THE li‘é ViVORELEASE 0F
ERDOGENGUSLY SYNTHESIZED '
CATECHOLAMSNES FROM THE CAT BRAIN
BY LATERAL HYPOTHALAMIC
SUMULAHOR ANB BY d-AMPHETRMWE
ADMENISTRMION

fiissefiafion far the fiegree 0% Ph. 9.. .
MECHEGRR S‘WE UHWERSEW
CfiURNG ems»; CHEEEEH
1974

 

This is to certify that the

thesis entitled

THE IN VIVO RELEASE OF ENDOGENOUSLY SYNTHESIZED
CATECHOLAMINES FROM THE CAT BRAIN BY LATERAL
HYPOTHALAMIC STIMULATION AND BY d-AMPHETAMINE
ADMINISTRATION

presented by

Chuang Chin Chiueh

has been accepted towards fulfillment
of the requirements for

Ph. D. degree in Pharmacology

 

 

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ABSTRACT
THE IN VIVO RELEASE OF ENDOGENOUSLY SYNTHESIZED CATECHOLAMINES

FROM THE CAT BRAIN BY LATERAL HYPOTHALAMIC STIMULATION
AND BY d-AMPHETAMINE ADMINISTRATION

By
Chuang Chin Chiueh

The primary aim of this investigation was to demonstrate the
jn_vjvg_release of endogenously synthesized dopamine from the nigro-
striatal neurons in response to appropriate stimuli. V

A cerebroventricular perfusion technique was utilized to detect
the efflux of endogenously synthesized dopamine and/or norepinephrine
from the cat brain into a ventricular perfusate. Catecholamine stores in
the brain were labeled using an intraventricular injection of radioactive
precursors or a continuous infusion of 3H-tyrosine of high specific
activity. The perfusing cerebrospinal fluid superfused the caudate nucleus,
septum, hypothalamus and thalamus and was collected at 5 or 10 min inter-
vals from a catheter placed at the aqueduct. Perfusates and brain tissues
were analyzed for labeled catecholamines using a combination of ion-
exchange and alumina adsorption chromatography.

Radioactive tyrosine or dopa, administered intraventricularly, was
converted to catecholamines in brain tissues lining the cerebroventricular
system; most of the labeled catecholamine found was located in the caudate
nucleus on the side of injection and consisted of only dopamine. Electrical
stimulation at the coordinates of A TO, L 3, H —3.5 in the lateral
hypothalamus increased the conversion of 14C—tyrosine to dopamine in

the ipsilateral caudate nucleus, whereas a chronic lesion at these

Chuang Chin Chiueh

sites reduced the synthesis of dopamine. The endogenous content of
dopamine was also lowered on the side of the lesion. _
A The cerebroventricular system was perfused with cerebrospinal

fluid starting several hours after an intraventricular injection of

3 3

H-dopa or H-tyrosine; endogenously synthesized radioactive catechol-

amines were identified in the perfusing effluent and consisted pri-
marily of dopamine. Electrical stimulation of the lateral hypothalamus,

which was shown to contain the dopaminergic nigrostriatal neurons,

3

increased the efflux of H-dopamine and 3H-norepinephrine. The

absolute amount of labeled catecholamines in the perfusate after an

intraventricular injection of 3H-dopa was several times higher than that

3H-tyrosine. The addition of d—amphetamine (0.3 to 30 ug/ml) to

3

after
the perfusing fluid caused a large increase in the efflux of H-dopamine
and a small increase in the efflux of 3H-norepinephrine. When 9:

amphetamine was infused into the third ventricle through the lateral

3H-dopa, no

ventricle contralateral to the side for injection of
significant increase in the efflux of 3H-dopamine was found.

Most of the lateral hypothalamic stimulation-induced efflux of
3H—dopamine and virtually all the efflux of 3H-norepinephrine originated
from structures lining the anterior horn of the lateral ventricle.
Electrolytic or 6-hydroxydopamine-induced lesions of the same sites in
the lateral hypothalamus decreased the content of caudate dopamine and
septal norepinephrine on the side of lesion.

Three to four months after a unilateral electrolytic lesion of
the lateral hypothalamus, cats showed a tendency to circle toward the
lesioned side when startled. ‘Apomorphine and L-dopa increased the circling

toward the lesioned side in a dose-related manner. Pretreatment with

Chuang Chin Chiueh
haloperidol caused a shift to the right of the dose-response curve of
' apomorphine. g:Amphetamine increased ipsilateral circling in cats
with chronic lesions, this effect was partially blocked by pretreatment
with a-methyltyrosine.

In order to monitor the release of newly synthesized catechol-
amines from the cat brain jn_vjvg, 3H-tyrosine of high specific
activity (greater than 50 c/mmole) was infused continuously into the
cerebroventricular system. The problems inherent in experiments using

3

a continuous infusion of the cerebroventricular system with H-tyrosine

of high specific activity were largely overcome in the present study by

3H-tyrosine immediately before use and by

carefully purifying the
utilizing a combination of weak cation-exchange (Bio-Rex 70) and alumina
adsorption chromatography to selectively isolate the endogenously

3H-norepinephrine. With this procedure it

synthesized 3H-dopamine and
was possible to detect a few thousand dpm of 3H-dopamine in a ventricular
perfusate containing 30 to 80 million dpm of 3H-tyrosine.

At the end of 3 to 4 hours of 3H-tyrosine perfusion the relative
distribution of labeled catecholamines in the caudate nucleus, septum
and hypothalamus was 94:2:4. The caudate nucleus contained only dopamine.
Over 95% of the labeled catecholamines in the ventricular perfusates
consisted of dopamine; the spontaneous efflux of this amine was slightly
decreased by the addition of a-methyltyrosine to the perfusing fluid.
Intraventricular administration of gramphetamine caused a dose-related

3

efflux of H-dopamine previously or newly synthesized during the con-

tinuous infusion of 3H-tyrosine. a—Methyltyrosine added to the perfusing

3H-tyrosine

solution 90 min prior to and during the perfusion of
inhibited the biosynthesis of catecholamines in the brain and thus

blocked the gramphetamine-induced release of brain dopamine.

Chuang Chin Chiueh

Two hours after the start of the 3H-tyrosine perfusion, g;
amphetamine was added to the perfusing fluid for an additional two
hours. The increased efflux of 3H-dopamine induced by amphetamine
declined slowly to reach a steady level 60 min after the addition of
amphetamine to the perfusing fluid. The rate of decline was
accelerated if a-methyltyrosine was also added to the perfusing
solution; the initial response of d:amphetamine, however, was not
altered by this drug. The addition of gramphetamine to the 3H-tyrosine
solution at the start of perfusion increased the rate of efflux of

3H-dopamine two to three fold over the control efflux.

newly synthesized
This response to g;amphetamine was completely blocked by the presence
of cemethyltyrosine in the perfusing solution but was not blocked or
decreased by treating the cat with reserpine 2 hours prior to the start
of the experiment in order to eliminate the storage pool of dopamine in
the caudate nucleus. Reserpine treatment effectively reduced the
endogenous and 3H-dopamine content of the caudate nucleus by 95%. The
intravenous injection of increasing doses of g7amphetamine elicited a

3

' dose-related increase in the efflux of newly synthesized H-dopamine in

the reserpineetreated cat. After reserpine treatment, the amount of
3H-dopamine released by amphetamine was higher than the content of

labeled amines remaining in the brain. 0n the other hand, the peripheral
pressor effect of gramphetamine was blocked following reserpine treatment.
Thus, newly synthesized striatal dopamine plays a more important role in

the maintenance of amine release by g;amphetamine than does mobilization

of stored dopamine.

THE IN VIVO RELEASE OF ENDOGENOUSLY SYNTHESIZED CATECHOLAMINES
FROM THE CAT BRAIN BY LATERAL HYPOTHALAMIC STIMULATION
AND BY d—AMPHETAMINE ADMINISTRATION

By

Chuang Chin Chiueh

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of
DOCTOR OF PHILOSOPHY
Department of Pharmacology

1974

to my wife, Hsun Lan

11

ACKNOWLEDGMENTS

The author wishes to thank Dr. K. E. Moore for his advice
and encouragement throughout the course of this study.

He acknowledges the constructive assistance of Dr. T. M.
Brody, Dr. G. L. Gebber, Dr. R. H. Rech, Dr. J. E. Thornburg
and Dr. M. E. Neichsel in the preparation of this thesis.

He is very grateful to Mrs. Mirdza Gramatins, Mrs. Susan'
Stahl and Miss Nanette Friedle for their excellent technical
"assistance.

The author would like to express his appreciation to Dr.

C. C. Chang for his valuable advice.

TABLE OF CONTENTS

INTRODUCTION . . . . ...................... l

A. Criteria for striatal dopamine as a neuro-

transmitter of nigrostriatal pathway in the

brain . . . . . ...... . . . . . . l
B. Electrical stimulation- induced release of

endogenously synthesized dopamine from the

brain in vivo . . . . . . . ..... 2
C. Effects “OT’Emphetamine on the release of

stored and newly synthesized striatal

dopamine . ......... . ...... 4
D. The primary aim of the present study ....... 6
METHODS.................. .......... 8
A. Cerebroventricular perfusion technique . ..... 8
B. Administration of radioactive dopamine or
its precursors . . . . . . . . . . . . . . . . 10
C. Purification of radioactive tyrosine . . . . . . . ll
D. Separation and analysis of catecholamines . . . .,. 15
E. Electrical stimulation of the nigrostriatal
pathway in the lateral hypothalamus . . . . . . . . 20
F. Electrolytic or 6Ehydroxydopamine-induced
lesions of the nigrostriatal neurons in the
lateral hypothalamus . . . ..... . . . . . 21
G. Histological examination of lesions and
electrode placements . . . . . . ......... 21
H. Behavioral study . . . . . . . . . . . . . . . . . 22
1. Drugs and chemicals . .............. . 22
J. Statistical analysis ............... 24
RESULTS . . . . . . . . . . . . . . . . . . . . . . ...... 25

1- Acute labeling experiments with radioactive pre-
cursors of dopamine . . . . . . . . . . . . . . ..... 25

A. Control experiments with the intraventricular
injection of 3H-dopamine . ............ 25

1. Dose response curve of d- and l-

isomers of amphetamine on the efflux
of exogenously administered 3H- -dopamine . . 28

iv

TABLE OF CONTENTS (Continued . . . .)
Page

2. The stimulating sites in the lateral
hypothalamus effective in increasing
the efflux of exogenously .
administered 3H-dopamine ........... 32

8. Electrical stimulation and gfamphetamine
administration induce release of endo-
genously synthesized 4-catechols from the
brain after labeling the nigrostriatal
_ terminals with 14C-tyrosine ............. 36

1. Effects of lesion or stimulation of
the diencephalic nigrostriatal path-
way on the conversion of 14C-tyrosine
to T4C-dopamine in the caudate nucleus . . . . 36
2. The concomitaat release of endogenously
synthesized C-catechols and exogenously
administered 3H-dopamine from the brain
evoked by electrical stimulation and by
d-amphetamine administration after
intraventricular injection of 14C-tyrosine
and 3H-dopamine ..... . . . . . . . . . . 41

C. The release of endogenously synthesized 3H-
catecholamines from cat brain following the
ntraventricular injection of 3H-tyrosine or
H-dopa . . . . . . . . . . . . . . . . . ...... 44

l. Ig_vivo release of endogenously

synthesized 3H-dopamine from cat brain

following electrical stimulation of

lateral hypothalamus and intra-

ventricular infusion of g7amphetamine . . . . 45
2. Effects of contralateral or ipsilateral

stimulation of the lateral hypothalamus

or the intraventricular administration

of gramphetamine on the release of

endogenously synthesized 3H-catecholamines:

Demonstration of the releasing sites ..... 5l
3. Effects of the combination of low frequency

stimulation and g:amphetamine administra-

tion on the gfflux of endogenously

synthesized H-catecholamines . . . ..... 63

II. Electrolytic or 6-hydroxydopamine-induced lesion of
the ascending monoaminergic fibers in the lateral
hypothalamus . . . . . . . . . . . . . . . . . . . . . . . 66

A. Biochemical and histological evaluation of
the lesions of the lateral hypothalamus . ... . . . . 66

V

TABLE OF CONTENTS (Continued . . . .)

III.

Effects of apomorphine, L-dopa and g:

amphetamine on the locomotor asymmetry of

cats with a chronic unilateral lesion of

ascending monoaminergic fibers . . . . . .......

tudies employing the continuous infusion of
H-tyrosine ..... . . . . . . . . . . .........

A.

The distribution of 3H-dopamine and 3H-
norepinephrine in brain tissues and in
cerebroventricular perfusates following a con-
tinuous cerebroventricular perfusion with

3H- -tyrosine . ....................
Effects of intravenous or intraventricular
administration of d- amphetamine on the

efflux of 3H- -dopam1ne during the continuous

infusion of 3H- tyrosine ....... . . . . . .
Effects of a-methyltyrosine on the d-
amphetamine-induced efflux of endogenously
synthesized or exogenously administered

3H-dopamine . . . . . . . . . . . . . . . ......

l.

Pretreatment with a-methyltyrosine on

the biosynthesis of 3H-catecholamines

and on the d—amphetamine-induced

efflux of 3H-dopamine following the

continuous infusion of 3H-tyrosine . . . . . .

. Effects of a-methyltyrosine on the

release of endogenously synthesized
3H-dopamine from the caudate nucleus

during the continuous infusion of

d- -amphetamine . ..... . . . .......

3. Effects of a-methyltyrosine on the

Sfflux of endogenously synthesized

H -dopamine evoked by pulse intraventricular
administration of d- -amphetamine . .......
Failure of a-methyTtyrosine to alter the

d- amphetamine-induced efflux of exogenously
administered 3H- -dopamine from the caudate
nucleus jn_vivo . . . . . . . . . . . . . . . .

Effects of reserpine and a-methyltyrosine on
the d-amphetamine-induced efflux of striatal
dopamine newly synthesized from continously

infused 3H- tyrosine . . . . . . . . . . . . . . . .

I.

Effects of reserpine and a-methyltyrosine

on the efflux of newly synthesized striatal
dopamine evoked by the continuous intra-
ventricular infusion of g:amphetamine . . . .

vi

Page

73

83

84

9O

95

95

95

98

100

. 103

. 108

TABLE OF CONTENTS (Continued . . . .)

Page
2. Effects of reserpine on the central
dopamine releasing action and peripheral
vasopressor action induced by the intra-
venous injections of increasing dose of
g7amphetamine ................. 110
DISCUSSION .......................... . . 118

A. Methodological problems in specifically and

intensively labeling the nigrostriatal dopaminer-

gic neurons in the brain ............... 118
B. The specificity of the coordinates for the

stimulation of the nigrostriatal pathway in

the lateral hypothalamus ............... 121
C. In vivo release of endogenously synthesized

cate cholamines from cat brain by electrical

stimulation ............... 122
D. Lesion of the ascending monoaminergic pathway

in the lateral hypothalamus ....... . ..... 123
E. Selective release of striatal dopamine by

d- -amphetamine . . . . . ............. 127

F. 'Effects of d- -amphetamine on the release of
"stored" and "newly synthesized" dopamine

from the nigrostriatal neurons . . . . ........ 128
SUMMARY AND CONCLUSION ........... . .......... 133
BIBLIOGRAPHY ........................... 139

vii

Table

10.

LIST OF TABLES

The recovery of authentic 3H-dopamine using the
weak cation-exchange and alumina adsorption
chromatographic method ......... . ....... .

The contamination of 3H-tyrosine in dopamine
fraction . . . . . . . . . . . . . . . . .........

Conversion of 14C-tyrosine to dopamine in the caudate
nucleus two weeks after placement of a unilateral
lesion in the lateral hypothalamus . . . . ........

Conversion of 14C-tyrosine and 14C-dopa to dopamine
in the cat caudate nucleus during electrical
stimulation of the lateral hypothalamus ........ .

Effects of electrical stimulation of the nigro-

striatal pathway and the intraventricular

administration of gyamphe amine on the release of
exogenously dministered H-dopamine and endogenously
synthesized 4C-catechols . . . . . . . . ........

The metabolites of 3H-tyrosine in the periods
immediately before and during electrical stimulation
of lateral hypothalamus . . . . . . . . . . . . . . . . .

Retention of 3H-dopamine in the caudate ngcleus
gallowing intraventricular injections of H-dopa or
"-terSine o o o o o o o o o o o o ooooooooooo

he increased efflux of endogenously synthesized

-catecholamines in response to ipsilateral or
contralateral stimulation of the lateral hypothalamus
or the intraventricular infusion of gramphetamine

The effects of lesions in the lateral hypothalamus
on the contents of dopamine and norepinephrine in
the ipsilateral caudate nucleus and septum of cats . . . .

The effects of unilateral 6—hydroxydopamine injection

into the lateral hypothalamus on the monoamine
contents in ipsilateral caudate nucleus and septum . . . .

viii

Page

l7

T8

37

39

43

50

56

59

67

69

LIST OF TABLES (Continued . . . .)

Table
11.

12.

l3.

14.

15.

16.

17.

18.

Page

Relative distribution of 3H-dopamine and

3H-norepinephrine in tissues bordering the

lateral and third cerebral ventricles following

perfusion of 3H-tyrosine .......... . ...... . 88

The increased efflux of endogenously synthesized

3H-dopamine from the cat brain in response to
intraventricular infusions of increasing concen-
trations of gramphetamine . . ........ . ...... 94

Effects of a-methyltyrosine on the radioactive
compounds in tissues lining the lateral and third
gerebral ventricles following perfusion with
-tyrosine ........................ 97

Failure of a-methyltyrosine to alter gramphetamine-
induced efflux of endogenously synthesized
H-dopamine from cat brain ................. 102

Failure of a-methyltyrosine to alter gfamphetamine-
3nduced efflux of exogenously administered
3H-dopamine and endogenously synthesized

' H-norepinephrine from cat brain . . . ........... 106

Effects of a-methyltyrosine on the contents of

radioactive and endogenous dopamine in the caudate

nucleus after intraventricular administration of

3H-dopamine ........................ 107

Summary of the efflux of newly synthesized

3H-dopamine from the caudate nucleus of reserpine

treated cats evoked by intravenous injections of

increasing doses of g:amphetamine . . . . . ........ 114

Effects of reserpine on the contents of endogenous
dopamine and radioactive dopamine synthesized from
H-tyrosine in the caudate nucleus following intra-
ventricular and intravenous administration of
g:amphetamine . . . . ................... 115

ix

LIST OF FIGURES

Figure . » Page

1. Schematic representation of the sagittal view at
L3 of a cat brain prepared for cerebroventricular
perfusion .................... . . . . 9

2. Methods for separation of dopamine and norepinephrine
from amino acid precursors:
A. Acute labeling experiment
B. Continuous labeling experiment ........... 12

3. Effects of electrical stimulation of the lateral
hypothalamus and intraventricular administration
of d-amphetamine on the release of exogenously
amenistered 3H-dopamine from the cat brain . . ..... 26

4. Efflux of 3H-dopamine from cat brain in response to
intraventricular infusion of increasing concentra-
tions of g:amphetamine . . . . . . . . . . . . . . . . . 29

5. Efflux of exogenously administered 3H-dopamine
from cat brain in response to intraventricular
infusion of cumulative concentrations of
g:amphetamine . . . . . . . . . . . . . ......... 30

6. The increased efflux of exogenously administered
H-dopamine from the cat brain in response to
intraventricular infusions of g: and 1:amphetamine . . . 31

7. Frontal section of the cat brain at the level of
posterior hypothalamus depicting electrode tract . . . . 33

8. Release of exogenously administered 3H-dopamine in
response to stimulation at sites in the lateral
hypothalamus . . . . . . . . . . . . . . . . . . . . . . 34

9. Typical efflux pattern of exogenously administered
3H-dopamine and endogenously synthesized 14C-
catechols evoked by electrical stimulation and by
administration of g:amphetamine . . . . . . . . . . . . 42

10. The washout curve of 3H-tyrosine and 3H-dopa from
the brain into the cerebroventricular perfusates . . . . 46

X

LIST OF FIGURES (Continued . . . .)

Figure
11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

2].

Effects of electrical stimulation of the lateral
ypothalamus on the release from the brain of
H-dopamine and 3H-norepinephrine synthesized from

3H-tyrosine or 3H-dopa . ................

Effects of g:amphetamine on the Sfflux of 3H-
catecholamines synthesized from

Effects of d-amphetamine on the efflux of

3H-catech013mines synthesized from 3H-dopa . ......

Effects of contralteral or ipsilateral stimulation
of the lateral hypothalamus or the intraventricular
infusion of d-amphetamine on the release of '

endogenouslyrsynthesized 3H-catecholamines ....... .

Frontal section of cat brain at the level of
posterior hypothalamus depicting stimulating

electrode tracts . . . . . . . . . . . . . . . .....

voked release of endogenously synthesized
H-dopamine and 3H-norepinephrine from the cat
brain by low frequency electrical stimulation and

by administration of gramphetamine . . . . . . . . . .

Histological section of the cat brain: Demonstration

of the effective lesion sites in the lateral

hypothal amus O O O O O O O O O O I O I O O O O O O O O 0

Diagram of the 6-hydroxydopamine lesion of ascending
monoaminergic neurons in the lateral hypothalamus of
cat brain '

A. At the level of A 10

B. At the level of A 8 ........ . . . . . . . . .

The effect of haloperidol on the apomorphine-induced
turning behavior in cats with a chronic unilateral
lesion of ascending monoaminergic fibers in the

lateral hypothalamus . . . . . . . . . .........

The dose response curve of L-dopa on turning be-
havior in cats with chronic unilateral lesions of
the ascending monoaminergic fibers in the lateral

hypothalamus . . . . . . . . . . . . . . . . . . . . . . .

The effect of gramphetamine on turning behavior of
cats with a chronic unilateral lesion of the
ascending monoaminergic neurons in the lateral

hypothalamus . . ....................

xi

H-tyrosine ......

Page

48

52

54

6O

62

64

68

7O

74

77

79

LIST OF FIGURES (Continued . . . .)

.Figure

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

Page
The effect of o-methyltyrosine on the g;amphetamine-
induced ipsilateral turning behavior in cats with
chronic unilateral lesions of the ascending mono-
aminergic neurons ..................... 81
Separation of 3H-catecholamines in the brain tissues
lining the lageral and third ventricles after the
perfusion of H-tyrosine.
A. Caudate nucleus
B. Septum and Hypothalamus ................ 85
Separation of 3H-catecholamines in the cerebroventricular
perfusate collected during the administration of
‘gfamphetamine .......... . ............ 89
Efflux of endogenously synthesized 3H-dopamine in
response to the intravenous injection of
g;amphetamine . . . . ................... 91

Efflux of endogenously synthesized 3H-dopamine from
the caudate nucleus in response to intraventricular
infusions of increasing concentrations of gramphetamine . . 92

Blockade of d-amphetamine-induced efflux of endo-
genously synthesized 3H-dopamine from the cat brain by
pretreatment with a-methyltyrosine . . . . . . . . . . . . 96

Effects of a-methyltygosine on the release of endo-

genously synthesized H-dopamine from the cat caudate

nucleus during continuous g7amphetamine infusion in_

v1 v0. 0 0 O O O O O O O I O O O O O O O O O 0000000 O 99

Effects of g:amphetamine on the efflux of endo-‘
genously synthesized 3H-dopamine before and after
the addition of a-methyltyrosine to the perfusing CSF . . . 101

Failure of a-methyltyrosine to alter gramphetamine-
nduced efflux of exogenously administered
H-dopamine from cat brain jn_vivo . ........ . . . 104

Failure of a-methyltyrosine (5xlO'4M) to alter
gramphetamine-induced efflux of exogenously
administered 3H-dopamine from cat brain jn_vivo ...... 105

Effects of reserpine and a-methyltyrosine on the

d-amphetamine-induced release of newly synthesized
abpamine from the caudate nucleus in vivo ......... 109

xii

LIST OF FIGURES (Continued . . . .)

