THESIS

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thesis entitled
Comparison of Hypothalamic Serotonergic Activity in
Relation to Feeding and Strain Differences in Dietary
Obesity Susceptible (OM) and Resistant (SSB/Pl) Rats

presented by

Thomas Karr Custer

has been accepted towards fulfillment
of the requirements for

MS degree in m. .

 

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Major professor

Date _Eehnuar_y_ll,_1984

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COMPARISON OF HYPOTHALAMIC SEROTONERGIC ACTIVITY IN
RELATION TO FEEDING AND STRAIN DIFFERENCES IN DIETARY
OBESITY SUSCEPTIBLE (ON) AND RESISTANT (SSS/PL) RATS

BY

Thomas Karr Custer

A THESIS

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

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1983

ABSTRACT
COMPARISON OF HYPOTHALAMIC SEROTONERGIC ACTIVITY IN
RELATION TO FEEDING AND STRAIN DIFFERENCES IN DIETARY
OBESITY SUSCEPTIBLE (OM) AND RESISTANT (SSB/PL) RATS
By

Thomas Karr Custer

Serotonergic stimulation has been linked with reduced
energy intake in rats. Whether hypothalamic serotonergic
activity measured by 5-HT production is greater in SSB/Pl
(S) rats (dietary obesity resistant) than Osborne-Mendell
(OM) rats (non-dietary obesity resistant) was tested. Each
strain was divided into three groups: Fed Drug (pargyline,
a MAO inhibitor), Non-Fed and Fed Sham. All rats were fed
an energy-dense, high-fat diet and were adapted to eat a 2-
hour meal every 2“ hours. 5-HT accumulation was measured at
0, 20, AD, and 60 minutes post drug or sham injection to
calculate 5-HT accumulation rate (b1L. S rats ate less food
than the OM during a 20 minute period prior to killing
(p < CLOS). Greater serotonergic activity was indicated in
S Non-Fed rats (23.86 ng/g/min) than S Fed (12308 ng/g/min)
(p < 0.10), while the 0M Non-Fed vs. Fed showed no differ-
ence (15.94 and 12.87 ng/g/min respectively) thus giving

qualified support to the hypothesis.

Dedicated to

my parents.

ii

ACKNOWLEDGMENTS

I would like to thank Dr. Schemmel and Dr. Romsos for
their patient assistance in the day-to-day problems that
arise in carving out a research project. Also, I would like
to thank my other committee members, Dr. Beck and Dr. Bond,
for their ideas which were essential for the planning and
carrying out of this project. .Also, I would like to thank
Dr. Vandertuig for helping me troubleshoot problems on the
HPLC and Dr. Gill for applying his expertise to my
statistical problems.

This research was supported, in part, by an AURG.

iii

TABLE OF CONTENTS

LIST OF TABLES o o o o o o o I o o o o o o o. o o o 0
LIST OF FIGURES . . . . . . . . . . . . . . . . . .
INTRODUCTION 0 o o o o o o o o o o 0 o o o o c o _o 0

LITERATURE REVIEW . . . . . . . . . . . . . . . . .
5-HT Metabolism . . . . . . . . . . . . . . . .

Drug Studies . . . . . . . . . . . . . . . . .
Brain Lesions or 5-HT Depletion . . . . .
Serotonergic Stimulation and Food Choices

Diet Studies . . . . . . . . . . . . . . . . .

Conclusions . . . . . . . . . . . . . . . . . .

AN APPROACH TO ASSESSING SEROTONERGIC ACTIVITY . . .
Serotonergic Activity and Feeding . . . . . . .
The Dietary Model of Obesity . . . . . . . . .
Measuring Serotonergic Activity . . . . . . . .

Objectives 0 I O O O O O O O O O O O O O O O 0

MATERIALS AND METHODS . . . . . . . . . . . . . . .
Experimental DeSign . . . . . . . . . . . . . .
Food Intake . . . . .°. . . . . . . . . . . . .
Injection and.Sacrifice . . . . . . . . . . . .
Dissection . . . . . . . . . . . . . . . . . .

iv

vi

vii

(D004:

11

13
13
14
1A
18

19
19
23
23
24

MATERIALS AND METHODS (Cont.)

Sample Analysis .

Statistics . .

RESULTS . . . . . . .
Body Weights . .
Food Intakes . .
5-HT Accumulation

5-HIAA Decline .

Hypothalamus Weights
Mean Level At Each Time

O-Time Values

60 Min. Values

Effect of Pargyline .

DISCUSSION . . . . . .

Omission of Fed Sham

Choice of Statistical

SUMMARY & CONCLUSIONS

POSSIBLE FOLLOW-UP STUDIES

APPENDIX . . . . . . .

BIBLIOGRAPHY . . . . .

Method

2a
26

28
28
28
28
42
A7
147
A7
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53

5A
59
59

60

62

6A

67

LIST OF TABLES

TABLE 1 - Composition of the High Fat Diet . . . . .

TABLE 2 - Comparison of Mean 5-HT Accumulation Rate
in OM and S Rats with Different Fed & Drug
States 0 O O O O 0 O O O O O 0 I I O I O 0

TABLE 3 - ANOVA for Estimations of D1 or 5-HT
Accumulation Rate Based on One Sample Per
Time Paint 0 O O O O O O O O O I O O O O 0

TABLE 4 - Comparison of 5-HIAA Decline Over Time in
OM and S Rats with Different Fed and Drug
States 0 O O O O O O O O O O O O O O O O 0

TABLE 5 - Amount of 5-HT (ng/gm) in Hypothalamus at

TABLE

TABLE

Zero Time in OM & S Rats . . . . . .
ANOVA for 0 Time Values of 5-HT . .

Mean ¢S.E.M. at Zero Time 5-HIAA Levels

in OM & S Rats . . . . . .,. . . . .

ANOVA for 0 Time Values in all Treatment

Groups or 5-HIAA o o o o o o o o o 0

Amount of 5-HT (ng/gm) in the Hypothalamus
60 Min. Post Injection in OM and S Rats

ANOVA of Values in all Treatment Groups

60 Min. Post Injection 5-HT Levels .

vi

50
50

51

51

52

52

'FIGURE
FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE
FIGURE

FIGURE

10

Diagram of Metabolic Pathways of

LIST OF FIGURES

S-Hydroxytryptamine (5-HT)

Illustration of Cross Classified Factors

in Experimental Design .

Fed vs. Non-Fed Comparison of 5-HT
Accumulation in the S Rat After Injection

of Pargyline .

Fed vs. Non-Fed Comparison of 5-HT
Accumulation Rate in the OM Rat After

Injection of Pargyline .

Strain Comparison of 5-HT Accumulation
Rate in the Fed State After Injection of

Pargyline

Strain Comparison of S-HT Accumulation
in the Fed State After Injection of

Vehicle .

Strain Comparison of 5-HT Accumulation in
the Non-Fed State After Injection of

Pargyline

Fed Drug vs. Fed Sham Comparison of 5-HT
Accumulation in the S Rat

Fed Drug vs. Fed Sham Comparison of 5-HT
Accumulation in OM Rats

Bar Graph of Mean $8.0. 5-HT Accumu-
lated in Each Treatment Group at Each

Time Point

vii

22

31

33

35.

