1 is mediated differently in cerebellar granule cells. Characterizing the source
2+

of Ca

release of phase 1 or the involvement of other divalent cations was beyond the scope of

this dissertation. However, those studies could be completed in the future and modeled after
work done by Denny and Atchison (1994).
54

Understanding the consequences of long-term exposure to low-doses of environmental
toxicants is critical in advancing the field of toxicology. Humans are most commonly exposed to
low doses of a toxicant throughout their lifetime. Therefore studies must be designed to begin to
parse out the health impact(s) of such exposures. In this study our goal was to understand the
effects of long-term exposure to MeHg on VGCC function. We found that 72h exposure to 20nM
2+

MeHg caused a statistically significant decrease in KCl-induced Ca

influx. Twenty four hour
2+

MeHg treatment (30nM) in PC12 cells caused the block of both peak and end type Ca

current

(Shafer et al., 2002). Our data support these findings and suggest that prolonged exposure to
nanomolar concentrations of MeHg results in the blockade of VGCCs. Acute MeHg treatment
2+

has been shown to block Ca

current through L- and N-type VGCCs in PC12 cells (Shafer et

al., 1990) as well as block of KCl-induced

45

2+

Ca

influx in neuronal synaptosomes (Atchison et

al., 1986; Shafer and Atchison, 1989). The similarity in response suggests that MeHg acts in the
same way to block VGCCs in both acute and chronic treatments. Upon further investigation, we
found that L-type VGCCs are more vulnerable to MeHg-induced block than were N-type
2+

VGCCs. Following 48h MeHg treatment L-type VGCCs had significantly decreased Ca influx, whereas N-type VGCCs were only affected following a 72h MeHg exposure. MeHg
blocks VGCCs in a time- and subtype-specific manner. L-type VGCCs must have a unique
characteristic(s) that makes them vulnerable to block by MeHg such as the amino acid
composition or duration of channel opening.
2+

Acute MeHg treatment increases [Ca ]i in SH-SY5Y cells. We wanted to determine if
2+

prolonged MeHg exposure caused increased resting [Ca ]i. Following a 72h MeHg treatment,

55

2+

differentiated SH-SH5Y cells had significantly higher resting [Ca ]i than did untreated cells.
2+

The source of excess cytosolic Ca
2+

Ca

is unknown, but we hypothesize that it is due to intracellular

release from the SER or mitochondria, similar to what occurs in phase 1 of acute treatment.
2+

Increased resting [Ca ]i may result in depolarization of the mitochondria causing reduced ATP
2+

production. This would further hinder the neurons ability to handle Ca

via ATP-dependent

2+

pathways such as SERCA and the plasma membrane Ca -ATPase. These events may lead to
cell death.
2+

This study demonstrated that acute MeHg treatment causes a biphasic increase in [Ca ]i
differentiated SH-SY5Y cells similar to what occurs in NG108-15 cells. VGCCs contribute to
the onset of phase 2 in SH-SY5Y cells. In support of our hypothesis long-term exposure caused
2+

decreased KCl-induced Ca

influx in a time- and VGCC subtype-dependent manner and caused

2+

increased resting [Ca ]i. This work supports previous findings that MeHg causes a block of
2+

VGCCs (Atchison et al., 1986; Shafer et al., 1990). Ca

signaling plays a critical role in

numerous neuronal processes. Disruption of VGCC function as well as mitochondrial function
2+

due to increased [Ca ]i might explain known cellular susceptibilities which lead to the
characteristic cell-type specific damage following MeHg exposure.

56

CHAPTER 3
STRIATAL SLICES FROM AGED ANIMALS ARE MORE SUSCEPTIBLE TO
METHYLMERCURY-INDUCED CALCIUM INCREASE THAN YOUNG ANIMALS

57

A) Abstract
MeHg is a potent environmental neurotoxicant that impairs several neurological functions
2+

upon short and long term exposure. Disruption of [Ca ]i homeostasis is an early and crucial
event in MeHg induced cytotoxicity. The cerebellum is a primary target of MeHg-induced
cytotoxicity resulting in loss of the granule cell layer. The striatum is another potential target due
2+

to high expression of Cav1.3, an L-type Ca

channel. The developmental effects of MeHg are

well established, however the effects on aged individuals have not been characterized. As
2+

neuronal [Ca ]i homeostasis is significantly altered during aging, aged neurons may be
particularly susceptible to MeHg. The objective of this study was to examine MeHg-induced
2+

[Ca ]i dysregulation in striatal MSNs of aged mice. Changes in the relative fluorescence (RF)
2+

of Fluo4, a [Ca ]i indicator, were measured by laser scanning confocal microscopy every 5 min
during 45 min of perfusion with 20μM MeHg, or every 3 min during a 21 min treatment with
100μM MeHg from striatal slices from male ICR mice aged 1 month (mo), 9 mo or 20.5 mo.
Striatal slices from 9 and 21mo mice treated with 20μM MeHg had a significantly hastened timeto-maximum fluorescence compared to 1mo. There was no difference in response when slices
were treated with 100μM MeHg. We also compared the time-to-peak fluorescence between
cerebellar granule cells and striatal medium spiny neurons and found no difference between their
responses. This study is the first to demonstrate that aged mice are more susceptible to MeHg2+

induced Ca -dysregulation than young animals.

58

B) Introduction
Methylmercury is an environmental neurotoxicant that causes cell type specific damage
to the CNS upon short and long term exposure. There have been two well-known catastrophic
MeHg poisonings that have contributed to our knowledge about MeHg poisoning. The first
occurred in Minamata, Japan in the 1950's and the second in Iraq in 1973. In Minamata,
industrial waste that contained MeHg was dumped into the nearby bay where it accumulated in
the fish, which lived in the bay. Fishermen and their families who consumed the contaminated
seafood were consequently exposed to MeHg over a period on many years. In Iraq, wheat seed
which was coated with a MeHg containing fungicide that was supposed to planted was instead
used to make bread. In this exposure people whom consumed the bread were exposed to a high
concentration of MeHg. Victims of both occurrences presented with the same signs and
symptoms: cerebellar ataxia, parasthesia, constriction of the visual field, dysarthria, and loss of
hearing (Bakir, 1973; Harada, 1968).
Cerebellar granule cells are a very sensitive target for MeHg-induced cytotoxicity
2+

(Hunter and Russel, 1954) and the disruption of [Ca ]i is an early and crucial event in that
2+

process. Acute MeHg treatment causes a biphasic increase in [Ca ]i. The first phase is due to
2+

release of intracellular stores of Ca

2+

and the second phase is due to extracellular Ca

entry

(Hare and Atchison, 1995; Marty and Atchison, 1997; Limke and Atchison, 2002). Cotreatment
with L- or N-type VGCC antagonists will delay the onset of phase 1 and 2 and delay cytotoxicity
(Marty and Atchison, 1997; Marty et al., 1998). VGCC antagonists have also been shown to
delay the onset of behavioral deficits (Bailey et al., 2013) induced by MeHg and mortality in vivo

59

2+

(Sakamoto et al., 1996). These data demonstrate the modulation of Ca -homeostasis is a central
event in MeHg-induced neuronal dysfunction and subsequent cytotoxicity.
MSNs are the principal neuron of the striatum. They play a very important role in
modulating the activity of the basal ganglia. Their main inputs come from the cortex
(glutamatergic) and the midbrain (dopaminergic) (Smith and Bolam, 1990). Altered function of
the basal ganglia is associated with a number of disease states including Parkinson’s and
Huntington’s disease (Chuhma, et al., 2011). Proper functioning of the MSNs rely on their ability
to modulate both the glutamatergic and dopaminergic signals they receive. Changes in
neurotransmission of their primary inputs would result in altered function and output of the basal
ganglia (Giraultm, 2012; Kreitzer and Malenka, 2008). MeHg alters neurotransmission of both
dopaminergic and glutamatergic neurons. Acute exposure to dopmaminergic neurons causes
increased dopamine release (Dreiem et al., 2009; Tiernan et al., 2013). The same has been shown
for glutamatergic neurons (Farina et al., 2011). Lesions occur in the striatum following acute
application of MeHg in rats (O’Kusky et al., 1988; O’Kusky and McGeer 1989; Wakabayashi et
2+

al., 1995; Sakamoto et al., 1998). A combination of MeHg-induced [Ca ]i-increase and altered
neurotransmission in the striatum may induce neuronal dysfunction resulting in cytotoxicity in
the striatum.
2+

As neuronal [Ca ]i homeostasis is significantly altered during aging, aged neurons may
be particularly susceptible to MeHg. During the aging process many changes occur that alter a
2+

neurons ability to efficiently produce ATP (Toescu, 2005) and buffer changes in [Ca ]i
(Toescu, 2007; Toescu and Vreugdenhil, 2010). These changes influence an aged neuron’s
2+

2+

ability to respond to changes in [Ca ]i leaving them vulnerable to stressors that alter [Ca ]i

60

homeostasis such as MeHg. The objective of this study was to examine the responses of striatal
MSNs from 1mo (young), 9mo (adult), and 21mo (aged) animals to acute MeHg treatment. We
2+

hypothesized that aging will increase the striatum’s susceptibility to MeHg-induced Ca
dysregulation.

61

C) Materials and methods
Chemicals and solutions
Methylmercuric (II) chloride was obtained from Aldrich Chemical (Milwaukee, WI). Fluo4-NW
was obtained from Invitrogen (Carlsbad, CA). TPEN was obtained from Sigma Aldrich (St.
Louis, MO). Slicing solution contained (in mM): 222.5 sucrose, 2.5 KCl, 1.25 KH2PO4, 26
NaHCO3, 25 D-glucose, 5 MgCl2, and 0.5 CaCl2 (pH 7.3-7.4 upon aeration with 95% O2/5%
CO2. ACSF contained the following changes in salt composition (in mM): 125 NaCl, 1 MgCl2,
and 2 CaCl2. Alternative ACSF contained the following changes in salt composition (in mM):
110 sucrose, 62.5 NaCl, and 20 D-glucose.
Slice preparation
All animal procedures adhered to NIH guidelines and were approved by the MSU institutional
animal use and care committee. Adult male ICR (Harlan Laboratories) 1mo, 9-9.5 mo and 2121.5 mo mice were utilized for this study. Following anesthesia with isoflurane, mice were
perfused intra-cardially with ice-cold slicing solution. Mice were decapitated, and the whole
brain was removed and placed in ice-cold slicing solution. The forebrain was sliced into 200µm
coronal sections using a Leica VT100S vibratome (Leica Microsystems Inc., Bannockburn, IL).
Slices were transferred to an incubation chamber containing oxygenated alternative ACSF and
incubated for 30min at 37°C and then at 23-25°C until use.
Methylmercury treatment
All MeHg solutions were prepared on the day of experiment from a 10mM stock solution. MeHg
concentrations of 20 and 100μM were chosen based on the acute in vivo exposure to MeHg in
Iraq where 90% of the patients who presented with cerebellar ataxia had a blood [Hg] of 19µM

62

(Bakir et al., 1973). Also, previous work from our lab demonstrated that acute exposure to MeHg
2+

causes a concentration-dependent increase in [Ca ]i in a cerebellar slice (Yuan and Atchison,
2007). Relatively high MeHg concentrations are needed in acutely isolated brain slices compared
to cells in culture because of the extensive ability of MeHg to bind to the slice non-specifically
thereby reducing its effective concentration.
Confocal imaging
Slices were loaded for >30min at 23-25°C in 1x Fluo4-NW with 2.5 mM probenecid and 5μM
TPEN. Probenecid was added to the loading solution to inhibit organic-anion transporters, which
2+

can extrude dye. MeHg treatment alters intracellular [Zn ] (Denny and Atchison, 1994) which
2+

can bind to fura-2 and skew Ca
2+

chelate Zn

measurements. TPEN was added to the loading solution to
2+

prior to initiating Ca
2+

commenced. Intracellular Ca

recording. Immediately following loading imaging

imaging was performed on a Leica confocal microscope. A

region containing at least 10 fluo-4 loaded cells was located to perform imaging. The z-series
was optimized and a 3μm step size was used. A pretreatment z-series was recorded at time 2+

5min. Confocal images of Fluo-4 ([Ca ]i) fluorescence (excited at 488nm, emission >512nm)
were obtained with a 40x water immersion objective (numerical aperature 0.75) fitted to a Leica
DM LFSA (Leica Optics, Bannockburn, IL). Images (512x512 pixels, xyz or xyzt scan mode)
were collected before and during exposure to 20µM MeHg (scanned every 5m for 45m) or
100µM MeHg (scanned every 3m for 21m). All experiments were completed at room
temperature (23-25°C). Changes in Fluo4 fluorescence were analyzed using Leica software.
Fluorescence intensity data from several regions of interest in the same slice were averaged.

