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H3538

HIGAN STATE UNIVE ERS

Illll III IIIHIHIIIIIIIIII

31293 00914 5255

IHIIIII

 

 

 

 

 

 

 

 

 

 

 

 

 

 

This is to certify that the

dissertation entitled
Behavioral, Cellular and Metabolic fiomponents of Drug
Tolerance to Pentobarbital-Induced Hypothermia and Ataxia

presented by

Deborah Ruth MacKenzie-Taylor

has been accepted towards fulfillment
of the requirements for

Ph.D. Pharmacology and

Toxicology/Neuroscience

‘ E
Ma jo professor

degree in

 

 

Date November 15, 1990

 

MS U i: an Affirmative Action/Equal Opportunity Institution 0-12771

 

 

LIBRARY
{Mlchigan State
University

 

 

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or betore data duo.

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MSU Is An Affirmative Action’Equol Opportunity Institution
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fi*_ . r_._4-._._~— , -_—-t

BEHAVIORAL, CELLULAR AND METABOLIC COMPONENTS OF DRUG

TOLERANCE TO PENTOBARBITAL-INDUCED HYPOTHERNIA AND ATAXIA

BY
Deborah Ruth MacKenzie-Taylor

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/Neuroscience

1990

é¢7" / ‘J’u‘

ABSTRACT

BEHAVIORAL, CELLULAR AND NETABOLIC CONPONENTB OI’ DRUG
TOLERANCE TO PENTOEAREITAL-INDUCED “POTEERNIA AND ATAXIA

BY
Deborah Ruth MacKenzie-Taylor

The development of behavioral tolerance to pentobarbital-
induced hypothermia and ataxia, as separate from cellular and
metabolic tolerances, was established. Pentobarbital was
administered to 4 groups of rats, 2 groups of which received
intermittent (INT) i.p. treatment. One of these (INT/EXP;
intermittently-treated, drug-deficit experienced animals)
experienced the drug effects by testing for rotarod
performance (RR, ataxia measure) and body temperature (BT,
hypothermia measure) after drug injection. The other
(INT/NonEXP; intermittently-treated animals that were
protected from experience) was tested for R & BT before
receiving drug and then prevented from drug- induced
hypothermia by maintenance of body temperature and ataxia
experience by a towel-wrap restraint after drug
administration. Two groups received chronic treatment (i.p.
and in ground chow), one with drug-effect experience (CHR/EXP)
and one prevented from experiencing drug effects (CHR/NonEXP) .
Results of a Postchronic Test, with all groups experiencing
the drug effects, was compared to Prechronic Test effects to
assess .the degree of tolerance. The INT/EXP group

demonstrated behavioral tolerance for drug-induced

hypothermia, but not for drug-induced ataxia. The INT/NonEXP
group actually showed increased sensitivity to pentobarbital-
induced.ataxian Prominent tolerance was noted in.both.chronic
groups for hypothermia and ataxia, without any difference
between them. After Postchronic Testing, chronic treatment
was discontinued for 9 days (withdrawal) and then, 10 days of
extinction training (vehicle-behavioral testing) was
conducted. The two intermittent groups demonstrated no change
in the hypothermia or ataxia during the Postwithdrawal and
Postextinction Drug Tests. However, in CHR/EXP rats tolerance
to both hypothermia and ataxia was decreased at the
Postwithdrawal Test, with a greater loss at the Postextinction
Test. CHR/NonEXP animals showed a prominent loss of tolerance
at the Postwithdrawal Test only. Brain concentrations of
pentobarbital in identically-treated rats (up to the
Postchronic Period) yielded evidence of metabolic tolerance
in the two chronic treatment groups. Evidence for cellular
tolerance was also produced in these two groups when the brain
concentrations were correlated with the hypothermia.
Behavioral tolerance to drug effects was expressed after both
chronic and intermittent pentobarbital treatment and existed

separate from cellular and metabolic tolerance.

To my loving husband, Allan, for his support and
encouragement, without whom this dissertation, and all
it stands for, would not have been possible.

Also, to Lars, whose life taught me the importance of

understanding pharmacological intervention, how it can
increase the quality of life and how it can shorten life.

iv

ACRNOULEDGENENTS

I would like to thank Cynthia Clingan, Neelu Shukla,
Staci Haarer and Clare Casey for their technical and
managerial assistance required for this study. I would also
like to acknowledge the many student workers (army of
undergrads, as it were) who made it possible to conduct this
study without automation, some of whom are Suzanne Rado,
Cecilia Ho, Rhonda Norton, Jacquie Stoudt, Scott Giraud,
Leslie Tilletson, Traci Kimbrough, Robyn Turner, MaryJo
Charron, Hussein Huraibi, and Debbie Burkholder. If I have
forgotten anyone, please forgive me. I would also like to
thank Marlee Langham and Rob Durham for their help with
computer programs and their patience with me using their
equipment.

Most of all, I would like to thank Dr. Richard Rech for
his patience, guidance, and inspiration. Dr. Rech has been
more than a mentor, he is a friend.

LIST 0'
LIST 0’
LIST or

Chapter

Chapter

Chapter

Chapter

Chapter
Chapter

L18? 0?

Page
THBBOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO Vii
rIGma...OOOOOOOOOOOOOOOOOOOOCOOO0.00.00.00.00 ix
ABBREVIATIONS AND NOMENCLAIURB...... ........... xv
1. ImbaflIONOOOOO0.0.0.0000...OOOOOOOOOOOOO 1
speCific AimSOOOOOOOOOOOOO ........ O... 6
2. "TIOMOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.0... 8
3. “TEDDBOIOCOOOOOOOOOOOCOO000...... ..... O... 16
subjects - FOOd - Hangingooooooooooooo 16
Training.0.00.0.0...OOOOOOOOOOOOOOOOO. 16
Dmg DOSing.OOOOOOOOOOOOOOOOOOOOOOOOO. 17
Treatment and Testing Schedule........ 18
Analysis of Brain and Serum
Pentobarbital Concentrations......... 22
statistical AnaIYSiSOOOOOOOOOOOOO0.... 24
4. “BOLTSOOOOOOIOOOOOOOOOOOOOOOOOOO0.0.000... 25
Hypothermia Tolerance................. 25
Ataxia Tolerance...................... 31
Pentobarbital Brain Concentration
and Behavior Comparisons............. 34
5. DISCOBBIO'.OOOOOOOOOOOOOOOOOOOOOO00....O... 84
6. BUIIIR! AND COUCLUBIONB.................... 97

“IsmaBOOOOCOOIOCCOOOOOOOOOOOOOOOOOOOOOIOOOC 100

”mxxl. Additional DataOOCOOCOOOOOO00.0.0000...0.. 109

1??me 3. Statistical Values........................ 112

vi

Page

Table 1. Schedule of Treatment Periods and
Pentobarbital Exposure for All Four
GrouPSOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 19

Table 2. Pentobarbital Postchronic Log Dose-
Response Regression Coefficients,
Hypothermia Duration and Peak,
Ataxia Duration and Peak........... ....... 35

Table 3. Pentobarbital (40 mg/kg) Hypothermia,
Ataxia, and Brain Concentration Group
Means:Soeomooooo00.000000000000000...one 37

Table 4. Number of Subjects for Each Test (N) and

Group (n)OOOOOOOOOOOO0.0000000000000000000 109
Table 5. PENTOBARBITAL CHRONIC TREATMENT PERIOD

mSING PAI%OOOOOOOOOOOOOOOOOOOO0......OO. 110
Table 6. PENTOBARBITAL TEST DOSES for Postchronic

vs. Postwithdrawal vs. Postextinction
TestSOOOOOOO0.0.0.0.0000...0.0.0.0000...O. 111

Table 7. PENTOBARBITAL HYPOTHERHIA, Prechronic
and Postchronic F-values and
PtObability “VBISOOOOOOOOOOOOO0......O... 112

Table 8. PENTOBARBITAL HYPOTHERMIA, Postchronic
vs. Postwithdrawal vs. Postextinction
Tests F-values and Probability Levels..... 113

Table 9. PENTOBARBITAL ATAXIA, Prechronic vs.
Postchronic and EXP vs. NonEXP,
Probability Levels for Duration, Peak
and Total................................. 114

vii

LI§I_QI_ILEL£§_122351331§1

Table 10.