37

39

A1

AA

49

INTRODUCTION

Reports of the role of serotonin as a neurotransmitter
that mediates satiety have become more common in the scien-
tific and lay press recently. The relationship of seroto-
nergic activity and food intake has been studied through two
major routes: pharmacological manipulation of serotonergic
activity and the effect of diet on serotonin (5-HT) levels
in the brain. Before discussing these two aspects, basic 5-

HT metabolism will be reviewed.

LITERATURE REVIEW

W

The precursor to serotonin, tryptophan (Trp), an essen-
tial amino acid, crosses the blood brain barrier by means of
active transport (see Figure 1). Trp competes with other
neutral amino acids (naa) for this transport so the rate at
which it is carried into the neuron is dependent on the
Trpznaa ratio (Wurtman et al., '76; Curson '81; Boadle-
Biber, '82). Inside the neuron trptophan hydroxylase (TH)
catalyzes the first step in the production of 5-HT by
hydroxylation of Trp to form 5-HTP. Then an aromatic amino
acid decarboxylase catalyzes the step to 5-hydroxytryptamine

(5-HT). The step catalyzed by tryptophan hydroxylase is

Figure 1: Diagram of the Metabolic Pathways of
5-Hydroxytryptamine (5-HT)

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thought to be the rate limiting step and the synthesis rate
is dependent on two factors: 1) substrate availability
since the enzyme is not saturated at physiological levels,
and 2) neuronal activity which can induce the tryptophan
hydroxylase intrinsic activity (Hamond et al., '81; Boadle-
Biber, '82). The 5-HT is transported to the neuron membrane
where it is released upon depolarization into the synapse
where it binds its receptor on an adjacent neuron. After 5-
HT dissociates from the receptor, most of it is taken up
again by the neuron and transformed by Monoamine Oxidase
(MAO) to 5-hydroxyindole acetic acid (5-HIAA) which is then
transported out of the neuron and away from the central

nervous system.

W

Fenfluramine, a prototypic serotonergic drug, meta-
chlorophenylpiperazine and quipazine have a high affinity
for 5-HT receptors and will cause dose dependent anorexia
(Samanin et alt, '80). Pretreatment with a 5-HT antagonist,
methergoline, will reduce the effects of these drugs. Com-
parison of fenfluramine with amphetamine, which causes ano-
rexia through catecholaminergic pathways, has indicated that
two pathways may mediate satiety (Garattini, '81). Lesions
of noradrenergic nerve terminals in the hypothalamus will
block most of the effects of amphetamine but not those of
fenfluramine (Garattini, '81). The reverse is true when
serotonergic neurons are destroyed by raphe lesions. A

comparison of amphetamine and fenfluramine anorexia by

5
Blundel et a1” ('76) indicated that amphetamine affects
hunger or the motivation to begin eating, while fenfluramine
affects satiety or the motivation to discontinue feeding.

Blundel et a1. ('76) measured the rate and frequency of
food intake by rats given amphetamine or fenfluramine. Am-
phetamine delayed the onset of the first meal, while with
fenfluramine, food intake began as usual but the meal size
was reduced. Amphetamine-induced-delay of meal onset is
interpreted as an effect on the initiation of feeding while
fenfluramine induced decrease in meal size is interpreted as
an effect on satiety. Burton et a1. ('81) replicated the
temporal pattern of fenfluramine with an apparatus similar
to Blundel et a1” ('76) that recorded the number of pellets
consumed per minute and correlated observed feeding behavior
changes with decreased intake. While Blundel et alt ('76)
found that food intake decreased toward the end of the meal,
they reported that food intake was decreased throughout the
meal.

Grinker et al. ('79), in testing whether the Zucker
obese rat has less precise control over food intake than the
lean Zucker rat, found that amphetamine tended to reduce
food intake by decreasing meal frequency, while fenfluramine
tended to decrease meal size, but the differences were not
significant because both drugs had some effect on each meal
parameter.

Altogether, the studies on the temporal effects of
fenfluramine vs. amphetamine provide a strong indication

that serotonergic pathways may mediate food intake through

6

cessation of feeding or satiety, while catacholaminergic
pathways may mediate food intake through feeding onset.

BLa1n_Lgs12n§_gz_5=fl1_ngnletign, Depletion of brain 5-
HT by use of drugs such as parachlorophenylalamine (p-CPA),
5-6-Dihydroxytryptophane (5-6-DHT), or 5-7-Dihydroxytrypto-
phane (5-7-DHT) or by lesions can cause a reduction in
satiety or hyperphagia in rats (Blundel, et alt, “79)
through these methods often show inconsistent results. For
example, Ashley et a1. ('79) used 5-7-DHT, p-CPA or thermal
raphe lesions in rats and found that, given a choice-of high
or low protein intake, protein intake decreased with each
treatment but energy intake was not affected. However,
Waldbillig et a1. ('81) gave microinfusions of D-7-DHT into
the ventralateral hypothalamus as opposed to ventricular
injections into the brain used by Anderson ('79) and found
that rats consumed more kcals and gained more weight com-
pared to controls when fed a high fat diet. Two possible
reasons for the differences between these studies are 1) the
5-7-DHT was administered to a more specific area in the
Waldbillig study and 2) the different diets given may have
affected foods intake. Possibly, serotonergic pathways
mediating energy intake were less able to respond to the
calorie-dense diet used by Waldbillig while the less
calorie-dense diet used by Ashley, et a1. ('79) would not
have challenged the pathways.

SenaIanensic_SLinniaiian_ann_Eacn_Engigas. Wurtman and
Wurtman ('77) allowed Sprague-Dawley rats to select food

intake from two isocarloric diets that were high or low in

7
protein with equal amounts of fat. When these animals were
given the drug fenfluramine, which enhances or stimulates
serotonergic transmission, the rats decreased their energy
intake by decreasing the food consumed from the high carbo-
hydrate food cup while protein intake was maintained.

In a series of trials, the rats were presented with two
food cups and the proportions of fat, protein and carbohy-
drate varied with each trial. When given fenfluramine, the
rats always chose to decrease energy intake by decreasing
intake from the food cups high in carbohydrate and main-
tained intake from the food cups high in protein regardless
of the proportions (Wurtman & Wurtman, '79). When total
grams of protein and carbohydrate and Kcals consumed from
all cups in a trial were calculated, the protein intake was
similar to control while energy intake and grams of carbo-
hydrate decreased significantly in rats given fenfluramine.
Changes in fat were not clearly reported and were implied to
be incidental to carbohydrate and protein choices since the
diets were made isocaloric by adjusting fat content. Fur-
thermore, when the animals were given a choice between three
cups where the fat level varied (1--high protein, high
carbohydrate, moderate fat; 2--low protein, high carbohy-
drate, moderate fat; 3--low protein, low carbohydrate, high
fat) they still decreased carbohydrate and maintained pro-
tein intake when given fenfluramine.-

However, diet choices were not quite the same when
Sprague-Dawley rats were presented with three food cups with

pure rations of fat, protein and carbohydrate (each with an

8
appropriate amount of vitamins and minerals) and were given
fenfluramine (Orthen-Gambill & Kanereck, '82). When the
three rations were isocaloric, fenfluramine caused a reduc-
tion in energy intake by decreasing fat intake while carbo-
hydrate and protein were not significantly reduced compared
to controls. When the fat component was made more energy
dense, energy intake was reduced by decreasing fat and
protein intake. One other difference in the design of these
two studies is that Orthen-Gambill gave three doses of
fenfluramine, 1.5, 3.0, and 6.0 mg/kg, and averaged the
response while Wurtman et al. ('79) only observed the ef-
fects of one dose at 2.5 mg/kg. The responses at higher
doses in the Orthen-Gambill study showed the difference
between the studies most distinctly.