Figure
33.

34.

Page

Efflux of newly synthesized 3H-dopamine from the

caudate nucleus of a reserpine-pretreated cat in

response to the cumulative doses of the intravenous

injections of g:amphetamine . . . . . . . . . . . ..... 112

Effects of the cumulative doses of the intravenous

injections of dyamphetamine on the blood pressure
of the reserpine pretreated cat . . . . . ......... 116

xiii

INTRODUCTION

A. Criteria for striatal dopamine as a neurotransmitter of the nigro-
striatal pathway in the brain.

Substantial evidence obtained within the past decade suggests
that dopamine is a neurotransmitter of an ascending nigrostriatal
neuronal system in mammalian brain (see review by Hornykiewicz, 1966;
Barbeau and McDowell, 1970; Iversen, 1973). For example, biochemical
studies have demonstrated a relatively high concentration of dopamine
in the caudate nucleus and midbrain areas (Carlsson gt 11., 1958;
Bertler and Rosengren, 1959; Carlsson, 1959). The enzymes necessary
for the biosynthesis of dopamine are present in these brain tissues
(Masuoka gt_al,, 1963; Goldstein §£_El:, 1969; McGeer gt_al,, 1971;
Fyrb gt 31,, 1972; kufelt gt_al,, 1973). Dopamine-B-hydroxylase,
which catalyzes dopamine to norepinephrine, is not located in the
caudate nucleus (Hartman and Udenfriend, 1972). The direct connection
of the substantia nigra with the striatum has been demonstrated by the
fluorescence histochemical technique (Andén gt_al,, 1964a; Hdkfelt-
and Ungerstedt, 1969; Nobin and Bjdrklund, 1973) and by retrograde
degeneration studies (Bédard gt al,, 1969; Moore et_al,, 1971).
Electrophysiological studies have also demonstrated that the caudate
neurons are sensitive to dopamine and nigral stimulation (Bloom et_al,,
1965; McLennan and York, 1967; Connor, 1970; Hull gt a1,, 1970). Thus,
dopamine satisfies most of the criteria for a neurotransmitter listed

by Bloom and Giarman (1968) and by Andén gt_al, (1969). However, one
1

critical criterion that should be fulfilled by a substance before it
can be considered to have the role of neurotransmitter agent remains
to be demonstrated unequivocally for dopamine; that is, this amine
should be released from terminals of nigrostriatal neurons in response
to appropriate stimuli (Hornykiewicz, 1966). Recently, Ng gt 21,
(1971) have reported that dopamine synthesized from labeled precursors
in the nigrostriatal terminals is released from slices of caudate
nucleus upon electrical field stimulation. Since dopamine does not
pass through the blood-brain-barrier, special techniques have been
utilized to monitor the jn_vivg_release of this amine from the brain;
these have included the use of push-pull cannulation (Gaddum, 1961),
modified cup superfusion (Mitchell, 1966; Besson gt_al,, 1971) and the
cerebroventricular perfusion technique (Carmichael gt_al,, 1964; Carr

and Moore, 1969).

B. Electrical stimulation-induced release of the endogenously syn-
thesized dopamine from the brain in vivo.

Using Gaddum's push-pull cannulation technique, McLennan (1964)
attempted to detect the release of endogenous dopamine from the I
caudate nucleus. He reported that the dopamine efflux from the
caudate nucleus could be enhanced by stimulation of the thalamic
centromedian nucleus but not of the substantia nigra. Subsequently he
reported that the release of dopamine from the putamen could be in-
creased by nigral stimulation (McLennan, 1965). These results, however,
are suspect because the amount of dopamine released was below the level
of sensitivity for fluorescent analysis of this amine. Portig and

Vogt (1969), utilizing a cerebroventricular perfusion technique,

found an increase in the efflux of homovanillic acid, a deaminated
O-methylated metabolite of dopamine, but failed to detect a consist-
ent release of dopamine during electrical stimulation of the sub-
stantia nigra. They suggested that another technique must be devised
to analyze for dopamine because the amounts of amine and metabolite
are at the lower limits of chemical methods available for their
detection in the brain perfusates.

Initial experiments in our laboratory also revealed that it was
not possible to detect endogenous dopamine in cerebroventricular
perfusates of the cat using fluorometric procedures (Moore, K.E., un-
published observation), so that it was necessary to resort to a
radioactive tracer technique. In early reports from this laboratory,
tissues lining the cerebroventricular system were labeled by intra-
ventricular injections of 3H-norepinephrine or 3H-dopamine. It was
found that the labeled catecholamines were located primarily in the
caudate nucleus (Carr and Moore, 1969). It was subsequently demon-
strated that electrical stimulation of the caudate nucleus, substantia
nigra and the ascending nigrostriatal fiber tract elicited a frequency-
and intensity- related release of exogenously administered 3H-dopamine
into the cerebroventricular perfusate (Von Voigtlander and Moore,
197la,b). It was also demonstrated that exogenously administered
3H-catecholamines could be released by drugs, for example, by the
intraventricular or intravenous administration of d;amphetamine (Carr
and Moore, 1970; Von Voigtlander and Moore, 1973a). Although the
intraventricularly administered radioactive catecholamines are taken

up by catecholaminergic neurons lining the cerebroventricles (Glowinski

4

gt 21:9 1966), the amines appearing in the perfusate probably do not
originate exclusively from these neurons. For example, labeled
catecholamines may be taken up by and released from serotonergic
neurons because of the lack of chemical specificity in the mechanism
for uptake into various monoaminergic neurons (Kopin, 1968).

Since tyrosine hydroxylase is located in nigrostriatal neurons
(Moore _e_i_;_§_1_., 1971; Goldstein fig” 1969; Fyrd e_t_al., 1972) and in
other catecholaminergic neurons, any radioactive catecholamines
detected in the brain perfusate following the administration of
radioactive tyrosine must originate from these neurons. Attempts
to demonstrate the in.vjyg_release of dopamine synthesized from radio-
active precursors during stimulation of the caudate nucleus (McKenzie
and Gordon, 1972; Roth gt 31,, 1969) and other brain regions (Riddell
and Szerb, 1971) have been generally unsuccessful. Nevertheless,
Riddell and Szerb (1971) and McKenzie et_al, (1972), using a push-pull
cannulation technique, have detected release evoked by a variety of
drugs of catechols synthesized from radioactive precursors. Besson
gt_gl, (1971), using a modified cup superfusion technique, detected

3H-tyrosine of high specific

the release of dopamine synthesized from
activity from the caudate nucleus during the administration of drugs
such as lyamphetamine. Although push-pull cannulation or the modified
cup technique can be utilized to perfuse a small brain area, the tissue
that is perfused is probably damaged by the implantation of the push-

pull cannula or by the modified surface cup procedure (Chase and Kopin.1958)-

C. Effects of amphetamine on the release of stored and newly synthe-

sized striatal dopamine.

Several explanations have been offered to explain the mechan-
ism Of amphetamine-induced central stimulant actions. It has been
suggested that amphetamine reacts directly with catecholaminergic
receptors in the brain (Smith, 1963). On the other hand, Stein (1964)
and Rech (1964) proposed that amphetamine exerts excitatory actions by
releasing catecholamines from nerve terminals in the brain. Glowinski
_e_ta_1_. (1966) suggested that amphetamine inhibits amine uptake pro-
cesses and brain monoamine oxidase, leading to an increase in the
concentration of amines in the synaptic cleft. The central stimulant
actions of g:amphetamine can be blocked by a-methyltyrosine, a drug
which interferes with the synthesis of catecholamines (Nagatsu gt_al,,
1964; Spector gt_al,, 1965). It has been proposed that blockade of
amine synthesis and actions of amphetamine by a-methyltyrosine are
causally related (Heissman gt_al,, 1966; Dingle et_al,, 1967; Sulser
gt 21,. 1968; Dominic and Moore, 1969). Furthermore, reserpine, a drug
which reduces the brain content of amines (Brodie gt_al,, 1957;
Carlsson, 1959; Holzbauer and Vogt, 1956; Rech gt_al,, 1968), did not
affect but, on the contrary, augmented the behavioral stimulant actions
of amphetamine (Smith, 1963; Stolk and Rech, 1967). Combined pre-
treatment with reserpine and a-methyltyrosine completely abolished the
central stimulant effects of amphetamine (Rech and Stolk, 1970).
Accordingly, it has been proposed that the anti-amphetamine property of
a-methyltyrosine results from the ability of this drug to compromise a
small functional pool catecholamines which is maintained continuously
by synthesis. Accumulated evidence has led to the hypothesis that

g:amphetamine exerts its stimulant actions by releasing brain dopamine

and/or norepinephrine from this newly synthesized pool (Sulser gt_al,,
1968; Rech st 31,, 1968; Besson gt__l,, 1969; Moore et_al,, 1970).
However, this mechanism of the anti-amphetamine actions of a-methyl-
tyrosine has recently been questioned by Enna gt_al, (1973), who
reported that a-methyltyrosine interferes with amphetamine-induced
release of amines from brain slices. Regardless of the exact mechan-
ism by which amphetamine alters the dynamics of brain catecholamines,
a number of reports have suggested that amphetamine acts upon striatal
dopaminergic neurons rather than noradrenergic neurons to exert theY
locomotor stimulation and stereotyped behaviors (Van Rossum and
Hurkmans, 1964; Ernst, 1967; Creese and Iversen, 1973; Costa gt_al,,
1972; Thornburg and Moore, 1973a).) The innvitrg_release of newly syn-
thesized dopamine by gramphetamine from rat striatal slice has been
reported by Besson _e_t__a_l_. (1969). Furthermore, Besson g_t_31_. (1973)

3H-dopamine newly synthesized from 3H-tyrosine is

have reported that
preferentially released spontaneously into the perfusate of a cup placed

upon the exposed cat caudate nucleus.

0. The primary aim of the present study.

The primary aim of the present investigation was to demonstrate
jg_g1!g release of dopamine from terminals of nigrostriatal neurons in
response to appropriate stimuli. A cerebroventricular perfusion tech-
nique and intraventricular administration of radioactive precursors of
catecholamines were utilized to investigate the effects of gramphetamine
and electrical stimulation on the efflux of endogenously synthesized
dopamine and/or norepinephrine from the cat brain. The results

demonstrate that gyamphetamine selectively releases newly synthesized

dopamine from the nigrostriatal neurons in the caudate nucleus 3n
vivo. The endogenously synthesized dopamine and norepinephrine can
be also released from the brain by electrical stimulation of ascend-

ing monoaminergic fiber tracts in the lateral hypothalamus.

METHODS

A. Cerebroventricular perfusion technique.

The perfusion technique used was modified from that described
previously (Von Voigtlander and Moore, 1971a; Carr and Moore, 1969).
Mongrel cats of either sex (2 to 3.5 kg) were anesthetized with
methoxyflurane and then prepared for ventricular perfusion by placing -
an inflow cannula in the anterior horn of the lateral ventricle
(A 16.5, L 3.5, H 7.5; Snider and Niemer, 1961) and an outflow catheter
at the cerebral aqueduct (Figure 1). In experiments concerned with
electrical stimulation of the nigrostriatal pathway, the spinal cord
was sectioned at the level of C1 in order to prevent stimulation-
induced pressor effects and to immobilize the subject; anesthesia was
then withdrawn and the cat maintained by artificial respiration. All
incisions and pressure points were treated with a local anesthetic
(lidocaine) before recovery from general anesthesia. In other experi-
ments in which the brain catecholamine stores were labeled with a pulse

3 3H—tyrosine,

injection of H-dopamine or by a continuous infusion of
the animals were anesthetized with sodium pentobarbital (30 to 40 mg/kg,
i,p.) instead of methoxyflurane. Artificial cerebrospinal fluid (CSF)
containing NaCl, 129 mM; NaHC03, 24.4 mM; KCl, 2.9 mM; CaC12.2H20,

1.3 mM; MgC12.6H20, 0.8 mM; NazHPO4, 0.5 mM and saturated with 95% 02
and 5% CO2 to buffer the pH at 7.4 (Pappenheimer gt_gl,, 1962) was
infused into the inflow cannula at a constant rate of either 0.6 or

0.2 ml/min with a Harvard infusion pump (Model 975). The ventricular

8

CSF STIM

 

 

'fi.‘

 

Figure 1. Schematic representation of the sagittal view
at L 3 of a cat brain prepared for cerebroventricular perfusion.

The cerebroventricular system is depicted in crosshatching;
the third ventricle is illustrated at level of L 0. Artificial
CSF is infused into the inflow cannula which is stereotaxically
implanted into the lateral ventricle, and the outflow catheter
is inserted at the aqueduct. A bipolar electrode (STIM) is
placed in the lateral hypothalamus. SN=Substantia nigra;
CN=Caudate nucleus.

10

perfusate was collected from the catheter in the aqueduct every 5 or 10
min in 15 ml centrifuge tubes containing 0.1 m1 of 1N acetic acid,

0.1 ml of 0.15% disodium ethylenediamine tetraacetate (NazEDTA) and

0.1 m1 of a solution containing 100 u g/ml of dopamine.

At the termination of the experiment, the cat was sacrificed
by a rapid intravenous injection of sodium pentobarbital. The brain
was removed and placed on ice. The right and left caudate nuclei,
septa and hypothalami were rapidly dissected,weighed and kept at
~27°C until analyzed for catecholamines. In some experiments the
brain tissue was fixed in 10% formalin for histological examination of
the electrode placement. In experiments in which radioactive pre-
cursors were infused intraarterially, the cerebrovasulcar system was

perfused with 0.9% NaCl prior to removing the brain.

B. Administration of radioactive dopamine or its precursors.

In experiments concerned with the effects of the unilateral
nigrostriatal stimulation (400pA, 1 msec, 30 Hz for 30 sec of each min
for 90 min) on the conversion of radioactive tyrosine and dopa (3,4-
dihydroxyphenylalanine) to dopamine in the caudate nucleus, 20uc of

‘4 14C-dopa was dis-

purified and 1yophilized C-tyrosine or 10 uc of
solved in 3 m1 of a 0.9% NaCl solution and infused into both carotid
arteries at a constant rate of 0.1 ml/min during the last 30 min of the

stimulation period.

In experiments concerned with the efflux from the cerebral
ventricles of radioactive dopamine synthesized following the acute
injection of radioactive precursors, the following procedures pertained:

3

160 uc of purified and lyophilized H-tyrosine was dissolved in 20 ul

11

of artificial CSF and injected slowly (over a 5 min period) into a
lateral ventricle through the lateral ventricular cannula 3 hr prior
to the start of ventricular perfusion; 20 uc of 3H-dopa was injected
slowly (over a 5 min period) into the lateral ventricle in a volume
of 20 pl every 30 min for five injections. Ninety min after the last

3H-dopa, perfusion of the cerebroventricular system with

injection of
artificial CSF (0.6 m1/min) was begun.

Catecholamine stores in tissues lining the cerebroventricular
system were also labeled using a continuous infusion (0.2 ml/min) of

3H-tyrosine (either 20 uc/ml or 12.5 uc/ml) in experiments-

purified
concerned with the effects of drugs on the efflux of newly synthesized
dopamine.

In experiments concerned with the efflux of exogenously

3H-dopamine from the caudate nucleus, 3H-dopamine (5 uc

administered
in 10 ul) was injected slowly (over a 2 min period) into the lateral
ventricle 15 min prior to the start of perfusion (0.6 or 0.2 ml/min,

Carr and Moore, 1970).

C. Purification of radioactive tyrosine.

In experiments concerned with the pulse injection of labeled
tyrosine, radioactive tyrosine was purified and 1yophilized according
to the method described by Heiner and Rabadjija (1968). In experiments
in which radioactive tyrosine was continuously infused into the
cerebroventricular system, 3H-tyrosine of high specific activity
(greater than 50 c/mmole) was purified immediately before the start
of perfusion because of the problem that 3H-tyrosine may be converted
to dopa in the presence of tritium of high specific activity (Evans,

1955; Naldeck, 1971). One mc of 3H-tyrosine was mixed with 2 ml of

12

Figure 2. Methods for separation of dopamine and norepine-
phrine from amino acid precursors:

A. Acute labeling experiment.

B. Continuous labeling experiment.

Radioactive dopamine and/or its precursors were injected into
the lateral ventricle in a volume less than 30 ul (in A. acute
labeling experiment) or continuously infused into the cerebroventrie
cular system at a constant rate (in B. continuous labeling experi-
ment) in order to label the stores of catecholamines in the brain.
Samples of brain tissue and cerebroventricular perfusate were extr-
acted with acids and analyzed for radioactivity in dopamine and
norepinephrine fractions by using alumina adsorption and ion-
exchange chromatography. HAc=acetic acid; HC104=perchloric acid;
HCl=hydrochloric acid.

13

A. ACUTE LABELING EXPERIMENTS

PERFUSATE (in HAc)

 

 

 

TISSUE EXTRACT (in H0104)
ALUMINA ADSORPTION
pH 8.4
0.2N HAc ELUATE 1N HCl ELUATE EFFLUENT
norepinephrine dopa tyrosine
dopamine deaminated catechols O-methylated
l J noncatechols

 

ION EXCHANGE CHROMATOGRAPHY
(AG 50HX4, Na+, 200-400 mesh)
pH 6.5

l
V

EFFLUENT 1N HCl ELUATE 2N HCl ELUATE
dopa norepinephrine dopamine

deaminated
catechols

 

 

B.

EFFLUENT
tyrosine
dopa

14

Figure 2 (cont'd)
CONTINUOUS LABELING EXPERIMENTS

PERFUSATE (in HAc)
TISSUE EXTRACT (in HClO4)

ION EXCHANGE CHROMATOGRAPHY
(Bio-Rex 70, Na+, 200-400 mesh)

pH 6.5

0.5N HAc ELUATE
norepinephrine
dopamine

deaminated catechols O-methylated amines

ALUMINA ADSORPTION
pH 8.4

e | C
I 1
0.2N HAc ELUATE EFFLUENT

norepinephrine O-methylated
dopamine amines

ION EXCHANGE CHROMATOGRAPHY
(AG 50wx4, Na+, 200-400 mesh)
pH 6.5

 

 

 

F

1N HC1 ELUATE
norepinephrine

 

2N HC1 ELUATE EFFLUENT
dopamine

15

artificial CSF and placed on a column containing Bio-Rex 70 resin
(Na+, 200-400 mesh, 26 mm2 x 25 mm, Bio-Rad Lab., Richmond, Ca.) to
remove amine impurities (for example, tyramine). The effluent and
subsequent 7 ml wash with artificial CSF were placed directly on a
second column containing washed aluminum oxide (pH 8.4, 26 mm2 x

25 mm, Noelm, Eschwege, Germany) to remove the catechol impurities
(mainly dopa). The alumina column was washed with 5 m1 of sodium
'acetate buffer (pH 8.4) before use. The effluent containing purified

3H-tyrosine was then diluted with artificial CSF to the desired con-

centrations (either 20 uc/ml or 12.5 uc/ml).

0. Separation and analysis of catecholamines.

The perfusates and brain samples were analyzed for catechol-
amines by alumina adsorption and ion-exchange chromatography using
modifications of procedures described previously (Bhatnagar and Moore,
1970; Carr and Moore, 1969; Barchas gt_al,, 1972; Figure 2 A and B).

In experiments in which the brain was labeled with a pulse
intraventricular administration of radioactive dopamine and/or its
amino acid precursors, total radioactivity of the perfusate was deter-
mined by adding 0.1 m1 of the perfusate to counting vials containing
10 m1 of 0.5% 2,5-diphenyloxazole in 7 parts of toluene and 3 parts of
95% ethanol. (The remaining perfusate was adjusted to pH 8.4 with 1 m1
of 0.5M tris hydroxymethyl aminomethane (Trizma base, Sigma Chemical
Co., St. Louis, Mo.) and shaken with 200 mg of washed alumina for
10 min. The alumina was then washed twice with 5 ml of redistilled

imater. The catecholamines were eluted from the alumina by shaking with

7 lnl of 0.2N acetic acid for 10 min; the radioactivity was determined

 

16

by adding a 0.1 ml aliquot to counting vials containing 10 ml of 0.5%
2,5-diphenyloxazole scintillation solution.

A In experiments concerned with dopa and/or deaminated catechol
~ metabolites, the acidic catechols were eluted from the alumina with
1 ml of 1N HC1. Norepinephrine, dopamine and deaminated catechols in
the acid eluates were then separated on a strong cation-exchange resin
(AG 50HX4, Na+, 200-400 mesh, Bio-Rad Lab., Richmond, Ca.). The
alumina eluates were adjusted to pH 6.5 and placed on columns contain-

2

ing AG 50HX4 resin (Na+, 200-400 mesh, 26 mm x 50 mm). The columns

were washed with 15 ml of 0.1M phosphate buffer (pH 6.5, in 0.1%
NazEDTA). The effluent and subsequent wash with 10 ml of 0.1M
phosphate buffer represented the deaminated catechols and dopa. After
washing the column with 5 ml of redistilled water and 3 ml of 1N HC1,
norepinephrine and dopamine were eluted with 8 m1 of 1N HC1 and 12 m1
of 2N HC1 respectively. One ml samples of the HC1 eluates were added
to counting vials containing 10 m1 of PCS phosphor solution
(Amersham/Searle, Arlington Heights, Ill.) and counted in a Beckman
LS-100 liquid scintillation counter. Counting efficiency was about 30%.

3 3 3H-dopamine were 74.212.2,

Recoveries for H-tyrosine, H-dopa and
81.510.07 and 65.413.6 % respectively; no corrections were made for
these recoveries.

Brain tissue samples were homogenized in 10 ml of cold 0.4N
perchloric acid and analyzed for radioactive catecholamines as des-
cribed above for the perfusates. An aliquot of the acetic acid
eluate from the alumina was analyzed for endogenous dopamine and/or
-norepinephrine (Moore and Rech, 1967; Chang, 1964). An aliquot of the

alumina effluent was also analyzed for endogenous tyrosine (Naalkes

and Udenfriend, 1957).

17

Table l. The recovery of authentic 3H-dopamine using the weak
cation-exchange and alumina adsorption chromatographic method.

 

 

3H- .
Number Total 3H-dopamine Radioactivity in Recovery
dopamine fraction
N' T (dpm) BRae (dpm) [BRae/T] x 100 (%)
. 1 11,001 8,723 79.29
2 11,970 9,174 76.64
3 12,438 8,406 67.58
4 11,324 4,896 43.23
5 11,000 8,321 75.64
6 12,200 8,280 67.86
7 13,400 11,297 84.30
Mean 11,904 8,442 70.64
:S.E. i330 :713 $5.09

 

3H-Dopamine standards were added to a solution containing 1.9 m1
of CSF, 0.1 ml of 0.15% Na EDTA and 0.1 ml of 1N acetic acid. Samples
were analyzed for dopamine by the method described in Figure 28 (see
also METHODS). Dopamine (0.1 ml of lOOug/ml)was also added as a cold
carrier. BRae=catecholamine fraction of a combination purification
method of weak cation-exchange and alumina adsorption chromatography.