63

Background fluorescence was then subtracted and normalized to the pre-treatment control (n=1).
The time-to-peak fluorescence for each region of interest was determined, and averaged to
determine the mean time-to-peak fluorescence for each slice.
Statistics
Comparisons of mean time-to-peak fluorescence were made using one-way ANOVA followed
by Tukey-Kramer multiple comparisons post-hoc test, p<0.05.

64

Relative Fluorescence

A.

1mo

9mo

21mo

1.50
1.30
1.10
0.90
0.70
0.50

0

5 10 15 20 25 30 35 40 45
Time (min)

T ime-to-Peak (min)

B.

21m

9mo

1mo
50
40

*

30

*

20
10
0

Figure 3.1. Striatal slices from aged animals are more susceptible to 20μM MeHg than
slices from 1mo animals. Striatal slices from 1mo, 9mo, and 21mo mice were loaded with Fluo4
and superfused with 20μM MeHg for 45min. (A) Relative Flou4 fluorescence (F/F0) of striatal
slices from 1mo, 9mo, and 21mo mice to 20μM MeHg treatment. (B) The time-to-peak
fluorescence was quantified for each group. Striatal slices from 9 and 21mo mice had a
significantly decreased time-to-peak compared to slices from 1mo animals. Data are means ±
SEM (n≥4) * = significantly different than 1mo animals (p<0.05, One-Way ANOVA, Tukey’s
post-test).

65

D) Results
a. Confocal recordings
2+

The influence of age on the susceptibility to MeHg-induced Ca

dysregulation was

investigated by performing acute MeHg treatment on striatal slices from different age mice and
2+

monitoring changes in [Ca ]i with Fluo-4. Striatal slices from 1, 9, and 21mo mice were
superfused with 20μM MeHg for 45min. Images were recorded every 5 min for 45min (Figure
3.1A). Responses from multiple cells in each slice were recorded, and the time-to-peak
fluorescence was determined. Slices from 9 and 21mo old mice had statistically significant faster
time-to-peak fluorescence than did those slices from 1mo old mice (Figure 3.1B). Because there
was no difference in response between the striatal slices from 9 and 21mo old animals we
compared the response striatal slices from 1 and 9mo animals to 100μM MeHg. Striatal slices
from 1 and 9mo old animals were superfused with 100μM MeHg for 21min (Figure 3.2A). There
was no difference in the time-to-peak fluorescence between 1 and 9mo animals (Figure 3.2B).
2+

These data demonstrate that the susceptibility of striatal MSNs to MeHg-induced Ca dysregulation increases with age. However, when striatal MSNs are exposed to a very high
[MeHg] age does not influence their response.
b. Comparative responses of striatal and cerebellar slices to acute methylmercury
Cerebellar granule cells are among the most sensitive cell types to MeHg cytotoxicity
(Hunter and Russell, 1954). To determine if striatal MSNs were more or less sensitive to MeHg2+

induced Ca

dysregulation than cerebellar granule cells, we compared the time-to-peak

fluorescence. Confocal recordings of cerebellar granule cells was completed by a former member
of the Atchison lab (Dr. Erin Wakeling). Treatments, recordings, and data analysis were

66

Relative Fluorescence

A.

9mo

1mo
2.00
1.70
1.40
1.10
0.80
0.50

0

3

6

9

12 15 18 21

Time (min)

T ime-to-Peak (min)

B.

1mo

9mo

30
20
10
0

Figure 3.2. Age had no effect on the response of striatal slices to 100μM MeHg treatment.
Striatal slices from 1 and 9mo animals were loaded with Fluo4 and superfused with 100μM
MeHg for 21min. (A) Relative Fluo4 fluorescence (F/F0) response of striatal slices from 1 and
9mo mice to 100μM MeHg treatment. (B) There was no difference in the time-to-peak effect
between 1 and 9mo striatal slices to 100μM MeHg treatment. Data are means ± SEM (n≥4).

67

completed in the same manner allowing for comparison between the two regions. The time-topeak fluorescence of striatal and cerebellar slices to 20 and 100μM were compared and no
differences were detected (Table 3.1). This study was the first to compare the relative
susceptibility of cerebellar granule cells and striatal MSNs at different ages to acute MeHg
treatment. We demonstrated that 1) MeHg disrupts intracellular calcium statistically significantly
faster in aged animals compared to young and 2) striatal MSNs and cerebellar granule cells are
2+

equally sensitive to MeHg-induced [Ca ]i changes in 1, 9, and 21mo animals when they are
treated with 20 or 100μM MeHg .

68

Cerebellar
Granule Cells

Striatal
Medium Spiny Neurons

1mo

42.5 ± 1.4

43.8 ± 1.3

9mo

29.3 ± 4.5

27.2 ± 2.2

21mo

21.3 ± 5.2

27 ± 2.7

1mo

18 ± 1.2

18.6 ± 1.7

9mo

11.1 ± 2.6

18 ± 1.7

20μM MeHg

100μM MeHg

Table 3.1. No difference in time-to-peak fluorescence between cerebellar granule cells and
striatal medium spiny neurons. The time-to-peak fluoresce was determined for cerebellar
granule cells and striatal medium spiny neurons in response to 20μM and 100μM MeHg. No
differences were detected between the time-to-peak fluorescence (min) in the cerebellum and
striatum at any age. Data are expressed at means ± SEM. (n ≥4).

69

D) Discussion
2+

Disruption of [Ca ]i is an early event in neuronal MeHg toxicity (Hare et al., 1993;
2+

Marty and Atchison, 1997). An inability to regulate changes in [Ca ]i may increase a neuron’s
susceptibility to MeHg-induced damage. The breakdown of cellular processes involved in
maintaining neuronal Ca

2+

homeostasis is associated with aging (Toescu and Vreugdenhil,

2010). Results of this study indicate that adult and aged neurons are more susceptible to MeHg2+

induced Ca -dysregulation than young neurons. Striatal slices from 1, 9, and 21mo old animals
2+

were treated with 20μM MeHg and changes in [Ca ]i were monitored using Fluo4. The time-topeak fluorescence of striatal slices from 9 and 21mo old animals occurred approximately 20min
faster for than 1mo old animals. These data indicate that as animals age the striatal MSNs are
2+

more sensitive to MeHg-induced Ca -dysregulation. However when striatal slices were treated
with 100μM MeHg there was no difference in response. These data demonstrate that when
exposed to a very high [MeHg] striatal MSNs respond the same, no matter their age.
2+

This study was the first to compare the Ca i response of young and aged animals to
acute MeHg exposure. Neuronal aging has been associated with the breakdown of numerous
2+

cellular processes involved with Ca

regulation and ATP production. Due to these changes,

aged neurons are less able to respond to neuronal stressors (Toescu, 2005; Toescu and
Vreugdenhil, 2010). Thus, it is not surprising that aged neurons are more sensitive to MeHg2+

induced Ca -dysregulation. Acute exposure to MeHg triggers a series of events that neurons
2+

with an impaired ability to buffer changes in [Ca ]i would not be able to withstand. Acute

70

2+

treatment has been shown to cause release of intracellular stores of [Ca ]i (Hare and Atchison,
1995; Limke and Atchison, 2002). In order to recover from this first MeHg-induced increase in
2+

2+

[Ca ]i, the neuron would need to be able to use energy-dependent pathways to pump Ca

into

the SER via the smooth endoplasmic reticulum ATPase (SERCA) or out of the cell via the
plasma membrane ATPase. Aged neurons have decreased expression of both of these ATPases
(Toescu, 2007; Toescu and Vreugdenhil, 2010) and their mitochondria are depolarized. This
2+

would decrease their ability to produce ATP and buffer increases in [Ca ]i (Toescu, 2005). It
may be that the inability of aged neurons to respond to the increased need for ATP to handle
2+

large fluxes in [Ca ]i is what makes them more sensitive to MeHg.
We also compared the time-to-peak fluorescence of striatal MSNs and cerebellar granule
cells to 20μM and 100μM MeHg and found no differences in response. Cerebellar granule cells
are a sensitive target of MeHg-induced cytotoxicity (Hunter and Russell, 1954). It was surprising
to find no differences in the response time between the MSN and granule cells. This indicates
that MSNs are a sensitive target of MeHg-induced cytotoxicity. Although we found no
differences in response time, more comparative studies need to be completed to fully understand
if MSNs are also a sensitive target or if this was just a circumstance of the system used in this
2+

study. We monitored changes in Ca

2+

every 3min or 5min using the Ca

sensitive fluorophore

2+

Fluo4. This method is effective to determining spatial changes in Ca , however we are not able
2+

to continuously monitor local changes in Ca
2+

changes in Ca

in a cell by cell manner. Continuously monitoring

throughout the entire treatment period may have parsed out temporal changes in

71

2+

Ca , and differences may have been found between brain regions. However, based on this study
cerebellar granule cells and striatal MSNs are equally sensitive to 20μM and 100μM MeHg.
This study was the first to demonstrate an age-dependent effect on neuronal susceptibility
2+

to MeHg-induced Ca

-dysregulation. We also compared the Ca2+ responses of striatal MSNs

and cerebellar granule cells to acute MeHg treatment and found them to be equally sensitive to
2+

MeHg-induced Ca -dysregulation. We have demonstrated the need to characterize responses of
2+

not only young animals but aged animals to toxicants that disrupt Ca -regulation to make
decisions about safe exposure levels in all ages of the population. These results imply that
interaction of the natural aging process with exposure to environmental toxicants is a potential
concern for public health, particularly for a society with increasing life-span.

