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EVALUATION OF DEXTROAMPHETAMINE-INDUCED BODY TEMPERATURE CHANGES IN RATS

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Authors Broadie, Larry Lewis, 1940-

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71-2489

BROADIE, Larry Lewis, 1940-EVALUATION OF DEXTROAMPHETAMINE-INDUCED BODY TEMPERATURE CHANGES IN RATS.

University of Arizona, Ph.D., 1970 Pharmacology

University Microfilms, A XEROX Company, Ann Arbor, Michigan

THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED

EVALUATION OF DEXTROAMPHETAMINE-INDUCED

BODY TEMPERATURE CHANGES IN RATS

by

Larry Lewis Broadie

A Dissertation submitted to the Faculty of the

COLLEGE OF PHARMACY

In Partial Fulfillment of the Requirements For the Degree of

DOCTOR OF PHILOSOPHY WITH A MAJOR IN PHARMACOLOGY

In the Graduate College

THE UNIVERSITY OF ARIZONA

19 7 0

THE UNIVERSITY OF ARIZONA

GRADUATE COLLEGE

I hereby recommend that this dissertation prepared under my

direction by Larry Lewis Broadie

entitled Evaluation of Dextroamphetamine-induced Body

Temperature Changes in Rats

be accepted as fulfilling the dissertation requirement of the

degree of Doctor of Philosophy

7̂ $ ' P O

Dissertation Director Date

After Inspection of the dissertation, the following members

of the Final Examination Committee concur in its approval and

recommend its acceptance:*

<OL£&*U-c>fc

• C?.\ vujimA

S~z,st- 7O

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jfcTfiielapprovel aiid acceptance is contingent on the candidate's adequate performance and defense of this dissertation at the final oral examination. The inclusion of this sheet bound Into the library copy of the dissertation is evidence of satisfactory performance at the final examination.

STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.

S1GNE

ACKNOWLEDGEMENTS

The author wishes to express sincere gratitude to Dr. Lincoln

Chin for his counsel and guidance, especially during the preparation of

this manuscript.

The author also wishes to express his appreciation to Dr. Albert

L. Picchioni and Dean Willis R. Brewer for their efforts and concern.

Finally, the author is indebted to his wife, Barbara, for her

continual encouragement and understanding.

Ill

TABLE OF CONTENTS

Page

LIST OF ILLUSTRATIONS vii

LIST OF TABLES x

ABSTRACT ' . . xi

1. INTRODUCTION 1

2. THE EFFECT OF INTRAVENTRICULARLY AND INTRAPERITONEALLY ADMINISTERED DEXTROAMPHETAMINE ON BODY TEMPERATURE OF RATS . . 4

Materials and Methods . . . . . 5 Animals and Housing . . 5 Administration of Drugs 5 Measurement of Body Temperature 6 Assay of Tritiated Dextroamphetamine 6 Surgical Techniques 7 Statistical Procedures 7

Results 7 Body Temperature Changes after Intraventricular

Dextroamphetamine in Restrained Rats 7 Body Temperature Changes after Intraventricular

Dextroamphetamine in Unrestrained Rats 8 Body Temperature Changes after Intraperitoneal

Dextroamphetamine in Unrestrained Rats 8 Brain Concentrations of Dextroamphetamine Sulfate ... 8 Comparison of Body Temperature Changes after

Equivalent Intraperitoneal and Intraventricular Doses of Dextroamphetamine at Various Ambient Temperatures ..... 9

Effects of Adrenergic Blocking Drugs on Body Temperature Responses to Dextroamphetamine .... 10

Effect of Pithing on Body Temperature Response to Dextroamphetamine 10

Effect of Adrenalectomy on Body Temperature Response to Dextroamphetamine ..... 11

Discussion 11

3. BEHAVIORAL AND PHYSIOLOGICAL FACTORS ASSOCIATED WITH DEXTROAMPHETAMINE-INDUCED BODY TEMPERATURE CHANGES 28

Materials and Methods 30 Measurement of Oxygen Consumption 30

lv

V

TABLE OF CONTENTS--Continued

Page

Measurement of Spontaneous Activity 31 Quantification of Sniffing Behavior 31

Results 31 Effects of Dextroamphetamine on Oxygen

Consumption 32 Effects of Dextroamphetamine on Spontaneous

Motor Activity ......... 32 Evaluation of Sniffing Behavior ....... 33

Discussion 34

4. THE ROLE OF CENTRAL MONOAMINES IN DEXTROAMPHETAMINE-1NDUCED HYPOTHERMIA 43

Materials and Methods 47 Effects of Monoamine-depleting Agents on

Dextroamphetamine-induced Body Temperat u r e C h a n g e s . . . . . 4 7

Brain Monoamine Levels after Single Intraventricular or Intraperitoneal Administration of Dextroamphetamine . ..... 48

Effects of Repeated Intraventricular Injections of Dextroamphetamine on Hypothermic Response ... 49

Brain Monoamine Levels at Peak Hypothermic Effect Induced by the Last of Three Repeated Intraventricular Injections of Dextroamphetamine .... 49

Brain Monoamine Levels at Time of Recovery of Body Temperature after Repeated Intraventricular Injections of Dextroamphetamine 49

Spectrophotofluorometric Assay of Catecholamines and 5-Hydroxytryptamine 50

Hlstochemical Fluorescence Technique 50 Results 51

Effects of Monoamine-depleting Agents on Dextroamphetamine 'Induced Body Temperature Changes ... 51

Brain Monoamine Levels after Single Intraventricular or Intraperitoneal Administration of Dextroamphetamine 51

Effects of Repeated Intraventricular Injections of Dextroamphetamine on Hypothermic Response ... 52

Brain Monoamine Levels at Peak Hypothermic Effect Induced by the Last of Three Repeated Intraventricular Injections of Dextroamphetamine 52

Brain Monoamine Levels at Time of Recovery of Body Temperature Following Repeated Intraventricular Injections of Dextroamphetamine 53

Discussion 53

vi

TABLE OF CONTEtTTS-- Continued

Page

5. GENERAL DISCUSSION 69

APPENDIX A: INTRACEREBROVENTRICULAR TECHNIQUE 80

APPENDIX B: PREPARATION OF DRUGS 85

APPENDIX C: ASSAY FOR BRAIN DEXTROAMPHETAMINE 88

APPENDIX D: STATISTICAL PROCEDURES 93

APPENDIX E: SPECTROPHOTOFLUOROMETRIC ASSAY OF 5-HYDROXY-TRYPTAMINE, NOREPINEPHRINE AND DOPAMINE 95

APPENDIX F: HISTOCHEMICAL FLUORESCENCE TECHNIQUE FOR DIRECT OBSERVATION. OF NOREPINEPHRINE, DOPAMINE AND 5-HYDROXYTRYPTAMINE 109

LIST OF REFERENCES 118

LIST OF ILLUSTRATIONS

Figure Page

1. Effect of Intraventricular Amphetamine on Body Temperature of Restrained Rats at an Ambient Temperature of 25°C ... 12

2. Effect of Intraventricular Amphetamine on Body Temperature of Unrestrained Rats at an Ambient Temperature of 25°C . . 13

3. Concentration of Amphetamine in Rat Brain Following Intraperitoneal and Intraventricular Administration at an Ambient Temperature of 25°C 15

A. Effect of Intraperitoneal Amphetamine on Body Temperature of Rats at an Ambient Temperature of 25°C 16

5. Effect of Intraperitoneal Amphetamine on Body Temperature of Rats at Various Ambient Temperatures 17

6. Effect of Intraventricular Amphetamine on Body Temperature of Rats at Various Ambient Temperatures 18

7. Effect of Phenoxybenzamine and Propranolol on Hypothermia Produced by Intraventricular Amphetamine in Rats at an Ambient Temperature of 25°C 19

8. Effect of Propranolol on Hyperthermia Produced by Intraperitoneal Amphetamine in Rats at Ambient Temperatures of 25° and 20°C 20

9. Effect of Phenoxybenzamine on Hyperthermia Produced by Intraperitoneal Amphetamine in Rats at Ambient Temperatures of 25° and 20°C 21

10. Effect of Intraperitoneal Amphetamine on Body Temperature Change in Pithed Rats at an Ambient Temperature of 26°C 22

11. Effect of Adrenalectomy on Hyperthermia Produced by Intraperitoneal Amphetamine at an Ambient Temperature of 2S°C 23

12. Effect of Intraperitoneal and Intraventricular Amphetamine on Oxygen Consumption Rate of Rats at an Ambient Temperature of 25°C 35

vii

viii

LIST OF ILLUSTRATIONS--Continued

Figure Page

13. Effect of Intraperitoneal Amphetamine on Spontaneous Activity of Rats at an Ambient Temperature of 25°C .... 36

14. Effect of Intraventricular Amphetamine on Spontaneous Activity of Rats at an Ambient Temperature of 25°C .... 37

15. Effect of Intraperitoneal and Intraventricular Amphetamine on Sniffing Behavior of Rats at an Ambient Temperature of 25°C 38

16. Body Temperature Response to Intraventricular Amphetamine at an Ambient Temperature of 25°C in Rats Pretreated with Ro 4-1284 or a Combination of Alpha-methyl-m-tyrosine (aMMT) and Alpha-methyl-p-tyrosine (aMPT) .... 56

17. Body Temperature Response to Intraperitoneal Amphetamine at an Ambient Temperature of 25°C in Rats pretreated with Ro 4-1284 or a combination of Alpha-methyl-m-tyrosine (aMMT) and Alpha-methyl-p-tyrosine (aMPT) .... 57

18. Effect of Intraventricular and Intraperitoneal Amphetamine on Fluorescence of Anterior Hypothalamic Nucleus Peri-vent ricularis in the Rat at an Ambient Temperature of 25°C 59

19. Effect of Intraventricular and Intraperitoneal Amphetamine on Fluorescence of Nucleus Caudatus Putamen (NCP) and Globus Pallidus (GP) in the Rat at an Ambient Temperature of 25°C 60

20. Peak Hypothermic Responses to Repeated Intraventricular Injections of Amphetamine at an Ambient Temperature of 25°C 61

21. Fluorescence in Anterior Hypothalamic Nucleus Periven-tricularis and Nucleus Caudatus Putamen at Time of Peak Hypothermia after Multiple Intraventricular Injections of A-nphetamine at an Ambient Temperature of 25°C 62

22. Fluorescence in Anterior Hypothalamic Nucleus Periven-tricularis at Time of Body Temperature Recovery after Multiple Intraventricular Injections of Amphetamine at an Ambient Temperature of 25°C 63

lx

LIST OF ILLUSTRATIONS--Continued

Figure Page

23. Fluorescence in Nucleus Caudatus Futamen at Time of Body Temperature Recovery after Multiple Intraventricular Injections of Amphetamine at an Ambient Temperature of 25°C 64

LIST OF TABLES

Table Page

1. Effect of Intraperitoneal Amphetamine on Body Temperature of Rats at an Ambient Temperature of 25°C 14

2. Effect of Ambient Temperature of 6°C, Propranolol and Adrenalectomy on Sniffing Behavior Following Intraperitoneal Amphetamine in Rats 39

3. Effect of Ro 4-1284 on Whole Brain Norepinephrine, Dopamine and 5-Hydroxytryptamine Concentrations in Rats at an Ambient Temperature of 25°C 54

4. Effect of p-Chlorophenylalanine and ?vO /:-1284 on Hypothermia Produced by Intraventricular Amphetamine at an Ambient Temperature of 25°C 55

5. Effect of Intraventricular and Intraperitoneal Amphetamine on Whole Brain Norepinephrine, Dopamine and 5-Hydroxy-tryptamine Concentrations in Rats at an Ambient Temperature of 25°C 58

x

ABSTRACT

There Is general agreement that amphetamine characteristically

produces hyperthermia in rats. However, there is disagreement whether

this effect is due to the central or peripheral action of the drug.

Dextroamphetamine was administered to rats by intraventricular or intra

peritoneal injection in an effort to resolve this controversy. Various

manipulative and pretreatment procedures were employed to modify dextro

amphetamine -induced body temperature responses to obtain evidence of the

relative roles of central and peripheral action of dextroamphetamine in

this regard. Additional studies were conducted to examine possible cor

relation between physiological or behavioral responses and temperature

changes and to examine certain endogenous neurochemicals as probable

mediators of the central hypothermic action of dextroamphetamine.

Intraventricular injection of dextroamphetamine in doses of 4,

100, and 400 meg at 25°C ambient temperature produced hyperthermia, hy

pothermia, or initial hypothermia followed by secondary hyperthermia,

respectively, whereas intraperitoneal injection of 2 mg/kg of the drug

produced hyperthermia. Intraperitoneal injection of 2 mg/kg of dextro

amphetamine and intraventricular Injection of 100 meg produced the same

brain concentration of the drug 30 minutes postadmlnistration. These

two doses are considered as "equivalent doses." Hypothermia appeared to

be the predominant central effect of dextroamphetamine, whereas hyper

thermia appeared to be mainly caused by the peripheral effect of dextro

amphetamine. It was suggested that systemic dextroamphetamine

xi

xll

simultaneously activates peripheral hyperthermic and central hypothermic

mechanisms. Support for this hypothesis was provided by observations

which suggest that high ambient temperature reduces body heat dissipa

tion and allows the hyperthermic action of the drug to become manifested,

whereas low ambient temperature enhances heat dissipation and allows the

hypothermic action of the drug to become manifested. Additionally, ad

renergic blocking agents appeared to antagonize the peripheral hyper

thermic effect of systemic dextroamphetamine and unmask the central

hypothermic effect of the drug. The predominant hyperthermic effect of

dextroamphetamine is apparently of peripheral origin because the drug

retards the rate of progressive hypothermia caused by ablation of the

central nervous system and because the absence of adrenal catecholamines

prevents the hyperthermic effect of dextroamphetamine.

Attempts to correlate physiological or behavioral responses and

dextroamphetamine-induced temperature changes yielded the initial im

pression that changes in spontaneous motor activity and oxygen consump

tion correlated with changes in body temperature, whereas sniffing

behavior did not. Subsequently, overt observations seemed to contradict

the initial findings and Implied a lack of correlation. It was ration

alized that various physiological or behavioral responses may be more

appropriately correlated with body heat generation or dissipation rather

than with changes in body temperature per se.

The probable role of norepinephrine, dopamine, and/or 5-hydroxy-

tryptamlne In the central hypothermic action of dextroamphetamine was

studied by observing the effect of depletion of these amines on the body

temperature effect of dextroamphetamine. All three of the biogenic

xiii

amines were simultaneously depleted with Ro 4-1284, 5-hydroxytryptamine

was depleted with p-chlorophenylalanine, and the two catecholamines were

simultaneously depleted with the combined use of alpha-methyl-m-tyrosine

and alpha-methyl-p-tyrosine. These depletion studies suggested that the

central hypothermic action of dextroamphetamine is mediated, at least in

part, by one or both of the catecholamines.

The Importance of one or both of the catecholamines in the cen

tral hypothermic effect of dextroamphetamine was further investigated

through the use of hlstochemlcal fluorescence analyses of specific areas

of the brain following single or repeated Injections of the drug. Of

the two catecholamines considered, norepinephrine appeared to be impor

tant in mediation of the central hypothermic action of dextroampheta

mine. This concept is particularly appealing because norepinephrine is

the predominant monoamine In the anterior hypothalamus and because this

region is considered to be important in thermoregulation.

CHAPTER 1

INTRODUCTION

Amphetamine has been repeatedly shown to elevate body tempera

ture (Haffner, 1938; Haas, 1939; Simonyi and Szentgyorgyi, 1949; Harrison,

Ambrus and Ambrus, 1952; Cheymol and Levassort, 1957; Lessin and Parkes,

1957; Klissiunis and Dosi, 1959; Tedeschi et al., 1959; Askew, 1962;

Belenky and Vltolina, 1962; Morpurgo and Theobald, 1965; Mantegazza,

Naimzada and Riva, 1968a; Gessa, Clay and Brodie, 1969; Slater and

Turnbull, 1969). However, the role of the central nervous system in

this response is not clearly defined. Amphetamine has been described as

having predominantly ergotropic activity (Brodie, Spector and Shore,

1959), thus leading to the conclusion that the hyperthermia may be pro

duced by stimulation of adrenergic receptors in the brain (Euler, 1961).

Indeed, elevation of brain amphetamine levels is reported to be associ

ated with a prolonged hyperthermic response in rats (Valzelli, Consolo

and Morpurgo, 1966; Valzelli et al., 1968). In an attempt to describe

the central site of action of amphetamine, Belenky and Vltolina (1962)

administered amphetamine to cats and rabbits in which lesions had been

produced at various levels in the brain. They concluded from their re

sults that the site of initiation of the hyperthermic response was in

the region of the diencephalon.

A different conclusion was reached by" Gessa, Clay and Brodie

(1969) who related amphetamine-induced hyperthermia to the mobilization

1

2

of free fatty acids through the release of norepinephrine from neurons

in adipose tissue. Since hyperthermia was not altered by a ganglionic

blocking agent these investigators concluded that amphetamine produces

hyperthermia by a peripheral action. Hence, a controversy exists re

garding the relative Importance of central and peripheral mechanisms in

amphetamine-induced hyperthermia.

The effect of other sympathomimetic amines on body temperature,

particularly the catecholamines, has also been studied. The nature of

body temperature changes induced by catecholamines appears to vary with

factors such as animal species and route of administration. For exam

ple, peripherally administered norepinephrine or epinephrine is reported

to produce hyperthermia in rats (Feldberg and Lotti, 1967; Jori ,

Paglialunga and Garattini, 1967) and rabbits (Masek and Raskova, 1964);

hypothermia in chicks (Allen and Marley, 1967) and monkeys (Essex,

1952); and both hyperthermia and hypothermia in dogs (Essex, 1952). In-

tracerebroventricular administration of these catecholamines is reported

to produce hyperthermia in rabbits (Euler, Linder and Myrin, 1943;

Cooper, Cranston and Honour, 1965) and sheep (Bllgh, 1966a); hypothermia

in mice (Brittain, 1966; Brittain and Handley, 1967), oxen (Findlay and

Thompson, 1968), cats (Feldberg and Myers, 1963, 1964a, 1964b), dogs

(Feldberg, Hellon and Myers, 1966), and monkeys (Feldberg, Hellon and

Lotti, 1967); and both hyperthermia and hypothermia in rats (Feldberg

and Lotti, 1967; Myers and Yaksh, 1968; Slater and Turnbull, 1969).

Since catecholamines are known to cross the blood brain barrier

with difficulty (Axelrod, Weil-Malherbe and Tomchlck, 1959; Weil-Malherbe,

Witby and Axelrod, 1961; Samorajski and Marks, 1962), it could be

3

justifiably argued that peripheral administration of catecholamines

produces hyperthermia by peripheral mechanisms whereas intracerebroven-

trlcular injection produces hyperthermia or hypothermia by central

mechanisms. On the other hand, dextroamphetamine, a noncatechol sympa

thomimetic amine, readily penetrates the blood brain barrier (Young and

Gordon, 1962; Fuller and Hines, 1967; Rosso, Dolfini and Franchl, 1968;

Malckel et al., 1969) and simultaneously produces central and peripheral

actions. Hence, a similar statement can not be made about dextroam

phetamine with respect to its body temperature effects and the contro

versy persists. The present project was conducted in order to help

resolve the controversy regarding the relative roles of central and pe

ripheral mechanisms in dextroamphetamine-Induced body temperature changes

in rats.