18

Table 2. The contamination of 3H-tyrosine in dopamine fraction.

 

Number Total radioactivity

of 3H-tyrosine

3H-Radioactivity in % of total radio-
dopamine fraction

activity

 

N T (dpm) BRae (dpm) [BRae/T] x 100 (%) .

3 9,808,000i191,000 8.0i2.7 0.000077i0.000026

3 19,853,750i414,800 23.7i6.6 0.000115i0.000034

3 39,128,000t530,000 56.2i16.4 0.000140i0.000044

12 9,000,000 to - 0.000110i0.000020
40,000,000

 

Different concentrations of 3H-tyrosine standards were added to a
solution containing 1.9 m1 of CSF, 0.1 m1 of 100 ug/ml dopamine, 0.1
ml of 1N acetic acid and 0.1 m1 of 0.15% NazEDTA. Samples went through
the chromatographic column of Bio-Rex 70 and alumina adsorption as
described in Figure 2B. BRae=catecholamine fraction of a combination
purification method of weak cation-exchange and alumina adsorption

chromatography.

19

In experiments with the continuous infusion of 3H-tyrosine,
the total radioactivity of perfusate effluent was determined by add-i
ing 10 ul of the perfusate to 10 ml of 0.5% 2,5-diphenyloxazole
scintillation solution. 3H-Catecholamines were separated from
3H-tyrosine by a combination of weak cation-exchange and alumina
adsorption chromatography. The cerebroventricular perfusates were
adjusted to pH 6.5 with 0.25 ml of 1M phosphate buffer (pH 8.0) and
placed on the columns containing Bio-Rex 70 resin (Na+, 200-400 mesh,

2 x 30 mm), which were prepared according to the method of

26 mm
Barchas gt_gl, (1972). The effluent and subsequent 15 m1 wash with
0.02M phosphate buffer (pH 6.5, 0.1% NaZEDTA) containing 3H-tyrosine
and deaminated metabolites were discarded. After washing the column
with 3 ml of redistilled water and 0.5 ml of 0.5N acetic acid,
3H-catecholamines were eluted with 3 ml of 0.5N acetic acid. The
eluates were then adjusted to pH 8.4 with 1.5 ml of 1M Trizma base

and shaken with 200 mg of washed alumina for 10 min. The effluent was
discarded and the alumina was washed 3 times with 5 m1 of redistilled
water. 3H-Catecholamines were eluted from the alumina with 1 ml of
0.2N acetic acid and the radioactivity was determined by adding the
total eluate to a counting vial containing 10 ml of PCS phosphor
solution. Recovery for 3H-dopamine was 70.6: 5.1% (Table l).
3H-Tyrosine contamination in the alumina eluate was 0.00011 1

0.00002% of the total radioactivity (Table 2). No corrections were

3H-tyrosine) in

made for the recovery or for the contamination (mainly
the alumina eluate. In some experiments, after the addition of 10 ug

each of norepinephrine and dopamine as carriers, the alumina eluates

20

were pooled and separated into norepinephrine and dopamine fractions
using an ion-exchange column of AG 50wx4 resin (Na+, 200-400 mesh, 1
26 mm2 x 50 mm) as described above, except the HC1 eluates were
collected in 1 ml fractions. An aliquot (0.9 ml) of each HC1 eluate
was added to a vial containing 10 ml of PCS phosphor solution for
determination of the radioactivity. The remaining HC1 eluate was
assayed for authentic norepinephrine and dopamine as described by

Chang (1964).

Brain tissue samples were homogenized with 3 ml of 0.4N
perchloric acid; the supernatant was adjusted to pH 8.4 with 2.5 m1 of
Trizma base (1M) and analyzed for radioactive catecholamines by alumina
adsorption and by weak cation-exchange chromatography as described
above for perfusates, except that catecholamines were eluted from the
alumina with 4 m1 of 0.2N acetic acid.

Serotonin in the brain sample was determined by the method des-
cribed by Cox and Perhach (1973). Briefly, serotonin was extracted
from the alumina effluent by the acidified n-butanol solvent extraction

method (Chang, 1964) and was quantified fluorometrically following

reaction with o-phthaldialdehyde.

E. Electrical stimulation of the nigrostriatalgpathway in the lateral
hypothalamus.

The parameters used for electrical stimulation of the nigro-
striatal pathway in the lateral hypothalamus have been described pre-
viously by Von Voigtlander and Moore (1971b). Briefly, a bipolar
stainless steel electrode (0.5 mm exposed tips, 0.5 mm separation,

NE 200, David Kopf Instruments, Tujunga, Ca.) was placed at A 10, L 3,

21

H -3.5 (Snider and Niemer, 1961). A rectangular wave of 400 uA
intensity, 0.5 or 1.0 msec duration and 3 or 30 Hz frequency was
applied to the electrode continuously for 5 or 10 min or inter-
mittently (30 sec of each min) for 90 min with a Grass stimulator

(Model S-4) and a Grass constant current unit (Model ccUIA).

F. Electrolytic or 6-hydroxydopamine—induced lesions of the nigro-
striatal pathway in the lateral hypothalamus.

Electrolytic lesions of the nigrostriatal fiber tracts were
performed in the pentobarbital sodium anesthetized cat as described
previously by Von Voigtlander and Moore (1973a). Chemical lesions
of the catecholaminergic fibers in the lateral hypothalamus were made
by injecting 6-hydroxydopamine into the brain. Four pl of a solution
containing 0.5 mg/ml ascorbic acid and 24 ug 6-hydroxydopamine was
injected slowly (1 ul/min) into the brain through a 26 gauge needle
which was mounted to the electrode holder of the David Kopf stereo-
taxic instrument and aimed at A 10, L 3, H -3.5. Extensive care of
the animals (such as lodging separately in a warm and clean area) was
necessary during the first two days following lesions of the lateral
hypothalamus. Procaine penicillin G (150,000 I.U.) was injected

intramuscularly immediately following the lesion.

0. Histological examination of lesions and electrode placements.
After sacrificing the cat with an overdose of pentobarbital

sodium (i.p. or i.v.), the brain was quickly removed and the caudate

nuclei were dissected for biochemical analysis. The rest of the

brain was fixed in a 10% formalin solution for at leaSt 2 weeks. The

 

22

specimen was frozen and 40 u sections were prepared with an
American Optic microtome (Model A0 880). A series of brain sections
was collected and mounted on a gelatinized glass plate and stained with
cresyl violet acetate (Humason, 1967).
The locations of the lesions or of the electrode tips were
determined under a dissecting microscope with an eyepiece micrometer. l#_

Diagrams of brain sections were copied directly from photographs.

H. Behavioral study.

 

The circling behavior of cats with unilateral chronic lateral

“Ill

hypothalamic lesions was observed after leaving the cat in a box L_;'
(1 m3) with a side window and illuminated from the top. The cat was

placed in the box 60 min prior to the start of the injection of drugs;

the rate of circling during the last 30 min period Served as the con-

trol. Drugs were prepared for injection immediately before use.

After injection of the drug, the number of times the cat made a full

3600 turn toward the lesioned or control side was recorded every 30 min.

Behaviors other than circling motor activity were also recorded through-

out the course of the experiment.

I. Druggiand chemicals.

L-Tyrosine-14C (uniformly labeled, 374 mc/mmole), DL-3,4-di-
hydroxyphenylalanine-2-14C (9.3 mc/mmole) and L-tyrosine-3,5-3H
(30.6 to 69.4 c/mmole) were purchased from New England Nuclear (Boston.
Ma.). The purity of the radioactive compounds was checked before use
by thin layer chromatography on cellulose (Eastman chromagram 6065)

with the solvent system of 1N acetic acid, n-butanol and 95% ethanol

23

(30:104:30 by volume, ascending for 2 1/2 hours). The cellulose
plate was sprayed with 0.1% ninhydrin (Nin-Sol, Pierce Chemical Co.,
Rockford, Ill.) to determine the Rf values of labeled and authentic
samples. Sections (0.5 cm) of the plate were extracted with 0.1 ml
of 0.2N acetic acid in counting vials; the radioactivity was sub-
sequently determined after adding 10 ml of 0.5% 2,5-diphenyloxazole
scintillation solution.
g:Amphetamine sulfate or l;amphetamine sulfate (Smith, Kline

and French Laboratories, Philadelphia, Pa.) were dissolved in
artificial CSF immediately before use. l.-a-Methyltyrosine (aMT,
Merck, Sharp and Dohme Research Laboratories, West Point, Pa.) was
dissolved in warm redistilled water and this solution was utilized to
make the artificial CSF (5 x 10‘6 or 4 x 10'4 M o-MT in CSF).
Reserpine was dissolved in about 0.1 ml of glacial acetic acid and
diluted to a concentration of 1 mg/ml with redistilled water.
6-Hydroxydopamine HBr (Regis Chemical Co., Chicago, Ill.) was dis-
solved in a solution containing 0.5 mg/ml ascorbic acid. Apomorphine
HC1 (Eli Lilly and Co., Indianapolis, Ind.) was dissolved in 0.9% NaCl.
a-Hydrazinomethyldopa (HMD, MK-486, Merck, Sharp and Dohme Research
Laboratories, West Point, Pa.) was suspended in 1% methylcellulose.
L-B-3,4-IHhydroxyphenylalanine methylester HCl was purchased from
Sigma Chemical Co. (St. Louis, Mo.) and dissolved in 0.9% NaCl
immediately before use. Cresyl violet acetate was obtained from
Allied Chemical Co. (Morristown, N.J.).

L-[G-BHJ-3,4-Dihydroxyphenylalanine (10.38 c/mmole, 3H-dopa) was

purchased from New England Nuclear (Boston, Ma.).

24

J. Statistical analysis

The data are reported as the mean t the standard error of
mean (S.E.) obtained from at least three experiments. Statistical
comparisons were made by the Student's t_test for paired or group
samples. P values of less than 0.05 were considered to be statistic-

ally significant (Goldstein, 1964).

 

 

RESULTS

1. Acute labeling experiments with radioactive precursors of dopamine.

A. Control experiments with the intraventricular injection of

3H-dopamine.

 

Previous studies have demonstrated that, after labeling the
caudate nucleus with 3H-dopamine, the intraventricular administration of
gramphetamine or electrical stimulation in the area of the diencephalic

3H-dopamine into the

nigrostriatal neurons increased the efflux of
ventricular perfusates (Carr and Moore, 1970; Von Voigtlander and
Moore, 197la,b, 1973a).

In order to establish a fundamental base for studies with
radioactive precursors, initial experiments were performed to confirm
and modify these earlier studies. The cerebroventricular perfusion
technique described by Von Voigtlander and Moore (1971b) was modified
in the following manner: the duration of the collection periods and
of electrical stimulation was increased from 2 to 10 min and the rate of
perfusion was decreased from 0.5 to 0.2 ml/min. The effects of
electrical stimulation and g;amphetamine administration on the efflux
of exogenously administered 3H-dopamine using this modified method are
summarized in Figure 3. Electrical stimulation near the medial border

of the internal capsule at the level of the posterior lateral hypothalamus

markedly increased the efflux of exogenously administered 3H-dopamine;

25

26

Figure 3. Effects of electrical stimulation of the lateral
hypothalamus and intraventricular administration of dramphetamine
on the release of exogenously administered 3H-dopamine from the
cat brain.

Fifteen min after the intraventricular injection of 3H-dopamine
(5 uc in 10 ul), the cerebroventricular system was perfused with
CSF at 0.2 ml/min and perfusates were collected every 10 min for
analysis of radioactivity. Electrical stimulation (square wave
pulses of 30 Hz, 0.5 msec, 400 HA, _) was applied to regions
in the lateral hypothalamus (A 10, L 3, H -2.5, -3.5 and -4.5)
with a bipolar electrode. ngmphetamine (d-A, 30 ug/ml, E2221)
was added to the perfusing CSF during a single ten min period.
The efflux of 3H-dopamine during the electrical stimulation and
the infusion of d-amphetamine are significantly increased (p<.05).
The height of eaEh bar and vertical line represents the mean
i l S.E. as determined from 5 experiments.

18

_.n
N

3H-DOPAMINE (dpm x 1,000)

\O

on

27

 

H-D

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

STIMULATION

 

 

-3.5 d-A
-2.5 —4.5 (30)
'___L__JlllflllnllI. I er*:a i 1

 

 

 

160 180 200 220 240 260
PERFUSION TIME (min)

 

U:

 

 

28

the optimal site for stimulation was A 10, L 3, H -3.5. Intra-‘
ventricular infusion of g:amphetamine (30 ug/ml) caused a seven-
fold increase in the efflux of 3H-dopamine. The magnitude of the

release of 3

H-dopamine in response to intraventricular administration
of gramphetamine was considerably greater than that previously reported
(Carr and Moore, 1970; Von Voigtlander and Moore, 1973a). During
lateral hypothalamic stimulation in the nonanesthetized spinal cats

‘ in the present study, a marked bilateral mydriasis and a moving of the

lower jaw of the subject were also noted.

A—l. Dose response curve of gfand l;isomers of amphetamine on the
efflux of exogenously administered 3H-dopamine.

Since g:amphetamine at a concentration of 30 ug/ml, was far
more effective in this experiment than in previous studies, the dose-
response curve of gramphetamine on the efflux of exogenously
. administered 3H-dopamine was determined. The addition of 5 min pulses
of increasing concentrations of gramphetamine (0.03, 0.3, 3 and 30 pg/ml
gramphetamine base) to the perfusing CSF (0.6 mllmin) at 25 min
intervals caused dose-related increases in the efflux of 3H-dopamine.
The results of a single experiment, typical of 3 other experiments, are
depicted in Figure 4. A similar dose-dependent increase in the efflux
of 3H-dopamine was also noted when the same increasing concentrations
of gramphetaminewere added to the CSF during 4 consecutive collection
periods (Figure 5). Similar experiments were performed with 1;
amphetamine. The increased efflux of 3H-dopamine induced by the two
isomers of amphetamine is summarized in Figure 6. The minimally

effective concentration of lfamphetamine to cause a significant increase

29

 

 

 

 

 

 

 

 

32

l 3H-0 1

A24b ..
O
O
‘1
>c
E
Q.

:3 15 F 1
Lu
ES
2'.
<2
0..

8 8,. ' I -i

(gt L4
d-A
.03 .3 3 30
OFJJl-SALAHIILELAIAHI'J‘
30. 60 90 120 .
' PERFUSION TIME (min)
Figure 4. Efflux of 3H-dopamine from cat brain in response

to intraventricular infusion of increasing concentrations of
g:amphetamine.

The catecholamine stores of the cat brain were labeled using
an intraventricular injection of 3H-dopamine (5 uc in 10 pl) 15
min prior to the start of perfusion of CSF (0.6 ml/min). After a
30 min period of washout, samples (3 ml/5 min) of perfusate were
analyzed far 3H-dopamine. The height of each bar represents the
amount of H-dopamine in a single 5 min sample of perfusing CSF
effluent. The times that g;amphetamine (d-A, 0.03 to 30 ug/ml)
were added to the perfusing CSF are noted on the abscissa.

30

40

(p
N
T
1

 

N
p
T
1

 

l6e .

3H-DOPAMINE (dpm X 1,000)

d-A
.03 3
.3 3O
.tr-r—T-T—fillg_
150 160 170 180 190 200
PERFUSION TIME (min)

 

 

 

 

 

Figure 5. Efflux of exogenously administered 3H-dopamine
from cat brain in response to intraventricular infusion of
cumulative concentrations of gramphetamine.

The catecholamine stores of the cat brain were labeled with
an intraventricular injection of 3H- -dopamine (5 HC in 10 ul) 15
min prior to the start of perfusion. After a 2 1/2 hr of washout,
samples of perfusate were analy yzed for 3H -dopamine. The height of
each bar represents the mean H- -dopamine content in a 5 min
sample of perfusing CSF and the vertical line depicts 1 S. E. of
that mean as determined from 4 experiments. The addition of d- -am-
phetamine (d- A, 0. 03 to 30 ng/ml) to the perfusing CSF of three
consecutive 5 min period is noted on the abscissa.

31

 

 

 

 

§ F 311.11 1

:2

5.18 - -

'U

E:

z b u

E?

g: d-A

e,- 12 . .

m3:

LL.

0 - ..

X

E}

3: 6 . -

LLI

53

g L. .-

93 ' 1-A

E. 0 AK .l
.03 3 3 30

AMPHETAMINE tug/m1)

Figure 6. The increased efflux of exogenously administered
3H-dopamine from the cat brain in response to intraventricular
infusions of g; and lfamphetamine.

Each point represents the mean net increase in efflux of
3H-dopamine induced by infusions of d: or l;amphetamine (d-A or
l-A) in experiments similiar to those depicted in Figure 5. The
vertical lines represent 1 S.E. as determined in 4 experiments.
Where no line is drawn, the S.E. was less than the radius of the
symbol. Solid symbols indicate a significant increase in the
efflux of 3H-dopamine (p <.05). "Net increased in 3H-dopamine
efflux" is the content of 3H-dopamine during the drug infusion
less the content of 3H-dopamine during the collection period
immediately before the addition of the drug.

32

in the release of 3H—dopamine was approximately 10 times greater than.
that of gramphetamine, 0.3 and 0.03 ug/ml respectively. When equal
concentrations of the amphetamine isomers were infused, the grisomer
released appr0ximately 3 times more 3H-dopamine than did the lyisomer.
Thus, the relative potencies of g: and 17amphetamine on dopaminergic
dynamics obtained in the present studies are in agreement with those
reported previously by Ferris gt_al,(1972), Harris and Baldessarini
(1973 ) and Thornburg and Moore (1973b) jg £1532 and by Von Voigtlander
and Moore (1973a) jp_vjvg, but contrast with the results of Coyle and
Snyder (1969) and Taylor and Snyder (1971). The difficulty in pre-
vious studies (Carr and Moore, 1970; Von Voigtlander and Moore, 1973a) in
demonstrating the release of 3H-dopamine by low concentrations of
amphetamine (smaller than 30 ug/ml) may have been due to the fact that

amphetamine is readily oxidized in the oxygenated CSF.

A-2. The stimulating sites in the lateral hypothalamus effective in
increasing the efflux of exogenously administered 3H-dopamine.

3H-dopamine induced by

The release of exogenously administered
electrical stimulation is dependent upon the frequency of stimulation.
Stimulation with 30 to 50 Hz results in a maximal release of 3H-dopamine
when the electrode is placed at A 10, L 3, H -3.5 (Figure 3; Von
Voigtlander and Moore, 1971b). In order to locate precisely the
effective site for optimal release of 3H-dopamine induced by electrical
stimulation of the nigrostriatal pathway, histological examinations of
the electrode tract were performed. Figure 7 shows the location of

the tip of an electrode in the lateral hypothalamus of the cat brain.

Figure 8 illustrates the results of a series of experiments in which

 

Figure 7. Frontal section of the cat brain at the level of
posterior hypothalamus depicting electrode tract.

Frozen sections (40 u thickness) of formalin fixed cat brain
were stained with cresyl violet acetate. The tip of a stimulating
electrode shown in the lateral hypothalamus is at coordinates of
A 10, L 3. H-3.5. '

34

Figure 8. Release of exogenously administered 3H-dopamine'
in response to stimulation at sites in the lateral hypothalamus.

The catecholamine stores of the cat brain were labeled with
3H-dopamine (5 uc in 10 ul) and the cerebroventricular system
was perfused with CSF at a constant rate of 0.6 ml/min. Samples
of perfusate were collected every 5 min and analyzed for H-do-
pamine. The symbols denote the placement of the tip of the
stimulating electrode as determined from histological sections
such as the one illustrated in Figure 7. Values present the in-
creased amounts of efflux of 3H-dopamine evoked by appling
square wave pulses (30 Hz, 0.5 msec, 400 uA) to the electrode
during a single period of collection after one hour of washout.

F =Fornix '

IC =Internal Capsule

MT =Mammilotha1amic Tract

OT =0ptic Tract

VMHN=Ventromedial Hypothalamic Nucleus

IIIV=Third Ventricle

(min)

%

 

I i V +

 

 

S

"o

8
po-ooqq

III A /

 

 

 

 

36

the lateral hypothalamus was stimulated (30Hz, 0.5 msec, 400 uA)
during a single 5 min collection period after labeling the caudate
nucleus with 3H-dopamine. After an intraventricular injeCtion of
3H-dopamine (5 uc in 10 pl), the amount of 3H-dopamine released by
electrical stimulation at various sites ranged from 2,000 to 20,000
dpm. The stimulation sites which evoked the highest amount of
3H-dopamine efflux (higher than 12,000 dpm) were located in the area
of L 3 and H-3 to -3.5 at the level of A 8 to A 10. Electrical
stimulation at L 3, H-l, L 2.5, H-2.5, and L 4.5, H-2.5 caused a
relatively small increase in the release of 3H-dopamine (2,000 to

3

6,000 dpm). Thus, the greatest increase in H-dopamine release was

evoked by stimulation at the coordinates of A 8 to 10, L 3, H-2.5 to
i -3.5, confirming earlier results of Von Voigtlander and Moore (1971b).
This site for stimulating the nigrostriatal pathway was in agreement

with the results of a subsequent lesion study (see section II).

8. Electrical stimulation and d-amphetamine administration induced

14C-catechols from the brain after

14

release of endogenously synthesized
labelinggthe nigrostriatal terminals with C-tyrosine.
B-l. Effects of lesion or stimulation of the diencephalic nigro-

striatal pathway on the conversion of 14C-tyrosine to 14C-dopamine in

the caudate nucleus.

3

The results of experiments with H-dopamine suggest that this

amine acts as the neurotransmitter in the nigrostriatal pathway, but

3H-dopamine, when

the results must be tempered by the fact that
administered into the lateral ventricle, is probably not taken up by and

subsequently released exclusively from dopaminergic neurons (Kopin,

37

Table 3. Conversion of 14C-tyrosine to dopamine in the caudate
nucleus two weeks after placement of a unilateral lesion in the lateral
hypothalamus.

 

Control Lesioned

 

Total radioactivity 161i55 51:7.4*
("C/9) r-

 

% of(total radioactivity
% )
Alumina effluent
'Alumina eluate
Dopamine
Dowex effluent

._l._ag1

NOOO-A
O—‘NU‘I
O-h
31'!!-

3‘-

\iwmm
1+ 1+ |+ 1+
cod—a
#00001

0'1
a.
AP" .

 

Endo enous content
ug/g)
Tyrosine 21.0
Dopamine 10.1

OWN
tow
1+ H-
-'O
°_.;,,

 

Unilateral lesions were performed at the level of lateral hypo-
thalamus (A 10, L 3, H -2.5 to -3.5) with a D. C. current (3 mA 60
sec) applied to a stainless steel electrode. TWO weeks later, 14C-ty-
rosine (10 uc in 20 pl) was injected intraventricularly into both
lateral ventricles. Cats were sacrificed 90 min later and the caudate
nuclei were dissected and analyzed for 14C-dopamine and endogenous
levels of tyrosine and dopamine. Animals were anesthetized with
pentobarbital sodium during periods of lesion and injection. Values
represent mean i 1 S.E. of 5 experiments. * represents significant
decrease at p < .05; ** represents significant increase at p < .05.