72

CHAPTER 4
CHRONIC METHYLMERCURY TREATMENT ALTERS STRIATAL CALCIUM
REGULATION AND SYNAPTOSOME MITOCHONDRIAL FUNCTION

73

A) Abstract
The acute effects of MeHg have been investigated extensively however the effects of
chronic low-dose exposure have not been characterized. The data presented in this chapter are
part of a comprehensive study of the behavioral, molecular, and neurophysiological effects of
chronic MeHg exposure in malb Balb/c mice. The ability of an L-type VGCC blocker isradipine
(Isr), to prevent or delay MeHg-induced toxicity was also investigated. The objective of this
study was to investigate the effects of chronic low-dose MeHg treatment on mitochondrial
2+

function of NSDA neuron synaptosomes and Ca

regulation of striatal MSNs and whether co-

treatment with the VGCC antagonist, Isr, would prevent MeHg-induced neuronal dysfunction.
Male Balb/c mice were given free access to 0 or 6.25 ppm Hg as MeHg in their drinking water
and 0 or 2 ppm isradipine in their feed. Treatments did not alter mouse weight and caused no
overt toxicity. Twelve mo MeHg treatment resulted in a small decrease in striatal synaptosome
2+

mitochondrial ATP production and a significantly faster [Ca ]i-increase in response to AMPA
receptor activation. Co-treatment with the VGCC antagonist Isr resulted in a significant increase
in ATP production compared to Isr or MeHg treatment alone. Together these results suggest a
role for MeHg-induced disruption of striatal synaptosome mitochondrial function and MSN
AMPA receptor function. These results will be integrated with other results from this project to
provide a more comprehensive understanding of the effects of long-term exposure to low-dose
MeHg on neuronal function.

74

B) Introduction
Previous chapters established that MeHg is an environmental neurotoxicant of
contemporary concern, due to the exposure to low levels of MeHg throughout ones lifetime. Our
current understanding of MeHg-induced toxicity has largely been derived from acute treatment
studies (for review see: Atchison and Hare, 1994; Limke et al., 2004; Farina et al., 2011). While
these studies have discovered many of the targets of MeHg following acute treatment we do not
know if the same series of events occur following chronic exposure to MeHg. Therefore, the
effects of chronic exposure to MeHg are not well understood. A limited number of studies have
investigated changes in neuronal function following prolonged exposure to MeHg (Bourdineaud
et al, 2008; Huang et al., 2008; Cambier et al., 2009; Sokolowski et al., 2011). These studies
support previous work demonstrating that long-term exposure to MeHg decreases mitochondrial
respiration resulting in decreased ATP production (Bourdineaud et al., 2008; Cambier et al.,
2009) as well as induces oxidative stress (Huang et al., 2008) and mitochondrial dependent
apoptosis (Sokolowski et al., 2011).
In the adult brain SNc neurons are spontaneously active, firing actions potentials in the
absence of synaptic input. The generation of action potentials is reliant on the opening of Cav1.3
2+

channels to allow Ca

+

influx as opposed to the usual reliance on Na entry (Chan et al., 2009).

The autonomous firing of action potentials in SNc neurons results in repeated fluctuations of
2+

[Ca ]i which is then buffered by the SER or mitochondria or removed from the cytosol via the
+

2+

plasma membrane Na /Ca

2+

exchanger or Ca -ATPase. MeHg disrupts L-type VGCC function
2+

(shown in Chapter 2; Peng and Atchison, 2002; Hajela et al., 2003), [Ca ]i regulation (Marty
2+

2+

and Atchison, 1997) and Ca m and SER Ca

regulation (Levesque and Atchison, 1991; Hare
75

2+

and Atchison, 1995; Limke and Atchison 2002). The reliance of SNc neurons on Ca
2+

L-type Ca

2+

channels and resultant continuous stress on Ca

entry via

buffering organelles SNc neurons

may make them a sensitive target of MeHg toxicity.
2+

Striatal MSNs are also potential sensitive targets for MeHg-induced Ca -dysregulation.
They serve an important role in modulating the activity of the basal ganglia and alterations in the
function of VGCCs or glutamate receptors, specifically AMPA and NMDA, could significantly
alter the output of the basal ganglia. MeHg-induced disruption of VGCC function was discussed
in the previous chapter. It also modifies glutamate receptor function and expression. The
overstimulation of glutamate receptors has been implicated in MeHg-induced excitotoxicity
(Aschner et al., 2000; Liu et al., 2012). Exposure to MeHg sensitizes AMPA receptors resulting
in increased Ca2+ influx upon receptor activation (Johnson et al., 2011). Antagonism of NMDA
receptors protects primary motor (Ramanathan and Atchison, 2011) and cortical neurons (Xu et
2+

al., 2013) as well as SH-SY5Y (Petroni et al., 2011) cells from MeHg-induced Ca

dysregulation and cell death. A reduction of NMDA receptor expression in mouse cortical
neurons (Xu et al., 2013) and in the basal ganglia, cerebellum, brain stem and occipital cortex of
wild mink (Basu et al., 2007) was associated with exposure to MeHg. MeHg clearly disrupts
VGCC and glutamate receptor function. We expect that MeHg will interact with VGCCs to
2+

reduce KCl-induced Ca

influx; this will be prevented by co-treatment with Isr. Chronic MeHg
2+

treatment will also alter AMPA and NMDA receptor function causing increased Ca
upon channel activation.

76

influx

The goal of the work described here was to expose mice to an environmentally relevant
2+

[MeHg] and investigate MeHg-induced changes in Ca

regulation, neurotransmission,

mitochondrial function, and gene expression in susceptible brain regions. We also sought to
determine whether co-treatment with Isr prevents or delays MeHg-induced toxicity. This was a
highly collaborative study performed in our laboratory and involving multiple measures. The
data presented in this chapter are solely the work investigating the effects of chronic MeHg
2+

treatment on striatal synaptosome mitochondrial function and striatal Ca

regulation. We report

that chronic exposure to MeHg sensitizes AMPA receptors on MSNs resulting in a faster
response time. We also found striatal synaptosomal mitochondria increased ATP production in
response to chronic exposure to Isr and MeHg.

77

C) Materials and methods
Chemicals and solutions
Methylmercuric (II) chloride was obtained from Aldrich Chemical (Milwaukee, WI).
Fura-2AM, TPEN, AMPA, NMDA, and pluronic acid were obtained from Sigma-Aldrich
(Milwaukee, WI). Probenecid was obtained from Molecular Probes (Eugene, OR). Oligomycin,
FCCP, antimycin A, and rotenone were obtained from Seahorse Biosciences (North Billerica,
MA). Percoll was obtained from GE Healthcare (Pittsburg, PA).
Animal care and treatments
All animal procedures complied with the National Institute of Health guidelines on
animal care and are approved by Michigan State University Institutional Animal Use and Care
Committee. Male Balb/c mice (Harlan, Fredrick, MD) were utilized for this study. The use of
only male mice simplified the study; gender-specific differences between adult-onset
neurotoxicity are currently unknown. Animals arrived at 8wks of age and were allowed to
acclimate to their new environment for 1wk. Mice were housed in pairs and fed standard feed
(Harland, Teklab 18% Protein Global Rodent Diet). They were randomly assigned a treatment
group; Con/Con (No isradipine/No MeHg), Con/MeHg (No isradipine/6.25ppm MeHg), Isr/Con
(2ppm Isr/No MeHg), or Isr/MeHg (2ppm Isr/6.25ppm MeHg). MeHg and Isr treatments started
when animals were 12 wks old. Mice had free access to 6.25ppm MeHg in their drinking water
and food pellets, which contained 0 or 2ppm Isr for a period of 6 or 12mo. Isr was purchased
from VWR (Chicago, IL) and sent to Harlan to be mixed with standard feed. An inert yellow dye
was added to the Isr diet to distinguish it from the standard feed. Animal cages were coded with
colors to blind experiments. All mice were monitored 7 days a week. Mice were weighed and
their extent of hind limb cross was assessed 3 times a week (M-W-F). The mean body weight

78

and hind limb cross score per week was determined and then averaged with all mice within the
same treatment group. The extent of hind limb cross was assigned a score based on severity. A
score of 0, 1, or 2 were assigned to no cross, partial cross, and complete cross, respectively. The
animal’s ability to remain on an accelerating rotarod was tested 3 times a week (T-W-Th) every
4wks for the first 6mo of treatment and then every 2wks for the duration of the treatment. The
rotarod was programmed as a continuously accelerating ramp: 1 - 40 rpm in 120 sec. The latency
to fall for each mouse was recorded and the best time per week was selected for each mouse and
then averaged for each treatment group.
Tissue collection
Upon completion of each treatment period, mice were anaesthetized with isoflurane. To
ensure that mice were under anesthesia their blink reflex was checked, and when they no longer
blinked they were determined sufficiently unconscious for intra-cardiac perfusion. The chest
cavity was opened and 1mL of trunk blood was collected for Hg analysis. Then the mouse was
perfused with ice-cold slicing solution, which contained (in mM): 222.5 sucrose, 2.5 KCl, 1.25
KH2PO4, 26 NaHCO3, 25 D-glucose, 5 MgCl2, and 0.5 CaCl2 (pH 7.3-7.4 upon aeration with
95% O2/5% CO2). The mouse was then decapitated and the whole brain removed and placed in
ice-cold slicing solution. Blood samples were stored at -80°C until analysis. Total Hg
concentration was determined using cold-vapor atomic absorption spectrometry by the
Diagnostic Center for Population and Animal Health at Michigan State University.
Striatal synaptosome isolation
Striatal synaptosome isolation protocol was adapted from Dunkley et al. (2008). The
striatum was dissected from the whole brain and placed in cold mitochondrial isolation buffer
(MIB). MIB contained in mM: 300 sucrose, 10 HEPES, 1 EGTA, pH 7.4. The striatum was
79

homogenized in 2mL of MIB using a Teflon homogenizer. Following homogenization, the tissue
was placed in a centrifuge tube and 6mL of MIB was added bringing the volume to 8mL. The
homogenized tissue was spun at 20,890 x g for 10 min at 4°C using a Sorval SM-24 rotor. The
supernatant was removed and pellet resuspended in 3mL of 3% (v/v, Percoll/Percoll Buffer)
Percoll. Percoll buffer contained (in mM): 300 sucrose, 10 HEPES, 1 EGTA, pH 7.4. This was
carefully added on top of a Percoll gradient of 10% (v/v) and 23% (v/v) Percoll. The gradient
was centrifuged at 31,650 x g for 10m at 4°C. This resulted in the production of 4 bands. Band 3,
which contained the synaptosomes, was removed and added to 8mL of MIB and then spun at
17,800 x g for 10m at 4°C. The supernatant was removed and the pellet was resuspended in
sucrose buffer. The synaptosomes were placed in a 2ml tube and spun using a microcentrifuge at
10,000 x g for 5m at 4°C. The supernatant was removed and protein content determined using
the Bradford Assay.
Mitochondrial function analysis
Mitochondrial function was assessed using the extracellular flux analyzer (XF-24)
(Seahorse Biosciences, North Billerica, MA). The XF-24 simultaneously measures extracellular
flux of oxygen (oxygen consumption rate (OCR)) and protons (extracellular acidification rate
(ECAR)) of striatal synaptosomes. The synaptosome plating density and chemical concentrations
were optimized before starting the experiments. Synaptosomes (10μg/well) were plated in XF-24
plates using ionic buffer which contained (in mM): 20 HEPES, 10 D-glucose, 1.2 Na2HPO4, 1
MgCl2, 5 NaHCO3, 5 KCl, 140 NaCl, pH 7.4. In order to assure that synaptosomes remained
attached to the plate throughout the duration of the mitochondrial function assay the plate was
centrifuged at 3,400 x g for 1h at 4°C. Ionic buffer was then removed and replaced with 750μL

