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INVOLVEMENT OF DOPAMINE AND 5—HYDROXYTRYPTAMINE NEURONAL

SYSTEMS IN THE BEHAVIORAL EFFECTS OF HALLUCINOGENS

By

Randall Lee Commissaris

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Pharmacology and Toxicology

1981

 

 

€//éfi”8 7

ABSTRACT

Involvement of Dopamine and 5—Hydroxytryptamine Neuronal
Systems in the Behavioral Effects of Hallucinogens

by

Randall Lee Commissaris

The effects of a number of hallucinogens and non—hallucinogenic
agents were examined in rats performing on a fixed ratio-40 (FR—40)
operant schedule for food reinforcement. Many psychoactive agents
disrupt this behavior. However, hallucinogens disrupt FR—40 respond-
ing uniquely, characterized by periods of non-responding or ”pausing"
interspersed between periods of responding close to the control rate.
A number of non-hallucinogenic psychoactive drugs examined disrupted
this behavior with a pattern of slowed and erratic intrasession re—
sponse rates rather than pausing. The development of a lO—second
pause interval counter allowed for the quantification of this dose—
dependent "pausing" produced by the hallucinogens. Since the non-
hallucinogens examined in these early studies produced increases in
"pausing" only at doses which decreased response rates dramatically,
the pause interval counter was used to classify agents as similar or
dissimilar to hallucinogens.

The neurotransmitter basis for the "pause" effect of the hallu—
cinogens was explored. Studies employing the neurotoxin 6—hydroxy—

dopamine, the catecholamine synthesis inhibitor a-methyljpftyrosine,

 

Randall Lee Commissaris
the neuroleptics haloperidol and chlorpromazine and the dopamine
releasing agent dfamphetamine were conducted. These treatments failed
to alter the pause-inducing effects of 2,S-dimethoxy—A—methylamphet—
amine (DOM) or gflysergic acid diethylamide (LSD), suggesting that
the "pause" effect does not appear to be mediated via brain catechol-
amine mechanisms.

A second line of investigation into the neurotransmitter basis
for the effects of the hallucinogens centered on the role of 5—hydroxy-
tryptamine (S—HT). Initial studies were conducted employing the
intraventricular administration of the neurotoxin 5,7-dihydroxytrypt-
amine (5,7-DHT) or systemic administration of the 5-HT synthesis
inhibitor pfchlorophenylalanine to decrease whole brain 5—HT concen-
trations. In these studies, depletion of whole brain S—HT potentiated
the effects of LSD, DOM and mescaline equally. These studies impli-
cated S—HT neurons in the mechanism of action of the hallucinogens and
suggested similarities in this mechanism.

In an effort to localize the site of the hallucinogen—S-HT
neuronal interaction, local injections of 5,7—DHT were made into
nucleus accumbens and septal nuclei. These failed to alter the "pause"
effect of DOM, LSD or mescaline. Injection of 5,7—DHT into the medial
forebrain bundle caused only moderate depletion of 5—HT in forebrain
areas, slightly potentiated the FR—4O disruption by LSD, attenuated
somewhat the influence of DOM, while not altering the "pause" effect
by mescaline.

A number of drug interaction studies were carried out using puta-

tive S—HT agonists and antagonists. Initial studies employing these

Randall Lee Commissaris
agents alone revealed two important findings: 1) the putative 5—HT
agonists quipazine and mfchlorophenylpiperazine (MCPP) produced a
dose—dependent disruption of FR—4O behavior characterized by ”pausing",
and 2) the putative S—HT antagonists methergoline and cinanserin alone
actually decreased the "pausing" observed in control sessions. Thus,
"pausing" produced by the hallucinogens may relate to S—HT agonistic
properties of these agents. The "pausing" produced by MCPP or quipa-
zine was found to be additive to the effects produced by DOM. Pre—
treatment with cinanserin or methergoline shifted the dose—response
curves of indolealkylamine (LSD—type) hallucinogens about 2— to 4—fold
to the right. These 5—HT antagonists (particularly methergoline)
caused a much greater shift to the right in the dose—response curves
for the phenethylamine (DOM—type) hallucinogens, however. Reducing
the methergoline pretreatment from 1.0 mg/kg to 0.1 mg/kg decreased
the extent of the shift in the DOM dose—response curve for disrupting
FR—4O responding. Methergoline antagonized the "pause" effects of
quipazine in a manner similar to that observed with DOM.

The results support a role of brain 5—HT receptors in the pattern
of disruption of FR—40 responding induced by the indole and phenethyl—
amine hallucinogens. They further suggest that the two classes exert
their actions on S—HT neuronal functions by somewhat different mecha—

nisms.

ACKNOWLEDGEMENTS

The author extends his sincere thanks to Dr. Theodore M. Brody,
Dr. Walter K. Beagley and Dr. Gerard L. Gebber of his graduate
committee for their support and constructive comments throughout
the course of these studies. The author extends a special thanks to
Dr. Kenneth E. Moore of his committee for his assistance throughout
these studies, particularly in their early stages. The author is
grateful for the assistance and consultation of a number of indivi—
duals throughout these studies, in particular Richard Alper, Lisa
Bero, John Gordon, Russell Owen, Charles Rewa and Barbara Stetler.
Lastly, the author sincerely thanks Dr. Richard H. Rech for his
advice, encouragement and support throughout the course of these

studies.

ii

 

 

ACKNOWLEDGEMENTS

LIST OF TABLES

LIST OF FIGURES

GENERAL INTRODUCTION

I.

II.

III.

STATEMENT

MATERIALS

I.
II.
III.
IV.
V.
VI.
VII.
VIII.

RESULTS

TABLE OF CONTENTS

 

 

Page
ii

vi

 

 

S—Hydroxytryptamine (S—HT) Neurons and the Effects of
Hallucinogens

A. Anatomy of 5—HT neurons

B. Hallucinogen interactions with S—HT neurons ———————

 

 

Dopamine (DA) Neurons and the Effects of Hallucinogens—
A. Anatomy of DA neurons
B. Hallucinogen interactions with DA neurons —————————

 

Techniques for Disrupting S—HT or DA Neuronal Activity—
A. Neurotoxin treatments
B. Interactions with neuroactive drugs ———————————————

 

 

 

1. Receptor antagonists
2. Receptor agonists
3. Synthesis inhibitors

 

 

OF PURPOSE

AND GENERAL METHODS

 

 

Behavioral Paradigm
Subjects
Apparatus

Behavioral Procedure
Stereotaxic Procedures
Neurochemical Analyses
Statistical Analyses
Drugs

 

 

 

 

 

 

 

 

I.

Quantitation of the Disruptive Effects of Hallucinogens
and Other Psychoactive Agents on Fixed Ratio-4O (FR-40)
Operant Responding with the Pause Interval Timer ———————

viii

O‘\

l3
l3
l6

l8
19
20

21
22

23

24

24
24
25

27
27

30

33

33

 

TABLE OF CONTENTS (continued)

RESULTS (continued)

II.

III.

UOUUS>

Introduction

 

 

Methods
. Results
. Discussion

 

 

The Role of Catecholamines in the FR—40 Disruptive

Effects of Hallucinogens
A. Introduction
B. Methods
Neurotoxin study
Drug interaction studies
C. Results
Neurotoxin study
Drug interaction studies
D. Discussion

1.
2.

l.
2.

 

 

 

 

 

 

 

 

 

Page

33
33
34
44

52
52
52
52
53
54
54
59
69

The Role of 5-HT Neurons in the FR—4O Disruptive Effects

of Hallucinogens
A. Neurotoxin Studies

1.
20

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Introduction
Methods
a. Intraventricular 5,7-DHT (5,7—DHT) -------
b. Administration of 5,7—DHT into specific
brain nuclei
1) 5,7-DHT administration into the
septum
2) 5,7-DHT administration into the
nucleus accumbens
c. Administration of 5,7-DHT into the
medial forebrain bundle (MFB) -----------
Results
a. Intraventricular 5,7—DHT
b. Administration of 5,7-DHT into specific
brain nuclei
1) 5,7—DHT administration into the
septum
2) 5,7—DHT administration into the
nucleus accumbens
c. Administration of 5,7—DHT into the MFB--
Discussion '
a. Intraventricular 5,7—DHT
b. Administration of 5,7-DHT into specific
brain nuclei
c. Administration of 5,7-DHT into the MFB——

iv

81
82
82
82
82

83
83
84
84
85
85
91
91
97
97
105
105

110
111

 

TABLE OF CONTENTS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

RESULTS (continued)
III. B. PCPA Studies
1. Introduction
2. Methods
3. Results
4. Discussion
C. Interactions Between Hallucinogens and Putative
5—HT Agonists
1. Introduction
2. Methods
3. Results
4. Discussion
D. Interactions Between Hallucinogens and Putative
S-HT Antagonists
1. Introduction
2. Methods
3. Results
4. Discussion
IV. Additional Behavioral Studies—
A. Introduction
B. Methods
1. Intraventricular 5,7-DHT and the effects of
various agents on punished and unpunished
responding
2. 5—HT agonists and antagonists and the effects
of hallucinogens on punished and unpunished
responding
C. Results
1. Intraventricular 5,7-DHT and the effects of
various agents on punished and unpunished
responding
2. 5—HT agonists and antagonists and the effects
of hallucinogens on punished and unpunished
responding
D. ' Discussion
SUMMARY AND GENERAL DISCUSSI N
BIBILIOGRAPHY

 

Page

112
112
112
113
121

121
121
123
123
129

129
129
132
133
148
154

154
154

154

157
158

158

166
176
178

192

Table

10

11

12

LIST OF TABLES

Administration parameters for FR—4O neurotoxin studies-

Drugs used in FR—40 operant studies

 

Relationship between changes in reinforcements and in—
creases in pausing induced by hallucinogens and non—
hallucinogenic psychoactive drugs

 

Effects of intraventricular 6-OHDA administration on
the concentrations of 5—HT, DA and NE in various brain
regions

 

Effects of vehicle or 6—OHDA administration on control
FRr4O operant response parameters

 

The effects of a—methylfpftyrosine on the characteris-
tics of FR—4O operant responding

 

The effects of chlorpromazine on FR—4O operant respond-
ing

 

Effects of intraventricular 5,7-DHT administration on
the concentrations of 5—HT and NE in various brain
regions

 

The effects of intraventricular or 5,7-DHT administra-
tion on control FR—40 operant response parameters ——————

The effects of 5,7—DHT administration into the septum
on the concentrations of 5-HT and NE in various brain
regions

 

The effects of 5,7—DHT administration into the septum
on the characteristics of FR—40 responding

 

The effects of 5,7—DHT administration into the nucleus
accumbens on the concentrations of 5-HT and DA in the
nucleus accumbens and striatum

 

vi

Page

28

31

45

55

56

66

72

86

87

96

98

101

 

LIST OF TABLES (continued)

Table

l3

14

15

16

17

18

19

20

21

22

23

Page

The effects of 5,7—DHT administration into the nucleus
accumbens on the characteristics of FR—4O Operant
responding 102

 

The effects of 5,7-DHT administration into the MFB on
regional brain amine concentrations 106

 

The effects of 5,7-DHT administration into the MFB on
the characteristics of control FRe40 operant responding 107

The concentrations of 5-HT, NE and DA in various brain
regions following administration of PCPA (100 mg/kg/
day) for 3 days (PCPA-DOM study) 118

 

The concentrations of 5—HT, NE and DA in various brain
regions following administration of PCPA (100 mg/kg/
day) for 3 days (PCPA-LSD study) 122

 

Relationship of changes in reinforcements to changes in
pausing induced by the putative S—HT agonists quipazine
and MCPP 128

 

Effects of cinanserin on the characteristics of FR—40
operant responding 134

 

Effects of methergoline on the characteristics of FR—40
operant responding 135

 

Effects of intraventricular 5,7-DHT administration on
the concentrations of 5-HT, DA and NE in various brain
regions (conflict procedure) 159

 

The effects of 5,7-DHT treatment on control conditioned
suppression performance 160

 

The effects of methergoline on conditioned suppression
responding 169

 

vii

 

Figure

1

7a

7b

10

11

12

13

LIST OF FIGURES

 

 

 

Page
The chemical structures of the indolealkylamines LSD,
DMT and 5—HT 2
The chemical structures of the phenethylamines DOM,
mescaline and DA 4
Anatomical distribution of 5—HT neurons ———————————————— 7
Anatomical distribution of DA neurons 14
Cumulative recordings illustrating the effects of d:
amphetamine and LSD on FR—40 operant responding ———————— 35

Quantitation of the effects of LSD and dfamphetamine on
the characteristics of FR—40 operant responding ———————— 38

The effects of hallucinogens on FR—40 operant respon—
ding 40

 

The dose—relationships of LSD and DOM to the longest
and second—longest periods of non—responding produced
in FR—4O sessions 42

 

The effects of LSD on FR—40 responding alone or in com-
bination with a threshold dose of dfamphetamine ———————— 46

The effects of DOM on FR—40 responding alone or in com—
bination with a threshold dose of dfamphetamine ———————— 48

The effects of DOM on FR—40 responding alone or in com—
bination with a threshold dose of LSD or mescaline ————— 50

Cumulative recordings illustrating the effects of
saline, d—amphetamine and DOM on FR—40 responding in

one vehiEle pretreated and one 6—OHDA-treated rat —————— 57

The effects of DOM on FR—40 responding in vehicle- and

 

 

6—OHDA—treated rats 60

The effects of LSD on FR—40 responding in vehicle— and

6-OHDA—treated rats 62
viii

LIST OF FIGURES (continued)

Figure

14

15

16

17

18

19

20

21

22

23

24

25

26

The effects of dfamphetamine on FR—40 responding in
vehicle— and 6—OHDA—treated rats

 

The effects of LSD on FR—4O responding alone or in com—
bination with a threshold dose of a-methyl—pftyrosine——

The effects of dfamphetamine on FR—40 responding alone
or in combination with a threshold dose of d—methyl—pf
tyrosine

 

The effects of DOM on FR—40 responding alone or in com—
bination with a threshold dose of chlorpromazine or
haloperidol

 

The effects of LSD on FR—40 responding alone or in com—
bination with a threshold dose of chlorpromazine ———————

The effects of dfamphetamine on FR—40 responding alone
or in combination with a threshold dose of chlorproma-
zine

 

The effects of gfamphetamine on FR—40 responding alone
or in combination with 1.0 mg/kg chlorpromazine ————————

Cumulative recordings from one vehicle—treated subject

and one intraventricular 5,7—DHT—treated subject illus-
trating the effect of saline, LSD (100 pg/kg) and phe—

nobarbital (FEB; 25 mg/kg) administration on FR—40 re—

spending

 

The effect of LSD, DOM and mescaline on FR—4O operant
responding in vehicle— or intraventricular 5,7—DHT—
treated rats

 

The effects of phenobarbital on FR-40 operant respond-
ing in vehicle— and intraventricular 5,7—DHT—treated
rats

 

The effects of LSD and DOM on FR—40 responding in rats
treated with 5,7—DHT or its vehicle into the septum——-—

The effects of hallucinogens on FR—40 responding in
rats treated with 5,7—DHT or its vehicle into the
nucleus L

 

The effects of hallucinogens on FR—40 responding in
rats treated with 5,7—DHT or its vehicle into the MFB——

Page

64

67

70

73

75

77

79

89

92

94

99

103

108

 

 

LIST OF FIGURES (continued)

Figure

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

 

 

 

 

 

 

 

 

Page
The effects of PCPA treatment on the FR—40 response to
DOM 114
Cumulative recordings illustrating the response pat—
terns of four rats (B—l, B—2, A—l, A—2) receiving the
schedule of drug treatments as indicated in Figure 27—— 116
The effects of PCPA pretreatment on the FR—40 response
to LSD 119
The effects of quipazine on the characteristics of FR-
40 responding 124
The effects of MCPP on the characteristics of FR—40
responding 126
The effects of DOM on FR—40 responding alone or in com-
bination with threshold doses of quipazine and MCPP-——— 130
Cinanserin antagonism of the effects of LSD and DOM on
FR—40 responding 136
Cumulative recordings illustrating the effects of
various treatments on FR—40 operant responding ————————— 139
Methergoline antagonism of the effects of indolealkyl-
amine hallucinogens 142
Methergoline antagonism of the effects of phenethyl-
amine hallucinogens 144
The effects of methergoline on the disruption of FR—40
operant responding produced by dfamphetamine and pheno—
barbital 146
Antagonism of the effects of DOM by methergoline ——————— 149
Antagonism of the effects of quipazine by methergoline— 151
The effects of pentobarbital on conditioned suppression
of drinking in rats before and after 5,7-DHT treatment— 162
The effects of methaqualone on conditioned suppression
of drinking in rats before and after 5,7—DHT ——————————— 164

 

 

LIST OF FIGURES (continued)

Figure

42

43

44

45

 

 

Page
The effects of LSD and DOM on conditioned suppression
of drinking in rats before and after 5,7-DHT ——————————— 167
Antagonism of the effects of LSD on punished and un—
punished responding by methergoline 178
Antagonism of the effects of DOM on punished and un-
punished responding by methergoline 172
Antagonism of the effects of quipazine on punished and
unpunished responding by methergoline —————————————————— 175

xi

 

GENERAL INTRODUCTION

Hallucinations often occur in persons with certain mental dis—
orders. They can also be drug—induced. The precise mechanism(s) for
the production of hallucinations from either cause is unclear, al—
though interactions with dopamine (DA) and/or 5—hydroxytryptamine (5—
HT) neurons in the brain have often been proposed (see reviews by
Brawley and Duffield, 1972; Jacobs, 1978).