38
1968). ~For example, earlier studies of Carr and Moore (1970) and
Von Voigtlander and Moore (1971c) using these procedures indicated
that electrical stimulation and amphetamine administration also can

3H-norepinephrine. To

cause release of exogenously administered
overcome this problem. attempts were made to label dopamine stores in
the caudate nucleus by administering its radioactive precursor.

Since tyrosine hydroxylase in the caudate nucleus, the enzyme
catalyzes the conversion of tyrosine to dopa, is located exclusively
within the dopaminergic neurons (Goldstein et_al,, 1969; Fyr6 gt 21,,
1972), dopamine synthesized jg_§jtu_from radioactive tyrosine in the
caudate nucleus should be located in these neurons. After intra-
ventricular administration, 14C-tyrosine was taken up by the caudate
nucleus and converted to dopamine in the nigrostriatal neurons (Table
3). TWO weeks after an electrolytic lesion was placed in the lateral
hypothalamus (A 10, L 3, H -2.5 to -3.5), the percentage of total
radioactivity representing dopamine in the caudate nucleus decreased
from 10.3 1 0.8 to 3.8 i 1.0%, but the content of endogenous dopamine
was only reduced from 10.1 i 0.7 to 6.9 i 1.1 ug/g (Table 3). This
difference in the reduction of the content of endogenous and radio-

14C-tyrosine administered

active dopamine may be due to the fact that
into the lateral ventricle was distributed only to the caudate tissue
bording the ventricle. Nevertheless, these results suggest that
dopaminergic nigrostriatal fibers ascend through the areas (A 10, L 3,
H -2.5 to -3.5) in the lateral hypothalamus.

Tyrosine hydroxylase catalyzes the rate limiting step in the

biosynthesis of catecholamines (Nagatsu §t_al,, 1964). Stimulation

39

.Amo. v av

muwm umpepaswumco: we» co can» cmwemcm xppcmuwwpcmwm wee mcwm umumpzswpm on» :o mmapm> a
.eaouioep cue: mpcmswcmaxm a can mnemocxuiue saw: mucmswcmaxm m soc; umcwsgwpmu mm

.m.m _ H cams mgu ucmmmcamc mm=Fm> .uowcma cowpmpkeppm «:9 yo manages om pmmp on» mcwcau
mmwcmucm uwpoceo Lyon one? nmmzmcw mm: Au: opv mgovio F co Au: omv mnemocxwiuep .mmuacws
om cow ounces comm mo omm om cow N: om um Aomms P .<x ooav mwmpza cmyamcmpmc owmmzaocos
;p_z aFFMmee_w:= team—sewum we: magepeguoa»; chmpeF ecu cw amzsuea Pepercpmocmwz on»

 

. . eo._n ~.m_ N~._H w.op Am\m:v mcwmocza mzocmmoucm
om.onom.__ om.oaoe..P m~.onm_.mp -.oaoe..P Am\mav weaseaoe msoeomooem
o.Nnm.m_ m.oan.mp am.onm.m m.oa~.e ARV oo_ x mpeooe\oeWEaaoe-ue_H
mmmnmem.m ANN._nNPo.e amemnmmo.m eomnmem.m Am\2aev oeeeaaoc-uep

oop.mnmmm.m_ pom.mnmom.mp Nun.mnmom.om moo.mnnom.¢m Am\Eavv xuw>wuumowumg pouch

 

cmpcpzswum Focucou cope—asepm Focpcoo
eaooioep mnemocxpiuep

.m=Ee_e;uoa»: chmme asp eo cowuepzswum Feuwcuumpm mcecav mampuzc
mgeuaeu ueo we» cw mewseaou on maouiuep use mcvmocxuiuep mo cowmco>cou .e «Pooh

40

of peripheral sympathetic nerves accelerates norepinephrine synthesis
from tyrosine but not from dopa (Gordon et_al,, 1966; Sedvall and .
Kopin, 1967). Attempts were made initially to increase dopamine

. synthesis in the caudate nucleus by electrical stimulation of the
nigrostriatal pathway in the lateral hypothalamus. Results of such
experiments using intraarterially injected 14C-tyrosine are summarized

14C-Tyrosine (20 pc) was infused into each carotid artery

in Table 4.
for 30 minutes while the nigrostriatal neurons were stimulated on one
side. Electrical stimulation did not influence the amount of total

radioactivity or the endogenous contents of dopamine or tyrosine in

14

the caudate nucleus but increased the amount of C-dopamine 0n the

stimulated side from approximately 6% to 8.5% of the total radio-
activity on the non-stimulated side. In another experiment of similar
design, 10 uc of 14C-dopa was infused into each carotid artery.
Stimulation of the nigrostriatal neurons at the level of the lateral
hypothalamus did not alter the amount of 14C-dopamine formed from

' 14C-dopa; the percentage of total radioactivity represented by dopamine
was 19.7 t 0.8% on the nonstimulated side and 19.2 f 2.0% on the-
stimulated side (Table 4, N=5). The percentage of total radioactivity

14

represented by dopamine after C-dopa is greater than after

14C-tyrosine, 19% and 6% respectively. Since the enzyme dopa
decarboxylase is also located in glial cells,blood vessels and
serotonergic neurons within the caudate (kufelt gt_§l,. 1973). much 0f

14

the 14C-dopamine formed from C-dopa may not have been localized in

dopaminergic neurons.

41

B-2. The concomitant release of endogenously synthesized 14C-catechols

3

and exogenously administered H-dopamine from the brain evoked by

electrical stimulation and by gramphetamine administration after intra-

14

ventricular injection of C-tyrosine and 3H-dopamine.

Despite the fact that dopamine stores in the nigrostriatal

neurons in the caudate nucleus could be labeled by intraarterial in-

14C-precursors, it was not possible to detect significant

14

'fusions of
quantities of C-amines in the cerebroventricular perfusate. This
probably resulted because the dopamine stores were labeled diffusely
throughout the caudate nucleus, whereas dopamine released into the
CSF originated only from tissues adjacent to the lateral ventricle.

To specifically label catecholaminergic neurons in this region a series

14

of experiments was performed in which C—tyrosine (20 uc) and

3H-dopamine (5 uc) were injected into the lateral ventricle 70 minutes
and 15 minutes, respectively, prior to the start of perfusion. The
perfusates contained such low amounts of radioactivity that it was not
possible to accurately separate the alumina eluate (catechol fraction)
into amines and deaminated catechols. It was necessary, therefore,

to report the efflux of radioactivity as "catechols". 'Essentially the
same results as in Figure 3 were obtained when the dopamine stores in

14

the caudate nucleus were labeled by administration of C-tyrosine.

The efflux of 14C-catechols and 3H-dopamine was similar after electrical
stimulation of regions in the lateral hypothalamus or after g;

amphetamine administration (Figure 9). Despite the fact that the

14

caudate nucleus retained 34,565 1 11,724 dpm/g of C-dopamine, the

14

resting efflux of C-catechols was low and variable and it was

42

-30

 

 

 

 

 

N
O

3HeDOPAMINE (dpm x 1,000)

N
T
J

 

'- ---1

 

an---”

 

r-
I
I
I
1
i

emu.

L
.—a
O

 

 

 

2-------q

r-..----

 

---‘4c-CATECHOLS (dpm X 1.000)

I
.----J

 

 

 

r
I
I
I
.l

--- .1

STIMULATION L.._l d—A
—2.5-3.5-4.5 (30)

l I I I

.. .1
160 180 200 220 240 260

 

  

 

 

 

 
 

PERFUSION TIME (min)

Figure 9. Typical efflux pattern of exogenously administered
H-dopamine and endogenously synthesized 4C-catechols evoked by
electrical stimulation and by administration of g;amphetamine.

The catecholamine stores in the brain were labeled with 3H-dopamine
(5 uc in 10 hi) and 4C-tyrosine (20 uc in 30 n1) 15 min and 70 min
respectively prior to the start of perfusion (0.2 ml/min, collected ~
every 10 min). The experiment was then carried out in the manner
described in Figure 3. The double labeled samples were counted in
a Packard Tricarb spectrometer which was programmed for double
labeled counting (Model 3380, Absolute activity analyzer 544).

43

Table 5. Effects of electrical stimulation Of nigrostriatal path-
way and the intraventricular administration of 07amphetamine on the
release of exogenously administered 3H-dopamine and endogenously synthe-
sized 4C-catechols.

 

Perfusion fractions 14C-Catechols 3H-Dopamine

 

 

(10 min) ( % ) ( % )
Before** 100 100
Electrical During AlO,L3,H-2.5 160:19* 234:28*
stimulation H-3.5 200:22* 390:105*
(30 Hz, 0.5 H-4.5 170141 247:64*
msec, 400 HA)
After lst period 173:55 165:54
2nd period 65:19 95:20
Before*** 100 100
g:Amphetamine During 630:94* 731:119*
(30 u9/ml)
After lst period 6061120* 7571157*
20d period 481:228 417i41*

 

Summary Of the double labeled experiments involving 3H-dopamine and
14C-tyrosine as the one illustrated in Figure 9. Numbers represent the
mean i 1 S.E. of catechols and dopamine in the ventricular perfusate
expressed as a percentage of the respective control values.

*Values that are significantly higher than the corresponding control
(p < .05) as determined by Student's t_test, paired comparison.

**The mean of 3 control periods prior to electrical stimulation; 100%
represents 240:60 dpm of 14C-catechols and 2,840:380 dpm Of 3H-dopamine
respectively (N=7).

**fThe mean of the control period before the administration of dfam-
phetam1ne(30 u /m1); 100% represents 94:52 dpm of 14C-catechols and 3,000
$1,050 dpm of H-dopamine respectively (N=5).

44
necessary for a statistical analysis to report the data on a percent-
age basis. The data summarized in Table 5 clearly show a concomitant

3H-dopamine and endogenously

release of exogenously administered
synthesized 14C-catechols into the cerebroventricular perfusates
during electrical stimulation (30Hz, 0.5 msec, 400 uA) of nigrostriatal
pathway (at A 10, L 3, H-2.5, -3.5 and -4.5) and gramphetamine

administration (30 ug/ml).

3H-catecholamines from cat

3

C. The release of endogenouslysynthesized

brain following the intraventricular injection of H-tyrosine or

3H-dopa.

The difficulties in detecting the release of 14C-dopamine syn-
thesized 1g_situ appeared related to the relatively low specific

14C-tyrosine and the percentage Of the activated nigro-

activity of the
striatal neurons. The high endogenous Content of tyrosine in the
caudate nucleus (Table 4, approximately 15 ug/g or 10‘4M) coupled with

14C-tyrosine (less than 400 mc/mmole) so

the low speCific activity of
diluted the radioactive amino acid‘ that it was not possible to
intensely label the dopamine stores in the caudate nucleus. Accord-
ingly, despite problems inherent with the stability and purification of
3H-tyrosine (Besson gt_al;.,‘1969,1971; WaldeCk, 1971), attempts were
made to label dopamine stores in the brain using an intraventricular
injection of high specific activity 3H-tyrosine or 3H-dopa. With this
approach, it was demonstrated that electriCal stimulation of regions
of the lateral hypothalamus previously shown to contain fibers of the
nigrostriatal pathway elicited release 0f dopamine synthesized 1n_§jtg_

from 3H-precursors.

45

C-1. Ig_vjvo_release Of endogenously synthesized 3H-dopamine from
cat brain following electrical stimulation of the lateral hypothalamus
and intraventricular infusion of gramphetamine.

Previous results (see section A-2, B-1 and B-2) indicated
that the lateral hypothalamus at coordinates of A 8 to 10, L 3, H-2.5
to -3.5 contains fibers of the nigrostriatal pathway. Electrical
stimulation of these regions for five minutes several hours after
3H-tyrosine or 3H-dopa had been injected into the ipsilateral lateral
ventricle resulted in an increased efflux of 3H-catecholamines. The
pattern of resting efflux of tritium was essentially the same using

either 3

H-tyrosine or 3H-dopa, but the absolute amounts of radio-
activity released were about 6 to 8 times greater following 3H-dopa
administration than following 3H-tyrosine (Figure 10). Electrical
stimulation (30 Hz, 0.5 msec, 400 uA) significantly increased (P<.05)
the efflux of 3H-dopamine and 3H-norepinephrine following injections
of either 3H-tyrosine or 3H-dopa (Figure 11). The contents of

3

catecholamines and deaminated catechols synthesized from H-tyrosine

in the perfusate samples before and during the electrical stimulation

are shown in Table 6. Electrical stimulation did not influence the

3H-tyrosine in the perfusate, but increased significantly

3

amount of

the amounts of H-dopamine, 3H-norepinephrine and deaminated catechols

by 25, 34 and 12% respectively.

When catecholamine stores in the brain were labeled using an

3

intraventricular injection of H-tyrosine, the efflux of 3H-dopamine

increased progressively as the amphetamine concentration in the

perfusing CSF increased from 0.3 to 30 ug/ml. In contrast, a

3

significant increase (P<.05) in H-norepinephrine efflux was observed

46

Figure 10. The washout curve of 3H-tyrosine and 3H-dopa from
the brain into the cerebroventricular perfusates.

Three hours after the intraventricular injection of 3H-tyrosine
(160 uc) or ninety min aftgr the last injection of five intra-
ventricular injections of H-dopa (20 ac every 30 min, total dose
of 100 uc), the cerebroventricular system was perfused with CSF
(0.6 ml/min). Samples of perfusate were collected every 5 min and
total radioactivity was determined. Each symbol and vertical line
represents mean i l S.E. as determined from 3 experiments with
3H-tyrosine and 5 experiments with 3H-dopa. The coordinate is

expressed in log scale.

(he)

3H-00PA

1,600

800

400 _

200

100

50

 

47

 

 

Ri :2:
_ W
+\+M\i\i\+\+
__ \+\\? (I
i + \¢\+
L L t . . n 1 A 10

PERFUSION TIME (min)

48

Figure 11. Effects of electrical stimulation of the lateral
hypothalamus on the release from th brain Of 3H—d3pamine and
3H-norepinephrine synthesized from H-tyrosine or H-dopa.

In experiments depicted in the panel on the left, 3H-tyrosine
(160 uc, 30.6 c/mmole) was injected into the lateral ventricle 3
hours before the start of perfusion. 0n the right, 3H—dopa (100
uc, 9 c/mmole) was injected into the lateral ventricle in divided
doses; 20 uc every 30 min; 90 min after the last injection
perfusion of the cerebroventricular system was begun. Artificial
CSF was infused at 0.6 ml/min and samples were collected every
5 min. The solid bar on the abscissa indicates that the nigro-
striatal pathway and other fiber tracts in the lateral hypo-
thalamus (A 10, L 3, H—3.5) were stimulated with a monophasic
rectangular pulse (400 pA, 0.5 msec) at 30 Hz for 5 min. The
height of each bar represents the mean radioactivity in a 5 min
sample of perfusate, the vertical line represents 1 S.E. of the
mean as determined from 9 experiments with H-tyrosine and 7
experiments with 3H-dopa. represents 3H-dopamine (D); ----
represents 3H-norepinephrine (NE).

 

49

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

20

10
PERFUSION TIME(min)

m . q . d a a
w .......
J, “at!
a
.11 r-
.
n ............. L
.r 11111
A .
wu. ....... 1--..
D l a-
. 1.1.
H . E
3 J D .r: N
r p p n r LIIIUI-llllllllllb 0
a. 9. no no ,6 .4 9. AU
1| 1| 1|
a
flooo P x smog mmzH2<oozume<u-=m
. . . rm 4
“llllllllllldi lllll
1--
r:
.
o
1"
mm 1 ............ h
I L orll
c. , a
O 1111111111 .
R .
Y “:1.
i 111
. .
H D E 1111.
3 i . N."- v!
r . . 11.1---iiiiuiiiiu 0
no no no no
0. nu nu
aw 1. ac

Asaev mmzH=<oo=ome<u-=m

PERFUSION TIME(min)

50

Table 6. The metabolites of 3H-tyrosine in the periods immediately
before and during electrical stimulation of lateral hypothalamus.

 

 

Five min 3H—Total Tyrosine Deaminated Norepinephrine Dopamine
periods catechols -
(dpm)
Before 64,518 55,938 6,081 227 318
stimulation :9,834 :8,641 :966 :23 :33
During 67,687 55,650 7,306* 305* 399*
stimulation :8,427 :7,581 :788 :16 :28

 

3H-Tyrosine (160 uC, 30.6 c/mmole) was injected into the lateral
ventricle 3 hours before the start of perfusion (see Figure 11 for
detail). Artificial CSF was infused at O. 6 ml/min and samples were
collected every 5 min. The nigrostriatal and other fiber tracts in
the lateral hypothalamus were stimulated at 30 Hz (400 pA, 0. 5 msec)
for 5 min (see Figure 11). Values represent mean :1 S. E. as deter-
mined from 9 experiments.

*represents significant increase at p <.05 .

51
only after infusion of the highest concentration of amphetamine
(30 ug/ml, Figure 12). A similar pattern Of release was seen when
catecholamine stores were labeled using an intraventricular injection

3

of H-dopa (Figure 13). The addition of gfamphetamine (3 ug/ml) to

the perfusing CSF for a single five minute perfusion period caused a

3

significant increase (P<.05) in the efflux of H-catecholamines.

While electrical stimulation released approximately equal amounts Of
3H-norepinephrine and 3H-dopamine (see Figure 11), gramphetamine caused
a relatively greater efflux of the latter amine. The radioactivity in
the caudate nuclei at the termination of the experiments described
above is tabulated in Table 7. Following intraventricular administra-~
tion of 3H-tyrosine, approximately 6% of the total radioactivity in the

3H-dopamine. This is approximately the

14

caudate nucleus consisted of
same percentage that was obtained in similar experiments with C-tyrosine
(Table 4). Following the administration of 3H-dopa a higher percentage
of total radioactivity was represented by 3H-dopamine (12%). The size
of 3H-dopamine stores in the caudate nucleus after 3H-dopa administra-

tion (100 pc) was about 15 times higher than that obtained following

3H-tyrosine administration (160 uc). This may account for the fact

that the absolute amounts of radioactive catecholamines released follow-

3

ing H-dopa administration were approximately 5-10 times greater than

those released following 3H-tyrosine administration.

C-2. Effects of contralateral or ipsilateral stimulation Of the

lateral hypothalamus or the intraventricular administration Of g:

3

amphetamine on the release Of endogenously synthesized H-catecholamines:

Demonstration Of the releasing sites.

52

3 Figure 12. Effects of dramphetamine on the efflux of
H-catecholamines synthesized from 3H-tyrosine.

Three hours after intraventricular injection Of 3H-tyrosine
(160 pc, 30.6 c/mmole) the cerebroventricular system was perfused
with CSF. Seventy-five min after the start Of perfusion pro-
gressively increasing concentrations Of d-amphetamine (d-A, 0.3,
3 and 30 ug/ml) were added to the perfusihg CSF during 3
successive collection periods. The height of each bar represents
the mean radioactivity in a 5 min sample Of perfusate, and the
vertical line represents 1 S.E. of the mean as determined from
3 experimen 5. represents 3H-dopamine (D) and —---
represents H-norepinephrine (NE).

 

53

. d

_

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

i .‘lll‘llll‘ll'NN‘
-
«Illa!
.
.1MH¢ .4
.
71101
.
1 1..

 

 

 

 

1 0
8
q l
. \II
11111 .. 1 0 .1.
n 6 m
.0003:- (
.
....... . L E
u. M
.1111 I
n. 0 T
111111 i. .. 4
.1- 0 N
. 3 0
rlll I I
o. 3 M
II “.1--- O F
i. A ._ 2 R
E I'll. n - E
N .71 d 3 fl. D.
I ,, r'J O
S .
0 u. ..
mm e rs; . no
T. I .r. 1-
u o
H 11L ..
3 II. D m.-
L‘lllll'I'll I 0
W P h n .-
0 0 0 0 0 0
0 0 0 0 0
0 8 6 4 2
’
Asaev mmsz<Sozome<u -z.

m

54

3 Figure 13. Effects of dramphgtamine on the efflux of
H-catecholamines synthesized from H-dopa.

Ninety min after the last injection of 5 intraventricular
injections of 3H-dOpa (20 no every 30 min, total dose of 100 pc,
9 c/mmole) the cerebroventricular system was perfused with CSF
(0.6 ml/min). TWO hours after the start of perfusion, dfamphe-
tamine (3 ug/ml, d-A) was added to the perfusing CSF for one
collection period. The height of each bar represents the mean
radioactivity in a 5 min sample of perfusate, and the vertical
line represents 1 S.E. of the mean as determined from 5
gxperiments. represents 3H-dopamine (D); ---- represents

H-norepinephrine (NE).

 

 

3H—DOPA -

 

 

 

 

 

 

 

 

F

a H u
1 ...... r
-
r!
.
.
VJ
.
r-
n
.
’llt
_
.T
.
_
1 '3'----
.
1|- )
_ 3
.
T... A
u .
ili 11. d
_
fi"
.
1 E.
D 711 N
c

30
PERFUSION TIME(min)

20

10

 

 

-... n I .Il'l

l.

55

 

20

16

2 8
.1.

Aooo.e x Ede: mmszeoozume<u.zm

56

Table 7. Retention of 3H-dopamine in the caudate nucleus following
intraventricular injections of 3H-dopa or 3H-tyrosine. .

 

 

dpm/g % of total radioactivity
3H-OOPA
Total radioactivity 19,077,000:3,095,000 100
Dopamine 2,261,000:610,000 12.34:2.76
3H-TYROSINE
Total radioactivity 2,116,000:585,000 100
Dopamine l4l,000:51,000 5.85:0.85

 

The caudate nuclei were dissected and analyzed for dopamine after
2 1/2 to 3 hours of perfusion; details of injection of radioactive iso—
topes and the perfusion are described in the legends to Figure 11, 12
and 13. Values represent the mean i 1 S.E. as determined from 5 exper-
iments with 3H-dopa and 3 experiments with 3H-tyrosine.

57

In the cerebroventricular perfusion experiments of this study,
the anterior horn of the lateral ventricle and the entire third
ventricle were perfused. Therefore, catecholamines detected in the
perstate_ may be released from the septum and the caudate nucleus into
the lateral ventricle and from the hypothalamus or thalamus into the
third ventricle. When a small volume (10-20 ul) of radioactive dopamine
is injected into the lateral ventricle most of the radioactivity
accumulates in the head of the ipsilateral caudate nucleus and in the
septum, while lesser amounts accumulate in the hypothalamus;
essentially no radioactivity appears in structures lining the contra-
lateral cerebral ventricle (Carr and Moore, 1969). The same was found
to be true in the present study when the radioactive precursors,

3

3H-tyrosine (see Table 11) or H-dopa, were injected into the lateral

ventricle. In experiments using 3H-dopa the relative distribution of
radioactivity in the ipsilateral caudate nucleus, ipsilateral septum,
ipsilateral hypothalamus and contralateral hypothalamus was 100:10:4:

3H-tyrosine), respectively; detectable quantities

4.3 (see Table 11 for
of radioactivity did not diffuse into the contralateral lateral
ventricle. In order to determine the source of the endogenously
synthesized catecholamines, 3H-dopa was injected into a lateral
ventricle and CSF was infused into both lateral ventricles and collected
from a catheter at the aqueduct in the usual manner. Since significant

3H-dopa do not diffuse into the contralateral lateral

quantities of
ventricle, the efflux of 3H-catecholamines in response to contralateral
stimuli (either by electrical stimulation of the nigrostriatal pathway
or by the infusion of gramphetamine) must originate from brain

structures lining the third ventricle. 0n the other hand, ipsilateral

58

3H-catecholamines from structures bordering the

stimuli could release
lateral and the third ventricles. The difference in radioactivity
in response to ipsilateral and contralateral stimuli should represent
3H—catecholamines released from structures lining the ipsilateral
lateral ventricle, the caudate nucleus and septum.

