Regulation of Mammalian Hibernation. John Thomas Burns Louisiana State University and Agricultural & Mechanical College

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1977 Regulation of Mammalian Hibernation. John Thomas Burns Louisiana State University and Agricultural & Mechanical College

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University Microfilms International 300 North 2eeb Road Ann Arbor. Michigan 48106 USA St John's Road. Tyler s Green High Wycombe. Bucks. England HP10 8HR BURNS, John Thomas, 1943- REGULATION OF MAMMALIAN HIBERNATION,

Louisiana State University and Agricultural and Mechanical College, Ph.D., 1977 Zoology

Xerox University MicrofilmsAnn , Arbor, Michigan 46106 Regulation of Mammalian Hibernation

A Dissertation

Submitted to the Graduate Faculty of the Louisiana state University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy

The Department of Zoology and Physiology

by John Thomas Burns B.A., Wabash College, 1965 M.S., Louisiana State University, 1968 August 1977 EXAMINATION AND THESIS REPORT

Candidate: John Thomas Burns

Major Field: Vertebrate Zoology

T itle of T hesis: Regulation of Mammalian Hibernation

Approved: Jlbf U. M ajor Professor and Chairman

) if h )/, r Dean I of the Graduate School

EXAMINING COMMITTEE: o

Date of Examination: J u n e 2 8 , 1977 ACKNOWLEDGEMENTS

Dr. Albert H. Meier encouraged and helped me through

out my graduate education. His concise logic and his

ability to design meaningful experiments continue to be

an inspiration to me. Most of the following studies were based on his ideas.

My graduate committee worked diligently to provide

just criticism of this dissertation. Several persons helped catch ground squirrels, including Paul Shepard,

Pat and Debbie Burns, and my wife Anne. Anne has been a

loving wife in addition to working to supply most of our

income. My parents and family also have been a source of encour agement and support. TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ...... ii

LIST OF FIGURES ...... iv

ABSTRACT ...... vi

INTRODUCTION ...... I

MATERIALS AND METHODS ...... 13

RESULTS ...... 24

DISCUSSION ...... 53

LITERATURE C I T E D ...... 65

VITA ...... 75

iii LIST OF FIGURES

Figure Page

1* Daily rhythm in insulin's lethal effects in golden hamsters (Mesocricetus auratus) ...... 26

2. Hibernation in alloxan-treated and saline-treated thirteen-lined ground squirrels (Citellus tridecemlineatus) . . . 28

3. Summer hypothermia in thiouracil- treated and untreated thirteen-lined ground squirrels (Citellus tridecem lineatus ) 31

4. Concentrations of plasma corticosterone at 6 times of day in thirteen-lined ground squirrels (Citellus tridecem lineatus ) maintained on a 14-hour daily photoperiod during February ...... 3 3

5. Concentrations of plasma corticosterone at 6 times of day in thirteen-lined ground squirrels (Citellus tridecem lineatus ) maintained on a 12-hour daily photoperiod during Ma y ...... 35

6. Plasma corticosterone concentrations in male and female thirteen-lined ground squirrels (Citellus tridecemlineatus) sampled throughout the day during February and M a y ...... 37

7. Hibernation in thirteen-lined ground squirrels (Citellus tridecemlineatus) during 27 days following injections of prolactin at either 12 or 20 hours after corticosterone injections ...... 39

8. PCPA effects on hibernation in thirteen- lined ground squirrels (Citellus tri decemlineatus) ...... 42

iv Figure Page

9. Arousal from hibernation in thirteen- lined ground squirrels (Citellus tridecemlineatus) 2 days after handling, saline inj ections, or PCPA injections .... 44

10. PCPA effects on the body weight of male golden hamsters (Mesocricetus auratus) ...... 47

11. PCPA effects on the body weights of male and female thirteen-lined ground squirrels (Citellus tridecemlineatus) .... 49

12. PCPA effects on the weight of the paired ovaries, the paired oviducts, and the abdominal fat pads of golden hamsters (Mesocricetus auratus) ...... 51

13. Serotonergic and noradrenergic pathways involved in the regulation of hypothermia and hibernation ...... 63

v ABSTRACT

Neural and endocrine mechanisms were investigatea with

regard to the regulation of hibernation in thirteen-lined

ground squirrels (Citellus tridecemlineatus) and golden

hamsters (Mesocricetus auratus). Hibernation in thirteen-

lined ground squirrels was inhibited with injections of

alloxan (prevents insulin production), p-chlorophenylalanine

(PCPA) (blocks serotonin synthesis), and two different

temporal relationships of corticosterone and prolactin.

Prolactin injections given 20 hours after daily injections

of corticosterone for 11 days during the hibernation season

prevented hibernation. Prolactin given 12 hours after cor

ticosterone markedly reduced the occurrence of hibernation.

This effect indicates that a temporal synergism of corticos

terone and prolactin may be part of a circannual mechanism

that regulates seasonal physiological and behavioral changes

in the thirteen-lined ground squirrel. Plasma corticosterone

concentrations were greater in May females than in February

female ground squirrels and were more constant throughout the

day. Alloxan decreases the production of insulin that promotes brain serotonin levels by facilitating the trans

port of tryptophan (a precursor of serotonin) into the brain.

All results can be interpreted in terms of serotonergic neural mechanisms. A dampened corticosterone rhythm is characteristic of low thyroid activity, which may be expected when brain serotonin levels are high. High thyroid activity may contribute to an inability of thirteen-lined ground squirrels to hibernate during summer; thiouracil treatment allowed the occurrence of hypothermia during summer. PCPA caused weight loss, abdominal fat pad reduction, and weight gains in ovaries and oviducts, changes that contrast with those that occur before and during hibernation.

The role of neurotransmitters in the neural control of hibernation was tested in golden hamsters. Profound hypo thermia (6°C esophageal temperature) in cold exposed golden hamsters was induced in animals within hours following the injections of ct-methyl-p-tyrosine (blocking agent for the synthesis of noradrenalin), pargyline, and fluoxetine

(agents that enhance serotonergic activities). Injections of the drugs singly or in combinations of two were ineffec tive. Return of the hamsters to room temperature within 4 to 10 hours after induction of hypothermia allowed rewarming and survival. These results suggest that an active seroton ergic system and an inactive noradrenergic system are neces sary for the natural hypothermia of hibernation. The drug- induced hypothermia may have important medical applications.

vii INTRODUCTION

Mammalian hibernation is a physiological and behavioral adaptation by which animals may avoid the hardships (e.g., cold and lack of food) of the winter environment outside the hibernaculum. Mammalian hibernation is found in various species including ground squirrels, chipmunks, dormice, birchmice, woodchucks, bats, hedgehogs, tenrecs, and lemurs

(Lyman, 1963). The diverse phylogeny of hibernators may indicate that the ability to hibernate has developed in dependently in several groups and that hibernation results from the modification of mechanisms already present in nonhibernators. Unlike the poikilotherm in hibernation, the mammal (or bird) in hibernation has the capacity to produce sufficient heat to rewarm and arouse from hibernation in a cold environment. When subjected to temperatures below 0°C the hibernating mammal can increase its metabolic rate

(without arousing) and thus avoid freezing. In fact, hibernation often occurs in bouts or sessions of a few days followed by arousal and return to hibernation. Thus, the mammalian hibernator is an animal that has a wide range of thermoregulatory abilities (for reviews of hibernation see Kayaer, 1961; Lyman, 1963; Fisher, et al., 1967;

Mrosovsky, 1971; South, ^ t ^ l . » 1972; Davis, 1976).