The most specific types of hallucinogens are divided into two
general classes based on their chemical structures. Indolealkylamine
hallucinogens, of which dflysergic acid diethylamide (LSD) and N,N—
dimethyltryptamine (DMT) are members, resemble the indolealkylamine
neurotransmitter 5—HT (Figure 1). 2,5—Dimethoxy—4—methy1amphetamine
(DOM) and mescaline are members of the phenylethylamine hallucinogen
class; members of this class resemble the catecholamine neurotrans—
mitter DA (Figure 2). Because of the similarities between the struc—
tures of the hallucinogens and these neurotransmitters, it is tempting
to speculate that the effects of the indolealkylamine and phenylethyl—
amine hallucinogens are mediated through 5—HT and DA neuronal systems,
respectively. However, considerable behavioral experimental evidence
has suggested that agents of both classes produce effects related to
both 5-HT and DA neuronal interactions (Brawley and Duffield, 1972;

Jacobs, 1978).

Figure 1. The chemical structures of the indolealkylamine hallucino—
gens d—lysergic acid diethylamide (LSD) and N,N—dimethy1tryptamine (DMT)
and the indolealkylamine neurotransmitter 5—hydroxytryptamine (5—HT) .

 

 

C H _
2 5 N CH3
NHZ
LSD \
1
CH3\ ICH3 N
N
5-HT
» \
N

DMT

 

Figure 1

 

 

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6
In addition to the phenethylamine and indolealkylamine halluci—

nogens, atropine—like anticholinergic agents have been reported to
produce hallucinations in man (Byck, 1975). The effects of these
agents differ from the "classical" hallucinogens in that the atropine—
like compounds produce hallucinations characterized by confusion,
delusions and memory disturbances. The effects of these atropine—like
agents will not be examined in these studies. Drugs in other classes
may also induce hallucinations under certain conditions. However,
their primary pharmacology generally focuses on some other effects on

bodily functions; therefore, they will not be considered here.

I. 5—HT Neurons and the Effects of Hallucinogens

A. Anatomy of 5-HT Neurons (Figure 3)

 

Cell bodies of 5—HT neurons in the brain are located pri—
marily in the dorsal and median raphé nuclei of the brainstem (Cooper
gt_al,, 1974). Axons from many of these cells form a major portion of
the medial forebrain bundle (MFB; Lorden_gt.al., 1979), which projects
to many areas of the brain receiving S—HT input.

B. Hallucinogen Interactions with S—HT Neurons

Alterations in the activity of 5—HT neurons and receptors
have been implicated in the mechanism of action of hallucinogens.
Studies demonstrating the antagonism of 5—HT—induced contractions of
gut strips by LSD in_yit£g led to the early hypothesis that LSD
produced hallucinations by acting as an antagonist of 5—HT receptors

in the brain (Gaddum, 1953; Wooley and Shaw, 1954). Since S—HT is

generally regarded as having an inhibitory modulating effect on many

 

 

 

 

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9

areas of the brain (Aghajanian and Wang, 1978; Bloom SE Ei" 1972;
Haigler and Aghajanian, 1977), the putative S—HT antagonist could
produce hallucinations via disinhibition. Recent evidence, as dis-
cussed below, has failed to provide much support for this hypothesis.

Microiontophoretic and electrophysiological studies by
Aghajanian and co—workers (Aghajanian_etlal., 1970, 1972, 1975; Agha—
janian and Haigler, 1974, 1975; DeMontigny and Aghajanian, 1977;
Haigler and Aghajanian, 1973, 1977) have suggested that LSD is an
agonist at 5—HT receptors on both the cell bodies of neurons in the
raphé nuclei (autoreceptors) and on postsynaptic sites at the projec—
tions of these neurons. The effects of systemically administered LSD
are greatest at these autoreceptors, where they inhibit S—HT neuronal
activity (Aghajanian §£_a1,, 1975; Aghajanian and Haigler, 1974, 1975;
DeMontigny and Aghajanian, 1977; Haigler and Aghajanian, 1973, 1977).
Brom—LSD, a non-hallucinogenic analogue of LSD, is devoid of this
action on 5—HT autoreceptors (Aghajanian, 1976). Many other halluci-
nogens have been shown to activate, albeit indirectly, the 5—HT
autoreceptors to decrease 5—HT neuronal activity (Aghajanian E£.§l::
1970; Haigler and Aghajanian, 1973). It has been proposed, therefore,
that the effects of hallucinogens are due to activation of 5—HT auto—
receptors which inhibits 5-HT neuronal activity and disinhibits other
areas of the brain.

Recent reports by Trulson and co—workers, however, have sug—
gested that activation of 5—HT autoreceptors may not be responsible
for producing the behavioral effects of hallucinogens. Chronic admi—

nistration of LSD has been reported to produce rapid tolerance (3—5

 

10

days) to the behavioral effects of LSD and other hallucinogens (Freed—
man gt_§13, 1963; Appel and Freedman, 1968; Winter, 1971; Rech, 1975).
Trulson gt 31. (1977a) found that chronic administration of LSD to
rats in doses which have been reported to produce dramatic tolerance
as measured by a number of behavioral tests did not change the effects
of LSD on the activity of single cells in the midbrain raphé nucleus.
These studies have been repeated in unanesthetized, freely—moving
cats (Trulson and Jacobs, 1978), in which it was reported that the
aberrant grooming patterns produced by LSD dissipated over the course
of repeated administrations of the drug (tolerance). However, the
effects of LSD to inhibit raphé unit activity in these same animals
persisted. These data suggest that the behavioral effects of hallu—
cinogens may not be mediated solely by the activation of 5—HT auto—
receptors. The results of work by Rogawski and Aghajanian (1979) have
further impugned the raphé inhibition hypothesis as the theoretical
basis for the mechanism of action for the hallucinogens. These latter
investigators found that the non—hallucinogenic LSD analogue lisuride
inhibits raphé neuronal activity similar to LSD. Unfortunately, in
these studies the effect of lisuride on the activity of post—synaptic
5—HT neurons was not examined. This is a critical omission since the
raphé inhibition hypothesis was based on the relative potency of LSD
for these two sites (autoreceptors vs. post—synaptic receptors).

Behavioral studies by Minnema gt_§1, (1980) have cast even
further doubt on the raphé inhibition hypothesis. These investiga—

tors, using a drug—discrimination technique (described below), have

 

11
found that administration of LSD directly into the median/dorsal raphé
produces surprisingly weak discriminative effects in subjects trained
to discriminate systemic LSD from saline. Presumably, if raphé
autoreceptor activation is sufficient for the discriminable effects of
LSD, then extremely low doses would be required by this local applica—
tion. In defense of the technique, similar studies employing the
administration of morphine have shown that minute quantities of morphine
placed into the periaqueductal gray area of the brain (presumed site
of morphine action) in subjects trained to discriminate systemic
morphine from saline produces lever—pressing almost exclusively on the
morphine-bar (Krynock and Rosecrans, 1979). These reports also suggest
that the raphé inhibition hypothesis for the behavioral effects of
hallucinogens is questionable.

Considerable evidence has accumulated to suggest that hallu-
cinogens act as postsynaptic agonists at 5—HT receptors. Much of the
behavioral evidence for the 5-HT agonistic actions of hallucinogens
comes from stimulus control experiments in the laboratories of Appel,
Rosecrans and Winter (Cameron and Appel, 1973; Winter, 1974; Schecter
and Rosecrans, 1972). In a two—bar test cage rats were trained to
discriminate between the effects of a hallucinogen and saline. When
a hallucinogen was administered, bar pressing on bar A produced food
reinforcement; when saline was administered, bar pressing on bar B
produced food reinforcement. Rats trained on this task will perform
at a level of at least 95% correct responses. To summarize the
results of many recent studies, rats trained to discriminate the
effects of an hallucinogen (of either class) from those of saline

generalize universally to all phenethylamine and indolealkylamine

12
hallucinogens examined but not to the non—hallucinogens dfamphetamine,
apomorphine and methylphenidate (Glennon 35 gl., 1979, 1980; Schechter
and Rosecrans, 1972; Winter, 1975, 1978, 1980; Silverman and Ho, 1980;
Kuhn £5 31., 1978; Shannon, 1980; Jarbe, 1980; Tilson e£_a1., 1975a;
Browne and Ho, 1975). These effects are selectively blocked by the
putative 5—HT antagonists cinanserin, methysergide, methiothepin and
BC—105, but not by the DA antagonists butaclamol and haloperidol
(Glennon_g£.al., 1979; Winter, 1969, 1978, 1980; Kuhn §£_a1,, 1978;
Silverman and Ho, 1980; Browne and Ho, 1975).

Studies by Andén g g. (1968; 1971; 1974) have shown that
the exaggerated extensor reflex in reserpine—treated rats (specific
for 5—HT agonists) can be produced by the hallucinogens LSD, DMT, DOM
and mescaline. Joseph and Appel (1977) reported that the behavioral
effects of LSD on operant responding are potentiated following deple—
tion of 5—HT produced by either pfchlorophenylalanine (PCPA; 5—HT
synthesis inhibitor) or intraventricular administration of 5,7—di—
hydroxytryptamine (5,7—DHT), an agent which selectively destroys 5—HT
neurons in the brain. Since recent reports have demonstrated that
"denervation supersensitivity" occurs in the 5—HT system following
5,7-DHT administration (Nelson gt 31., 1978), these data also may
support a 5—HT agonist theory for the mechanism of action of LSD.
Thus, there is considerable evidence to propose that hallucinogens may
act as agonists at 5—HT receptors in the brain.

Hallucinogens have also been reported to increase responding

that is normally suppressed by punishment (Schoenfeld, 1976). This

 

l3

influence is not nearly as prominent as with the classical ”anti—
anxiety" agents of the benzodiazepine and barbiturate classes, but the
effects are reproducible. These effects have been suggested to relate
to interactions of hallucinogens with 5—HT neurons (Schoenfeld, 1976).

Further evidence for the 5-HT agonistic actions of halluci—
nogens comes from studies with the putative 5—HT agonist, quipazine.
This compound produces head twitches in mice similar to LSD (Vetulani
g£_§1,, 1980) and has been shown to produce disruptions of complex
operant behavior in rats (multiple schedule: fixed ratio-15, fixed
interval—60 seconds) similar to the disruptions produced by LSD
(Poling and Appel, 1978). In addition, stimulus control experiments
by Kuhn et a1. (1978) have indicated that the cue produced by hallu—
cinogens generalizes to quipazine. Quipazine has also been reported
to produce stimulus cues; similarly, this cue has been shown to gener—
alize to hallucinogens and to be antagonized by the 5-HT antagonists
BC—105, cinanserin, cyproheptadine, methiothepin and methysergide
(White 3,531., 1977; Winter, 1979).

In summary, evidence from a number of studies has suggested
that hallucinogens of both the phenethylamine and indolealkylamine

classes produce effects that are mediated via S-HT neuronal systems.

11. DA Neurons and the Effects of Hallucinogens

A. Anatomy of DA Neurons (Figure 4)

 

In contrast to the 5—HT neuronal system, in which axons
radiate out from the cell bodies in the raphé nuclei to innervate

nearly all areas of the brain, the DA neuronal pathways are composed

 

 

 

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16
of three distinct systems, the nigrostriatal, the tuberoinfundibular
and the mesolimbic systems. Each DA system is characterized by the
distinct location of the cell bodies and the distinct structure(s)
which are innervated.

DA neurons in the zona compacta of the substantia nigra give
rise to axons which innervate the striatum. This nigrostriatal DA
system has been implicated in the stereotypic behavior observed follow—
ing agents which cause activation of DA receptors. DA—containing
neurons are also found in the arcuate nucleus of the hypothalamus,
which project to the median eminence; these neurons release DA into
the hypophyseal portal bloodstream; this DA acts on the anterior pitui—
tary to inhibit the release of prolactin. The last DA system is the
mesolimbic system; cells from the ventral tegmental region of the
brain project to a number of areas including the septum, the amygdala,
the olfactory tubercles and the nucleus accumbens. These regions are
part of the limbic system and are presumed to be important in the
control of emotions. It is obviously this mesolimbic DA system which
is of greatest interest in investigations on the effects of hallucino—
gens.

B. Hallucinogen Interactions with Dopamine Neurons

 

There is a good deal of evidence to suggest that hallucino—
gens can act as agonists at DA receptors in the brain. Hallucinogens
have been reported to stimulate motor activity and produce stereotyped
behavior (Koella EE.§l3, 1964; Tilson_gt.al., 1975b; Yamamoto and
Ueki, 1975). Both of these functions are presumed to be regulated by

DA neuronal activity. Moreover, the use of neuroleptics (DA antagonists)

 

17
in the clinical treatment of "bad trips" produced by hallucinogens
(Jaffe, 1975) suggests that the effects of hallucinogens may be
mediated through DA neurons. In addition, Marrazzi and co—workers
(Halasz 25 al., 1969; Marrazzi and Huang, 1979) have shown that the
inhibition of cortical unit firing produced by the intravenous admi—
nistration of LSD is attenuated by the neuroleptic chlorpromazine.

There is evidence to indicate that hallucinogens can act as
both direct and indirect agonists at DA receptors. Evidence that LSD
is a direct agonist at DA receptors in the brain comes from studies in
which some of the effects of LSD were shown to be pharmacologically
similar to apomorphine: 1) the reversal of reserpine-induced depres—
sion of motor activity, even after treatment with the putative S—HT
antagonist methysergide (Menon et_al,, 1977); 2) contralateral turning
following unilateral pretreatment with 6—hydroxydopamine (6-OHDA; an
agent which selectively destroys DA neurons) into the substantia nigra
(Trulson_e£‘al., 1977b); 3) autoinhibition of the activity of DA cells
in the substantia nigra (Christoph §£_al., 1977). In addition,
Nichols (1976) has noted strong similarities in the chemical struc-
tures of LSD and apomorphine.

Evidence regarding other hallucinogens suggests possible
indirect agonistic effects at DA neurons. Mescaline and DOM produced
ipsilateral turning in animals with unilateral 6—OHDA lesions (Trulson
§5_g1,, 1977b). Moreover, Vrbanac et a1. (1975) have shown that DOM
releases catecholamines from the brain. In producing these effects
these hallucinogens resemble dfamphetamine, a compound known for its

actions as an indirect DA agonist (Rech and Stolk, 1970; Chieuh and

 

 

18
Moore, 1973) and also capable of producing hallucinations in man under
chronic conditions of abuse (Innes and Nickerson, 1975; Nielsen £5
21,, 1980).

Thus, there is experimental data to suggest that both 5—HT
and DA neurons may be important in mediating the behavioral effects of
the indolealkylamine and phenylethylamine hallucinogens. The proposed
mechanisms are not mutually exclusive, however, as the effects of
these agents may be mediated by influences on both of these neuronal

systems.

III. Techniques Used for Disrupting 5—HT or DA Neuronal Activity

Several techniques have been used to disrupt 5—HT or DA neuronal
activity. These procedures include electrolytic lesions, surgical
procedures such as axotomy or surgical isolation, and administration
of short—acting neuroactive drugs (receptor agonists and antagonists;
synthesis inhibitors). More recently, the neurotoxins 6—OHDA and 5,7—
DHT have been used to produce relatively long—lasting and specific
destruction of catecholamine and 5—HT nerve terminals, respectively
(Bloom g£_a1,, 1969; Breese and Traylor, 1970, 1971; Uretsky and Iver—
sen, 1970; Baumgarten e£_alf, 1973; BjBrklund et_al,, 1974, 1975;

Daly EE_§1:, 1974).

Electrolytic lesions or surgical procedures (axotomy, surgical
isolation) can be used to alter neuronal activity, but these tech-
niques generally do not destroy only one neuronal system. The poten—
tial for nonspecific effects in the techniques mentioned above pre-

cludes their use in studies designed to determine the role(s) of DA

 

 

l9

and 5—HT in the manifestation of the behavioral effects of hallucino-

gens.

A. Neurotoxin Treatments

 

Recent developments in the use of the neurotoxins 5,7—DHT
and 6—OHDA have greatly enhanced their selective toxicity for 5-HT and
DA neurons, respectively. The neurotoxins are initially taken up by
presynaptic nerve terminals, presumably by the amine reuptake mecha—
nisms in these neurons. Once inside the presynaptic terminal, they
produce destruction of the terminal by a mechanism which is still
unknown. Since uptake into the presynaptic terminal is a critical
step in the neurotoxicity of these agents, selective monoamine reup—
take blocking agents would be expected to alter the extent of disrup—
tion by preventing the uptake of the neurotoxin into the neurons.
Perhaps the best example of this effect is the use of the tricyclic
antidepressants desipramine and protriptyline to protect norepinephrine
(NE) neurons from the neurotoxicity of both 5,7—DHT and 6—OHDA (Bj6rk—
lund et_§1., 1975; Gerson and Baldessarini, 1975; Hole et_al., 1976;
Breese and Howard, 1971). Another manipulation which has been reported
to enhance the cytotoxicity of the neurotoxins is treatment with a
monoamine oxidase inhibitor. Monoamine oxidase is the intraneuronal
enzyme responsible for breakdown of monoamines following their reup—
take into the presynaptic terminal. It is thought that this enzyme may
also be capable of metabolizing the neurotoxins as well, thus reducing
their activity. Pargyline, a monoamine oxidase inhibitor, has been
shown to enhance the cytotoxicity of these neurotoxins (Breese et 31.,

1978) presumably by delaying their metabolism to less active agents.