CHAPTER 2

THE EFFECT OF INTRAVENTRICULAR!^ AND INTRAPERITONEAL ADMINISTERED DEXTROAMPHETAMINE ON BODY TEMPERATURE OF RATS

Although numerous published investigations involving the periph

eral injection of amphetamine exist, relatively few investigations have

been reported in which the drug has been administered intracerebrally.

Intraventricular, intracisternal or intrahypothalamic administration of

amphetamine has been reported to produce lethargy, mydriasis, tachypnea

and panting (Gaddum and Vogt, 1956), excitation (Leimdorfer, 1950), ano

rexia (Booth, 1968), inhibition of phenylbenzoquinone-induced writhing,

piloerection, excitation (high dose) and depression (low dose) (Davis

and Horlington, 1964). However, a survey of the literature failed to

reveal reports concerning the influence of intraventricularly adminis

tered amphetamine on body temperature.

Since peripherally administered amphetamine has been hypothe

sized to cause hyperthermia by a central mechanism (Euler, 1961; Belenky

and Vitolinat 1962; Valzelli et al., 1966, 1968) and since proposed

thermoregulatory sites in the hypothalamus are in close proximity to

cerebrospinal fluid in the third cerebral ventricle (Bllgh, 1966b), it

appeared logical to use the intraventricular route of administration as

a means of studying the role of the central nervous system In ampheta

mine-induced body temperature changes. In the present study the tempera

ture responses to intraventricularly and Intraperitoneally administered

dextroamphetamine are compared. It was hoped that small doses of

4

5

dextroamphetamine given by intraventricular injection can produce prima

rily central actions with little or no peripheral actions.

Materials and Methods

Animals and Housing

Male Sprague-Dawley rats (Holtzman Co., Madison, Wisconsin),

weighing 225 to 275 g, were used throughout this Investigation and were

housed at 25 ±1°C with free access to water and food (Canine Checkers,

Ralston Purina Co.). Except for an Initial series of observations in

which restraint was employed (see Fig. 1), animals were removed from

their home cages for only brief periods during experimental manipula

tions such as injections or body temperature determinations. Animals

employed for the study reported in Fig. 1 were restrained in plastic rat

holders throughout the entire period of temperature determination. In

studies which Involved altered ambient temperatures, animals were trans

ferred to the new temperature environment In their home cages* Animals

were pre-conditioned at ambient temperatures of 30 + l°C, 6 ± 1°C and

20 +1°C for 1, 20 and 48 hours, respectively. The room in which experi

mental animals were housed was illuminated with incandescent lights from

6 A.M. to 6 P.M. daily and experiments were consistently performed be

tween 11 A.M. and 5 P.M.

Administration of Drugs

Drugs were administered by Intraventricular, Intraperitoneal and

subcutaneous injections. Intraventricular injections were made by means

of permanently Implanted cannulas in the right lateral cerebral

ventricle (Grunden and Linburn, 1969). Specifications of the materials

and details of the implantation and injection techniques are presented

in Appendix A. Details concerning the preparation of drug solutions for

this investigation are presented in Appendix B.

Measurement of Body Temperature

Body temperature was monitored by means of a telethermometer

(Yellow Springs Instrument Co.) with the thermistor probe inserted in

the rectum to a depth of 5 cm. Thirty-five seconds were allowed for

equilibration. Since handling and the procedures of injection are known

to cause alterations in body temperature (Brown and Julian, 1968), ani

mals were routinely subjected to body temperature determinations 3 times

at 30-minute intervals prior to dextroamphetamine or saline administra

tion. The third body temperature determination, made immediately before

injection, served as the reference temperature for subsequent body tem

perature changes.

Assay of Tritiated Dextroamphetamine

Rat6 were injected intraventricularly with 100 meg of tritiated

dextroamphetamine sulfate or intraperitoneally with 2 mg/kg of tritiated

dextroamphetamine sulfate. Thirty, 60 and 120 minutes after injection

the animals were killed and brain tissue was assayed for tritiated dex

troamphetamine sulfate. The assay was performed as described by Fuller

and Hines (1967). Details of the materials and procedure are presented

In Appendix C.

Surgical Techniques

Ablation of the central nervous system. Animals were anesthe

tized with methohexital and prepared by tracheal intubation for artifi

cial respiration. A large needle was quickly passed through the orbital

cavity into the brain and down the spinal column. The pithed animals

were then attached to a respirator (model V5KG, E & M Instrument Co.)

and respired at a rate of 66 cycles per minute with a 1:1 inspiration:

expiration ratio and an applied pressure of 15 cm water. The ambient

temperature was maintained at 26+0.2°C during this series of experi

ment s.

Adrenalectomy. Bilateral adrenalectomy was performed as de

scribed by D'Amour, Blood and Belden (1965). Animals were allowed to

recover at least two weeks before they were employed for experimental

studies.

Statistical Procedures

Data were statistically analyzed by means of Student's t_ test

(Snedecor and Cochran, 1967). Details of the calculations are presented

in Appendix D.

Results

Body Temperature Changes after Intraventricular Dextroamphetamine in Restrained Rats

Four meg of intraventricularly administered dextroamphetamine

resulted in hyperthermia in restrained rats, whereas 12, 20, or 100 meg

produced dose-related hypothermia (Fig. 1).

8

Body Temperature Changes after Intraventricular Dextroamphetamine in Unrestrained Rats

Four meg of intraventricularly administered dextroamphetamine

produced a modest but significant (P<0.01) increase in body temperature

at 30 minutes (Fig. 2). One hundred meg of dextroamphetamine produced

hypothermia which reached a peak at 30 minutes, showed partial recovery

at 60 minutes and returned to control levels at 120 minutes. In Com

parison, 400 meg of dextroamphetamine produced hypothermia no greater

than that produced by 100 meg at the 30-minute time period. However,

the body temperature of animals treated with the larger dose returned to

pre-injection levels more rapidly (in 60 minutes) and was elevated above

normal at 120 minutes.

Body Temperature Changes after Intraperitoneal Dextroamphetamine in Unrestrained Rats

Intraperitoneal administration of 100 meg of dextroamphetamine

produced no change in body temperature; whereas 200 meg and 400 meg pro

duced hyperthermia at 30 minutes postadministratlon (Table 1).

Brain Concentrations of Dextroamphetamine Sulfate

The brain concentrations of dextroamphetamine sulfate at se

lected times after intraventricular (100 meg) or intraperitoneal

(2 mg/kg) administration of dextroamphetamine sulfate are presented in

Fig. 3. At 30 minutes the brain concentrations for the 2 routes of ad

ministration were equivalent. By 60 minutes postadministratlon, the

mean brain concentration of dextroamphetamine after Intraventricular in

jection had decreased markedly, whereas the mean brain concentration

after Intraperitoneal injection was not significantly different

9

(P>0.05) from the 30-rainute value. By 120 minutes, the mean brain con

centration of dextroamphetamine after intraventricular injection was

still significantly less (P<0.01) than that after intraperitoneal

Injection, although both values were less than 20 per cent of the re

spective 30-minute values. Since these respective doses of dextroam

phetamine resulted in equivalent brain concentrations at 30 minutes

postadministration, they were chosen for further studies and will be re

ferred to as "equivalent" doses.

Comparison of Body Temperature Changes after Equivalent Intraperitoneal and Intraventricular Doses of Dextroamphetamine at Various Ambient Temperatures

Intraperitoneal administration of dextroamphetamine (2 mg/kg)

resulted in hyperthermia which reached a peak effect at 60 minutes

(Fig. 4). In a separate study involving the use of a series of ambient

temperatures (Fig. 5), the same dose of dextroamphetamine produced an

elevation of body temperature of approximately 1.1°C and 0.4°C at ambi

ent temperatures of 25°C and 20°C, respectively. In contrast, at an

ambient temperature of 6°C intraperitoneal dextroamphetamine injection

caused pronounced hypothermia. Control animals injected intraperito-

neally with saline solution showed relatively stable body temperatures

at these different environmental temperatures.

At an ambient temperature of 25°C, intraventricular injection of

dextroamphetamine produced hypothermia (Fig. 6). This hypothermic ef

fect was augmented by an ambient temperature of 6°C but was antagonized

by an ambient temperature of 30°C. Control animals given intraventricu

lar injections of saline showed moderate hyperthermia at an ambient

10

temperature of 30°C but showed no change In body temperature at ambient

temperatures of 25°C or 6°C.

Effects of Adrenergic Blocking Drugs on Body Temperature Responses to Dext roamphet amine

Propranolol In a dose of 10 mg/kg failed to block hypothermia

induced by intraventricular administration of dextroamphetamine at an

ambient temperature of 25°C (Fig. 7). On the other hand, it antagonized

the hyperthermic effect of peripherally administered dextroamphetamine

at an ambient temperature of 25°C and caused peripherally administered

dextroamphetamine to produce a moderate hypothermia, rather than hyper

thermia, at an ambient temperature of 20°C (Fig. 8).

Phenoxybenzamine in a dose of 10 mg/kg diminished by approxi

mately 50 per cent the hypothermia produced by intraventricular adminis

tration of dextroamphetamine at an ambient temperature of 25°C (compare

Figs. 7 and 6). Similar to propranolol, phenoxybenzamine also antago

nized the hyperthermic effect of intraperitoneal dextroamphetamine at an

ambient temperature of 25°C and reversed the temperature response pro

duced by intraperitoneal dextroamphetamine at an ambient temperature of

20°C (Fig. 9).

Effect of Pithing on Body Temperature Response to Dextroamphetamine

Destruction of the central nervous system of rats results in a

continuous fall in body temperature. Intraperitoneal administration of

dextroamphetamine perceptibly retarded this process (Fig. 10).

11

Effect of Adrenalectomy on Body Temperature Response to Dextroamphetamine

Bilateral adrenalectomy exerted no change on body temperature of

rats at an ambient temperature of 25°C. However, intraperitoneal admin

istration of dextroamphetamine In adrenalectomlzed rats failed to pro

duce hyperthermia (Fig. 11).

Discussion

Catecholamines administered intraventricularly to rats in low

doses produce moderate hyperthermia, whereas larger doses produce hypo

thermia (Feldberg and Lotti, 1967; Myers and Yaksh, 1968; Slater and

Turnbull, 1969). In the present study Involving restrained rats in

jected intraventricularly with dextroamphetamine, the lowest dose of

dextroamphetamine produced hyperthermia, whereas the larger doses (up to

100 meg) produced hypothermia. Similar results were obtained with in

traventricular injections of dextroamphetamine in unrestrained rats

(this and all subsequent body temperature studies were performed on un

restrained rats in order to permit possible correlation with data ob

tained from locomotor activity studies reported in a later chapter).

Very large doses of intraventricularly administered dextroamphetamine

produced three unexpected effects: (1) 400 meg of dextroamphetamine

produced no greater hypothermia than 100 meg, (2) hypothermia was of

shorter duration with the larger dose, and (3) with the larger dose a

secondary hyperthermia became apparent at 2 hours postadministratlon.

These anomalous effects on body temperature might be explained on the

basis of interplay between central hyperthermic and hypothermic mecha

nisms and peripheral hyperthermic mechanisms. Centrally mediated

12

O SALINE (N-3) • 4meg AMPHETAMINE {N-2> A I2mcg AMPHETAMINE (N«2) • 20meg AMPHETAMINE (N* 2) • 100meg AMPHETAMINE (N«3)

24

TIME (MINUTES)

Figure 1. Effect of Intraventricular Amphetamine on Body Temperature of Restrained Rats at an Ambient Temperature of 25°C

Dextroamphetamine sulfate or 0.9% NaCl solution was injected into the lateral cerebral ventricle in a volume of 5 mcl at time 0. The amount of dextroamphetamine is expressed as the sulfate salt.

13

O SALINE (N<7) * 4meg AMPHETAMINE (N«4) • 100meg AMPHETAMINE (N»5) • 400meg AMPHETAMINE (N>5)

U

z>

£ K ItJ a 3 UJ l->-o o m - 0 . 5

UJ o z X u

- 1 . 5

2.0

SO 60

TIME (MINUTES)

Figure 2. Effect of Intraventricular Amphetamine on Body Temperature of Unrestrained Rats at an Ambient Temperature of 25 C

Dextroamphetamine sulfate or 0.9% NaCl solution was Injected into the lateral cerebral ventricle in a volume of 5 mcl at time 0. The amount of dextroamphetamine is expressed as the sulfate salt. Four meg produced significant (P<0.01) hyperthermia at 30 minutes, whereas 100 and 400 meg produced significant (P<0.001) hypothermia at 30 minutes. By 60 minutes the value for 400 meg is not significantly different (P>0.05) from the value for saline-treated rats but is significantly different (P<0.05) from value for 100 meg. By 120 minutes the value for 400 meg is significantly higher (P<0.05) than control. Vertical lines indicate + S.E.M.

Table 1

Effect of Intraperitoneal Amphetamine on Body Temperature of Rats at an Ambient Temperature of 25°C

TREATMENT® CHANGE IN BODY TEMPERATURE (°C) (30 minutes after treatment)

Saline -0.13 + 0.16 (N=5)

100 meg Amphetamine -0.06 ± 0.09 (N=5 )

200 meg Amphetamine +0.46 + 0.06b

(N=4)

AOO meg Amphetamine +0.66 + 0.10c

(N=5)

a Dextroamphetamine sulfate (dose expressed as salt) or 0.9% NaCl solution was injected intraperitoneally in a volume of 2 ml/kg.

b Significantly different from saline treatment (P<0.05)

c Significantly different from saline treatment (P<0.01)

15

2.5

2.0

1.9

o o E 1.0

0.5

• AMPHETAMINE ip |N"B) A AMPHETAMINE ivt(N>6)

30 i

60 120 TIME(MINUTES)

Figure 3. Concentration of Amphetamine in Rat Brain Following Intraperitoneal and Inttfaventricular Administration at an Ambient Temperature of 25°C

Intraperitoneally (ip)» dextroamphetamine sulfate (2 mg/kg) was injected in a volume of 2 ml/kg at time 0. Intraventriculariy (ivt), dextroamphetamine sulfate (100 njcg) was injected in a volume of 5 mcl at time 0. The ordinate indicates ntcg of dextroamphetamine sulfate per g of tissue. Values for the 2 routes are not significantly different (P>0„5) at 30 minutes. At 60 and 120 minutes the values were significantly different from each other (P<0.001 and P<0.01, respectively). Vertical lines Indicate + S.E.M.

16

O SALINE (N>8)

• AMPHETAMINE (N* 7)

Ili

UJ

+ 0.5

-0.5

30 60

TIME (MINUTES)

Figure A. Effect of Intraperitoneal Amphetamine on Body Temperature of Rats at an Ambient Temperature of 25°C

Dextroamphetamine sulfate (2 rog/kg) or 0.9% NaCl solution was injected intraperitoneally in a volume of 2 ml/kg at time 0. Dextroamphetamine produced significant (P<0.001) hyperthermia at all time periods shown. Vertical lines indicate + S.E.M.

17

+ I .5

HI CC O I-< CC UJ 0. z LJ

>-Q O ffi

UJ o z « X o

+ I .0

+ 0 5

-0.5

- I 0

- 1.5

-2.0

O SALINE ,25*C (N>6) • AMPHETAMINE, 25*C (N-5)

& SALINE, 20'C <N«8> A AMPHETAMINE, Z0*C(N*8)

• SALINE, 6*C (N>5)

• AMPHETAMINE, 6*C (N>7)

-i 60 30

TIME( MINUTES)

Figure 5* Effect of Intraperitoneal Amphetamine on Body Temperature of Rats at Various Ambient Temperatures

Dextroamphetamine sulfate (2 mg/kg) or 0.9% NaCl solution was injected lntraperitoneally in a volume of 2 ml/kg at time 0. At ambient temperatures of 25° and 6 C dextroamphetamine-treated animals showed significant (P<0.001) alterations in body temperature 30 and 60 minutes after injection compared to the corresponding saline controls. At an ambient temperature of 20°C dextroamphetamine-treated animals showed a significant increase in body temperature 30 and 60 minutes after injection compared to saline controls (P<0.05 and P<0.001, respectively). Vertical lines indicate + S.E.M.

18

+ 0 5

0 SALINE, 30°C (N»4) • AMPHETAMINE, 30* C(N«4|

A SALINE, 25*C (N-7) * AMPHETAMINE, 25*C (N-S>

• SALINE, 6* C (N» 5) • AMPHETAMINE, 6*C(N"6)

30 60

TIME (MINUTES)

Figure 6. Effect of Intraventricular Amphetamine on Body Temperature of Rats at Various Ambient Temperatures

Dextroamphetamine sulfate (100 meg) or 0.9% NaCl solution was injected into the lateral cerebral ventricle in a volume of 5 mcl at time 0. At ambient temperatures of 25° and 6°C dextroamphetamine-treated animals showed significant (P<0.001) reductions in body temperature compared to saline controls. Vertical lines indicate * S.E.M.

19

+ 0.5 -

O PHENOXYBENZAMINE + SALINE (N-4» • PHENOXYBENZAMINE + AMPHETAMINE tN-SI

A PROPRANOLOL + A PROPRANOLOL +•

SALINE (N«4) AMPHETAMINE (N-6)

60

TIME (MINUTES) 120

Figure 7. Effect of Phenoxybenzamine and Propranolol on Hypothermia Produced by Intraventricular Amphetamine in Rats at an Ambient Temperature of 25°C

Dextroamphetamine sulfate (100 meg) or 0.9% NaCl solution was injected into the lateral cerebral ventricle in a volume of 5 mcl at time 0. Phenoxybenzamine hydrochloride (10 mg/kg) was injected intraperitoneally 5 hours before dextroamphetamine or saline; propranolol hydrochloride (10 mg/kg) was injected subcutaneously 30 minutes before dextroamphetamine or saline. In both phenoxybenzamine and propranolol pretreated animals, dextroamphetamine produced significant (P<0.001) hypothermia at 30 minutes postadministration. Vertical lines indicate t S.E.M.

20

O SALINE, 25°C (N = 3) • AMPHETAMINE, 25"C (N=3)

A SALINE, 20°C (Na4) ^ • AMPHETAMINE, 20°C (N-4)

UJ a: o

£ + 0 . 5 a: UJ CL 5 UJ I-

5 o CQ

z - 0 . 5

UJ e> z < X o

60 30

TIME (MINUTES)

Figure 8. Effect of Propranolol on Hyperthermia Produced by Intraperitoneal Amphetamine in Rats at Ambient Temperatures of 25° and 20 C

Dextroamphetamine sulfate (2 mg/kg) or 0.9% NaCl solution was Injected intraperltoneally in a volume of 2 ml/kg at time 0. Propranolol hydrochloride (10 mg/kg) was injected subcutaneously 30 minutes before dextroamphetamine or saline. At an ambient temperature of 20°C dextro-amphetamlne-treated animals showed a significant decrease in body temperature 30 and 60 minutes after injection compared to saline controls (P<0.001 and P<0,05, respectively). At an ambient temperature of 25°C dextroamphetamine-treated animals showed no significant (P>0.05) change in body temperature compared to saline controls. Vertical lines indicate ± S.E.M.