80

incubation buffer which contained: 3.5mM KCl, 120mM NaCl, 1.3mM CaCl2, 0.4mM KH2PO4,
1.2mM Na2SO4, 2mM MgSO4, 15mM D-glucose, 10μM pyruvate, 4mg/ml bovine serum
albumin, pH 7.4. The plate was then placed in a CO2 free incubator at 37°C for 10m prior to the
start of assay. The plate was then placed in the XF-24 and mitochondrial function assessed. The
sequential application of 1μM oligomycin, 2μM FCCP, 1μM rotenone and 1μM antimycin A
metabolically perturbs mitochondrial function. Basal respiration, ATP production, maximum
respiration and spare capacity can be subsequently determined, respectively. This is
demonstrated in Figure 1.4. Oligomycin inhibits ATP synthase resulting in a reduction of
mitochondrial OCR by the fraction used to produce ATP. FCCP is an oxidative phosphorylation
uncoupling agent and forces the mitochondria to reach their maximum respiration rate. The
difference between maximum respiration and ATP production is considered the spare
mitochondrial capacity. Antimycin A and rotenone inhibit complexes I and III respectively,
which inhibits all mitochondrial respiration, allowing for determination of the small fraction of
non-mitochondrial respiration. Subtraction of the non-mitochondrial respiration from basal,
maximum, spare, and ATP production allows for a more accurate calculation of mitochondrial
respiration. Data are expressed as OCR (pMol/min).
Striatal slice Preparation
Slices were prepared in the same manner as described in Chapter 3. The forebrain was
glued on the cutting stage and placed in a chamber containing oxygenated sucrose-based slicing
solution. Coronal slices (200μm) containing the striatum were collected. Slicing solution
contained (in mM): 222.5 sucrose, 2.5 KCl, 1.25 KH2PO4, 26 NaHCO3, 25 D-glucose, 5 MgCl2,
and 0.5 CaCl2 (pH 7.3-7.4 upon aeration with 95% O2/5% CO2). Following slicing brain slices
81

were allowed to recover in oxygenated alternative solution for 30 min at 37°C. Alternative ACSF
contains the following changes in salt composition (in mM): 110 sucrose, 62.5 NaCl, and 20 Dglucose. After the 30min incubation in alternative solution slices were placed in ACSF until used
for an experiment. ACSF contains the following changes in salt composition (in mM): 125 NaCl,
1 MgCl2, and 2 CaCl2.
High speed calcium imaging
Images were collected using an inverted Nikon Eclipse TE 2000-U microscope coupled
to a Polychrome V light source (Till Photonics) and a high-speed electron multiplying CCD
camera (Ixon EM+, Andor, South Windsor, CT). Slices were loaded in 2mL of ACSF with
10μM Fura-2AM, 2.5mM probenecid, 5μM TPEN and 0.002% (v/v) pluronic acid for 45m in a
5% CO2, 37°C incubator. Probenecid was added to the loading solution to inhibit organic-anion
transporters, which can extrude dye. Pluronic acid was added to facilitate Fura-2AM entry into
2+

cells. MeHg treatment alters intracellular [Zn ] (Denny and Atchison, 1994) which can bind to
2+

fura-2 and skew Ca

2+

measurements. TPEN was added to the loading solution to chelate Zn

2+

prior to initiating Ca

+

recording. Following incubation, slices were washed with Mg -free

+

ACSF for 10m. Mg -free ACSF had all of the same components of ACSF except with no
2+

MgCl2. Fura-2 is a Ca

sensitive fluorophore which we utilized for ratiometric microscopy.

Fura-2 was excited at 340nm and 380nm (bound vs. unbound fura-2) and emits at 515nm.
Following the wash, regions of interest were selected in the plane of view and treatments began.
Slices were exposed to 25μM AMPA, 50μM NMDA + 10μM glycine and 40mM KCl. Each
treatment lasted for 2min followed by 8min of ACSF. Agonist concentrations were selected

82

based on previous work completed in our laboratory (Johnson et al., 2011). Fura-2 fluorescence
was continuously monitored. Image series were exported and then processed using Image J. In
Image J, background was subtracted using a 50 pixel rolling ball subtraction, and images were
registered to reduce movement between images. Regions of interest were then selected.
Ratiometric values were produced from individual 340nm and 380nm values. Only ratiometric
traces with a value less than 1 were used for data analysis. Ratiometric values greater than one
indicate a dead or damaged cell. All data are represented as relative fluorescence (fluorescence at
time 0/fluorescence at time x). Time-to-peak fluorescence was determined for each slice (n=1)
and then averaged to determine a mean time-to-peak for each treatment group.
Statistics
Statistical analysis of all behavior data was done using a Two-Way ANOVA followed by
Bonferonni post-test, p<0.05. All other comparisons were made using One-Way ANOVA
followed by Tukey-Kramer multiple comparisons post-hoc test, p<0.05.

83

Co n /M eHg

Co n /Co n

Weight (g)

A . 35

Isr/Co n

Isr/M eHg

6mo

30

25

20 0

5

10

15

20

25

MeHg Treatment (weeks )

Weight (g)

B. 35

12mo

30
25
20

0

5

10 15

20 25

30 35

40 45

50

MeHg Treatment (weeks )
Figure 4.1. Six or twelve month MeHg or Isr/MeHg treatments did not alter mouse weight.
Male balb/c mice were treated with 0ppm Isr/0ppm MeHg (Con/Con), 0ppm Isr/6.25ppm MeHg
(Con/MeHg), 2ppm Isr/0ppm MeHg (Isr/Con), 2ppm Isr/6.25ppm MeHg (Isr/MeHg) for 6 or
12mo. Throughout the treatment period mice were weighed three times a week and their mean
weight per week was determined. (A) Six or (B) twelve month treatments did not alter mouse
weight. Treatment and time had a significant effect on mouse weight following 6 and 12mo
treatments (Two-Way ANOVA, Bonferroni post-test, p<0.05) (n≥12). Data are expressed as
mean ± SEM.
84

Co n /Co n

Co n /M eHg

L atency to Fall (s)

A . 60

Isr/M eHg

Isr/Co n

6mo

53
46
39
32
25

0

4

8

12

16

20

24

M eHg Treatment (weeks )

L atency to Fall (s)

B. 60

12mo

53
46
39
32
25

0

20

30

40

50

M eHg Treatment (weeks )
Figure 4.2. Six or twelve month MeHg or Isr/MeHg treatments did not alter mouse
performance on the rotarod. Male balb/c mice were treated with 0ppm Isr/0ppm MeHg
(Con/Con), 0ppm Isr/6.25ppm MeHg (Con/MeHg), 2ppm Isr/0ppm MeHg (Isr/Con), 2ppm
85

(Figure 4.2 cont’d) Isr/6.25ppm MeHg (Isr/MeHg) for 6 or 12mo. During the first 6mo of
treatment mouse ability to remain on an accelerating rotarod was tested every 4wks and then
every 2wks for the duration of the treatment. (A) Six or (B) twelve month treatments did not alter
mouse ability to remain on an accelerating rotarod. Treatment and time had a significant effect
on mouse ability to remain on the accelerating rotorod following 6 and 12mo treatments (TwoWay ANOVA, Bonferroni post-test, p<0.05) (n≥12). Data are expressed as mean ± SEM.

86

Co n /Co n

Co n /M eHg

Isr/M eHg

Isr/Co n

A . 1.0
L imb Cross T es t

6mo
0.5

#

*

#

0.0
-0.5

0

5

10

15

20

25

MeHg Treatment (weeks )

B. 1.5
L imb Cross T es t

12mo

0.8

#

#

0.2

-0.5

0

5

10 15 20 25 30 35 40 45 50
MeHg Treatment (weeks )

Figure 4.3. Six or twelve month MeHg or Isr/MeHg treatments did cause a consistent
pattern of altered mouse limb cross. Male balb/c mice were treated with 0ppm Isr/0ppm MeHg
(Con/Con), 0ppm Isr/6.25ppm MeHg (Con/MeHg), 2ppm Isr/0ppm MeHg (Isr/Con), 2ppm
Isr/6.25ppm MeHg (Isr/MeHg) for 6 or 12mo. Throughout the treatment period the extent of
87

(Figure 4.3 cont’d) mouse hind limb cross was scored three times a week and their mean score
per week was determined. The extent of hind limb cross was assigned a score based on severity.
A score of 0, 1, or 2 were assigned to no cross, partial cross, and complete cross, respectively.
(A) Mean limb cross test scores for six month treatment. Con/Con hind limb score is
significantly different than Con/MeHg hind limb score on week 25. Con/Con hind limb score is
significantly different than Isr/MeHg hind limb score during weeks 8 and 9. (B) Mean limb
cross test scores for twelve month treatment. Con/Con hind limb score is significantly different
than Isr/MeHg hind limb score during weeks 39 and 48. Treatment and time had a significant
effect on mouse hind limb score (Two-Way ANOVA, Bonferonni post-test, p<0.05). * =
Con/Con is significantly different than Con/MeHg. # = Con/Con is significantly different than
Isr/MeHg. Data are expressed as mean ± SEM.(n≥12)

88

D) Results
a. Animal health and behavior
The goal of this study was to mimic a lifetime exposure to low levels of MeHg in the
2+

diet and assess alterations in striatal synaptosome mitochondrial function and striatal MSN Ca
regulation. Our collaborators at Auburn University performed pilot studies to determine both

[MeHg] and [Isr]. Mouse weights were monitored to determine if the treatments altered mouse
growth or weight gain and no differences were found (Figure 4.1A,B). This indicates that
animals from all treatment groups were eating and drinking similar amounts regardless of their
treatment. Previous work has demonstrated that adult exposure to MeHg alters motor
coordination and hind limb strength as assessed by the animal’s ability to remain on a rotarod
and extent of hind limb cross (Kobayashi et al., 1981; Bellum et al., 2007; Huang et al., 2008;
Johnson et al., 2011). Six or twelve mo treatments did not alter mouse performance on the
accelerating rotarod (Figure 4.2A, B). Following the 6mo treatment there was no consistent
pattern of effect as assessed by the hind limb cross test. However significant differences were
found between Con/Con and Con/MeHg groups on week 25 and between Con/Con and
Isr/MeHg groups on weeks 8 and 9 (Figure 4.3A). Following the 12mo treatment there was no
consistent pattern of treatment effects. There were significant differences found between
Con/Con and Isr/MeHg on weeks 39 and 48 (Figure 4.3B). Taken together these data indicate
treatments caused no overt toxicity or behavioral deficits.
b. Trunk blood Hg concentration
Trunk blood Hg content was analyzed to determine if Con/MeHg and Isr/MeHg mice
accumulated Hg throughout the treatment period. Following the 6mo and 12mo treatment period

89

6mo

12

Hg (ppm)

9
6
4
1
-2

12

Co n /Co n Co n /MeHg

Isr/Co n

Isr/MeHg

12mo

Hg (ppm)