1l‘c-|cl l-l-||
o '

 

 

20

These agents do not directly affect the post—synaptic neuron;
however, compounds which activate post-synaptic receptors (receptor
agonists, amino acid precursors) have greater activity in neurotoxin—
treated animals as compared to controls (Ungerstedt, 1971; Daly £5
31,, 1974; Stewart et_al,, 1976; Trulson_et‘al., 1976). In agreement
with this "behavioral supersensitivity" is the observation that the
number of radiolabelled binding sites (receptor sites) increases
following administration of 5,7-DHT (Nelson EE.21:’ 1978). Converse—
ly, agents which require a functional (releasable) presynaptic terminal
in order to produce their effects (indirect agonists) are considerably
less effective following these neurotoxin treatments.

B. Interactions with Neuroactive Drugs

 

As mentioned above, a number of agents with established
mechanism(s) of action used clinically and/or experimentally can be
employed as aides in determining the involvement of 5—HT and/or DA
neurons in the behavioral effects of hallucinogens. These agents
include receptor antagonists, receptor agonists and drugs that inhibit
the synthesis of 5—HT or DA.

1. Receptor antagonists. Haloperidol and chlorpromazine

 

are both clinically effective antipsychotic neuroleptics which presu—
mably act as DA receptor antagonists (Byck, 1975). The list in the
experimental literature of putative 5—HT antagonists is long, but
selecting potent and selective antagonists at central 5—HT receptors
is a difficult task. A number of compounds including cinanserin,

mianserin, methysergide and cyproheptadine have been reported to

 

 

21
possess peripheral S-HT antagonist properties, but reports regarding
their central 5-HT antagonistic actions are inconsistent (Jacoby_gt
31,, 1978; Vargaftig g£_a1,, 1971). In addition, these agents produce
various non-specific effects, presumably through interactions with
other neuronal systems. Methergoline (l—methyl—8-carbobenzyloxyamino—
methyl-lOa-ergoline) recently has been reported to be a potent and
relatively selective central 5—HT antagonist (Samanin §£_a1,, 1977,
1979, 1980; Fuxe 35 31., 1975).

2. Receptor Agonists. Direct activation of DA receptors

 

by the compounds piribedil and apomorphine produces, at low to moder—
ate doses, stereotyped behavior and aphagia (McGeer e5 31., 1978).
Since these two effects greatly interfere with operant responding for
food reinforcement, these agents are likely to be ineffective as tools
to study the neuronal basis for the effects of hallucinogens in a FR-
40 appetitive procedure. The psychomotor stimulant dfamphetamine has
been proposed to produce many of its behavioral effects by enhancing
the release of DA from nerve terminals (Rech and Stolk, 1970; Chieuh
and Moore, 1973). Although this agent also produces aphagia and
stereotyped responding, these effects generally require doses greater
than those which produce stimulation of motor activity and disruption
of operant behaviors (Fibiger_e£.a1., 1973).

Quipazine has been proposed to act as a 5—HT agonist
(Rodriguez e£_a1,, 1973), although a number of additional neurochemi—
cal effects have been reported to be produced by this agent. As
described above, this agent produces a number of behavioral effects

similar to the hallucinogens (Vetulani EE.§£°’ 1980; White SE 31.,

 

22
1977; Winter, 1978, 1979). mehlorophenylpiperazine (MCPP) is a
recently developed tool for studying 5—HT neurons. This agent is an
extremely effective drug in suppressing feeding behavior, an effect
which is presumably related to S—HT agonistic neuronal activity (Sama—
nin e£_§1,, 1979, 1980).

3. Synthesis Inhibitors. Inhibition of catecholamine

 

synthesis by a—methyljpftyrosine (aMT) is an extremely selective and
potent technique for determining the role of stored catecholamines in
mediating the effects of a particular agent (Rech 35 31., 1966;
Spector 31.31,, 1967). Although NE synthesis is also altered by aMT
treatment, this effect on NE neurons usually requires larger doses
over a longer period and is less dramatic than the effects of aMT on
DA synthesis. This differential susceptibility presumably relates to
the relatively slow turnover of brain NE compared to that of DA
(Demarest EE_a1,, 1979).

The phenylalanine analogue PCPA has been used as an
effective tool for selectively decreasing stored 5—HT concentrations
(Koe and Weissman, 1966) and to examine the role of 5—HT neurons in a
number of behaviors (Robichaud and Sledge, 1969; Tenen, 1967; Geller

and Blum, 1970; Tilson and Rech, 1974).

 

 

STATEMENT OF PURPOSE

The purpose of the present study was to determine if 5—HT or DA
neuronal systems play a role in certain aspects of the behavioral
effects of hallucinogens in rats. The behavioral effects of both
indolealkylamine and phenethylamine hallucinogens were examined in
untreated animals and in those in which 5—HT or DA neuronal activity
was selectively disrupted by systemic pretreatment with 5—HT or DA
synthesis inhibitors, receptor agonists and antagonists and by the

intracerebral administration of the neurotoxins 6—OHDA and 5,7—DHT.

23

 

MATERIALS AND GENERAL METHODS

I. Behavioral Paradigm

 

Since the prominent effects of hallucinogens in man are percep—
tual and subjective in nature, assessment of the effects of these
agents in non-human subjects is extremely difficult. Although hallu-
cinogens have been shown to produce a number of behavioral effects in
experimental animals, many of these effects are unsuitable as models
of the human pharmacology because they are produced only by high doses
or are not easily quantifiable. One behavioral test which satisfies
these criteria of sensitivity and quantifiability is the fixed ratio
(FR) schedule of operant responding in rats, originally described by
Ferster and Skinner (1957). Most of the behavioral data presented
here are based on the disruptive effects of various drugs on respond—
ing in the FR—40 operant paradigm. The question of selectivity of
this test in differentiating the effects of hallucinogens from those
of a number of non—hallucinogenic psychoactive agents has been a
source of contention in previous studies (Appel and Freedman, 1965;

Rech g£_a1,, 1975) and is also addressed in this study.

11. Subjects

Subjects in all of the FR—40 studies were male Sprague-Dawley

rats (Spartan Farms, Haslett, MI). These subjects were housed singly

24

 

25
in a room with a 12—hour day—night light cycle (lights on 0700—1900
hr) and maintained at approximately 70-80 percent of their free—

feeding weights.

III. Apparatus

Behavioral testing was conducted between 0700 and 1900 hr in four
standard operant chambers (LVE 143—20—215) equipped with food pellet
dispensers. These chambers were located in sound—attenuating boxes and
contained a single lever which required a force of 10—15 g to acti—
vate. All experimental events were controlled by electromechanical
programming circuits and responses were recorded on electromagnetic
counters and cumulative recorders. Two parameters were monitored in
the operant sessions: (1) the number of reinforcements obtained, a
reflection of the average response rate, and (2) the periods of non—
responding during operant sessions. To accomplish this second measure—
ment, a lO—second "pause" interval counter was incorporated into the
program as described below.

Each response by the subject reset a 10—second timer. If the
animal responded before 10 seconds elapsed, the timer reset and the
program continued. If the animal failed to respond during this 10—
second interval, a count was registered and the timer automatically
reset. Therefore, the number of counts registered by the pause inter—
val timer was an index of the extent of non—responding in terms of
cumulated 10 second pause intervals.

The pause interval in these studies was selected to be 10 seconds
for a number of reasons. First, 10 seconds is about the limit of the

resolution possible on the cumulative records. Second, with an

 

26
interval shorter than 10 seconds, visual double—checking of the pause
intervals against a cumulative record would not have been possible.
Moreover, in pilot studies utilizing shorter intervals (5—sec, l—sec),
the "turnaround" time of the electromechanical programming equipment
became prohibitively slow and introduced error into the measure. This
effect on turnaround time often resulted in the development of a true
negative; 12g., a situation in which the pausing of an animal is
underestimated by the number of pause intervals produced due to the
slow turnaround time of the timer. It was also found that longer
durations (9&5'9 20 seconds) failed to detect many of the "mini—pauses"
which occurred during control sessions (usually following the delivery
of the food pellet). Since baseline values in this measure are the
major advantage of this technique over the "zero baseline” techniques
of a number of researchers in this field (Kovacic and Domino, 1976;
Kovacic et_a1,, 1978; Ruffing £5 31., 1979; Ruffing and Domino, 1980)
it was felt that the intervals longer than 10 seconds offered a less

refined analysis (another true negative).

IV. Behavioral Procedure

 

The subjects were first trained to respond on a continuous rein—
forcement (CRF) schedule for food reinforcement (45 mg Noyes Pellets).
Daily sessions were 40 minutes in duration. Each animal was run at
the same time of day and in the same cage seven days a week. After
the subjects were responding on the CRF schedule (approximately 2-4
days) a FR schedule was introduced and gradually (2—3 weeks) increased

to FR—40. After an additional 2—3 weeks of control FR—40 sessions,

behavioral testing was begun.

 

 

27
In all experiments the order of doses and drugs administered was
randomized for each subject. All test drugs were administered intra—
peritoneally. In these studies drug test days were preceded by at
least three non—drug days, unless otherwise specified, to avoid the

development of tolerance.

V. Stereotaxic Procedures

 

In a number of studies the neurotoxins 5,7—DHT and 6—OHDA were
administered into various brain regions in order to destroy 5—HT or cate—
cholamine neurons, respectively. These subjects were then trained to
perform in the FR—40 paradigm and the effects of various hallucinogens
and non—hallucinogens were determined. Subjects in the neurotoxin
studies weighed 150—200 gm at the time of neurotoxin administration.
Prior to stereotaxic surgery all subjects were anesthetized with
Equithesin (3 ml/kg); neurotoxins or vehicle (0.1% ascorbate in
saline) were administered through a 30 gauge stainless steel cannula.
All neurotoxin administrations were done bilaterally, with the excep—
tion of the septal—lesion study (unilateral structure). Table 1 lists
the stereotaxic coordinates (Konig and Klippel, 1967), neurotoxin
concentrations, volumes, infusion rates and pretreatments used in all

of the neurotoxin studies to be presented.

VI. Neurochemical Analyses

 

Following completion of behavioral testing in the neurotoxin
studies and studies involving PCPA administration the subjects were

sacrificed, their brains were removed and the concentrations of 5—HT,

 

28

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moumnflwuooo
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H mgm<H

 

29
NE, DA and the DA metabolite dihydroxyphenylacetic acid (DOPAC) in
various regions were determined. Concentrations in large brain
regions (1.3., cortex, hippocampus, hypothalamus and striatum) were
analyzed by fluorimetric procedures (Curzon and Green, 1970; Chang,
1964); these analytical procedures were used in the intraventricular
neurotoxin studies, in the study employing the administration of 5,7—
DHT into the medial forebrain bundle, and in the studies employing
PCPA administration. In studies employing the administration of 5,7—
DHT into discrete brain regions (where tissue mass limits quantifica—
tion using fluorimetric procedures; e,g,, septum and nucleus accumbens),
the concentrations of 5—HT, NE, DA and DOPAC were determined by a
modification of the high performance liquid chromatography (HPLC) tech—
nique of Felice et a1. (1978) using electrochemical detection (Lyness
eE_a1,, 1980). In the study employing 6—OHDA administration in the
nucleus accumbens, DA concentrations in this structure and the sub—
stantia nigra were determined using the radioenzymatic procedure of
Umezu and Moore (1979); 5—HT concentrations in this instance were

analyzed by the HPLC technique cited above for the septum.

VII. Statistical Analyses

 

Statistical analyses were conducted as described by Klugh (1974).
Drug effects were assessed by comparing the data from test days to the
average of the three days prior to the test day (baseline). The
Student's Eftest for paired data was used to evaluate the effects of
individual doses of the drugs. Dose—response relationships were

compared by analysis of variance (in a block design, when appropriate).

 

 

30
The effects of the neurotoxins and PCPA on regional biogenic amine
concentrations were determined by Student's Eftest for unpaired data.
In all statistical evaluations p<0.05 was used as the criterion for

statistical significance.

VIII. Drugs
The drugs employed in these studies are listed in Table 2, which

indicates the supplier and chemical form used.

 

Drugs Used in the FR—40 Operant Studies

31

TABLE 2

 

 

Drug Abbrev. Dosage Form Supplier
alpha—methyl—p- uMT methyl ester Merck, West
tyrosine Point, PA
dfamphetamine de sulfate Sigma Chemical,
St. Louis, MO
chlorpromazine CPZ hydrochloride Smith, Kline and
French Labs,
Philadelphia
cinanserin CIN hydrochloride Squibb
cocaine COC hydrochloride Mallinckrodt,
St. Louis, MO
desipramine DMI hydrochloride Merrel Ind.,
Cincinnati, OH
5,7—dihydroxytrypt— 5,7—DHT creatinine sulfate Sigma Chemical
tamine

2,5—dimethoxy—4— DOM hydrochloride NIDA
methylamphetamine

N,N—dimethyltrypt- DMT free base NIDA
amine

haloperidol HALO free base McNeil Labs,

Ft. Washington
6—hydroxydopamine 6—OHDA hydrobromide Sigma Chemical
dflysergic acid LSD tartrate NIDA

diethylamide
meta—chlorophenyl— MCPP hydrochloride Farmitalia Labs,
piperazine Milan, Italy
methaqualone MQ free base W.H. Rorer, Inc.

Ft. Washington

methergoline MTG free base Farmitalia Labs

 

 

TABLE 2 (Continued)

32

 

 

Drug Abbrev. Dosage Form Supplier
mescaline MESC hydrochloride NIDA
naloxone NALOX hydrochloride Endo Labs, Garden

City, NY
para—chlorophenyl— PCPA free base Aldrich Chemical
alanine

pargyline -—— hydrochloride Sigma Chemical
pentobarbital PB sodium Sigma Chemical
phenobarbital PhB sodium Sigma Chemical
quipazine QUIP hydrochloride Miles Labs,

Elkhart, IN

 

 

W

RESULTS

Part I. Quantitation of the Disruptive Effects of Hallucinogens and
Other Psychoactive Agents on FR—40 Operant Responding with
the Pause Interval Timer (Counter)

A. Introduction
Hallucinogens produce a disruption of FR—40 behavior character—

ized by periods of nonresponding or "pausing" (Freedman §£_a1., 1963;

Appel and Freedman, 1964, 1965; Appel_e£{a1., 1970; Rech g£_a1,,

1975). This pausing occurs at doses which do not necessarily produce

sedation or ataxia in these animals. Literature reports regarding the

"pause—producing" effects of other psychoactive agents are somewhat

less clear. Appel and Freedman (1965) have reported that the psycho—

active stimulant dfamphetamine (1.0 mg/kg) produces ”pausing" in FR
responding. However, Rech e£_a1, (1975) have also reported that the
same dose of this agent produces a pattern of disrupted FR—responding
characterized by slowed and erratic response rates not characterized
by ”pausing". Unfortunately, in both of the above studies the con—
clusions were made on the basis of cumulative records obtained from
single subjects. With the use of the lO—second pause interval counter
(described above) to quantitative FR—40 "pausing", we now feel that we
can indeed resolve this controversy.
B. Methods
In subjects which had achieved stable FR—40 baseline response

rates, the effects of a number of psychoactive agents were determined.

33

 

 

34
These agents included the hallucinogens DMT, DOM, LSD and mescaline,
the central nervous system (CNS) stimulants dfamphetamine and cocaine,
the CNS depressant phenobarbital, and the neuroleptic chlorpromazine.
To further investigate the possible specificity of the "pausing"
produced by the hallucinogens versus the disruption produced by Sf
amphetamine, a series of drug interaction studies were undertaken. In
the first phase of this experiment, the effects of various doses of
LSD or DOM alone or in combination with 0.25 mg/kg dfamphetamine were
determined. This dose of dfamphetamine is at the threshold for the
response—rate decreasing effects of this agent. In the second phase
of this experiment, the effects of various doses of DOM alone or in
combination with 5.0 mg/kg mescaline and 20 ug/kg LSD were determined.
C. Results

Figure 5 illustrates with cumulative recordings the results
commonly obtained with saline, LSD (100 ug/kg) or dfamphetamine (1.0
mg/kg) administration immediately prior to the session. Control FR—40
sessions, the response patterns of which were not altered by saline
administration, are characterized by a rapid, constant rate of respond—
ing with occasional brief pauses throughout the session. These brief
pauses often, but not always, follow the delivery of a food pellet.
Typically dfamphetamine produces a pattern of disrupted FR—40 respond—
ing characterized by slowed and erratic response rates without a
significant increase in the extent of "pausing". This pattern of
disruption results in a decrease in reinforcements received without

any appreciable change in the number of pause intervals produced.

 

 

 

35

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m muswflm

 

 

1% is

 

 

 

 

37
LSD, on the other hand, produces a pattern of disruption characterized
by ”pausing"; this pattern of disruption results in a decrease in
reinforcements received and a concomitant increase in the number of
pause intervals produced.

Quantification of these effects of LSD and dfamphetamine in
groups of subjects is shown in Figure 6. LSD produces a dose—depen—
dent decrease in reinforcements received and a dose—dependent increase
in pause intervals. demphetamine also produces a dose—dependent
decrease in reinforcements received; however, unlike LSD, this agent
does not produce a dose—dependent increase in pause intervals.