21

O SALINE, 25*C (N«5) • AMPHETAMINE, 25'C (N-5)

A SALINE, 20#C (N-4)

A AMPHETAMINE, 20*C (N»4)

U t. + 10

Ul oc

a 00 UJ +0 5 Q. 5 Ul I-

>-Q O CD

Z

UJ e> z < X

- 0 5

o

30 60

TIME (MINUTES)

Figure 9. Effect of Phenoxybenzamine on Hyperthermia Produced by Intra peritoneal Amphetamine in Rats at Ambient Temperatures of 25° and 20 C

Dextroamphetamine sulfate (2 mg/kg) or 0.9% NaCl solution was injected intraperitoneally in a volume of 2 mg/kg at time 0. Phenoxybenzamine hydrochloride (10 mg/kg) was injected intraperitoneally 5 hours before dextroamphetamine or saline. At an ambient temperature of 20°C dextroamphetamine -treated animals showed a significant (P<0.05) decrease in body temperature 60 minutes after injection compared to saline control. At an ambient temperature of 2S°C dextroamphetamine-treated animals showed a significant (P<0.05) increase in body temperature 30 minutes after injection compared to saline control. Vertical lines indicate t S.E.M.

22

SALINE (N-S) AMPHETAMINE (N>5)

U • r Ui ac => Si oc UJ a. z UJ l—

-2

>-O o ffi z

UI CD z x -4 O

-5

10 20 60 30 40 50

TIME AFTER PITHING(MINUTES)

k Figure 10. Effect of Intraperitoneal Amphetamine on Body Temperature Change in Pithed Rats at an Ambient Temperature of 26°C

Dextroamphetamine sulfate (2 ml/kg) or 0.9% NaCl solution was injected intraperitoneally In a volume of 2 ml/kg immediately after pithing. Dextroamphetamine significantly retarded the process of hypothermia due to pithing at the 40-, 50- and 60-minute time periods (P<0.01) P<0.01 and P<0.05, respectively). Vertical lines indicate + S.E.M.

23

O SALINE (N-6) • AMPHETAMINE (N-5)

A SALINE, Adrtnalcclomiztd (N«3) A AMPHETAMINE, Adrenol«ctomiz6d IN"4)

O

UJ

5

£ 0. 2 UJ H

+0.5

5 3 Z

UJ o z < X o

-0.5

60

TIME (MINUTES)

Figure 11. Effect of Adrenalectomy on Hyperthermia Produced by Intraperitoneal Amphetamine at an Ambient Temperature of 25°C

Dextroamphetamine sulfate (2 mg/kg) or 0.9% NaCl solution was injected intraperitoneally in a volume of 2 ml/kg at time 0. Body temperature changes for dextroamphetamine and saline in non-adrenalectomized animals are taken from Fig. 5. Body temperature response to dextroamphetamine in adrenaiectomlzed animals is not significantly different (P>0.5) from saline control. Vertical lines indicate + S.H.M.

hyperthermic and hypothermic mechanisms have been demonstrated by sev

eral investigators (Keller, 1963; Jacobson and Squires, 1964; Rewerskl

and Jori, 1967). The present study suggests that both of these central

mechanisms are activated by dextroamphetamine and catecholamines but

that the hyperthermic mechanism is more sensitive and may be activated

by low doses of sympathomimetic amines, whereas the hypothermic mecha

nism requires larger doses. Once Initiated, however, the hypothermic

response appears to mask the centrally-induced hyperthermic response.

Although intraventricular doses of 100 meg and 400 meg of dex

troamphetamine both produce hypothermia, the shorter duration and

secondary hyperthermia associated with the larger dose may be due to

peripherally-induced hyperthermia consequent to escape of dextroampheta

mine into the systemic circulation. This explanation seems plausible

since a peripheral hyperthermic mechanism which responds to sympathomi

metic amines has been reported (Gordon et al., 1966; Bernard!, Paglialunga

and Jori, 1968; Cowell and Davey, 1968; Cowell, 1969; Gessa, Clay and

Brodie, 1969). The failure of 100 meg of dextroamphetamine intraven

tricular^ to produce secondary hyperthermia may have resulted from

inadequate escape into the systemic circulation. This suggestion of

inadequate escape is supported by a lack of hyperthermia following In

traperitoneal administration of 100 meg of dextroamphetamine and the

production of hyperthermia following intraperitoneal administration of

both 200 meg and 400 meg of dextroamphetamine. The above hypothesis im

plies that dextroamphetamine in adequate doses, regardless of route of

administration, may simultaneously activate central hyperthermic, cen

tral hypothermic and peripheral hyperthermic mechanisms.

25

To teBt this proposed central-peripheral Interaction of dextro

amphetamine, body temperature changes In rats were studied following the

Intraperitoneal Injection of a dose of dextroamphetamine (2 mg/kg) which

yielded the same brain concentration of drug as that produced by intra

ventricular administration of 100 meg of the amine. Since body tempera

ture represents a balance between heat production by the body and heat

loss from the body (Du Bois, 1939; Mount, 1960; Hammel and Hardy» 1963;

Borison and Clark, 1967; Hemingway and Price, 1968), altered ambient

temperatures were employed for the purpose of modifying this balance.

Variation of the ambient temperature produced no changes In body tem

perature of control animals injected lntraperitoneally with normal sa

line. In comparison, intraperitoneal Injection of dextroamphetamine

caused an elevation or a reduction of body temperature which was depen

dent upon the environmental temperature. Furthermore, intraventricular

injection of an equivalent dose of dextroamphetamine produced a reduc

tion of body temperature at all ambient temperatures tested. These

observations suggest that systemic injection of dextroamphetamine simul

taneously activates both hyperthermic and hypothermic mechanisms, thus

allowing the ambient temperature to become an important determinant of

body temperature. On the other hand, intraventricular injection of dex

troamphetamine, at the dose employed, predominantly activates central

hypothermic mechanisms, thus alteration of ambient temperature does not

qualitatively affect body temperature changes.

Adrenergic blocking agents were more effective In antagonizing

hyperthermia induced by peripherally Injected dextroamphetamine than hy

pothermia Induced by intraventrlcularly injected dextroamphetamine. At

26

an ambient temperature of 20°C propranolol and phenoxybenzamlne not only

prevented hyperthermia induced by peripheral administration of dextroam

phetamine but modified the response so that hypothermia ensued. This

"reversal" of body temperature change is presumably the result of re

moval of peripheral opposition (i.e., hyperthermia) to the central hypo

thermic component of dextroamphetamine action.

Pithing produced a precipitous fall In body temperature which

was effectively retarded by peripheral administration of dextroampheta

mine. This observation further supports the concept of a peripheral hy

perthermic action of dextroamphetamine since the central nervous system

is obliterated in pithed animals.

Because peripheral administration of epinephrine produced hyper

thermia (Masek and Raskova, 1964; Feldberg and Lottl, 1967) and because

dextroamphetamine has been shown to release epinephrine from the adrenal

gland (Rubin and Jaanus, 1966; Harvey, Sulkowski and Weenlg, 1968), it

was Important to Investigate the role which endogenous epinephrine might

play in hyperthermia produced by peripherally administered dextroam

phetamine. Since adrenalectomy blocked the hyperthermic response to

dextroamphetamine It is suggested that epinephrine liberated from the

adrenal medulla plays an important role in dextroamphetamine-induced hy

perthermia (perhaps through the release of free fatty acids). Because

adrenocortlcosterolds potentiate free fatty acid release by epinephrine

(Ingle, 1956; Reshef and Shapiro, 1960; Shafrlr and Steinberg, 1960),

the possibility exists that total adrenalectomy may interfere with dex

troamphetamine-induced hyperthermia through deprivation of corticoster

oids. However, since adrenalectomized rats showed normal growth without

27

supplementation with exogenous corticosteroids or saline and since rats

are known to have accumulations of tissues'similar to adrenocortical

tissue (Gorbman and Bern, 1962), it appears that there is no deficiency

of corticosteroids. Therefore, lack of adrenal epinephrine appears im

portant in preventing dextroamphetamine-induced hyperthermia.

The data presented in this section support the hypothesis that

dextroamphetamine may simultaneously activate central hyperthermic, cen

tral hypothermic and peripheral hyperthermic mechanisms.

CHAPTER 3

BEHAVIORAL AND PHYSIOLOGICAL FACTORS ASSOCIATED WITH DEXTROAMPHETAMINE-INDUCED BODY TEMPERATURE CHANGES

Numerous behavioral and physiological changes occur in associa

tion with alteration of body temperature (Du Bois, 1939; Euler, 1961).

Both peripheral and intracerebral administrations of amphetamine, which

alter body temperature, produce many behavioral, physiological and bio

chemical changes. Peripheral administration of amphetamine has been re

ported to produce stimulation of motor activity (Greenblatt and Osterberg,

1961; Moore, 1964; Rech, 1964; Stein, 1964; Morpurgo and Theobald, 1965;

Smith, 1965; Wenzel and Broadie, 1966; Clark, Blackman and Preston,

1967), stereotyped sniffing and gnawing (Mennear, 1965; Randrup and

Scheel-Kruger, 1966: Ernest, 1967; Fog, Randrup and Pakkenberg, 1967;

Morpurgo and Theobald, 1967; Randrup and Munkvad, 1967; Arnfred and

Randrup, 1968), elevation of oxygen consumption (Gyermek, 1950; Obal,

Mozes and Erdei, 1955; Zalls, Lundberg and Knutson, 1967), hypoglycemia

(Moore, Sawdy and Shaul, 1965; Moore, 1966a, 1966b; Clark et al., 1967),

hyperglycemia (Mantegazza, Naimzada and Rlva, 1968b), increased fat mo

bilization (Fasslna, 1966; Gessa, Clay and Brodie, 1969), depletion of

glycogen stores (Moore, 1966a) and alterations of tissue and plasma

electrolyte concentrations (Moore, 1966c). Intraventricular, intracls-

ternal or intrahypo thai antic administration of amphetamine has been re

ported to produce lethargy, mydriasis, tachypnea and panting (Gaddum and

Vogt, 1956), excitation (Leimdorfer, 1950), anorexia (Booth, 1968),

28

29

inhibition of phenylbenzoqulnone-induced writhing, piloerection, excita

tion (high dose) and depression (low dose) (Davis and Horlington, 1964).

i Two of the above parameters have been widely studied as causal

factors in drug-induced body temperature changes. Some investigators

have suggested that increased oxygen consumption or motor activity is

responsible for hyperthermia after administration of amphetamine or

other central nervous system stimulants (Sokoloff et al., 1957; Hardinge

and Peterson, 1964; Morpurgo and Theobald, 1965; Clark et al., 1967;

Zalis et al., 1967). Other investigators report poor correlation be

tween body temperature changes and increases in oxygen Consumption or

motor activity (Allen and Marley, 1967; Mount and Willmott, 1967;

Mantegazza et al.» 1968a; Turner and Spencer, 1968; Gessa, Cho, Clay,

Tagllamonte and Brodle, 1969). In view of this controversy, It was

considered important to study behavioral and physiological responses

which occurred In association with alterations of body temperature fol

lowing intraventricular injection of dextroamphetamine. Another justi

fication for such evaluations is provided by the apparent association

between behavioral and physiological events and alterations of body tem

perature during heating or cooling of the hypothalamus. For example,

elevation of hypothalamic temperature has been reported to produce de

pression (Hemingway et al., 1940; Euler, 1961). Kahn (1904) reported an

"almost narcotic effect" from heating carotid blood. Local hypothalamic

heating also causes peripheral vasodilation (Hemingway et al., 1940) and

reduction of body temperature (Magoun et al., 1938; Andersson, Ekman,

Gale and Sundsten, 1963; Andersson, Gale and Ohga, 1963; Hammel et al.,

1963; Edinger, Elsenman and Brobeck, 1969). On the other hand,

30

hypothalamic cooling causes peripheral vasoconstriction (Kruger et al.,

1959; Hamrnel, Hardy and Fusco, 1960; Andersson et al., 1964), elevation

of oxygen consumption (Jacobson and Squires, 1964) and elevation of body

temperature (Andersson, Ekman, Gale and Sundsten, 1963; Andersson, Gale

and Ohga, 1963). Since intraventricular injection of dextroamphetamine

has been shown to reduce body temperature it was considered important to

investigate the effect of Intraventricular injection of dextroampheta

mine on oxygen consumption and spontaneous activity. In addition,

stereotyped sniffing induced by peripheral administration of dextroam

phetamine was compared with the response induced by intraventricular ad

ministration of dextroamphetamine.


Where applicable, materials and methods used in Chapter 2 were

also used in this section. All studies in this section were performed

at an ambient temperature of 25°C. Techniques unique to this section

are described below.

Measurement of Oxygen Consumption

Oxygen consumption was determined with a Minute Oxygen Uptake

Servo Equipment Spirometer (model 160, Custom Engineering and Develop

ment Co*, St. Louis, Missouri). Animals were placed into the upper por

tion of a glass desiccator which was maintained at 25 t1°C by passing

cooled air (21-22°C) over the sealed chamber. The consumed oxygen was

replaced by the spirometer through a tubing connected to the desiccator

lid, and expired carbon dioxide and water were absorbed by potassium

hydroxide solution (187L) and a calcium sulfate-soda lime mixture.

31

Animals were protected from these chemicals by two layers of copper

screen (18 mesh). Oxygen consumption was measured for two consecutive

15-minute intervals, beginning 5 minutes after injection of saline or

dextroamphetamine. The rate of oxygen consumption was expressed as

ml/kg/mln.

Measurement of Spontaneous Activity

Spontaneous motor activity of individual rats was recorded with

an Ultra-Sonic/Electrostatic Grid Motion Detector (Alton Electronics Co.,

Gainesville, Florida). The home cage was placed in a sound-Insulated

box, and the transmitting and receiving transducers were positioned in

the box but outside of the cage. Sensitivity of the motion detector was

adjusted to record head movements but not breathing movements. Sponta

neous activity was continously recorded by means of a digital counter.

Cumulative spontaneous activity was noted at the end of each 10-mlnute

interval during a 1 hour period following saline or dextroamphetamine

injection.

Quantification of Sniffing Behavior

Evaluation of sniffing behavior was made by observing the number

of seconds per 60-second time period that the animal was engaged in

sniffing activity. Sniffing activity was defined as exploratory move

ments associated with alternating dorsi- and ventro-flexion of the neck.

Results

Approximately 5 minutes after dextroamphetamine was administered

lntraventricularly animals displayed a typical posture, of approximately

32

15 minutes duration, characterized by an elongation of the body with the

ventral surface of the body flattened against the floor of the cage.

Animals showed little mobility during this period. This effect was not

seen after intraperitoneal administration of dextroamphetamine or after

intraventricular or intraperitoneal administration of saline.

Effects of Dextroamphetamine on Oxygen Consumption

Oxygen consumption was significantly (P<0.05) increased (25%)

during the second 15-minute time interval following intraperitoneal ad

ministration of dextroamphetamine (2 mg/kg), although no alteration of

oxygen consumption was seen during the first 15-mlnute measurement (Fig.

12). Oxygen consumption was significantly (P<0.05) decreased (18%)

during the first 15-minute time interval following intraventricular ad

ministration of dextroamphetamine (100 meg), but oxygen consumption dur

ing the second 15-minute time interval was not different from control

values.

Effects of Dextroamphetamine on Spontaneous Motor Activity

Intraperitoneally administered dextroamphetamine increased cumu

lative spontaneous activity approximately 300 per cent during the 60-

mlnute observation (Fig. 13). The spontaneous activity of control

animals was highest during the initial (exploratory) 10 minute counting

interval and then declined with each succeeding interval to a very low

level* In contrast, the spontaneous activity of drug-treated animals

showed a high level (comparable to the initial value for control ani

mals) which was sustained throughout the 60-minute observation. In the

33

final measurement Intervals, activity of the drug-treated animals was

greater than for saline controls.

Intraventricularly administered dextroamphetamine had very lit

tle effect on cumulative spontaneous activity (reduction of 16%) during

the 60-minute observation period (Fig. 14). The spontaneous activity of

control animals was highest during the Initial 10-minute counting inter

vals and then declined with each succeeding interval to a very low

level. In contrast, the spontaneous activity of drug-treated animals

was low (25% that of the corresponding control value) during the initial

10-minute interval. This level of activity was maintained throughout

the 60-minute observation, however, and during the final measurement in

tervals activity of the drug-treated animals was greater than for saline

controls.

Evaluation of Sniffing Behavior

The administration of dextroamphetamine by either intraperito

neal or intraventricular injection stimulated sniffing behavior (Fig.

15). Intraperitoneal injection of dextroamphetamine caused almost con

tinuous sniffing behavior during the 30- and 60-minute time periods.

This response was diminished approximately 50 per cent at 120 minutes

postadmlnistration. Intraventricular injection of dextroamphetamine

also stimulated sniffing behavior during the 30- and 60-minute time peri

ods, but to a lesser degree. At 120 minutes postadmlnistration, the

sniffing activity had subsided and the animals treated intraventricu

larly with dextroamphetamine behaved no differently than saline-injected

controls. Testing at an ambient temperature of 6°C, pretreatment with

34

propranolol, or adrenalectomy did not alter the sniffing behavior pro

duced by intraperitoneal dextroamphetamine (Table 2).

Discussion

It has long been recognized that constancy of body temperature

in homeothermic animals is maintained by a balance between heat genera

tion and heat loss (Du Bols, 1939; Mount, 1960; Hammel and Hardy, 1963;

Borison and Clark, 1967; Hemingway and Price, 1968). Changes in oxygen

consumption rate and in spontaneous motor activity are evaluated in the

present study since these functions are associated with changes in heat

generative processes. The rate of oxygen consumption, which is a

reflection of the overall metabolic rate, is increased when heat gen

erative processess are stimulated and decreased when heat generative

processes are depressed.

Intraperitoneal administration of dextroamphetamine, which in

duces hyperthermia, caused an increase in oxygen consumption. These

observations are in agreement with the reports of Gyermek (1950) and

Zalis et al. (1967) and imply that a causal relationship may exist

between oxygen consumption rate and body temperature changes. That

increased oxygen consumption may be an important factor in dextroam-

phetamlne-induced hyperthermia is further suggested by the report that

adrenalectomy, which prevented dextroamphetamine-induced hyperthermia

(see previous Chapter), reduced the elevation of oxygen consumption

caused by administration of racemic amphetamine (Gyermek, 1950). Fur

thermore, in the present investigation intraventricular administration

of dextroamphetamine produced both hypothermia and a decrease in oxygen

35

40

SO

ZO

I 0

O* JC

INTRAPERITONEAL

ill rfl

10

I

Jl

10 MLINE AMPHETAMINE •AUttE AMPHETAMINE

«o

Jo

O " o

Ld (9 >o >-

O

10

INTRAVENTRICULAR

10

\h

•ALINE AMPHETAMINE

(»; 9-20 mfo )

k

to

h

•AUHK AMPHETAMINE

(1:20-59 mlit)

Figure 12. Effect of Intraperitoneal and Intraventricular Amphetamine on Oxygen Consumption Rate of Rats at an Ambient Temperature of 25°C

Intraperitoneally, dextroamphetamine sulfate (2 mg/kg) or 0.9% NaCl solution was injected in a volume of 2 ml/kg. Intraventricularly, dextroamphetamine sulfate (100 meg) or 0.9% NaCl solution was injected in a volume of 5 mcl. Oxygen consumption rate was determined during 2 consecutive 15-minute intervals, 5 to 20 minutes and 20 to 35 minutes after Injection (t: 5-20 min and t: 20-35 min, respectively). Number in box equals N. Adenotes significant difference from corresponding saline control (P<0.05). Vertical lines indicate + S.E.M.