1 2

The study of the regulation of mammalian hibernation developed rather slowly, probably due in part to the lack of appropriate instrumentation (Lyman, 1963)- Mammalian hibernation has received very little attention in comparison to the study of bird migration. Some of the most informative studies of mammalian hibernation have been investigations of endocrinological changes during the annual cycle of hibernators. Despite these studies of hormonal functions, no particular hormone has been shown to be responsible for hibernation and no general theory based on endocrine changes has been accepted.

A hypothermia referred to as "artificial hibernation" was induced with insulin and moderate cold in woodchucks

(Cassidy, Dworkin, and Finney, 1925; Dworkin and Finney,

1927), with insulin and Mg in hedgehogs (Suomalainen, 1939), and with insulin in hamsters (Suomalainen and Petri, 1952).

However, these hypothermic states were more similar to the loss of body temperature just before death than natural hibernation (Popovic, 1960). Moreover a recent study reports very low levels of plasma insulin in the hibernat ing hedgehog (Senturia and Johansson, 1974). Nonetheless, some modification of the technique of giving insulin injec tions might induce a state more similar to hibernation.

The time of day when hormones are injected is of 3 significance- The finding of a daily rhythm in the pigeon cropsac bioassay for prolactin (Burns and Meier, 1971,*

Meier, Burns, Davis, and John, 1971) made me aware of this fact. An additional understanding of insulin-induced hypothermia might result from injecting insulin at differ ent times of the day in golden hamsters (Expt. I).

If insulin producing cells of the islets of Langerhans are damaged by alloxan treatment (Dunn and Letchie, 1943;

Lukens, 1948; Lazarow, 1954) hibernation does not occur in the golden hamster (Mesocricetus auratus) (Helle, 1953).

The statement has been made (e.g., Kayser, 1961) that alloxan prevents hibernation only in species with nearly normal levels of blood glucose during hibernation. The thirteen-lined ground squirrel (Citellus tridecemlineatus) develops lower blood sugar levels during hibernation

(Lyman, 1943; Stuckey, 1942) and thus should not hibernate after alloxan treatment. Because of limited information, alloxan injections were given to hibernating thirteen-lined ground squirrels to determine if hibernation would continue

(Expt. II).

The thyroid differs in hibernators and nonhibernators in its response to cold. Cold exposure stimulated the thyroid in rats (Hoffman and Zarrow, 1958; Deane and

Lyman, 1954) but not in thirteen-lined ground squirrels 4

(Hoffman and Zarrow, 1958) nor in golden hamsters (Deane

and Lyman, 1954). The thyroid has a marked annual cycle

in various animals including the thirteen-lined ground

squirrel (e.g., Hulbert and Hudson, 1976) and other hiber-

nators (for review, Popovic, 1960) . Adler (1926) fed

hibernators dried thyroid glands and prevented their

hibernation. Popovic (1955) confirmed this finding in

European ground squirrels (Citellus citellus). On the

other hand, treatment of ground squirrels with thiouracil

(a goitrogen) might allow hypothermia to occur during the

summer; i.e., perhaps an active thyroid during the summer

hinders the development of hypothermia. An experiment

with thirteen-lined ground squirrels tested this

possibility (Expt. Ill).

The adrenal gland functions during hibernation and

arousal, with the adrenal medulla aiding in the spring

arousal of the thirteen-lined ground squirrel. Medullec-

tomized ground squirrels (a remnant of cortical tissue was

left i n situ) were unable to arouse from hibernation and

subsequently died (Hoffman and Hester, 1965). The adrenal

cortex is necessary for hibernation because adrenalectomized

European ground squirrels (Citellus citellus) fail to hibernate (Popovic and Vidovic, 1951; Vidovic and Popovic,

1954). Either a cortical graft to the anterior chamber of 5 the eye (Vidovic and Popovic, 1954) or deoxycorticosterone or cortisol injections for 2 or 3 days (Popovic, Vidovic, and Vidovic, 1957) enabled hibernation to take place.

Deoxycorticosterone or cortisol similarly permitted hibernation in the adrenalectomized European hamster

(Cricetus cricetus) (Kayser and Petrovic, 1958).

Daily rhythms of hormones are of functional importance.

Daily rhythms of corticoids (e.g., Meier and Fivizzani,

1975) and prolactin (e.g., Meier, Burns, and Dusseau,

1969) have a central role in the occurrence and scheduling of a myriad of seasonal physiological states and behaviors in a wide selection of vertebrates: For example, body fat gain or loss in Gulf killifish, the green anole (Meier,

Trobec, Joseph, and John, 1971), and the white-throated sparrow (Meier and Martin, 1971; Meier, Martin, and MacGregor,

1971), locomotor activity and orientation (Martin and

Meier, 1973), and reproductive sensitivity (Meier, Martin, and MacGregor, 1971) in white-throated sparrow, and the red eft water drive (Meier, Garcia, and Joseph, 1971). A regulatory system of changing relationships between daily hormone rhythms (a "temporal synergism," Meier, Trobec,

Joseph, and John, 1971) could exist in hibernators. To gather evidence concerning temporal synergisms, corticos- 6 terone assays were performed on blood plasma collected throughout the day from thirteen-lined ground squirrels

(Expt. IV). Corticosterone is the major corticoid for

Citellus tridecemlineatus (Huibregtse, Gunville, and Ungar,

1971) .

In addition to using hormone assays to ascertain the existence of seasonal changes of the daily rhythms of the temporal synergi sm, another approach is to inject hormones in different temporal relationships to induce seasonal physiological states or behaviors. For example, in the white-throated sparrow, corticosterone and prolactin were injected in various temporal relationships to elicit seasonal conditions including gonadal growth (Meier, Martin, and MacGregor, 1971), fat storage (Meier and Martin, 1971), nocturnal restlessness (Meier, 1969), and migratory orientation (Martin and Meier, 197 3). To test for a role of temporal synergisms in the thirteen-lined ground squirrel, a study was made of the effects of daily injections of 2 different temporal relationships of corticosterone and prolactin on the onset of hibernation (Expt. V).

Because neurotransmitters regulate the production and release of hormones, the role of neurotransmitters in hibernation was investigated. Serotonin, a neurotransmitter, has received some attention from investigators of hiber 7 nation. Spafford and PengelXey (1971) used an inhibitor of serotonin synthesis, PCPA (p-chlorophenylalanine), to prevent hibernation in the golden-mantled ground squirrel

(Citellus lateralis). Because hibernation is investigated so often in thirteen-lined ground squirrels, an experi ment with PCPA was performed to determine if serotonin is required for their hibernation (Expt. VI).