Effects similar to those observed with LSD (dose—dependent de—
crease in reinforcements paralleled by an increase in pause intervals)
have been observed with the other hallucinogens investigated, DMT, DOM
and mescaline. Figure 7a illustrates the dose—dependent "pausing"
produced by these agents. It should be noted that the relative
potency of these agents to produce "pausing" is similar to their
potency as hallucinogens in man. It should also be noted that this
dose—dependent increase in the duration of total pausing produced by
both LSD and DOM is related primarily to a single long pause and not
to a series of shorter pauses (Figure 7b). On the other hand, the
stimulant cocaine, the depressant phenobarbital and the neuroleptic
chlorpromazine have been shown to produce dose—dependent decreases in
reinforcements without significantly increasing pausing until gross
excitatory, ataxic or sedative doses are administered. In this

respect these agents resemble dfamphetamine.

 

 

38

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Since extremely high doses of any of these psychoactive agents
can produce complete disruption of FR240 responding, the best demon—
stration of this difference between the hallucinogens and non—hallu—
cinogens investigated can be obtained when comparing the extent of
pausing at doses which decrease reinforcements received to approxi—
mately 50% of control. Table 3 illustrates that the hallucinogens
typically produce large (70—100 over baseline) increases in pause
intervals at doses which decrease response rates to approximately 50%
of control. On the other hand, dfamphetamine, cocaine, phenobarbital
and chlorpromazine typically do not produce large increases in pausing
at or near ED50 doses for decreasing reinforcements. We have occa—
sionally observed "pausing" following administration of lower doses of
the latter three agents, but this is clearly the exception and not the
rule.

The results of the drug interaction studies, illustrated in
Figures 8 and 9, indicated that co-administration of a threshold dose
for the response—rate decreasing effects of_dfamphetamine did not
alter the dose—dependent "pausing" produced by either the indolealkyl—
amine LSD or the phenethylamine DOM. Figure 10 illustrates that the
dose—dependent "pausing" produced by DOM is enhanced by the admini—
stration of threshold doses of either the indolealkylamine LSD or the
phenethylamine mescaline.

D. Discussion

Using the pause interval measurement in conjunction with response

rates, the dose—dependent "pausing" produced by hallucinogens, alone

or in combination, can be distinguished from the slowed and erratic

 

 

45

TABLE 3

Relationship Between Changes in Reinforcements and Increases in
Pausing Induced by Hallucinogens and Non—Hallucinogenic

Psychoactive Drugs

 

1
Drug and Dose N Percent of Contro

Increase in Number

 

Reinforcements of Pause Intervals

Hallucinogens

0.5 mg/kg DOM 8 44: 8* 941138

100 ug/kg LSD 8 45211-1E 102121=E

1.8 mg/kg DMT 8 522108 721.14..

7.1 mg/kg Mescaline 8 59i 6* 69i 6*
Non—Hallucinogens

1.0 mg/kg dramphetamine 8 54$ 9* l8il6

25 mg/kg phenobarbital 8 63ill* l4i13

0.5 mg/kg chlorpromazine 7 67¢ 8* 17i10

30 mg/kg cocaine 4 46f 9* 27:21

 

Each value represents the mean i S.E.M. Percent of control rein—
forcements and change in pause intervals were determined as described
in Methods. See Methods for details of drug treatments.

*p<0.05, Student's Eftest for paired values.

 

 

46

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response rates produced by a number of psychoactive non—hallucinogenic
drugs. Since co-administration of dfamphetamine failed to alter the
"pausing" produced by either LSD or DOM, this agent clearly does not

manifest the same spectrum of activity as do the hallucinogens.

Part II. The Role of Catecholamines in the FR—40 Disruptive Effects
of Hallucinogens

A. Introduction

As reviewed in the General Introduction, there is considerable
evidence to suggest that many of the behavioral effects of hallucino—
gens are mediated through catecholaminergic neurons in the brain.
Members of both the indolealkylamine and phenethylamine classes of
hallucinogens stimulate motor activity (Koella_e£‘a1., 1964; Tilson 3E
31,, 1975b; Yamamoto and Ueki, 1975) and, at higher doses, produce
stereotyped responding (Koella E£.§l', 1964). Hallucinogens of both
classes produce rotational behavior following unilateral destruction
of dopaminergic neurons in the substantia nigra produced by 6—OHDA
(Pieri E£_a1., 1974; Trulson 33 a1., 1977b). These behavioral effects
of hallucinogens, which are also observed following administration of
the psychomotor stimulant dfamphetamine (Tilson EE.§£': 1975b; Weston
and Overstreet, 1976; Trulson SE a1., 1977b), are presumed to be
mediated by activation of catecholaminergic systems in the brain.

B. Methods
1. Neurotoxin study
The purpose of the first study in this section was to deter—

mine the role of catecholaminergic neuronal systems in the effects of

 

 

53

hallucinogens and dfamphetamine on FR—40 operant responding. LSD was
chosen as the prototype indolealkylamine hallucinogen. The ampheta—
mine analogue DOM was chosen as a representative member of the phen—
ethylamine class of hallucinogens.

In this study, eight subjects received 6—OHDA and eight
subjects received vehicle intraventricularly as detailed in Table l.
The subjects were allowed to recover from surgery, were trained to
respond on the FR—40 schedule, and the effects of various doses of
LSD, DOM and dfamphetamine were determined. Two of the 6—OHDA—treated
subjects failed to reach stable FR—40 response levels and were there—
fore excluded from all aspects of the study. Following completion of
the behavioral testing, the animals were sacrificed, their brains were
removed, and the concentrations of DA, 5—HT and NE were determined by
fluorimetric procedures as described in Materials and General Methods
(Chang, 1964; Curzon and Green, 1970).

2. Drug interaction studies

 

In addition to the neurotoxin study, a number of drug inter—
action studies further investigating the role of catecholamines,
particularly DA, in the disruptive effects of the hallucinogens were
conducted. In these studies, the effects of the indirect DA agonist
dfamphetamine, the catecholamine synthesis inhibitor dMT and the
neuroleptics chlorpromazine and haloperidol on the dose—dependent

"pausing" produced by the hallucinogens LSD and DOM were determined.

 

 

54
C. Results
1. Neurotoxin study

Table 4 summarizes the results of the neurochemical deter—
minations in these subjects. 6—OHDA treatment significantly decreased
DA and NE concentrations in all regions tested without altering the
concentrations of 5—HT in any of the regions examined.

Table 5 indicates the effects of 6—OHDA—induced destruction
of catecholamine neurons on control FR—40 responding. As described
above, control FR—40 responding in vehicle-treated subjects was charac—
terized by a rapid, constant rate of responding (approximately 100
responses/min) throughout the session. 6—OHDA treatment decreased the
rate of responding, significantly reducing the number of reinforce—
ments obtained, without significantly altering the number of pause
intervals produced. Comparison of the effects of 6—OHDA treatment
with other agents which disrupt FR—40 responding in regard to these
two parameters of FR—40 behavior (Table 3 above) indicates that 6-OHDA
is similar to dfamphetamine, cocaine, chlorpromazine and phenobarbital,
but different from the hallucinogens.

Cumulative records illustrating the effects of saline, d;
amphetamine and DOM in a vehicle— and a 6—OHDA—treated subject are
depicted in Figure 11. In the vehicle—treated subject dfamphetamine
and DOM produced dose—dependent disruptions of FR—40 operant respond—
ing which are both qualitatively and quantitatively different. 9f
Amphetamine typically produced erratic intrasession response rates,
while DOM produced "pausing". Destruction of catecholamine neurons

with 6-OHDA decreased the rate of responding relative to control, but

 

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56

TABLE 5

Effects of Vehicle or 6—OHDA Administration on
Control FR—40 Operant Response Parameters

 

 

Reinforcements Pause Intervals
Vehicle 96115 56:12
6—OHDA 512 7* 81112
(53) (144)

 

Each value represents the mean i S.E.M. obtained
from six 6—OHDA—treated or eight vehicle—treated animals.
Average response parameters for each animal were deter-
mined as the mean of the 30—40 control (no injection)
FR—40 sessions throughout the study. Numbers in paren—
theses represent percent of control (vehicle—treated)
values.

*p<0.05, Student's Eftest.

 

 

57

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59

produced neither pausing nor erratic intrasession response rates. In
the 6—OHDA—treated subjects the disruptive effects of dfamphetamine
were greatly attenuated, while the effects of DOM were not altered.

Quantitative assessment of the effects of DOM on FR—40
responding in vehicle— and 6—OHDA—treated subjects is illustrated in
Figure 12. DOM produced a dose—dependent increase in pause intervals
and a dose—dependent decrease in reinforcements obtained; these
effects were not altered by 6—OHDA treatment. Figure 13 illustrates
the effects of LSD on FR—40 responding in vehicle- and 6—OHDA—treated
subjects. As with DOM, this hallucinogen produced a dose—dependent
increase in pause intervals and a decrease in reinforcements obtained.
Again, 6—OHDA treatment failed to alter these effects. Figure 14
quantitates the effects of dramphetamine on FR—40 operant responding
with and without the neurotoxin pretreatment. In vehicle—treated
subjects dfamphetamine produced a dose—dependent decrease in rein—
forcements obtained. Unlike the hallucinogens, this drug did not
produce a significant change in pause intervals. Moreover, the
effects of dfamphetamine on response rate were significantly attenu—
ated by 6—OHDA treatment.

2. Drug interaction studies

 

As described above in Part I, co—administration of d7
amphetamine at a threshold dose for FR—40 disruptive effects (0.25
mg/kg) did not alter the dose—dependent "pausing” produced by either
LSD or DOM (Figures 8 and 9). Similarly, administration of dMT at a
dose which did not alter FR—40 responding pe£_§g (see Table 6) failed

to alter the dose-dependent "pausing" produced by LSD (Figure 15).

 

 

 

60

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61

 

 

 

 

 

 

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62

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64

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66

TABLE 6

The Effects of a—MethyljpjTyrosine (uMT) on the
Characteristics of FR—40 Operant Responding

 

 

T t t Percent of Change in
rea men Control Reinforcements Pause Intervals
50 mg/kg dMT + 40' 7 95i3 6i4

100 mg/kg aMT + 40' 8 99i4 4:4

 

   

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67

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69
Surprisingly, this uMT treatment did not alter the FR—40 disruptive
effects of dfamphetamine (Figure 16).

Lastly, administration of chlorpromazine (for the effects of
chlorpromazine alone on FR-40 responding, see Table 7) clearly did not
attenuate the FR—40 pause—producing effects of DOM or LSD (Figures 17
and 18). Again, however, this treatment regimen with chlorpromazine
failed to alter the disruptive effects of dfamphetamine (Figures 19
and 20). Similarly, haloperidol was found to have no effect on the
dose—dependent pausing produced by DOM (Figure 17).

D. Discussion

 

These data suggest that the catecholamines are not involved
in FR—40 disruptive effects of the hallucinogens LSD or DOM. The
results of the 6—OHDA study suggest that normal catecholamine neuronal
activity is required for the disruptive effects of dfamphetamine. In
contrast to these findings are those of Petersen and Sparber (1974),
who reported that 6-0HDA treatment failed to alter the FR disruptive
effects of dfamphetamine. However, these investigators found that 22
weeks after 6—OHDA (200 pg; intraventricular administration) NE con—
centrations were reduced to approximately 50 percent in all brain
areas and DA concentrations in the striatum (the only area examined
for DA) were not altered. These authors therefore concluded that NE
neurons were not involved in the FR—disruptive effects of dfamphet—
amine. It would appear, then, that the attenuation by 6—OHDA of the
dfamphetamine-induced disruption of FR—4O operant behavior is due to

the destruction of DA and not NE neurons in the brain. This finding

 

 

70

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72

TABLE 7

The Effects of chlorpromazine on FR—40 Operant Responding

 

 

Dose PERCENT CONTROL CHANGE IN
(mg/kg) REINFORCEMENTS PAUSE INTERVALS
0.25 1041 5 5: 8
0.5 104i 5 —21i 72
1.0 67f 8* 17110
2.0 34:11* 83117*

 

*p<0.05, Student's E—test.

 

73

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74

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77

Figure 19. The effects of dfamphetamine on FR—40 responding alone or
in combination with a threshold dose of chlorpromazine. The change in
pause intervals (circles) and percent of control reinforcements re—
ceived (squares) after various doses of dfamphetamine alone (open sym—
bols) or in combination with 0.5 mg/kg chlorpromazine (filled symbols)
are plotted. This dose of chlorpromazine alone, plotted in the far
left portion of the figure, did not increase the extent of pausing.
Each symbol and vertical bar represents the mean i S.E.M. for eight
subjects. chlorpromazine pretreatment did not alter the FR—4O
disrupting effects of dfamphetamine. See Figure 6 legend for further
details.

78

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79

Figure 20. The effects of dfamphetamine on FR—40 responding alone or
in combination with 1.0 mg/kg chlorpromazine. The change in pause in-
tervals (circles) and percent of control reinforcements (squares) after
various doses of d—amphetamine alone (open symbols) or in combination
with 1.0 mg/kg chIorpromazine (filled symbols) are plotted. The effects
of this dose of chlorpromazine alone are plotted on the far left portion
of each panel. Each symbol and vertical bar represents the mean i S.E.M.
for eight subjects. Chlorpromazine pretreatment did not alter the FR—40
disrupting effects of dfamphetamine. See Figure 6 legend for further
details.

80

 

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81
is consistent with the established mechanism of action of dfamphet-
amine (1,3,, release of catecholamines, in particular DA, in the
brain; Chiueh and Moore, 1973; Rech and Stolk, 1970). These data are
also in agreement with those of Fibiger e£_a1. (1973) and Roberts and
Fibiger (1975) who reported that the stimulant, taste aversion— and
stereotypie—inducing properties of dfamphetamine are markedly attenu-
ated by 6-OHDA treatment.

The aMT and chlorpromazine studies, however, do not support
the contention that the FR—40 disruptive effects of gfamphetamine are
mediated via alterations in DA neuronal activity. These data are
perplexing in that they do not agree with effects observed in 6—OHDA—
treated rats. However, the time allowed for the aMT effect to develop
may not have been optimal (Rech e£.a1., 1966). Clearly, further
experimentation in this area would be of great help in solving this

enigma.

Part III. The Role of 5—HT Neurons in the FR-40 Disruptive Effects of
Hallucinogens

A number of studies were conducted to investigate the role of
5—HT neurons in the FR—40 disruptive effects of the hallucinogens.
These approaches included neurotoxin experiments, studies involving
the use of the 5—HT synthesis inhibitor PCPA, and interactions employ—

ing putative 5-HT agonists and antagonists.

 

82
A. Neurotoxin Studies
1. Introduction
As discussed in the General Introduction, the destruction of
5—HT neurons induced by intraventricular administration of the 5—HT
neurotoxin 5,7-DHT enhances the effects of single doses of LSD and
mescaline on FR operant responding in rats (Appel g; g1., 1977; Joseph
and Appel, 1977). However, dose—response analyses for the effects of
these agents in 5,7—DHT—treated rats were not conducted in these pre—
vious studies. Therefore, a number of experiments were conducted to
investigate the effects of 5,7—DHT administration on the disruption of
FR—40 operant behavior produced by a full range of doses for a number
of agents. These studies utilized the intraventricular administration
of 5,7—DHT to destroy 5—HT neurons throughout the brain and more
localized administration of 5,7—DHT in an effort to determine the site
or sites of action of the hallucinogens.
2. Methods

a. Intraventricular 5,7-DHT. In the first of a series of

 

studies utilizing the 5—HT neurotoxin 5,7-DHT, subjects received
either 5,7—DHT or its vehicle intraventricularly as detailed in Table
1. Desipramine and pargyline pretreatments were employed in this
study to enhance the selectivity and efficacy, respectively, of the
neurotoxic effect (Breese 25.31., 1978; Bj6rklund SE 31., 1975).
Following recovery from surgery, the subjects were trained to respond
on the FR—40 schedule and the effects of various doses of LSD, DOM,
mescaline and phenobarbital were determined. The hallucinogens were

administered immediately prior to the start of the FR-40 session;

 

83
phenobarbital was administered thirty minutes prior to the start of
the session.

Twenty—four hours after the last test dose, the subjects
were sacrificed, their brains were removed, and the concentrations of
5—HT and NE in selected brain regions (cortex, hippocampus, hypothala—
mus and striatum) were determined by fluorimetric procedures (Chang,
1964; Curzon and Green, 1970). In addition, the concentrations of 5—
HT and the dopamine metabolite DOPAC were determined in the septum using
high performance liquid chromatography with electrochemical detection
(Lyness 21.31., 1980).

b. Administration of 5,7—DHT into specific brain nuclei.

 

Additional studies were conducted utilizing local injections of 5,7—
DHT in an attempt to define the site(s) of action of the hallucino—
gens. These studies concentrated on the septum and the nucleus accum—
bens because these structures are a part of the limbic system and re—
ceive a relatively dense 5—HT input from the raphé neurons.

1) 5,7—DHT administration into the septum. In this

 

study the subjects were randomly assigned to one of two groups and
received either 5,7—DHT (4 ug/ul, 2 ul; n=8) or its vehicle (n=8) into
the septum. See Table l for parameters of neurotoxin administration.
Following recovery from surgery, the subjects were trained to respond
on the FR—40 schedule and the effects of LSD and DOM were determined.
The effects of 5,7—DHT administration into the septum on the concen-
trations of NE and 5—HT in the septum, hippocampus and occipital cortex
were determined in comparably—treated animals. These latter subjects

were sacrificed two weeks after administration of the neurotoxin.

 

84

2) 5,7—DHT administration into the nucleus accumbens.