Table 11.

Table 12.

PENTOBARBITAL ATAXIA, Prechronic vs.
Postchronic Time Course Mann-Whitney U
Test PrObability uve18O O O O O O O O O O O O O O O O O O O 115

PENTOBARBITAL ATAXIA, Postchronic EXP vs.
NonEXP Time Course Mann-Whitney U Test
PrObability IgveISOOOOOOOOOOOOOOOOOOOOOOOO 116

PENTOBARBITAL POSTCHRONIC LOG DOSE-

RESPONSE REGRESSION LINE PARALLELISM,

T-test Values for Hypothermia Duration

and Peak, Ataxia Duration and Peak........ 117

viii

Page

Figure 1. Hypothermia tolerance development to
a 20 mg/kg pentobarbital dose after
intermittent drug treatment. Time
course of change in body temperature

is.e.mOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 39

Figure 2. Hypothermia tolerance development to
a 28 mg/kg pentobarbital dose after
intermittent drug treatment. Time
course of change in body temperature
t s.e.m............................... ...... 40

Figure 3. Hypothermia tolerance development to
a 40 mg/kg pentobarbital dose after
intermittent drug treatment. Time
course of change in body temperature

is.eOnOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 41

Figure 4. Hypothermia tolerance development to
a 28 mg/kg pentobarbital dose after
chronic drug treatment. Time course
of change in body temperature i s.e.m. ...... 42

Figure 5. Hypothermia tolerance development to
a 40 mg/kg pentobarbital dose after
chronic drug treatment. Time course
of change in body temperature 1 s.e.m....... 43

Figure 6. Hypothermia tolerance development to
an 80 mg/kg pentobarbital dose after
chronic drug treatment. Time course
of change in body temperature 1 s.e.m....... 44

ix

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

10.

11.

12.

13.

14.

Pentobarbital tolerance development

to peak hypothermia i s.e.m. after
intermittent or chronic drug
treatment........................... ........

Pentobarbital tolerance development
to duration of hypothermia i s.e.m.
after intermittent or chronic drug
treatment...................................

Pentobarbital tolerance development

to total hypothermia (see text for

details) 1 s.e.m. after intermittent

or chronic drug treatment........... ..... ...

Hypothermia time course for a 28

mg/kg pentobarbital test dose.

Comparison of intermittent vs.

chronic drug treatment. Change in

body temperature 1 s.e.m.......... ..... .....

Hypothermia time course for a 40

mg/kg pentobarbital test dose.

Comparison of intermittent vs.

chronic drug treatment. Change in

body temperature i s.e.m............ ........

Loss of pentobarbital tolerance to

peak hypothermia i s.e.m. after a
withdrawal period and extinction
trials............................. .........

Loss of pentobarbital tolerance to

duration of hypothermia i s.e.m.

after a_withdrawal period and

extinction trials................. ..........

Loss of pentobarbital tolerance to

total hypothermia i s.e.m. after a
withdrawal period and extinction
trials............................ ......... .

Page

45

46

47

48

49

50

51

52

Page

Figure 15. INT/EXP loss of pentobarbital
tolerance to hypothermia after a
withdrawal period and extinction
trials. Time course of change in
body temperature i s.e.m........... ..... .... 53

Figure 16. INT/NonEXP loss of pentobarbital
tolerance to hypothermia after a
withdrawal period and extinction
trials. Time course of change in
body temperature i s.e.m............. ....... 54

Figure 17. CHR/EXP loss of pentobarbital
tolerance to hypothermia after a
withdrawal period and extinction
trials. Time course of change in
body temperature i s.e.m.................... 55

Figure 18. CHR/NonEXP loss of pentobarbital
tolerance to hypothermia after a
withdrawal period and extinction
trials. Time course of change in
body temperature 1 s.e.m............... ..... 56

Figure 19. Ataxia tolerance development to a 20
mg/kg pentobarbital test dose after
intermittent drug treatment. Time
course of rotarod performance 1

s.eOmOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 57

Figure 20. Ataxia tolerance development to a 28
mg/kg pentobarbital test dose after
intermittent drug treatment. Time
course of rotarod performance 1

s.eOmOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 58

Figure 21. Ataxia tolerance development to a 40
mg/kg pentobarbital test dose after
intermittent drug treatment. Time
course of rotarod performance 1

sOeOMOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 59

xi

Page

Figure 22. Ataxia tolerance development to a 28
mg/kg pentobarbital test dose after
chronic drug treatment. Time course
of rotarod performance 1 s.e.m........ ..... . 60

Figure 23. Ataxia tolerance development to a 40
mg/kg pentobarbital test dose after
chronic drug treatment. Time course
of rotarod performance 1 s.e.m....... ....... 61

Figure 24. Ataxia tolerance development to an
80 mg/kg pentobarbital test dose
after chronic drug treatment. Time
course of rotarod performance t s.e.m ..... .. 62

Figure 25. Pentobarbital tolerance development
to peak ataxia t s.e.m. after
intermittent or chronic drug
treatment................................... 63

Figure 26. Pentobarbital tolerance development
to duration of ataxia i s.e.m. after
intermittent or chronic drug
treatment........................... ....... . 64

Figure 27. Pentobarbital tolerance development
to total ataxia i s.e.m. after
intermittent or chronic drug
treatment................................... 65

Figure 28. Ataxia time course for a 28 mg/kg
pentobarbital test dose. Comparison
of intermittent vs. chronic drug
treatment. Change in rotarod
performance 1 s.e.m.............. ........... 66

Figure 29. Ataxia time course for a 40 mg/kg
pentobarbital test dose. Comparison
of intermittent vs. chronic drug
treatment. Change in rotarod
performance 1 s.e.m......................... 67

xii

Page

Figure 30. Loss of pentobarbital tolerance to
duration of ataxia i s.e.m. after a
withdrawal period and extinction
trials......................... ............. 68

Figure 31. Loss of pentobarbital tolerance to
total ataxia i s.e.m. after a
withdrawal period and extinction
trials............................. ......... 69

Figure 32. INT/EXP loss of pentobarbital
tolerance to ataxia after a
withdrawal period and extinction
trials. Time course of rotarod
performance 1 s.e.m......................... 70

Figure 33. INT/NonEXP loss of pentobarbital
tolerance to ataxia after a
withdrawal period and extinction
trials. Time course of rotarod
performance 1 s.e.m......................... 71

Figure 34. CHR/EXP loss of pentobarbital
tolerance to ataxia after a
withdrawal period and extinction
trials. Time course of rotarod
performance 1 s.e.m......................... 72

Figure 35. CHR/NonEXP loss of pentobarbital
tolerance to ataxia after a
withdrawal period and extinction
trials. Time course of rotarod
performance 1 s.e.m......................... 73

Figure 36. Pentobarbital log dose-response
regression analysis for peak
hypothermia t s.e.m............. ............ 74

Figure 37. Pentobarbital log dose-response

regression analysis for duration
of hypothermia i s.e.m................ ...... 75

xiii

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

38.