36

• SALINE CN-5)

AMPHETAMINE (N>6)

10

9

? 8 O

X 7

CO h-Z z> o o

o <

6

5

4

> 3

2

I » #

r*

V.

ft

V.

K<

J: \V

i;

& o-lb 10-20 20-30 30-4o 40-30 90-60

TIME(MINUTES)

Figure 13. Effect of Intraperitoneal Amphetamine on Spontaneous Activity of Rats at an Ambient Temperature of 25°C

Dextroamphetamine sulfate (2 mg/kg) or 0.9% NaCl solution was injected intraperitonally in a volume of 2 ml/kg at time 0. Spontaneous activity was cumulated for each 10-minute interval for I hour, o denotes significant difference from saline control (P<0.01). • denotes significant difference from saline control (P<0.001). Vertical lines indicate + S.E.M.

37

10

9

8

>< 7

ro

O

CO I- 1

O 5 o

>- 4 K

> 3 I-<-> 0 < 2

I

I

0-10

• SALINE (N»4)

AMPHETAMINE (N»6)

y.

& $

A 3 $

1 10-20 20-30 30-40 40-50 50-60

TIME (MINUTES)

Figure 14. Effect of Intraventricular Amphetamine on Spontaneous Activity of Rats at an Ambient Temperature of 25 C

Dextroamphetamine sulfate (100 meg) or 0.9% NaCl solution was injected Into the lateral cerebral ventricle In a volume of 5 mcl at time 0. Spontaneous activity was cumulated for each 10-minute interval for 1 hour, o denotes significant difference from saline control (P<0.01). Adenotes significant difference from saline control (P<0.05). Vertical lines indicate + S.E.M.

38

O SALINE ip (N*8) • AMPHETAMINE ip (N• 10)

£ SALINE ivt (N*9) • AMPHETAMINE ivl (N 9)

UJ 30

60

TIME( MINUTES) 120

Figure 15. Effect of Intraperitoneal and Intraventricular Amphetamine on Sniffing Behavior of Rats at an Ambient Temperature of 25°C

Intraperltoneally (ip), dextroamphetamine sulfate (2 mg/kg) or 0.9X NaCl solution was Injected in a volume of 2 ml/kg at time 0. Intraventricular^ (lvt), dextroamphetamine sulfate (100 meg) or 0.9% NaCl solution was injected Into the lateral cerebral ventricle in a volume of 5 tncl at time 0. Intraperitoneal dextroamphetamine produced significant (P<0.001) enhancement of sniffing behavior at all time periods. Intraventricular dextroamphetamine produced significant enhancement at the 30- and 60-minute time periods (P<0.001 and P<0.01, respectively). Vertical lines indicate + S.E.M.

39

Table 2

Effect of Ambient Temperature of 6°C, Propranolol and Adrenalectomy on Sniffing Behavior Following Intraperitoneal Amphetamine in Rats

TREATMENT® SNIFFING SCORE (seconds in 60)

Saline (25°C)c 3 + 2b

(N=8 )

Amphetamine (25°C) 56+1 ( N= 10 )

Saline (6 C) 2*1 ( N=3 )

Amphetamine (6°C) 58+2 (N=7)

Propranolol + Saline (25°C) 1+1 (N=3 )

Propranolol + Amphetamine (25°C) 56+1 (N=4)

Adrenalectomy + Saline (25°C) 2+1 (N=3 )

Adrenalectomy + Amphetamine (25°C) 56 J 1 (N=4)

a Dextroamphetamine sulfate (2 rag/kg) or 0.97. NaCl solution was injected intraperitoneally in a volume of 2 ml/kg 30 minutes before sniffing score evaluation. Propranolol hydrochloride (10 mg/kg) was injected subcutaneously 30 minutes before saline or dextroamphetamine.

b Value represents mean sniffing score i- S.E.M.

c Ambient temperature

40

consumption. This last observation provides an additional basis for

suggesting that the body temperature effects of dextroamphetamine are at

least partially due to its actions on oxygen consumption rate.

Spontaneous motor activity of test animals is presumably the re

sult of interaction between environmental factors (i.e., manipulations,

process of injection, familiarization with the new environment of the

counting box) and dextroamphetamine treatment. Hence, the difference

between each test result and its corresponding control result should

represent the drug effect, a positive value Indicating stimulation and a

negative value indicating depression of spontaneous motor activity.

Consideration of these calculated values reveal that intraperitoneal in

jection of dextroamphetamine caused stimulation of spontaneous motor ac

tivity which developed gradually and became pronounced and sustained.

In comparison, intraventricular injection of dextroamphetamine caused a

biphasic response. Spontaneous motor activity was initially depressed,

gradually returned to near normal by the third and fourth observation

periods, and was moderately stimulated during the remainder of the test.

These spontaneous motor activity observations appear to correlate with

dextroamphetamine-induced body temperature changes noted In the previous

Chapter. It was noted that at an environmental temperature of 25°C In

traperitoneal Injection of dextroamphetamine produced hyperthermia which

reached a peak value at 30 minutes postadministration and was sustained.

On the other hand, intraventricular injection of dextroamphetamine pro

duced hypothermia which reached a peak value at 30 minutes postadmlnis-

tration and was partially recovered at 60 minutes postadministration.

41

It is also noteworthy that moderate local heating of the hypo

thalamus causes sedation (Magoun et al.» 1938; Andersson, Gale and Ohga,

1963; Hammel et al., 1963; Edinger et al., 1969). These observations

lend further support to the concept of correlation between depression

of spontaneous activity and the production of hypothermia.

Sniffingp a characteristic dextroamphetamine-induced behavioral

effect, can be readily evaluated without disturbing the animal. This

parameter was quantified during some of the body temperature investiga

tions. Both intraventricularly and intraperitoneally administered dex

troamphetamine produced an increase in sniffing behavior, the duration

of which appeared to correspond with the sojourn of dextroamphetamine in

brain tissue (see Fig. 4). Sniffing behavior seems to be unrelated to

changes in body temperature since treatments which prevent hyperthermia

produced by peripherally injected dextroamphetamine did not affect snif

fing behavior. This 1b not unexpected since sniffing behavior is re

ported to be a centrally-mediated phenomenon (Laursen, 1962; Ernest and

Smelik, 1966; Fog, 1967), whereas as pointed out in Chapter 2, dextroam

phetamine -induced hyperthermia may result from activation of peripheral

mechanisms.

Of the 3 physiological and behavioral parameters which were

evaluated in this section, oxygen consumption rate and spontaneous motor

activity show the best correlations with dextroamphetamine-induced tem

perature changes. Since changes in spontaneous motor activity may be at

least partially responsible for corresponding changes in oxygen consump

tion rate, it is not surprising that they both correlate with body

temperature changes produced by dextroamphetamine. In view of the

apparently similar effects produced by moderate local heating of the

hypothalamus and Intraventricular Injection of dextroamphetamine (1.

sedation and hypothermia), the hypothalamus Is suggested as an lmpor

tant site for the hypothermic action of dextroamphetamine.

CHAPTER 4

THE ROLE OF CENTRAL MONOAMINES IN DEXTROAMPHETAMINE-INDUCED HYPOTHERMIA

The sympathomimetic activity of amphetamine is prevalently

thought to be mediated through the libration of norepinephrine (Burn

and Rand, 1958; Burn, I960; Trendelenberg» 1963; Harvey et al., 1968).

The role of norepinephrine in the action of amphetamine on the central

nervous system is less clearly established (Van Rossum, Van der Schoot

and Hurkmans, 1962; Moore and Lariviere, 1963, Moore, 1964; Stein, 1964;

Carlsson, Linqvist, Dahlstrom, Fuxe and Masuoka, 1965; Hanson, 1966;

Mennear and Rudzik, 1966; Corrodi, Fuxe and Hokfelt, 1967; Littleton,

1967; Morpurgo and Theobald, 1967). Since dextroamphetamine releases

norepinephrine from brain tissue following intraventricular administra

tion (Carr and Moore, 1969a) and peripheral administration (Moore,

1963a, 1963b, 1964; Smith, 1965; Littleton, 1967; Fuxe and Ungerstedt,

1968; Gropetti and Costa, 1969; Welch and Welch, 1969), and since intra

ventricular administration of norepinphrlne causes alterations of body

temperature (Feldberg and Lottl, 1967; Myers and Yaksh, 1968) similar to

those following intraventricular administration of dextroamphetamine

(see Chapter 2), it would appear that norepinephrine may be a central

mediator In the hypothermic action of dextroamphetamine.

Another catecholamine, dopamine, has been implicated as a media

tor of various behavioral effects of dextroamphetamine (Van Rossum and

Hurkmans, 1964; Ernest and Smelik, 1966; Randrup and Scheel-Kruger,

43

44

1966; Ernest, 1967; Fog, 1967). Administered intraventricularly it

caused hyperthermia (Myers and Yaksh, 1968). Dextroamphetamine has been

shown to release dopamine into the ventricular perfusate after intraven

tricular administration (Carr and Moore, 1969b). Peripheral administra

tion of dextroamphetamine has been reported to increase (Smith, 1965;

Lewander, 1968a; Littleton, 1967; Welch and Welch, 1969) and decrease

(Lewander, 1968b; Welch and Welch, 1969) brain concentrations of this

catecholamine. The increase was seen following Injection of small doses

of dextroamphetamine whereas the decrease was seen following injection

of large or repeated doses.

A third monoamine which might be a mediator of dextroampheta-

mine-induced hypothermia is 5-hydroxytryptamine. This amine has been

reported to produce hypothermia when it is administered by the intra

ventricular route to rats (Feldberg and Lotti, 1967; Myers and Yaksh,

1968). In addition, brain concentrations of 5-hydroxytryptamine have

been reported to be elevated by dextroamphetamine (McLean and McCartney,

1961; Smith, 1965; Welch and Welch, 1969).

Different types of studies have been used to relate alterations

in body temperature with endogenous chemicals. For example, drugs which

alter brain norepinephrine, dopamine and 5-hydroxytryptamine levels were

administered and body temperature was subsequently monitored (Ingenito

and Bonnycastle, 1967a; Somerville and Whittle, 1967; Shellenberger and

Elder, 1967, 1968). In general, studies of this type have shown poor

correlations between whole brain amine levels and alteration of body

temperature.

45

Another approach has been to expose animals to altered environ

mental temperature and monitor tissue levels or turnover of suspected

mediators. These studies report norepinephrine synthesis In peripheral

tissues to be enhanced at reduced environmental temperatures (Ollverio

and Stjarne, 1965; Corrodi and Malmfors, 1966; Goldstein and Nakajima,

1966; Gordon et al., 1966). Some studies have reported little or no

change In metabolism of brain norepinephrine and 5-hydroxytryptamine

during cold exposure (Corrodi, et al., 1967; Reid et al., 1968; Ingenito

and Bonnycastle, 1967b; Ingenito, 1968). Others have reported the con

centration of brain norepinephrine to be reduced in cold-stressed ani

mals (Levi and Maynert, 1964; Maynert and Levi, 1964). With heat

stress, the metabolism of brain norepinephrine and 5-hydroxytryptamine

is increased (Corrodi et al., 1967; Shellenberger and Elder, 1967; Reid

et al., 1968; Simmonds and Iversen, 1969).

An additional approach has involved direct administration of the

proposed mediator. Work of this type has gained wide acceptance since

Feldberg and Myers (1964a) proposed the concept that the body tempera

ture is related to a balance between 5-hydroxytryptamine and norepineph

rine in the hypothalamus.

Two of the principles described above will be applied in the

present investigation. In one approach, tissue levels of catecholamines

and/or 5-hydroxytryptamine will be reduced by commonly used depleting

agents* and dextroamphetamine will then be administered intraventricu

lar^ to determine the effect of monoamine defecit on the usual hypo

thermic effect. This investigation will attempt to establish which, if

any, of these monoamines are important in mediating the body temperature

46

response Induced by intraventricular administration of dextroampheta

mine. The drugs used for depleting tissue catecholamines and 5-hydroxy

tryptamine are Ro 4-1284, p-chlorophenylalanine, and a combination of

alpha-methyl-m-tyrosine with alpha-methyl-p-tyrosine. Ro 4-1284, a ben-

zoquinolizine, produces a rapid reduction of brain norepinephrine, dopa

mine and 5-hydroxytryptamine levels. The levels of these monoamines are

maximally reduced from 1 to 3 hours postadministration and are recovered

to approximately 85 per cent of normal by 12 hours postadministration

(Pletscher, 1957; Quinn, Shore and Brodie, 1959; Pletscher, Burkard and

Gey, 1964; Carlsson and Lindqvist, 1966; Pletscher and Da Prada, 1966;

Pletscher and Bartholini, 1967; Jobe, 1970). p-Chlorophenylalanine, a

hydroxylase inhibitor which is more selective for tryptophan hydroxylase

than for tyrosine hydroxylase (Koe and Weissman, 1966), produces a

marked reduction of brain 5-hydroxytryptamine levels but only a modest

reduction of brain catecholamines from 1 to 4 days postadministration

(Koe and Weissman, 1966; Jequier, Lovenberg and Sjoerdsma, 1967;

Lovenberg, Jequier and Sjoerdsma, 1968; Sato et al., 1967; Welch and

Welch, 1967; Jobe, 1970). Alpha-methyl-m-tyrosine, 12 hours postadmin

istration, reduced brain norepinephrine and dopamine levels to less than

60 per cent of normal while leaving brain 5-hydroxytryptamine at near

normal levels (Hess et al., 1961; Porter, Totaro and Leiby, 1961;

Carlsson, Falck and Hillarp, 1962; Carlsson, Dahlstrom, Fuxe and Hillarp,

1965; Fuxe, 1965; Shore, Alpers and Busfield, 1966; Jobe, 1970). Alpha-

methyl-p-tyrosine, a tyrosine hydroxylase inhibitor, reduced brain nor

epinephrine and dopamine levels (Nagatsu, Levitt and Udenfriend, 1964;

Levitt et al., 1965; Rech, Borys and Moore, 1966; Spector, Sjoerdsma and

Udenfriend, 1965; Weissman and Koe, 1967; Jobe, 1970). The combination

of alpha-methyl-m«tyrosine and repeated doses of alpha-methyl-p-tyrosine

has been reported to produce marked lowering of brain norepinephrine and

dopamine with no effect on brain 5-hydroxytryptaraine (Jobe, 1970).

Another approach used in this section of the investigation in

volves a study of the effect of dextroamphetamine administration on

brain monoamine levels and its relationship to dextroamphetamine-induced

hypothermia. Repeated systemic administrations of dextroamphetamine at

short intervals cause the development of tachyphylaxis to peripheral re

sponses* a phenomenon which is associated with decreases in peripheral

catecholamine levels (Burn and Rand, 1958; Cession-Fossion, 1963; Harvey

et al., 1968). Analogous studies to observe the effect of repeated in

traventricular injections of dextroamphetamine on centrally mediated

hypothermia and on brain monoamine levels may provide additional infor

mation regarding the relative Importance of the various monoamines in

dextroamphetamine-induced hypothermia.


Where applicable, materials and methods used in Chapter 2 were

also used in this section. All studies in this section were performed

at an ambient temperature of 25°C. Techniques unique to this section

are described below.

Effects of Monoamine-depleting Agents on Dextroamphetamine-induced Body Temperature Changes

Monoamine-depleting agents were Injected into rats, an appro

priate interval of time was allowed to elapse, dextroamphetamine was

48

injected intraventricularly or Intraperitoneally, and body temperature

was determined. Simultaneous depletion of brain norepinephrine, dopa

mine and 5-hydroxytryptamine was accomplished by administration of

Ro 4-1284 (10 mg/kg) 1 hour before testing with dextroamphetamine.

Twelve-hour pretreatment with the benzoquinollzine was employed to de

termine the body temperature effect of dextroamphetamine at a time when

monoamines have recovered to near normal levels. Whole brain monoamine

levels were assayed by the spectrophotofluorometric technique described

below. Depletion of brain 5-hydroxytryptamine (with modest depletion of

catecholamines) was induced by intraperitoneal injection of p-chloro-

phenylalanine (300 mg/kg) 3 days before testing with dextroamphetamine.

Simultaneous depletion of both norepinephrine and dopamine was achieved

by combination treatment with a single dose of alpha-methyl-m-tyrosine

(500 mg/kg) and repeated doses of alpha-methyl-p-tyrosine (80 mg/kg).

The former drug was injected 12 hours and the latter drug was Injected

12, 8 and 4 hours before testing with dextroamphetamine.

Brain Monoamine Levels after Single Intraventricular or Intraperitoneal Administration of Dextroamphetamine

Rats were injected intraventricularly or intraperitoneally with

saline or dextroamphetamine solutions as described previously. Whole

brain concentrations of norepinephrine, dopamine, or 5-hydroxytryptamine

of some animals were analyzed 30 minutes postadministration by the spec

trophotof luorometric technique described below. Various parts of brain

from other animals were analyzed for monoamine concentration by the his-

tochemical fluorescence technique described below.

49

Effects of Repeated Intraventricular Injections of Dextroamphetamine on Hypothermic Response

Rats were injected intraventricularly with saline or dextroam

phetamine and body temperature responses were recorded at various times.

When the body temperature of dextroamphetamine-treated animals returned

to preinjectlon levels, saline and dextroamphetamine treatments were re

peated in control and test animals, respectively, and body temperature

was again monitored. This procedure was repeated until hypothermia was

nonsignificant.

Brain Monoamine Levels at Peak Hypothermic Effect Induced by the Last of Three Repeated Intraventricular Injections of Dextroamphetamine

Rats were administered 3 intraventricular injections of saline

or dextroamphetamine solution at 2-hour intervals (corresponding to time

for recovery from hypothermia). Thirty minutes after the final injec

tion, a time which corresponds to peak hypothermia, the anterior hypo

thalamic nucleus periventricularis and nucleus caudatus putamen were

analyzed by the histochemical fluorescence technique described below.

Brain Monoamine Levels at Time of Recovery of Body Temperature after Repeated Intraventricular Injections of Dextroamphetamine

Rats were administered up to 4 intraventricular injections of

saline or dextroamphetamine solution at 2-hour intervals between injec

tions 1, 2 and 3 and at a 1-hour interval between Injections 3 and 4.

These time intervals, including the 1-hour interval after the fourth in

jection, correspond to the time of recovery from hypothermia Induced by

multiple injections of dextroamphetamine. Fluorescence in the anterior

hypothalamic nucleus periventricularis and nucleus caudatus putamen was

50

analyzed at the following times: (1) before dextroamphetamine or saline

injection, (2)2 hours after the second dextroamphetamine or saline in

jection, and (3) 1 hour after the fourth dextroamphetamine or saline

injection.

Spectrophotofluorometric Assay of Catecholamines and 5-Hydroxytryptamine

A spectrophotofluorometric technique was used for biochemical

determinations of whole brain norepinephrine, dopamine and 5-hydroxy-

tryptamine. The extraction technique was a modification of the methods

described by Wiegand and Perry (1961) and Mead and Finger (1961). Assay

procedures for detection of norepinephrine, dopamine and 5-hydroxytryp

tamine were similar to those of Shore and Olin (1958), Carlsson and

Waldeck (1958) and Bogdanski et al. (1956), respectively. A detailed

account of the spectrophotofluorometric procedure is presented in Appen

dix £.