Kamberi (1973) concluded in a review that serotonin and melatonin decrease the release of pituitary luteinizing hormone (LH) and follicle stimulating hormone (FSH) and increase prolactin release (by inhibiting the hypothalamic neurohormones). Small doses of dopamine or large doses of noradrenalin (or adrenalin) increase LH and FSH and decrease prolactin release (by causing the release of the neuro hormones) (Kamberi, 1973) . In the LH-mediated process of ovulation, serotonin blocked ovulation in rats when injected systematically just prior to the daily "critical period" when progesterone induces ovulation (O'Steen,

1964). This effect also resulted from 5-hydroxytryptophan

(serotonin precursor) injections. PCPA, a serotonin synthesis inhibitor, stimulated super-ovulation when injected just prior to the critical period (Kordon, et al.,

1968). The function of serotonin in LH and FSH release was reviewed by Kordon and Glowinski (1972). 8

On the other hand, prolactin secretion increased after

serotonin administration (intracerebroventricular) in rats

(Kamberi,

treatment (intravenous infusions) in human beings (Mac Indoe

and Turkington, 1973). Either PCPA or methysergide (blocks

serotonin receptors) prevents the rise in prolactin levels

induced by suckling (Kordon, et _al., 1973; Gallo, Rabbi, and

Moberg, 1975). Fluoxetine, which enhances serotonin's

effectiveness by inhibiting serotonin reuptake, potentiated

the increase of serum prolactin levels induced by the action

of 5-hydroxytryptophan in rats (Krulich, 1975; Clemens,

Sawyer, and Cerimele, 1977). Growth hormone secretion also

increased in rats after serotonin injections (intraventricu-

lar) (Collu, tit _al., 1972) and the concentration of serum

growth hormone was greatly raised following 5-hydroxy-L-

tryptophan injections (intraperitoneal) (Smythe and Lazarus,

1973). Growth hormone release is inhibited by dopamine

(Collu, Fraschini, and Martini, 1973).

In general, the inhibition of serotonergic processes may be expected to diminish conditions that are dependent

upon the presence of prolactin or growth hormone and to

stimulate or allow conditions requiring the gonadotropins.

Inasmuch as obesity and gonadal inhibition are correlated with hibernation, a study was made of PCPA's effect on the 9

body weight of thirteen-lined ground squirrels, and on the

body weight, abdominal fat pad weight, paired ovary and

paired oviduct weights in golden hamsters (Expt. VII).

Serotonin and norepinephrine are involved in tempera

ture regulation (Vogt, 1954; Brodie and Shore, 1957). The

two neurotransmitters have antagonistic actions (Bligh,

1966a, b; Feldberg and Myers, 1963, 1964, 1965). Serotonin

injected into mice caused a dose related decline in body

temperature (Lessin and Parkes, 1957). Serotonin trig

gered heat loss and norepinephrine caused heat production

in the rat, goat, sheep, and ox, whereas, such injections

had just the opposite effects (for unknown reasons) in the

chicken, dog, cat, and monkey (Myers, 1970).

Various drugs that influence serotonin and norepineph

rine synthesis or activity also modify body temperature.

Changes in body temperature of as much as 6 centigrade degrees occurred after treatments with drugs (see the discussion for details) including PCPA (p-chlorophenyla-

lanine), which inhibits serotonin synthesis by inhibiting

tryptophan hydroxylase; fluoxetine, which enhances sero

tonin's effectiveness by preventing serotonin reuptake;

alpha-methyl-p-tyrosine, which inhibits norepinephrine

synthesis by inhibiting tyrosine hydroxylase; and reserpine 10

and pargyline, which inhibit monoamine oxidase (MAO).

PCPA elevated the body temperature of rats (McDonald

and Mueller, 1968) and prevented its decline in response

to morphine (Haubrich and Blake, 1971). The decline in body temperature in response to morphine was enhanced and prolonged by fluoxetine (Fuller and Baker, 1974). Reser- pine or alpha-methyl-p-tyrosine slightly lowered the rectal

temperature of rats (Williams and Moberg, 1975). Pargyline enhanced the decline in temperature in mice due to seroto nin injections (Ritzmann and Tabakoff, 197 6).

The natural hypothermia of mammalian hibernation and its relationship to serotonin is the subject of a few recent papers. The highest levels of a seasonal variation in brain serotonin content in the European hedgehog (Erin- aceus europaeus) occurred during hibernation (Uuspaa, 1963).

Serotonin content increased in the brain of the pale bat

(Antrozous pallidus) when hibernation occurred (Shaskan,

1969). However, in the Arctic ground squirrel (Citellus undulatus), the only significant change was a fall in brain serotonin levels during arousal (Feist and Galster, 1974).

A correlation was reported between higher brain serotonin levels and the ability to hibernate in both the golden- mantled ground squirrel (Citellus lateralis) and the round tailed ground squirrel (Citellus tereticaudus) and lower 11 brain serotonin levels and the inability to hibernate in

the antelope ground squirrel (Citellus leucurus? (Spafford

and Pengelley, 1971).

Arousal from hibernation is associated with a fall in

serotonergic activity coupled with a rise in noradrenergic

activity. The prevention of either of these changes can

interfere with arousal. Melatonin injections, which in

crease serotonin levels in the rat brain (Anton-tay, et al .,

1968), resulted in fewer arousals and longer bouts of hiber nation in the golden-mantled ground squirrel (Palmer and

Riedesel, 1976). Serotonin or 5-hydroxytryptophan injec

tions slowed the arousal of the suslik (Citellus erythro- genys major) (Popova, 1975; Popova and Kudryavtseva, 1975), and the inhibition of norepinephrine with alpha-methyl-p- tyrosine (a-MPT) slowed arousal in the golden hamster

(Mesocricetus auratus) (Feist, 1970). On the other hand, a precursor of norepinephrine, L-dopa, aroused the hiber nating hedgehog (Erinaceus europaeus) (Uuspaa, 1963).

The hypothermia of hibernation may depend on dissimilar levels of brain serotonin and noradrenalin. By simultane ously manipulating the antagonistic serotonergic and nor adrenergic mechanisms with drugs, a deep hypothermia might be induced in a cold-exposed hibernator. Attaining this condition may require a stimulation of the serotonergic 12

activity combined with an inhibition of the noradrenergic

activity. Therefore, 3 drugs, ct-MPT, pargyline, and

fluoxetine were selected for injection in golden hamsters

(Expt. VIII). Additional tests were made of each drug given alone and of the 3 possible combinations of 2 drugs.