These drug studies indicate that fenfluramine produces
an anorectic effect through serotonergic pathways. However,
whether the anorexia is mediated through an effect on fat,
protein or carbohydrate selection is not clear and seems
contingent on the types of diet presented. Studies which
use dietary methods to affect serotonin levels to get a
measure of serotonergic activity, present a different pic-

ture of serotonin's role in feeding.

121mm

Correlation of plasma Trp to neutral naa ratio and
brain 5-HT with feeding has indicated that brain levels of 5-
HT are inversely related to protein intake while there is no

relationship with energy intake (Anderson, '79). Woodger et

9
al. ('79) reported that when streptozotocin-induced diabetic
rats and control rats were offered a choice of 10 and 605
casein diets and individual groups had 0, 1.u5, and “.551
Trp added to the diet, the protein intake of both diabetic
and control rats was inversely related to plasma Trp:naa
ratio, brain Trp, and brain 5-HT while no relationship was
seen with energy intake. Therefore, increasing Trp consump-
tion was associated with increased brain 5-HT and Trp, and
decreased protein intake. It appears from this study that
insulin sufficiency, which has an effect on Trp:naa is not
essential for affecting changes in brain Trp and 5-HT from
dietary influences. Woodger et al. concluded that the
plasma amino acid ratio of Trp:naa plays a major role in
affecting S-HT synthesis although the results cannot be
taken as a cause and effect relationship.

In a study of self-selected meal composition on the
relation of circadian rhythms to plasma and brain Trp, and
brain 5-HT, Li et a1” ('82) found that plasma Trp:naa and
brain Trp showed significant circadian rhythms. Comparison
of these rhythms to corresponding food circadian choices
indicated an inverse correlation to protein intake, while
carbohydrate and energy consumption were not found to be
correlated. In the second part of this study, the animals
were sacrificed 20 minutes after a meal, plasma Trp:naa, and.
5 hydroxyindole (the whole brain 5-HT and 5 HIAA quantities
combined) were inversely related to protein intake. Li, et
a1. ('82) concluded that plasma Trp:naa ratio, brain Trp and

5 hydroxyindole concentrations are under continual influence

10
of food choice and may act as part of the feedback mechanism
that regulates short-term food selection but not-energy
intake.

Studies that are similar to the above studies of
Woodger et al. ('79) and Li et al. ('82) have not always
produced consistent results. When there was an alteration
in the amount of dietary fat, lean and obese (ob/ob) mice
consumed different amounts of protein. Decreased plasma
levels of Trp:naa was associated with increased protein
intake (Chee et al., '81). In an extension of this study,
Romsos-et alt ('82) reported that, with a similar feeding
regime, the different levels of long-term protein intake
were not related to changes in brain 5-HT in the obese and
lean (ob/ob) mice. When the diet was supplemented with Trp,
no significant changes in protein and energy intake were
observed. In a similar study by Peters & Harper ('81),
where Sprague-Dawley rats were allowed to choose between
isocaloric high and low protein diets, no correlation between
long-term protein intake and brain S-HT levels was found.

The reason for these inconsistent results within diet
studies is hard to explain. Since all the studies assayed
whole brain levels of Trp and 5-HT, it may be necessary to
examine more discrete brain regions because it is possible
that different nerve bundles may have different responses to

the same stimuli.

11
muons

The pharmacological studies and the dietary studies
indicate that 5-HT has some role in the mediation of energy
and macro nutrient intake. The inconsistencies within and
between them point to the complex nature of the CNS and
that the role of serotonergic pathways in food intake still
needs to be clarified.

The two types of studies may produce inconsistent re-
sults because they have very different approaches, each with
its own weakness. Drug studies show gross effects of sero-
tonergic stimulation or depletion which may produce an un-
physiological state, while diet studies only show a correla-
tion of dietary intake and brain levels of 5-HT, plasma
Trp:naa ratio and brain Trp levels. Basic physiological
theory suggests that brain levels of a neurotransmitter or
its precursor are not reliable indicators of neuron activity
or of the amount of transmitter released into the synapse
(Henry & Ternaux,'81; Boadle-Biber,'823 Curzon,'81).
Wistar rats injected with p-chlorophenylalanine (p-CPA),
which inhibits the synthesis of 5-HT, were found one month
later to have the same resting brain brain 5-HT levels as
control animals (Alexander, et al., '80). When the rats
were injected with a MAO inhibitor, the accumulation of
brain 5-HT for rats injected with p-CPA was much less than
controls. This could indicate that, even with different
synthesis rates, the neuron maintains a certain level of
neurotransmitter and the same amount of 5-HT would be re-

leased into the synapse. To present conclusive findings on

12
the role of serotonin in food intake, a more direct measure

of neuron activity than base 5-HT levels is needed.

AN APPROACH TO ASSESSING SEROTONERGIC ACTIVITY

Wading

No study reviewed to date has measured the effect of
feeding on serotonergic neuron activity which is the key to
linking a neuro-pathway to a physical event. Two studies
comparing S-HT levels in laboratory animals with different
food intakes have been reviewed.

Different S-HT levels in obese Zucker rats, which
greater or smaller were hyperphagic, and non-obese Zucker
rats were found in the mesencephalon but not in the hypo-
thalamus, cortex, hippocampus, corpus striatum, remaining

diencephalon, pans-medulla or cerebellum of Zucker

 

(Finkelstein et al., '82). Trp was decreased in the cortex,
hippocampus, corpus striatum, hypothalamus and diencephalon.
Also, the free plasma Trp was reduced in the obese rat and
was cited as a possible cause for the reduction in brain
Trp. The association of decreased 5-HT levels in the mesen-
cephalon, the Trp in several brain areas in the obese Zucker
rat indicates little about actual serotonergic activity.

Genetically obese hyperglycemic mice had increased
whole brain S-HT, total plasma Trp and total plasma-free Trp
(Garthwaite, et al., '79). These findings are hard to
compare with the findings of Finkelstein, et al.(U79)
because 5-HT was assayed in the whole brain and

13

1A
hyperglycemic mice were used as opposed to rats. Also, food
intakes for the mice were not reported in the Garthwaite
study. This study, like Finkelstein's et al. ('82), can
only associate S-HT levels with obesity and differences in

food intake.

W
The dietary obesity model (Mickelsen et al., 1955) was

used here to determine if serotonergic activity has any
relation to energy intake. Evaluation of several strains of
rats for obesity has provided two strains, the Osborne
Mendel (0M) and the S 5B/Pl (S) which respond differently to
a high fat diet (Schemmel et al., '70). When fed a high fat
diet, 0M rats consumed more kcals, retained more energy per
kcal consumed and gained more weight than S rats (Schemmel
et al., '72; Schemmel 8: Mickelsen, '73). When both strains
were fed a high carbohydrate diet--less calorically dense
than the high fat diet--the differences in food intake and
weight gain were much less (Schemmel, '70). In view of
serotonin's role in appetite control, the different response
to caloric density between strains presents the question:

Do the two strains have different serotonergic activity that
can be associated with their different food intakes?