9
6
4
1
-2

Con/Con Con/MeHg

Isr/Con

Isr/MeHg

Figure 4.4. Six and twelve month MeHg treatment caused blood Hg accumulation. Male
balb/c mice were treated with 0ppm Isr/0ppm MeHg (Con/Con), 0ppm Isr/6.25ppm MeHg
(Con/MeHg), 2ppm Isr/0ppm MeHg (Isr/Con), 2ppm Isr/6.25ppm MeHg (Isr/MeHg) for 6 or
12mo. Following anaesthesia with isofluorane truck blood was collected and later analyzed for
Hg content. MeHg and Isr/MeHg caused significant Hg accumulation following (A) 6mo and (B)
12mo treatment (One-Way ANOVA, Tukey’s post-test, p<0.05) (n=4).
90

Con/MeHg mice had a trunk blood Hg level of 7.5ppm and 7.6ppm, respectively. Similarly, the
Isr/MeHg mice had a trunk blood Hg level of 6.6ppm and 8.7ppm following the 6 and 12mo
treatment period (Figure 4.4A,B). Trunk blood Hg concentrations from mice treated with MeHg
in their drinking water have been found to be similar with levels of Hg in the brainstem (Johnson
et al., 2011). A similar study treating mice for 7 wks with MeHg via oral gavage demonstrated
that trunk blood Hg levels were similar to those found in the cerebral cortex, cerebellar cortex,
and brainstem (Huang et al., 2008). Based on these studies we predict that similar levels of Hg
would be found in the striatum.
c. Mitochondrial function
Chronic MeHg treatment had no effect on striatal synaptosome mitochondrial
bioenergetics following 6 or 12mo treatment (Figure 4.5). Addition of oligomycin, FCCP, and
antimycin A/rotenone allowed for the determination of mitochondrial basal respiration, ATP
production, maximum respiration, and spare capacity. Six-mo treatment did change any of these
measures of mitochondrial function (Figure 4.6A-D). This length of MeHg treatment may not
have been long enough to induce mitochondrial dysfunction or striatal synaptosome
mitochondria are not a sensitive target of MeHg-induced dysfunction. Similarly following the
12mo treatment no changes occured between groups in their basal respiration, maximum
respiration, or spare capacity (Figure 4.7A-C). However, the 12mo Con/MeHg treatment caused
a small decrease in ATP production while the Isr/MeHg treatment caused a significant increase
in ATP production compared to the Isr/Con and Con/MeHg groups (Figure 4.8A). To determine
if this change in ATP production was specific to striatal synaptosome the mitochondrial function
of cortical synaptosomes was also measured. The same increase in ATP production was seen in
the Isr/MeHg group (Figure 4.8B) indicating that it was not a region-specific effect. MeHg

91

Co n /Co n

OCR (pMoles/min)

A.

Co n /M eHg

800

Isr/Co n

O ligomyc in FC C P

Isr/M eHg

A ntimyc inA /
R ote none

640
480
320
160
0

0

18

36

54

72

90

Time (min)

OCR (pMoles/min)

B. 800

A ntimyc inA /
FC C P R ote none

Oligomycin

640
480
320
160
0

0

18

36

54

72

90

Time (min)
Figure 4.5. MeHg or Isr/MeHg treatment did not alter mitochondrial bioenergetics.
Mitochondrial bioenergetics of striatal synaptosomes was determined following treatment period.
Oxygen consumption rate (OCR, pMoles/min) before and after the addition of oligomycin
(1µM), Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (2µM), antimycin A
(1µM), and rotenone (1µM). No differences were detected in OCR between treatment groups
following (A) 6mo or (B) 12mo treatment period. Data are means ± SEM (n≥4).
92

Basal Respiration

OCR (pMol/min)

A . 300
240
180
120
60
0

Co n /Co n

OCR (pMol/min)

B. 80
70
60
50
40
30
20
10
0

Co n /M eHg

Isr/Co n

Isr/M eHg

ATP Production

Co n /Co n

Co n /M eHg

Isr/Co n

Isr/M eHg

Figure 4.6. Mitochondrial function was unaltered following 6mo MeHg or Isr/MeHg cotreatment. Quantification of mitochondrial basal respiration, ATP production, maximum
respiration and spare capacity striatal synaptosomes. MeHg or Isr/MeHg co-treatment caused no
significant differences in (A) basal respiration (B) ATP production (C) maximal respiration or
(D) spare capacity. Data are means ± SEM (n≥5).
93

(Figure 4.6 cont’d)

OCR (pMol/min)

C . 700

Maximum Res piration

600
500
400
300
200
100
0

Co n /Co n

OCR (pMol/min)

D . 400

Co n /M eHg

Isr/Co n

Isr/M eHg

Spare Capacity

300
200
100
0

Co n /Co n

Co n /M eHg

94

Isr/Co n

Isr/M eHg

OCR (pMol/min)

A . 400
300
200
100
0

B. 700
OCR (pMol/min)

Bas al Res piration

600

Co n /Co n Co n /M eHg Isr/Co n

Isr/M eHg

Maximum Res piration

500
400
300
200
100
0

Co n /Co n Co n /M eHg Isr/Co n

Isr/M eHg

Figure 4.7. Mitochondrial function was unaltered following 12mo MeHg or Isr/MeHg cotreatment. Quantification of striatal synaptosome mitochondrial basal respiration, maximum
respiration, and spare capacity. MeHg or Isr/MeHg co-treatment caused no significant
differences in (A) basal respiration (B) maximal respiration or (C) spare capacity. Data are
means ± SEM (n≥5).
95

(Figure 4.7 cont’d)

OCR (pMol/min)

C . 300

Spare Capacity

200
100
0

Co n /Co n Co n /M eHg Isr/Co n

96

Isr/M eHg

Striatal ATP Production

OCR (pMol/min)

A . 125

*

100

*

75
50
25
0

Co n /Co n

Co n /M eHg

Isr/Co n

Isr/M eHg

Cortical ATP Production

*

OCR (pMol/min)

B. 125

*

100
75
50
25
0

Co n /Co n Co n /M eHg

Isr/Co n

Isr/M eHg

Figure 4.8. Twelve month Isr/MeHg treatment caused a significant increase in ATP
production in both striatal and cortical synaptosome mitochondria. Quantification of (A)
striatal and (B) cortical synaptosome ATP production. 12mo Isr/MeHg treatment caused a
significant increase in ATP production compared to both Isr/Con and Con/MeHg treatment
groups. * = significant difference (One-Way ANOVA, Tukey’s post-test, p<0.05)(n≥5). Data are
means ± SEM.
97

treatment may induce mitochondrial dysfunction in specific cell types as seen in the decrease in
ATP production of striatal synaptosomes following 12mo treatment. However, when animals are
co-treated with Isr the mitochondria are able to meet the increased ATP demand that accounts for
the increased ATP production.
2+

d. High speed [Ca

] i imaging
2+

To determine if chronic MeHg treatment altered Ca

entry pathways, striatal slices from

12mo treated animals were exposed to high [KCl] (40mM) and the glutamate receptor agonists,
NMDA and AMPA. Slices from all treatment groups responded similarly to 40mM KCl; there
was no difference between the fura-2 response, time-to-peak, or amplitude of response (Figure
4.9A-C). These data are in contrast to what was seen in Chapter 2 in which prolonged MeHg
2+

treatment inhibited KCl-induced Ca

influx in SH-SY5Y cells. This may be due to a

compensatory mechanism that increases VGCC expression in striatal neurons in order to account
for blocked VGCCs. The striatal response to NMDA was variable between treatments groups
and amongst striatal slices within the same treatment group. Many times slices did not respond to
NMDA, which resulted in an ‘n’ value less than 3. No differences were found between the timeto-peak or amplitude of response to NMDA (Figure 4.10A-C). The response of striatal slices to
AMPA was also recorded (Figure 4.11A). The Isr/Con and Isr/MeHg groups appeared to respond
in a biphasic manner with a first phase being a fast low amplitude response followed by a more
prolonged high amplitude response. All treatment groups responded to AMPA and had a
2+

sustained increase in [Ca ]i that did not return to baseline. AMPA receptors desensitize quickly,
2+

2+

which should result in an increase in [Ca ]i followed by a decrease in [Ca ]i resulting in a
return to baseline. This did not occur in any treatment group. The Con/Con group began to return
98

to baseline, however that was never reached. Work done by Tseng et al. (2007) demonstrated
2+

that a sustained depolarization in striatal MSNs could be mediated by Ca

influx through

NMDA receptors. Taken together this suggests that application of AMPA is sufficient to
depolarize the MSN membrane and cause activation of NMDA receptors resulting in a sustained
2+

increase in [Ca ]i. There was no difference between treatment groups in their amplitude of
response (Figure 4.11C). However, the time-to-peak fluorescence of the Con/MeHg group was
significantly faster than the Con/Con group (Figure 4.11B). This suggests that 12mo MeHg
treatment causes an increase in AMPA receptor expression or causes a sensitization of AMPA
receptors to AMPA resulting in a faster response time.

99

Co n /Co n

Co n /M eHg

Isr/Co n

Isr/M eHg

Rel. fura-2 R(F 340 /F 380 )

A.
1.75
1.55
1.35
1.15
0.95
0.75

0

100

200

300

400

500

Time (s )
Figure 4.9. Twelve month MeHg or Isr/MeHg did not alter striatal response to KCl2+
induced [Ca ]i increase. Striatal slices were loaded with fura-2AM and then exposed to 40mM
KCl for 2min followed by 8min of ACSF. Fura-2 fluorescence was continuously monitored.
Data are presented as relative fura-2 fluorescence (Fluorescence at time 0/fluorescence at time
2+
x). An increase in fura-2 fluorescence corresponds to an increase in [Ca ]i. Time-to-peak
2+

fluorescence was determine for each treatment group. (A) Striatal [Ca ]i response to 40mM
KCl.(B) Time-to-peak and (C) amplitude of response was quantified. No Differences were
detected between treatment groups. Data are means ± SEM (n≥3).

100

(Figure 4.9 cont’d)

Co n /Co n

Co n /M eHg

T ime-to-Peak (s )

B. 500
400
300
200
100
0

Amplitude (Arbitrary Units )

C.
0.75
0.60
0.45
0.30
0.15
0.00

101

Isr/Co n

Isr/M eHg

Co n /M eHg

Co n /Co n

Rel. fura-2 R(F 340 /F 380 )

A.

Isr/Co n

Isr/M eHg

2.00
1.60
1.20
0.80
0.40
0.00

0

100 200 300 400 500
Time (s )

Figure 4.10. Twelve month MeHg or Isr/MeHg did not alter striatal response to NMDA2+

induced [Ca ]i increase. Striatal slices were loaded with fura-2AM and then exposed to 50μM
NMDA and 10μM glycine for 2min followed by 8min of ACSF. Fura-2 fluorescence was
continuously monitored. Data are presented as relative fura-2 fluorescence (Fluorescence at time
0/fluorescence at time x). An increase in fura-2 fluorescence corresponds to an increase in
2+

2+

[Ca ]i. Time-to-peak fluorescence was determine for each treatment group. (A) Striatal [Ca ]i
response to 50μM NMDA.(B) Time-to-peak and (C) amplitude of response was quantified.
Many slices did not respond to NMDA application resulting in an n value less than 3.