39.

40.

41.

42.

43.

44.

45.

Pentobarbital log dose-response
regression analysis for total

hypothermia i s.e.m.............. ..... ......

Pentobarbital log dose-response
regression analysis for peak

ataXiaiSOeOmOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

Pentobarbital log dose-response
regression analysis for duration

at ataXiais.eOmOOOOOOOOOOOOOOOOOOOOO OOOOOO

Pentobarbital log dose-response
regression analysis for total

ataxia:BOeOmOOOOOOOOOOOOOOOOOOOOO OOOOOOO OO

Pentobarbital brain concentrations
s.e.m. as a function of time after
injection. Comparison of
intermittent vs. chronic drug

i

treatmentOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

Representation of ANCOVA, mean of
the pentobarbital brain
concentration multiplied by the

hypothermia t s.e.m.........................

Regression analysis of mean
hypothermia i s.e.m. as a function
of mean pentobarbital brain
concentration 1 s.e.m. Comparison
of EXP vs. NonEXP.................

Tolerance development to total
ataxia i s.e.m. during prechronic
days 1-3, prechronic days 4-6, and
after intermittent drug treatment
with or without drug-effect

experiencaOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

xiv

Page

76

78

79

80

81

82

83

 

Treatment Groups:

INT/IX? - intermittent drug exposure (i.p. injection every
4th day) during the Chronic Treatment Period /
drug deficit experience (ataxia-rotarod testing
and hypothermia-body temperature measurements)

Ina/nonnxr - intermittent drug exposure (i.p. injection
every 4th day) during the Chronic Treatment
Period / protection from drug deficit
experience by prevention of ambulation with a
towel wrap and body temperature maintenance
with a heat lamp

CHI/RIP - chronic drug exposure (daily i.p. injection plus
drug in ground chow) during the Chronic Treatment
Period / drug deficit experience (ataxia-rotarod
testing and hypothermia-body temperature
measurements)

CHR/ROnRXP - chronic drug exposure (daily i.p. injection
plus drug in ground chow) during the Chronic
Treatment Period / protection from drug deficit
experience by prevention of ambulation with a
towel wrap and body temperature maintenance
with a heat lamp

Behavioral manipulations:

RR - rotarod testing

8T - body temperature measurement

RR 5 RT - rotarod testing (every 15 minutes) and body
temperature measurements (every 10 minutes) for
two hours

3T! - protection from drug deficit experience by prevention

of ambulation with a towel wrap and body temperature
maintenance with a heat lamp

 

Prechronic - measurements obtained from INT/EXP and CHR/EXP
during the Pre-chronic Dose-response Test
Period
Postchronic - measurements obtained for all groups to a
Dose-Response Test after the Chronic Treatment
Period
Postwithdrawal - measurements obtained for all groups to a
single test dose after a 10-day withdrawal
period
Postextinction - measurements obtained for all groups to a
single test dose after 10 days of
extinction.training
i.p. - intraperitoneal
rpm - revolutions per minute
i.d. - inner diameter
AIOVA - analysis of variance
ARCOVA - analysis of covariance

C8 - conditioned stimulus, classical 'Pavlovian
conditioning' - e.g. bell

0C8 - unconditioned stimulus, classical 'Pavlovian
conditioning' - e.g. food

xvi

Adaptation to changes in environment is inherent to life.
Adaptations may be manifested structurally, physiologically,
biochemically, and/or behaviorally. Drug administration is
an example of environmental change. Adaptation to drug
effects can produce drug tolerance and dependence in some
situations, while it can produce drug sensitization in other
situations (LeBlanc and Cappell, 1977).

Drug tolerance is a phenomenon in which repeated
administration of a drug is accompanied by a decrease in a
drug effect, requiring an increased amount of drug to produce
the same effect (Kalant,et al., 1971: Krasnegor, 1978; LeBlanc
and Cappell, 1977; Schuster, 1978). A parallel shift to the
right in the dose-response curve demonstrates drug tolerance
(Goudie and Griffiths, 1986; Schuster, 1978).

A given drug generally produces multiple responses with
multiple mechanisms for these responses. Drug tolerance
likewise has multiple mechanisms, resulting in various levels
of tolerance for each drug effect. Drug tolerance has been
split into two major mechanistic categories: dispositional

and functional (Goldstein, 1979: Goudie and Griffiths, 1986:

2
Kalant et al., 1971: Krasnegor, 1978; LeBlanc and Cappell,
1977; Schuster, 1978).

Dispositional or pharmacokinetic tolerance occurs in
chronically-treated subjects when a decreased drug
concentration reaches the site of drug action as compared to
control subjects. This may be produced by changes in drug
absorption, distribution, metabolism, and/or excretion
(Krasnegor, 1978: Schuster, 1978) . Examples of mechanisms for
dispositional tolerance which more rapidly decrease the plasma
concentration of the drug include induction of drug
metabolizing enzymes, increased production of non-target
tissue binding proteins, or increased elimination at the
kidney.

Functional tolerance is a decrease in the functional
effect of the drug without a decrease in drug concentration
at the site of action. The traditional concept of functional
tolerance is that of cellular tolerance. Cellular or
pharmacodynamic tolerance occurs when there is a decreased
response of the target cells to the same concentration of the
drug at the site of drug action that initially produced
greater deficits. Possible mechanisms may include changes in
drug or neurotransmitter receptor numbers, changes in the
production or release of second messengers, or changes in
levels of endogenous chemicals which act at the drug receptor
or for which availability to their receptors is affected by

the drug .

3

It has been demonstrated that some types of functional
drug tolerance are affected by manipulations that also alter
associative learning/conditioning. Examples of these
manipulations are environmental conditioning, task practice,
reinforcement loss, stimulus strength, etc. , all involving
stimulus and/or response expectancies. It has been suggested
by some researchers that there is a separate tolerance
component, behavioral tolerance, which is due to a. 'more
specific development of learned behavioral changes that allow
the organism to accommodate to the drugged condition' (Tang
and Falk, 1978).

It is, presumably, behavioral tolerance that can be
altered by stimulus and response expectancies rather than the
classical cellular tolerance defined as a target-cell
physiological change altered by the presence of the drug.
Behavioral tolerance involves a mechanism similar to
behavioral conditioning or learning. Development of this type
of tolerance requires an association between the experience
of the drug effect and specific behaviors which are affected.
Behavioral tolerance is thought to be an adaptation to the
alterations in behavioral stimuli or response patterns caused
by the drug effects. Classical cellular and dispositional
tolerances are adaptations to the drug and/or its proximate
physiological effects. These adaptations can be expressed in
terms of decreased neuronal sensitivity without requiring

drug-deficit experience for cellular tolerance or reduced

4
delivery of drug to the site of action for dispositional
tolerance.

Adaptations may be useful and/or harmful. Drug tolerance
complicates many therapeutic applications (Schuster, 1978).
Tolerance may develop to both wanted and unwanted drug
effects. An understanding of what behavioral contributions
are made to the tolerance of certain drug effects may be
useful in therapeutic drug management by allowing an approach
toward enhancing or diminishing tolerance development to some
drug effects.