Effects of contralateral or ipsilateral stimulation of nigro-
striatal pathways or intraventricular administration of amphetamine
on the release of 3H-catecholamines synthesized from 3H-dopa are
summarized in Table 8; a typical experiment is illustrated in Figure
14. The sites of electrical stimulation of the typical experiment are
shown in Figure 15. Electrical stimulation of the nigrostriatal path-
way ipsilateral to the injection of 3H-dopa elicited an increase in
the efflux of 3H-dopamine of approximately 7,000 dpm, whereas
stimulation in the contralateral hypothalamus caused an increase of
only 2,000 dpm. The difference in radioactivity in response to '
ipsilateral and contralateral stimulation suggests that at least two-
thirds of the released dopamine originates from structures lining the
lateral ventricle and that less than one-third of the dopamine is
released from structures lining the third ventricle. Infusion of g:
amphetamine into the ipsilateral or contralateral lateral ventricle
caused increases in the efflux of 3H-dopamine of approximately ll,000 dpm,
and 800 dpm respectively. Therefore, it would appear, that over 95%
of the endogenously synthesized dopamine released by amphetamine
originates from structures bordering the lateral ventricle (probably
the caudate nucleus).

Ipsilateral electrical stimulation in the lateral hypothalamus

or infusion of dramphetamine elicited a relatively small but

59

Table 8. The increased efflux of endogenously synthesized 3H-cate-
cholamines in response to ipsilateral or contralateral stimulation of
the lateral hypothalamus or the intraventricular infusion of gramphetamine.

 

 

 

Potential ncreased amg unt of amines
sites of release H- -Dopamine SH- -Norepinephrine
'(cerebroventricle) (dpm/5 min) .

Hypothalamic stimulation - g7=
ipsi- L+III 6,879:2,754* l,l36il85* T
contra- III 2,233:l,287* ~651198

d-amphetamine infusion ;
ipsi- L+III ll,273:2,l09* 844:l98* '
contra- III 786:596 -166:3l9

 

Values represent the mean +l S. E. (N= 3) of "change" in efflux of
3W catecholamines in response to stimuli applied to the same side (ipsi)
or to the opposite side (contra) of the 3H- -dopa injections. "Change"
means the 3H- amines in the 5 min perfusate sample during electrical
stimulation or d-amphetamine infusion less the 3H- -amines in the 5 min
perfusate sample immediately prior to application of the stimuli. See
legend to Figure l4 for additional details. L= lateral ventricle;

‘ III=third ventricle.
*represents significant increase at p <.05 .

. F

 

60

Figure l4. Effects of contralateral or ipsilateral stimu-
lation of the lateral hypothalamus or the intraventricular infu-
sion of d-amphetamine on the release of endogenously synthesized
3H-catecholamines.

Five injections of 3H-d0pa (zone in 20 ul) were made into a
lateral ventricle at 30 min intervals. Ninety min after the last
injection both lateral ventricle were perfused with CSF at a rate
of 0.3 ml/min and perfusate samples were collected every 5 min.
Solid bars on the abscissa represent electrical stimulation of
the nigrostriatal pathway and other fiber tracts in the lateral
hypothalamus (A l0, L 3, H-3.5) contralateral or ipsilateral to
the injection of 3H-dopa. Open bars on the abscissa represent
infusions of d-amphetamine (3 ug/ml) contralateral or ipsi ateral
to the injection of 3H-dopa. represents 3H-dopamine ( H-D);
---- represents 3H-norepinephrine (3H-NE).

 

6i

 

: J
1 .
5.-.!1.) . . .J' r . l .

 

q
.1
u
u
an
-
an

3H-DOPA

 

 

H-NE
F_—

-‘Illl'
ll‘l‘“Ill"l|"l‘lll‘l‘
'l‘l“"‘lI

l4
2
10 _
8

6 4 2

100

75

 

 

 

    

 

25

PERFUSION TIME (min)

62

 

Figure 15. Frontal section of cat brain at the level of
posterior hypothalamus depicting stimulating electrode tracts.

At the termination of the experiment of the one illustrated
in Figure l4, animal was sacrificed with an overdose of pento-
barbital sodium and the cauda e nuclei were dissected for the
determination of H-dopa and H-dopamine. The rest of the brain
was fixed in formaline and frozen sections (40 u thickness) were
stained with cresyl violet acetate. The tip of the electrodes
are at the coordinates of A 8, L 3, H -3.

 

63

significant-(P<.05) increase in the efflux of norepinephrine; there

was no increase in the efflux of this amine evoked by contralateral
electrical stimulation or contralateral administration of gramphetamine.
These results suggest that most of the norepinephrine synthesized from
3H-dopa in the cerebroventricular perfusates originates from brain

structures lining the lateral ventricle (probably the septum).

C-3. Effects of the combination of low frequency stimulation and d:
amphetamine administration on the efflux of endogenously synthesized
3H-catecholamines.

Comparison of the abilities of low frequency stimulation of the
specific regions in the lateral hypothalamus and gramphetamine to

3Wdopa is

increase the efflux of 3H-catecholamines synthesized from
shown in Figure 16. Low frequency stimulation of the lateral hypo-
thalamus (3 Hz, 0.5 msec, 400 uA; A 10, L 3, H-3.5) induced a lesser

3Wdopamine and 3H-norepinephrine than that by high fre-

efflux of
quency stimulation (30Hz); the net increased efflux of 3H-dopamine was
reduced from 5,185 1 2,621 dpm/5 min (N=7) to 790 i 135 dpm/5 min (N=3),
whereas 3Wnorepinephrine release was decreased from 3,429 f 954

dpm/5 min (N=7) to 1,383 f 310 dpm/5 min (N=3). The g:amphetamine—
induced efflux of 3H-dopamine and 3H-norepinephrine synthesized from
3H-dopa (N=4) was 13,366 i 1,964 dpm/5 min and 1,291": 396 dpm/5min,
respectively. The combination of dyamphetamine and low frequency

3H-dopamine

stimulation caused an additional increase in efflux of
(14,947 1 2,099 dpm/5min, N=4) and 3H-norepinephrine (5.368 1 1,083

dpm/5min, N=4). When reported as a percentage of control (Figure 16),

 

64

Figure 16. Evoked release of endogenously synthesized
3H-dopamine and 3H-norepinephrine from the cat brain by low
frequency electrical stimulation and by administration of
dramphetamine.

3H-Dopa (100 uc) was injected into the lateral ventricle as
described in Figure 14. Ninety min after the last injection, the
cerebroventricular system was perfused with CSF at a rate of 0.6
ml/min and perfusate samples were collected every 5 min.
ngmphetamine infusion (d-A, 3 pg/ml) or electrical stimulation
of lateral hypothalamus (STIM, 3 Hz, 0.5 msec, 400 uA) at A 10,
L 3, H-3.5 were applied separately or in combination in random
order. The height of each bar and vertical line represents mean
i l S.E. as determined from 3 to 4 experiments. The control
values for 3H-dopamine in periods immediately before stimulation,
gramphetamine and stimulation plus gramphetamine were 3,647 i
1,003 (N=3), 3,343i908 (N=4) and 3,790:921 (N=4) dpm/5 min
respectively. The corresponding control values for 3H-norepine-
phrine were 1,77lt375 (N=3, STIM), 2,153i457 (N=4,d-A) and
1,879i4l3 dpm/5 min (N=4, d-A + STIM) respectively.

% OF CONTROL

 

 

 

65

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1,000 3
F 311- DOPAMINE H-NOREPINEPHRINE

800 _.
1
400 _
200 — 'T -r

0

dA - + .. 1.

STIM + - + —

 

 

 

66

the results clearly demonstrate that d;amphetamine selectively
released endogenously synthesized dopamine rather than norepinephrine;
in contrast low frequency stimulation induced approximately an 80%

increase in norepinephrine efflux and a 30% increase in dopamine efflux.

II. Electrolytic or 6-hydroxydopamine-induced lesion of the ascending

 

 

 

monoaminergic fibers in the lateral hypothalamus. if?“
A. Biochemical and histological evaluation of the lesions of the 3
lateral hypothalamus. :
Two weeks after an electrolytic lesion (3mA,60$ec) or the F
intracerebral injection of 6-hydroxydopamine in the region of the E_:J

posterior lateral hypothalamus (A 8 to 10, L 3, H-3.5), the dopamine
concentration of the caudate nucleus and the norepinephrine concentra-
tion of the septum were lowered on the lesioned side (Table 9). When
an electrolytic lesion was made in the anterior hypothalamus (A 12-14,
L 3, H-3.5), the norepinephrine content of the ipsilateral septum was
lowered, although the dopamine content of caudate nucleus was un-
altered(Table 9). Figure 17 is a photograph of a frontal section at
the level of the lateral hypothalamus of a 6-hydroxydopamine-injected
catbrain in which the lesion was apparent upon gross examination.
Microscopic examination of the lesioned area revealed gliosis surr0und-
ing the liquefaction cavity, thus suggesting that 6-hydroxydopamine
destroyed the cells in the area by a nonspecific cytotoxic action.
Table 10 shows the monoamine contents in the ipsilateral caudate nucleus
and septum of two experiments two weeks after an unilateral injection

of 6-hydroxydopamine into the posterior lateral hypothalamus. The

 

 

67

Table 9. The effects of lesions in the lateral hypothalamus on
the contents of dopamine and norepinephrine in the ipsilateral caudate
nucleus and septum of cats.

 

 

CONTROL LESION 0F HYPOTHALAMUS
Anterior Posterior
(A 12 to 14) (A 8 to 10)

 

Electrolytic Electrolytic 6-Hydroxy-

 

dopamine
(pg/g)
Dopamine of 11.4:0.4 10.9:0.5 6.9:l.l* 5.1:l.9*
caudate nucleus (10) (5) (6) (6)
Norepinephrine 1.41:0.10 0.76:0.15* 0.6li0.15* 0.46i0.12*
of septum (7) (6) (3) (7)

 

TWo weeks after a unilateral lesion of the anterior or posterior
hypothalamus in cats, the caudate nuclei and septa were dissected
and analyzed for endogenous catecholamines. Electrolytic hypothalamic
~ lesions of cats were made by applying a 0.0. current (3 mA, 60 sec)
to a stainless steel electrode placed stereotaxically in the anterior
hypothalamus (A12 to 14, L 3, H -2.5 to -3.5) or posterior hypo-
thalamus (A 8 to 10, L 3, H -2.5 to -3.5). 6-Hydroxydopamine (6 09/01)
was dissolved in a solution containing ascorbic acid and injected
(4 ul) stereotaxically into the brain regions at A 10, L 3,_H -3.5.
Lesions were made under pentobarbital anesthesia. Values represent
mean i l S.E. and the experimental number is denoted in parentheses.

*significantly decreased as compared to the control group (p <.05).

 

 

68

 

Figure 17. Histological section of the cat brain:
Demonstration of the effective lesion sites in the lateral
hypothalamus.

6- Hydroxydopamine (24 09 in 4 pl of 0. 5 mg/ml ascorbic acid)
was injected into the lateral hypothalamus (A 10, L 3, H -3. 5)
under pentobarbital anesthesia. Two weeks after the unilateral
intracerebral injection of 6-hydroxydopamine, the cat was
sacrificed and the caudate nuclei were dissected for analysis
of dopamine. The rest of the brain was fixed in formaline and
analyzed for the site of lesion (40 u thickness, frozen section,
cresyl violet acetate stain). Note that the tract of the needle
lies in the corpus callosum and the lesion is characterized by
cytotoxic liquefaction and gliosis.

 

 

 

69

Table 10. The effects of unilateral 6-hydroxydopamine injection
into the lateral hypothalamus on the monoamine contents in ipsilateral

caudate nucleus and septum.

 

 

6-Hydroxydopamine lesion Caudate nucleus Septum
Dopamine Serotonin Norepinephrine
(HQ/9)
, s:

012573 Lesioned side . 2.21 0.0 0.50 1

Control side 10.49 0.9 1.91 §
021273 Lesioned side 1.66 0.3 0.78 7

Control side 8.82 1.2 1.61

 

 

1.

TWo weeks after the intracerebral injection of 6-hydroxydopamine 1

(24 ug) into the lateral hypothalamus (A 10, L 3, H-3.5), cats were

sacrificed with an overdose of pentobarbital sodium. Caudate nuclei

and septa were analyzed for monoamine content. A control lesion in

cat 021273 was made by injecting 6-hydroxydopamine into the thalamus.

The opening 0f the injecting needle faced posterially so that the

lesion extended from A 10 to A 8. The diagrams of the lesioned site

in the cross section of the cat brain are also shown in Figure 18.

70

Figure 18. Diagram of the 6—hydroxydopamine lesion of
ascending monoaminergic neurons in the lateral hypothalamus of
cat brain.

A. At the level of A 10.

B. At the level of A 8.

The sites of the 6-hydroxydopamine-induced lesions in the
lateral hypothalamus of two cats (same forebrain as were analyzed
biochemically, see Table 10) were examined histologically. The '
diagrams were copied directly from photographs of the brain slices
(see also Figure 17).

71

Figure 18 A. at the level of A 10.
012573

 

    

Control Lesioned

 

 

 

 

021273

 

   
  

Control Lesioned

, (Sham lesion)

A 10

 

 

 

 

72

Figure 18 B. at the level_of A 8.

   

 

73

location of the lesions is illustrated in Figure 18(A;B). A 6~
hydroxydopamine-induced lesion in the posterior hypothalamus (A 8 to

10, L 3, H -3.5) decreased the monoamine concentrations in the

ipsilateral caudate nucleus and septum. The dopamine concentration of the
caudate nucleus was decreased to 20% of control and the norepinephrine
concentration of the septum was reduced to about 25 to 50% of control.

The serotonin concentration in the caudate nucleus completely dis- 1
appeared. Thus, the hypothalamic coordinates (A 10, L 3, H-2.5 to

-3.5) are not as selective for the nigrostriatal pathway as has been

 

reported previously (Von Voigtlander and Moore, 1971b; 1973a).

 

B. Effects of apomorphine, L-dopa and d-amphetamine on the locomotor
asymmetry of cats with a chronic unilateral lesion of ascending mono-
aminergic fibers.

Two to four months after an electrolytic lesion of the lateral
hypothalamus at A 10, L 3, H-3.5,a series of experiments was performed
to investigate the physiological functions of the ascending mono-
aminergic pathways in the cat. Although a lesion at A 10, L 3, H-3.5
failed to show a selective reduction of dopamine in the caudate nucleus,
it was hoped that some effects of the lesion could be obtained by
using pharmacological agents which exert actions through a dopaminergic
mechanism. Figure 19 illustrates the dose-response curve of apomorphine
on the motor behavior of cats with unilateral hypothalamic lesions.

In control cats, apomorphine had little effect on the motor activity
but produced stereotyped behaviors, for example: licking, biting,
. sniffing and exploratory behaviors. Nausea and vomiting were also

noted after apomorphine administration. Administration of apomorphine

 

[F—Hau- m "gnu

 

 

74

Figure 19. The effect of haloperidol on the apomorphine-
induced turning behavior in cats with a chronic unilateral lesion
of ascending monoaminergic fibers in the lateral hypothalamus.

Two to three months after placement of unilateral electrolytic
lesions in the lateral hypothalamus (A 10, L3, H -2.5 and -3.5;
3 mA 0.0., 60 sec at each site), cats (N=5) were injected subcu-
taneously with doses of apomorphrine (0.1 to 3.2 mg/kg) given at
30 min intervals. Haloperidol (.05 mg/kg, i.p.) was injected 60
min prior to administration of apomorphine. No contralateral
turning was observed during the experiment. Each symbol represents
the mean of total count of 360° circling toward the lesioned side
during a 30 min interval; vertical bar represents 1 S.E. of the
mean as determined from 5 experiments.

 

IPSILATERAL TURNING BEHAVIOR (counts/30min)

 

75

 

 

 

100 r z
0 CONTROL
80 . ' HALOPERIDOL
‘- (.OSmg/ kg) _
60 L, .s
40 __ z
20 _ .4
O L H M .1
L 1 . l 1 J l J I 1
TIME -60 -30 120 150 180(min)

1.1 1.21.41.11.03.

APOMORPHINE (mg/kg 5 .c.)

5 L 7. Cut.“ .17
,0

’ K} 'I.

 

 

 

76

caused a dose-related increase in the number of 3600 turns made by
the cat toward the lesioned side; no contralateral turns were noted.
Haloperidol, an antagonist acting at dopaminergic receptors (Andén
£3 31., 1967), shifted the dose-response curve for apomorphine to the
right, thereby increasing the minimal effective dose of apomorphine
from 0.1 to 1.6 mg/kg. These results suggest that a dopaminergic
mechanism of the brain was impaired by the lesion of the lateral
hypothalamus. Turning toward the lesioned side suggests that dopaminer-
gic receptors on the side of lesion do not develop supersensitivity; on
the contrary, these receptors may undergo transsynaptic degeneration
three to four months after the lesion. The dose-response curve for
the effect of L-dopa upon the circling of cats with unilateral
lesions of the lateral hypothalamus is illustrated in Figure 20.
Sixty minutes after administration of a-hydrazinomethyldopa (HMD;MK486,
50mg/kg i.p.), a peripheral dopa decarboxylase inhibitory
agent, the cats showed no change in motor activibm but L-dopa (25 to
100 mg/kg) significantly increased ipsilateral circling.

ngmphetamine (2 mg/kg, i.p.) also significantly increased the
ipsilateral circling (Figure 21). The time of peak effect was at
about 2 hours after the administration of this drug; the responses
lasted for 3 hours. The response of the chronic lesioned cats to d:
amphetamine was more variable than to L-dopa or apomorphine.
Occasionally contralateral circling was also noted 2 hours after the
injection of dfamphetamine. Nevertheless, pretreatment with a-
methyltyrosine (100 mg/kg, i.p.) two hours before the administration of

gramphetamine reduced gramphetamine-induced ipsilateral circling to

77

Figure 20. The dose response curve of L-dopa on turning
behavior in cats with chronic unilateral lesions of the ascending
monoaminergic fibers in the lateral hypothalamus.

Three to four months after the unilateral electrolytic lesion
of brain regions in the lateral hypothalamus, cats were pretreated
with a-hydrazinomethyldopa (HMD, 50 mg/kg, i.p.). Sixty min after
the injection of HMD, L-dopa methylester (12,5 mg/kg, N=3; 25
mg/kg, N=4; 100 mg/kg, N=5) was injected intraperitoneally and
the cats were tested for turning behavior. Each symbol and vertical
line represents mean: 1 S.E. Where no line is drawn, the S.E.
was less than the radius of the symbol. + represents ipsilateral
circling toward the lesioned side; - indicates contralateral
turning.

TURNING BEHAVIOR (COUNTS / 30 min)

80

6O

40

20

+

.‘

L {}HMD
l l
-90 -60

 

78

 

100mg/kg

 

 

 

 

25mg/kg

 

 

° 12.5mg/k9 I

A.———‘ _——_‘l——_— ‘fiQr—-19r”’*§

’ L-DOPA _.
l l

0

 

l l 1 .LJ
30 60 90 120 150

TIME (min)

L
-30

79

Figure 21. The effect of dfamphetamine on turning behavior
of cats with a chronic unilateral lesion of the ascending mono-
aminergic neurons in the lateral hypothalamus.

Seventy to eighty days after the unilateral lesion of the
ascending monoaminergic neurons with the electrolytic methods as
described above (see Figure 19 and Table 9), gfamphetamine sulfate
(d-A, 2 mg/kg, i.p.) was administered to four cats with chronic
lesions of the lateral hypothalamus. Each symbol represents the
mean of turning of 4 experiments.

8O

 

120

_ . W

0 0
8 4

AC_EOM\WBC=OUV eoH><Imm wszmae seeme<42mafi

 

120 150 180 210

TIME (min)

90
fl d-A (2mg/kg i.p.)

60

30

-30

81

Figure 22. The effect of a-methyltyrosine on the gfamphe-
tamine-induced ipsilateral turning behavior in cats with chronic
unilateral lesions of the ascending monoaminergic neurons.

The cat most sensitive to gfamphetamine (Figure 21) was
treated with a-methyltyrosine methylester (100 mg/kg, i.p.).
No contralateral circling was observed in this typical experi-
ment. Two hours after the administration of a-methyltyrosine,
d7amphetamine sulfate (d-A, 2 mg/kg) was injected and tested
for the turning score.

IPSILATERAL TURNING BEHAVIOR (counts/30min)

280

240

200

160

120

80

4O

 

 

82

-*- CONTROL
.muuquT(100mg/kg i.p.2hr)

 

 

TIME (min)

9 d-A (2mg/ kg 1UP)

”O.
E)
l l I l _J
I ~30 0 30 60 90 120 150 180

 

83

one-half of control (Figure 22). The ability of gfamphetamine to
increase ipsilateral circling of the chronic lesioned cats may relate
to the ability of this drug to release brain dopamine, presumably

newly synthesized striatal dopamine.

III. Studies employing the continuous infusion of 3Wtyrosine.

Behavioral studies suggest that the newly synthesized dopamine
of the striatum plays a more important role than brain norepinephrine
in the gramphetamine-induced locomotor stimulation (Costa gt_al,,
1972; Thornburg and Moore, 1973b; see also Figure 22). Nevertheless,
there is as yet no direct biochemical evidence for this hypothesis.

In order to monitor the efflux of newly synthesized dopamine
from the brain jn_vjvg, carefully purified 3H-tyrosine was continously
infused into the cerebroventricular system at a constant rate. A
combination of weak cation exchange and alumina adsorption chromato-
graphy was utilized to separate a few thousand dpm of radioactive

catecholamines from 30 to 80 million dpm of 3

H-tyrosine in the perfusate
samples. Using a similar procedure, Besson gt_al, (1973) found that
dopamine recently synthesized from 3H-tyrosine was preferentially
released spontaneously into the perfusate collected from a cup placed
upon the exposed cat caudate nucleus. Therefore, the ability of
gramphetamine to release striatal dopamine from a "stored" or a "newly

synthesized" pool was examined using the continuous infusion method

with carefully purified 3H-tyrosine of high specific activity.

84

A. The distribution of 3Wdopamine and 3H—norepinephrine in brain

tissues and in cerebroventricular perfusates following a continuous

3H-tyrosine.

cerebroventricular perfusion with
After perfusing the lateral and third cerebral ventricles with
3H-tyrosine (20 uc/ml) at a constant rate of 0.2 m1/min for four

3 3

hours, the distribution of H—dopamine and H-norepinephrine syn-

thesized from 3

H-tyrosine in tissues lining the cerebroventricular
system was determined (Figure 23A and B, Table 11). The caudate
nucleus contained about 93.7% of the total brain 3Wcatecholamines
which consisted only of 3H-dopamine. Of the 3Wcatecholamines in the
septum and hypothalamus, 84% and 60% respectively were found in the

3

dopamine fraction. Chromatographic separation of H-catecholamine

fractions from the a—methyltyrosine-treated brain revealed that a-
methyltyrosine completely blocked the conversion of 3H-tyrosine to
3H-dopamine (Figure 23A).