Also, PCPA was given to some animals before treatment with

all 3 drugs to see if it blocked any effects. MATERIALS AND METHODS

General information is followed by specific details

for each experiment. Thirteen-lined ground squirrels

(Citellus tridecemlineatus) were housed in separate cages

(15 x 17 x 34 cm). Golden hamsters (Mesocricetus auratus)

were housed individually in a cage (15 x 17 x 24 cm) or

in groups of 2 or 3 to a cage (15 x 35 x 34 c m). Purina

rat chow and water were provided ad libitum. The room

temperature was approximately 22°C unless stated otherwise.

All time information was recorded as C.S.T. Rectal and

esophageal temperatures were determined with a telether

mometer (Yellow Springs Instrument Co.) equipped with a

small animal thermistor probe. The chemicals injected were obtained from sigma Chemical Company, St. Louis,

Missouri, except for the following: fluoxetine, Lilly

110140 or 3-(p-trifluromethylphenoxy)-N-methyl-3-phenyl- propylamine hydrochloride, was a gift from the Lilly

Research Laboratories, Indianapolis, Indiana, and ovine prolactin (NIH-P-S-12, 1 mg = 35 International Units) was a gift of the Endocrinology Study Section of the

National Institutes of Health.

13 14

Insulin (Expt. I)

Thirty-two female golden hamsters, 10 to 11 months

old, were maintained on a 10-hour daily photoperiod

(0700 to 1700). The hamsters were housed 2 or 3 to a

cage. Insulin injections (10 International Units of bovine insulin of 0.1 ml of 0.85% saline given i.p.) were made at the following times: 0400 (7 animals), 1000 (8

animals), 1600 (8 animals), and 2200 (9 animals).

Injections were begun on October 29, 1976 and continued daily for 9 days.

Alloxan (Expt. II)

The 12 thirteen-lined ground squirrels were adults collected from the golf course at North Texas

State University in Denton, Texas during the first week of August 1976. The squirrels were maintained on a 14-hour daily photoperiod (0500 - 1900). To allow for hibernation, the caged squirrels were placed into refrigerators on

October 8, 1976. Hibernation occurred in each squirrel by October 16, 1976. The control group (3 males and

3 females) received injections of 0.85% saline and the experimental group (3 males and 3 females) received injec tions of alloxan in 0.85% saline. When alloxan injections 15

were given, the control group received 0.85% saline injec

tions of an equal volume. Injections were made daily

between 1100 and 1300. Alloxan injections were single

doses or simple multiples of 12.5 mg alloxan in 0.1 of

0.85% saline. Each alloxan-treated squirrel received the

following: 10/24/76, 10/26/76, and 10/28/76, single doses;

10/30/76, a quadruple dose; 11/3/76, a double dose; 11/5/76,

a single dose. Until 11/7/76, daily checks (between 1100

and 1300) for hibernation were made and the rectal tempera

ture of inactive squirrels was determined.

Thiouracil (Expt. Ill)

The thirteen-lined ground squirrels were adult

females collected from the golf course at the Leavenworth

Country Club, Leavenworth, Kansas in early April, 1973.

The squirrels were maintained on a 12-hour daily photo

period (0600 - 1800). A control group of 6 squirrels

received normal drinking water and an experimental group

of 6 squirrels received 1% thiouracil in the drinking water beginning July 22, 197 3. After two days, the caged squirrels were placed in dark refrigerators (4 to 8°C) and checked

(5 times) for hypothermia (between 1100 and 1300) every

two or three days until August 8, 197 3. The rectal temper

ature of inactive squirrels was determined by use, in this 16

experiment only, of a phyaiograph equipped with a thermistor

bridge and a rectal probe. Squirrels with a rectal temper

ature between 4 and 11°C were considered to be hypothermic.

Corticosterone Levels (Expt. IV)

Thirteen-lined ground squirrels were sampled through

out the day to measure levels of plasma corticosterone in

February and May 1973. The February ground squirrels were

30 adults (8 males and 22 females) collected in June 1972

from the golf course at Leavenworth Country Club at

Leavenworth, Kansas. They were maintained on a 14-hour

daily photoperiod (0600 - 2000). Six groups of 5 squirrels

were established (each group had only 1 or 2 males). A

different group was bled at each of six times of the day.

The dates and times for the samples were as follows:

February 17, 1973 at 0700, 1100, 1500, 1900, 2300, and

February 18, 1973 at 0300.

The May ground squirrels were 35 adults (18 males

and 17 females) collected in early April 197 3 from the

golf course at Leavenworth Country Club. They were maintained on a 12-hour daily photoperiod (0600 - 1800).

Six groups of squirrels were established with the groups

as similar as possible in number and sex ratio. A

different group was bled at each of six times of the day. Tfte dates and times for the samples were as follows

May 14, 1973 at 0600, 1000, 1400, 1800, 2200, and May 15,

1973 at 0200.

The blood was collected in heparinized capillary

tubes after cutting the tip of the tail with a razor

blade. Capillary tubes were plugged with clay and

centrifuged for 20 minutes. Plasma was stored in a

freezer until assays were made. Plasma corticosterone

levels were determined by a slightly modified version

of a competitive protein-binding radioassay technique

(CBG) first described by Murphy (1967). Human plasma was the source of CBG. Samples were extracted with

ethanol. Florisil was the absorbant for labeled corti

costerone and plasma corticosteroids. For each sample

a triplicate determination was made of corticoid levels;

these values were averaged. Tritium counting was with a

Beckman Liquid System and with a toluene scintillation solution.

Corticosterone and Prolactin (Expt. V)

The thirteen-lined ground squirrels were adults collected from the golf course at North Texas State

University in Denton, Texas during the first week in

August 1976. They were maintained initially on a 14-hour 18

daily photoperiod (0600 - 2000) and then placed on continu

ous light beginning September 1, 1976. To observe the

effects of daily injections of corticosterone and prolactin

in two different temporal relationships (a 12-hour and a

20-hour relationship) 3 groups of ground squirrels were

studied. Each group had 4 males and 4 females, except

for the 20-hour relationship group that lacked 1 female.

One group received no injections, a second group received

injections of corticosterone at 1800 and prolactin at

0600, and the third group received injections of corti

costerone at 1800 and prolactin at 1400. Corticos

terone (200 gg/squirrel) was injected every other day at

1800 (beginning on September 15, 1976). Ovine prolac

tin (200 V g/squirrel) was injected daily (beginning Sep

tember 16, 1976) at 1800 (12-hour relation) or 1400 (20- hour relation). within 12 days each experimental group received 6 alternate-day corticosterone injections and

11 daily injections of prolactin. Hie day after the last

injections, the ground squirrels were placed in 4 dark o refrigerators (4 to 8 C) . Each refrigerator contained

2 squirrels from each group (except for one refrigerator with only one squirrel of the 20-hour relationship).

Purina rat chow and water were provided ad libitum.

After 5 days of no disturbances, the squirrels were 19

checked for hibernation every other day from October 3 to

October 29, 1976. Rectal temperature was measured to

confirm the state of hibernation in inactive squirrels.

PCPA and Arousal (Expt. VI)

The 13 thirteen-lined ground squirrels were adults

collected from the golf course at North Texas State Univer

sity in Denton, Texas during the first week of August 1976.