Histochemical Fluorescence Technique

The histochemical fluorescence technique for viewing norepineph

rine, dopamine and 5-hydroxytryptamine used in the present work is a

modification of the method reported by Falck and Owman (1965). The

areas of the brain employed in the present studies included the anterior

hypothalamic nucleus periventricularis, the fluorescence of which is

primarily norepinephrine (Falck, 1964; Dahlstrom, Fuxe and Hillarp, 1965;

Friede, 1966); the nucleus caudatus putamen, the fluorescence of which

is primarily due to dopamine (Fuxe, 1965); and the globus pallidus, the

fluorescence of which is primarily due to 5-hydroxytryptamine (Fuxe,

51

Hokfelt and Ungerstedt, 1968). A description of the histochemical tech

nique Is presented in Appendix F.

Results

Effects of Monoamine-depleting Agents on Dextroamphetamine-induced Body Temperature Changes

One-hour pretreatment with Ro 4-1284 reduced whole brain norepi

nephrine, dopamine and 5-hydroxytryptamine by 59, 72 and 73 per cent,

respectively (Table 3). This pretreatment antagonized the hypothermic

effect of intraventricularly administered dextroamphetamine (Table 4,

compare Fig. 16 with Fig. 2) and the hyperthermic effect of intraperito-

neally administered dextroamphetamine (compare Fig. 17 with Fig. 4).

Antagonism of the hypothermic action of dextroamphetamine was no longer

apparent 12 hours after pretreatment (Table 4). Pretreatment with

p-chlorophenylalanine did not affect the body temperature change induced

by intraventricular administration of dextroamphetamine (Table 4).

Catecholamine depletion by combination pretreatment with alpha-methyl-m-

tyroslne and alpha-methyl-p-tyrosine partially antagonized both the

hypothermic effect of intraventricularly administered dextroamphetamine

(compare Fig. 16 with Fig. 2) and the hyperthermic effect of intraperi-

toneally administered dextroamphetamine (compare Fig. 17 with Fig. 4).

Brain Monoamine Levels after Single Intraventricular or Intraperitoneal Administration of Dextroamphetamine

Intraventricular or intraperitoneal administration of dextroam

phetamine produced no significant (P>0.1) alteration in whole brain

concentration of norepinephrine, dopamine or 5-hydroxytryptamine 30

52

minutes after injection (Table 5). However, fluorescence in the ante

rior hypothalamic nucleus periventricularis was noticeably reduced 30

minutes after injection and was normal by 60 minutes postadministration

(Fig. 18). Administration of dextroamphetamine by either intraventricu

lar or intraperitoneal injection produced no change in fluorescence in

the nucleus caudatus putamen or globus pallidus 30 minutes after treat

ment (Fig. 19).

Effects of Repeated Intraventricular Injections of Dextroamphetamine on Hypothermic Response

Repeated intraventricular injections of dextroamphetamine pro

duced a peak hypothermic response, 30 minutes after each Injection,

which was progressively less pronounced (Fig. 20). The fifth injection

of dextroamphetamine produced a hypothermic response in test animals

which was not significantly different (P>0.1) from that of correspond

ing saline-treated controls (V, Fig. 20). It was also observed that

recovery of body temperature to preinjection level required 2 hours fol

lowing the first and second dextroamphetamine injections. This recovery

time was reduced to 1 hour following the third, fourth and fifth Injec

tions of dextroamphetamine.

Brain Monoamine Levels at Peak Hypothermic Effect Induced by the Last of Three Repeated Intraventricular Injections of Dextroamphetamine

Analysis of brain tissue 30 minutes after the last of 3 repeated

intraventricular injections of dextroamphetamine revealed reduction in

fluorescence in the anterior hypothalamic nucleus periventricularis

(compare A with B, Fig. 21). Triple intraventricular injection of dex

troamphetamine appears to be more effective in reducing fluorescence in

53

the hypothalamus than a single intraventricular injection of dextroam

phetamine (compare B» Fig. 21 with B, Fig. 18). In the case of the

nucleus caudatus putamen, 3 repeated intraventricular injections of dex

troamphetamine appear to have no effect on fluorescence (compare A1 with

B', Fig. 21).

Brain Monoamine Levels at Time of Recovery of Body Temperature Following Repeated Intraventricular Injections of Dextroamphetamine

Analysis of brain tissue at a time when body temperature had

recovered to preinjection levels following repeated intraventricular in

jections of dextroamphetamine revealed a progressive reduction of fluo

rescence in the anterior hypothalamic nucleus periventricularis (compare

A, B* and C1, Fig. 22). Fluorescence In the hypothalamus of controls

was also progressively reduced by repeated intraventricular injections

of saline solution (compare A, B and C, Fig. 22), but the reduction In

fluorescence was more pronounced in dextroamphetamlne-treated animals.

In contrast, fluorescence In the nucleus caudatus putamen was unchanged

by repeated intraventricular Injections of saline or dextroamphetamine

solution (Fig. 23).

Discussion

Ro 4-1284 markedly depleted norepinephrine, dopamine and 5-hy-

droxytryptamine and antagonized the hypothermic action of intraventricu-

larly administered dextroamphetamine. This antagonism was lost at a

time when brain levels of these monoamines were apparently recovered to

near normal. These observations imply that deficiency of 1 or more of

these monoamines may be responsible for the reduced effect of

54

Table 3

Effect of Ro 4-1284 on Whole Brain Norepinephrine, Dopamine and 5-Hydroxytryptamine Concentrations in Rats

at an Ambient Temperature of 25°C

TREATMENT3 NOREPINEPHRINE DOPAMINE 5-HYDROXYTRYPTAMINE (mcg/g tissue) (mcg/g tissue) (mcg/g tissue)

Saline 0.33 ± 0.02b 0.86 0.03 0.52 ± 0.03

Ro 4-1284 0.08 ± 0.01c 0.24 t 0.03° 0.14 + 0.02c

a Ro 4-1284 (10 mg/kg) or 0.9% NaCl solution was injected intraperito-neally 1 hour before sacrifice.

b Values represent mean concentration ± S.E.M. N equals 4 for all determinations.

c Significantly different from corresponding values In saline-treated animals (P<0.01).

Table 4

Effect of p-Chlorophenylalanine and Ro 4-1284 on Hypothermia Produced by Intraventricular Amphetamine at an

Ambient Temperature of 25°C

PRETREATMENT CHANGE IN BODY TEMPERATURE (°C) (30 minutes after amphetamine)3

Saline (1 hr)c -2.05 ±0.15b

(N=4)

Ro 4-1284 (1 hr)c -0.40 t 0.19e

(N=4)

Saline (12 hr)c -1.72 + 0.17 (N=4)

Ro 4-1284 (12 hr)c -1.45 ± 0.13 (N=6)

Vehicle (3 da)d -2.00 ± 0.18 (N=3)

p-Chlorophenylalanine (3 da)d -1.62 ± 0.27 (N=10)

a Dextroamphetamine sulfate (100 meg) was injected into the lateral cerebral ventricle in a volume of 5 mcl.

b Mean change in body temperature + S.E.M.

c Ro 4-1284 (10 mg/kg) or 0.9% NaCl solution was injected intraperitoneally 1 or 12 hours before dextroamphetamine .

d p-Chlorophenylalanine (300 mg/kg) or suspension vehicle (0.4% Tween 80, 1% carboxymethylcellulose, 0.97. NaCl) was injected intraperitoneally 3 days before dextroamphetamine.

e Value taken from data in Fig. 16. Significantly different from saline control (P<0.001)

56

A R04-I284 4 SALINE (N-4) A R04-L264 + AMPHETAMINE (N-4)

• O.MMT and aMPT + SALINE (N-4) • o»MMT ond aMPT + AMPHETAMINE (N-5) U

+ 0.5 Ui oz

6 ac UJ a. 2 UJ H

>•

§ "0 5 CO

z UJ o z < X o

60 30

TIME(MINUTES)

Figure 16. Body Temperature Response to Intraventricular Amphetamine at an Ambient Temperature of 25°C in Rats Pretreated with Ro 4-1284 or a Combination of Alpha-methyl-m-tyrosine (aMMT) and Alpha-methyl-p-tyrosine (aMPT)

Ro 4-1284 (10 mg/kg) was injected intraperitoneally 1 hour before saline or amphetamine. aMMT (500 mg/kg) was injected intraperitoneally 12 hours before saline or amphetamine; aMPT (80 mg/kg) was injected intraperitoneally 12, 8 and 4 hours before saline or amphetamine. Dextroamphetamine sulfate (100 meg) or 0.9% NaCl solution was injected into the lateral cerebral ventricle in a volume of 5 mcl at time 0. Vertical lines indicate ± S.E.M.

57

O o

UJ cc. 3

5 a: UJ o. 2E UJ

>-o o ffi

UJ o z < u

A R04-IZ84 + SALINE (N-3) A R04-I284 + AMPHETAMINE (N»7> • ammf and ampt + SALINE (N>3) • am ml and ampt + AMPHETAMINE <N«6>

+ 0.5

-0.5

i 30

—i 60

TIME (MINUTES)

Figure 17. Body Temperature Response to Intraperitoneal Amphetamine at an Ambient Temperature of 25°C in Rats Pretreated with Ro 4-1284 or a Combination of Alpha-methyl-m-tyrosine (aMMT) and Alpha-methyl-p-tyrosine (aMPT)

Ro 4-1284 (10 mg/kg) was injected intraperitoneally I hour before saline or amphetamine. aMMT (500 mg/kg) was injected intraperitoneally 12 hours before saline or amphetamine; aMPT (80 mg/kg) was injected intraperitoneally 12, 8 and 4 hours before saline or amphetamine. Dextroamphetamine sulfate (2 mg/kg) or 0.97a NaCl solution was injected intraperitoneally in a volume of 2 ml/kg at time 0. Vertical lines indicate ± S.E.M.

58

Table 5

Effect of Intraventricular and Intraperitoneal Amphetamine on Whole Brain Norepinephrine, Dopamine and 5-Hydroxytryptamine Concentrations in Rats at an Ambient Temperature of 25°C

TREATMENT NOREPINEPHRINE DOPAMINE 5-HYDROXYTRYPTAMINE (mcg/g tissue) (mcg/g tissue) (mcg/g tissue)

Saline (ivt)a 0.36 ± 0.03c 0.91 ± 0.06 0.49 ± 0.03

Amphetamine (ivt)a 0.40 + 0.02 1.01 + 0.04 0.50 + 0.02

Saline <ip)b 0.37 + 0.02 0.78 + 0.06 0.44 ± 0.02

Amphetamine (ip)b 0.42 + 0.03 1.01 + 0.11 0.48 + 0.01

a Dextroamphetamine sulfate (100 meg) or 0.9% NaCl solution was injected into the lateral cerebral ventricle in a volume of 5 mcl 30 minutes before sacrifice.

b Dextroamphetamine sulfate (2 mg/kg) or 0.9% NaCl solution was injected lntraperitoneally in a volume of 2 ml/kg 30 minutes before sacrifice.

c Mean concentration ± S.E.M. N equals 4 to 6 for each determination.

Figure 18. Effect of Intraventricular and Intraperitoneal Amphetamine on Fluorescence of Anterior Hypothalamic Nucleus Periventricularis in the Rat at an Ambient Temperature of 25°C

Intraventricularly (ivt), dextroamphetamine sulfate (100 meg) or 0.9% NaCl solution in a volume of 5 mcl was injected in A, B, C and D. Intraperitoneally (ip), dextroamphetamine sulfate (2 mg/kg) or 0.9% NaCl solution in a volume of 2 mg/kg was injected in A', B', C* and D'.

Key to figure:

A 30 min after ivt saline B 30 min after ivt amphetamine C 60 min after ivt amphetamine D 120 min after ivt amphetamine

A' 30 min after ip saline B* 30 min after ip amphetamine C1 60 min after ip amphetamine D1 120 min after ip amphetamine

59

IV T IP

A

B

c

D

Figure 18. Effect of Amphetamine on Hypothalamic Fluorescence

Figure 19. Effect of Intraventricular and Intraperitoneal Amphetamine on Fluorescence of Nucleus Caudatus Putamen (NCP) and Globus Pallidus (GP) in the Rat at an Ambient Temperature of 25°C

Intraventricularly (ivt), dextroamphetamine sulfate (1Q0 meg) or 0,9% NaCl solution in a volume of 5 mcl was injected in A, Bt C and D 30 minutes before sacrifice. Intraperitoneally (ip), dextroamphetamine sulfate (2 mg/kg) or 0.9% NaCl solution in a volume of 2 ml/kg was injected in A1, B', C' and D1 30 minutes before sacrifice.

Key to figure:

A ivt saline B ivt amphetamine

A' ip saline B' ip amphetamine

C ivt saline D ivt amphetamine

C' D'

ip saline ip amphetamine

60

IVT IP

A

e

NC!It

c

Figure 19. Amphetamine on Nucleus Caudatus Putamen and Globus Pallidus

61

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t£J AMPHETAMINE (N-8>

• SALINE (N-4)

Figure 20. Peak Hypothermic Responses to Repeated Intraventricular Injections of Amphetamine at an Ambient Temperature of 25°C

Dextroamphetamine sulfate (100 meg) or 0.9% NaCl solution in a volume of 5 mcl was injected into the lateral cerebral ventricle at 2-hour intervals for doses 2 and 3 and at 1-hour Intervals for doses 4 and 5. These times correspond to the time of body temperature recovery from dextroamphetamine -induced hypothermia. Values in the figure represent the mean body temperature change + S.E.M. 30 minutes after saline or amphetamine injection. All hypothermic effects except that following injection V are significantly different (P<0.001) from saline control.

62

A

B

Figure 21. Fluorescence in Anterior Hypothalamic Nucleus Periventricularis and Nucleus Caudatus Putamen at Time of Peak Hypothermia after Multiple Intraventricular Injections of Amphetamine at an Ambient

0 Temperature of 25 C

Dextroamphetamine sulfate (100 meg) or 0.9% NaCl solution in a volume of 5 mel was injected into the lateral cerebral ventricle at 2-hour intervals for 3 injections. These times correspond to the time of body temperature recovery from dextroamphetamine-induced hypothermia. Animals were sacrificed 30 minutes after third injection.

Key to figure:

A hypothalamus (saline) A' caudate nucleus (saline) B hypothalamus (amphetamine) B' caudate nucleus (amphetamine)

A

B

c c'

Figure 22. Fluorescence in Anterior Hypothalamic Nucleus Periventricularis at Time of Body Temperature Recovery after Multiple Intraventricular Injections of Amphetamine at an Ambient Temperature of 25°C

63

Dextroamphetamine sulfate (100 meg) or 0.9% NaCl solution in a volume of 5 mel was injected into the lateral cerebral ventricle at 2-hour intervals for doses 2 and 3 and at 1-hour interval for dose 4. These times, including 1 hour after the fourth dose, correspond to the time of body temperature recovery from dextroamphetamine-induced hypothermia.

Key to figure:

A no injection

B 2 hours after second saline injection B' 2 hours after second amphetamine injection

c 1 hour after fourth saline injection C' 1 hour after fourth amphetamine injection

A

B a'

c

Figure 23. Fluorescence in Nucleus Caudatus Putamen at Time of Body Temperature Recovery after Multiple Intraventricular Injections of Amphetamine at an Ambient Temperature of 25°C

Dextroamphetamine sulfate (100 meg) or 0.9% NaCl solution in a volume

64

of 5 mel was injected into the lateral cerebral ventricle at 2-hour intervals for doses 2 and 3 and at 1-hour interval for dose 4. These times, including 1 hour after the fourth dose, correspond to the time of body temperature recovery from dextroamphetamine-induced hypothermia.

Key to figure:

A no injection

B 2 hours after second saline injection B' 2 hours after second amphetamine injection

c 1 hour after fourth saline injection C' 1 hour after fourth amphetamine injection

dextroamphetamine on body temperature. Marked reduction of brain 5-hy

droxy try pt amine concentrations with slight reduction of catecholamine

levels (1.e., p-chlorophenylalanine pretreatment) failed to produce an

tagonism of the hypothermic effect produced by intraventricular injec

tion of dextroamphetamine. In contrast, depletion of catecholamines

without depletion of 5-hydroxytryptamine (I.e., alpha-methyl-ni-tyroslne

and alpha-methyl-p-tyrosine pretreatment) caused approximately AO per

cent reduction In the hypothermic response induced by intraventricular

injection of dextroamphetamine. These data suggest that the catechola

mines may participate in mediating the central hypothermic action of

dextroamphetamine and that 5-hydroxytryptamine may be of little or no

Importance In this regard.

A number of published observations lend support to the pre

sumption that a central hypothermic action of dextroamphetamine is me

diated through the release of endogenous catecholamines. For example,

it has been demonstrated that intraventricular injection of norepineph

rine can cause hypothermia (Feldberg and Lottl, 1967; Myers and Yaksh,

1968) and that injection of dextroamphetamine into the cerebrospinal

space releases norepinephrine into the cerebrospinal fluid (Carr and

Moore, 1969a). Also, dextroamphetamine releases catecholamines in such

a manner as to form metabolites which are produced primarily by cate

chol -o-methyl transferase rather than by monoamine oxidase (Glowlnskl,

Snyder and Axelrod, 1965; Glowlnskl, Axelrod and Iversen, 1966; Fuxe and

Ungerstedt, 1968), suggesting that these released endogenous catechola

mines have access to receptor sites In the brain.

66

If catecholamines are to be accepted as possible mediators of

dextroamphetamine-lnduced hypothermia, it is important to correlate al

teration in concentration or turnover of catecholamines in brain tissue

with reduction in body temperature. Thirty minutes after intraventricu

lar or intraperitoneal administration of dextroamphetamine) spectropho-

tofluorometric analyses showed no change in the concentration of whole

brain norepinephrine, dopamine and 5-hydroxytryptamine. However, histo-

chemlcal fluorescence analyses of specific areas of the brain demonstra

ted a reduction of norepinephrine in the anterior hypothalamic nucleus

periventricularis, but no change in the concentration of dopamine in the

nucleus caudatus putamen or of 5-hydroxytryptamine in the globus palli-

dus. The anterior region of the hypothalamus is considered important in

thermoregulation (Meyer, 1913; Teague and Ranson, 1936; Jacobson and

Squires, 1961; Bligh, 1966b), and reduction of norepinephrine in this

area correlates with peak hypothermia induced by intraventricular injec

tion of dextroamphetamine. The observation that intraperitoneal admin

istration of dextroamphetamine also reduces norepinephrine levels in the

hypothalamus appears to coincide with the postulation that systemic ad

ministration not only activates peripheral hyperthermic mechanisms but

also central hypothermic mechanisms (see Chapter 2).

Analyses of the nucleus caudatus putamen and globus pallidus

were based on the assumption that if dopamine and/or 5-hydroxytryptamine

were mediators of dextroamphetamine in production of hypothermia,

changes in amine level in these areas would reflect similar changes in

brain areas involved with thermoregulation, On the basis of this as

sumption, there is no change in the concentration of dopamine or

67

5-hydroxytryptamlne in thermoregulatory areas during dextroamphetamine -

induced hypothermia. Of the 3 amines under consideration, it would ap

pear that norepinephrine is the most likely candidate as a mediator for

hypothermia produced by dextroamphetamine.