In the initial studies, chromatographic separation of the
3H-catecholamine fractions (see Figure 2B) of the perfusate before and
during the administration of amphetamine revealed that 96% of the

3

radioactive amines consisted of H—dopamine and only 4% consisted of

3Wnorepinephrine (Figure 24). This procedure was utilized throughout
the course of the study and clearly demonstrated that the 3H-catechol-
amine fraction collected during the administration of amphetamine con-
sisted of 96.4 f 1.9% 3H-dopamine and only 3.6 f 1.9% 3H-norepinephrine
(N=4). Thus, in the following results the radioactivity in the

catecholamine fractions, after the combination of ion-exchange and

alumina adsorption chromatographic separations (see Figure 2B), was

85

Figure 23. Separation of 3H-catecholamines in the brain
tissues lining the lateral and third ventricles after the per-
fusion of 3H-tyrosine.

A. Caudate nucleus.

B. Septum and Hypothalamus.

After perfusing the cerebroventricular system of the cat
with 3H-tyrosine (20 uc/ml, 0.2 ml/min) for four hours, the
tissues bordering the ventricular system were dissected and
analyzed for 3H-catecholamines (see Figure 2B). Aliquots of
the acid eluate in the catecholamine fraction were further
separated into dopamine and norepinephrine fractions by using
a strong cation-exchange chromagographic column (Dowex AG
SOWX4, Na+, ZOO-400 mesh, 26 mm x 50 mm). The eluates of
this column were collected in 1 m1 fractions and analyzed for
radioactivity (o——-o; A, Caudate nucleus; B, Septum and Hypo-
thalamus) and for authentic catecholamines (o---o; B, Authentic
NE and D). The 3H-dopamine peak in the caudate nucleus com-
pletely disappeared when a-methyltyrosine (4xlO‘4M) was infused
into the brain one hour prior to and during the perfusion of
3Wtyrosine (o-——o; A, Caudate nucleus).

3H-CATECHOLAMINES '(dpm x 100)

86

Figure 23A

CAUDATE NUCLEUS

o 311- TYROSINE
150 _. o 3W TYROSINE+6MT .2

120 ‘ '4

 

 

90— —
O
O
60L. ..
O
O
301. _
C
O
O
O
L -'. .
o—o—o—o—o— ‘ We
0 O.

l llllllJlllJ
'1NHc1 2 4 6 8 lOZNHCl 2 4 6 8 10 12(m1)

_ ——

 

ELUATE

87

 

 

 

 

Figure 238
F - 100
AUTHENTIC D
F‘ NE ’0‘!) -
NE .0 d -,
b D o". "'0. I! '1. -160
-. i \.
“ . . 0 <5, .2
.' ‘-_ .I \,
__ 43 C1, / ‘3. _420
o": "0,. g ‘0" 0-~ 0‘0-
. .. . .. .. .. ...... .0
__ C><><3 ()mC»<>43~CL<ylg' C><3 C>C>13 C>() f_ O
r—' ..18
'- —1
SEPTUM
“ -12
6
1‘ 1—
1— J
L. .- O
1
18
F' AM '1
_ HYPOTHAL us 0 1
T “‘12
r- .3
_ _1 6
L waoo’C/D J 0

 

 

llllllllJLlllJlllJlLll
lNHCl 2 4 6 8 10 2NHC12 4 6 8 10 12(m1)

ELUATE

FLUORESCENCE UNIT

3H-CATECHOLAMINE (dpm x 100)

88 '

Table 11. Relative distribution of 3H-dopamine and 3H-norepine-
phrine in tissues bordering the lateral and third cerebral ventricles
following perfusion of 3H-tyrosine.

 

 

% of total Relative % of 3H-amines
3 brain N in the tissue N
H-catecholamines 3H-D 3H-NE
7T7) (%)

Caudate nucleus 93.72:1.12 4 100:0 O 6
Septum 2.34:0.47 4 83.67 22.32 l6.33:2.32 4
Right hypothalamus 1.96:0.51 4
Left hypothalamus 1.97e0.60 4 3 59°8 40-2 1

 

After perfusing the cerebroventricles for 4 hours with CSF contain-
ing 3H-tyrosine (20 uc/ml, 0.2 ml/min), tissues lining the lateral and
third ventricles were analyzed for 3H-dopamine and H-norepinephrine.
The total brain 3H-catecholamines content was 103,736i30,244 dpm. Values
represent mean t l S.E. N represents experimental number. The relative
percentage of 3H-dopamine and 3H-norepinephrine in the tissue was
calculated from the amount of dpm of radioactivity under the area of
dopamine and norepinephrine chromatogram as depicted in Figure 23.

89

 

 

 

 

3 6 081373! ‘90
2
X
Lu
5 u
29, Z
w
m U
UJ ‘_ -1 (I)
Z 60 (a:
z , 9
< ' .1
6 I].
3: .9
U , Lu
1‘: 2 L ,. - so E
4t -' ,
L.) . o
o
I _ '5
m . ”0‘ Q
~ 9
I ° it 6
0- -O--o--o- ‘O-oo 000 JO

1-
p
L
b

11:11.61; i‘é‘iNécd‘i‘é O 10 12
ELUATE (ml)

 

Figure 24. Separation of 3H-catecholamines in the cerebro-
ventricular perfusate collected during the administration of
gramphetamine.

Aliquots of the 3H-catecholamine fraction of perfusates obtained
during the administration of dfamphetamine sulfate were pooled and
separated into dopamine and norepinephrine by ion-exchange chromato-
graphy (o-—-o, see METHODS for detail). Authentic cold norepinephrine
(o ----- o) and dopamine (o---o) were identified by fluorometric analysis

(Chang, 1964).

90
reported as 3H-dopamine.

B. Effects of the intravenous or intraventricular administration of
d-amphetamine on the efflux of 3H-dopamine during the continuous in-

3H-tyrosine.

fusion of

Following two and one-half hours of perfusion with 3Wtyrosine,
the intravenous injection of gramphetamine sulfate (2 mg/kg)
significantly increased (p<.05) the efflux of endogenously synthesized
3H-dopamine (Figure 25). The dramphetamine-induced efflux of dopamine
synthesized from 3H-tyrosine was sustained for more than one hour.

The presence of d-methyltyrosine (4 x 10'4M) in the perfusion fluid
from the start of perfusion completely blocked the response of
amphetamine. Intravenous injection of gyamphetamine also produced a
sustained increase in the mean blood pressure and pulse pressure;
thus, in the subsequent studies, dfamphetamine was added directly to
the perfusing CSF which contained 3Wtyrosine (12.5 uc/ml).

The addition of 10 min pulses of increasing concentrations of
.dfamphetamine sulfate (1.1 x 10'7 to 1.1 x 10'3M) to the perfusing CSF
at 40 min intervals caused a dose-related increase in the efflux of
3H-dopamine synthesized from 3H-tyrosine,whereas no increase was noted

in the total radioactivity. The results of a single experiment which

is typical of 3 experiments is depicted in Figure 26. The g;

3H-dopamine in periods

amphetamine-induced increase in the efflux of
during and immediately following the administration of this drug is
summarized in Table 12. The minimally effective concentration of g;
amphetamine to cause a significant increase in the release of newly

synthesized 3H-dopamine from the dopaminergic nerve endings in the

91

_3 u-rvnosme
m—3w-rvnos I u: + on"

 

 

 

 

 

 

 

 

61' 1
1
6‘ _ .
9.
X
S
2 4’ ‘
LU
Z . .
z
<
0..
O
i’
I
6')
..._. i _ - "II-"'1 M, ,
J
4A!“
l 1 _ L 1 l l l #1
140 I60 I80 200 220
PERFUSION TIME (min)
Figure 25. Efflux of endogenously synthesized 3H-dopamine

in response to the intravenous injection of dfamphetamine.

The cerebroventricular system of the cat brain was continu-
ously infused with 3H-tyrosine containing CSF (20 uc/ml) for
4 hours at a rate of 0.2 ml/min. After 160 min of perfusion of
3H-tyrosine, gfamphetamine sulfate (2 mg/kg) was dissolved in
3 m1 of saline and injected slowly (l m1/min) into the femoral
vein as indicated by the solid bar on the abscissa. Each bar
represents the mean t l S.E. of the results obtained from 4
experiments ( ). ---- represents the result of one experi-
ment in which a-methyltyrosine (4x10'4M) was perfused into the‘
cerebroventricular system 1 hour prior to and during the
perfusion of 3H-tyrosine.

 

92

Figure 26. Efflux of endogenously synthesized 3H-dopamine
from the caudate nucleus in response to intraventricular infusions
of increasing concentrations of g;amphetamine.

CSF containing 3H-tyrosine (12.5 uc/ml) was continuously
infused into the lateral ventricle at a constant rage of 0.2 ml/min
for 4 hours. The height of each bar represents the H-dopamine
content in a 10 min perfusate sample. The times that g7amphetamine
(0.03 to 300 ug/ml) was added to the perfusing solution are noted
on the abscissa by the open bar.

3H-DOPAMINE (dpm x 1,000)

93

14
3H-TYROSINE 1

._.II

12 _ (_1

 

10 _ _
‘7

 

 

L

L A A A A A #4 A A A A A 4 1 J 1 A L J A A L J

TIME 20 4O 6O 80 100 120 140 160 180 200 220 240(m1n)

 

 

 

 

 

 

 

O

d-AIIJ C2] CI] [:3 [:3
.03 .3 3 30 300

94

Table 12. The increased efflux of endogenously synthesized
H-dopamine from the cat brain in response to intraventricular
infusions of increasing concentrations of g:amphetamine.

 

 

 

d-Amphetamine d-Amphetamine Increased content of 3H-dopamine
(base, L1g/ml) (sulfate, M) (dpm/10 min) (dpm/20 min)
0.03 1.1x10-7 141334 173347
0.3 1.1x1O-g 1,7862629 2,5173943
3.0 1.1x10'4 3,057i612 4,168:1,l35
30.0 1.1x10‘ 5,635i298 9,9522259
300.0 1.1x10-3 9,115:1,445 l4,796:3,022

 

Values are a summary of 3 experiments similar to the one illus-
trated in Figure 26. The increased efflux of H-dopamine following the
intraventricular perfusion of 3H-tyrosine represents the mean net
increase of 3H—dopamine in the period (dpm/10 min) or periods during
and immediately afteg (dpm/20 min) the additions of d;amphetamine
less the amounts of H-dopamine during the period immediately before
the additiOn of each drug solution. Values represent the mean i 1 S.E.
as determined from 3 experiments and are significantly increased

p <.05 .

95

caudate nucleus was approximately lO-7M (p<.05).

C. Effects of a-methyltyrosine on the d-amphetamine-induced efflux of

endogenously synthesized or exogenously administered 3H-dopamine.

C-l. Effects of pretreatment with a-methyltyrosine on the biosynthesis
of 3H-catecholamines and on the gramphetamine-induced efflux of
3Wdopamine following the continuous infusion of 3H-tyrosine.

3H-dopamine synthesized

The gramphetamine-induced efflux of
during the continuous infusion of 3H-tyrosine was completely blocked
by the addition of a-methyltyrosine to the perfusing CSF (Figure 27).
The distribution of 3H-tyrosine and 3H-catecholamines in the tissues
lining the lateral and third ventricles after four hours of perfusion
with CSF or with CSF containing 4 x 10'4M a-methyltyrosine is summarized
in Table 13. a-Methyltyrosine did not significantly alter (p>.05) the
amount of total radioactivity (primarily 3Wtyrosine) but significantly
reduced (p<.05) the content of 3H-catecholamines in all tissues. The
inability of gramphetamine to increase the efflux of endogenously

synthesized 3H-dopamine is undoubtedly due to the fact that the

tissues contained essentially no 3H-catecholamines available for release.

C-2. Effects of a-methyltyrosine on the release of endogenously
synthesized 3H-dopamine from the caudate nucleus during the continuous
infusion of g;amphetamine.

After the cerebroventricles were perfused with a solution con—
taining 3H-tyrosine (12.5 uc/ml, 0.2 ml/min) for 2 1/2 hours, addition
of gramphetamine sulfate (1.1 x lO'SM) to the perfusing solution during

 

 

——- 3H-TYROSINE
--------- 3H—TYROSINE+¢MT
25 _ J
20 _ .
g; 15 _ -
X
E
D.
:1
u) 10 . -
Z
E?
<
D.
O
C)
(113': 5 _ q
1*":1
0 1 1 ll

 

 

 

 

 

80 100 120 140 160 180
PERFUSION TIME (min)

Figure 27. Blockade of dramphetamine-induced efflux of
endogenously synthesized 3Wdopamine from the cat brain by
pretreatment with a-methyltyFOSlne-

CSF containing 3H-tyrosine (20 pc/ml) was contonuously infused
into the cerebroventricular system for 3 hours at a constant rate
of 0. 2 ml/min3 Perfusate samples were collected every 10 min and
analyged for 3H -dopamine. The height of each bar represents the
mean H- -dopamine content (-——— control, N= 4) and vertical line
represents 1 S. E. In the latter experiments, d-methyltyrosine
(""'" aMT 4x10 4M, N= 3) was infused into the ventricular system
starting 90 min prior to and during the infusion of H- -tyrosine.
Two hours after the start of perfusion, d- -amphetamine was added
to the perfusing CSF for 10 min as indicated by the open bar on

the abscissa (d- A, 1. 1 x 10 ' 5M).

97

Table 13. Effects of a-methyltyrosine on the radioactive compounds
in tissues linigg the lateral and third cerebral ventricles following
perfusion with H-tyrosine.

 

3

Tissue Total radioactivity H-catecholamines

 

 

weight Control dMT Control dMT
(mg) (dpm/tissue)
Caudate nucleus 213 3,625,050 2,716,333 97,317 3,137*
:10 :936,753 :390,610 :29,506 :846
Septum 75 1,083,870 1,045,136 3,095 203*
:4 :307,200 :95,859 :881 :43
Right hypothalamus 104 690,120 858,900 1,350 120*
:11 :197,365 :257,256 :351 :20 .
Left hypothalamus 101 697,690 943,500 1,430 110*
:12 :204,412 :315,359 :538 :10

 

After perfusing the ventricles for 4 hours with 25F containing
H-tyrosine, with or without o-methyltyrosine (4x10‘ M), tissues lining
the lateral and third ventricles were analyzed for total radioactivity
(primarily 3H-tyrosine) and for 3H-catecholamines. Tissue contents of
radioactive compounds are expressed as dpm per tissue and values
represent mean : l S.E. as determined from 4 control and 3 a-methyl-
tyrosine experiments. Tissue weights are expressed in mg (N=7).

*statistically different from control (p <.05).

98

the rest of the experimental period resulted in an increase in the
efflux of 3H-dopamine. The efflux of dopamine evoked by g;amphetamine
declined 20 min after the addition of this drug to the perfusing fluid;
a steady rate of efflux at levels of 50% of the initial rate was
reached after about 60 min (Figure 28). When a-methyltyrosine was
added to the perfusing solution 10 min prior to the infusion of
amphetamine, the initial release of 3Wdopamine by g7amphetamine was
not significantly diminished (p>.05), but there was a marked decrease
(p<.05) in the amount of 3H-dopamine released in the perfusing periods
60 min after g;amphetamine (Figure 28). After reaching a steady state
of efflux, gramphetamine released only about one-third as much
3H-dopamine in the presence of a-methyltyrosine as it did in the
absence of a-methyltyrosine.

a-Methyltyrosine blocks the central stimulant actions of g;
amphetamine in animals (Weissman gt_al,, 1966; Rech §t_gl,, 1968).
The mechanism of this effect of a-methyltyrosine is not known, but it
has been postulated to be due to the inhibition of the biosynthesis of
catecholamines (Weissman gt_al,, 1966) or to the prevention of release
of amines from nerve terminals (Enna gt_gl,, 1973). The relative
importance of these two mechanisms for the anti-amphetamine actions of

a-methyltyrosine was tested in the following experiments.

C-3. Effects of a-methyltyrosine on the efflux of endogenously syn-
thesized 3H-dopamine evoked by pulse intraventricular administration of
gfamphetamine.

Catecholamine stores were labeled by adding 3H-tyrosine to the

perfusing CSF. Two hours after the start of perfusion, ten minute

99

 

 

 

 

3H-DOPAM1NE (dpmxio3)
b

 

 

 

 

 

 

 

 

 

8L0 110 140 I70 200
PERFUSION 1th (min)
Figure 28. Effects of a-methyltyrosine on the release of

endogenously synthesized 3H-dopamine from the cat caudate nucleus
during continuous g;amphetamine infusion ig_vivo.

The caudate nuclei were continuously perfused with CSF
containing 3H- tyrosine (12. 5 uc/ml) for 3 hours at a constant
rate of O. 2 ml/min. The cerebroventricular pgrfusing effluent
was collected every 10 min and analyzed for H-dopamine. The
height of each bar and the vertical line represents mean i
1 S. E. of 3H- dopamine content as determined from 4 control
experiments (———-) and 4 d-methyltyrosine experiments ( ------- ).

In the latter experiments, d-methyltyrosine (4x10"4 M) was added
to the perfusing fluid 10 min prior to and during the d- amphet—
amine infusion. d- -Amphetamine (d- A, Bug/ml) was added to the
perfusing CSF 100 min after the start of 3H- -tyrosine infusion,
as indicated by the open bar on the abscissa.

100

pulses of dyamphetamine were added to CSF prior to and after the
addition Of a-methyltyrosine. A typical experiment on the effects

of gramphetamine on the efflux of endogenously synthesized 3Wdopamine
before and after the addition of a-methyltyrosine to the perfusing

CSF is illustrated in Figure 29 and a summary of 3 similar experiments
is presented in Table 14. The results indicate that once the stores
of catecholamines were labeled, a-methyltyrosine (4 x 10'4M) did not

significantly alter (p>.05) the ability of dramphetamine (3 ug/ml;

5

1.1 x 10' M) to increase the efflux of endogenously synthesized

3H-dopamine, but it did decrease the spontaneous efflux of this amine.

3

These results also suggest that the initial release of H-dopamine in

reSponse to the administration of g7amphetamine originated predominately

from a "storage pool" of 3H-dopamine.

C-4. Failure of a-methyltyrosine to alter the g7amphetamine-induced

3

efflux of exogenously administered H-dopamine from the caudate nucleus

1 vivo.

Enna gt g1. (1973) reported that 5 x 10'6

M a-methyltyrosine
reduced the gfamphetamine-induced efflux of radioactivity from rat
brain slices which had been preincubated with 3H-a-methy'l-Ey-tyramine or
3H-norepinephrine. The ability of a-methyltyrosine to alter the g;

3

amphetamine-induced efflux of exogenously administered H-dopamine was

also investigated in the present study by using the jg_xjvg_brain
perfusion technique. After labeling the dopamine stores in the

caudate nucleus with 3H-dopamine, the addition of 5 x 10'6M a—
methyltyrosine (the same concentration used by Enna gt_al,, 1973) to

the perfusing CSF did not alter the ability of amphetamine to increase

101

 

 

 

 

 

 

3
T — 311-1111051111:
8 '1 3H—TYROSINE+01MT
O ........
C2. 5
x
E 2 - -1
o. 2 E
3 3 §
OJ ___ i f
z
< :
‘l a
8 1 _ "*1 _
i:
(V)
"".—'——.
—A d-A
O ., I J (1—1 _1 l l l I 1 1 :1 1 l 1 jj
60 100 140 180 220

PERFUSION TIME (min)

Figure 29. Effects of g;amphetamine on the efflux of
endogenously synthesized 3H-dopamine before and after the
addition of d-methyltyrosine to the perfusing CSF.

CSF containing 3H-tyrosine (20 uc/ml), without (-———)
and with ( ...... ) d-methyltyrosine (4x10-4M), was infused into
the cerebroventricular system for 4 hours gt a rate of 0.2
ml/min. The height of each bar represents H-dopamine in a 10
min collection Eample of perfusate. d7Amphetamine sulfate
(d-A, 1.1 x 10' M) was added to the perfusing CSF for 10 min
as indicated by the open bar on the abscissa.

102

Table 14. Failure of d-methyltyrosine to alter dramphetamine-
induced efflux of endogenously synthesized 3H-dopamine from cat brain.

 

 

Control d—Methyltyrosine
_ (4x10-4M)
Efflux of 3H-dopamine: (dpm/20 min)
1. Before g7amphetamine. 1,346: 76 918:116*
2. After g;amphetamine 4,392:252 4,053:243
3. Induced by g7amphetamine 3,046:318 3,135:128

(2 - 1)

 

Data are a summary of 3 experiments similar to the one illustrated
in Figure 29. 3H-Dopamine (dpm/20 min) in the two collection periods
before the addition of g7amphetamine (1.) and in the collection periods
during and immediately after the addition of d-amphetamine sulfate
(1.1xlO‘5M) to the perfusing CSF (2.) are reparted as means : l S. E.

*statistical decrease from control (p <.05).

103

the efflux of 3H-dopamine from the cat brain jg_vjvg, One of three
experiments, which was typical of the other two, is illustrated in
Figure 30. Essentially the same negative results were obtained when
the experiment was repeated using a 100 fold higher concentration of

a-methyltyrosine (p>.05, Figure 31). The increased efflux of

3 3

exogenously administered H-dopamine and endogenously synthesized H-
norepinephrine induced by amphetamine in these experiments is sum-
marized in Table 15. With each of the 4 pulses of dyamphetamine

(3 ug/ml; 1.1 x 10-5M) there was a significant increase (p<.05) in the
efflux of both catecholamines, although the response declined pro-
gressively with each administration. There was, however, no significant
difference (p>.05) in the amphetamine-induced efflux of 3H-dopamine
between the control and the experiment in which o-methyltyrosine was
added to the perfusing CSF. Thus, a-methyltyrosine, even at a con-
centration which was 100 times that used in the 15.31359 study, did not
alter the ability of amphetamine to increase the efflux of
catecholamines from brain in 1139, The effects of a-methyltyrosine on

3

the endogenous and H-dopamine contents in the caudate nucleus are sum-

marized in Table 16. a-Methyltyrosine did not alter the content of

3Wdopamine, but did reduce slightly the endo-

total radioactivity or
genous content of dopamine (p<.05). These results suggest that a-
methyltyrosine reduces the synthesis of endogenous dopamine but does

not influence the release or retention of radioactive catecholamines.
0. Effects of reserpine and a-methyltyrosine on the d-amphetamine-
indUced efflux Of Striatal’dopamine'newLyggynthesized from c0ntinuoUsly

infused'3H-tyrosine.

104

20 r H-D .
-—-CONTROL 6
dMT(5X10' M)

 

16 _

12 .

 

 

 

3H-DOPAMINE (dpm x 1,000)
00

    

d-A d-Ai d-A

0.111H111H11Lf—111l

3O 60 90 120’

 

 

 

PERFUSION TIME (min)

Figure 30. Failure of d-methyltyrosine to alter d- amphetamine-
induced efflux of exogenously administered 3H- -dopamine from cat brain
in vivo.

Five no of 3H-dopamine were injected into the right lateral cerebral

ventricle. The right lateral and third ventricles were then perfused
with normal CSF (-——-). After 30 min of washout, perfusate samples were
collected _gvery 5 min and analyzed for 3H- ~dopamine. d- Amphetamine sulfate

(1.1 x 10' M) was added to the perfusing CSF for one—5 min collection
period at 50, 75 and 100 min after the start of perfusion, as indicate
on the abscissa by the open bars. The same procedure was repreated in

the contralateral lateral ventricle except that the CSF containing

5 x 10'5M d-methyltyrosine (°°'°).