102

(Figure 4.10 cont’d)

Co n /Co n

Co n /M eHg

T ime-to-Peak (s )

B. 600
500
400
300
200
100
0

Amplitude (Arbitrary Units )

C.
1.00
0.80
0.60
0.40
0.20
0.00

103

Isr/Co n

Isr/M eHg

Rel. fura-2 R(F 340 /F 380 )

A.

Co n /Co n

Co n /M eHg

Isr/Co n

Isr/M eHg

3.00
2.46
1.92
1.38
0.84
0.30

0

100

200

300

400

500

Time (s)

Figure 4.11. Twelve month MeHg treatment caused a significanltly decreased time-to-peak
fluorescence following application of AMPA. Striatal slices were loaded with fura-2AM and
then exposed to 25μM AMPA for 2min followed by 8min of ACSF. Fura-2 fluorescence was
continuously monitored. Data are presented as relative fura-2 fluorescence (Fluorescence at time
0/fluorescence at time x). An increase in fura-2 fluorescence corresponds to an increase in
2+

2+

[Ca ]i. Time-to-peak fluorescence was determine for each treatment group. (A) Striatal [Ca ]i
response to AMPA (B) 12mo MeHg treatment caused a significantly faster time-to-peak
fluorescence in response to AMPA (p<0.05, unpaired student t-test, n≥3). (C) Amplitude of
response was not affected by treatment. Data are means ± SEM (n≥3).

104

(Figure 4.11 cont’d)

Co n /Co n

Co n /M eHg

T ime-to-Peak (s )

B. 400
300
200

*

100
0

Amplitude (Arbitrary Units )

C.
1.50
1.20
0.90
0.60
0.30
0.00

105

Isr/Co n

Isr/M eHg

D) Discussion
The objective of this study was to determine whether chronic adult-onset exposure to
2+

low-dose MeHg would alter mitochondrial function or Ca

regulation and if co-treatment with a

L-type VGCC antagonist would prevent or delay those effects. Studies focused on striatal
2+

synaptosome mitochondrial function and striatal MSN Ca

regulation. The NSDA cell bodies

lie in the SNc and project to the striatum where they release DA. They are autonomously active
2+

and firing of action potentials is due to Ca

influx through Cav1.3 channels (Bonci et al, 1998;
2+

Ping and Shepard, 1996; Puopolo et al., 2007). Due to the continual fluctuations in [Ca ]i we
hypothesized that chronic MeHg treatment would result in decreased mitochondrial function and
Isr would prevent or delay those effects. In Chapter 3 we established that striatal MSNs are
2+

susceptible to MeHg-induced Ca
2+

exposure would disrupt Ca

dysregulation. Therefore we hypothesized that chronic MeHg

regulation and Isr co-treatment would prevent effects associated

with VGCCs.
Results of this study are consistent with the following conclusions. First, chronic
exposure to Isr/MeHg treatment causes increased ATP demand in both striatal and cortical
2+

synaptosomes, an effect consistent with MeHg-induced [Ca ]i increase. Second, chronic
2+

exposure to MeHg results in a faster time-to-peak of [Ca ]i in response to AMPA, indicative of
sensitized or increased expression of AMPA receptors.
This was the first study of its kind to expose adult mice to an environmentally relevant
dose of MeHg for 12mo and investigate the potential effects in a wide range of tissues and end
points. This was a highly collaborative study with researchers investigating the effects of MeHg
106

2+

in a variety of tissues on Ca

regulation, neurotransmission, and ion-channel expression. The

work presented in this chapter was a small piece to a larger puzzle that will be assembled to
provide a better understanding of a lifetime exposure to a low dose of MeHg on neuronal
function. Previous studies have investigated the long-term effects of an environmentally relevant
dose of MeHg on oxidative stress, mitochondrial function, and cell death (Huang et al., 2008;
Bourdineaud et al., 2008; Cambier et al., 2009; Bourdineaud et al., 2011; Sokolowski et al.,
2011), however these studies lack the integrative nature of ours and do not provide an overall
understanding of what occurs following a long-term exposure to MeHg.
This study demonstrates that following a 6mo exposure MeHg in adult mice there were
no significant differences in striatal synaptosome mitochondrial function. However, there was a
slight change in ATP production that is worthy of discussion. ATP production of both Isr/Con
and Isr/MeHg groups were slightly lower than the non-Isr treated groups. This is consistent with
previous reports demonstrating that dihydropyridines slow the utilization of ATP (Sauter and
Rudin, 1987; Rudin and Sauter, 1989). These studies demonstrated that dihydropyridines slow
ATP consumption not by changing the rate of ATP production but by reducing its consumption
in a rat model of global ischemia (Rudin and Sauter, 1989).
In contrast to the 6mo treatment, 12mo MeHg treatment caused a small reduction in ATP
production in striatal synaptosomes that was not seen in cortical synaptosomes. Although the
reduction in ATP was not significant if the power of study had been increased we may have
found a significant difference. It is interesting that this decrease was seen solely in the striatal
synaptosomes and not in the cortical synaptosomes. This supports our hypothesis that SNc
neurons may be uniquely sensitive to MeHg-induced damage. Numerous studies have shown
acute exposure to MeHg causes mitochondrial dysfunction resulting in increased ROS (Oyama et

107

al., 1994; Yee and Choi, 1996; Polunas et al., 2011), loss of mitochondrial membrane potential
(Bondy and McKee, 1991; Hare and Atchison, 1992; Limke and Atchison, 2002) and a reduction
of ATP production (Sone et al., 1977; Verity et al., 1975). Studies, which used a low-dose and
long-term exposure, also found that MeHg caused a mitochondrial dependent increase in ROS
(Sokolowski et al., 2011; Mori et al., 2011), reduction in mitochondrial respiration (Bourdineaud
et al, 2008) and ATP production (Cambier et al., 2009). Our data demonstrate that an adult onset
low-dose 12mo exposure results in decreased ATP production. This reduction may be due to a
loss of the mitochondrial membrane potential, which drives ATP production (Hill et al., 2012) or
MeHg-induced damage to the electron transport chain (Yee and Choi, 1996; Mori et al., 2011)
causing a reduction in ATP production or a combination of the two. Experiments utilizing single
synaptosome fluorescent imaging (Choi et al., 2009) to measure mitochondrial membrane
potential could address these possibilities.
Twelve-month treatment with Isr/MeHg resulted in a significant increase in both striatal
and cortical synaptosome ATP production. Isr is a vasodilator and is known to increase cerebral
blood flow (Rudin and Sauter, 1989). The pharmacokinetics of Isr have also been shown to
change with age in humans due to reduced liver function resulting in increased bioavailability,
prolonged elimination half life, and increased peak plasma concentrations (Schachter, 1991). No
adjustments were made in [MeHg] or [Isr] to accommodate potential changes in metabolism in
the mice due to advanced age by the end of the study (15mo). Thus, Isr treatment may have
increased cerebral blood flow throughout the duration of the study resulting in increased
exposure of all brain regions to MeHg. This might lead to higher neuronal stress due to
2+

elevations in [Ca ]i increasing ATP demand. Therefore, the Isr/MeHg synaptosomal
mitochondria increased ATP production to account for increased demand. This theory could be

108

tested by analyzing Hg content in frozen brain samples from a representative sample of mice
from each treatment group.
In an attempt to investigate that effects of chronic MeHg treatment on the function of
2+

2+

VGCCs and Ca -permeable glutamate receptors, Ca

imaging experiments were performed in

striatal slices. As discussed earlier, there are 2 major subsets of MSNs which cannot be
differentiated based on morphology. The striatonigral MSNs predominantly express the D1
receptor, which is positively coupled to adenylyl cyclase, and activation causes an increase in
2+

[Ca ]i (Surmeier et al., 2007). The striatopallidal MSNs mainly express the D2 receptor and are
coupled to Gi/o which inhibits adenylyl cyclase and reduces the opening of VGCCs (Surmeier et
al., 2007). We were unable to identify if we were collecting data from striatonigral or
2+

striatopallidal MSNs in these Ca

imaging experiments. Regardless, we found that 12mo
2+

exposure to MeHg resulted in a faster time-to-peak of [Ca ]i in response to AMPA. This could
be due to sensitization of the AMPA receptor to agonists, or increased AMPA receptor
expression. A similar response was seen in a study investigating the possible contribution of
MeHg to the onset of an amyotrophic lateral sclerosis- (ALS) like phenotype in genetically2+

susceptible mice. Intracellular Ca

fluorescence recordings in brainstem slices from mice
2+

exposed to MeHg for 120 days had enhanced Ca

influx in response to AMPA application

(Johnson et al., 2011). Therefore, chronic MeHg may sensitize the AMPA receptor by inducing a
2+

conformational change allowing more Ca

to enter the cell upon activation. However, the

alternative possibility is that chronic MeHg caused increased receptor expression. Activation of
the D1 receptor in striatonigral MSNs enhances the expression of both AMPA and NMDA
109

receptors (Surmeier et al., 2007). Exposure to MeHg induces DA release (Faro et al., 2007;
Dreiem et al., 2009) at SNc nerve terminals. Therefore chronic MeHg treatment may cause
persistent increases in DA release, which activate post-synaptic D1 receptors on MSNs and result
in increased AMPA receptor expression. Isr treatment inhibits DA release (Nakane et al., 1995)
and prevents the MeHg-induced change in AMPA receptor expression.
In conclusion, this study demonstrated that 12mo exposure to low-dose MeHg does not
significantly alter striatal synaptosome mitochondrial function. However co-treatment with the
VGCC antagonist Isr results in significantly increased ATP production consistent with MeHg2+

induced Ca -dysregulation causing an increased ATP demand. We also demonstrated that 12mo
exposure altered AMPA receptor response supporting a role for MeHg-induced alterations in
glutamate signaling. These results support a role for the interaction of MeHg with the basal
ganglia altering its function.

110

CHAPTER FIVE
SUMMARY AND CONCLUSIONS

111

SUMMARY AND CONCLUSIONS
This thesis presents the results of experiments designed to understand the effects of
2+

chronic exposure to low levels of MeHg on Ca

regulation and mitochondrial function. I knew
2+

from the literature that acute exposure to MeHg alters neuronal Ca i regulation (Komulainen
and Bondy, 1987; Denny and Atchison, 1994; Marty and Atchison, 1997; Edwards et al., 2005),
VGCC function (Shafer et al., 1990; Shafer and Atchison, 1991; Sirois and Atchison 2000; Peng
et al., 2002; Yuan et al., 2005), mitochondrial function (Levesque and Atchison, 1991; Limke
and Atchison, 2002), and induces cell death (Marty and Atchison, 1998; Edwards et al., 2005).
The central hypothesis guiding these studies was that MeHg toxicity is due to its dysregulation of
2+

the Ca

2+

homeostasis system, mainly by disrupting mitochondrial and Ca -permeable ion

channel function. Based on our in vitro experiments we found that prolonged exposure to lownanomolar MeHg causes a time- and subtype-dependent block of VGCCs, as well as increased
2+

resting [Ca ]i. This is shown in Figure 5.1. The in vivo experiments suggest that MeHg interacts
with the post-synaptic response to glutamate at MSN AMPA receptors. Following Isr/MeHg
treatment, mitochondrial ATP production was increased in striatal synaptosomes. This suggests
that chronic exposure to MeHg alters neuronal glutamatergic neurotransmission and ATP
demand. This is shown in Figure 5.2.
2+

In the first set of experiments, Ca i regulation and L- and N-type VGCC function in
differentiated SH-SY5Y cells were investigated following acute and prolonged MeHg exposure.
Overall this study demonstrated that differentiated SH-SY5Y cells respond to acute MeHg
exposure in the characteristic biphasic manner. This response has been demonstrated in

112

Figure 5.1. A schematic representation of the effects of prolonged exposure to MeHg in
differentiated SH-SY5Y cells. Exposure of differentiated SH-SY5Y cells with 20nM MeHg
2+
resulted in a time- and VGCC subtype-dependent block of depolarization-dependent [Ca ]i
2+

increase. MeHg blocked KCl-induced Ca

increase through L-type VGCCs after a 48h

2+

exposure and through both L- and N-type VGCCs after 72h exposure. The resting [Ca ]i was
also significantly increased following 72h MeHg exposure.