They were maintained on a 14-hour daily photoperiod (0500 -

1900) until changed to a 12-hour daily photoperiod (0500 -

1700) on October 25, 1976. After they were transferred to

dark refrigerators (3 to 8°C) on January 12, 1977 , hiber

nation occurred within the following 8-day period in each

squirrel. The squirrels were checked every other day between 1100 and 1300 and the rectal temperature of inactive

animals was taken.

Between January the 20th and February the 5th, the

3 controls (males) and the 10 experimentals (4 males and

6 females), respectively, received injections of 0.4 ml of 0.85% saline or injections of 80 mg of PCPA (DL-p-

chlorophenylalanine) in 0.4 ml of 0.85% saline. During

a 16-day period each control received 7 or 8 injections of saline and each experimental received 2 to 5 injections of PCPA. The number of injections varied because PCPA 20

injections were skipped unless the squirrel returned to hibernation. Whenever PCPA injections were given to the experimentals, an equivalent proportion of the controls were given saline injections. All injections were given between 1100 and 1300.

Other PCPA Effects (Expt. VII)

Three experiments were performed on thirteen-lined ground squirrels (Experiment ji) and golden hamsters

(Experiments b and c). The thirteen-lined ground squirrels were adults collected from the golf course at North Texas State University in Denton, Texas during the first week of August 1976. The squirrels were main tained on a 14-hour daily photoperiod (0500 - 1900) until the L:D regimen was changed to a 12-hour daily photoperiod

(0600 - 1800) on October 25, 1976. The hamsters, 22 to 26 weeks old, were housed 2 or 3 to a cage and maintained on a 10-hour daily photoperiod (0800 - 1800).

Experiment a determined the effect of PCPA (DL-p- chlorophenylalanine) on body weight of ground squirrels.

Three males and 3 females received injections of 0.5 ml of 0.85% saline and 4 males and 3 females received injections of 100 mg PCPA in 0.5 ml of 0.85% saline.

Injections were given at 1100 on December 30, 1976, 21

January 1 and January 3, 1977. The animals were weighed

before {December 30) and after {January 4) the experimental

treatment.

Experiment b determined the effect of PCPA on body

weight of male golden hamsters. Eighteen hamsters were

subdivided into 3 groups; 6 animals were untreated,

4 animals were given saline injections {0.5 ml of 0.85%),

and 8 animals were given PCPA injections (100 mg in 0.5 ml

of 0.85% saline). Injections were at 0800 on January 17

and 20, 1977. Body weights were taken before {January 16)

and after (January 23) the experimental period.

Experiment c determined the effect of PCPA on body

weights and on weights of reproductive organs and abdominal

fat pads of female golden hamsters. Eighteen hamsters were

divided into 3 equal groups: One received no treatments

or handling, another received injections of 0.5 ml of

0.85% saline, and another received injections of 7 5 mg

of PCPA in 0.5 ml of 0.85% saline. Injections were given

on December 18, 21, and 24, 1976 at approximately 1300.

The hamsters were weighed on December 16 and were killed

on December 26, 1976, to weigh the reproductive organs and

abdominal fat pads. Inasmuch as some body weight loss may have to be considered, organ weights were compared in terms

of organ weight per 100 g original body weight. 22

ct-MPT, Pargyline, and Fluoxetine (Expt. VIII)

The golden hamsters were adult males and females (100 to 150 g B.W.) maintained on a 10-hour daily photoperiod

(0700 - 1700). From March 15 to April 16, 1977, 20 hamsters

(13 males and 7 females) were tested in small groups during various 2- to 3-day periods. The treatment (given 1 or 2 days) consisted of intraperitoneal injections of all the following: 10 mg alpha-methyl-p-tyrosine (a-MPT), 8 mg pargyline, and 5 mg fluoxetine in 0.2 ml, 0.2 ml, and 0.5 ml of 0.85% saline, respectively. Injections were between

1200 and 1400. Additionally, one group of 3 female ham sters was pretreated with PCPA injections (100 mg PCPA in 0.5 ml of 0.85% saline) on each of the two days prior to a 3-drug treatment. Within an hour the hamsters were placed in a dark 6°C refrigerator with Purina rat chow and water available ad libitum.

Additional hamsters were used to study the effect of the individual drugs or their various combinations. Groups of hamsters received injections as follows: only saline; a-MPT; pargyline; fluoxetine; a-MPT and pargyline; a-MPT and fluoxetine; pargyline and fluoxetine; a-MPT, pargyline, and fluoxetine; PCPA pre-treatment and then a-MPT, pargy line, and fluoxetine. Each of the 9 groups had 4 male 23 hamsters plus female hamsters as follows: saline (4 females); a-MPT, pargyline, and fluoxetine (5 females);

PCPA pre-treatment and a-MPT, pargyline, and fluoxetine

(5 females). The PCPA pre-treatment consisted of 3 injec tions of 100 mg of PCPA in 0.5 ml of 0.85% saline given

(at 1000) for 3 days. The individual drugs and their combinations (in the doses already specified) were injected

(at 1200 to 1400) on May 15 and 16, 1977. After the injec tions on May 15, the hamsters were placed in a cold room

(6°C) with Purina rat chow and water provided ^ad 1ibiturn.

In these experiments, checks of the animals were made every 3 or 4 hours (but none from 0000 to 0800). Esophageal temperatures (2 to 3 cm down the throat) of inactive ham sters were determined. (Minor temperature changes were not monitored and only inactive hamsters were checked for hypothermia.) RESULTS

Data were analyzed with an analysis of variance for the daily variation in corticosterone levels (Expt. IV) and the

Student's _t test for all other statistical comparisons.

Insulin (Expt. I)

Insulin injections at 2200 killed all hamsters in this group within the 9-day period of daily injections (Fig. 1).

Injections at 1600 did not cause death of any member of this group. The 0400 and 1000 times for injection had intermediate effects inasmuch as some deaths occurred in each of these groups. Hamsters died from the 5th through the 9th day of injection.

Alloxan (Expt. II)

In the alloxan-treated group, 5 of 6 squirrels were active at one or more of the daily checks and the mean per cent time spent in hibernation was 57 + 13% (Fig. 2). One hundred per cent hibernation occurred in saline-treated squirrels inasmuch as none were observed to be active during the 8 day period. The "mean per cent time spent in hiberna tion, " was based on the per cent of the 8 checks that each squirrel in a group was in hibernation. The occurrence of

24 25

Fig. 1. Daily rhythm in insulin's lethal effects in

golden hamsters (Mesocricetus auratus). The

hamsters were on a 10-hour daily photoperiod. 9 Mesocr ice t us au ra tus

0400 1000 1600 2200 Time of Insulin Injection 27

Fig. 2. Hibernation in alloxan-treated and saline-treated

thirteen-lined ground squirrels (Citellus

tridecemlineatus). Time in Hibernation 100 - 6 -- o N otos Alloxan Controls r fTi e ■ c tr e d s u l l e t i C 1 j l t a e n 1 29

hibernation was less (P < .005) in the alloxan-treated group.