Many drugs (such as opiates, barbiturates, alcohol) which
have actions on the central nervous system and produce drug
tolerance upon repeated administration also produce physical
dependence and an accompanying withdrawal syndrome when
chronic drug treatment ceases. Although these two phenomena
do not always exist together, there is some correlation
between physical dependence and functional tolerance with many
drugs. The overt physiological changes which produce cellular
tolerance remain in effect for a relatively short time after
discontinuation of drug administration before re-adaptation
occurs. Most of the symptoms of withdrawal syndromes appear
to be rebound effects relating to compensations developed
during chronic drug treatment. Often, these symptoms are
alleviated by administration of the chronically-administered
drug or another drug in the same class. Thus, the
physiological changes which also produce drug tolerance may
be the manifestations which produce withdrawal symptoms upon

1 5
sudden discontinuation of drug administration (Dews, 1978;
Schuster, 1978).

Drug dependence also may have a behavioral conditioning
component which is related to behavioral tolerance (Balster,
1985; O'Brien et al., 1986; Siegel, 1983). This relationship
of drug tolerance and drug dependence appears to play a part
in drug addiction. Cappell and LeBlanc (1981) construe drug
tolerance and drug dependence as independent variables to
which drug consumption is a dependent variable. Psychological
factors are important in initiating and 'maintaining the
repeated drug administration in human drug abuse leading to
drug tolerance and drug dependence, but drug tolerance and
drug dependence are also important in perpetuating and
increasing drug consumption. Therefore, an understanding of
the factors involved in drug tolerance, which also may be
involved in drug dependence, is important for understanding
and combatting drug abuse (Krasnegor, 1978; Schuster, 1978).

Although dispositional tolerance and cellular tolerance
are accepted as two separate physiological adaptations, the
relationship of behavioral tolerance to these other types of
tolerance has not been established. Previous studies of
behavioral tolerance have looked at what type of behavioral
manipulations can produce and affect this type of tolerance,
but none have attempted to demonstrate the separation of
behavioral and cellular tolerance. This study was designed to
determine if some types of behavioral tolerance are 1)

developed due to repetitions of drug experience with the

6
behaviors in question without the necessity of chronic
administration; and 2) separable from cellular tolerance,
which theoretically would not require repeated experiences of
drug exposure during performance of the behaviors. Many
components of drug tolerance may be involved in the total
tolerance to a single drug effect. These components may
include dispositional, functional-cellular and functional-
behavioral types of tolerance. The goals of this study were
to determine what components exist within the total drug
tolerance for the motor ataxic and hypothermic effects of
sodium pentobarbital. More specifically, is there a separable
component that fits the definition of behavioral tolerance and
what contribution does this behavioral tolerance make to the
total tolerance observed after both intermittent and chronic

pentobarbital administration?

W:

1) To develop tolerance to the hypothermia and ataxia
effects of pentobarbital after both chronic and
intermittent drug treatment.

2) To attribute the tolerance developed to these
pentobarbital-induced deficits within each
experimental group either to learned adaptations
based on experience and/or to cellular/metabolic
tolerance due to chronic exposure to the drug.

3) To determine the presence of cellular and metabolic

types of tolerance in the chronic treatment groups.

4)

7
To determine whether the cellular and/or metabolic
types of tolerance are attributable to chronic drug
treatment without requiring specific drug
experience, and whether behavioral tolerance is
attributable to drug-deficit experience without

requiring development of cellular tolerance.

Simple behaviors were studied for ease of testing
sufficiently large numbers of subjects without long training
periods, allowing for the time-course of drug effects to be
determined, and for more widespread significance to normally
observed drug effects. Both a physiological drug effect
(hypothermia) and a behavioral drug effect (ataxia) were
chosen. Both of these deficits are controlled by the central
nervous system. The hypothermia was measured by recording the
rectal temperature and the ataxia measured by rotarod
performance.

The purpose of this study was to separate behavioral,
cellular, and metabolic tolerance to an extent that the
existence and autonomy of each could be examined. Separation
of the components was attempted by the design of the study
that incorporates exclusionary requisites. Four groups of
animals with the following types of tolerance development were
produced: (INT/EXP) behavioral tolerance only; (CHR/EXP)
behavioral and cellular/metabolic tolerance; (INT/NonEXP) no
tolerance; (CHR/NonEXP) cellular/metabolic tolerance only.
The existence of behavioral tolerance was determined by

comparing the tolerance developed in INT/EXP and INT/NonEXP

9

for intermittent treatment and in CHR/EXP and CHR/NonEXP for
chronic treatment. The existence of cellular/metabolic
combined tolerance was determined by comparing INT/EXP to
CHR/EXP and INT/NonEXP to CHR/NonEXP. Separation of the
cellular and metabolic components was produced through
analysis of brain concentration time courses and their
associated behaviors. In this manner the existence of
metabolic tolerance was determined by comparing the brain
concentration time courses of INT/EXP and INT/NonEXP to those
of CHR/EXP and CHR/NonEXP. The existence of cellular
tolerance was determined by comparing the behavioral deficits
produced by the corresponding brain concentrations of INT/EXP
to those of CHR/EXP and of INT/NonEXP to those of CHR/NonEXP.

Both cellular and metabolic tolerance development
require a continuous exposure to the drug for an extended
period. A dosing frequency greater than every other day is
required to produce either cellular or metabolic tolerance for
pentobarbital (Belknap et al., 1977; Cappell et al., 1981;
Okamoto et al., 1986) . Cellular/metabolic tolerances were
eliminated in the present study in two groups of rats by an
intermittent dosing schedule of one dose every four days.

Behavioral tolerance was minimized in two groups of rats
by attenuating the experience to the drug effects to be
measured. Minimizing the experience to the motor ataxia was
accomplished by immobilizing the animals in a towel wrap.
Minimizing the experience to the hypothermia was attained by

maintaining the body temperature at the predrug control

10
temperature with the aid of a heat lamp similar to the
procedure used by Alkana et al. (1983).

The chronic exposure groups were maintained on
pentobarbital by adding the drug to their ground chow. This
level of drug is not enough to produce overt effects even
though it is adequate (along with the daily injected.drug) for
chronic maintenance.

The first part of this study was designed to determine
the contribution and autonomy of behavioral tolerance to the
total tolerance. Since it was not possible to separate out
the metabolic tolerance from the cellular tolerance with the
design used in this part of the study, these two components
are referred to together as cellular/metabolic tolerance. The
design included four groups of rats: INT/EXP with experience
to drug effects, intermittent drug exposure and development
of behavioral tolerance only; CHR/EXP with experience to the
drug effects, chronic drug exposure, and development of both
behavioral and cellular/metabolic tolerances; INT/NonEXP*with
no experience to the drug effects, intermittent drug exposure
and no tolerance development; and CHR/NonEXP with no
experience to the drug effects, chronic drug exposure and
development of cellular/metabolic tolerance only. Thus, a
comparison between INT/EXP and INT/NonEXP should determine if
behavioral tolerance has been developed and whether it is
separable from cellular/metabolic tolerance. A comparison
between CHR/EXP and CHR/NonEXP should determine the relative

contribution of behavioral tolerance to the total tolerance

11
development after chronic administration in drug-experienced
rats.