The importance of endogenous norepinephrine in the central hypo

thermic action of dextroamphetamine is further supported by the observa

tions involving repeated intraventricular injections of dextroamphetamine

at close intervals. Each injection of drug produced a fall in body tem

perature which was less pronounced than the preceding response. After

the fifth injection the hypothermic response was nonsignificant. In

conjunction with the body temperature study, 1 set of hlstochemlcal

fluorescence observations revealed that norepinephrine concentration in

the hypothalamus was less 30 minutes after the third intraventricular

injection of dextroamphetamine than 30 minutes after a single intraven

tricular injection of dextroamphetamine. A sequence of 3 injections

also gave rise to less reduction in body temperature than did the sin

gle injection. As in the case following a single injection of dextro

amphetamine, repeated Injections produced no change in the concentration

of dopamine in the nucleus caudatus putamen. Another set of observa*

tions revealed that successive intraventricular administrations of dex

troamphetamine, after recovery from hypothermia, caused a progressive

decline of norepinephrine concentration in the hypothalamus but no

change in the concentration of dopamine in the nucleus caudatus puta-

men. The progressive depletion of norepinephrine appears to correlate

well with the progressive decrease in hypothermic response.

68

The observations reported in this Chapter do not completely rule

out dopamine or 5-hydroxytryptamine as mediators for dextroamphetamine

in the production of hypothermia. There is a possibility that dopamine

and/or 5-hydroxytryptamine concentrations may be reduced in brain areas

involved in thermoregulation and yet such changes may not be reflected

by analyses of the nucleus cadatus putamen or the globus pallidus.

Also, the turnover rate of certain blochemicals has shown correlation

with physiological phenomena in the absence of absolute change in con

centration (Garattini, Glacolone and Valzelli, 1967; Engel et al., 1968;

Reid et al*, 1968; Slmmonds and Iversen, 1969). Such a relationship may

exist for dopamine or 5-hydroxytryptamine with regard to dextroampheta

mine -induced hypothermia. However, of the 3 monoamines considered, the

evidence presented in this study provides most support for norepineph

rine aB a mediator for dextroamphetamine-lnduced hypothermia.

CHAPTER 5

GENERAL DISCUSSION

Although the hyperthermic effect of systemically administered

amphetamine has been uniformly reported by numerous investigators, the

mechanism by which hyperthermia is produced remains controversial. Some

Investigators claim the effect to be central in origin (Brodie et al.,

1959; Euler, 1961; Belenky and Vitolina, 1962; Valzelli et al., 1966,

1968), whereas others claim the effect to be peripheral in origin

(Gessa, Clay and Brodie, 1969; Slater and Turnbull, 1969). Since the

drug is distributed throughout the body (Young and Gordon, 1962; Fuller

and Hines, 1967; Rosso et al., 1968; Maickel et al., 1969), its effect

on body temperature may be due to simultaneous actions on central and

peripheral tissues. In the present investigation the effects of intra

ventricular^ and intraperitoneally injected dextroamphetamine on body

temperature were studied. It was hoped that this investigation would

reveal more definitive information concerning the actions of dextroam

phetamine in producing body temperature changes. It was anticipated

that small intraventricular injections of dextroamphetamine would evoke

a relatively pure central action.

Intraventricular administration of graded doses of dextroam

phetamine to rats produced alterations in body temperature which do not

appear to describe a conventional dose-response relationship. For ex

ample, at an ambient temperature of 25°C, a small dose (4 meg) produced

69

a transient elevation of body temperature, whereas an intermediate dose

{100 meg) produced hypothermia and a large dose (400 meg) produced Ini

tial hypothermia followed by secondary hyperthermia. These unusual

dose-response observations may be tentatively explained on the basis of

central and peripheral actions of dextroamphetamine. The central ac

tions of dextroamphetamine may involve 1 or both of 2 proposed hypo

thalamic temperature regulating centers, i.e. the heat gain center and

the heat loss center (Meyer, 1913; Teague and Ranson, 1936; Jacobson and

Squires, 1961; Keller, 1963; Bligh, 1966a, 1966b). It is postulated

that the heat gain center is more sensitive than the heat loss center

and that the small dose of lntraventrlcularly administered dextroam

phetamine may activate the former without activating the latter. It is

further postulated that the intermediate dose may activate both hypo

thalamic centers, but the effects of the heat loss center mask the

effects of the heat gain center.

Support for the concept that the intermediate dose of dextroam

phetamine activates the heat loss center is provided by preliminary

observations in this laboratory. It was noted that the surface tempera

ture of the rat tail is increased at a time when body temperature is

reduced by lntraventrlcularly administered dextroamphetamine. Activa

tion of the heat loss center is reported to cause peripheral vasodila

tion, increased surface temperature and augmented loss of body heat

(Andersson, Grant and Larson, 1956; Andersson and Persson, 1957;

Andersson, Persson and Strom, 1960). The initial hypothermia following

intraventricular administration of the large dose of dextroamphetamine

may be explained on the same basis as the hypothermia produced by the

71

intermediate dose of dextroamphetamine. The secondary hyperthermia is

probably caused by escape of dextroamphetamine Into the systemic circu

lation to activate peripheral hyperthermic mechanisms.

It was observed in the current investigation that intraperito

neal injection of 100 meg of dextroamphetamine produced no change in

body temperature, whereas intraperitoneal injection of 20Q and 400 meg

of dextroamphetamine produced hyperthermia. These observations suggest

that the temperature effects of small and intermediate do$es of intra

ventricular^ injected dextroamphetamine are mainly, if n<>t solely,

central in origin. However, because the central hypothermic effect

masks the central hyperthermic effect, the central action of dextroam

phetamine may be considered predominantly hypothermic in nature. It was

also shown in the current investigation that intraperitoneal administra

tion of 2 mg/kg of dextroamphetamine produced a brain concentration of

dextroamphetamine equivalent to that produced by intraventricular admin

istration of the intermediate dose (100 meg) of dextroamphetamine. Pe

ripheral administration of this equivalent dose of dextroamphetamine

caused hyperthermia despite a brain concentration of dextvoamphetamine

which can produce hypothermia. On the basis of these observations It

may be postulated that systemlcally administered dextroamphetamine si

multaneously activated peripheral hyperthermic and central hypothermic

mechanisms. At an ambient temperature of 25°C the peripheral hyperther

mic effect exceeded the central hypothermic effect and hyperthermia was

observed. Plausibility of such a postulatlon is strengthened by reports

that hypothermia induced by intraventricular injection of norepinephrine

Is antagonized by peripheral administration of dextroamphetamine

72

(Brittaln, 1966) or norepinephrine (Cowell and Davey, 1968). If the

concept that systemic dextroamphetamine simultaneously activates periph

eral hyperthermic and central hypothermic mechanisms is correct, it

should be possible to unmask the hypothermic action of peripherally in

jected dextroamphetamine by overcoming the hyperthermia through en

hancing the dissipation of body heat.

Alterations in ambient temperature influence the rate of body

heat loss (Euler, 1961; Borison and Clark, 1967; Hemingway and Price,

1968). It can be expected that higher ambient temperatures would reduce

the rate of body heat dissipation and lower ambient temperatures would

enhance the rate of body heat dissipation. Intraventricular injection

of the intermediate dose of dextroamphetamine (100 meg) consistently

caused hypothermia which varied with the ambient temperatures employed

in the test. In contrast, intraperitoneal injection of the matching

dose of dextroamphetamine (2 mg/kg) caused hyperthermia at 25° and 20°C

but caused hypothermia at an ambient temperature of 6°C. In comparison,

the body temperature of control animals was constant at these 3 ambient

temperatures. It may be logically rationalized that at the higher ambi

ent temperatures (25° and 20°C) heat dissipation was low, the hyperther

mic action of dextroamphetamine predominated and hyperthermia was

observed. At the lowest ambient temperature (6°C) heat dissipation was

greatly enhanced, hypothermic action of dextroamphetamine predominated

and hypothermia was observed. These studies provide further support to

the concept that peripherally administered dextroamphetamine simultane

ously activates central hypothermic and peripheral hyperthermic mecha

nisms.

73

An additional study which served to dissociate the simultaneous

hyperthermic and hypothermic actions of systemically administered dex

troamphetamine involved the use of propranolol and phenoxybenzamine at

ambient temperatures of 20° and 25°C. Propranolol and phenoxybenzamine

are adrenergic blocking drugs which have the ability to antagonize

amphetamine-induced hyperthermia (Wolf and George, 1964; Mantegazza

et al., 1968a). It was found in the current study that the adrenergic

blocking drugs exerted no overt effect on the body temperature of con

trol animals at either ambient temperature. Also, at an ambient tem

perature of 25°C they were more effective in antagonizing hyperthermia

caused by peripheral injection of dextroamphetamine than hypothermia

caused by intraventricular injection of dextroamphetamine. But at 20°C,

an ambient temperature at which intraperitoneal injection of dextroam

phetamine typically causes hyperthermia, pretreatment with the adrenergic

blocking drugs caused dextroamphetamine to produce hypothermia. The

"reversal" of body temperature is apparently due to inhibition of the

peripheral hyperthermic effect of dextroamphetamine, which resulted in

unmasking of the concurrent central hypothermic action of dextroampheta

mine. These observations further strengthen the concept that system

ically administered dextroamphetamine simultaneously activates peripheral

hyperthermic and central hypothermic mechanisms.

Since dextroamphetamine is distributed throughout the body,

Including the central nervous system, It was important to substantiate

the concept that the hyperthermic effect of dextroamphetamine is due to

its action on peripheral tissues. A study, in which the central nervous

system of rats was destroyed, showed that intraperitoneal injection of

74

dextroamphetamine effectively retarded the rate of fall in body tempera

ture following ablation of the brain and spinal cord. This study pro

vided evidence that dextroamphetamine produces hyperthermia, at least

in part, by acting on peripheral tissues. Another study, in which the

hyperthermic action of dextroamphetamine was examined, involved consid

eration of the role of the adrenal glands in mediating the hyperthermic

action of dextroamphetamine. Peripheral administration of epinephrine

is reported to cause hyperthermia in rats (Masek and Raskova, 1964;

Feldberg and Lotti, 1967). This action must be due mainly, if not ex

clusively, to peripheral mechanisms because catecholamines do not readily

cross the blood brain barrier (Axelrod et al., 1959; Weil-Malherbe

et al., 1961; Samorajski and Marks, 1962). Since dextroamphetamine

releases epinephrine from the adrenal glands (Harvey et al., 1968; Rubin

and Jaanus, 1966) and since the present study showed that adrenalectomy

prevented the hyperthermic effect of intraperltoneally administered dex

troamphetamine, it could be concluded that the hyperthermic response in

duced by dextroamphetamine is, to a large extent, mediated peripherally

by the release of epinephrine from the adrenal glands. Also, it has

been reported that peripheral neuronal norepinephrine is responsible for

mediating the hyperthermic action of dextroamphetamine (Gessa, Clay and

Brodle, 1969). Although it is not known which catecholamine is rela

tively more important in mediating the hyperthermic action of dextroam

phetamine, there is convincing evidence that the hyperthermic action of

dextroamphetamine is indirect and is primarily peripheral In origin.

Various physiological and behavioral responses are known to

occur in association with induced temperature changes (Du Bols, 1939;

75

Euler, 1961)* Because some of these responses may be Involved In tem

perature regulation, a study was undertaken to determine if there Is a

correlation between certain of these physiological or behavioral re

sponses and dextroamphetamlne-induced body temperature changes. It

was found that peripheral administration of dextroamphetamine at an

ambient temperature of 25°C induced hyperthermia and caused an Increase

In spontaneous motor activity, oxygen consumption and sniffing behavior.

Intraventricular administration of dextroamphetamine produced hypother

mia and caused a reduction of spontaneous motor activity and oxygen con

sumption, but a moderate increase in sniffing behavior. The initial

impression was that changes In spontaneous motor activity and oxygen

consumption appeared to correlate well with changes in body temperature,

whereas sniffing behavior did not correlate with changes in body tem

perature. Subsequently, it was observed that intraperitoneal injection

of dextroamphetamine in normal rats at an ambient temperature of 6°C and

in adrenalectomlzed rats at an ambient temperature of 25°C produced a

reduction in body temperature and no change in body temperature, respec

tively. Yet, on the basis of overt observations both procedures appeared

to increase spontaneous motor activity. These later observations seem to

contradict the initial findings and seem to imply a lack of correlation

between spontaneous motor activity and dextroamphetamine-induced body

tenqierature changes. However, It should be recalled that body tempera

ture Is the result of heat generating and heat dissipating processes and

that systemically administered dextroamphetamine simultaneously activates

both processes. Therefore, enhancement of a process may result in over

riding of the opposing process, whereas inhibition of a process may

76

result in domination by the opposing process. From these considera

tions » it appears logical to suggest that various physiological re

sponses, such as increase or decrease in spontaneous motor activity and

Increase or decrease in oxygen consumption, may be more appropriately

correlated with heat generation or heat dissipation rather than with

changes in body temperature per se.

Because the administration of dextroamphetamine is reported to

modify the brain concentration of norepinephrine (Moore, 1964; Smith,

1965J Fuxe and Ungerstedt, 1968; Gropetti and Costa, 1969), dopamine

(Smith, 1965; Littleton, 1967; Lewander, 1968a, 1968b; Welch and Welch,

1969) and 5-hydroxytryptamine (McLean and McCartney, 1961; Smith, 1965;

Welch and Welch, 1969) and because body temperature is modified by

intraventricular administration of norepinephrine (Feldberg and Lotti,

1967; Myers and Yaksh, 1968), dopamine (Myers and Yaksh, 1968) and 5-hy

droxytryptamine (Feldberg and Lotti, 1967; Myers and Yaksh, 1968), it is

reasonable to speculate that 1 or more of these biogenic amines may be

lnqjortant in mediating the central body temperature effects of dextroam

phetamine. The relative importance of the 3 biogenic amines was exam

ined by means of a study in which chemical agents were employed to

deplete individual or combinations of the monoamines by inhibiting their

synthesis or by enhancing their release before testing with dextroam

phetamine. It was observed that release and depletion of all 3 monoa

mines with the benzoqulnollzlne Ro 4-1284 antagonized the hypothermic

action of intraventricularly injected dextroamphetamine. Depletion of

5-hydroxytryptamine with the tryptophan hydroxylase inhibitor p-chloro-

phenylalanine failed to antagonize the hypothermic effect of

77

lntraventricularly administered dextroamphetamine. On the other hand,

depletion of norepinephrine and dopamine with the combined use of alpha-

methyl -m-tyrosine , an agent which releases catecholamines, and alpha-

methyl -p -tyrosine, an agent which inhibits tyrosine hydroxylase,

decreased dextroamphetamine-induced hypothermia by 40 per cent. These

observations suggest that the central hypothermic action of dextroam

phetamine is mediated, at least in part, by 1 or both of the catechola

mines and that 5-hydroxytryptamine is not involved.

An additional study was performed to substantiate the possible

role of 1 or both of the catecholamines as central mediators of dex

troamphetamine -induced hypothermia. In this study histochemical fluo

rescence analyses were made of specific areas of the brain following

administration of dextroamphetamine. Thirty minutes after intraven

tricular or intraperitoneal administration, a time when peak hypothermic

activity may be expected, dextroamphetamine caused a reduction of nor

epinephrine In the anterior hypothalamic nucleus, but no change in the

concentration of dopamine in the nucleus caudatus putaraen or of 5-hy

droxytryptamine in the globus pallidus. Each of the amines exists as

the predominant amine in each of the respective areas examined (Fuxe,

1965; Frlede, 1966; Fuxe, Hokfelt and Ungerstedt, 1968). It was assumed

that changes In the fluorescence of a given area reflects mainly a

change of the predominant amine. Furthermore, It was assumed that if

these amines were mediators of dextroamphetamine in the production of

hypothermia, changes in amine level In any of these areas would reflect

similar changes in brain areas Involved with thermoregulation. Because

norepinephrine was the only 1 of the 3 amines whose concentration was

78

reduced, it was postulated that norepinephrine is Important In mediating

the central hypothermic action of dextroamphetamine. The postulation

Is particularly appealing because norepinephrine is the predominant

monoamine in the anterior hypothalamus and because this region is con

sidered to be important in thermoregulation (Meyer, 1913; Teague and

Ranson, 1936; Jacobson and Squires, 1961; Bllgh, 1966b). Further sup

port for this concept was provided by repeated intraventricular injec

tions of dextroamphetamine at close intervals. Repeated intraventricular

injections of dextroamphetamine resulted in a progressive development

of tachyphylaxis to dextroamphetamlne-lnduced hypothermia. These in

jections also caused a progressive reduction in the concentration of

norepinephrine in the anterior hypothalamus but no change in the con

centration of dopamine in the caudate nucleus. Hence, there appears to

be a correlation between changes In the degree of hypothermia and the

relative concentration of norepinephrine in the anterior hypothalamus.

This correlation further supports the postulation that norepinephrine is

important in mediating the central hypothermic action of dextroampheta

mine.

The findings of the present investigation may be summarized as

follows:

1. The central action of dextroamphetamine on body temperature is pre

dominantly hypothermic. There is a central hyperthermic component which

is believed to be readily masked by the hypothermic component.

2. The peripheral action of dextroamphetamine on body temperature is

hyperthermic.

3. The central hypothermic action of dextroamphetamine is believed to

79

be mediated to a large extent by norepinephrine, whereas the peripheral

hyperthermic action Is mediated at least partially by adrenal epineph

rine.

4. Various physiological or behavioral responses, such as motor ac

tivity and oxygen consumption, Induced by dextroamphetamine which may

participate in thermoregulation are probably more appropriately corre

lated with heat generation or heat dissipation rather than with changes

in body temperature per se.

5* Dextroamphetamine, administered systemically» is distributed through

out the body and simultaneously activates central hypothermic and periph

eral hyperthermic mechanisms. Consequently, the direction and extent

of body temperature change after systemic dextroamphetamine administra

tion depends on extraneous factors which may Increase or decrease the

rate of heat dissipation.

APPENDIX A

INTRACEREBROVENTRICULAR TECHNIQUE

Equipment

A. Implantation apparatus: The apparatus consists of 2 basic com-

ponents, the animal securing device and the cannula implantation

component. The securing device is composed of a wooden platform

and a metal incisor bar. The animal is secured to the platform

and the upper incisors are placed over the bar. The incisor bar

is attached to the base of a ring stand (E & M Instrument Co.)

and is perpendicular to the saggital plane of the animal. A

myograph tension adjustor (E & M Instrument Co.) Is positioned

on the ring stand so that adjustment moves a horizontally sus

pended bar in a vertical plane. A pair of curved forceps Is

attached to the horizontal bar with a Drinker clamp (A. H.

Thomas Co., Inc.). The forceps are used to hold the cannula.

Vertical adjustment of the cannula is accomplished by manipu

lation of the muscle tension adjustor. Lateral adjustment is

accomplished by rotation of the entire muscle tension adjustor

on the metal shaft of the ring stand.

B. Dental drill: model number 73, Foredom Electric Co., Inc.

C. Doriot hand piece: model 42, Foredom Electric Co., Inc.

D. Dental burr: number 700 uarbide, number 3 carbide, S. S. White

Co.