Thiouracil (Expt. Ill)

Some days of hypothermia occurred in all 6 thiouracil-

treated ground squirrels and in 4 of 6 untreated ground

squirrels. The occurrence of hypothermia was greater (P <

.05) in thiouracil-treated ground squirrels (Fig. 3).

Corticosterone Levels (Expt. IV)

The February plasma corticosterone levels were constant

throughout the 24-hour period in the thirteen-lined ground

squirrels (Fig. 4). No significant variation was found.

The mean levels of corticosterone for males and females in

February were not significantly different (Fig. 6). The May plasma corticosterone levels showed some variation in levels

(but not significant) throughout the 24-hour period (Fig. 5).

The May levels of corticosterone for females were signifi cantly higher (P < .05) than February levels for females

(Fig. 6).

Corticosterone and Prolactin (Expt. V)

Hibernation was partially inhibited or blocked entirely in thirteen-lined ground squirrels receiving, respectively,

12-hour or 20-hour relationships of corticosterone and 30

Fig. 3. Summer hypothermia in thiouracil-treated and

untreated thirteen-lined ground squirrels

(Citellus tridecemlineatus). The mean of the

daily occurrence of hypothermia is shown. Squirrels in Hypothermia (Mean Daily % )

K> Ui NJ in O in

CD O O n

CZ 3 cx ro i"D

c: 32

Fig. 4. Concentrations of plasma corticosterone at

6 times of day in thirteen-lined ground

squirrels (Citellus tridecemlineatus) maintained

on a 14-hour daily photoperiod during February. rn C")

Plasma Corticosterone (ng/ml) 100 25 50 75 0700 February

1100 1500 ie f Day of Time

iels tridecemlineatus Citellus 1900 2300

0300 34

Fig. 5. Concentrations of plasma corticosterone at 6

times of day in thirteen-1ined ground squirrels

(Citellus trideceml ineatus) maintained on a

12-hour daily photoperiod during May. Plasma Corticosterone (ng/ml) 100 0 5 25 75 - i S 0600 May

1000

1400 me o Day of e im T

iels tridecemlineatus Citellus 1800

2200

0200 m in

Plasma Corticosterone (ng/ml) 100 50 25 75 0600 May

1000

1400 me o Day of e im T

iels tridecemlineatus Citellus 1800

2200

0200 36

Fig. 6. Plasma corticosterone concentrations in male

and female thirteen-lined ground squirrels

(Citellus tridecemlineatus) sampled throughout

the day during February and May. \ Plasma Corticosterone (nDO 50 25 75 Februa iels decemlineatus i f t Citellus May 38

Fig. 7. Hibernation in thirteen-lined ground squirrels

{Citellus tridecemlineatus) during 27 days

following injections of prolactin at either

12 or 20 hours after corticosterone injections.

The mean of the daily occurrence of hibernation

is shown.

Citellus tndecemlineatus

TO Q

TO 75 a?

o SE- ro 5 0 a a> iIm J

on 25 CD r h 3 O' CO No - 9 7 8 None Untreated CP-12 CP-20 40 prolactin (Fig. 7). Ground squirrels hibernating at least sometime during the 27-day period (after injections were stopped) included 7 of 8 untreated squirrels, 2 of 8 re ceiving the 12-hour relationship, and 0 of 7 receiving the

20-hour relationship. There was a greater mean per cent squirrels in hibernation in the 12-hour relationship com pared with the uninjected controls (P < .001), in the 20- hour relationship compared with the uninjected controls

(P < .001), and in the 12-hour relationship compared with the 20-hour relationship (P < .001). During hibernation the rectal temperatures were approximately 6° to 10°C.

Following the experiment, all the squirrels were alive and those hibernating aroused from hibernation when they were returned to room temperature (22°c).

PCPA and Arousal (Expt. VI)

PCPA-treated ground squirrels hibernated approximately

1/3 as much as the saline-injected controls during the 16- day period of injections (Fig. 8). Figure 9 gives a summary of the per cent times that ground squirrels were aroused 2 days after handling, saline injections, or PCPA injections of hibernating individuals. The saline injections did not seem to alter the per cent arousal from that observed in squirrels only handled. Arousal was observed in 75% of the 41

Fig. 8. PCPA effects on hibernation in thirteen-lined

ground squirrels (Citellus tridecemlineatus).

Either 7 or 8 saline injections or 2 to 5 PCPA

injections were given to each squirrel. Citellus tridecemlineatus

100 OuO

50 cr GO - uninjected - saline 25 -PCPA

Day 2-4 6 -8 10-12 14-16 18-20 22-24 43

Fig. 9. Arousal from hibernation in thirteen-lined ground

squirrels (Citellus tridecemlineatus) 2 days after

handling, saline injections, or PCPA injections. % Arousal from Hibernation 50 25 75 - - No-38 Handled Saline Saline Handled s u t a e n i l m e c e d i r t s u l l e t i C PCPA 45

PCPA-injected squirrels and in only 12.5% of the saline- injected squirrels.

Other PCPA Effects (Expt. VII)

PCPA treatment resulted in a significant decrease in body weight in male (P < .025) and female (P < .025) thirteen

-lined ground squirrels (Fig. 11) and in male golden hamsters

(P < .001) (Fig. 10). Body weights were not significantly changed in either saline-injected or uninjected animals.

Weights of paired ovaries (P < .005) and paired oviducts

(P < .005) of PCPA-treated hamsters were greater than those of saline-injected controls but weights of abdominal fat pads were lower (P < .005) than those of saline-injected controls (Fig. 12). Saline-injected controls and uninjected controls did not differ significantly with regard to the weights of paired ovaries, paired oviducts, or abdominal fat pad s .

a-MPT, Pargyline, and Fluoxetine (Expt- VIII)

The combination of a-MPT, pargyline, and fluoxetine produced a profound hypothermia in 27 out of 29 hamsters subjected to a temperature of 6°C. The esophageal temper atures were as low as 6°C, a temperature at which there were no visible respiratory movements. The paws and nose became 46

Fig. 10. PCPA effects on the body weight of male golden

hamsters (Mesocricetus auratus). 47

d Mesocricetus aurat us

100 SE —\— r i 00 Oi 75

o CD

.2 50

No. = 6 4 5

Uninjected Saline PCPA 48

Fig. 11. PCPA effects on the body weights of male and

female thirteen-lined ground squirrels (Citellus

tridecemlineatus}. Initial Body Weight otos CA otos PCPA Controls PCPA Controls iels rdecemlneat s tu a e lin m e c e trid Citellus 49 50

Fig. 12. PCPA effects on the weight of the paired

ovaries, the paired oviducts, and the

abdominal fat pads of golden hamsters

(Mesocricetus auratus). Weight Per lOOg Body Weight -30 -20 -40 mg -40 20 S.E., Ovaries Paired -30 -20 10 40 mg 40 Oviducts Paired 85PCPA 5 8 * M e s o c r i c e t u s Abdominal □ Umnjected □ □ Saline □ a Pad Fat aurat us aurat

51 bright pink and (at the lowest temperatures) the only move ment was a slight trembling of the front feet. When hypo

thermic hamsters were returned to room temperature (22°C)

they aroused and became active in about two hours, but

their esophageal temperature often remained less than 30°c

for a day or more. Hamsters left in profound hypothermia

for more than 4 to 10 hours did not survive. Four days after the drug-induced hypothermia and their return to room temperature, 4 male hamsters were once again placed in the

6°C cold room. One of 4 hamsters returned to hypothermia

{12°c) after 2 days but the other 3 remained active even after 5 days.