The experimental design involved a training period, an
acclimation.period, a preliminary or prechronic dose-response
determination, a tolerance development period, a postchronic
dose-response determination, a withdrawal period, a
postwithdrawal single dose-effect test, an extinction period,
a postextinction single dose-effect test, a retention.period,
and a retention single dose-effect test (see Table 1).

The training period allowed training for the rotarod
performance as well as introduction to the towel wrap and
rectal temperature procedure. The animals were then
habituated 'to the two-hour towel wrap, repeated rectal
temperature measurements and rotarod performance tests during
the first six days of the study to allow subjects to adjust
to the stress of this schedule. All groups received the same
non-drug experiences to eliminate any differences due to
maintenance and testing treatments.

The preliminary dose-response determination was made in
duplicate from the animals in INT/EXP and CHR/EXP during the
second six days. The animals in INT/NonEXP and CHR/NonEXP
received the same drug exposure at this time but experience
was minimized by the towel wrap and temperature maintenance
with a heat lamp. Three doses of pentobarbital were
administered in a random order and then repeated. Matched
pairs of subjects were assigned (INT/EXP to INT/NonEXP and

CHR/EXP to CHR/NonEXP) to receive the same dosing order.

12

During the tolerance development period the animals in
CHR/EXP and CHR/NonEXP received daily i.p. pentobarbital
injections and pentobarbital in their ground chow for
initiation and maintenance of the cellular/metabolic
tolerance. The animals in INT/EXP and INT/NonEXP received
i.p. saline for three«days, then an i.p. dose of pentobarbital
on the fourth day and ground chow without pentobarbital.
During the three saline days for INT/EXP and INT/NonEXP all
animals in all groups were wrapped in the towel during the
treatment time.

Rotarod performance was tested for test-dose effects over
the tolerance development period in INT/EXP and CHR/EXP
(experienced), but was monitored only after vehicle during
this period in INT/NonEXP and CHR/NonEXP (nonexperienced).
On the fourth day the animals in INT/EXP and CHR/EXP were
tested after pentobarbital administration and the animals in
INT/NonEXP and CHR/NonEXP were tested after saline
administration. In order to preserve the equivalent items of
treatment of all animals, the animals in INT/EXP and CHR/EXP
received saline and then were towel wrapped, and the animals
in INT/NonEXP and CHR/NonEXP received pentobarbital and then
were towel wrapped on the fourth day also.

This four-day cycle was continued for 36 days, allowing
for nine spaced days of the pentobarbital-testing experience
for the animals in. INT/EXP and CHR/EXP, as well as 36
continuous days of chronic pentobarbital exposure for the

animals in CHR/EXP and CHR/NonEXPu The tolerance development

13

was maximized during these 36 days by the accelerating chronic
dosing method of Okamoto and Boisse (1981) for both the chow
exposure for the animals in CHR/EXP and CHR/NonEXP and for the
i.p. pentobarbital administration for all groups. The matched
pairs from INT/EXP and INT/NonEXP as well as CHR/EXP and
CHR/NonEXP were assigned for equivalent dosing increases for
the i.p. pentobarbital administration.

The postchronic dose-response determinations, testing for
drug effects on both body temperature and rotarod performance,
were made for all groups to measure tolerance development in
each group. Three doses were administered in a random order
to the matched pairs. The doses in the intermittent treatment
groups (INT/EXP and INT/NonEkP) were the same as the
prechronic determination (20, 28 and 40 mg/kg). Due to the
large amount of cellular/metabolic tolerance produced in the
chronic treatment groups (CHR/EXP and CHR/NonEXP) the
postchronic test doses were shifted upward in dose (28, 40,
and 80 mg/kg) . This was done in an attempt to produce
effective doses comparable: to those in the intermittent
treatment animals in order to achieve maximum sensitivity for
distinguishing behavioral tolerance in these groups.

The dose of pentobarbital that produced a just-
significant, short-lived effect (15-45 minutes in length) was
chosen for each animal from the postchronic dose determination
data. This dose was used subsequently as the test dose for the

postwithdrawal and postextinction tests (see below).

14

The pentbbarbital administration, both by injection and
in the ground chow, was then discontinued and the animals were
monitored for withdrawal signs. A ten-day withdrawal period
was allotted for cellular tolerance and metabolic tolerance
to dissipate completely (Okamoto et al., 1976). All animals
in all groups were then tested with a single test dose of
pentobarbital (indicated above) to determine the amount of
total tolerance that was lost after withdrawal. Any remaining
tolerance at this test is presumed to derive from a behavioral
tolerance mechanism.

Removal of the behavioral component of tolerance was
calculated to be achieved by application of ten days of
extinction trials, since behavioral tolerance is similar in
characteristics to conditioning/learning phenomena. Loss of
any behavioral tolerance was then determined by postextinction
testing with the single pentobarbital test dose.

The determination of cellular and metabolic tolerance
components was achieved in the second part of the study. The
training period, acclimation period, prechronic dose-response
determination, and tolerance development period were repeated
in new rats assigned to the same groups. A time-course for
brain concentrations was determined by sacrificing the animals
at various times after drug injection on day 49, immediately
after a final ataxia and hypothermia measurement was obtained.
The brain pentobarbital concentrations of the intermittent
animals were compared with the chronic animals to determine

the relative contribution of the metabolic tolerance component

15
to the total tolerance observed in the chronic groups. An
analysis of covariance was used to statistically control the
brain levels, therefore effectively eliminating the metabolic
tolerance. This allowed the existence of cellular tolerance
to be ascertained by comparing between experienced INT/EXP and
CHR/EXP, as well as between nonexperienced INT/NonEXP and
CI-IR/NonEXP. Thus, an accounting of the significance of
metabolic and/or cellular tolerance components induced by

chronic drug treatment was accomplished.

Masts - £9.94 - Housing

Male Sprague-Dawley rats (Harlan) weighing 175-200 grams
at the start of the study were maintained on a regular light-
dark cycle (dark between 7 p.m. and 7 a.m.) in temperature-
and humidity-controlled animal quarters. Animals were housed
individually with water available ad libitum. Food, as Lab
Blox’, was available ad libitum, except during the tolerance
development period (days 13-48) , when ground chow with or
without pentobarbital was available ad libitum only from 5
p.m. to 7 a.m. If the body weight of a subject receiving
pentobarbital dropped below 80% of the control diet animals'
body weights, a supplement of Very Vanilla Sego’ was made

available between 3 p.m. and 5 p.m.

I . .

After allowing the animals to accommodate to the
facilities, they were trained for 3-5 days on the rotarod
(RR) . This involves the subjects' learning to walk on an
elevated rotating cylinder (18 cm width, 10.5 cm diameter, and
9 rpm), the training criterion being maintenance on the

rotarod for at least 180 seconds for three consecutive trials

16

l7
(Rech et al., 1966; Akera et al., 1973; Commissaris and Rech,
1983).

During these training days the animals were also
introduced and adapted to the towel wrap and rectal
temperature probe. The towel wrap (BTW) involves securing the
animal snuggly, but not tightly, in a hand towel with Accoo
clips to render the animal immobile. Body temperature (BT)
was determined using a telethermometer (Yellow Springs Model
46 TUC) with a plastic coated probe which was inserted into
the rectum to a distance of 4 cm for 40 seconds (Commissaris

et al., 1982).