HEW

All the studies reviewed here, which examined the rela-
tion of serotonin to some aspect of food intake, used either
indirect approaches to measure serotonergic activity or

observed feeding behavior under the influence of

15
pharmacologic serotonergic stimulation. More direct methods
for measuring neuron activity, which involve steady state
and non-steady state techniques, are now being used. Steady
state methods use radioactive labeling of 5-HT, 5-HTP, or
Trp, or a stable isotope of 02 to follow the production of
5-HT and 5-HIAA (Neckers, '82). Non-steady state methods'
involve inhibition of a metabolic step in 5-HT production or
catabolism (see Figure 1) and use the relative rate of
increase or decrease of a 5-HT metabolite as an indicator of
production or breakdown which are in part related to sero-
tonergic activity (Hamon, et al., '81).

In this study, MAO inhibition by pargyline was chosen
as the method to assess serotonergic activity because it is
as accurate a measure of 5-HT production as other non-steady
state methods (Morot-Gaundry et al. '74). Comparison of
pargyline with pheniprazine, another MAO inhibitor, produced
similar rates of 5-HT accumulation in the mouse brain 10
minutes after drug injection. Morot-Gaundry et al. ('7A)
concluded that the two drugs produced the same response and
that, though the drugs tested may not provide an accurate
estimate of actual 5-HT turnover, they can be used to esti-
mate relative changes in 5-HT turnover in various treatment
conditions.

MAO inhibition with pargyline is rapid and causes a
linear accumulation of 5-HT for at least the first 60 min-
utes after injection (Johnson & Crowley, '82; Morot-Gaundry
et 31”, '78). Using another inhibitor that acts on decarb-

oxylase production of 5-HT from 5-HTP (see Figure 1) would

16
produce a linear accumulation of 5-HTP at a rate similar to
MAO inhibition. However, Morot-Gaundry et a1” ('7“) found
that the response stays linear for only 30 minutes.

VanLoon et al. ('81) compared four methods of 5-HT
turnover estimation in the hypothalamus: MAO inhibition
induced increase of 5-HT and decrease of 5-HIAA, S-HTP
accumulation after decarboxylase inhibition with m-hydroxy-
benzylhydrazine and accumulation of 5-HIAA after probenecid
induced block of 5-HIAA transport at one time point post
injection. The four methods showed very similar estimates,
though S-HIAA accumulation from probenecid transport in-
hibitor was lowest but consistent with the other accumula-
tion of disappearance rates. Though no linear accumulation
measure was one, this suggests that MAO inhibition is a
useful and a state of the art tool for assessing 5-HT
accumulation.

So MAO inhibition by pargyline was used in this study
since it provides a longer 5-HT accumulation time which
would help to show relative differences in accumulation
rates, as well as allow for easier sample collection and
since it is as effective as pheniprazine. The major assump-
tions in using pargyline as an MAO inhibitor are that com-
plete and rapid inhibition of MAO occurs; that accumulated
S-HT does not diffuse away from the CNS; that 5-HTP decarb-
oxylation is not significantly inhibited; and that signifi-
cant end product inhibition does not occur within 60

minutes.

17

A relative indication of serotonergic activity can be
detected through a measure of 5—HT production since there is
good evidence that tryptophan hydroxylase (TH) activity and
possibly tryptophan uptake can be induced with increased
serotonergic activity (Hamon, et al., '81; Henry A Ternaux,
'81). Since TH is not saturated at physiological levels,
the intrinsic activity of the carrier could play a role in
5-HT synthesis. Hamon et al. ('81) notes that changes in 5-
HT synthesis, due to changes in brain Trp levels, may happen
independently of changes in plasma Trp:naa because the car-
rier rate can be induced by a drug, dibutyrylcyclic-AMP, and
because normal circadian fluctuation in S-HT synthesis ob-
served using brain slices are associated with fluctuations
in the intrinsic activity of the carrier. TH activity is
thought to be induced from nerve depolarization which allows
Caz influx resulting in activation of a protein kinase. The
protein kinase may then activate the enzyme or a cofactor
(Hamon, et al., '81; Boadle-Biber, '82). Whether the Trp
uptake or TH is stimulated, the net result would be observed
in 5-HT accumulation. One possible disadvantage of relying
on 5-HT production to indicate neuron activity is that the
uptake and TH intrinsic rates may not always be induced in
the same direction, though there are indications that the
Trp carrier activity and TH intrinsic rate may be induced
simultaneously. Hamon et a1" ('81) found that the same
substance which stimulates the Trp carrier also stimulates
TH. This gives support to the hypothesis that TH and the

carrier may be affected in the same manner under a given

18
condition. So, S-HT production is used here as a relative
measure of TH activity which in turn reflects neuron
activity.
The hypothalamus was chosen as the region to assay for
S-HT activity since it has been shown to have major control
over feeding behavior and has a high amount of serotonergic

nerve terminals (Leibowitz, '80).

0111311123

The overall objective of this study was to examine the
relationship of serotonergic activity to satiety and food, or
energy, intake. The major hypothesis to be tested was that
when fed an energy dense (i.e., high fat) diet, the S rat
will eat less and have a greater serotonergic activity in
response to feeding than the 0M rat. A secondary hypothesis
was that serotonergic activity will increase in response to

feeding.

MATERIALS AND METHODS

The rats were male Osborne-Mendell (0M) and SSB/Pl (S)
that were 7 to 8 weeks of age at the time of sacrifice.
Both strains of rats were obtained from a breeding colony at
MSU, FSHN. After weaning, at 21-24 days, the animals were
housed in suspended individual metalcages and kept in an
animal room lighted from 0700 to 1900. All animals had chow
available while in litters, then at 21-24 days they were
weaned and fed a high fat diet (Table 1) (Schemmel, et al.
'82). After one week of an libitnm feeding of the high fat
diet, the food cups were placed in the cages each day at
1900 and removed from the cages each day at 2100 to give a
two-hour feeding period at the beginning of the dark cycle.
The rats were meal fed for approximately 2 1/2 weeks before

killing and 5-HT assay.

Wain

Three factors were involved in the experimental design:
Strain, Fed State and Drug, which results in eight possible
groups (Figure 2). The Fed rats were given their food cups
for the first 20 minutes of their usual feeding period.
Then, they were injected with vehicle or pargyline within 10
minutes after feeding. The Non-Fed rats were not allowed to
feed prior to injection. Thus, they were killed after
having been not fed for the customary 22 hours.

19

20

TABLE 1

Composition of the high fat diet

 

 

 

High FatsDiet WtAlOOfigm
caseina 32.0
vitamin m'x 1.5
cellulose d 3.0
mineral mix 6.0
d1 methionine .0.4
cerelose 7.1
corn oil 5.0f
hydrogenated shortening 45.0
6.062kca14gi
Mimi.
pro 21
CHO 5
fat 74

aHigh protein casein, purchased from Teklad Test Diets,
Madison, WI.