 

In this study the subjects were first trained to respond on the FR-40
schedule. They were then randomly assigned to one of two groups and
were administered either 5,7—DHT (8 ug/2 ul; n=6) or its vehicle (n=5)
bilaterally into the nucleus accumbens. See Table 1 for the para—
meters of the neurotoxin treatment. Following recovery of the subjects
from surgery, FR—40 sessions were reinstated. After approximately one
week responding stabilized and the effects of LSD, DOM, mescaline and
dfamphetamine were determined. The subjects were then sacrificed and
the concentrations of 5-HT and DA in the nucleus accumbens and striatum
were determined as detailed in Materials and General Methods.

c. Administration of 5,7—DHT into the medial forebrain

 

bundle (MFB). As described in the General Introduction, the processes
of the raphé nuclei extend forward to various forebrain structures via
the MFB. 5,7—DHT administration into the MFB has been shown to damage
selectively 5—HT neurons in these fiber tracts (Lorden EE.E£', 1978).
Therefore, a study was conducted to determine the effects of neuro—
toxin administration into this site on the behavioral disruption
produced by a number of hallucinogens.

Neurochemical lesions were placed bilaterally in the
MFB as detailed in Table 1. Following recovery from surgery, the
subjects were trained to respond on the FR—4O schedule and the disrup—
tive effects of various doses of LSD, DOM and mescaline were deter—
mined. Five days after completion of the behavioral testing the

animals were sacrificed by decapitation, their brains removed, and the

 

85
hypothalamus, hippocampus, striatum, and cortex dissected out and
weighed. Fluorimetric procedures were utilized to analyze 5—HT in all
four regions as described by Curzon and Green (1970). Concentrations
of NE and DA were likewise analyzed fluorimetrically as described by
Chang (1964).
3. Results

a. Intraventricular 5,7—DHT. Intraventricular admini—

 

stration of 5,7—DHT significantly decreased 5-HT concentrations in the
cortex, hippocampus, hypothalamus, and striatum, while NE concentra—
tions in those regions examined were unaltered relative to vehicle—
treated controls (Table 8). Concentrations of 5—HT in the septum of
5,7—DHT-treated subjects were not different from blank measurements at
the level of sensitivity of the HPLC method (<3 ng/mg protein).

These values were significantly different from vehicle—treated values
of 54:3 ng/mg protein (p<0.05, 95% confidence limits). Septal DOPAC
concentrations in the 5,7—DHT treated subjects, 4.0:0.9 ng/mg protein,
were not significantly different from vehicle—treated subjects,
4.3:0.4 ng/mg protein.

As described above, control FR—40 responding is charac—
terized by a rapid, constant rate of responding throughout the session,
with brief pauses following the delivery of the food pellet reinforce—
ments. In the present study vehicle—treated subjects received 104:6
reinforcements and produced 70:13 pause intervals in control FR—40
sessions. 5,7—DHT treatment did not significantly alter these charac—
teristics of FR—4O responding (Table 9). As described previously,

administration of the hallucinogens invariably resulted in a cessation

 

86

TABLE 8

Effects of Intraventricular 5,7—DHT Administration on
the Concentrations of 5—HT and NE in Various Brain Regions

 

 

 

5-HT NE
Vehicle 5,7—DHT Vehicle 5,7-DHT
Cortex 340:29 53:18* 311:12 389:47
(16) (125)
Hippocampus 279:15 42f 8* 364i21 36li32
(15) (99)
Hypothalamus 836:40 282:6l* 1796:79 1751:189
(34) (97)
Striatum 408:27 53:18* n.d. n.d.
(l3)

 

Data are expressed as ng/gm wet tissue weight as deter—
mined fluorimetrically. Each value represents the mean

: S.E.M. obtained from four 5,7-DHT—treated (180 ug/lO

p1) or six vehicle-treated animals. Numbers in parenthe—
ses represent concentration of amine in 5,7—DHT—treated
rats expressed as a percentage of vehicle-treated controls.

n.d. — amine concentration not determined.

*p<0.05 Student's Eftest.

 

87

TABLE 9

The Effects of Vehicle or 5,7—DHT Administration on
Control FR—4O Operant Response Parameters

 

 

TREATMENT REINFORCEMENTS PAUSE INTERVALS
Vehicle 104:6 70i13
5,7—DHT 81:6 73:19

Percent of
vehicle value 77% 105%

 

Each value represents the mean : S.E.M. obtained from four
5,7—DHT—treated (180 pg 5,7—DHT/10 pl) or six vehicle—treated
subjects. Average response parameters for each subject were
determined as the mean of the control (no injection) FR—40
sessions throughout the study. No significant differences
between vehicle— and 5,7—DHT-treated animals were found.

 

 

88
of FR—40 responding for some portion of the test session, followed by
reinstatement of responding at or very near the control response rate
("pausing”). This pattern of disruption resulted in a decrease in
reinforcements received and a correlated increase in the number of
pause intervals produced. Occasional viewing of the performing sub—
jects through a wide angle lens in the door of the sound—attenuated
boxes revealed no overt signs of altered behavior. Administration of
these agents to other animals not trained in FR—40 did not result in
ataxia or a loss of motor function. Therefore, this pausing is pre—
sumably not due to motor deficits. On the other hand, phenobarbital
most often produced slowed, erratic response rates throughout the
session, with no clear—cut "pausing”. This pattern of disruption also
resulted in a decrease in reinforcements received, but with little or
no effect on the number of pause intervals produced. Higher doses
(35, 50 mg/kg) would produce "pausing" in some animals, but this
was associated with ataxia and motor deficits. Cumulative recordings
illustrating the effects of saline, LSD and phenobarbital in vehicle—
and 5,7—DHT—treated subjects are shown in Figure 21.

In vehicle—treated subjects, all four agents produced
effects ranging from little or no change in reinforcements received at
the lowest doses to nearly complete disruption of FR—40 behavior at
the highest doses. The hallucinogens also produced a concomitant
increase in pause intervals which was well correlated with the decrease
in reinforcements received, as observed earlier in control subjects
receiving these doses of the hallucinogens. This increase in pause

intervals was between 70—100 counts over baseline values following

 

 

89

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HN onsmHn

 

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91
doses of the hallucinogens which decreased reinforcements by nearly 50
percent (50 pg/kg LSD, 0.5 mg/kg DOM and 10.0 mg/kg mescaline). In
contrast, the 25.0 mg/kg dose of phenobarbital, which decreased rein—
forcements received to less than 70 percent of control, had no effect
on the number of pause intervals produced. Moreover, the 50.0 mg/kg
dose of this latter agent, while decreasing reinforcements to less
than 20 percent of control, still produced a mean increase of less
than 60 pause intervals.

In the 5,7—DHT treated subjects, the effects of the
hallucinogens on both reinforcements received and pause intervals
produced were potentiated (Figure 22 illustrates the effects on pause
intervals). Moreover, these two measures were still well correlated
for the hallucinogens in the 5,7—DHT—treated subjects. The effects of
phenobarbital on the change in pause intervals and percent of control
reinforcements received were not altered by 5,7—DHT treatment (Figure
23). In the 5,7—DHT—treated subjects injected with this agent, as
with the vehicle—treated subjects, there was a dissociation between
the decrease in reinforcements received and the change in pause
intervals produced. For example, the 25.0 mg/kg dose decreased rein—
forcements received by almost 25 percent but actually produced a
tendency to decrease, not increase, the number of pause intervals.

b. Administration of 5,7—DHT into specific brain nuclei

 

l) 5,7—DHT administration into the septum. The

 

effects of 5,7—DHT administration into the septum on regional brain

amine concentrations are shown in Table 10. Concentrations of 5—HT

 

 

92

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96

TABLE 10

The Effects of 5,7—DHT Administration into the
Septum on the Concentrations of 5—HT and NE in
Various Brain Regions

 

 

 

5—HT NE

Vehicle 5,7—DHT Vehicle 5,7—DHT

Hippocampus 256:15 82: 2* 235: 9 253:19

(32) (108)

Septum 55: 4 l4: 3* ____ _--_
(25)

Occipital Cortex 243:17 99:15* 299:28 224: 6

(41) (75)

 

Data are expressed in ng amine/gm tissue wet weight for
hippocampus and occipital cortex, as determined fluorimetri—
cally. Septal values represent ng amine/mg protein, as de—
termined by HPLC. Each value represents the mean : S.E.M.
for four subjects. Numbers in parentheses represent concen—
tration of amine in 5,7—DHT-treated animals expressed as a
percentage of vehicle—treated animals.

*p<0 . 05 , Student ‘ s E—test .

 

97
in the septum, hippocampus and occipital cortex were significantly
decreased; NE concentrations in the hippocampus and occipital cortex
were not altered by administration of 5,7-DHT into the septum. Table
11 illustrates that 5,7—DHT administration into the septum did not
alter the characteristics of FR—40 responding. The effects of LSD and
DOM in these two groups are shown in Figure 24. Both agents produced
dose—dependent pausing in vehicle—treated subjects; these effects were
not altered by 5,7—DHT administration into the septum.

2) 5,7—DHT administration into the nucleus accumbens.

 

The effects of 5,7—DHT administration into the nucleus accumbens on
regional brain amine concentrations are shown in Table 12. 5—HT
concentrations were significantly decreased in the nucleus accumbens,
while DA concentrations in this region were not changed. Administra—
tion of 5,7—DHT into this nucleus had no effect on the concentrations
of 5—HT or DA in the nearby striatum. Thus, it appears that local
administration produced a relatively selective destruction of 5—HT
nerve terminals in the nucleus accumbens alone. Table 13 illustrates
that this disruption of 5—HT input into the nucleus accumbens did not
alter the characteristics of control FR—40 responding. The effects of
the hallucinogens LSD, DOM and mescaline in these two groups of sub-
jects are shown in Figure 25. All three agents produced dose—depen—
dent pausing in vehicle—treated subjects. These effects were not
altered by 5,7—DHT administration into the nucleus accumbens.

c. Administration of 5,7-DHT into the MFB. 5,7—DHT

 

injection into the MFB significantly decreased the concentrations of

 

98

TABLE 11

Effects of 5,7-DHT Administration into the Septum on
the Characteristics of Control FR—40 Operant Responding

 

 

Reinforcements Pause Intervals
Vehicle 85:10 66:10
5,7—DHT 70: 5 78: 7
Percent of Control 82 109

 

Each value represents the mean : S.E.M. obtained from seven
5,7—DHT treated or eight vehicle—treated subjects. Average
response parameters for each subject were determined as the
mean of the control (no injection) FR—40 sessions through—
out the study. No significant differences between vehicle—
and 5,7—DHT—treated subjects were found.

 

 

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101

TABLE 12

The effects of 5,7—DHT administration into the nucleus
accumbens on the concentration of 5—HT and DA in
the nucleus accumbens and striatum

 

 

5—HT DA
Vehicle 5,7—DHT Vehicle 5,7—DHT
N. Accumbens ll.5:0.9 2.7:0.3* 84:4 98:6
(23) (117)
Striatum 6.6:0.4 5.6:O.5 102:5 101:6
(85) (99)

 

Data are expressed in ng amine/mg protein, as determined by
HPLC. Each value represents the mean : S.E.M. for six
(5,7—DHT—treated) or 5 (vehicle—treated) subjects. Numbers
in parentheses represent concentration of amine in 5,7—DHT-
treated animals expressed as a percentage of vehicle—treated
animals.

*p<0.05, Student's Eftest.

 

 

102

TABLE 13

The Effects of 5,7—DHT Administration into the
Nucleus Accumbens on the Characteristics of
FR—40 Operant Responding

 

 

Reinforcements Pause Intervals
Vehicle 104: 8 39:8
5,7—DHT 117:11 31:7
(113) (79)

 

Each value represents the mean : S.E.M. obtained from
six (5,7—DHT—treated) or five (vehicle—treated) subjects.
Average response parameters for each subject were deter—
mined as the mean of the control (no injection) FR—40
sessions throughout the study. No significant differences
between vehicle— and 5,7—DHT—treated subjects were found.

 

 

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104

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105
5—HT in the cortex, hippocampus, and hypothalamus (Table 14). Concen—
trations of 5—HT in the cortex and hippocampus were reduced to fifty
percent, while the concentration of 5—HT in the hypothalamus was re-
duced to only 70 percent; striatal 5—HT concentrations were not signi—
ficantly altered by 5,7—DHT administration into the MFB. Similarly,
DA and NE concentrations in all areas examined were not altered by
this treatment.

Administration of 5,7—DHT into the MFB did not change
control FR—40 operant responding as measured by either total rein—
forcements obtained or pause intervals produced in control sessions
(Table 15). The hallucinogens, as before, produced a dose—dependent
increase in pause intervals (Figure 26). The effects of LSD were
potentiated by administration of 5,7—DHT into the MFB, while the
effects of DOM were attenuated in these animals and the disruptive
effects of mescaline were not altered by this 5,7—DHT treatment.

4. Discussion

a. Intraventricular 5,7—DHT. As described above, the

 

hallucinogens produced a disruption of FR—40 behavior characterized by
periods of non—responding. This dose—related effect was dramatically
potentiated by 5,7—DHT pretreatment to about the same extent for LSD,
DOM and mescaline. The effects of phenobarbital were considerably
different, however. In vehicle—treated animals this agent produced
disruptions of behavior characterized by periods of erratic respond—
ing, a finding which is consistent with the data shown in Table 3.
This pattern of disruption produced by phenobarbital results in a

decrease in reinforcements obtained similar to that observed with the

 

106

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107

TABLE 15

The Effects of 5,7—DHT Administration into the MFB on
the Characteristics of Control FR—40
Operant Responding

 

 

Reinforcements Pause Intervals
Vehicle 95: 5 36: 6
5,7-DHT 96:18 48:15
(101) (133)

 

Each value represents the mean : S.E.M. obtained from four
(5,7—DHT—treated) or eight (vehicle—treated) subjects.
Average response parameters for each subject were deter—
mined as the mean of the control (no injection) FR—40
sessions throughout the study. No significant differences
between vehicle— and 5,7—DHT—treated subjects were found.

 

 

 

 

108

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110
hallucinogens, but without "pausing". Destruction of 5—HT neurons by
5,7-DHT treatment did not alter the effects of phenobarbital.

These data indicate that central 5—HT systems are
important in the disruptive effects of the hallucinogens LSD, DOM and
mescaline. Moreover, these effects seem to be somewhat specific,
since 5,7—DHT treatment failed to alter the disruptive effects of
phenobarbital. These findings extend the earlier studies by Appel 35
a1, (1977) and Joseph and Appel (1977) in which it was reported that
5,7—DHT treatment enhanced the FR disruptive effects of single doses
of LSD and mescaline.

b. Administration of 5,7—DHT into specific brain nuclei.

 

0n the basis of the above results, it appears that the 5—HT neurons
innervating either the septum or nucleus accumbens are not critically
important for the manifestation of the behavioral effects of halluci-
nogens. It is quite possible, moreover, that there is no single brain
region which, upon destruction of the 5—HT input, will result in an
alteration of the behavioral effects of the hallucinogens. Before
this statement can be strongly supported a number of additional brain
regions should also be investigated utilizing the local administration
of 5,7—DHT. Unfortunately, pilot studies have indicated that a number
of brain regions, for unknown reasons, appear to be relatively resi—
stant to the selective 5—HT-depleting effects of local 5,7—DHT admini—
stration. These areas include the dorsal and median raphe nuclei

and the amygdala.

 

 

111

c. Administration of 5,7—DHT into the MFB. As antici—

 

pated, 5,7—DHT administration injected into the MFB produced different
neurochemical effects when compared to intraventricular injection.
Injection of 5,7—DHT into the MFB produced moderate decreases in the
concentrations of 5—HT in the cortex and hippocampus, with only a
slight decrease in the hypothalamus. There was no significant change
in striatal 5—HT concentration in these animals. Intraventricular
5,7—DHT produced large (80—90%) decreases in S—HT concentrations in
all of these areas (see Table 8 above). These differences in the
pattern of 5—HT depletion produced by either intraventricular or MFB
5,7—DHT administration presumably relate to the differences in the
response to the various drugs. Indeed, treatment probably also
depends on the route of administration and the quantity administered.
Intraventricular injection of the neurotoxin potentiated the disrup—
tive effects of LSD, DOM and mescaline to a similar extent (see
Figure 22 above). Injection of 5,7—DHT into the MFB potentiated the
effects of LSD while the effects of DOM were attenuated, the effects
of mescaline being unchanged. Both neurotoxin treatments suggest a
role of 5—HT neuronal systems in the behavioral effects of hallucino—
gens. The more specific treatment with 5,7—DHT injection into the MFB
yields evidence that the brain sites and/or mechanisms of action of

these hallucinogens may differ slightly.

 

112
B. PCPA Studies
1. Introduction

As mentioned above, pretreatment with the 5-HT synthesis
inhibitor PCPA has been reported to potentiate the behavioral effects
of the hallucinogens LSD and mescaline, but not those of dfamphetamine
(Appel and Freedman, 1964; Appel $1.31., 1968, 1970, 1977; Joseph and
Appel, 1977). The present study replicates the findings reported with
LSD and extends them to the hallucinogenic amphetamine analogue DOM.