DIQQ_DQ§inQ

Solutions of sodium pentobarbital in 0.9% saline were
administered i.p. at a volume of one ml/kg. Test doses,
varied by group (see below), were 0 (vehicle), 20, 28, 40, and
80 mg/kg. During the chronic administration period (days 13-
48) the drug dosing was gradually increased (Okamoto and
Boisse, 1981), starting at 30 mg/kg, in 3 mg/kg increments,
as tolerance was displayed in INT/EXP and CHR/EXP on the RR
and BT testing days. Sodium pentobarbital was also
administered in ground chow placed in weighted cups at an
initial dose of two mg/g during the chronic administration
days 12-48. Gradual increases in the dose of pentobarbital
in the ground chow were made as the subjects consumed 80% or

more of the amount consumed by control diet rats.

18
WW
Subjects were randomly divided into four groups of 12
rats each (Ii-=48); INT/EXP, INT/NonEXP, CHR/EXP, and
CHR/NonEXP. INT/EXP and CHR/EXP experienced the motor
deficits and the hypothermia produced after pentobarbital
administration while performing on the rotarod and without
body temperature maintenance (RR & BT) . INT/NonEXP and
CHR/NonEXP did not experience the motor deficits or
hypothermia after pentobarbital administration because they
were rendered immobile by the towel wrap while their body
temperature was maintained within control range through the
application of heat lamps (BTW). INT/EXP and INT/NonEXP
received intermittent administration (every fourth day) of
pentobarbital, while CHR/EXP and CHR/NonEXP received chronic
administration of pentobarbital, during the tolerance
development period of the study. The entire seven periods of
sequential treatment lasted 72 days.

The subjects were manipulated as follows (see also Table

Minimum: Rats in INT/EXP and
CHR/EXP received vehicle at 10 a.m., then were

wrapped in the body towel and had their body
temperature maintained within the control range for
two hours of monitoring the body temperature every
ten minutes (body towel wrap or "BTW") . Rats in
INT/NonEXP and CI-IR/NonEXP received vehicle at 8

a.m., then were tested on the rotarod every 15

19

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20
minutes and had their body temperatures monitored
every ten minutes for two hours (rotarod and body

temperature or RR & BT).

- - - s :
Rats in INT/EXP and CHR/EXP received twice each of
three doses of pentobarbital (20, 28, and 40 mg/kg
i.p.) randomly assigned, one dose/day, at 8 a.m.
and then were tested for RR 5 BT. Rats in
INT/NonEXP and CHR/NonEXP received twice each of
the three doses of pentobarbital also but at 10 a.m.
followed by BTW.
Ig§p9n§g__;g§§__pg;iggl: Rats in INT/EXP and
INT/NonEXP*received control ground chow from.5 p.m.
to 7 a.m., while rats in CHR/EXP and CHR/NonEXP

received ground chow containing pentobarbital (2

mg/g initially) over the same period.

WWW: Rats
in INT/EXP and INT/NonEXP received i.p vehicle at
8 a.m. followed by BTW. Rats in CHR/EXP and
CHR/NonEXP received i.p pentobarbital (starting at
30 mg/kg) at 10 a.m. followed by BTW.
Di2E_1éi_ZQI_Zil_Z§l_lZl_§§l_in_iii_sn§_1§_12h22n1§
3g§;_ggy§1: Rats in INT/EXP and CHR/EXP received
i.p vehicle at 8 a.m. followed by BTW, then i.p.

pentobarbital at 10 a.m. followed by testing of RR

21

& BT. Rats in INT/NonEXP and CHR/NonEXP received
i.p. vehicle at 8 a.m. followed by testing of RR &
BT, then i.p. pentobarbital at 10 a.m. followed by
BTW.
WW:
Rats in all groups received three doses of
pentobarbital (20, 28, and 40 mg/kg for INT/EXP and
INT/NonEXP; 28, 40, and 80 mg/kg for CHR/EXP and
CHR/NonEXP) randomly assigned to one dose/day,
following which RR & BT were tested. From the
measurements obtained from this dose-response test
period, a test dose was chosen for each rat which
produced only a short-lived behavioral deficit.
This calculated dose was used as the test dose for
the Postwithdrawal test and Postextinction test.
WW1: Rats in all groups
were maintained in their home cages without drug
treatment and monitored for withdrawal signs (such
as hyperexcitability, tremor, spasticity,
hyperthermia, loss of body weight, convulsions).
MW: Rats in all
groups received the above-calculated i.p.
pentobarbital test dose followed by R & BT testing.

WM: Rats in all groups
received i.p. vehicle followed by RR 8 BT testing.

WW: Rats in all

22
groups received the i.p. pentobarbital test dose
followed by RR & BT testing.
WW: Rats in all groups
received the i.p. pentobarbital test dose followed

by RR & BT testing.

&!2 ‘ f -’ = a ! 1!! ‘1mu "! 092,:_ a O! ‘1 ,2 .-:=

The above treatment and testing schedules were repeated
in other rats to day 48. Each group contained 20-21 rats.
Four rats from each group were started at one time with three
to four days in between starting times for each set. On day
49 from the start of the treatment schedules, all groups
received 40 mg/kg of pentobarbital and then were tested for
RR 8 BT. Rats were sacrificed by decapitation in groups of
at least four (one from each group of INT/EXP, INT/NonEXP,
CHR/EXP, and CHR/NonEXP) each at 15, 30, 60, and 120 minutes
immediately after the last BT recording and RR test (within
five minutes). The trunk blood and the brain were collected‘
from each subject and frozen at -90 'C until time was
available for extraction of the pentobarbital and gas
chromatographic analysis of the sample extracts.

Extraction of the pentobarbital from the brain was
modified from the methods of Belknap et al., 1977, and
Commissaris et al., 1982. The brain samples were thawed,
weighed, and homogenized in two volumes of 0.1 N HCl. A 500
pl sample of the brain homogenate was then added to a 50 ml

silanized centrifuge tube containing one ml distilled water

23
and 2.5 pg amobarbital (in 100 pl methanol) as an internal
standard for the gas chromatographic analysis. Then 2 ml of
0.4 N HCl was added and the sample was sonicated. Chloroform
(15 ml) was added and the sample was vortexed for 30 seconds
then centrifuged. The bottom (chloroform) layer was removed
with a transfer pipet and filtered through anhydrous sodium,
sulfate into a second 50 ml silanized centrifuge tube. Next
5 ml of 1.0 N'NaOH were added then the sample was vortexed and
centrifuged. The chloroform layer was then removed with a
transfer pipet and discarded. HCl (5 ml of 1.0 N) was added
and the sample was vortexed. Next 10 ml of chloroform were
added and the sample was vortexed and centrifuged. The bottom
(chloroform) layer was filtered through anhydrous sodium
sulfate into a 15 ml conical tube. The sample was then dried
with a gentle stream of nitrogen. Finally, 25 pl of TMAH
(trimethylaniliniumhydroxide in methanol; MethElute‘, Pierce
Biochemicals) were added prior to injection of the sample onto
the gas chromatographic column, 4 mm id packed with SP2250'
(Supelco) in a Varian Aerograph 2400 gas chromatograph with
an HP 3392A integrater. The carrier gas was nitrogen with a
flow rate of 60 ml/min. The injection port temperature was
250 'C and the column temperature was 180 'C. Detection was
by flame ionization at 250 'C with a mixed dry air and
hydrogen flame. Pentobarbital determination was by peak area
ratio method with the 2.5 pg amount of amobarbital as an
internal standard. A pentobarbital recovery of 95% or

greater after the extraction was achieved for the tissue

24
samples. A pentobarbital standard curve was generated that

was linear between 0.5 and 120 pg/sample.