Hypothermia was prevented in 10 of 12 hamsters that received PCPA prior to injection of a-MPT, pargyline, and fluoxetine. Neither individual drugs nor combinations of the two drugs led to profound hypothermia. DISCUSSION

A unifying hypothesis for interpreting these diverse results centers on serotonergic and noradrenergic neural regulation. A fundamental objective is the understanding of the relationship of the hormone injections, hormone assays, or drug injections to the role of neurotransmitters, especially serotonin, in the development of seasonal hiber nation. Because of the wide implications of such a general approach, undoubtedly additional published studies could be cited to support the conclusions made*

The experiment with insulin injections at different times of the day gave unexpected results (Expt. I). Rather than causing hypothermia (other than a fall in temperature just before death), insulin injections killed 14 of 32 ham sters. A daily rhythm was observed inasmuch as during a brief period from 1600 to 2200 the effect of injected insulin changed from apparently harmless to lethal (see Fig. 1).

In human males awake from approximately 0700 until 2300, insulin injected at 1000 (early during the activity period) caused the greatest hypoglycemia (Sensi and Capani, 1976).

The relatively high doses of insulin injected during the first part of the nocturnal period in hamsters may have caused a harmful hypoglycemia. Other daily rhythms of

53 54 deleterious effects include a rhythm in the susceptibility to seizures caused by sound or electricity in mice (Schreiber and Sdhlesinger, 1971). The timing of seizures was corre lated with low brain serotonin levels at night. Further more, genetic strains of mice with greater susceptibility to seizures had lower brain serotonin levels (Schlesinger,

Boggan, and Freedman, 1965). A daily rhythm was found in the serotonin content of the brainstem of golden hamsters

(Morgan, et. _al., 1976) and, perhaps coincidentally, the lowest levels were at 2200, a time when insulin was most lethal.

Results more applicable for the study of hibernation were obtained by inhibiting insulin with alloxan (Expt. I).

Alloxan treatment caused a reduction in the time spent in hibernation (see Fig. 2). Specific role(s) of insulin in hibernation remained undefined. The blocked function may have been related to changes in blood glucose levels that occur with hibernation. For example, a fall of 16.7 mg% in plasma glucose level was noted in thirteen-lined ground squirrels when they entered hibernation (Lyman, 1943). A nearly 60 mg% drop in plasma glucose level occurred in the

Eastern Chipmunk (Tamias striatus) when it entered hypo thermia after summer cold exposure (Woodward and Condrin,

1945). Insulin may not have caused these changes, inasmuch 55

as other hormones (growth hormone, adrenalin, corticoster

oids, glucagon) influence (stimulate hyperglycemia) blood

glucose levels.

Insulin may have a role in hibernation by way of its

effect on brain serotonin concentration. Insulin raises

the levels of plasma tryptophan (and lowers the concentra

tion of the competing neutral amino acids). This process

allows for greater transport of tryptophan into the brain

and an increase in brain serotonin (Femstrom and Wurtman,

1972 a, b; for review, Wurtman and Fernstrom, 1975). There

fore, alloxan treatment would be expected to reduce the move

ment of tryptophan into the brain and thereby reduce brain

serotonin levels. Brain serotonin allows for hibernation,

as evidenced by a suppression of hibernation in the golden-

mantled ground squirrel (Spafford and Pengelley, 1971) and

in the thirteen-lined ground squirrel (Expt. VI) treated

with PCPA, a serotonin synthesis inhibitor. If alloxan

treatment does impair the movement of tryptophan into the brain, the net effect would be the same as PCPA treatment,

i.e., an interference with the production of brain serotonin

and an inhibition of hibernation.

Thiouracil's effect on hypothermia in thirteen-lined

ground squirrels (Expt. Ill) also may indirectly involve

actions of serotonin. A greater tendency for summer 56 hypothermia in thiouracil-treated ground squirrels (Fig. 3) suggests that an active thyroid during the summer hinders the development of hypothermia. In addition to an annual cycle of thyroidal activity, there is also an annual rhythm in brain serotonin concentration in at least several hibernators.

In fact the two seasonal rhythms may be functionally related.

The levels of brain serotonin reach an annual high during the fall or early winter, as documented for the European hedge hog (Erinaceus europaeus) (Uuspaa, 1963), just when the thy roid is most inactive. Increased serotonin leads to a lower release of thyrotropin-releasing hormone (TRH) in mice (Grim and Reichlin, 1973) and the intravenous infusion of L-trypto phan (the fundamental precursor of serotonin) causes a fall in plasma TSH levels in man (Mac Indoe and Turkington, 197 3) .

During hibernation the thyroid does not respond to cold.

Perhaps related is the fact that the thyroidal response to cold is depressed markedly in rats given 5-hydroxytryptophan

(the immediate precursor to serotonin) (Onaya and Hashizume,

1976).

The data collected on corticosterone levels in the thirteen-lined ground squirrels (Expt. IV) are too few to show a seasonal change in phase of daily rhythms character istic of a temporal synergism of hormones. The higher levels of plasma corticosterone in May female ground squirrels as 57

compared with February female ground squirrels (see Fig. 6)

show that the seasonal levels may vary. Although a statis

tically significant rhythm is not present, the May data have

some variation (see Fig. 5).

The February group (see Fig. 4) is of interest because

relatively unchanging levels of cortisol during the day is characteristic of hypothyroidism in man (Martin, Mintz, and

Tamagaki, 1963). Additionally, thyroxin is required for the express ‘.on of the circadian rhythm of corticosterone in rats

(Meier, 1976). Hypothyroidism present in thirteen-lined ground squirrels during the winter (Hoffman and Zarrow,

1958; Hulbert and Hudson, 1976) could account for the dam pened corticosterone rhythm I observed in the February ground squirrels.

The second approach to a study of temporal synergisms, the injections of corticosterone and prolactin in different temporal relationships, provides some evidence for temporal synergisms in the regulation of the annual cycle of thirteen- lined ground squirrels (Expt. V). The occurrence of hiber nation was reduced as a result of corticosterone and prolac tin given in a 12-hour relationship and was completely inhib ited by the 20-hour relationship (see Fig. 7). In golden hamsters the 12-hour relationship of corticosterone and prolactin induced the lowest ovarian weights and intermediate 58 amounts of abdominal fat, whereas the 20—hour relationship induced the highest ovarian weights and the lowest amounts of abdominal fat (Joseph and Meier, 1974). Inasmuch as hibernation is characterized by reduced gonads and heavy fat stores, for hibernation to take place in hamsters with the

20-hour relationship would be less appropriate than in ham sters with the 12-hour relationship of corticosterone and prolactin. Unfortunately I did not have sufficient ground squirrels to determine whether corticosterone and prolactin lead to similar effects on gonads and amounts of abdominal fat.