80

81

E. Cannula: The cannula is composed of 2 basic components, the

outer guide which directs the tip of the injecting needle into

the lateral ventricle and the inner stainless steel shaft which

is removed when the injecting needle is inserted. The shaft

maintains an open passage way for insertion of the injecting

needle and prevents leakage of cerebrospinal fluid. The outer

guide is made from a 23 gauge hypodermic needle. A small high

speed rotary motor fitted with a beveled grinding stone is used

to reduce the size of the hub and length of the shaft. When

completed the overall length of the cannula (shaft and hub) is

8 mm and the length of the shaft is 5 mm. The hub is ground so

that 2 flat surfaces are opposite each other. A groove approxi

mately 1 mm wide and 0.5 mm deep completely encircles the hub

and is positioned 0.5 mm above the juncture of the shaft and the

hub. The flat surfaces facilitate proper attachment to the can

nula holding component of the implantation apparatus. The

groove assures firm attachment to the orothodontic resin. The

inner stainless steel shaft is made from a 30 gauge hypodermic

needle. The dimensions of this needle are reduced with the same

equipment as described above. When completed the overall length

(shaft and hub) Is 10 nun and the length of the shaft is 8 mm.

The grinding procedure closed the lumen of the shaft. The re

duced hub is used to facilitate removal and replacement of the

inner shaft.

F. Injecting needle: The injecting needle is made from a blunt, 30

gauge 8 mm hypodermic needle. The size of the hub is reduced to

82

a length of 3 mm. The width at the juncture of the shaft and

the hub Is 2.5 mm. From this point the hub is beveled so that

It is generally cone shaped. A 15 inch polyethylene tube

(PE 20) is forced onto the cone and cemented in place with Epoxy

resin. The open end of the polyethylene tube is attached to a

10 mcl syringe (Hamilton Co., Inc.).

II. Materials

A. Iodochlorhydroxyquin powder: Vioform, Ciba Pharmaceutical

Products, Inc.

B. Methohexital: Brevital Sodium, Eli Lilly and Co.

C. Orthodontic resin: L. D. Caulk Co.

111. Implantation of cannula

A. Anesthetize a 180-220 g rat by intravenous administration of

methohexital.

B. Clip hair from dorsal surface of head.

C. Make mldsaggltal incision from the eyes to lambda.

D. Retract skin to provide ample working area.

E. Remove periosteum from exposed skull by scraping.

F. Drill a hole through the skull at a point 1.5 to 2.0 mm lateral

and 1.0 mm caudal to the bregma with a number 700 dental burr.

G. Approximately 9.5 mm from this hole drill 3 additional holes so

that they describe a triangle with the original hole (drilled in

step III.F) in the center. Keep all holes at least 1.5 mm

from the saggital suture since excessive bleeding occurs if the

saggital sinus is ruptured.

Enlarge all 4 holes with a number 3 dental burr. Avoid rupture

of the dura mater.

Place a stainless steel machine screw (0-80 X 0.32 cm, 1/8 inch)

in each hole drilled in step III.G. Screws should not penetrate

beyond ventral surface of skull bone.

Insert a sharp 26 gauge hypodermic needle through the center

hole and penetrate the dura mater. Avoid excessive damage to

brain tissue.

Mount a cannula on the holding component of the implantation

apparatus.

Position animal and cannula so that the cannula is perpendicular

to the skull at the point of the center hole.

Lower the tip of cannula 4 mm below the dorsal surface of the

skul1.

Quickly cement the cannula In place by application of a small

amount of orthodontic resin to the base of the exposed cannula

so that contact is made with the skull and at least 1 screw.

Walt 20 to 30 seconds and carefully detach holding component

from cannula.

Immediately elevate holding component and rotate away from ani

mal.

Apply additional resin so that contact is made between cannula

and all 3 stainless steel screws.

Allow 2 to 3 minutes for resin to harden.

Apply a small quantity of lodochlorhydroxyquin powder to ex

posed subdermal tissue*

84

T. House animals individually.

U. Allow at least 5 days for recovery.

IV. Intraventricular administration of drugs

A. Remove inner shaft from cannula.

B. Insert injecting needle into cannula opening.

C. Inject solution at the rate of 10 mcl per minute.

D. Slowly remove injecting needle.

E. Quickly replace inner shaft of cannula.

V. Confirmation of proper implantation

A. Inject 5 mcl of permanent black ink through cannula.

B. Decapitate animal and remove brain.

C. Dissect away the cortex near the path of the cannula.

D. Uniform staining of lateral ventricle wall suggests proper Im

plantation of cannula.

E. Perform saggital section.

P. Proper implantation is further assured if ink outlines third and

fourth ventricles.

APPENDIX B

PREPARATION OF DRUGS

D,L-Alpha-methyl-m-tyrosine (Nutritional Biochemicals Corp.)

Type of preparation: Solution

Vehicle: 0.9% NaCl solution

Solution strength: 500 mg/10 ml

D,L-Alpha-methyl-p-tyrosine (Regis Chemical Co.)

Type of preparation: Suspension


Dose: 80 mg/kg

Suspension strength: 80 mg/5 ml

Dextroamphetamine sulfate (Dexedrine Sulfate, Smith, Kline & French Labs.) for intraventricular use


Vehicle: Deionized water (solution adjusted to osmolarity equal to 0.9% NaCl)

Dose: 4 meg (as salt)

Solution strength: 4 meg/5 mcl


Solution strength: 12 mcg/5 mcl

85


Solution strength: 20 meg/mel



Vehicle: Delonlzed water



Dextroamphetamine sulfate (Dexedrlne Sulfate, Smith, Kline & French Labs.) for intraperitoneal use



Dose: 2 mg/kg


D,L-p-chlorophenylalanlne (Chas. Pfizer & Company, Inc.)

Type of preparation: Suspension

Vehicle: 0.4% Tween 80, 1% carboxymethylcellulose, 0.9% NaCl

Dose: 300 mg/kg

Suspension strength: 300 mg/5 ml

Phenoxybenzamine hydrochloride (Dlbenzyllne Injection, Smith, Kline French Labs.)


Vehicle: Delonlzed water (1 part Dlbenzyllne Injection diluted with 9 parts dionized water)

Dose: 10 mg/kg


Propranolol hydrochloride (Inderal, Ayerst Laboratories)



Dose: 10 mg/kg


Ro 4-1284 (Hoffman-LaRoche, Inc.)



Dose: 10 mg/kg


APPENDIX C

ASSAY FOR BRAIN DEXTROAMPHETAMINE

1. Equipment

A. Centrifuge: Servall, type SS-3, Ivan Sorvall, Inc.

B. Glass Homogenizer: Duall, size C, Kontes Glass Co.

C. Liquid scintillation counting system: model 314, Packard

Instrument Co.

D. Mechanical shaker: Eberbach Corporation

E. Scintillation counting vial: Spectravial, model 3334 Nylon

vial. New England Nuclear

II. Materials

A. Benzene: analytical reagent grade, Mallinckrodt Chemical Works

B. Dextroamphetamine-H^ (G) sulfate: New England Nuclear

C. 2,5-Diphenyloxazole (PPO): scintillation grade, Packard

Instrument Co.

D. Hydrochloric acid: analytical reagent grade, Mallinckrodt

Chemical Works

E. Perchloric acid: analyzed reagent grade, J. T. Baker

Chemical Co.

F. 2,2-p-Phenylenebis-(5-phenyloxazole) (POPOP): scintillation

grade, Packard Instrument Co.

G. Sodium hydroxide: analyzed reagent grade, J. T. Baker

Chemical Co.

88

89

H. Toluene: analytical reagent grade, Mallinckrodt Chemical Works

III. Solutions

A. Isotope solutions

1. Dextroamphetamine stock solutions for standards

a. 10 meg standard: Ten meg of dextroamphetamine sulfate

is contained in each 0.1 ml of solution. The tritium

label is diluted 1:10,000 in this solution; the vehicle

is 0.01N HC1.

b. 3 meg standard: Three meg of dextroamphetamine sulfate

is contained in each 0.1 ml of solution. The tritium

label is diluted 1:10,000 in this solution; the vehicle

is 0.01N HC1.

c. 1 meg standard: One meg of dextroamphetamine sulfate is

contained in each 0.1 ml of solution. The tritium label

is diluted 1:10,000 in this solution; the vehicle is

0.01N HC1.

2. Dextroamphetamine stock solutions for injection

a. Solution for intraperitoneal injection: One mg/ml of

dextroanqphetamine sulfate is contained in the solution

for injection. The tritium label is diluted 1:10,000;

the vehicle is 0.9% NaCl solution.

b. Solution for intraventricular injection: Twenty mg/ml

of dextroamphetamine sulfate is contained in the solu

tion for injection. The tritium label is diluted

1:10,000; osmolarlty of the solution Is adjusted to

90

equal that of 0.9% NaCl solution.

B. Solutions for extraction procedure

1. Hydrochloric acid: 0.1N

2. Perchloric acid: 30% w/v

3. Sodium hydroxide: ION

4. Washed benzene: Keep an excess of deionized water in

container of benzene.

C. Scintillator solution

1. Place 2.0 g FPO into glass-stoppered container.

2. Add 50 mg POFOP and 500 ml toluene

3. Protect from light and store at 5°C.

IV. Procedure

A. Decapitate animal and dissect out brain.

B. Expose lateral ventricles with saggital incisions through cortex

approximately 2 mm lateral to midline.

C. Flood ventricles and outer surface of brain with 25 ml of cold

0.9% NaCl.

D. Drain surface liquid from brain and freeze brain on dry ice.

Store at -30°C until step E below.

E. Weigh tissue to nearest 10 mg.

F. Homogenize tissue with A volumes of 0.1N HC1 in motor-driven

chilled glass homogenizer. Assume brain tissue has a density of

1 g/ml. For standards substitute 0.1 ml of appropriate stock

solution (step 1II.A.1) per g of brain tissue for the same

amount of 0.1N HC1.

91

G. Add 30% perchloric acid to make a final concentration of 6%

perchloric acid. Mix thoroughly.

H. Transfer contents of homogenlzer to a 60 ml glass-stoppered

bottle.

I. Centrifuge at 1000 X gravity for 8 minutes.

J. Transfer 1 ml of supernatant to a second 60 ml glass-stoppered

bottle containing 0.2 ml 10N NaOH and 3 ml washed benzene.

K. Shake for 10 minutes on mechanical shaker at 220 oscillations

per minute.

L. Centrifuge at 1000 X gravity for 4 minutes.

M. Transfer 2 ml of benzene (upper) phase to scintillation counting

vial containing 10 ml of scintillator solution.

N. Quantitate the tritium decay with a liquid scintillation

counting system.

0. Calculate dextroamphetamine concentration In brain tissue by the

method described in V below.

Calculation of dextroamphetamine concentration in brain

A. Determine theoretical decays for entire brain

Theoretical decays (X) = decays per minute recorded for sample (A) (B) (C)

where

A» ^ .1 (2/3) (0.4) (0.95) [(6.25) (brain weight in g)J

The term in brackets represents the fraction of the whole

sample transferred to benzene. The 2/3 represents the

92

fraction transferred from benzene to scintillation solution.

The 0.4 and 0.95 represent approximate counting efficiency

and extraction efficiency, respectively.

B = the dilution factor for the tritium label (1/10,000)

C = correction factor for decomposed dextroamphetamine sulfate

This value reflects a decomposition of approximately 1.5%

per month. If 1 month had elapsed since dextroamphetamine-H3

sulfate was assayed by the manufacturer, then the value for

C would be 0.985.

B. Determine amount of dextroamphetamine sulfate in brain tissue

Amount of dextroamphetamine sulfate = 5 mce 5.36 x 107 6

The 5.36 x 10^ represents the theoretical decays of 1 meg of

dextroamphetamine-H sulfate.

C. Determine concentration of dextroamphetamine sulfate in brain

tissue

dextroamphetamine sulfate = amount of dextroamphetamine sulfate brain weight in g

APPENDIX D

STATISTICAL PROCEDURES

Student's t^ Test

Calculations were performed according to the following formula

|Xy _ iz|

= ~ 2 i_±_ + Jjfn*

where

p (-t -a

t = the calculated Student's t_ value

x = the mean value of a treatment

2 Sp— the pooled variance

n ° the number of replicates per sample

and

y and z = the particular treatments in question.

93

94

The standard error of the mean (S.E.M.) was calculated according

to the following formula:

S.E.M. - bX ' X>2"1 | n(n - 1) I

where

X = the value of a replicate within a sample

x = the mean of a sample

and

n = the number of replicates per sample.

Numerical data In this work are reported as the mean + S.E.M.

In line drawings and bar graphs the S.E.M. is indicated by a vertical

bracketed line. The difference between 2 means is reported to be

significant only when the calculated Student's t value is larger than

that value of the Student's t_ distribution where the probability (P)

of a larger t_ value is less than 0.05.

APPENDIX E

SPECTROPHOTOFLUOROMETRIC ASSAY OF 5-HYDROXY-TRYPTAMINE, NOREPINEPHRINE AND DOPAMINE

I. Equipment

A. Glass homogenizers: Duall, size D, Kontes Glass Co.

B. Centrifuge: Servall type SS-3, Ivan Sorvall, Inc.

C. Cells: fused quartz, 5 ml., American Instrument Co.

D. Spectrophotofluorometer: Aminco-Bowman, American Instrument Co

(photomultiplier tube, IP21, slit size, 3/16"; slit arrangement

No. 5)

E. Dopamine resin columns: The dopamine resin columns are made

from 14 mm glass tubing with a constriction on one end which is

10 mm long and has a 5 mm Inside diameter. A sintered glass

column 5 mm in diameter and 2.5 mm In length is fused into the

distal end of the constriction. The overall length of the dopa

mine resin column is 120 mm with the top 110 mm serving as a

reservoir for solutions and the lower 10 mm constriction serv

ing as a resin compartment.

II. Materials

A. L-Ascorbic acid: reagent grade, Fisher Scientific Co.

B. 1-Butanol: reagent grade, Fisher Scientific Co.

C. Dopamine hydrochloride: Calblochem

95

96

D. Glacial acetic acid: analytical reagent grade, Malllnckrodt

Chemical Works

E. n-Heptane: pure grade, Phillips Petroleum Co.

F. 5-Hydroxytryptamine creatinine sulfate: Calbiochem

G. Hydrochloric acid: analytical reagent grade, Malllnckrodt

Chemical Works

H. Iodine: analytical reagent grade, Malllnckrodt Chemical Works

I. Norepinephrine bltartrate, monohydrate: Winthrop Laboratories

J. Parafilm "M": American Can and Co.

K. Potassium chloride: analytical reagent grade, Malllnckrodt

Chemical Works

L. Potassium iodide: analytical reagent grade, Malllnckrodt

Chemical Works

M. Potassium phosphate, monobasic: analytical reagent grade,

Malllnckrodt Chemical Works

N. Resin: Dowex 50W-X4 resin, 200-400 mesh, hydrogen ionic form,

J. T. Baker Chemical Co.

0. Sodium acetate, trihydrate: analytical reagent grade,


P. Sodium hydroxide: analytical reagent grade, Malllnckrodt

Chemical Works

Q. Sodium phosphate, dibasic: analytical reagent grade,


R. Sodium phosphate, dibasic, heptahydrate: analytical reagent

grade, Malllnckrodt Chemical Works

97

S. Sodium phosphate, monobasic, monohydrate: analytical reagent

grade, Mallinckrodt Chemical Works

T. Sodium sulfite, heptahydrate: analyzed reagent grade,

J. T. Baker Chemical Co.

U. Sodium thiosulfate, pentahydrate: analytical reagent grade,

Mallinckrodt Chemical Works

III. Preparation of cation exchange resin, sodium ionic form (Moore and

Stein, 1951)

A. Place 1 pound of moist Dowex 50W-X4 resin into a Buchner funnel.

B. Wash with 4N HC1 until filtrate is almost colorless*

C. Wash resin twice with distilled water.

D. Wash resin with 2N NaOH until filtrate is alkaline.

E. Place the resin in about 3 times its volume of IN NaOH and sus

pend over a steam bath.

F. Heat approximately 3 hours and shake periodically.

G. Allow resin to settle.

H. Decant and discard liquid.

I. Add 3 volumes of hot IN NaOH and heat with shaking as in steps

III.E-H above.

J. Repeat step III.l 3 times.

K. Wash resin with distilled water until filtrate is neutral.

L. Pass resin through a 120 mesh screen with 6000 to 8000 ml of

distilled water.

M. Collect the screened resin and store as the moist sodium salt in

an air-tight container.

98

IV. Preparation of solutions

A. Stock solutions for monamlne standards: Solutions may be

stored several weeks If refrigerated and protected from light.

1. 5-Hydroxytryptamine: Weigh 23.007 mg of 5-hydroxytryptamlne

creatinine sulfate and dilute to 100 ml vlth 0.01N HCl.

This solution contains 100 meg of 5-hydroxytryptamlne base

per ml.

2. Norepinephrine: Weigh 19.935 mg of norepinephrine bitar-

trate, monohydrate and dilute to 100 ml with 0.01N HCl.

This solution contains 100 meg of norepinephrine base per

ml.

3. Dopamine: Weigh 12.38 mg of dopamine hydrochloride and

dilute to 100 ml with 0.01N HCl. This solution contains

100 meg of dopamine base per ml.

B. Salt-saturated butanol solution

1. Place 2500 ml of 1-butanol into a 4000 ml separatory funnel.

2. Add 1000 ml of 0.01N HCl.

3. Shake vigorously for 2 minutes; allow phase separation.

4. Discard lower phase.

5. Place approximately 120 g of NaCl into a 500 ml glass-

stoppered flask.

6. Add approximately 300 ml of the butanol phase from step 3

above.

7. Agitate on mechanical shaker for 30 minutes.

8. Allow phase separation.

99

9. Decant and store top layer (salt-saturated butanol) in air

tight glass-stoppered flasks.

C. Solutions for assay of norepinephrine and dopamine

1. Acetate buffer

a. Place 1 volume of 2N acetic acid in glass-stoppered

flask.

b. Add 2 volumes of 2N sodium acetate.

c. Mix thoroughly and store at 5°C.

2. Norepinephrine iodine solution

a. Dissolve 1.27 g of iodine in 100 ml of absolute ethanol.

b. Store at 5°C in light-resistant glass-stoppered flask.

3. Sodium thiosulfate solution

a. Dissolve 1.24 g of sodium thiosulfate, pentahydrate in

100 ml of deionized water. Properly prepared solutions

are assured when 1.875 + 0.025 ml of the sodium thio

sulfate solution decolorizes 1.00 ml of the norepineph

rine iodine solution.

4. Alkaline ascorbate solution

a. Dissolve 50 mg of ascorbic acid in 5 ml of deionized

water.

b. Add 10 ml of 5N NaOH; mix carefully.

5. Dopamine buffer number 1

a. Place 2.5940 g of monobasic sodium phosphate, mono-

hydrate and 1.8700 g of dibasic sodium phosphate, hepta-

hydrate into a 1000 ml volumetric flask.

b. Dilute to 1000 ml with deionized water.

100


a. Place 1.8440 g of monobasic sodium phosphate mono-

hydrate, 4.2500 g of dibasic sodium phosphate

heptahydrate, and 74.560 g of potassium chloride Into

a 1000 ml volumetric flask.

b. Dilute to 1000 ml with deionlzed water.


a. Place 4.259 g of dibasic sodium phosphate and 9.520 g

of monobasic potassium phosphate into a 1000 ml volume

tric flask.

b. Dilute to 1000 ml with deionlzed water.