A.O.A.C. purchased from Tekled Test Diets, Madison, WI.
Purchased from Teklad Test Diets, Madison, WI.

AIM-76, purchased from Teklad Test Diets, Madison, WI.
Purchased from Corn Industry Division of CPC Interna-
ional Co., Englewood, NJ.

Crisco, purchased from Procter and Gamble, Cincinnati,
OH.

(0000‘

HI

21

Figure 2: Illustration of Cross Classified Factors
in Experimental Design

22

mm EEG—filial: 12m

!—Drug

Fed e:
/ ‘Sham
OM
\ "Dl’ug
Non-Fed:

~Sham

 

““Drug

Feds:
S
\ ADrug
Non-Fed":Z

—Sham

Figure 2

23

EQQQ_InLaK£

Each animal was assign a food cup. The cups were
weighted at least twice a week and whenever diet was added
to the cups. Food intake was measured in terms of grams of
food removed from the cup. Due to the solid nature of the
high fat diet, diet was not lost due to spillage. Average
food intake per day for an individual rat was calculated by
averaging its food intake over a 4 to 8 day period before
killing. The individual daily average was used to calculate
the overall daily average for each strain. The four Fed
test groups were given their food cups at 1900 hours and the
cups were removed after 20 minutes. To calculate the 20
minute meal size, the cups were weighed before and after the

meal.

Wine

At the time of sample collection, a group of 8 rats--4
from each strain in a given treatment group (14%, Fed Sham,

Fed Drug, Non-Fed Drug, eth--were injected with pargyline

or vehicle, then sacrificed at 0, 20, 40, or 60 minutes post
injection. The rats were injected in reverse chronological
order (that is, the 60 minute rat was injected first) so
that the 0 time animal was injected last and killed within
30 seconds after injection. Each strain was injected this
way so that the sacrifice times were staggered. The injec-
tions were i.p. and were either 0.9% saline (vehicle) or
pargyline (Sigma Corp.) at 75 mg/kg in 0.9% saline. Approx-

imately 40 mg of pargyline were dissolved in 1.0 ml of 0.9%

24
saline and rats were injected with different volumes but no
volume exceeded 0.5 ml. The animals were injected and
sacrificed by decapitation in the animal room under red
lights between 1900 and 2030, the first hour 1 1/2 of the

usual feeding period.

Dissection

After decapitation, the brain was immediately removed
and the hypothalamus was dissected out using the procedure
described by Holman et a1" ('76). The hypothalamus was then
placed in a pretared plastic weighing boat, frozen on dry
ice and was held for up to 1 1/2 hours while sample col lec-
tion was being completed. The time between decapitation and

hypothalamus freezing was rarely greater than 3 minutes.

was

The process for 5-HT analysis used are very similar to
those described by Reinhard et al. ('80), Mefford ('80,
Mefford 8: Barchas ('80), and Shum et al. ('82). The hypo-
thalami were weighed frozen in the preweighed container and
then transferred to a glass homogenization tube that had 200
ul 0.1N perchloric acid and 50 ul of 1.75 ng/ul n-methyl 5-
HT (nm-SHT), the internal standard. After hand homogenizing
for about 60 seconds, the homogenization tube and pestle
were washed down with 100 ul of 0.1N perchloric acid and the
tube was covered with parafilm. The samples were homog-
enized in random order each time and were held at 40°F for
5-45 minutes until the rest of the sample were homoginized.

The homogenization tubes were centrifuged at 5,000 rpm in a

25
refrigerated Sorval RC-5 centrifuge for 10-12 minutes at 0°-
4°C. The supernatants were then decanted into a microfilter
centrifuge tube (B.A.S.) and centrifuged through a 0.2 um
microfilter for 1-2 minutes. The collected samples were
then pipetted into a disposable 20 x 20 cm culture tube,
placed on dry ice, stored at -40°C and assayed for 5-HT and
S-HIAA within 12 hours.

Due to high overall sample loss in the first half of
the samples, 80, and the second half of 80 samples received
slightly different processing. The second half was processed
as described above except 25 ul of 0u5% sodium bisulfate and
25 ul of 0m2M EDTA were added to the homogenization tube to
reduce sample loss. Also, after the samples were pipetted
into the disposable culture tube, they were placed on
crushed ice and assayed for 5-HT and 5-HIAA within 3 hours
without being frozen. The supernatant yield was estimated
to be 80-190 ul.

All samples were assayed for 5-HT and 5-HIAA by high
performance liquid chromatography with electrochemical de-
tection (LCEC) which has been shown to be an accurate and
efficient means of 5-HT quantification (Warsh et al., '80).
The LCEC system consisted of a B.A.S. LC 4 amperometric
detector with a Watman reverse phase column (OD 5-3). The
mobile phase consisted of monochloroacetic acid buffer
(0.1M, pH, 3.0) containing 0.012% sodiumoctyl sulfate and 7-
9% acetonitrile. The flow rate was set at approximately 7.5
ml/minute and the column was operated at ambient tempera-

ture. The electrochemical detector was set at 2nA/volt with

26
a potential of 0.56 volts with the recorder set to give a
full scale deflection with one volt.

The 5-HIAA, 5-HT, and N-M-SHT (N-Methyl-SHT) standards
were diluted in 0.1N perchloric acid to a concentration of
0.1 ng/ul. Curves for all standards had an R2 value of at
least .97. Due to rapid decay of the 5-HIAA standard it was
mixed frequently.

For each sample, the ng of 5-HT and 5-HIAA/gm hypo-
thalamus was calculated by solving the regression equation
(Y .-. b0 + b1(X)) for x, multiplying by ul dilution of total
sample/ul injected, adding the % loss indicated by nm-SHT
recovery, and dividing by the hypothalamus weight. The
ng/gram of hypothalamus at each time point was used to
calculate the slope (D1) of 5-HT accumulation and 5-HIAA

decline.

533311113:

The data were evaluated by two methods. A Bonferoni T
test for multiple comparisons was used because it could
incorporate the SSe for each slope into the test statistic.
Since the first and second set of animals received slightly
different processing, the slopes and variances of each test
group from each set were calculated, then the average means
and variances were averaged together to allow for the pos-
sible different influences of the processing differences. A
two-way analysis of variance on the slopes derived from each
sample collection was done to compare results with the

Bonferoni T.

27
To determine if any differences between base levels in
any of the groups existed, a two-way ANOVA was done on the 0
time value. A student's T test and a test of linearity was
done on each drug group 5-HT slope to test whether it was
different than 0 and to see whether a linear response was

produced.

RESULTS

W

During the final week before killing, and after rats
had adjusted for at least 1 1/2 weeks to the meal-feeding
schedule, S rats gained 31 11 gms per week while OM rats
gained 32 1 1 gms per week. When they were killed, mean
weights 1; SEM were 163 :1; 3 for the S and 181 1 3 for the

Osborne-Mendell rats.

W

Average food intake for the 2-hour feeding period was
8.8 g; 0.2 SEM gm/day for S rats and 9.5 3; 0.2 for CM rats.
The average 20 minute meal for the Fed test groups was 6.9
3 0.96 gms for S rats and 7.8 1; 0.4 for CM rats. OM rats ate
significantly more in both the 2-hour and the 20-minute meal
(P < 0.05) than S rats.