2. Methods

Seven subjects were used in the PCPA—DOM study and five
subjects were used in the PCPA—LSD study. The test doses of DOM and
LSD were 0.5 mg/kg and 50 pg/kg, respectively; these doses were chosen
because they produce a reliable increase in "pausing” which is clearly
sub—maximal. In these studies, days 1—3 were control FR—40 sessions.
On day 4 the subjects were given the test dose of either LSD or DOM
immediately prior to the start of the session. Days 5—7 were control
sessions. Again on day 8, the appropriate dose of the hallucinogen
was administered before the start of the FR—40 sessions. No drugs
were given before operant sessions on days 9—11. However, shortly
(approximately, 30 minutes) after each of these sessions all subjects
were administered 100 mg/kg PCPA intraperitoneally. On day 12, after
3 days of 100 mg/kg PCPA per day, the hallucinogens were again admini—
stered prior to the start of the session. For subjects in the PCPA—
DOM study, the animals were sacrificed approximately six hours after
the last session (day 12) and the concentrations of DA, 5-HT and NE in

various brain regions were determined by fluorimetric methods as

 

113
described in Materials and General Methods. Subjects in the PCPA-LSD
study were run in control FR—40 sessions on day 13; six hours later
these subjects were sacrificed and the brain amine analyses described
above were conducted.
3. Results

The behavioral results of the PCPA—DOM study are shown in
Figure 27. The number of pause intervals produced during control FR—
40 sessions (days 1—3, 5—7 and 9) did not change over the course of
the experiment. Daily administration of 100 mg/kg PCPA failed to
alter the number of pause intervals produced in subsequent control
sessions (days 10—11). Administration of a low dose (0.5 mg/kg) of
DOM produced a slight but significant increase in the number of pause
intervals (days 4, 8). After 3 days of PCPA administration, however,
this same dose of DOM (day 12) resulted in a significantly greater
"pause" than produced by DOM previously. Cumulative recordings ob—
tained from four subjects illustrating the above-mentioned results are
shown in Figure 28. Table 16 summarizes the biochemical data obtained
from these animals. 5-HT concentrations were significantly decreased
in all brain regions following PCPA treatment. A modest, though
significant, decrease in the concentration of NE in the hypothalamus
was observed following this treatment. Hippocampal NE and striatal DA
concentrations were not significantly different from control values.

The behavioral results of the PCPA—LSD study are shown in
Figure 29. Again, the number of pause intervals produced during
control FR—4O sessions (days 1-3, 5-7 and 9) did not change over the

course of the experiment. Daily administration of 100 mg/kg PCPA

 

 

 

114

Figure 27. The effects of PCPA pretreatment on the FR—4O response
to DOM. Ordinate: The number of reinforcements received (top panel)
and pause intervals produced (bottom panel) by various treatments.
Each point represents the mean : S.E.M. for seven rats. Abscissa:
Days of the study and various treatments. Days 1-3 and 5—7 and 9—11
were control days (no injection before session). DOM, 0.5 mg/kg,

was administered IP on days 4, 8 and 12 immediately before the start
of the 40 min FR—40 session. PCPA, 100 mg/kg, was administered IP 30
min after the FR—40 session on days 9—11. On day 12 the effects of
0.5 mg/kg DOM were determined 24 hr following the last PCPA injection.
*Significantly different (p<0.05) from control. The DOM response on
Day 12 (after PCPA) was significantly different from DOM responses

on Day 4 and 8.

115

 

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116

 

 

 

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121
failed to alter the number of pause intervals produced in subsequent
control sessions (days 10, ll and 13). Administration of a low dose
(50 ug/kg) of LSD produced a slight but significant increase in the
number of pause intervals (days 4, 8). After 3 days of PCPA admini—
stration, however, this same dose of LSD (day 12) resulted in a signi—
ficantly greater ”pause" than produced by LSD previously. Table 17
summarizes the biochemical data obtained from these animals. 5—HT
concentrations were significantly decreased in all brain regions
examined following PCPA treatment; DA and NE concentrations were
unaltered in this study.
4. Discussion

In summary, decreasing whole brain S—HT concentrations with
PCPA, while not affecting FR—40 operant responding by itself, poten—
tiated the pause—producing effects of both LSD and DOM. These data
correlate well with those provided earlier using intraventricular
administration of 5,7—DHT.

C. Interactions between Hallucinogens and Putative 5—HT Agonists

 

1. Introduction

Hallucinogens have been shown to produce effects similar to
those observed following administration of the putative S—HT agonists
quipazine and MCPP. LSD and quipazine both produce head twitching in
mice, although there seem to be some quantitative differences in the
effects of these drugs on this behavior (Vetulani et_al,, 1980). Both
quipazine and MCPP decrease free—feeding presumably through their 5—HT
agonistic properties (Samanin et_al., 1977, 1979); LSD has also been

reported to decrease free—feeding behavior in rats (Hamilton and

 

 

122

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123
Wilpizeski, 1961). Moreover, stimulus control experiments by Winter
(1978, 1979) and White E£.3l' (1977) have shown that hallucinogens
substitute for quipazine as a discriminative cue and these effects are
blocked by the putative 5—HT antagonist BC—lOS. In light of these
similarities a number of studies were undertaken to evaluate the FR—40
disruptive effects of these putative 5—HT agonists alone and in combi—
nation with various agents.
2. Methods

Various doses of the putative S-HT agonists quipazine and
MCPP were administered to subjects responding on the FR-AO schedule.
MCPP solutions were prepared fresh shortly (within an hour) before
injection and were administered ten minutes prior to the start of the
session. In another study involving the putative S—HT agonists, a
group of subjects were administered various doses of DOM alone or in
combination with 0.25 mg/kg MCPP or 0.25 mg/kg quipazine.

3. Results

As seen in Figures 30 and 31, both agents produced dose—
dependent increases in pausing and decreases in reinforcements re—
ceived. Table 18 illustrates the relationship between these two
parameters at near ED50 doses (1.0 mg/kg quipazine; 0.5 mg/kg MCPP)
for the response—rate decreasing effects. As can be seen in this
table, these agents produce large increases in pause intervals at
doses which decrease responding to near 50% of control. In this
respect, these agents resemble the hallucinogens and are different

from dfamphetamine, cocaine, phenobarbital and chlorpromazine as

 

 

 

 

124

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TABLE 18

Relationship of Changes in Reinforcements to Changes in
Pausing Induced by the Putative 5-HT Agonists
Quipazine and MCPP

 

 

Dr d D e N Percent of Change in
ug an OS Control Reinforcements Pause Intervals
1.0 mg/kg Quipazine 6 84f 9* 47: 9*
0.5 mg/kg MCPP 8 80i20* 55ill*

 

*p<0.05 compared to control values.

 

 

129
discussed earlier (see Table 3). Figure 32 illustrates that the
pause—producing effects of DOM were enhanced by pretreatment with
these agents.
4. Discussion
The two putative 5—HT agonists examined both produced dose—
dependent disruptions of FR—4O responding similar to that seen with
the hallucinogens. Comparison of pausing produced by these agents
with those induced by the hallucinogens and non—hallucinogens previously

examined (Table 3), at near—ED doses for response—rate decrease,

50
indicates that these agents resemble the hallucinogens in producing
pausing rather than slowed and erratic intrasession response rates.

In addition, the dose—dependent pausing produced by DOM was enhanced
by a threshold dose of either MCPP or quipazine. These data suggest
that the hallucinogens may be producing "pausing" through the activa—
tion of 5—HT receptors in the brain. These findings are in agreement
with other reports (Samanin et al., 1977, 1979; Hamilton and Wilpizel—
ski, 1961; Vetulani et_al,, 1980; Winter, 1978, 1979; White gt 31.,
1977) in which the effects of hallucinogens and putative S—HT agonists

were similar.

D. Interactions between Hallucinogens and Putative S—HT
Antagonists

 

1. Introduction
A number of studies have examined interactions of putative
S-HT antagonists on the behavioral effects of hallucinogens and puta—
tive S—HT agonists. Stimulus control experiments have shown that the

stimulus cues produced by quipazine and both phenethylamine and

 

   

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indolealkylamine hallucinogens are blocked by the 5—HT antagonists BC—
105, cinanserin, methysergide, methergoline and methiothepin (Jarbe,
1980; White e£_al., 1977; Winter, 1978, 1979, 1980; Glennon et 31.,

1979; Silverman and Ho, 1980). Preliminary reports by Rech et_al.
(1975) have shown that the 5-HT antagonist cinanserin blocks the FR-40
pause—producing effects of single doses of a number of hallucinogens
in both the phenethylamine and indolealkylamine classes. Sloviter gt
a1. (1980) have recently shown that the "5—HT syndrome" characterized
by side—to—side headweaving or head tremor, forepaw padding and
splayed hindlimbs produced by both indolealkylamine and phenethylamine
hallucinogens is blocked by the 5—HT antagonists methergoline, methy-
sergide and mianserin. In addition, Samanin_gt.al. (1977, 1979) have
reported that the food—intake suppressing effects of both quipazine
and MCPP are attenuated by the putative S—HT antagonist methergoline.
Surprisingly, these S-HT antagonists have never been reported to
produce any effects pe£_se on FR operant response rates. We, there—
fore, initiated a series of studies investigating the FR—40 effects of
a number of putative S—HT antagonists alone and in combination with
hallucinogens.
2. Methods

Various doses of the putative S—HT antagonists cinanserin
and methergoline were administered to subjects responding in the FR—40
paradigm. Cinanserin was injected 80 minutes and methergoline 180
minutes prior to the start of the session. In another series of
studies involving the putative S—HT antagonists, the FR—40 disruptive

effects of a number of hallucinogens and non—hallucinogens were

 

133
examined in control subjects or in subjects following pretreatment
with cinanserin or methergoline.

Initial studies utilized 1.0 mg/kg of the methergoline dose.
In these studies 16 subjects were used. Half of the subjects received
various doses of LSD (12.5—400 ug/kg), mescaline (5.0—28.4 mg/kg) and
dfamphetamine (0.25—2.0 mg/kg) alone and after pretreatment with
methergoline (180 minutes prior to testing). The other half received
various doses of DOM (0.125—4.0 mg/kg), DMT (1.0—8.0 mg/kg) and pheno—
barbital (12.5—50.0 mg/kg) alone and after methergoline pretreatment.
All subjects received methergoline alone at some point in the study.
The order of drugs and doses administered was completely randomized
for each subject. The hallucinogens and dramphetamine were admini—
stered immediately prior to the start of the FR—4O session; pheno—
barbital was administered thirty minutes prior to the start of the
session.

Subsequent studies investigated the interaction between 0.1
mg/kg methergoline and DOM, as well as the putative S—HT agonist
quipazine. In addition, an experiment was conducted in which a 20
mg/kg dose of cinanserin was paired with various doses of LSD and DOM.

3. Results

As can be seen in Tables 19 and 20, these putative S—HT
antagonists used did not produce "pausing”; in fact, the 20 mg/kg
cinanserin and 0.1 and 1.0 mg/kg methergoline doses actually produced
a modest, yet significant, decrease in ”pausing". Figure 33 illu—

strates that pretreatment with 20 mg/kg cinanserin antagonized the

 

 

134

TABLE 19

Effects of Cinanserin on FR—40 Operant Responding

 

 

Reinforcements Pause Intervals
Control 108i8 55i10
Cinanserin 97i6 39il3*
(90) (71)

 

Values represent mean i S.E.M. for 7 subjects. Cinanserin
(20 mg/kg) administered i.p. 80 minutes prior to the operant
session. Numbers in parentheses indicate percent of con—
trol for the values in treated subjects.

*p<0.05, Student's tftest for paired values.

 

 

 

135

TABLE 20

The Effects of Methergoline on FR—4O Operant Responding

 

 

Reinforcements Pause Intervals
Control 116i7 47i6
0.1 mg/kg Methergoline 129i7* 32i6*
(111) (68)
1.0 mg/kg Methergoline 126i7* 28i5*
(108) (60)

 

Values represent mean i S.E.M. for 8 subjects. Methergoline
was administered 180 minutes prior to the start of the operant
session. Numbers in parentheses indicate percent of control
for the values from treated subjects.

*p<0.05, Student's Eftest for paired values. Reinforcement

data normalized by square root transformation prior to stati-
stical analysis.

 

 

 

136

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138
pause—producing effects of both LSD and DOM, with about a 2-fold shift
in the LSD dose—response curve and about a 4—fold shift in the DOM
dose—response curve for the production of ”pausing”.

As described above, control FR—40 responding is characterized
by a rapid, constant rate of responding with a number of brief "mini-
pauses" throughout the session. These "minipauses" often but not
always follow the delivery of a food pellet. The response pattern of
an individual animal is depicted in Figure 34, panel A, and the charac-
teristics of control responding in all animals are summarized in Table
20. Methergoline treatment alone slightly increased the number of
reinforcements obtained and reduced the number of pause intervals
produced (Table 20; Figure 34, panel E).

The pattern and extent of disruption produced by LSD, DOM
and dfamphetamine alone or after methergoline pretreatment are illu—
strated in Figure 34. LSD alone (200 ug/kg; panel B) produced the
pattern of disrupted FR-40 responding characterized by a fairly long
period of non—responding, as described previously. Methergoline
pretreatment greatly reduced, but did not eliminate, the pause—producing
effect of this dose of LSD (panel F). DOM alone (2.0 mg/kg; panel C)
produced a prolonged period of non—responding; methergoline pretreat—
ment completely antagonized this effect (panel C). Administration of
dfamphetamine (1.0 mg/kg; panel D) produced a pattern of disruption
characterized by slowed, erratic intrasession response rates without
any clear—cut "pausing". Methergoline pretreatment did not alter this

pattern of disruption produced by dfamphetamine (panel H).

 

 

139

Figure 34. Cumulative recordings illustrating the effects of various
treatments on FR—40 responding. A: Saline; B: 200 ug/kg LSD; C:

2.0 mg/kg DOM; D: 1.0 mg/kg dfamphetamine (d—A). Treatments A—D

were administered by i.p. injection immediately prior to the start of
the FR—40 session. Treatments E, F, G and H denote methergoline
pretreatment (1.0 mg/kg, 180 min prior to the start of the session)

and administration of saline, LSD, DOM, or d—amphetamine, respectively,

as in A, B, C and D. See Figure 6 legend for further information.

140

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Quantification of the effects of methergoline pretreatment
on the disruption of FR—4O performance produced by the indolealkyl—
amine hallucinogens LSD and DMT can be seen in Figure 35. In control
animals these agents produced dose—dependent increases in pause inter—
vals and correlated dose—dependent decreases in reinforcements, as indi—
cated earlier. These hallucinogen—induced effects were significantly
attenuated but not blocked by methergoline pretreatment, as evidenced
by the shift to the right in the dose—response curves (p<0.05, by
factorial analysis of variance).

The effects of methergoline pretreatment on the FR—4O disrup—
tive effects of the phenethylamines DOM and mescaline are shown in
Figure 36. These agents administered alone also produced dramatic
dose—dependent increases in the number of pause intervals and dose—
dependent decreases in the number of reinforcements. Methergoline
pretreatment completely blocked the effects of these agents, even when
they were administered at supramaximal doses (4.0 mg/kg DOM; 28.4
mg/kg mescaline).

In control animals dfiamphetamine produced a dose—dependent
decrease in the number of reinforcements similar to that observed
following administration of the hallucinogens, but did not produce an
increase in pause intervals, except at the highest dose (Figure 37).
Examination of the cumulative records from these animals indicated
that (as shown in Figure 34, panel D) administration of dfamphetamine
generally produces slowed, erratic intrasession response rates not

characterized by pausing. The pattern of disruption produced by this

 

 

142

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148
agent results in a decrease in reinforcements with little or no change
in pause intervals except at higher doses. Methergoline pretreatment
did not significantly alter the effects of djamphetamine on reinforce—
ments and pause intervals.

Phenobarbital, like dfamphetamine, produced a dose—dependent
decrease in reinforcements without altering the number of pause inter—
vals until relatively high doses (35, 50 mg/kg) were administered
(Figure 37). Examination of the cumulative records indicated that, as
with dfamphetamine, administration of this agent also produced slow
and erratic response rates within the FR—4O session. Methergoline
pretreatment did not alter the disruptive effects of phenobarbital.

Figure 38 indicates that, if the doses of DOM are increased
sufficiently, the blockade produced by 1.0 mg/kg methergoline can
indeed be overcome. Moreover, the 0.1 mg/kg dose of methergoline is
also quite effective in antagonizing the pause—producing effects of
DOM, although this dose is significantly less effective than the 1.0
mg/kg methergoline dose. Similar studies with quipazine showed that
the effects of this drug are also antagonized by methergoline and that
this antagonism is similar to that observed with DOM; but different
from LSD (Figure 39). That is, 0.1 mg/kg methergoline, and to a
greater extent 1.0 mg/kg, produced a very dramatic shift to the right
in the dose—response curve for the pausing produced by quipazine.

4. Discussion

The data with cinanserin and methergoline alone illustrating

the decrease in pausing relative to control levels further support the

hypothesis that this pausing is produced by 5—HT agonistic actions and

 

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153

imply that the endogenous neurotransmitter exerts a tonic influence.
Moreover, the antagonism of the pause—producing effects of both LSD
and DOM by cinanserin further supports this S—HT agonistic hypothesis.
The difference in degree in the extent of antagonism of LSD and DOM by
cinanserin could be explained by either a difference in the affinities
of these agents for the receptor (presumably S—HT) occupied by cinan—
serin or a difference in the receptor populations upon which the two
hallucinogens act.

The findings with methergoline are in agreement with the
cinanserin data, indicating again that 5—HT agonistic actions are
involved in the pause—producing effects of the hallucinogens. More—
over, the slight difference in the antagonism of LSD vs, DOM observed
with cinanserin is considerably more apparent in the methergoline
studies and has been extended (with the DMT and mescaline interactions)
to suggest that these differences in the blockade by the putative 5-HT
antagonists are indeed class differences.