8. Dopamine iodine solution

a. Dissolve 5.00 g of K1 in 5.0 ml of deionlzed water.

b. Add 0.2540 g of iodine.

c. Agitate until solution is achieved.

d. Dilute to 100 ml with deionlzed water.

e. Store at 5°C in a light-resistant bottle.

9. Alkaline sodium sulfite solution

a. Dissolve 0.5040 g of sodium sulfite heptahydrate in

1 ml of deionlzed water.

b. Dilute to 10 ml with 5N NaOH.

V. Procedure

A. Preparation of glassware

1. Quartz cells

a. Handle cells with plastic-tipped tongs.

101

b. Rinse cells several times with delonlzed water. *

c. Soak in concentrated nitric acid for at least 10

minutes.

d. Remove from acid, rinse with delonlzed water, and

place in hot-air oven to dry.

e. Remove from oven and allow to cool before use.

2. Pipettes

a. Rinse inside and outside of pipette with distilled water.

b. Immerse pipettes in concentrated sulfuric acid contain

ing an excess of sodium nitrate. Store for at least 2

hours in this solution.

c. Remove pipettes from acid and wash with tap water.

d. Place in automatic pipette washer and rinse with tap

water for at least 2 hours.

e. Flush each pipette with judicious quantities of delon

lzed water.

f. Dry pipettes in hot-air oven and store in dust-free

area.

3. Dopamine resin columns

a. Rinse column with delonlzed water to remove resin from

sintered glass.

b. Reverse-flush columns with delonlzed water several

times.

c. Dry in hot-air oven and store In dust-free area.

4. Other glassware

a. Thoroughly rinse with tap water.

102

b. Clean with Tig soap, warm water, and brush.

c. Rinse with tap water.

d. Immerse In dilute HCl for at least 2 hours,

e. Rinse with large quantities of delonlzed water.

f. Dry in hot-air oven and store in a dust-free area.

B. Preliminary preparations for extraction of 5-hydroxytryptamine,

norepinephrine and dopamine

1. Prepare room for assay.

a. Adjust room temperature to approximately 22°C.

b. Remove dust from room.

2. Prepare salt-saturated butanol bottles.

a. Place 25 ml of Bait-saturated butanol into 12 60-ml

ground-glass stoppered bottles.

b. Add 2 g of NaCl to each bottle.

c. Add 3 ml of 0.01N HCl to the reagent blank bottle.

d. Add 2.5 ml of 0.01N HCl to the low standard bottle.

e. Add 2 ml of 0.01N HCl to the high standard bottle.

f. Bottles containing only salt and butanol are called

sanple bottles and will each receive 3 ml of homogenized

brain tissue (see step V.C.5).

g. Bottles may be stored for several days at 5°C.

h. Throughout the assay procedure maintain bottles at 5°C

or less whenever they are not in use.

3. Prepare heptane bottles.

a. Place 20 ml of n-heptane into 12 60-ml glass-stoppered

bottles.

103

b. Add 3 ml of 0.01N HCl to each bottle.

Extraction of 5-hydroxytryptamine, norepinephrine and dopamine

1. Decapitate animal.

2. Remove whole brain, rinse with distilled water, and blot

with filter paper.

3. Immediately weigh brain tissue to nearest 10 nig.

4. Homogenize brain in motor-driven chilled glass homogenlzers

with 2 volumes of cold 0.01N HCl. For this step the density

of the brain is assumed to be 1 g/ml.

5. Transfer 3 ml of the homogenate to the appropriate sample

bottle.

6. Secure lid, cover bottle with light-resistant black plastic,

and store on ice until step 9.

7. To prepare monoamine standard bottles transfer 1 ml of the

5-hydroxytryptamine and dopamine stock solutions and 0.5 ml

of the norepinephrine stock solution to a volumetric flask

and dilute to 100 ml with 0.01N HCl. This solution contains

1 meg each of 5-hydroxytryptamine and dopamine base and 0.5

meg of norepinephrine base per ml.

8. Transfer 0.5 ml of this diluted solution to the low-standard

bottle and 1 ml to the high-standard bottle. The low-

standard bottle now contains 0.5 meg each of 5-hydroxytryp-

tamlne and dopamine base and 0.25 meg of norepinephrine

base. The high standard bottle contains 1.0 meg each of

S-hydroxytryptamlne and dopamine base and 0.5 meg of

norepinephrine base.

104

9. Agitate bottles for 10 minutes on mechanical shaker.

10. Centrifuge for 5 minutes at a relative centrifugal force

of 600 X gravity.

11. Transfer 20 ml of the butanol (upper) phase to the corre

sponding heptane bottle.

12. Repeat steps 9 and 10.

13. Aspirate and discard most of the butanol-heptane (upper)

phase. The remaining 3 ml aqueous phase is the aqueous acid

extract.

D. Determination of 5-hydroxytryptamine concentration

1. Transfer 1 ml of the aqueous acid extract (see step V.C.13)

into a quartz cell.

2. Add 0.3 ml of concentrated HC1.

3. Agitate gently until homogeneity is achieved.

4. Determine fluorescence emission intensity with spectrophoto-

fluororaeter. Use an activation wavelength of 300 nm and a

detection wavelength of 545 nm.

5. Calculate 5-hydroxytryptamine concentration as described in

step VI.

E. Determination of norepinephrine concentration

1. Transfer 0.6 ml of aqueous acid extract from each bottle

(see step V.C.13) to an appropriate small test tube.

2. Collect the residual extract from each sample bottle used in

step E.l above.

3. Transfer 0.6 ml of the pooled residual extract to a small

105

test tube. This tube contains extract to be used as the

brain blank.

4. Add 0.2 ml of acetate buffer to each small test tube.

Agitate thoroughly.

5. Add 0.02 ml of norepinephrine iodine solution to each small

test tube except the 1 reserved for the brain blank. Agi

tate thoroughly.

6. After exactly 6 minutes add 0.04 ml of sodium thlosulfate

solution to each small test tube except the brain blank.

7. Immediately add 0.2 ml of alkaline ascorbate solution, pre

pared within 10 minutes prior to use, to each small tube

except brain blank.

8. Add 0.04 ml of sodium thlosulfate solution to brain blank.

Agitate thoroughly.

9. Add 0.02 ml of norepinephrine iodine solution to brain

blank. Agitate thoroughly.

10. Immediately add 0.2 ml of freshly prepared alkaline ascor

bate solution to brain blank. Agitate thoroughly.

11. Expose all tubes to strong fluorescent light for 45 minutes.

12. Transfer to quartz cells.

13. Determine fluorescence emission Intensity with spectrophoto-

fluoroneter. Use an activation wavelength of 400 nm and a


14. Calculate norepinephrine concentration as described in

step VI.

F. Determination of dopamine concentration

106

1. Place a lightly packed 2 mm column of Dowex 50W-X4, sodium

Ionic form Into each dopamine resin column.

2. Wash each column with 2 ml of deionlzed water (care should

be taken to avoid disturbing the resin when liquids are

added).

3. Place 1 ml of appropriate aqueous acid extract (see step

V.C.13) on the resin column.

4. Repeat step 2 above.

5. Discard solutions that pass through resin column.

6. Elute 3,4-dihydroxyphenylalanine by addition of dopamine

buffer number 1 to the column.

7. Discard eluate.

8. Position clean tubes for collection of dopamine containing

eluate in step 9 below.

9. Place 8 ml of dopamine buffer number 2 on the column and

collect eluate which contains dopamine.

10. Thoroughly agitate contents of collection tubes and transfer

3 ml of each solution into a separate silica test tube.

11. Add 0.5 ml of dopamine buffer number 3 to each silica test

tube.

12. Add 0.3 ml of deionlzed water to each silica test tube.

Agitate thoroughly.

13. Add 0.05 ml of dopamine iodine solution to each silica test

tube. Agitate thoroughly.

14. Exactly 5 minutes later add 0.5 ml of freshly prepared alka

line sodium sulfite solution. Agitate thoroughly.

107

15. Exactly 5 minutes after addition of sulfite solution add

0.6 ml of 5N acetic acid. Agitate thoroughly.

16. Seal each test tube with parafilm.

17. Expose to strong fluorescent light for 17 to 22 hours.

18. Transfer approximately 1.5 ml of solution from each silica

tube into a separate quartz cell.

19. Determine fluorescence emission intensity with spectrophoto-

fluorometer. Use an activation wavelength of 338 nm and a


20. Calculate dopamine concentration as described In step VI.

Calculation of monoamine concentration in brain

A >= The photomultlplier microphotometer reading for the high stand

ard minus the reagent blank reading

B = The photomultlplier microphotometer reading for the low standard

minus the reagent blank reading

C • The number of micrograms of monoamine in the low standard before

extraction

D « The photomultlplier microphotometer reading for each sample

minus the appropriate blank. Calculations for dopamine and

5-hydroxytryptemine employed the reagent blank rather than the

tissue blank since It has been shown In our laboratory that

Concentration of monoamine

where

108

there is no difference between the 2 values. Calculations for

norepinephrine employed the tissue (brain) blank.

APPENDIX F

HISTOCHEMICAL FLUORESCENCE TECHNIQUE FOR DIRECT OBSERVATION OF NOREPINEPHRINE,

DOPAMINE AND 5-HYDROXYTRYPTAMINE

Equipment

A. Isopentane cooling apparatus: This apparatus consists of 2

compartments. The outer compartment (Fluo-War Flask, 4000 ml,

Virtis Co., Inc.) contains liquid nitrogen. The Inner compart

ment (stainless steel beaker, 600 ml) suspends isopentane In the

liquid nitrogen. A small copper basket (14 mesh, 55 mm diameter,

35 mm height) is used to confine the tissue while freezing.

B. Temporary storage container for brain tissue: Fluo-War Flask,

8000 ml, Virtis Co., Inc.

C. Aluminum foil container: The container Is made from 3 thick

nesses of aluminum foil and is 63 mm long, 35 mm wide and 15 mm

deep.

D. Plastic tray: The bottom of the tray is made of polyethylene

plastic 6 ram thick, 136 mm long and 89 mm wide. The walls of

the tray which are 2.5 mm thick and 25 mm high describe an inner

compartment for brain tissue and a surrounding outer compartment

for granular phosphorus pentoxlde. The outer compartment is 83

mm wide and 121 mm long, whereas the inner compartment is 51 mm

wide and 81 mm long.

E. Glass lyophylization chamber: Freeze-drying flask with ground

110

glass flange, 1000 ml, Vlrtls Co., Inc. The top has a vented

19/38 ground glass outer joint. The chamber is held horizon

tally by a wooden rack.

F. Dry-air box: The box is made from stainless steel and is 600 mm

high, 500 mm long and 376 mm wide. It has a glass window for

viewing and 2 arm ports fitted with long surgical gloves for

manipulation of tissue.

G. Wire rack: The rack consists of an 18 mesh copper screen bottom

(130 mm diameter) on which 8 wire cylinders (15 mm height, 25 mm

diameter) are attached.

H. Paraformaldehyde humidifying chamber: Glass desiccator, 250 mm

inside diameter, Kimax, Owens-Illinois Glass Co. The proper

sulfuric acid solution is placed In the bottom of the desic

cator. Paraformaldehyde powder is held in a crystallization

dish (170 mm diameter, 100 mm height) which rests inside the

desiccator.

I. Formaldehyde reaction chamber: A glass desiccator (170 mm di

ameter) is fitted with a two-piece wooden clamp In order to

secure the lid during heating.

J. Constant temperature oven: Isotemp Oven, Fisher Scientific Co.

K. Vacuum pump: Duo Seal Vacuum Pump, Model 1405, Welch Scientific

Co. A 3-way glass stopcock connects the pump, lyophylizatlon

chamber and air intake. The air intake is fitted with a silica

gel column. A vacuum gauge (McLeod Gauge, Virtis Co., Inc.) is

placed between the pump and the stopcock. High vacuum rubber

tubing and metal hose clamps are used for the various

Ill

connections. A 19/38 ground glass joint connects the vacuum

system to the lid of the glass lyophylization chamber.

L. Embedding chamber: The embedding chamber consists of 2 halves

joined together with a 24/40 ground glass connector. The lower

half is a 125 ml flat-bottom boiling flask. The upper half,

which Is mounted with a vacuum outlet, has a chamber positioned

for short-term storage of brain tissue. A photograph of this

apparatus is shown by Falck and Owman (1965).

M. Metal cylinder: The metal cylinder is made from a polished

steel, nickel plated cork borer and is approximately 200 mm long

and 18 mm In diameter.

N. Tissue embedding medium: Paraplast, Van Waters and Rogers, Inc.

0. Microtome: American Optical Co.

P. Slide warmer: Chicago Surgical and Electrical Co.

Q. Glass slides: 1 mm, Corning Glass Works.

R. Cover glass: No. 0, Corning Glass Works.

S. Fluorescence microscope: Ortholux, model 250, E. Leltz, Inc.

T. Film: Black and white photographs were made with Kodak Tri-X

Fan 35 mm film.

11. Materials and solutions

A. High vacuum grease (silicone lubricant): Dow Corning Co.

B. Humidified paraformaldehyde: A 10 mm layer of paraformaldehyde

(Fisher Scientific Co.) Is placed in the crystallization dish

and subjected to a relative humidity of 58.3%, 70.4% or 97.5% in

the humidifying chamber for at least 5 days prior to use. These

112

humidities were obtained by use of sulfuric acid solutions

with densities equal to 1.3 g/ml, 1.25 g/ml or 1.05 g/ml,

respectively.

C. Isopentane: pure grade, Phillips Petroleum Company

D. Phosphorus pentoxide, granular: analyzed reagent grade, J. T.

Baker Chemical Co.

E. Phosphorus pentoxide, powder: Mallinckrodt Chemical Works

F. Silica gel: 6 to 16 mesh, Fisher Scientific Co.

III. Procedure

A. Lyophylization step

1. Decapitate animal.

2. Remove brain (steps 1 and 2 should require less than 2

minutes).

3. Remove brain area containing desired portion of hypothala

mus, nucleus caudatus putamen and globus pallidus. With a

scalpel make an anterior coronal section at the intersection

of the optic nerves and a posterior coronal section approxi

mately A mm from the first cut.

4. Place the brain tissue on a numbered copper plate so that

the anterior surface makes contact with the plate.

5. Stain the posterior side (now facing upward) with approxi

mately 1 mcl of eosln dye.

6. Drop tissue and plate into isopentane cooled with liquid

nitrogen. Both solid and liquid isopentane should be

present during the freezing procedure.

113

7. After 1 minute transfer tissue and plate to a 10 ml beaker

filled with liquid nitrogen.

8. For temporary storage place the 10 ml beaker in a large

container of liquid nitrogen. Tissues may be stored for

at least 24 hours In liquid nitrogen.

9. Transfer tissue and plate to the aluminum foil container

filled with liquid nitrogen. Care should be taken to keep

tissue covered with liquid nitrogen.

10. Fill outer compartment of the plastic tray with granular

phosphorus pentoxide,

11. Place the aluminum foil container into the Inner compartment

of the plastic tray.

12. Insert the plastic tray into the glass lyophylization

• chamber which has been stored In a freezing unit at less

than -45°C.

13. Place a thin film of high vacuum grease on the ground glass

lip of the chamber and its lid.

14. Grease and attach the vacuum pump connection to chamber lid.

15. Press lid into position on lyophylization chamber and at the

same time activate vacuum pump.

16. After the liquid nitrogen and residual lsopentane have

vaporized reduce the chamber pressure to less than 0.005

mm Hg.

17. Continue lyophylization for 3 days at a temperature of

approximately -30°C.

Formaldehyde treatment step

1. Remove glass chamber from freezing unit and slowly

adjust temperature of chamber to approximately 35°C.

2. Slowly release vacuum by allowing room air to enter the

chamber after passing through 6 X 0.5 inch silica gel

column.

3. Remove chamber lid and quickly transfer plastic tray to

dry-air box.

4. Carefully transfer the tissue to the appropriate basket

in the wire rack.

5. Place paraformaldehyde into formaldehyde reaction chamber

which has been heated to 80°C. Use the humidified paraform

aldehyde prepared in 58.3% relative humidity for development

of catecholamines. For development of 5-hydroxytryptamlne

use the humidified paraformaldehyde prepared in 70.4% rela

tive humidity.

6. Transfer wire rack from dry-air box to reaction chamber.

7. Secure reaction chamber lid and place in constant tempera

ture oven at 80°C for 1 hour.

8. Remove reaction chamber from oven and allow to cool in a

stream of room air for 30 minutes.

9. Slowly remove reaction chamber lid.

10. If 5-hydroxytryptamine is being developed repeat steps 5

through 9 above using a humidified paraformaldehyde prepared

at 91.57. relative humidity. If catecholamines are being de

veloped omit this step and proceed directly to step 11 below,

11. Transfer wire rack to vacuum desiccator containing powdered

115

phosphorus pentoxide. If overnight storage is desired re

duce desiccator pressure to less than 0.05 mm Hg and protect

from light. Before removing tissue from desiccator increase

internal pressure as specified in step 2 above.

Embedding step

1. Place an appropriate amount of tissue embedding medium into

lower half of embedding chamber. Melt in a water bath

heated to 60°C.

2. Transfer a tissue into the upper half of the embedding

chamber, connect upper portion to lower half of chamber and

reduce pressure to less than 0.05 mm Hg.

3. Tip the specimen into the liquefied tissue embedding medium

by tilting the chamber and tapping the upper half. Care

should be taken to avoid coating the inside wall of the

upper half of the chamber with liquefied embedding medium.

4. Infiltrate for 20 minutes.

5. Release vacuum and remove bottom half of chamber from water

bath.

6. Trap tissue (stained side up) inside the metal cylinder.

7. Cool lower half of chamber and cylinder with dry ice for

20 minutes.

8. Remove metal cylinder from chamber. The metal cylinder now

contains the tissue surrounded by solidified tissue embedd

ing medium.

9. Remove the tissue embedding medium and tissue from the

cylinder with aid of a glass rod. Tissues can now be stored

at room temperature for several weeks if protected from

light.

D. Fluorescence viewing step

1. Mount the embedded tissue on microtome so that sectioning

begins at the unstained surface.

2. Section tissue at a thickness of 10 microns.

3. When sectioning reaches a depth of 140 microns from the

anterior (unstained) surface, collect sections and place

on a dry slide. This portion corresponds to the section

which is 5780 microns anterior to the frontal zero plane

as described by Konig and Klippel (1963).

4. Transfer the slide to a slide warmer, maintained at 60° to

65°C, to allow the paraplast to melt and the sections f".o

spread. Remove from heat and allow to solidify.

5. Place cover glass over tissue and view with the aid of a

fluorescence microscope. With the third ventricle as a

guide, the anterior hypothalamic nucleus periventricularls

is viewed 0.8 to 0.9 mm dorsal to the ventral surface of the

brain. Intense green to yellow-green discrete fluorescence

appears in varicosities of nerve fibers in this region. The

nucleus caudatus putamen is viewed approximately 3 mm lat

eral to the third ventricle and 5.5 mm dorsal to the ventral

surface of the optic chiasm. This nucleus is characterized

by diffuse green to yellow-green fluorescence which con

trasts with the dark brain regions surrounding it. The

globus pallldus is found approximately 3 mm lateral to the

117

third ventricle and 3 ram dorsal to the ventral surface of

the optic chiasm. Discrete yellow fluorescence is seen in

this region.

6. Photograph the various areas soon after exposure to the

activating light since loss of some fluorescence occurs

after about 3 minutes.

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