113

Figure 5.2. Schematic diagram of the effects of MeHg on the striatal circuit. MeHg acts at
2+
striatal MSNs to causes increases in [Ca ]i as well as alter mitochondrial ATP production. It has
also been shown to cause increase in both DA and glutamate release. These actions along with
2+
potential direct interactions with AMPA receptors results in altered post-synaptic Ca response
to AMPA receptor activation.

114

cerebellar granule cells (Marty and Atchison, 1997), cerebellar purkinje cells (Edwards et al.,
2005), spinal motor neurons (Ramanathan and Atchison, 2011), and NG108-15 cells (Hare and
Atchison, 1995). Also, treatment with L- and N-type VGCC antagonists delayed the onset of
phase 2 in differentiated SH-SY5Y cells. The ability to delay the onset of phase 1 or phase 2
with VGCC antagonists varies amongst cell types. The addition of GVIA or Nif delays the onset
of both phase 1 and phase 2 in primary cerebellar granule cells and primary motor neurons
(Marty and Atchison 1997; Ramanathan and Atchison, 2011) however they delay only phase 2 in
NG108-15 (Hare and Atchison, 1995) and SH-SY5Y cells. The difference in response seen may
be due to the fact that primary neurons are more sensitive to acute MeHg treatment than
immortalized cells lines. They also differ in their ion channel expression, specifically in
expression of the P/Q-type VGCC. Cerebellar granule cells and motor neurons express the P/Qtype channel (Westenbrook et al., 1998; Bawa and Abbott, 2008) while the NG108-15 and SHSY5Y cells express negligible levels of P/Q VGCCs (Lukyanetz., 1998; Sousa et al., 2013). We
hypothesize that MeHg uses VGCCs as a portal of entry into the cell and differences in VGCC
expression may account for the differences in protection afforded by VGCC antagonists.
After characterizing the acute response of differentiated SH-SY5Y cells to MeHg we
characterized the effects of prolonged exposure to MeHg on VGCC function. We demonstrated
that prolonged exposure to low nanomolar MeHg causes a time- and subtype-dependent block of
VGCCs. This is consistent with previous studies, which found that exposure to MeHg blocked
2+

depolarization-dependent Ca

entry into forebrain synaptosomes and differentiated PC12 cells

(Atchison et al., 1986; Shafer et al., 1990; Shafer and Atchison., 1991; Shafer et al., 2002). The
2+

exact mechanism by which MeHg blocks Ca

entry via VGCCs is not currently understood.

Shafer and Atchison (1991) suggested that due to its lipophilicity MeHg may diffuse across the
115

2+

plasma membrane and then access the pore of the Ca

channel from the cytoplasmic side and

2+

block the entry of Ca . Acute treatment studies found that the block was not dependent upon
the configuration of the channel (Shafer et al., 1990) and could occur when the channel was in
the resting, activated, or inactivated state. In our experiments we found that L-type VGCCs were
more sensitive to block by MeHg than N-type channels. This suggests that L-type channels have
a unique characteristic which make them more vulnerable to MeHg-induced block, such as the
amount of time it spends open or amino acid composition. Our prolonged exposure experiments
also demonstrated that when exposed the nanomolar MeHg, the VGCC block does not occur
immediately. This differs from the acute treatment studies, which used low micromolar MeHg
and observed a block within seconds (Shafer et al., 1990; Shafer and Atchison, 1991). When at
very low [MeHg], the kinetics of its actions are much slower demonstrating that 1) it takes
longer to enter into the cell; 2) there are other cellular targets that MeHg interacts with prior to
altering VGCC function; or 3) intracellular [MeHg] needs to reach a certain level before it
interacts with VGCCs. Regardless of the exact mechanism by which MeHg blocks VGCCs these
effects will impact neurotransmission, further altering neuronal function.
This study could be strengthened with the addition of a number of experiments.
2+

Decreased KCl-induced Ca

influx may have been due to an altered concentration gradient

across the plasma membrane, altered VGCC expression, or block of the channel by MeHg. We
2+

found that prolonged exposure to nanomolar MeHg causes an increase in the resting [Ca ]i. An
increase in intracellular cations would depolarize the membrane altering the electrogenic
gradient. Experiments utilizing fluorescent potentiometric probes or an electrophysiology current
clamp could test for changes in membrane potential in treated and untreated cells. Prolonged

116

exposure to MeHg may also alter the expression of VGCCs. Gene expression of both N- and Ltype VGCCs could be measured using real-time polymerase chain reaction (RT-PCR). The
protein expression could be measured using western blot or immunocytochemistry. Although
western blots are a more quantitative method, the use of immunocytochemistry may prove to be
more useful in this situation. Only those channels located at the cell membrane are functional, so
omission of the permeabilization agent when performing immunocytochemistry would allow for
staining of channels located only at the cell membrane. However the availability of an antibody
that recognizes an extracellular component of the α1 (pore-forming subunit of VGCCs) subunit
may be the limiting factor in the utilization of this technique.
The next step for this project would be to perform the same MeHg treatments but add
both earlier and later time points. By adding more time points we would gain a better
understanding of the temporal action of MeHg. Is it blocking L-type channels immediately or
does it occur after a set period of time? At later time points is the same profile of block seen or
does the cell compensate by inserting more VGCCs into the plasma membrane? Investigation of
mitochondrial function and the dynamics of DA release and at each time point would create a
comprehensive overview of MeHg-induced neuronal changes following prolonged exposure.
Acute MeHg exposure in vitro alters mitochondrial function (Limke and Atchison, 2002) and
DA dynamics (Tiernan et al., 2013). We expect to find that mitochondrial function is decreased
in a time-dependent manner resulting in the loss of ATP production. We expect to find that
prolonged MeHg exposure would initially cause increased DA release followed by a reduction or
complete block of DA release. The block of DA release would occur at the same time that the Ntype channels were blocked, as they are known to be involved in neurotransmitter release
(Catterall, 2011). Completion of these experiments would provide a comprehensive overview of

117

changes in Ca2+ homeostasis and neurotransmission following prolonged MeHg exposure. Once
we have a better understanding of the temporal changes occurring following prolonged treatment
in vitro, we can design in vivo experiments to test if these events occur in the whole animal.
Experiments described in Chapter 3 were aimed at investigating whether age alters the
susceptibility of striatal MSNs to acute MeHg exposure. We found that aged MSNs are more
2+

susceptible than young MSNs to MeHg-induced Ca

dysregulation. This finding supports the

hypothesis that aged neurons are unable to handle neuronal stressors predominantly, those which
2+

interact with the Ca

2+

homeostasis system. Impairment of Ca

homeostasis, particularly

alterations in mitochondrial function have been associated with neuronal aging (Troescu and
Vreugdenhil, 2010). Aged neurons have depolarized mitochondria and reduced ATP production
2+

(Troescu, 2005). These factors would make them uniquely susceptible to MeHg-induced Ca
2+

dysregulation. Aged neurons may not be able to buffer increases in [Ca ]i due to depolarized
2+

mitochondria, and they are unable to extrude Ca

via ATP-dependent pathways due to

decreased availability to ATP. The experiments in Chapter 3 support these previous findings and
2+

suggest that a neuron’s propensity to handle toxicants which interfere with Ca i decreases with
age.
The work in Chapter 3 led us to a very interesting and unexpected finding. Following
2+

treatment with 20μM or 100μM MeHg, comparisons of time-to-peak Ca

response from 1, 9,

and 21mo MSNs were no different than those of cerebellar granule cells. Cerebellar granule cells
are extremely sensitive to MeHg toxicity (Hunter and Russell, 1954) and we expected that they
2+

would have a faster time-to-peak Ca i response than striatal MSNs. However, under the

118

conditions utilized in this study striatal MSNs and cerebellar granule cells are equally sensitive to
2+

MeHg-induced Ca

dysregulation. Cerebellar granule cells are more sensitive than cerebellar
2+

purkinje neurons (Edwards et al., 2005) to MeHg-induced changes in [Ca ]i and cytotoxicity.
Because the granule cell is a sensitive target of MeHg-cytotoxicity they have been used as a
2+

model to understand changes in [Ca ]i, mitochondrial function, and neurotransmission (for
review see: Denny and Atchison 1996; Limke et al., 2004; Farina et al., 2011). The role of MeHg
2+

toxicity in striatal MSNs, specifically its effect on Ca i regulation has not been studied. Lesions
occur in the striatum following administration of MeHg in rats (O’Kusky et al., 1988; O’Kusky
and McGeer, 1989; Wakabayashi et al., 1995; Sakamoto et al., 1998). However, our
understanding of the neurophysiological changes that occur in the striatum following MeHg
exposure are limited. We know that MeHg alters DA neurotransmission (Tsuzuki, 1982; Kallisch
and Racz, 1996; Faro et al., 1998, 2000, 2003, 2007), but very little is known about the direct
effects of MeHg on MSNs. This study was the first to demonstrate that acute MeHg treatment
2+

causes increased [Ca ]i on a similar time scale to cerebellar granule cells. Taken together this
study demonstrated that aged neurons are more susceptible to MeHg-induced damage than young
neurons. This is of particular importance in populations of people that rely on fish or marine
mammals as a main food source. Bjerregaard and Mulvad (2012) discussed the dietary habits of
Greenlanders in a recent paper. They demonstrated that between the daily consumption of fish
and marine mammals in a person 60yrs of age is double that of a 30yr old. Increased fish
consumption and consequent exposure to MeHg may lead to more pronounced neuronal damage
and accelerated neuronal aging. More studies need to be done to investigate the relationship

119

between aging and susceptibility to toxicants but a consideration must be made in reducing in the
reference dose for senior citizens.
A number of improvements could have been made to the experiments described in
2+

Chapter 3. We utilized confocal microscopy and the Ca

sensitive fluorophore, Fluo-4 to

2+

monitor spatial changes in [Ca ]i. Confocal microscopy allows for visualization through the
entire thickness of the slice (200μm) as opposed to only visualizing cells in one plane when
using an inverted microscope. However, a drawback of using confocal microscopy is the length
of time it takes to scan through the entirety of the slice. This limits the frequency at which you
can scan without causing significant photobleaching. Therefore the data represent spatial changes
2+

in [Ca ]i at relatively static time points. While spatial changes are helpful in understanding
2+

what is occurring in a general region we cannot continuously monitor changes in [Ca ]i as we
could in single cells. Utilization of high-speed imaging and fura-2 similar to that described in
Chapter 4, would allow us to monitor continuously changes in fluorescence. The temporal data
2+

obtained from those experiments would provide more precise tracking of changes in [Ca ]i.
This may tease out differences in responses between striatal MSNs and cerebellar granule cells.
2+

Resting [Ca ]i could also be calculated with the use of fura-2 because of the ability to make
ratiometric measurements. This would allow us to determine if one cell type starts at a
2+

significantly higher resting [Ca ]i causing it to be more susceptible to MeHg. The addition of
these experiments would provide more insight into neuronal changes due to aging and a more indepth view of the temporal events following acute MeHg exposure in each cell type.