These studies also demonstrate that the methergoline antago—
nism of the pausing produced by the 5—HT agonist quipazine is similar
in pattern to that seen when the antagonist was combined with the
phenethylamine DOM and dissimilar to that with the indolealkylamines
LSD and DMT (see above). Moreover, these studies also indicate that
this methergoline antagonism of the effects of DOM and quipazine is
indeed dependent on the dose of methergoline employed. These studies
suggest that members of the two classes of hallucinogens interact with

two receptor (presumably S-HT) populations differentiated by the

 

154
methergoline sensitivity. Alternatively, they may affect the same

receptor populations, but by very different mechanisms.

Part IV. Additional Behavioral Studies
A. Introduction

In addition to their disruption of FR—4O operant behavior, the
hallucinogens produce a number of other behavioral effects. One of
the reported effects involves the release of responding that is sup—
pressed by punishment. This release of punished responding has been
suggested to be related to alterations in the activity of brain S—HT
neurons (Schoenfeld, 1976).

Two studies were undertaken to explore the possible anti—conflict
effects of the hallucinogens. One study investigated the effects of
the hallucinogens in animals treated intraventricularly with 5,7—DHT
to destroy 5—HT neurons; the other study investigated the effects of
methergoline pretreatment on the behavioral effects of hallucinogens
in punishment-suppressed responding. In the first study, several
non—hallucinogenic agents with established anti—conflict activity
were also examined.

B. Methods

1. Intraventricular 5,7—DHT and the effects of various

 

agents on punished and unpunished responding. The purpose of the

 

first study was to determine the effects of pentobarbital, methaqua—
lone, LSD and DOM on punished and unpunished responding. The drugs
were tested first in control animals and later in the same animals

following destruction of 5-HT neurons with the neurotoxin 5,7—DHT.

155

The subjects were six female Sprague—Dawley rats (200—
225 g). These subjects were housed three per cage in a room with a
12—hour day-night cycle (lights on 0700—1900 hr). Although the sub—
jects had received previous drug treatments, a three—week drug—free
period was observed before the start of the present experiment.

Behavioral training and testing was conducted between
1400-1600 hr in the conditioned suppression chamber described by Ford
gt 31. (1979). The testing chamber was a rectangular box with plexi—
glass sides and a metal floor and top. Protruding from one wall was a
metal drinking tube. Seven—second tone periods were produced inter—
mittently (variable interval — 21 sec) throughout the lO—minute
session. During the latter 5 seconds of these 7—second periods con—
tact between the floor and the drinking tube closed a circuit which
resulted in the delivery of a 0.03 mA shock to the subject. All
experimental events were controlled by electromechanical programming
circuits and shocks were recorded on electromagnetic counters.
Attached to the metal drinking tube was a calibrated (0.5 ml units)
polyethylene drinking tube used to measure the volume of fluid drunk.

During training water—deprived subjects were first
placed in the experimental chamber and allowed to drink freely without
the shock contingency. After approximately one week of nonshock
sessions, the shock contingency was incorporated into the session.
Initially the shock inhibited all drinking. After several days,
however, all subjects came to drink stable volumes of water and
received a relatively constant number of shocks from day to day.

After responding had become stable for each rat, the effects of

156
various doses of LSD, DOM, pentobarbital and methaqualone were deter—
mined in each rat. All drugs were administered i.p. 10 minutes prior
to the start of the test session. The order of drugs and doses
administered was completely randomized for each subject. Drug test
days were separated by at least three nondrug sessions to avoid the
possibility of tolerance development.

After the effects of these agents were determined, the
subjects were anesthetized with equithesin (3 ml/kg) and placed in a
stereotaxic apparatus. All subjects received 5,7—DHT (200 ug/lO ul)
intraventricularly. All subjects also received 25 mg/kg desipramine
HCl 45 minutes prior to administration of the neurotoxin to protect
against the destruction of norepinephrine neurons (Bjorklund at al.,
1975). All subjects were allowed 3—5 days to recover from surgery
before behavioral testing was reinstated. After an additional week of
control sessions, water intake and the number of shocks received had
stabilized for each rat and the effects of LSD, DOM, pentobarbital and
methaqualone were again determined as described above.

Following completion of the post—lesion behavioral
analyses, the subjects and six untreated animals were sacrificed,
their brains were removed, and the concentrations of 5—HT, NE and DA
in a number of brain regions were determined by fluorimetric proce—
dures (Chang, 1964; Curzon and Green, 1970) as described in Materials
and General Methods.

Drug effects were assessed by comparing the data from

test days to the average of the three days prior to and three days

157
after the test day (baseline). Student's tftest for paired data was
used to evaluate the effects of individual doses of the agents used
and to evaluate the effects of 5,7-DHT treatment on baseline condi—
tioned suppression performance. Dose—response relationships were
examined by analysis of variance in a block design. In all statisti—
cal evaluations p<0.05 was used as the criterion for statistical sig—
nificance.

2. S—HT agonists and antagonists and the effects of hallu—

 

cinogens on punished and unpunished responding. The purpose of the

 

second study was to examine the effects of LSD, DOM and quipazine in
the conditioned suppression paradigm alone and after methergoline
pretreatment.

The subjects in this study were twelve drug—naive
female Sprague—Dawley (Spartan Farms, Haslett, MI) rats weighing
between 200—225 g at the start of the experiment. All subjects were
housed singly in a room with a 12—hr light—dark cycle (lights on
0700—1900 hr) and food-deprived to approximately 80% of their free—
feeding body weights.

Behavioral training and testing was conducted between
1500—1700 hr in the conditioned suppression chamber described above.
In these studies a chocolate—flavored liquid diet ("very" chocolate
Sego; Pet, St. Louis, MO) served as the liquid reinforcer, as opposed
to the water used in the previous study (see above).

During training the food—deprived subjects were placed

in the experimental chamber and allowed to consume liquid diet freely

158

without the shock contingency. After approximately one week of non—
shock sessions, the shock contingency was incorporated into the
session and training progressed as detailed above. After responding
had become stable for each rat, the effects of various doses of LSD,
DOM and quipazine were determined in each rat either alone or after
methergoline pretreatment. LSD, DOM and quipazine were administered
i.p. 10 minutes prior to the test session; methergoline was admini—
stered 180 minutes prior to the session. Drug effects were assessed
as described above. Student‘s tftest for paired data were used to
evaluate the effects of individual doses of the agents used. Dose—
response relationships were examined by analysis of variance in a
block design. In all statistical evaluations p<0.05 was used as the
criterion for statistical significance.

C. Results

1. Intraventricular 5,7-DHT and the effects of various

 

ggents on punished and unpunished responding. Pretreatment with 5,7—

 

DHT significantly decreased the concentration of 5-HT in all brain
areas examined, when these subjects were compared with untreated
control rats (Table 21). The concentrations of DA and/or NE in these
areas were not significantly altered. The disruption of 5—HT neuronal
activity did not significantly alter the number of shocks received in
post—lesion control sessions (Table 22). Water intake was signifi—
cantly greater after the 5,7—DHT treatment. However, this effect was
probably not due to the 5,7—DHT treatment BEE s3 since we have ob—

served that a similar group of six untreated subjects showed an

159

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160

TABLE 22

The Effects of 5,7—DHT Treatment on Control
Conditioned Suppression Performance

 

 

SHOCKS WATER ,
RECEIVED INTAKE (m1)
PRE-5,7—DHT 20:1 13.5:0.5
POST—5,7—DHT 25:4 15.0:0.5*
(123) (113)

 

Each value represents the mean i S.E.M. obtained

from six subjects before (PRE—5,7—DHT) or after
(POST—5,7—DHT) administration of 5,7-DHT. Average
response parameters for each animal were deter—

mined as the mean of the control (no injection)
sessions throughout the study. Numbers in
parentheses represent percent of control (PRE—5,7—DHT)
values.

*p<0.05, Student's tftest.

 

 

161
increase from l3.3i0.3 to l4.9i0.7 ml water consumed over the course
of two consecutive 3—month intervals (the time elapsed during the
present study).

Before 5,7—DHT treatment, pentobarbital produced a
dose—dependent increase in the number of shocks received (relative to
baseline) between 2.5 and 10 mg/kg, and a dose—dependent decrease in
water intake between 10 and 20 mg/kg (Figure 40). A decrease in
punished responding at 20 mg/kg was correlated with the highly seda—
tive effects of this dose. Maximal anti—conflict activity for pento—
barbital was observed at the 10.0 mg/kg dose, at which dose punished
responding was increased to greater than 600 percent of control.

Water intake was slightly depressed by this dose. Treatment with 5,7—
DHT did not significantly alter the effects of pentobarbital on either
punished or unpunished responding.

In control animals, methaqualone produced a dose—
dependent increase in punished responding between 2.5 and 10.0 mg/kg
(Figure 41). Maximal anti-conflict activity of this agent was also
obtained at the 10.0 mg/kg dose, which increased punished responding
to almost 400 percent of control. Water intake was not depressed by
this dose of methaqualone. A significant decrease in water intake was
observed at the 20.0 mg/kg dose in control animals, however. Treat—
ment with 5,7—DHT did not significantly alter the effects of methaqua—
lone on either punished or unpunished responding, although there was a
tendency for methaqualone to produce a greater increase in punished

responding after 5,7—DHT treatment.

 

   

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In control animals both LSD and DOM produced a dose-
dependent decrease in water intake (Figure 42). Many doses of these
agents produced a tendency for a modest increase in punished respond—
ing, but this effect was found to be significant only with the 25
ug/kg LSD and 0.35 mg/kg DOM doses. A trend for maximal anticonflict
activity was observed with the 35 ug/kg LSD and 0.5 mg/kg DOM doses,
at which doses mean values for punished responding were increased to
142 and 175 percent of control, respectively. Pretreatment with 5,7-
DHT attenuated the effects of both LSD and DOM to increase punished
responding over the entire range of doses. However, 5,7-DHT treatment
potentiated the dose—dependent depression of water intake observed
after both hallucinogens.

2. 5—HT agonists and antagonists and the effects of hallu—

 

cinogens 0n punished responding. Subjects in the conditioned sup—

 

pression paradigm utilizing liquid diet reinforcement received an
average of 15—25 shocks and consumed 15—20 ml fluid per session.
These control values, and the effects of various doses of methergoline
alone on punished and unpunished responding are shown in Table 23.

As can be seen, methergoline treatment alone did not alter punished
responding, but did reduce unpunished responses at the highest dose
tested (2.0 mg/kg). LSD (Figure 43) and DOM (Figure 44) in these
subjects produced a modest increase in punished responding at the 50
ug/kg and 0.5 mg/kg doses, respectively. Quipazine (Figure 45) pro—
duced a tendency for release of this behavior at the 1.0 mg/kg dose,
but this effect was not significant. All three agents produced dose—

dependent decreases in fluid consumption (Figures 43—45). Pretreatment

 

 

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169

TABLE 23

Effects of Methergoline on Conditioned Suppression Responding

 

 

Methergoline Punished Unpunished

Dose (mg/kg) Responding Responding
0.1 113i32 98f 3
0.25 75il9 110i 5
0.5 157i50 115il3
1.0 l47i30 100i 6
2.0 69:21 61: 6*

 

Values represent the mean i S.E.M. (percent of control) obtained
from six subjects for the number of shocks received (punished
responding) and the volume (in ml) of fluid drunk (unpunished
responding). Control values were l6i4 shocks/session and
l6.0il.0 ml liquid diet/session.

*p<0.05, Student's Eftest for paired values.

 

 

Figure 43. Antagonism of the effects of LSD on punished and unpunished
responding by methergoline. The effects of LSD alone (open symbols) or
after 1.0 mg/kg methergoline pretreatment (filled symbols) are plotted
on the basis of the percent of control shocks received (top panel) and
the percent of control fluid intake (bottom panel) in conditioned sup—

pression testing. Each symbol and vertical bar represent the mean i
S.E.M. obtained from six subjects. Methergoline pretreatment signifi—
cantly attenuated the fluid intake—decreasing effects of LSD, p<0.05,
Student's t—test for paired values. See Figure 40 legend for further
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174

Figure 45. Antagonism of the effects of quipazine on punished and
unpunished responding by methergoline. The effects of quipazine alone
(open symbols) or after 0.1 mg/kg (half—filled symbols) or 1.0 mg/kg
(filled symbols) methergoline are plotted on the percent of control
shocks received (top panel) and the percent of control fluid intake
(bottom panel) in conditioned suppression testing. One mg/kg methergo—
line, and to a lesser extent 0.1 mg/kg, significantly shifted the
dose—response curves to the right, p<0.05 by analysis of variance.
*p<0.05, Student's tftest for paired values. See Figure 40 legend

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176
with 1.0 mg/kg methergoline significantly attenuated the fluid—intake
decreasing effects of LSD (Figure 43). This same dose of methergoline
blocked the capacity of DOM and quipazine to decrease fluid intake up
to doses of 8.0 mg/kg of the agonists, but not for higher doses
(Figures 44 and 45). The lower dose of methergoline (0.1 mg/kg)
antagonized these effects of both DOM and quipazine to a lesser extent
than the 1.0 mg/kg dose. However, this antagonism was relatively much
greater than the antagonism of the LSD effects produced by 1.0 mg/kg
methergoline.

D. Discussion

 

In both the water reinforced and food—reinforced conflict proce-
dures, the hallucinogens (LSD and DOM) produced a mild increase in
punished responding. Quipazine also produced a tendency for an increase
in punished responding in the food—reinforced conflict procedure.

These effects were shown to be extremely weak in comparison to the
anxiolytics pentobarbital and diazepam (Kilts, 1979; Ford 33 al.,
1979). In addition to their varying effects on punished responding,
all the agents examined produced a dose—dependent decrease in un—
punished responding (fluid intake).

Neither the 5,7—DHT nor methergoline treatments (except at the
highest dose of methergoline) had any effects on punished or unpunished
responding. Also, neither the punishment—releasing nor the depressant
effects of pentobarbital or methaqualone were altered by 5,7—DHT
treatment. These data are in agreement with earlier results with

phenobarbital in FR—4O (Figure 23). The weak punishment—releasing

177
effects of both LSD and DOM were blocked by 5,7—DHT treatment; more—
over, the dose—dependent decreases in unpunished responding were
potentiated by 5,7—DHT treatment. These data are also in agreement
with earlier studies on FR—4O responding (Figure 22). Methergoline
pretreatment alone produced no discernible change in unpunished re—
sponding. The effects of the hallucinogens and quipazine on unpunished
responding, however, were antagonized by methergoline pretreatment.
Again, as observed in the FR—4O studies, the effects of LSD were
weakly (at best 2—fold) antagonized while those of DOM and quipazine
were antagonized considerably more effectively (8— to l6—fold; see
Figures 35, 36, 38 and 39). Also, as in the FR—4O studies (Figures 38
and 39), the methergoline antagonism of DOM and quipazine was found to
be dependent on the dose of methergoline used. These results in the
conditioned suppression paradigm provide no really new information;
rather, they corroborate the critical results of the earlier studies

conducted in the FR—40 paradigm.

SUMMARY AND GENERAL DISCUSSION

As had been reported by other investigators, the hallucinogens
produced a disruption of FR—4O operant behavior characterized by
periods of non-responding or "pausing" (Freedman gt al., 1963; Appel
and Freedman, 1964, 1965; Appel-gt'al., 1970; Rech et_al3, 1975).
Estimation of the pausing can be obtained by either the increase in
the number of pause intervals or the decrease in reinforcements pro—
duced. In contrast, the non—hallucinogenic psychoactive agents d7
amphetamine, phenobarbital, cocaine and chlorpromazine typically
produced disruptions of FR-4O operant behavior characterized by slowed
and erratic response rates within the operant session and not by
pausing. Although an occasional subject would be observed to "pause”
following administration of one of these agents (see also Appel and
Freedman, 1965), quantitation of the pausing in a group of these
subjects indicated that there was no increase in pause intervals
apparent, although a dose—dependent decrease in reinforcements re—
ceived (response rate) was observed. These findings are in agreement
with an earlier report by Rech_gtflal. (1975).

The capacity of the pause interval counter to differentiate the
hallucinogens from the four non—hallucinogens was most apparent at
near ED doses for the response—rate decreasing effects of all agents.

50

178

 

 

g

179
All four hallucinogens produced large increases in pausing at ED50
doses for a decrease in reinforcements, while the non-hallucinogens
were typically without significant effect on pause intervals at dose
levels reducing reinforcements to a 50 percent level.

Co—administration of a threshold dose of the stimulant dfamphet-
amine (for the response—rate decreasing effects) with various doses of
LSD or DOM failed to alter the dose—response curves for the pausing
produced by the hallucinogens. On the other hand, threshold doses of
either the indolealkylamine LSD or the phenethylamine mescaline shifted
the DOM dose-response curve to the right. These data further illu-
strate the capacity of the pause interval counter to differentiate the
hallucinogens from another class of psychoactive agents.

As discussed in the General Introduction, the hallucinogens
produce a number of behavioral effects that are presumably mediated
through interactions with catecholamines, particularly DA neurons
(Christoph et al., 1977; Koella §£_al,, 1964; Meltzer §E_al., 1978;
Menon g£_al,, 1977; Trulson_§tnal., l977b; Tilson gt_al,, 1975b;
Yamamoto and Ueki, I975). The data presented demonstrate that the FR—
40 pausing produced by hallucinogens is not mediated through DA
neurons. First, destruction of catecholamine neurons in the brain
with intraventricular 6—OHDA failed to alter the dose-dependent pausing
produced by either LSD or DOM. The FR-40 disrupting effects of if
amphetamine, in addition to being different in pattern from those
produced by the hallucinogens, were attenuated by 6—OHDA treatment.