W

Factorial and repeated measures analyses of variance
(ANOVAs) were used to analyze all of the body temperature data
and the rotarod duration of ataxia data. If the variances for
the groups being compared were nonhomogeneous, the non-
parametric Kruskal-Wallis or Mann-Whitney U tests were
performed. For the rotarod time course and peak effect data,
nonparametric Kruskal-Wallis and Mann-Whitney U tests were
employed. Regression analysis was performed on the
postchronic log dose-response data. Repeated measures ANOVA
was ‘used for' the brain. concentration. time course data.
Analysis of covariance (ANCOVA) was employed for the analysis
of the brain concentrations related to their corresponding

behavioral deficits.

For the number of subjects in each group, at each test,
see Table 4, Appendix A. For the final pentobarbital doses
reached from the maximally accelerated dosing during the
chronic treatment period by the rats of INT/EXP, INT/NonEXP,

CHR/EXP and CHR/NonEXP see Table 5, Appendix A.

11W

All hypothermia data were analyzed with ANOVA and Tukey's
test for' multiple comparisons. For' data in ‘which the
variances between groups being compared were nonhomogeneous,
a non-parametric (Kruskal Wallis or Mann-Whitney U) test was
used to confirm ANOVA results. For F values and probability
levels see Appendix 8.

Comparing the time course of the hypothermic effect of
pentobarbital after the tolerance development period in
INT/EXP and INT/NonEXP to that of the initial dose-response
determination reveals tolerance development after intermittent
treatment (See Figures 1, 2 and 3) . INT/EXP (Figure 1)
demonstrated significant tolerance at all time points except
the 90-minute time point for ‘the low' dose (20 mg/kg).

INT/NonEXP (Figure 1) demonstrated significant tolerance only

25

26

at 10-40 minutes for the low dose (20 mg/kg). For the middle
dose (28 mg/kg), significant tolerance was observed at 10-40
minutes and at 120 minutes for both INT/EXP and INT/NonEXP,
while INT/EXP also demonstrated tolerance at 80-110 minutes
(see Figure 2) . At the high dose (40 mg/kg) INT/EXP displayed
significant tolerance from 20-110 minutes, while INT/NonEXP
demonstrated significant tolerance from 10-90 minutes (see
Figure 3).

As demonstrated by the prechronic bars in Figures 7, 8
and 9, peak hypothermia, duration of hypothermia and total
hypothermia (approximation of area under the time course
curve) were reliable dose-dependent measurements of the
pentobarbital hypothermia effect. In accordance, these
measurements were also examined for tolerance development.

INT/EXP (Figure 7) also demonstrated a significant
tolerance with respect to the peak hypothermia produced by
pentobarbital after the low and high doses. INT/NonEXP
(Figure 7) displayed a significant tolerance with respect to
the peak hypothermia only at the high dose. There was a
significant decrease in the duration of the pentobarbital-
induced hypothermia after the low and middle doses for INT/EXP
(Figure 8) as compared to the prechronic controls. There was
no change in the duration of the pentobarbital hypothermia for
INT/NonEXP (Figure 8) at any of the three doses.

This demonstrates there‘ was a trend for increased
tolerance in INT/EXP over that observed in INT/NonEXP, which

was best demonstrated by the low-dose data. This°trend was

27

further demonstrated by the total hypothermia determination
(area under the curve approximation, Figure 9), which was an
addition of all hypothermia readings produced for each time
point from 10-120 minutes. Again, significant tolerance to
total hypothermia was demonstrated for INT/EXP (Figure 9) for
all three doses. INT/NonEXP (Figure 9) demonstrated
significant tolerance to total hypothermia only at the highest
dose.

No significant difference in the postchronic time course
for the hypothermic effect was observed between INT/EXP and
INT/NonEXP for the low (Figure 1) and middle (Figure 2) doses,
although there was a trend for INT/EXP to demonstrate less
hypothermia. This trend was reversed for the high dose
(Figure 3), and achieved significance at the 20-minute time
point where INT/NonEXP produced less hypothermia than INT/EXP.
There was no significant difference between INT/EXP and
INT/NonEXP for peak hypothermia (Figure 7), overall duration
of hypothermia (Figure 8) and total hypothermia (Figure 9) at
all three doses postchronically.

Comparing the dose-response time courses of the CHR
animals to those produced at the prechronic dose-response
determination demonstrates significant cellular/metabolic
tolerance (see Figures 4 through 6). The CHR animals received
larger amounts of pentobarbital (28, 40, and 80 mg/kg), in
essence a shifted dose-response curve due to the large amount
of cellular/metabolic tolerance in these animals. There was

significant tolerance from 20 to 120 minutes in both CHR/EXP

28

and CHR/NonEXP produced for the 28 mg/kg dose (Figure 4). The
40 mg/kg' dose (Figure 5) produced similar' results ‘with
significant tolerance at 20-120 minutes for both CHR/EXP and
CHR/NonEXP. Although there was no 80 mg/kg dose for the
prechronic measures, a significant difference would surely
have been produced compared to CHR/EXP and CHR/NonEXP body
temperature measures, since the 80 mg/kg dose in naive rats
is within the lethal range of the pentobarbital dose-response
curve (Barnes and Eltherington, 1973). The peak hypothermia
(Figure 7), overall duration of hypothermia (Figure 8) and
total hypothermia (Figure 9) for' CHR/EXP’ and CHR/NonEXP
demonstrated similar results as seen with the time courses.
Both CHR/EXP and CHR/NonEXP were significantly tolerant for
both measures at the 28 and 40 mg/kg doses, while the 80 mg/kg
measurements for CHR/EXP and CHR/NonEXP were similar to the
prechronic 40 mg/kg dose hypothermia levels.

Behavioral tolerance was not apparent in the CHR animals
at the postchronic dose-response determination (see Figures
4 through 6). CHR/EXP was not significantly different from
CHR/NonEXP at any time point of the time courses. There was
also no significant difference between EXP and NonEXP in the
CHR animals for peak hypothermia (Figure 7) , duration of
hypothermia (Figure 8) and total hypothermia (Figure 9) for
all three dose comparisons.

The postchronic test period tolerance observed for all
four groups can.be compared in Figure 10 for the 28 mg/kg dose

and Figure 11 for the 40 mg/kg dose. These time courses

29
demonstrate the large amount of cellular/metabolic tolerance
produced in the CHR groups as compared to the INT groups and
the prechronic testing.

Further characterization of the tolerance types involved
in the total tolerance seen for each group was attempted by
observing the loss of tolerance after a ten-day withdrawal
period and after a ten-day extinction training period. A test
dose was chosen for each subject based on their rotarod
performance during the Postchronic Testing (see Table 6,
Appendix A for individual doses). Comparisons were made
within each group of a postwithdrawal test and a
postextinction test to the postchronic test. Additionally,
to determine if a significant amount of tolerance was lost due
to extinction trials, the postextinction test was compared to
the postwithdrawal test.

INT/EXP demonstrated no tolerance loss after withdrawal
and extinction training for peak, duration, total and time
course of hypothermia (see Figures 12, 13, 14 and 15). This
would indicate that a conditioning-type of adaptation was not
present or that the extinction trials employed were not
sufficient to extinguish the conditioning-like behavioral
tolerance. The INT/NonEXP group showed no significant change
in the pentobarbital-induced hypothermia for peak, duration,
total or time course comparing postchronic, postwithdrawal and
postextinction tests, as would be expected (see Figures 12,

13, 14 and 16).