105

f ——- CONTROL 4
~ ----- dMT(5XlO'. M)

3H-DOPAMINE (dpm x 1.000)

 

 

 

 

I

 

 

 

0 E J_1 1 M I L 1 (ti-:54 1 _Ldlq—e 1 L ‘d-A
30 60 90 l20
PERFUSION TIME (min)

 

Figure 31. Failure of a-methyltyrosine (5 x lO‘4M) to alter
d- -amphetamine- ~induced efflux of exogenously administered 3H- -dopamine
from cat brain in vivo.

The height of each bar represents mean efflux of 3H-dopamine in
5 min perfusate samples and the vertical line represents l S. E. of
that mean based upon 4 experiments. The ventrigles were perfused
with CSF (-———) or with CSF containing 5 x l0“ M a-methyltyrosine
( "'). The open bars on the abscissa indicate the addition of
d- -amphetamine sulfate (d- A, l. l x 10 5M) to the perfusing CSF. See
legend of Figure 30 and METHODS for additional details.

106

Table l5. Failure of a-methyltyrosing to alter d- -amphetamine-
induced efflux of eéogenously administered H- -dopamine and endoge-
nously synthesized H- -norepinephrine from cat brain.

* Increased content of 3H-catecholamines in
cerebroventricular perfusate (dpm/5 min)

 

3H-Dopamine 3H-Norepinephrine

 

d- -Amphetamine Control aMT(5xl0'4M) Control aMT(5xl0’4M)
L3 Lie/ml)

lst infusion l7,879il.l75 16,762i3,5l9 648:70 512:98
2nd infusion lO,666il,683 8,3l7il,085 556ilO3 54li184
3rd infusion 6,046i675 6,752t668 231:38 295:38
4th infusion 4,889i604 5,506il,0l6 220:33 406i90

 

Artificial CSF with or without the addition of u-methyltyrosine
(leO M) was infused into the cerebroventricular system l5 min after
intravengricular injection of 5 uc of H -dopamine. d- Amphetamine sulfate
l. lxlO M) was added to the perfusing CSF for 5 min periods at 50,75,
100 and 125 min after the atart. of perfusion (see Figure 3l for detail).
*Increased content of H- -amines represents the difference between
the amount of 3H- -amines in the perfusate sample during the infusion of
d- -amphetamine less the amount of H- -amines in the perfusate sample imme-
diately prior to the infusion of this drug. Values represent the mean
+l S. E. of radioactivity as determined in 4 experiments.

__,-.j

 

107

Table l6. Effects of a-methyltyrosine on the contents of radio-
active and endogenous dopamgne in the caudate nucleus after intraven-
tricular administration of H-dopamine.

 

 

Control aMT(5xl0‘4M)
Weight of caudate nucleus 237:15 228:8
Endo§§§§g§ dopamine l0.65:0.70 8.44:0.27*
Total radioactivity 4,864,760:695,l16 4,57l,223:236,318
311-1133231143; 3,l92,453:494,869 2,971,521:189,202
(dgmfggpamine x l00(%) 65.3 :l.7 64.9:2.l

 

Total radioactivity

 

At the end of the experiments summarized in Figure 3l and Table 15
the caudate nuclei were analyzed for endogenous and 3H-dopamine. Values
represent means i l S.E. as determined from 4 experiments.

*significantly decreased from control (p <.05).

l08

D-l. Effects of reserpine and a—methyltyrosine on the efflux of newly
‘ synthesized striatal dopamine evoked by the continuous intraventricular
infusion of gfamphetamine.

The results of Besson gt_al,(l973) and those shown in Table l4

3

have suggested that newly synthesized H-dopamine is spontaneously

and preferentially released from the caudate nucleus jn_vivo. When
3H-tyrosine (l2.5 uc/ml, 0.2 ml/min) was infused into the cerebroventricular

3

system, the resting efflux of H-catecholamines, consisting of 96%

3H-dopamine, slowly increased during the 60 min perfusion period

 

(Figure 32). The addition of dramphetamine (0.3 pg/ml; 1.1 x lO'GM)
to the perfusing solution significantly increased (p<.05) the rate of L

3H-dopamine, but there was no increase in the rate of efflux

efflux of
while a-methyltyrosine was present in the perfusing solution. Indeed,
armethyltyrosine decreased the amounts of spontaneous efflux of newly
synthesized 3H-dopamine as well as abolished the ability of g:
amphetamine to enhance the rate of efflux (p<.05, Figure 32). Two
hours after an intravenous injection of reserpine (0.5 mg/kg,) the

3

addition of gramphetamine to the perfusing CSF containing H-tyrosine

caused an enhancement during the first 30 minute period above the rest-

3H-dopamine which was similar to that observed in control

ing efflux of
cats not treated with reserpine. The efflux of 3H-dopamine induced by
gramphetamine in the control experiment reached a steady level 30
minutes after the start of perfusion, but the gramphetamine-induced
efflux of 3H-dopamine following reserpine continued to increase.
Reserpine significantly decreased (p<.05) the endogenous and

3H-dopamine stores in the caudate nucleus (Table l8). Thus,

109

20 F 3
H-TYROSINE

a an" IA
. wuuour 45 i

      

 

 

 

a l ,1
K 1'
E
O.
3 ,
In 8 ~ ,’ i
E OOIYIOI.
z
<
m
o
o
| ,_ +\+/+/ ‘
(“I 4 /
(A I“?
”-0-.-. O“O" *‘O
0 LL A A l l 1 J
o 20 40 so
PERFUSION TIME (min)
Figure 32. Effects of reserpine and a-methyltyrosine on the

dyamphetamine-induced release of newly synthesized dopamine from
the caudate nucleus jn_vivo.

CSF containing 3H-tyrosine (l2.5 uc/ml) and gfamphetamine (dA,
l.lxlO'5M salt; 0.3 ug/ml base) was continuously infused into the
ventricular system for 60 min at a rate of 0.3 ml/min. Perfusates
were collected every 10 min and analyzed for H-dopamine. Each point
represents the mean and the vertical line represents lS.E. Where no
line is drawn, the S.E. was less than the radius of the symbol. o——-o
represents the perfusing CSF containing only 3H—tyrosine (control,
N=3). o——-o represents the perfusing CSF containing 3H-tyrosine and
d-amphetamine (N=4). o--~-o represents the perfusing CSF containing
3H-tyrosine, d-amphetamine and a-methyltyrosine (4xl0'4M, N=3).

In the reserpine experiments (o---o, dA and reserpine), reserpine
(0.5 mg/kq) was gnjected intravenously 2 hours prior to the initiation
of perfusion of H-tyrosine and gfamphetamine (N=4).

llO

reserpine effectively depleted the caudate nucleus of both endo-
genous and 3H-dopamine by 95% but had little effect on the g:

3H-dopamine. Despite the

amphetamine-induced release of newly formed
low concentration of dopamine in the caudate nuclei of reserpine-
treated cats, chromatographic separation of the acid eluate in
catecholamine fractions of pooled caudate nuclei revealed the presence
of a 3H-dopamine peak. There was no 3H-dopamine peak when the caudate
nuclei were perfused with a solution containing a-methyltyrosine and
3H-tyrosine (Figure 23A). These results suggest that g:amphetamine

is capable of releasing dopamine from a relative small "reserpine
resistant pool" of dopamine in the caudate nucleus which is maintained

primarily by ongoing synthesis.

D-2._ Effects of reserpine on the central dopamine releasing action and
the peripheral vasopressor action induced by the intravenous injections
of increasing doses of gramphetamine.

In the results presented thus far, most of the drugs were
administered intraventricularly. Although this route of administration
is not often used for behavioral studies, Taylor and Sulser (l973) have
recently reported that intraventricular injection of both 9: and 13am—
phetamine in rats caused increased motor activity and stereotyped
behaviors persisting for about 30 minutes. The effects of intravenous

3

injections of amphetamine on the efflux of brain H-dopamine synthesized

3H-tyrosine have been summarized in Figure25. In those experi-

from
ments, systemic administration of amphetamine produced a prolonged
pressor effect. In an effort to determine if the release of dopamine

might be dependent upon the pressor effect or the route of

.—‘

lll

administration of amphetamine, experiments were performed in which
gramphetamine was injected intravenously into cats pretreated with
reserpine (0.5 mg/kg, i.v.). Two hours after the administration of
reserpine, CSF containing 3H-tyrosine (12.5 uc/ml, 0.2 ml/min) was
infused into the lateral ventricle for four hours. 'One hour after the

3H-tyrosine perfusion increasing doses of g7amphetamine

start of the
sulfate (0.25 to 4.0 mg/kg), injected intravenously at 30 minute

intervals, produced a dose-related efflux of 3H-dopamine newly
3

.i
l",
J
5‘?
.31
g
l .

H-tyrosine. grAmphetamine did not alter the efflux
3

synthesized from

of total radioactivity (mainly H-tyrosine). Figure 33 illustrates

 

the typical experiment (also see Table 17). The minimal effective
dose of gramphetamine to cause a significant release of 3H-dopamine
from the reserpine-treated brain was about 0.5 mg/kg. At the end of
the experiment, the caudate nuclei contained only 3,000 dpm of
3H-dopamine and 80 ng of endogenous dopamine (less than 5% of the con-
trol level, Table l8). Since amphetamine released more 3H-dopamine than

3

was detected in the brain, the released H-dopamine must have originated

from a "newly synthesized pool" which was maintained by the ongoing
synthesis of this amine from tyrosine. The amphetamine-induced release
of striatal dopamine was not related to its peripheral pressor res-
ponses. After reserpine, only the first dose of geamphetamine (0.25
mg/kg)produced a pressor response; this dose failed to induce the
release of 3H-dopamine. Subsequent administration of higher doses of
g;amphetamine did not alter the blood pressure but caused the release
of dopamine from the brain. One of these experiments, which is typical

of two others, is illustrated in Figure 34.

ll2

Figure 33. Efflux of newly synthesized 3H-dopamine from
the caudate nucleus of a reserpine-pretreated cat in response
to the cumulative doses of the intravenous injections of
gbamphetamine.

Two hours after the intravenous injection of reserpine (0.5
mg/kg), CSF containing 3H-tyrosine (l2.5 uc/ml) was infused into
the lateral ventricle at a constant rate of 0.2 ml/min for 4
hours. Samples of perfusate were collected every l0 min and
analyzed for 3H-dopamine. The height of each bar represents the
mean concentration of 3H-dopamine in the perfusate determined
from two experiments. The times that gramphetamine sulfate
(0.25 to 4.0 mg/kg) was injected intravenously are noted on the
abscissa.

3H-DOPAMINE (dpm X 1,000)

113
14
(_ 3H-TYROSINE
RESERPINE PRETREATED(.5mg/kg i.v.)
12 __
1o _. {.2
81-—
61-
4 _.
2...
o L.
llllillllllllillllLl11114
TIME 20 4o 60 80 100 120 140 160 l80 200 220 240(min)
.25 .5 1.0 2.0 4.0
III III III III III

 

 

 

 

 

 

 

 

d-AMPHETAMINE (mg/kg i.v.)

 

_i

 

IF

Table l7.

ll4

Summary of the efflux of newly synthesized 3H-dopamine

from the caudate nucleus of reserpine treated cats evoked by intravenous
injections of increasing doses of g;amphetamine.

 

Perfusion time d-Amphetamine

3H-Tyrogine 3H-Dopamine

 

(min) _'(SO4, mg/kg) (dpmxlO /30 min) (dpm/30 min)
30-60 0 100.49t4.08 661:200
60-90 0.25 llO.62i5.l3 798i122
90-120 0.5 103.10i4.68 2,281i391*
120-150 1.0 110.06i4.59 4,684il,400*
150-180 2.0 102.56i2.68 12,918i2,873*

 

Values represent the mean i l S.E. determined from three experiments
similar to the one illustrated in Figure 33. Cats were pretreated with
reserpine (0.5 mg/kg, i.v.) two hours prior to the start of perfusion
of 3H-tyrosine (12.5 uc/ml, 0.2 ml/min).

*Values are significantly greater than control (p <.05).

ll5

Table l8. Effects of reserpine on the contengs of endogenous
dopamine and radioactive dopamine synthesized from H-tyrosine in
the caudate nucleus following intraventricular and intravenous
administration of g;amphetamine.

 

Control Reserpine treated

ng’ d-A
(intravent.) (Tlv.)

 

 

Height of caudate nucleus (mg) 23l:l0.l 246.2:l4.3 24l.3:9.9
Endogenous dopamine (pg/caudate) 2.51:0.06 0.09:0.0l* 0.08:0.04*
3H Dopamine (dpm/caudate) 63,066:6,29l 3,6l3:l,9ll* 3,100i721*
Experimental number 4 4 - , 3

 

Cats were pretreated with reserpine (0.5 mg/kg3 i. v. ) two hours
prior to the start of perfusion of CSF containing H- -tyrosine (12.5
uc/ml, 0. 2 ml/min). W A (intravent. ) represents the perfusing
solution containing 31amphetamine ( o. 3 ug/ml) as described previously
in Figure 32; d- A (i. v. ) represents the dopamine content in the

experiments of Figure 33 in which d- -amphetamine was injected intravenously

and cumulatively from O. 25 to 4 mg7kg. Values represent the mean i
l S. E. as determined from 3 to 4 experiments.
*denotes significantly decreased from control (p < .05).

 

116

Figure 34. Effects of the cumulative doses of the intra-
venous injections of gfamphetamine on the blood pressure of the
reserpine pretreated cat.

Two hours after the administration of reserpine (0.5 mg/kg,
i.v.), the arterial blood pressure of the animal was measured
with a Statham pressure transducer (PC23AC) and recorded on a
Grass polygraph (7PCPB). Increasing doses of d7amphetamine sulfate
(d-A, 0.25 to 4 mg/kg) were injected intravenously as described
in Figure 33 and Table l7.

BLOOD PRESSURE
(200 mmHg)

M 117 _ ”_MM '
. 10 min after RESERPINE (0.5 mig/Kg, 1.11. )1 512057.3CAi'
‘1 1 i i

 

i - ‘ ‘1'. K‘“ \ i .\ 1: Ni V
. .120 mi}! L 1 ‘

 

, ,.L

 

‘1

i d-AIIQ‘JSmngg)\\ . .7 .1 ,

 

l 250 min: (I

 
 

‘1 2 1‘

3 a. \ 1 in.“ .11 ‘1 1 1 1‘
1 i d-Aaiomg/Kgx \ ) W L L,
1 - ; - 1. 7 , . ; 1 1

i 280 min? T—(_ V

 
  

  

  

 

i d-.A12“.0mg/J.<21\ .7
1 ‘1 ' "

4 316111111?"

   

l V I
~. 1

‘1 1 1
1

‘ ._. -2,

g d-A(4.0mg/Kg1_\

TIME
(min)

DISCUSSION

A. Methodological Problems in specifically and intensively labeling_
thg_gjgrostriatal dopaminergic neurons in the brain

Limits in the sensitivity of chemical methods available for
the detection of putative neurotransmitters have plagued experiments
designed to detect the jn_glxg release of these substances from the
brain. Studies with catecholamines are no exception (McLennan, 1964;
McKenzie and Szerb, 1968; Portig and Vogt, l969). Accordingly, it has
been necessary to resort to radioactive tracer techniques to measure
these amines in the brain perfusate. Exogenously administered radio-
active catecholamines can be taken up by tissues lining the lateral
ventricle and subsequently released by a variety of stimuli (Carr and
Moore, 1970; Von Voigtlander and Moore, l973a), but the released
amines probably do not originate exclusively from catecholaminergic
neurons. Efforts have been made, therefore, to selectively label
amine stores exclusively within catecholaminergic neurons_by administer-

3H-dopa.

ing the radioactive precursors, 3H-tyrosine or
Since the content of endogenous tyrosine in the brain is
approximately l0'4M and the Km for tyrosine hydroxylase is l0'5M
(Levitt gt g1,, 1965), it is not possible to intensively label brain
catecholamine stores with 14C-tyrosine of low specific activity.
Difficulties in detecting release of catecholamines synthesized jg_§jty_

14

from C-tyrosine in the present (see Table 5) and in other studies

118

ll9

(Riddel and Szerb, 197l; Roth et_gl,, l969) were apparently related,
in part, to the dilution of 14C-tyrosine by the large endogenous con-
tent of this amino acid in the brain. '

In the past, numerous problems have been encountered in
attempting to label and then detect the release from the brain of

catecholamines synthesized jg_vivo from 3H-tyrosine. These problems

 

have been due to dopa-like impurities, to the unavailability of .g—5,
3H-tyrosine with high specific activity and to the difficulties in f
detecting a small amount of labeled amine in a large amount of ;
3H-tyrosine. These difficulties cited above were largely overcome in W

the present experiments by using 3H-tyrosine with very high specific
activity (higher than 50 c/mmole), by carefully purifying the 3H-
tyrosine immediately before use and by utilizing a combination of weak
cation-exchange resin and alumina adsorption chromatography to

3H-catecholamines.

selectively isolate the endogenously synthesized
With these procedures it is possible to detect a few thousand dpm of
3H-catecholamines in samples containing 30 to 80 million dpm of 3H-
tyrosine.

The catecholamine stores in the caudate nucleus can be labeled
intensively using an intraventricular injection of radioactive dopa
because of the low endogenous concentration of L-dopa in the brain.
Unfortunately, the ubiquitous distribution of aromatic L-amino acid
decarboxylase (mean and Rosengren, 1967; Hdkfelt‘et_gl,, 1973), which
catalyzes the conversion of L-dopa to dopamine, appears to limit the
use of radioactive dopa for selectively labeling dopaminergic neurons.

For example, L-dopa can be converted to dopamine in serotonergic

neurons, although it has been reported that at very low concentrations,

120

(lower than lO'7M) L-dopa is selectively taken up by dopaminergic
neurons (N9 2; 31,, 1971). In the present study, therefore, in an
effort to specifically label dopamine stores in the dopaminergic
neurons with 3H-dopa, this amino acid was injected in five divided
doses (2 x l0'9 mole) at 30 min intervals. Since immunohistochemical
studies have revealed that dopamine-B-hydroxylase is located exclusively
in the noradrenergic neurons in the brain, (Livett et_al,, l969; Fuxe
‘etugl,, l970; Coyle and Axelrod, 1972; Hartman and Udenfriend, l972),
any radioactive norepinephrine synthesized from labeled tyrosine, dopa
or dopamine must originate from noradrenergic neurons. Since the
caudate nucleus contains dopaminergic neurons and serotonergic neurons
but not noradrenergic neurons, the caudate dopaminergic neurons can be
labeled selectively only with radioactive tyrosine.

A study of the distribution of tyrosine hydroxylase activity
in the cat brain indicated that the region of highest activity was the
caudate nucleus, 98.5nmole/g/hr, whereas the activities of this enzyme
. in the thalamus and hypothalamus were much lower, 2.9 and 3.9 nmole/g/hr
respectively (McGeer gt 31,, 197l). Furthermore, the turnover of‘

' striatal dopamine was 40 times higher than that of tel-diencephalic
norepinephrine, 15:2 and 0.38:0.05 nmole/g/25 min respectively (Costa
$91., l972). Thus, despite the fact that tyrosine hydroxylase is

located in both dopaminergic and noradrenergic neurons, either intra-

3

ventricular injection or continuous infusion of H-tyrosine into the

cerebroventricular system produced an intensive and selective labeling
of the dopamine stores in the nigrostriatal neurons of the caudate

nucleus. Over 95% of total brain radioactive catecholamines was located

 

12l

in the caudate nucleus and consisted of only 3H-dopamine (see Table ll).

B. The specificity of the coordinates used for the stimulation of the
nigrostriatal_pathway in the lateral hypothalamus.

In order to establish dopamine as a neurotransmitter in the
ascending nigrostriatal pathway, one of the crucial experiments is to
demonstrate the release of endogenously synthesized dopamine upon
selective stimulation of these neurons. Recently, the dopaminergic
nigrostriatal pathway of the cat has been mapped by use of discrete
neuronal lesions in combination with the Fink-Heimer silver stain to
detect degenerating neurons (Bédard et_§l,, 1969; Moore et 31,,l97l).
Previousreports from this laboratory have shown that electrical
stimulation in the lateral hypothalamus increased the release of exo-
genously administered 3H-dopamine from the brain (Von Voigtlander and
Moore, 1971 a,b; 1973a). The results of the present experiments con-
firmed that stimulation at the stereotaxic coordinates of A 8 to lO, .

3

L3 and H-3.5 effectively released exogenously administered H-dopamine

from the caudate nucleus. Stimulation at these sites increased the

14C-tyrosine to dopamine in the ipsilateral caudate

conversion of
nucleus, whereas a lesion at these sites reduced the synthesis of
dopamine. Thus, the specific site in the lateral hypothalamus having
coordinates of A 8 to 10, L3, H-3.5 contains dopaminergic nigrostriatal
fibers. Therefore, this region was chosen for subsequent electrical
stimulation studies. Nevertheless, one should keep in mind that other

monoaminergic neurons (k) also ascend through the lateral hypothalamus

to the forebrain areas (Ungerstedt l97la, see also Table 9 and lo,

Moore and Heller, 1968).

l22

C. In vivo release of endogenously synthesized catecholamines from
cat brain by electrical stimulation.
After labeling the catecholamine stores in the brain with an

3 3H-catecholamines

intraventricular injection of H-tyrosine or 3H-dopa,
were identified in the cerebroventricular perfusate. Electrical
stimulation in the region of the lateral hypothalamus, previously

fi.“

shown to contain fibers of the nigrostriatal pathway, increased the

efflux of both 3H-dopamine and 3

H-norepinephrine to about the same
extent. With the cerebroventricular perfusion technique used in the
present study, the dopamine and norepinephrine could originate from

any structure lining the anterior horn of the lateral ventricle or

the third ventricle. Results of experiments in which both of the
lateral ventricles were perfused concurrently after labeling one side
of the caudate nucleus with 3H-dopa suggested that most of the dopamine
and virtually all of the norepinephrine which was released in response
to electrical stimulation of fiber tracts in the lateral hypothalamus
originate from tissue lining the lateral ventricle. The most obvious 1
sites of release would be the septum for norepinephrine and the caudate
nucleus for dopamine. Histochemical studies (Ungerstedt, 1971a; Nobin
and Bjdrklund,1973) have revealed that the dopaminergic neurons

ascend adjacent to the noradrenergic fibers in the medial forebrain
bundle, so it is not unexpected that stimulation in the lateral hypo-
thalamus should give rise to the release of both catecholamines. In-
deed, electrolytic and 6-hydroxydopamine-induced lesions at this site
in the lateral hypothalamus partially reduced both the dopamine content

in the caudate nucleus and the norepinephrine content in the septum

l23

(see Table 9 and l0). The present results suggest, therefore, that
stimulation at A10, L3 and H-2.5 to -3.5 is not as selective for
nigrostriatal neurons as has been reported previously (Von Voigtlander
and Moore, l97lb; 1973a). A lesion in the same site of the lateral
hypothalamus also reduced the content of serotonin in the caudate
nucleus. Labeled dopamine but not norepinephrine may be released,

in part, from serotonergic neurons in response to electrical stimula-
3

3

tion of lateral hypothalamus in experiments with H-dopamine or 3H-dopa.