One possible interpretation of the effect of the corti costerone and prolactin injections is that the various temporal relationships cause a greater or lesser shift in an endogenous circannual rhythm. According to this concept, the 20-hour relationship may have resulted in a shift or resetting of a circannual rhythm of hibernation so that hibernation was prevented from occurring at the normal season. If this model is valid, one could expect to find a temporal relationship of corticosterone and prolactin that would induce hibernation out of the normal season.

The temporal relationships of corticosterone and prolactin also may influence the functioning of the sero tonergic mechanisms, including the circadian and annual 59

rhythms in serotonergic activity. Corticosterone increased

tryptophan hydroxylase activity and thus increased brain

serotonin in the midbrain of the rat (Azmitia and McEwen,

1969). How the various temporal relationships of corticos

terone and prolactin might influence the serotonergic (and

noradrenergic) mechanisms remains unanswered, but the solu

tion could come from a greater understanding of the inter

relationships of hormones and neurotransmitter rhythms.

Another method of inhibiting hibernation in thirteen-

lined ground squirrels, in addition to alloxan and to

temporal synergisms of corticosterone and prolactin, was

through PCPA inhibition of tryptophan hydroxylase (Expt.

VI). The data (see Fig. 8) compare well with the results

reported for the golden-mantied ground squirrel (Spafford

and Pengelley, 1971). PCPA treatment caused a 50% increase

in thyroidal uptake in rats (De Prospo and Melgar,

1975). PCPA elevated the body temperature of rats (McDonald

and Mueller, 1968) and prevented the fall in temperature in response to morphine (Haubrich and Blake, 1971). PCPA also increased brain excitability (seizure susceptibility) in

rats and mice (for review, Marczynski, 1976). If these effects were to take place in PCPA-treated hibernators, they would be antagonistic to hibernation.

Effects of PCPA (Expt. VII) include loss of body weight 60

in ground squirrels (see Fig. 11) and hamsters (see Fig. 10)

as well as weight loss in the abdominal fat pads and weight

gain in paired ovaries and paired oviducts in golden hamsters

(see Fig. 12). These changes are not considered preparative

for hibernation. Alterations in body weight and organ weights may be caused by changes in hormone levels. For

example, prolactin increased fat pad weight in female golden hamsters (Joseph and Meier, 1974) and had lipogenic activi

ties in rat adipose tissue ^n vitro (Martin, Renold, and

Dagenais, 1958). Increased insulin production may cause the obesity following chronic growth hormone administration

(Mahler and Szabo, 1971) . Also, growth hormone administra tion for several days restored lipogenesis in hypophysectomized rats (Nejad, Chaikoff, and Hill, 1962). The gonado tropins have weight maintaining effects on the gonads (for review, Greep, 1961).

Hie changes in the PCPA-treated hamsters may have resulted from an impairment of the serotonergic processes involved in regulating hormones. Because they are important anabolic and lipogenic hormones, the inhibition of serotonin stimulation of prolactin and growth hormone release may have caused the decreases in body weight and abdominal fat pad weight. The increases in the weights of paired ovaries and paired oviducts may have resulted from the PCPA inhibition 61

of the serotonergic block of gonadotropin release (e.g.,

Kordon and Glowinski, 1972).

The neurotransmitters have been demonstrated to play a

role in the regulation of body temperature. Drugs that

affect neurotransmitters (see the Introduction for the mode of action) may cause declines, usually slight, in body tem perature. For example, a-MPT decreased body temperature

from 37°C to 32.9°C after 4 hours in golden hamsters kept at

4°C (Feist, 1970). Pargyline and serotonin caused a 6.5°C decreased compared with a 2.5°C decrease produced by sero tonin alone in mice kept at 22°C (Ritzman and Tabakoff, 1976).

Fluoxetine lowered and prolonged the body temperature decline resulting from morphine and caused a 3.5°C rather than a

2.5°C decline in rats (Fuller and Baker, 1974).

By potentiating serotonergic activity and inhibiting noradrenergic activity simultaneously with 3 drugs (a-MPT, pargyline, and fluoxetine), a severe hypothermia was induced in a cold-exposed golden hamsters (Expt. VIII). In this experiment, 31°C drops were observed in the esophageal temperatures of hamsters kept at an ambient temperature of

6°C. The occurrence of this profound hypothermia offers strong correlative evidence for a central hibernatory role of serotonergic mechanisms and an antagonistic noradrenergic mechanism (see Fig. 13). Further manipulation of these 62

Fig. 13. Serotonergic and noradrenergic pathways involved

in the regulation of hypothermia and hibernation. HIBERNATION

HEAT LOSS TYROSINE \ tyrosine hydroiy^ase OC-MPT

SEROTONIN DOPA I I 5hydroxytryptophan dopa decarboxylase dec arboiyiase \ 5-HYDROXYTRYPTOPHAN DOPAMINE \ / proi j ,,yplophan d opamine v hydroxylase a-hydroxylase \ TRYPTOPHAN NORADRENALIN / HEAT PRODUCTION

AROUSAL

o Ul 64 systems may induce a state even more like natural hiberna tion than the present example. One way to extend the dura tion of the hypothermia might include adjustments in the dose or the frequency of the drug injections. Another approach might center on the control of likely limiting factors such as blood glucose levels.

Further development of this method of inducing hypo thermia and its possible application to modify the body temperature in human beings may be of medical value. The lowering of body temperature is a treatment for various pathological states including cancer. Surgical procedures, especially heart and brain surgery, may depend on the suc cessful induction of hypothermia (Richardson, 1968). Hypo thermia can prolong the life of oxygen-depleted tissues by reducing the requirement for oxygen. Because expert skill, knowledge, and vigilance are required for the successful use of hypothermia as an operating room technique (Berry and

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John Thomas Burns was born in Richmond, Indiana on

February 22, 1943. He was intellectually stimulated by his father to follow whatever subjects drew his interest and he was encouraged to obtain a higher education.

Following graduation at Crawfordsville Senior High School in 1961, he entered Wabash College. At Wabash College he majored in Zoology and minored in Botany and received a

B.A . degree in 1965. The opportunity to do biological rhythm research with Dr. Albert H. Meier caused him to seek admission to Louisiana State University and he subsequently received a M.S. degree in Zoology from L.S.U. in 1968. For the next few years he taught in the Biology

Department at Baker University in Baldwin City, Kansas.

In June of 197 5 he was married to Anne Elizabeth Ramsey of Kansas City, Missouri.

At the present time Mr. Burns is a candidate for the

Ph.D. degree in Zoology at Louisiana State University-