W

Comparisons of 5-HT accumulation between the 2 strains
of rats and between the various treatments are presented in
Table 2 as average slopes + SEM and Figures 3-8 as graphs of
each comparison. In the Fed vs. Non-Fed Drug comparisons
(see Figures 3 a. 4) the S Non-Fed slope was 11.78 ng/g min.
greater than the S Fed slope (P < OCH” while the 0M Non-Fed
was 3.07 ng/gm min. greater than the 0M Fed (P > 0.10).

28

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30

Figure 3: Fed vs. Non-Fed Comparison of 5-HT Accumulation
in the S Rat After Injection of Pargyline.

5HT/HYPOTHALAMUS (nglg)

4400 A.

4008.

 

3600

3200

2800 .

2400 .

2000 .

1600 .

1200

 

' NONFED, b
----A FED

 

31

S DRUG

1= 23.86 ng/min

b1: 12.08 nol min -

 

' U

20 40 60
POST INJECTION (min)

Figure 3

32 ‘

Figure 4: Fed vs. Non-Fed Comparison of 5-HT Accumulation
Rate in the 0M Rat After Injection of Pargyline.

5HTIHYPOTHALAMJS (ng/g)

3600 b

 

3200

280

2400

2000

1800

1200

 

33

OM DRUG

"'— ‘3 NON FED, b1= 15.94ng/min

——- a FED, b1 =12.87 ng/min

 

I I l

0 20- 40 60

POST INJECTION (min)

Figure 4

34

Figure 5: Strain Comparison of 5-HT Accumulation Rate in
the Fed State After Injection of Pargyline.

5HT/HYPOTHALAMUS (nglg)

4000 _

—'-_

3600

3200 ,

2800 .

2400 .

2000 .

 

35

FED DRUG
a s . b1= 12.08ng/min

 

-....- . OM, b1=12.87ng/min

9 D

 

\

n
\
\
\
\
\
\
\
D
p

.9 55D DD.
’\
\

0 20 40 60

POST INJECTION (min)

Figure 5

36

Figure 6: Strain Comparison of 5-HT Accumulation Rate
in the Fed State After Injection of Vehicle.

5HT/HYPOTHALAMUS (ng/g)

 

37

FEDSHAM

——-O S b1 =3.80nglmin

3200 -----~ 0 0M h1 a1.31nglmin

 

 

 

 

 

280
2400 o o
. o
o o o
o . . g
200 o 0 __°__, .
:f' ___:IF ________ 0.. ________ ..°
0 o
1600 . o 3 2
o . .
o
0
ml .
4 I
O 20 4O 60

POST MOTION (min)

Figure 6

38

Figure 7: Strain Comparison of 5-HT Accumulation Rate in
the Non-Fed State After Injection of Pargyline.

5HTIHYPOTHALAMUS (ng/g)

4400

S

39

NON FED DRUG

. '—"—"' ' 3. b1 =23.86nglmin
---- 9 0M, b1215.94nglmin I

36001, a

3200

2800

24OO_

2000 .

 

 

fl T r I
0 20 40 so
POST INJECTION (min)

Figure 7

AO

Figure 8: Fed Drug vs. Fed Sham Comparison of 5-HT
Accumulation Rate in the S Rat.

 

 

 

41

 

 

 

s FED
4000,
-—~ DRUG b1=12.08ng/min
3600 . --- o SHAM b1= 3.08ng/min A
A
E"
a 3200. 4
5
a, A
g 2800. A
<
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5:: .
2400. A
8 . z/
1
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D 000 .. . _______ 43 ------ -:
I " ________ -2“ . .
IO :0
1600. '
O
. O
I2OOJ.
a -
o l I
20 40 go—

POST INJECTION (min)

Figure 8

 

42

In the Fed state the 5-HT accumulation rate was similar
in the 0M and S rat whether or not they were injected with
pargyline (Figures 5 & 6, respectively).

In the Non-Fed drug state the slope of the S rat was
7.92 ng/g min. greater than the 0M rat. However, due to
high variability, the difference was not significant.

Comparison of Fed drug vs. sham injections in the Fed
state within Strains (Figure 8 for S, and Figure 9 for CM)
produced a slope in the drug group that was 8.28 ng/g min.
greater and 11.56 ng/g min. greater in the S and 0M rat,
respectively. These differences in the 5-HT accumulation
rate were not significant.

The analysis of variance on individual slopes is pre-
sented in Table 3. The F value indicated a significant
variation in treatments. The Bonferoni T for nonorthoganal
multiple comparisons on the average b1 for a test group, did
not shOw a difference in any of the comparisons that were
done on slopes calculated for a whole test group. Only the
Non-Fed drug groups from both strains combined vs. the Fed
Sham groups from both strains were able to produce a signifi-

cant difference (p < 0.05).

5:fllAA.Decline

Comparisons of S-HIAA decline expressed as slope are
presented in Table 4. 'No comparison of strain within treat-
ment (Fed Drug, Non-Fed Drug, or Fed Sham) produced a dif-
ference with the Bonferoni T test. Nor did the slopes

appear to have any relation to their S-HT counterparts which

43

Figure 9: Fed State Drug vs. Sham Comparison of 5-HT
Accumulation Rate in CM Rats.

SHT/HYPOTHALAMUS (nglg)

360

3200

2800 .

2400 .

2000 .

1600 .

1200

 

K‘

—--- O

44

OM FED
DRUG b1=12.87ng/min

SHAM h1 = 1.31 ng/min

20 40
POST INJECTION (min)

Figure 9

b}.

 

45

TABLE 3

ANOVA for Estimations of b or
S-HT Accumulation Rate Based on
One Sample Per Time Point

 

Source of Variation df MS F

Strain l 74.46 .3560
Treatments 2 1200.32 5.739*
Interaction 2 214.00 1.0236

Error 30 209.14

46

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47
may be due to the large standard error in relation to the
absolute values of the slopes.

The Fed Drug vs. Non-Fed Drug treatment comparison
within strains shows no differences and do not appear related
to their 5-HT counterparts for reasons indicated above.

The Fed Drug vs. Fed Sham comparison did not show any
difference though the declines appear to be greater in the

drug group.

Wants
The S SB/P1 rat had an average hypothalamus weight of

47.1 g 0.9 mg while the 0M rat had 50.4 31.1 mg. The
differences were significant with 95% confidence (n = 154).

W

The mean 5-HT levels at each time point in each group 1
the standard deviation are presented in Figure 10.

W. The mean amounts 1 S.E.M. of 5-HT and
S-HIAA and ANOVA are presented in Tables 5 and 6,'respec-
tively. ANOVA results indicate that the values were similar
in each group for both 5-HT and S-HIAA.

QQ_flin._lalne§. The mean 60 minute post injection
values and the ANOVA is presented in Table 7. The F value
for treatments was significantly different. A Bonferoni T
test for the following four non-orthoganal comparisons was
done: Fed vs. Non-Fed strains combined, Fed Drug vs. Fed
Sham strains combined, S Fed Drug vs S Non-Fed Drug, and OM
Fed Drug vs. 0M Non-Fed Drug. The Fed Drug vs. Fed Sham

comparison was the only one that was significant (P < 0.01)

48

Figure 10. Bar Graph of Mean 1 3.0. S-HT Accumulated
in Each Treatment Group at Each Time Point.