After intraventricular administration of H-tyrosine, the
septum and the hypothalamus contained only 2 and 4% of the total brain
labeled catecholamines respectively; the amount of 3H-dopamine was
larger than that of 3H-norepinephrine in these tissues. The meso-
limbic dopamine axons from cell bodies in the interpeduncular nucleus
ascend with axons of the nigrostriatal neurons and then enter the
nucleus accumbens or the tuberculum olfactorium (Ungerstedt, l97la).
Thus, the septal dopamine is not a major source of dopamine in the.pre-
sent studies. However, electrical field stimulation of the lateral
hypothalamus increased the release of dopamine from the tissues lining
the third ventricle. The sites of release are presumably from the

'periventricular and the arcuate dopaminergic neurons (Bjdrklund and

Nobin, 1973).

D. Lesion of the ascending,monoaminergichathway in the lateral hypo: 4

thalamus.

The lesions of the lateral hypothalamus at coordinates of A8
to lQ,L3, H-3.5 caused reductions in the dopamine and serotonin content
of the ipsilateral caudate nucleus and norepinephrine content of the

septum. These results are in accordance with the results obtained

 

 

124

from the studies on the lateral hypothalamic stimulation-induced
release of endogenously synthesized catecholamines (see Figure ll)
which indicated that the site with coordinates of A10, L3, H-3.5 was
not selective for dopaminergic nigrostriatal neurons. Decreases of
forebrain norepinephrine and serotonin concentration by lateral hypo-
thalamic lesions in cats have been reported previously (Poirier gt_gl,,

1967; Heller gt_al,, 1966; Heller and Moore, 1968; Sourkes and F

Poirier, 1968; Moore et_gl,, 1965). Nevertheless, Parent and
Poirier (1969) and Von Voigtlander and Moore (l973a) have reported

that electrolytic lesions of the anterior lateral hypothalamus at

 

ERA _
W,
‘ 1";

the border of optical chiasm produced a selective decrease of the
dopamine content but not of the serotonin content of the ipsilateral
caudate nucleus. In the present studies, however, when lesions were
made in the anterior hypothalamus at A12 to 14, L3, H-3.5; the dopamine
content of caudate nucleus was not altered, although the norepinephrine
content of the septum was significantly decreased. The important
factors in the lesion experiments are the histological and biochemical
evaluation of the results. Unfortunately, because the earlier reports
of Von Voigtlander and Moore (1973a) did not include histological

data, the controversy cannot be resolved.

An attempt to selectively destroy the diencephalic dopaminergic
neurons by an intracerebral injection of 6-hydroxydopamine failed in
the present study. Similar to the results of the experiment using
electrolytic lesions, injection of 6-hydroxydopamine into the lateral
hypothalamus at coordinates of A10, L3, H-3.5 decreased the monoamine

content in the caudate nucleus and the septum on the side of lesion.

 

125

It has been proposed that injection of 6-hydroxydopamine into the
brain at low concentrations selectively destroys the nerVe terminals
of catecholaminergic neurons (Ungerstedt, 1971b). The selective
effects of 6-hydroxydopamine on catecholaminergic neurons have been
questioned recently by Cooper gt_gl, (1973) and Petitjean et_gl,,
(1972), who reported a concomitant decrease in the brain content of
serotonin in rats or cats after an intraventricular injection of 6-
hydroxydopamine. Intraventricular injection of 6-hydroxydopamine
reduced the content of both dopamine and serotonin in the caudate
nucleus of the cat by 50 to 60% of the control (Petitjean g3_gl,,
1972).. In the present study, injection of 6-hydroxydopamine into the
lateral hypothalamus produced a similar depletion of forebrain mono-

amine content. Any selectivity of 6-hydroxydopamine may be strictly

dependent upon the local concentration of the drug in the brain tissue.

However, the present histological examination revealed that intra-
cerebral injection of 6-hydroxydopamine, 24 pg in 4 pl, induced a non-
specific necrotic lesion in the lateral hypothalamus,thus suggesting
that the 6-hydroxydopamine-induced lesions were similar to the
electrolytic lesions. A 6-hydroxydopamine-induced necrosis (0.5 mm in
diameter) at the site of injection of 8 pg of 6-hydroxydopamine in

4 pl was reported initially by Ungerstedt (l97ld). Subsequent

histological and electronmicroscopic studies have revealed that

stereotaxic injection of 6-hydroxydopamine (4 pg in 4 pl, Sotelo §t_§1,

1973,0r 8 pg in 4 pl, Poirier st 31,, 1972) into the substantia nigra
of rats or cats induced a nonspecific necrotic lesion. Nevertheless,

Agid §t_gl,, (1973) suggested that, based upon the diffusion radius of

A ring-nu

 

126
this drug, only very small volumes and concentrations of 6-hydroxy-
_dopamine (smaller than 4 pg in 2 pl) allow for a specific degeneration
of dopaminergic neurons in the substantia nigra. Thus, the selectivity
of 6-hydroxydopamine on the lesion of catecholaminergic neurons
claimed by a number of studies appears to be, in part, dependent upon
this nonspecific action or upon the ability of neurons to selectively
accumulate this drug.

In the present experiments with nonanesthetized spinal cats,
electrical stimulation of the lateral hypothalamus at the coordinates
of A10, L3, H-3.5 elicited a bilateral mydriasis and a gnawing tendency
in addition to the increase in the efflux of endogenously synthesized
catecholamines from the forebrain. In the freely moving cat,
stimulation of the posterior hypothalamus at the border of the zona
incerta caused a smooth turning toward the contralateral side, a bi-
lateral mydriasis and a compulsive gnawing (Hess, 1956). The role of
caudate dopamine for stimulation-induced contralateral circling in cats
has been discussed recently (Cools, 1971; 1973). After lesioning the
monoaminergic fibers in the lateral hypothalamus, the animals circle
‘toward the side of the lesion when startled or otherwise stimulated.

In the present study, both apomorphine, a dopamine receptor

stimulating agent (Ernst, 1967; Andén g__al,, 1967), and L-dopa caused

a dose-dependent increase in circling toward the lesioned side. The
dose-response curve of apomorphine was shifted to right by haloperidol,

a proposed dopamine receptor blocking agent (Janssen, 1967; Andén gt_§l,,
1970), suggesting that a dopaminergic system is involved. Since the
lateral hypothalamic lesion is not specific for dopaminergic nigro-

striatal neurons, the involvement of other neuronal systems cannot be

 

n-

127

ignored. The rotational behavior after gramphetamine is partially '

blocked by a-methyltyrosine, suggesting that this action of

amphetamine is dependent upon ongoing synthesis of catecholamines,

presumably dopamine. The circling toward the side of lesion after

apomorphine, L-dopa and gramphetamine has been reported previously

(Andén et 31,, 1966; Andén, 1970). On the other hand, after producing

selective degeneration of the nigrostriatal dopaminergic system it was ”‘5‘
observed that apomorphine and L-dopa caused contralateral circling,
whereas gfamphetamine produced circling toward the lesioned side
(Ungerstedt, 1971b,c; Von Voigtlander and Moore, 1973b). One week
afteran unilateral ablation of the frontal cortex, g-amphetamine a..."
caused circling toward the lesioned side; but this drug caused a con-

tralateral circling two weeks after the lesion of cortex (Glick and

Greenstein, 1973). Thus, the controversy on the circling behavior

appears to be related to the development of postsynaptic super-

sensitivity and to the degree of degeneration of the dopaminergic’

nigrostriatal neurons.

E. Selective release of striatal dopamine by d-amphetamine.

The concept that amphetamine exerts its behavioral effects by
increasing the concentration of brain catecholamines in the synapse
(Stein, 1964; Rech, 1964; Moore et 31,, 1970) is supported in the
present experiments either by acute labeling with radioactive amino

3

acid precursors or continuous labeling with H-tyrosine. Following

3

intraventricular administration of H-tyrosine both intraventricular

infusion and intravenous injection of gfamphetamine significantly

increased the efflux of endogenously synthesized dopamine but not of

 

128

norepinephrine from the brain in a dose-related manner. Since
approximately 95% of the dopamine released by gramphetamine
originated from the lateral ventricle (Table 8) and about 96% of the
total brain radioactive dopamine is located in the caudate nucleus
(Table 10), it would appear that this drug acts at the terminals of
the dopaminergic nigrostriatal neurons in the caudate nucleus. The
results of the present study suggest that gramphetamine releases
endogenously synthesized striatal dopamine rather than norepinephrine
and provide direct evidence for the concept that amphetamine acts

on the striatal dopaminergic nerve terminals to exert the locomotor
stimulation and stereotyped behavior (Thornburg and Moore, 1973a;
Creese and Iversen, 1973; Ernst, 1967). It is possible that this
"selective" effect, results from the fact that dopaminergic terminals
adjacent to the ventricular system are more numerous, and thus are

more densely labeled than are the noradrenergic neurons. Nevertheless,

3H-dopamine content in the

reserpine treatment effectively reduced the
caudate nucleus by 95% but did not diminish the d:amphetamine-induced
efflux of 3H-dopamine (see Table 17 and 18). Another explanation for
the selective effect of gramphetamine on release of dopamine may be

the fact that this drug increases the turnover of the striatal dopamine
greater than the forebrain norepinephrine, from 15 to 27 and from 0.38

to 0.41 nmole/g/ZS min respectively (Costa 35.31,, 1972).

F. Effects of d-amphetamine on the release of "stored" and "newly

synthesized" dopamine from the nigrostriatal neurons

A number of reports have suggested that dramphetamine-induced

central stimulant actions appear to be dependent upon ongoing

 

 

 

129

catecholamine synthesis, but not upon the presence of granular
catecholamine stores (Moore gt 31,, 1970; Rech et_gl,, 1968).

Efforts have been made to determine the ability of amphetamine to
induce the release of newly synthesized dopamine from the brain during
the past ten years. The results of an jg_yjtgg_study by Besson ££;213

(1969) in which animals were treated with a high dose of‘gramphetamine

 

(5 mglkg, i.p.) suggested that this drug acts on the release or re- r‘,
uptake of newly synthesized dopamine. However, when radioactive 1
tyrosine was injected systemically after gramphetamine administration
(1 mg/kg, i.p., Costa and Groppetti, 1970) an increase in the striatal i

L:

radioactive dopamine content was observed. This argues against the
hypothesis that amphetamine acts by releasing newly synthesized dopamine.
In these earlier studies the efflux of newly synthesized dopamine from
the brain was not measured, but only the retention of dopamine newly
synthesized from radioactive tyrosine in the striatum was determined.
It has been proposed that a-methyltyrosine inhibits only the
biosynthesis of catecholamines and thus exerts its anti-amphetamine
effect by reducing newly synthesized amines in a "releasable pool"
(Weissman gt 21,, 1966). However, this mechanism of the anti-
amphetamine action of a-methyltyrosine has recently been questioned by
Enna et El: (1973) who reported that a-methyltyrosine blocked the
amphetamine-induced release of exogenously administered amines from
brain slices. The results of the present study, using an jg_ijg_brain
perfusion technique, do not support the "anti-release" action of a-
methyltyrosine. Intraventricular infusion of demethyltyrosine blocked

the synthesis of brain dopamine but did not interfere with the ability

 

130

of amphetamine to release endogenously synthesized or exogenously

3

administered H-dopamine (see Table 14, 15 and 16). ReSults of

experiments in which g;amphetamine and a-methyltyrosine were infused

3H-tyrosine perfusion

3

continuously two hours after the start of a
suggested that amphetamine initially released H-dopamine from a
storage pool but that continuous release was dependent upon the con-
tinued synthesis of this amine (see Figure 28 and 29). The failure

of a-methyltyrosine to block the initial release of 3H-dopamine induced
by g7amphetamine may be due, in part, to the equilibrium of the

3n-dopamine with the 3H-dopamine in

relative large amount of stored

the small functional pool.
Similar to the finding by Besson gt 31, (1973), the addition of

a-methyltyrosine to the perfusing CSF blocked the spontaneous release

3

of 3H-dopamine synthesized during the continuous infusion of H-

tyrosine (see Figure 29; Table 14). This suggested that newly syn-

3H-dopamine was preferentially released spontaneously from the

thesized
caudate nucleus. In the present study the addition of a low concentration
of geamphetamine (0.3 pg/ml, 1.1 x 10'6M) to the 3H-tyrosine solution at
the start of perfusion of the caudate nucleus increased the rate of-

3H-dopamine 2 to 3 fold over the control efflux (see

resting efflux of
Figure 32). The amphetamine response was completely blocked by the
presence of a-methyltyrosine but was not altered by pretreatment with
reserpine in order to eliminate the storage pool of dopamine in the
caudate nucleus. Despite the fact that the caudate nucleus retained only
5% of control of the endogenous nonlabeled and 3H-dopamine after

reserpine, intravenous injection of gramphetamine produced a dose-related

 

131

3H-dopamine‘during the 3H-tyrosine infusion (see Table 17).

3

release of
The total radioactivity of H-dopamine released by gramphetamine,
administered either intraventricularly or intravenously, was several
times higher than that retained in the brain, primarily located in the
caudate nucleus. These results clearly demonstrate that the dopamine

efflux induced by the intravenous injection of cumulative dosesof'g;

amphetamine is maintained mainly by the ongoing synthesis of this

3

amine from the continuously infused H-tyrosine. Since pretreatment

4m in IL“ Luna“ -q
’v

with reserpine did not impair the geamphetamine-induced efflux of

2. AL. .Ii

newly synthesized dopamine in the brain (see Figure 32 and 33), the .
persistence of the central actions of amphetamine in reserpine-treated L—Y
animals can no longer be interpreted to suggest that amphetamine is a
directly acting sympathomimetic which acts at central dopaminergic
receptor sites (Smith 1963; Van Rossum and Hurkmans, 1964).
Finally, it is interesting to note that the central effect of
‘gfamphetamine on the release of newly synthesized dopamine does not
diminish following repeated intravenous injections of increasing doses
of this drug whereas the peripheral pressor effect does diminish in the
reserpine-treated animal (see Figure 34). If reserpine acts on the '
catecholaminergic neurons of the central and of the peripheral nervous
system in a similar manner, there should be no dissociation between
the central and peripheral effects of amphetamine. The diminished
pressor effects with repeated injections of gyamphetamine in the
reserpine-treated cat may be related to the possibility that the

peripheral sympathetic functions are mainly maintained by reuptake or

 

reuse of released or circulating catecholamines rather than by the

132

newly synthesized amines as suggested by Chang and Chiueh (1969) and
Hedqvist and Stjarne (1969, cited by Stjflrne, 1971). It has been
proposed that dopamine-B-hydroxylase, the enzyme that catalyzes the
conversion of dopamine to norepinephrine, is highly localized in the
norepinephrine storage granules in adrenergic neurons (Stjflrne and
Lishajko, 1967; Livett ££;21:: 1969; Potter and Axelrod, 1963).
Reserpine administration blocks the uptake of dopamine into the
storage granule (Kirshner gt;al,, 1963), and hence the synthesis of
norepinephrine from precursors in the adrenergic tissues is also 1

blocked (Euler'g§;gl,, 1966, cited by Stjarne, 1966; Rutledge and

 

Weiner, 1967; Roth and Stone, 1968). In contrast, dopamine synthesis
in the brain continues after reserpine (Andén gt 31,, 1964b; Kopin and
Weise, 1968). Reserpine-induced inhibition of norepinephrine but not
of dopamine synthesis appears to be the most likely explanation for the
tolerance to the amphetamine-induced peripheral pressor effect in the
reserpinized cat. Thus, the availability of newly synthesized dopamine
in the brain, but not of norepinephrine in the periphery, following
reserpine administration may account for the differential effects on g:
amphetamine stimulation centrally and peripherally, and once more
emphasizes the relative importance of brain dopamine for the central

stimulant properties of gramphetamine.

SUMMARY AND CONCLUSION

The present study utilized a cerebroventricular perfusion
technique to detect the release of brain catecholamines synthesized
from radioactive precursors j__yjyp, The effects of electrical
stimulation of ascending monoaminergic fiber tracts such as the
nigrostriatal pathwayand the medial forebrain bundle in the lateral
hypothalamus and intravenous or intraventricular administration of g:
amphetamine on the release of endogenously synthesized catecholamines

were examined. The significant observations and conclusive remarks

of the present investigation are as fOllows.

A. A sensitive method for isolation and determination of
labeled and/or nonlabeled-catecholamine was developed in the present
study by modifications of the method of weak cation exchange resin
chromatography (Barchas £3 31,, 1972) and of the spectrophotofluorometric
assay for catecholamines (Chang, 1964). With this procedure,
it is possible to detect a few hundred dpm of labeled dopamine and/or
norepinephrine in samples containing 30 to 80 million dpm of 3H-
tyrosine or to measure samples of nonlabeled catecholamines containing

10 to 25 ng/ml.

B. After labeling the catecholamine stores in the brain

3 3

using an intraventricular injection of H-tyrosine or H-dopa,

electrical stimulation of ascending monoaminergic fiber tracts in the

133

 

 

134

lateral hypothalamus (stereotaxic coordinates A 10, L 3, H -3.5)

3H-catecholamines

increased the efflux of endogenously synthesized
from the brain into the ventricular perfusate. Since lesions of the
same sites in the lateral hypothalamus decreased the content of
dopamine and serotonin in the caudate nucleus and the content of
norepinephrine in the septum, most of the hypothalamic stimulation-
induced release of 3H-dopamine and 3H-norepinephrine originated from
the caudate nucleus and septum, respectively. Furthermore, lateral
hypothalamic lesions decreased the conversion of radioactive tyrosine
to dopamine and the endogenous dopamine content in the caudate nucleus
on the side of lesion. Thus, the hypothalamic stimulation-induced
,release of 3H-dopamine synthesized jp_§jtu_from 3H-tyrosine may have

originated, in part, from the terminals of the dopaminergic nigro-

striatal pathway in the caudate nucleus.

C. Intraventricular administration of geamphetamine increased
the efflux of exogenously administered and endogenously synthesized
radioactive dopamine from the cat brain in a dose-related manner.

The minimal effective concentration of gramphetamine needed to cause a

significant increase in the efflux of dopamine was about 10'7 to 10'6M.

1:Amphetamine was one-tenth as potent than geamphetamine in causing

3

the release of exogenously administered H-dopamine from the brain

jp_vivo. High concentrations of gramphetamine also increased the

efflux from the brain of labeled norepinephrine synthesized from intra-

3 3

ventricularly injected H-tyrosine, 3H-dopa or H-dopamine'but to a

lesser extent than the efflux of brain dopamine.

 

 

135

Since amphetamine induced release of more dopamine than nor-
epinephrine from the cat brain and over 95% of the dopamine was
released from brain structures lining the lateral ventricle, it is
obvious that gramphetamine acts at the nigrostriatal terminals in the
caudate nucleus to cause the release of endogenously synthesized

dopamine.

D. In an effort to monitor the release from the brain of

3

newly synthesized catecholamines, H-tyrosine of high specific

activity was continuously infused into the cerebroventricular system.

3

Radioactive catecholamines, which consisted primarily of‘ H-dopamine,

were identified in the ventricular perfusate during the perfusion of
3H-tyrosine. Intraventricular or intravenous administration of g:
amphetamine increased the efflux of labeled catecholamines newly or

3H-tyrosine. Over 96% of the amphetamine-

3

previously synthesized from

induced efflux of brain amines consisted of H-dopamine. After the

3

continuous infusion of H-tyrosine, approximately 94% of the total

labeled catecholamines in the brain was found in the caudate nucleus

3

and consisted of only H-dopamine. Thus, these results support the

data of the acute labeling experiments, suggesting that amphetamine

released endogenously synthesized dopamine from the caudate nucleus.

4

E. a-Methyltyrosine, at a concentration of 4x10" M ,

3

effectively blocked the synthesis of brain catecholamines from H-

tyrosine during the continuous intraventricular infusion of purified
3H-tyrosine (20 or 12.5 pc/ml, 0.2 ml/min). Therefore, the addition of

a-methyltyrosine to the perfusing solution of 3H-tyrosine completely

 

136

blocked the release of striatal dopamine induced by either intra-
ventricular (0.03 to 300 pg/ml) or intravenous administration of g;.

amphetamine (2 mg/kg, salt).

F. The addition of a-methyltyrosine (4x10'4M) to a perfusing
solution containing 3H-tyrosine failed to alter the initial response
to gramphetamine but slightly reduced the continuous release of 3H-
dopamine induced by geamphetamine. The addition of a-methyltyrosine
to the perfusing solution decreased the spontaneous efflux of dopamine.
a-Methyltyrosine (5x1o‘5M or 5x1o'4n) also failed to alter the
ability of gramphetamine to cause the release of brain 3H-norepinephrine
synthesized from 3H-dopamine and of 3H-dopamine after an intra-

3H-dopamine (5 pc).

ventricular injection of
Thus, a-methyltyrosine does not interfere with the g:
amphetamine-induced release from a "stored" pool of endogenously
synthesized or exogenously administered dopamine in 31:9, These
results also indicate that newly synthesized dopamine plays a more
important role than a stored pool of dopamine in the maintenance of
the spontaneous efflux of this amine and in the continuous amine

releasing action of geamphetamine.

G. The addition of gfamphetamine (0.3 pg/ml, equivalent to

3H-tyrosine in-

1.1x10'6M) to the perfusing solution at the start of
fusion increased the effluxof striatal 3H-dopamine 2 to 3 times over
the control. The amphetamine response was completely blocked by a-
methyltyrosine (4x10'4M) but not by reserpine (0.5 mg/kg, i.v., 2 hours

prior to the start of perfusion). In fact, gramphetamine released

137

more 3H-dopamine in reserpine-treated cats than in control cats.
After pretreatment with reserpine, intraventricular administration of
gramphetamine even at a low concentration of 0.3 pg/ml released more.
3H-dopamine into the ventricular perfusate than was retained in the
brain. Intravenous injection of increasing doses of gramphetamine 2
hours after the intravenous injection of reserpine (0.5 mg/kg) pro-

3H-dopamine during

duced a dose-related increase in the release of
the continuous infusion of 3H-tyrosine. The total amount of 3H-
dopamine in the perfusate was several times higher than that in the

brain.

 

Thus, these results suggest that amphetamine can release
striatal dopamine from a small newly synthesized pool which is

maintained mainly by the ongoing synthesis.

H. In control experiments, the intravenous injection of g;
amphetamine elicited concomitant increases in the release of brain
dopamine and the peripheral pressor response. After pretreatment
with reserpine, the peripheral pressor response but not the central
reSponse to gramphetamine was diminished.

The dissociation of the peripheral and central response of g;
amphetamine may be due to the fact that reserpine blocked the trans-
port mechanism of the storage granules and thus inhibited the synthesis
of norepinephrine but not of dopamine. Thus, the availability of
newly synthesized dopamine in the brain, but not of norepinephrine in
the periphery, following reserpine may account for the differential
effects of gramphetamine stimulation centrally and peripherally and

once more emphasizes the relative importance of brain dopamine for the

 

138
central stimulant properties of geamphetamine.

I. In conclusion, newly synthesized striatal dopamine plays

a more important role in the maintenance of the amine release by

3

.gramphetamine than does the mobilization of stored dopamine. H- '

3H-tyrosine in the nigrostriatal terminals

Dopamine synthesized from
can also be released by electrical stimulation of the fiber tracts of

these neurons in the lateral hypothalamus.

 

 

 

 

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