120

The objective of experiments described in Chapter 4 was to investigate the effects of
adult-onset chronic MeHg exposure on striatal synaptosome mitochondrial function and MSN
2+

Ca

regulation of exposure to MeHg. Contrary to our hypothesis we found that mitochondrial

function was unchanged following 6mo of exposure to MeHg or Isr/MeHg. However, ATP
production was slightly reduced in striatal synaptosomes but not in cortical synaptosomes
following 12mo MeHg treatment. This indicates that SNc neurons may be more sensitive to
MeHg-induced neuronal damage than other neuronal types. MeHg treatment disrupts
mitochondrial function and decreases ATP production following acute (Verity et al., 1975; Sone
et al., 1977; Kuznetsov et al., 1986; Levesque at Atchison, 1991) and long-term exposure
(Bourdineaud et al., 2008; Cambier et al., 2009; Kong et al., 2013). Based on these studies we
expected to find a significantly decreased ATP production following the 12mo MeHg treatment.
Changes in ATP production may not have occurred in our study for a number of reasons. First,
SNc synaptosomal mitochondria may not be a sensitive target for MeHg-induced damage. To
understand if this is the case we could perform a comparative study between cerebellar and SNc
mitochondria following chronic MeHg exposure. Another possibility is that SNc neurons are a
sensitive target and we did not see a change in mitochondrial function because our synaptosome
preparation contains a mixed population of nerve terminals. The striatum receives input from
both the SNc and cortex so the synaptosome preparation would contain SNc, cortical, and MSN
nerve terminals. It is possible that the SNc neurons were sensitive to MeHg-induced damage and
began to degenerate. At the 12mo time point there were fewer SNc synaptosomes present and
only the terminals of ‘healthy’ neurons present so no changes in ATP production were detected.
We could have avoided this problem by isolating the SNc terminals using magnetic microbeads
and performing mitochondrial function analysis on only those terminals. However, this technique

121

adds a significant cost to an already expensive study and experiment and was not pursued. We
could also perform immunohistochemistry in fixed striatal slices probing for tyrosine
hydroxylase to visualize if there was a loss of DA nerve terminals in the striatum. It could also
be a possibility that the [MeHg] or length of exposure does not disrupt mitochondrial function. If
we increased either of those variables we may find a disruption of mitochondrial function.
In Chapter 4 we also found that 12mo Isr/MeHg treatment caused a significant increase in
mitochondrial ATP production in both striatal and cortical synaptosomes. An increase in ATP
production could be thought of as being protective because the mitochondria are functional and
able to respond to the ATP demand. Isr is protective in a number of models of neurodegenerative
disease (Guzman et al., 2010; Ilijic et al., 2011), ischemia (Campbell et al., 1997), hypoxia
(Barhwal et al., 2009), stroke (Berjukow and Hering, 2001) and senescence (Yu et al., 2013).
2+

The protective action may be due to a combination of the blockade of L-type Ca

channels

(Schachter, 1991) and its antioxidant capacity (Takamura et al., 2000; Toniolo et al., 2002). Isr
2+

2+

+

has also been shown to disrupt Ca m by blocking the main Ca m exit pathway, Na -dependent
2+

release (Uceda et al., 1995). This may increase neuronal ATP demand because Ca

buffering

would need to occur through ATP dependent pathways such as SERCA and the plasma
2+

membrane Ca -ATPase. However, animals treated with Isr alone had no change in
2+

mitochondrial ATP production so any disruption in Ca

buffering due to Isr did not increase

ATP demand. Only when animals were co-treated with MeHg was there a significant increase
ATP production, suggesting that the increased demand is due to MeHg. Schafer et al (1990)
found that MeHg disrupts VGCC antagonist binding in PC12 cells. Therefore, it is possible that
instead of being protective co-treatment with Isr facilitated MeHg-induced damage. In the
122

presence of MeHg, Isr is unable to bind VGCCs offering no protection while increasing MeHg
distribution by increasing cerebral blood flow. Consequently, there is an increased demand for
2+

ATP due to MeHg-induced Ca -dysregulation. While we do not fully understand the interaction
between Isr and MeHg and the change in ATP production it raises the possibility that drugs
which increase cerebral blood flow cause increased neuronal exposure to toxicants. Drugs such
as Isr, are typically prescribed in adults who may be more sensitive to damage by neurotoxicants,
as seen in Chapter 3.
In Chapter 4 we also found that striatal MSNs from animals treated for 12mo with MeHg
2+

had a faster time-to-peak Ca

response following addition of AMPA than untreated animals.

This may be due to increased AMPA receptor expression via the mechanism discussed in
Chapter 4 or receptor sensitization, similar to what was seen by Johnson et al. (2011). The
striatum receives glutamatergic input from the cortex and dopaminergic input from the midbrain
(Surmeier et al., 2007). MeHg interacts with both dopaminergic (Faro et al., 2007; Dreiem et al.,
2009) and glutamatergic (Juarez et al., 2002; Farina et al., 2003) nerve terminals to induce
release. MeHg also inhibits glutamate uptake in cerebellar granule cells (Porciuncula et al., 2003)
and astrocytes (Aschner et al., 2000). While these studies demonstrate that MeHg causes increase
neurotransmitter release, electrophysiological studies have shown that acutely applied MeHg
causes a increase and then cessation of spontaneous synaptic current (Yuan and Atchison, 2007).
This suggests that while MeHg initially increases neurotransmitter release and subsequent postsynaptic current, the current is eventually blocked; possibly due to interactions of MeHg with the
post-synaptic receptor. Glutamatergic post-synaptic receptors are also involved in MeHg-induced
2+

Ca -dysregulation and cytotoxicity. Application of both NMDA and AMPA receptor

123

2+

antagonists delays the onset of MeHg induced Ca -dysregulation in spinal motor neurons
(Ramanathan et al., 2011) but not cerebellar granule cells (Marty and Atchison, 1997). Also,
application of MK-801, a NMDA receptor antagonist protects SH-SY5Y cells from MeHg
cytotoxicity (Petroni et al., 2011). These data support a role for MeHg’s interaction with postsynaptic glutamatergic receptors altering their function and possibly leading to altered
expression. RT-PCR or immunohistochemistry could be completed on striatal tissue to determine
if AMPA receptor expression was increased in MeHg-treated animals. Electrophysiological
single channel recordings could be performed to measure channel conductance and rates of
activation and inactivation to determine if MeHg altered the function of AMPA receptors.
We recognize that there are several limitations in these studies. Because animals had free
access to MeHg in their drinking water and Isr in their feed there was no control over the amount
or timing of their consumption. This may have introduced variability within and amongst
treatment groups. A better method would have been to inject mice with MeHg at the same time
on a daily basis as well as insert an osmotic pump, which would release Isr. An alternative to
administering Isr would be to use Cav1.3 knockout mice. These mice are commercially available
and do not have a lethal phenotype. These methods would greatly reduce the variability in the
study. Another improvement would be to increase the number of time-points. It is not possible to
understand the time course of changes in neuronal function by only sampling at 2 time-points, an
improved approach may be to sample 5-6 mice every month. Having more time-points would
allow for a better overall picture of alterations that occur in the neuron. However a deficiency of
this approach is the lack of statistical power that would be necessary by lowering the sample
size, or a major increase in cost if the number of animals was kept at the power level utilized in
the present study.
124

There are a number of experiments that would complement the work already completed.
In order to have a better understanding of the effects of chronic MeHg treatment on
mitochondrial function electron microscopy experiments could be completed to examine the
number and structure of synaptosomal mitochondria. Aging and exposure to toxicants has been
shown to alter the number of mitochondria present at the nerve terminal as well as their structure
(Pivovarova et al., 2013; Quiroz-Baez et al., 2013). Examination of the ultra-structural properties
of synaptosomal mitochondria would offer insight into whether the mitochondria are healthy or
if there are an increased number of mitochondria present to handle the increased demand to
2+

buffer Ca

2+

and produce ATP. Simultaneous Ca

imaging and electrophysiological experiments

similar to those done by Dryanovski et al. (2013) could be performed in both striatal MSNs and
2+

midbrain SNc neurons. This would greatly contribute to our understanding of both the Ca i as
well as any alterations in neurotransmission that occur due to chronic MeHg treatment. Upon
completion of electrophysiological recordings the intracellular contents of the cell could be
collected and single-cell RT-PCR (Toledo-Rodriguez and Markram, 2007) performed to
determine changes in gene expression. Another experiment that would contribute to the overall
understanding of MeHg-induced alterations in neuronal function would be the continuous
monitoring of DA and glutamate in the striatum by implementation of a microdialysis probe
(Bazzu et al., 2013). Completion of these experiments would provide a comprehensive overview
of MeHg-induced changes in neuronal function of the basal ganglia.
This work highlights the need to have a better understanding of the consequences of longterm exposure to environmental toxicants, especially in aged individuals. Due to the complexity
and cost of performing chronic treatment studies, the scientific field has largely avoided such
experiments. However, due to an aging population (Abbott et al., 2011) and an increased interest
125

in the possible role of environmental contaminants in the development of neurodegenerative
diseases (Johnson and Atchison, 2009; Vance et al., 2010; Williams et al., 2010; Cannon and
Greenamyre, 2011; Madison et al., 2012) scientists must begin to investigate the consequences of
long-term exposure to low levels of environmental toxicants. These studies demonstrate that
prolonged exposure to MeHg alters the function of VGCCs in vitro and AMPA receptors in vivo.
Alteration in the function of both VGCC and AMPA receptors could have detrimental effects on
neuronal function and ultimately the behavior or disease state of an individual. The role of MeHg
in the development of neurodegenerative and neuropsyciatric disorders is controversial.
However, it is clear that disruption of neuronal homeostatic synaptic plasticity leads to
neurological diseases (for review see: Wondolowshi and Dickman, 2013), and these studies
support a role for MeHg disrupting this process by alterting the function of key components of
neuronal synaptic plasticity.
In conclusion, this dissertation shows that prolonged exposure to MeHg in vitro causes a
time- and subtype-dependent block of VGCCs. It also causes a significant increase in resting
2+

[Ca ]i. We presented novel data demonstrated that striatal MSNs from aged animals are more
2+

susceptible to MeHg-induced Ca

2+

dysregulation than young animals. The MeHg-induced Ca

increase occurs at a similar time as cerebellar granule cells. Chronic exposure to low levels of
MeHg in vivo did not alter mitochondrial function. However, when animals were co-treated with
Isr and MeHg there was a significant increase in striatal synaptosome mitochondrial function.
2+

Finally, chronic exposure to MeHg altered postsynaptic Ca
activation.

126

response to AMPA receptor

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