49

 

 

 

  

 

 

 

 

 

I— J
' fi................
9
r
. ...
........‘
I.LJ.L.Q.IIAA
% I n
g 1
L L ...
' fv‘..........
.....
L J
r _'

 

 

0.
000000000...

I

[:3 NON-FED DRUG

E3723 FED DRUG

- FED SHAM

.9.

w......

 

 

l.

 
       

 

1P

 

OJ
3200
2800
240 T
200:.
1600

(616“) ShWV‘IVHlOdAH/lHQ

Figure 10

 

40

20

60

40

20

POST INJECTION (min)

Amount of 5-HT* in the Hypothalamus
at Zero Time in OM & S Rats

50

TABLE 5

 

5-HT

(No/g)
Treatment OM
Fed Drug 1967193* 21141118
Non-Fed Drug 20091156 20161164
Fed Sham 1807196 18331109

*Mean 1_S.E.M.

 

 

ANOVA for 0 Time S-HT Values

for All Treatment Groups

 

 

Source of Variation df MS F
Strain 1 1354 .0133
Treatments 2 9102.5 .0898
Interaction 2 796.3 .0079
Error 30 101,403

51

TABLE 6
Amount of 5-HIAA in

the Hypothalamus at Zero
Time in the OM & S Rats

S-HIAA in hypothalamus at 0 Time

 

(Ne/g)
Treatment OM S
Fed Drug 847.1346 884.1113
Non-Fed Drug 763.114S.3 740.1147.5
Fed Sham 708.1108.7 745.183.6

 

 

ANOVA for 0 Time S-HIAA Values
for All Treatment Groups

 

Source of Variation df MS F

Strain 1 2440.4 .00378
Treatments 2 6542.8 .10145
Interaction 2 3402.9 .00527

Error 30 645,157

52

 

TABLE 7

Amount of 5-HIAA in the Hypothalamus
at 60 Min. Post Injection
in OM & S Rats

 

OM S
Fed Drug 28321156 27921232
Non-Fed Drug 27991265 33871240
Fed Sham 17191164 2049194

 

 

ANOVA of 60 Min. Post Injection S-HT
Values in All Treatment Groups

 

 

Source of variation df MS F
Strain 1 388176 1.772
Treatments 2 3202928 14.618*
Interaction 2 195641 0.893
Error 30 219113

f , 2, 30

.05 = 3.32

53
while the S Fed vs. Non-Fed was only close to the critical

value.

Wrestling
The four drug-injected groups had slopes significantly

greater than 0 (P < 0.05) and no line was shown to be non-

linear (P < 0.05).

DISCUSSION

'Compared to a previous study (Schemmel unpublished
data) the average daily food intake for S rats was 93% of ad
libitum while 0M intake was 69% of ad libitum. The S rat
consumed more diet per gram of body weight than the OH in
this study which is consistent with ad libitum feeding. In
the Fed test groups the average 20 minute meal intake was
74% and 68% of the two hour meal for the 0M and S rats
respectively. This indicated that a considerable amount of
food had been consumed for both groups at the time of sample
collection.

The pattern of weight gain was also different when
compared to ad libitum weight gain. While both strains
continued to grow after adjusting to the meal feeding sched-
ule, the S and OM rats were 86% and 59% of their respective
ad libitum weights at 7 1/2 weeks of age.

The results can lend qualified support to the overall
hypothesis that, when sustained on a high (calorie dense)
diet, the S rat will consume less food and have greater
serotonergic activity in response to feeding than the 0M
rat. First, the S Non-Fed 5-HT accumulation tended to be
'significantly greater than the S Fed (p < 0.10). Second,
the OM Non-Fed accumulation was also greater than the 0M Fed
but the difference was less than the S Non-Fed vs. Fed

54

55

comparison and was not significant. Also, of equal
importance, the S rat ate 0:7 gms less food per day in the
2-hour feeding and 0.9 gms less during the 20-minute meal
periods than the 0M rat (p < 0.05). The conclusion that the
S rat had a greater serotonergic activity is qualified
because the strain comparison in the Non-Fed state indicated
that though there was a difference in the S accumulation
rate (23.86 ng/g/min) and the OM rate (15494 ng/g/min), it
was not significant.

The secondary hypothesis is rejected by these results.
They suggest that serotonergic activity, as measured by 5-HT
production, decreases with food intake. If both hypotheses
were true, a greater activity would be expected in the Fed
state of the strain that ate less. Since a greater pre-meal
serotonergic activity was associated with the strain that
consumed less diet, this could indicate that the greater the
serotonergic activity before a meal the smaller the meal
will be. This conclusion is inconsistent with the findings
of Blundel et alt ('76) which through examination of the
temporal effect of fenfluramine, suggested that serotonergic
activity increased after the onset of feeding. So the
results associate a greater serotonergic activity with the
animal that consumed less food and suggests that increased
serotonergic activity before feeding, instead of in response
to feeding, would cause decreased food intake.

It may be postulated that since the S rat had a greater
difference in 5-HT activity between the Fed vs. Non-Fed

state than the OM and ate less than the 0M rat, the S rat

56
may be more responsive to energy or food intake than the 0M
rat. These points may explain part of the mechanism which
enables the S rat to maintain the same energy intake when
fed a high fat--calorie dense--diet.

The lack of difference between the Fed Sham vs. Fed
Drug groups in each strain (Figures 8 and 9) is inconsistent
with the expected drug effects (Morot-Gaundry et al. '74,
VanLoon et al. '81) but does not necessarily indicate lack
of drug effectiveness. What the statistics imply here is
that the variance of the slopes is great enough that they
could be parallel. However, the observed slope trends indi-
cate that they are different and the Bonferoni comparison of
the levels of 5-HT at the 60 minute time point shows that
the Fed Drug groups accumulated more 5-HT than the Fed Sham
(p < 0.01). This shows that the drug was effective in
causing 5-HT to accumulate to levels significantly greater
than the Sham state.

Comparison of the results in this study with other
studies that have examined the relationship of serotonin and
appetite is difficult because no study has examined hypo-
thalamic serotonergic activity before and after food intake
by measuring 5-HT accumulation.

Finkelstein et al. ('81) has compared hypothalamic and
other brain region 5-HT levels in obese and non-obese Zucker
rats. They found approximately 1300 ng 5-HT per gm hypo-
thalamus of both types while in this study the average 0
time 5-HT level 1962 1 52 ng per gm in all groups. The

differences here may be, in part, because Finkelstein used a

 

57
flurometric assay method while this study used the LCEC
method. Garthwaite et a1" ('79) examined whole brain 5-HT
levels in obese (ob/ob) hyperglycemic mice but the results
cannot be compared to this study since the whole brain was
assayed and a flurometric method for assay was used.

In this study, no treatment differences were found when
ANOVA was done on base levels (O-time) of 5-HT while a
definite trend for a difference was found in the slope
comparison of the S Non-Fed vs. Fed groups. This finding
supports the earlier assertion that examination of neuro-
transmitter activity is more important than neurotransmitter
levels for assessing the role of a neuropathway in behavior.