This is in agreement with the presumed mechanism of action for this

 

180
agent (123., release of catecholamines from the brain; Rech and Stolk,
1970; Chiueh and Moore, 1973).

The lack of catecholamine involvement in the pause-producing
effects of the hallucinogens was further substantiated by the results
of the drug interaction studies using the neuroleptic agents chlorpro—
mazine and haloperidol and the catecholamine synthesis inhibitor aMT.
It has been reported previously that chlorpromazine actually enhances
some of the effects of LSD (Marrazzi and Huang, 1979; Halasz gt 31°:
1969). These data again suggest that, although hallucinogens may
produce a number of effects mediated by catecholamine systems, the FR-
40 pausing effect appears not to involve catecholamine neurons.

The results with aMT extend data from previous reports examining
one dose of LSD (Appel and Freedman, 1964; Appel_gtfla1., 1970) to an
entire dose-response analysis. However, a note of caution must be
added in evaluating the results from the chlorpromazine and aMT inter—
action studies, since these treatments also failed to alter the dose-
dependent disruption produced by dfamphetamine in the way that 6-OHDA
did. Since the 6-OHDA—induced attenuation of the dfamphetamine
disruption of FR—4O operant responding was earlier used as positive
evidence for disruption of normal catecholamine activity, it may be
suggested that these other treatments were without effect on catechol—
amine neurons. For example, it is possible that the pretreatment time
for dMT was not long enough to allow for optimal antagonism of the
effects of dfamphetamine, especially since they relate to disruptive
actions rather than response enhancing properties (Rech et al., 1966;

1968; Rech and Moore, 1968; Stolk and Rech, 1970.

 

 

 

181

Destruction of 5-HT neurons with the intraventricular admini—
stration of 5,7—DHT selectively potentiated the FR—4O disruptive
effects of both indolealkylamine and phenethylamine hallucinogens
equally, as evidenced by similar shifts in the dose-response curves.
These results extend earlier single—dose studies by investigators in
Appel's laboratory (Appel 25 a1., 1977; Joseph and Appel, 1977).
Intraventricular 5,7—DHT administration also selectively altered the
effects of hallucinogens on punished and unpunished responding, since
the dose-dependent decreases in fluid intake produced by LSD and DOM
were also potentiated by this 5,7—DHT treatment. These data strongly
imply that hallucinogens interact with 5-HT neurons and/or receptors
in the brain. Depletion of whole brain S-HT by treatment with PCPA
potentiated the FR—4O disruptive effects of LSD and mescaline, but not
dfamphetamine (Appel 35 al., 1977); the present studies replicate the
findings with LSD and extend them to the amphetamine analog, DOM.

Two approaches to the depletion of brain 5—HT concentrations have
yielded similar results;_ite., depletion of central S—HT enhances the
effects of both phenethylamine and indolealkylamine hallucinogens
equally. Several hypotheses can be advanced to explain these effects.

One possible explanation for these data is that, following 5,7-
DHT treatment, the hallucinogens are acting as agonists at supersensi—
tive 5—HT receptors. A number of investigators have suggested that
hallucinogens are agonists at postsynaptic central 5-HT receptors
(Andén et 31., 1968, 1971, 1974; Appel 35 31., 1977; Browne and Ho,
1975; G1ennon_g£_§l,, 1979; Joseph and Appel, 1977; Kuhn et_§13,

1978; Silverman and Ho, 1980; White_etflal., 1980; Winter, 1969, 1978,

 

i

 

182
1980). As indicated above, the putative S—HT antagonists methergoline
and cinanserin block the decrease in reinforcements and "pause—produ—
cing" effects of hallucinogens without attenuating the effects of £7
amphetamine or phenobarbital. Moreover, Nelson £3 31. (1978) have
reported an increase in the number of hippocampal S—HT binding sites
(receptor supersensitivity) following 5,7—DHT treatment. Therefore,
it may be postulated that (assuming the hallucinogens are acting as
postsynaptic agonists in control animals) the 5,7-DHT-treated subjects
exhibit a greater effect in response to these agonists as they interact
with an increased number of receptors. However, a number of other
drugs (228-, fenfluramine,_pfchloroamphetamine) activate post-synaptic
5—HT receptors, but they are not hallucinogenic in man (Trulson and
Jacobs, 1976a). Perhaps the hallucinogens produce a particular
pattern of regional post-synaptic receptor activation which differs
from that produced by fenfluramine and_pfchloroamphetamine, and this
difference accounts for the hallucinogenic effects.

Another possible explanation for these data relates to the hypo-
thesis for hallucinogenic drug actions originally proposed by Agha—
janian gt a1. (1975). According to this hypothesis, hallucinogens
activate autoreceptors on the cell bodies of 5-HT neurons in the raphé
nuclei (Aghajanian, 1976; Aghajanian_g£_al., 1970, 1972, 1975; Agha—
janian and Haigler, 1974, 1975; Aghajanian and Wang, 1978; DeMontigny
and Aghajanian, 1977; Haigler and Aghajanian, 1973, 1977) which in
turn results in a cessation of discharge of these neurons. Since many
central 5—HT neurons appear to be tonically inhibitory in nature

(Aghajanian and Wang, 1978; Bloom 35 31., 1972; Haigler and Aghajanian,

183

1977), attenuation of this discharge would result in "disinhibition"
in many forebrain areas, and presumably hallucinations, as higher
centers received excessive and inappropriate input. According to this
hypothesis, the threshold for the effects of the hallucinogens would
be determined by the concentration of drug required to inhibit raphé
cell firing. In 5,7-DHT treated animals, in which a large portion of
the 5-HT pathways to the forebrain have been destroyed with a re—
duction in the reserve of the system, this threshold may be lowered
relative to control subjects. Therefore, the effects of the hallu-
cinogens would be potentiated as described here. One major problem
with the hypothesis involving raphé neuron inhibition, however, is
that a number of non—hallucinogenic agents are also potent inhibitors
of raphé cell activity. This list includes the LSD analogue lisuride
(Rogawski and Aghajanian, 1979), the 5-HT precursor 5—hydroxytrypto—
phan (Trulson and Jacobs, 1976b) and the 5—HT releasing agents fen—
fluramine and pfchloroamphetamine (Sheard, 1974). Moreover, Trulson
§E_§1, (l977a) have demonstrated that hallucinogen—induced inhibition
of raphé cell activity persists despite the demonstration of tolerance
to the behaviorally disruptive effects of these agents in the same
animals. These discrepancies must be resolved through further experi-
mentation in order to assess the feasibility of this latter hypothesis
for explaining the central nervous actions of hallucinogenic drugs.

Localized neurotoxin administration was used in an attempt to
determine whether actions of these agents at post—synaptic 5—HT recep—
tors in the forebrain or autoreceptors in the raphé nuclei were criti—

cal for the production of this pausing. Unfortunately, these studies

 

184
proved to be inconclusive because a number of brain regions were, for
one reason or another, resistant to the selective neurotoxicity of
5,7—DHT. The nucleus accumbens and the septum, regions which had
demonstrated effective destruction of 5—HT neurons in pilot studies,
both proved to be negative in testing for a critical site of action
for the effects of the hallucinogens. If the parameters of neurotoxin
administration can be manipulated to provide adequate S—HT neuronal
destruction for the resistant brain regions, perhaps a number of
additional brain sites can be examined with similar designs in future
studies. Such possible sites include the amygdala and the median or
dorsal raphe nuclei, to name a few.

Administration of 5,7—DHT into the MFB produced a pattern of
central depletion that was different from that observed with intraven—
tricular administration of the neurotoxin. This infusion of 5,7—DHT
into the MFB also produced effects on the disruptive influences of the
hallucinogens that differed from those of intraventricular 5,7-DHT.
Instead of potentiating the effects of the hallucinogens equally, 5,7—
DHT into the MFB potentiated the effects of LSD and attenuated the
effects of DOM. The effects of mescaline, however, were unchanged by
this treatment. The LSD potentiation was not as great in magnitude as
that observed in the animals treated intraventricularly with 5,7—DHT.
Similarly, the attenuation of the effects of DOM was not outstanding.
Yet both were statistically significant. These data are the first
that clearly suggest that the various hallucinogens may disrupt behavior

by somewhat different mechanisms at various sites in the brain.

185
The putative S-HT agonists quipazine and MCPP both produced a
disruption of FR—4O operant responding characterized by pausing. This

effect is most apparent at the near-ED5 dose to reduce reinforcements

O
for each of these agents; at these doses pausing was significantly
increased (80 or more counts over baseline) to an extent similar to
that observed with the hallucinogens (see Table 3). These findings
with quipazine are in agreement with those of White_g£fl§1. (1977) and
Winter (1979), in which the discriminative stimulus cues of quipazine
were found to generalize to the hallucinogens. In addition to their
pause-producing effects, threshold doses of both quipazine and MCPP
shift the dose—response curve for the pause induced by DOM to the
left, much as did the hallucinogens LSD and mescaline (see Figures 10
and 32). Since quipazine and MCPP are presumed to be post-synaptic 5—
HT agonists, these data suggest more strongly that the pause—producing
effects of the hallucinogens may be the result of post-synaptic 5-HT
receptor activation.

The putative 5—HT antagonists cinanserin and methergoline, admi-
nistered alone, produced a surprising decrease in the duration of
pausing relative to control sessions. Methergoline actually increased
FR—4O response rates over control levels, while cinanserin had no
effect on response rates. These data suggest that the pausing pro—
duced during control FRr4O sessions may also be due to activation of
5—HT receptors by endogenous 5-HT activity. When administered in
combination with the hallucinogens, both cinanserin and methergoline
antagonized the pause-producing effects of all the hallucinogens

examined. However, there were class differences in the nature of this

 

186
antagonism. The dose—response curves for the indolealkylamines were
shifted at best 2—fold with either S-HT antagonist. The DOM dose-
response curve was shifted 3- to 4-fold to the right by the 20 mg/kg
cinanserin dose. Methergoline pretreatment blocked the effects of DOM
and mescaline to a greater extent, as the 1.0 mg/kg dose of the anta—
gonist produced at least a l6—fold shift in the DOM dose-response
curve. Thus, the effects of a normally supramaximal dose (28.4 mg/kg)
of mescaline were completely blocked by the 1.0 mg/kg methergoline
dose. This antagonism of the phenethylamine effects was found to be
dependent on the methergoline dose, as 0.1 mg/kg produced only about
an 8-fold shift in the dose—response curve for DOM. Quipazine, the
putative S-HT agonist, was found to be similar to the phenethylamines
regarding the nature of its interaction with methergoline. That is,
1.0 mg/kg methergoline produced a greater than 8—fold shift in the
dose-response curve for this agent to produce pausing, while the 0.1
mg/kg dose was somewhat less effective, producing a 4— to 6—fold
shift.

Similar differences in the interaction of methergoline with LSD
gs, DOM have been shown in the conditioned suppression paradigm.
Methergoline by itself had no significant effect on punished or un—
punished responding in the paradigm. The dose-dependent decrease in
fluid intake produced by LSD was shifted to the right approximately 2—
fold following pretreatment with the 1.0 mg/kg methergoline dose. As
in the FR—4O studies (above) this same dose of methergoline shifted
the dose—response curve for the fluid—intake decreasing effects of DOM

to the right greater than 8—fold; 0.1 mg/kg methergoline produced a

 

187
somewhat smaller (still 4—fold) shift in this dose—response curve.
Again, quipazine was found to be similar to DOM, and dissimilar to
LSD, in the pattern of antagonism of its effects by methergoline.

The majority of the literature reports regarding S-HT neurons and
the behavioral effects of hallucinogens and 5-HT agonists has supported
a common site of action and level of sensitivity (Auden gt 31., 1968,
1971, 1974; Appel gt 21°: 1977; Browne and Ho, 1975; Glennon 25 a1.,
1979; Kuhn_g£flal., 1978; Silverman and Ho, 1980; Schechter and Rose-
crans, 1972; Winter, 1969, 1978, 1980). However, differences in the
hallucinogenic drug classes and quipazine regarding their interactions
with 5-HT antagonists have been suggested by investigators using other
behavioral measures. For example, although head—twitching is produced
by both LSD and quipazine, the capacity of the putative 5-HT antago—
nist methysergide to block this effect is much greater for quipazine
than LSD (Vetulani gt_a1,, 1980). Moreover, although there have been
no reports of class differences in hallucinogens regarding their
discriminative stimulus properties in rats, a recent study by Jarbe
(1980) has suggested that pigeons trained to discriminate LSD will
generalize readily to other indolealkylamine hallucinogens but do not
generalize a strongly to the phenethylamine mescaline. Furthermore,
the LSD discriminative cue is not antagonized by methergoline. Unfor-
tunately, this paper did not address the capacity of methergoline to

antagonize the mescaline cue.

 

188

The results of the present experiments strongly implicate 5-HT
neurons in the behavioral effects of hallucinogens. However, there
are now clear—cut differences between the phenethylamine and indole-
alkylamine classes as shown by the methergoline studies. Much of the
data may be explained by postulating the presence of two sites of
action for the effects of hallucinogens on FR—4O responding. The first
type (type—I; Indolealkylamine) would be activated strongly by the
indolealkylamines LSD and DMT and may not interact with the phenethyl—
amines DOM and mescaline. The second type (type—P; phenethylamine)
would be activated strongly by DOM, mescaline and quipazine and possi—
bly more weakly by LSD and DMT. Activation of either or both receptor
sites is presumed to disrupt the balance of brain functions to favor
hallucinatory—type drug effects. Type—I receptors are apparently non-
responsive or only weakly responsive to methergoline blockade; type-P
receptors would be more effectively blocked by this agent. It is
further proposed that the type-P receptor represents a postsynaptic 5—
HT receptor, since the results of electrophysiological (Aghajanian 35
213, 1970, 1972, 1975; Aghajanian and Haigler, 1974, 1975; DeMontigny
and Aghajanian, 1977; Haigler and Aghajanian, 1973, 1977) and beha-
vioral (Anden 3331;, 1968, 1971, 1974; Browne and Ho, 1975; Glennon
et_a1,, 1979; Kuhn_et_a1., 1978; Silverman and Ho, 1980; Winter,1969,
1978, 1980) studies have suggested that the hallucinogens of both
classes activate post—synaptic receptors in the brain. Depletion of
whole brain 5—HT by intraventricular 5,7—DHT or systemic PCPA poten—

tiates the effects of LSD, DOM and mescaline equally. This potentiation

189
is most likely related to type—P receptor denervation supersensi-
tivity. Indeed, 5,7—DHT treatment has been shown to increase the
number of hippocampal (post—synaptic) S-HT binding sites (Nelson gt
31,, 1978). Working from this proposed model, one may explain the
weak antagonism of LSD and DMT by methergoline on the basis of actions
of the indolealkylamines on the methergoline—insensitive type—I recep-
tors. The question remains of course, what is the type-I receptor and
what is its normal function? Electrophysiological studies have shown
that the indolealkylamine hallucinogens have the capacity to directly
activate autoreceptors on the cell bodies of raphé neurons (Aghajanian
et_§1,, 1970, 1972, 1975; Aghajanian and Haigler, 1974, 1975; DeMon—
tigny and Aghajanian, 1977; Haigler and Aghajanian, 1973, 1977). The
phenethylamine mescaline and DOM are without such an effect directly
(Aghajanian gt al,, 1970; Haigler and Aghajanian, 1973). These data
can be interpreted to suggest that the type—I receptor site is the
autoreceptor on the raphe cells. Conversely, the type—P receptor
would involve neuronal inputs into the raphe neurons or would relate
to post-synaptic 5-HT receptors in the forebrain.

Alternative proposals are not difficult to imagine. Both classes
may exert their effects on postsynaptic 5—HT receptors in various
regions of the forebrain, but by different mechanisms. The indole
hallucinogens may bind at a receptor site that very effectively acti—
vates the effector changes,_iae., resulting in altered neuronal acti—
vity of the postsynaptic neurons. Methergoline may compete for this

site with some degree of affinity, but little intrinsic activity (i333,

190
a competitive antagonist). Phenethylamine—type hallucinogenic agents
may act at an allosteric site on the receptor to alter reactivity to
the normal transmitter or even to activate the receptor but in a less
direct manner. Methergoline, by blocking "downstream" from this site,
would be much more effective as an antagonist against this class,
since there would not be direct agonist-antagonist competition for the
same site. Analyses of these proposals will require careful, appro—
priately designed experimental approaches beyond the scope of the
current effort.

In summary, the present studies demonstrate that the FR—4O pause-
producing effects of phenethylamine and indolealkylamine hallucinogens
can be differentiated from the pattern of slowed and erratic intra-
session response rates produced by a number of non—hallucinogenic
psychoactive agents. The analyses combined the assessment of pausing,
through the use of the pause interval timer, and the number of rein—
forcements obtained. Studies employing the neurotoxin 6—OHDA, the
catecholamine synthesis inhibitor aMT, and receptor antagonists halo—
peridol and chlorpromazine have indicated that catecholamine neurons
appear not to be involved in this pausing manifested by the hallucino—
gens. Studies employing agents to produce comparable manipulations of
5—HT neurons indicated that the hallucinogens appear to produce
pausing through their agonistic actions at 5—HT receptors. Lastly,
studies with the 5—HT antagonist methergoline, in which it was shown
that this agent was more effective in antagonizing the effects of

phenethylamines than indolealkylamine hallucinogens, have indicated

191
that there appear to be at least slight differences in the mechanisms

by which agents of these two classes produce their effects.

 

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