In studies that examined the use of LCEC for assay of
5-HT, Reinhard et al. ('80) found 1080 1 80 ng/gm hypothala-
mus and Mefford & Barchas ('80) found 841 1.59 ng/gm hypo-
thalamus using an LCEC technique very similar to the one
used here. These values are almost half the average 0 time
amount found in this study, 1962 1 52 ng/gm hypothalamus,
but the S.E.M. is very similar.

Reports by Anderson ('79), Curzon ('81), Boadle-Biber
('82) and Wurtman & Fernstrom ('76) indicate that a high
protein (low carbohydrate) as opposed to a low protein (high
carbohydrateQ meal would decrease the availability of Trp
for neuronal uptake since the protein would increase the
amounts of (naa) that compete for uptake. In this study it
may be possible that the diet had this effect since it was
relatively high in protein and low in carbohydrate. Also,

the study by Li ('82) indicated that as the protein intake

58
of a meal increased 5-HT levels in the whole rat brain would
be decreased when they were meaSured 20 minutes after the
meal.

However, none of the reports reviewed to date has
measured 5-HT production before and after a high fat or any
other type of meal. Whether the meal caused a decrease in
5-HT production or whether the observed change was due to a
change in TH activity cannot be determined from this study.

Though 5-HIAA declined as expected in response the
pargyline, no differences in decline were detected with the
Bonferoni T test. The high variability, combined with the
lower stability of 5-HIAA as indicated by rapid decay of the
S-HIAA standard, may have caused the variation to be too
high to detect differences in 5-HIAA decline.

The greater hypothalamus weight in the 0M rat did not
appear to contribute extra 5-HT since the OM accumulation-
rates tended to be lower. The greater weight is consistent
with findings of Stone et al. ('81) that GM rats have
greater absolute cerebrum weights.

Individual animal variation, the assay technique, and
the method for assessing serotonergic activity may have
contributed to the large differences in individual sample
values at any given time point. Since an individual animal
had to be killed for each sample at each time point, the
different individual responses to the drug and each animal's
variation in enzyme activity would add some variation to the
results. The LCEC was very consistent and had a 1-3% error

with injection of standards, which is similar to previous

59
studies (Reinhard '80). Twenty to fifty percent sample
decay was seen with duplicate injections in the first set of
samples which was due to storage and freezing. Also, 30%
overall decay was observed to be caused by freezing. When
the samples were assayed immediately after processing and
antioxidants were added to the homogenate solution 0 to 3%
difference was seen in duplicate injections. The decay in
the first half of the samples used to calculate the results

could have induced a considerable degree of error.

W
In the preliminary sample collection, the Fed Sham

groups in each strain had slopes that were 0 and the Non-Fed
Sham groups with 2 values per time point showed that they
had a slope of 0. So, to conserve animals and time, the
Non-Fed Sham group was omitted from the study.
QhQi££_Q£_§Laiifiilnal_flfiihad

The Bonferoni T test is the method of choice because it
incorporates the variance about each test group slope into
the test statistic and more degrees of freedom can be used
than if ANOVA was done on the average slope (b1). Both of
these aspects increase the power of the Bonferoni test. An
ANOVA on individual slopes can only incorporate the variance‘
between slopes derived from one sample at 3 or 4 time
points. So, the degrees of freedom are much less than the
Bonferoni T and variation is likely to be greater since the

slopes are based on one value per time point.

SUMMARY & CONCLUSIONS

Pharmacological and feeding studies have linked sero-'
tonin with regulation of food intake in laboratory animals.
Whether serotonergic activity is different in two rat
strains, the Osborne-Mendell (0M) and S 5B/P1 (S), that
respond differently to a high fat diet and whether sero-
tonergic activity is increased after feeding are examined
here. Hypothalamic 5-HT turnover was measured in CM and S
rats by using an MAO inhibitor (pargyline) to assess the 5-
HT production rate. 5-HT production can be linked with
serotonergic activity since TH (Tryptophan Hydroxylase) can
be induced from neuronal activity.

Both strains were divided into 3 groups: Fed Drug,
Non-Fed Drug, and Fed Sham. All groups were fed a calorie-
dense, high fat diet and were adapted to eat their daily
food intake in a 2-hour meal at the start of the dark cycle.
The Fed groups were given the food cups for 20 minutes of
the usual 2-hour meal before being sacrificed, while the
Non-Fed groups were not allowed to feed before sacrifice.
At the time of sacrifice, the hypothalamus was removed,
weighed, and homogenized in 0.1 N perchloric acid with an
internal standard. The homogenate supernatant was assayed
for 5-HT using LCEC. After injection of drug or vehicle,
one rat from each strain in each treatment group was

60

61

sacrificed at 0, 20, 40, and 60 minutes post injection. The
rate of 5-HT production, or accumulation, was compared
between the groups to determine if there were differences in
serotonergic activity between strains and between the Fed
and Non-Fed state.

Both strains had greater 5-HT accumulation in the Non-
Fed state than in the Fed state. Only the S rats had a
significantly greater difference (P < 0.10)‘while the 0M
difference did not produce a significant trend. Strain
comparisons in the treatment groups produced no significant
trends through the S slope was greater than the 0M and both
strains had very similar slopes in the Fed state. The
ability of the S rat to show a significant difference in the
Non-Fed vs. Fed comparison while the 0M could not lends
support to the overall hypothesis that the S rat will have a
greater serotonergic activity than the OH. The Fed vs. Non-
Fed comparisons indicate that serotonergic activity is re-
duced after feeding and does not support the hypothesis that
serotonergic activity would increase. These conclusion are
preliminary and indicate that further strain comparisons of
5-HT activity may show some strain differences and further
evaluation of the relationship of food intake to serotoner-
gic activity is needed to clarify the role of serotonin

pathways in appetite regulation.

POSSIBLE FOLLOW-UP STUDIES

Follow-up studies that extend the design should incor-
porate the protein:carbohydrate ratio as a factor. This
would show whether the high protein diet actually suppressed
Trp production here since high carbohydrate intake has
been reported to cause more Trp uptake into the neuron
(Curzon '81; Wurtman & Fernstrom, '76). So, in the Fed
state, 5-HT accumulation from a high carbohydrate diet would
be expected to be greater than from a high fat diet and
possibly could show differences in 5-HT turnover between the
strains. Also, further trials should measure plasma Trp:naa
ratio and hypothalamic Trp levels to examine their effects
on 5-HT production.

In light of the feeding studies and drug studies that
evaluate food choices (Wurtman & Fernstrom, '79; Orthen-
Gambill & Kanareck, '82), it would be interesting to examine
the choices of fat, carbohydrate and protein between the two
strains with and without the administration of a serotoner-
gic drug. Also, the temporal pattern of food intake would
be another aspect that could be examined with relative ease.

To really compare serotonergic activity, one would want
to measure the amount of 5-HT released into the synapse.
Since reliable methods for this are still being developed,

we have to rely on more indirect measures. The present

62

63
method may not be as sensitive as needed to detect differ-
ence in serotonergic activity. This method may be more
sensitive if micro punches of specific nuclei regions were
sampled instead of the whole hypothalamus since it is possi-
ble that different nerve bundles could mediate different
aspects of feeding and thus have different responses to a

given stimuli which together may offset each other.

APPENDIX

64

 

 

 

 

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