30

Although the CHR groups showed no differences to indicate
there was behavioral tolerance development in the EXP group,
a comparison of the postchronic, postwithdrawal and
postextinction data for extent of hypothermia indicates that
a conditioning-like component was indeed present. For the
animals in CHR/EXP, a small trend (non-significant) for loss
of tolerance was observed for the peak hypothermia (Figure
12), overall duration of hypothermia (Figure 13) and total
hypothermia (Figure 14) at the postwithdrawal test, although
a significant loss of tolerance for all three measures was
seen for CHR/NonEXP after withdrawal.

A significant loss of tolerance was observed at the
postextinction test for CHR/EXP regarding all three measures,
while there was no significant additional loss in CHR/NonEXP
after extinction trials. The overall duration of hypothermia
(Figure 13) and total hypothermia (Figure 14) for CHR/EXP
showed significant tolerance loss between the postwithdrawal
and postextinction trials, which further supports a
conditioning-like component for this tolerance. The time
courses for CHR/EXP and CHR/NonEXP (Figures 17 and 18) also
demonstrate that CHR/EXP had lost significant tolerance after
withdrawal and additionally after extinction trials, while the
tolerance observed in CHR/NonEXP was significantly lost after
withdrawal with just a slight trend (non-significant) for

additional loss after extinction trials.

31

mm

Data on peak and duration aspects of pentobarbital ataxia
were analyzed with ANOVA and Tukey's test for multiple
comparisons. For data in which the variances between groups
being compared were nonhomogeneous, a non-parametric (Kruskal
Wallis or Mann-Whitney U) test was used to confirm ANOVA
results. For time-course data, the non-parametric Kruskal
Wallis and Mann-Whitney U tests were performed. For F values
(where appropriate) and probability levels see Appendix II.

Examination of the prechronic dose-response rotarod data
demonstrated a fast developing (probably behavioral) tolerance
by comparing the first three days to the last three days of
the six day period (see Figure 45) . This decreased drug-
induced ataxia became significant at the highest dose (40
mg/kg) . Recall that the second three days of this period
repeated the random drug dosing order of the first three days,
thus a change in dosing patterns does not complicate these
results. Due to this rapid tolerance development the
prechronic ataxia measures will only include the first three
days of the prechronic dose-response determination data.

Figures 19 through 21 depict the postchronic time courses
for the motor ataxia in the INT animals, compared with
prechronic ataxia measures. INT/EXP demonstrates no tolerance
over all three doses as compared to prechronic testing.
However, a significantly increased ataxia was produced in
INT/NonEXP as compared to prechronic testing for the two lower

doses (20 mg/kg and 28 mg/kg). This increased ataxia for

32
INT/NonEXP was also significant as compared to INT/EXP scores
at the middle dose (28 mg/kg) time points from 105-120
minutes.

The peak ataxia, duration of ataxia, and total ataxia
(approximation of the area under the time course curve) were
demonstrated as reliable dose-dependent measures of
pentobarbital ataxia by the prechronic data representations
in Figures 25, 26 and 27.

There was a significantly increased overall duration of
ataxia and a significantly decreased overall performance
(total ataxia) in INT/NonEXP as compared to the prechronic
testing and as compared to INT/EXP postchronic testing for the
low and middle doses (see Figure 26 and 27). Thus, in terms
of duration of ataxia and total ataxia, INT/NonEXP actually
demonstrated an enhanced effect on postchronic testing,
relative to the prechronic test. No similar enhancement of
these ataxia measures was observed for INT/EXP. No
significant differences were observed between the prechronic
testing and postchronic testing of INT/EXP and INT/NonEXP for
the peak ataxia (Figure 25), although trends similar to the
duration of ataxia and total ataxia effects were noted.

The CHR animals manifested significant tolerance
development when compared to the 28 and 40 mq/kg prechronic
ataxia measures. This ataxia tolerance was observed with the
time courses (Figures 22 and 23), as well as the duration of

ataxia (Figure 26) and total ataxia (Figure 27).

33

Although there was no significant difference between
CHR/EXP and CHR/NonEXP at any dose postchronically, the CHR
animals demonstrated a slight, non-significant trend for
behavioral tolerance to pentobarbital-induced ataxia for the
28 and 40 mg/kg dose time courses (see Figures 22 and 23).
There was also a trend (non-significant) at these doses for
CHR/EXP to display more tolerance than CHR/NonEXP with respect
to peak ataxia (Figure 25), duration.of ataxia (Figure 26) and
total ataxia (Figure 27), which was reversed at the 80 mg/kg
dose.

The cellular/metabolic tolerance to ataxia for the CHR
animals was depicted in Figures 28 and.29. These time courses
clearly demonstrate the degree of tolerance in CHR/EXP and
CHR/NonEXP for the 28 and 40 mg/kg doses as compared to the
lack of tolerance in INT/EXP and INT/NonEXP (actually,
enhanced effect in INT/NonEXP).

The postwithdrawal and postextinction tests for INT/EXP
and INT/NonEXP failed to demonstrate any loss of tolerance to
the pentobarbital ataxia (see Figures 30-33).

Although there was only a slight trend for behavioral
tolerance demonstrated postchronically for CHR/EXP, the
postwithdrawal and postextinction tests provided affirmation
of that behavioral component (see Figures 30, 31 and 34) .
After withdrawal CHR/EXP’demonstrated some tolerance loss, as
would be expected for the cellular/metabolic component, but
a significant amount of additional tolerance was lost after

extinction trials (see Figures 30, 31 and 34). CHR/NonEXP

34
demonstrated significant tolerance loss after’withdrawal, but
little additional loss after extinction trials (see Figures
30, 31 and 35), indicating that only cellular/metabolic
tolerance was predominant at the postchronic test.

The regression coefficients (Table 2) from the log dose-
response graphs (Figures 36 through 41) were compared. There
were no significant differences between any regression
coefficients in any of the six graphs. There was, however, a
slight ‘trend, for' CHR/EXP, CHR/NonEXP, and INT/NonEXP 'to
produce a decrease in slope as compared to INT/EXP for all
four’graphs and as compared to the Prechronic data in the peak
effect graphs (Figures 36 and 38). A trend for a very
similar, also non-significant, change in slope was observed
in the duration of hypothermia (Figure 37), hypothermia peak
(Figure 36), total hypothermia (Figure 38), ataxia peak
(Figure 39) and
total ataxia (Figure 41) for the NonEXP groups. The common
bond for these trends was that all three groups (CHR/EXP,
INT/NonEXP, and CHR/NonEXP) had experience with the drug in
the towel wrap. INT/NonEXP and CHR/NonEXP, which displayed
very similar slopes in five graphs, had only experienced the
drug within the towel wrap. CHR/EXP had slopes which were
less effected and more similar'to INT/EXP, perhaps due to some

experience with the drug outside the towel wrap.

35

2A§L§_Z

Pentobarbital Postchronic Log Dose-
Response Regression Coefficients
Hypothermia Duration and Peak,
Ataxia Duration and Peak

 

 

 

Hypothermia Ataxia
Groups Peak Duration Peak Duration
Prechronic -5.85 176.8 -55.7 166.6
INT/EXP -5.48 254.1 -74.2 199.4
CHR/EXP -4.97 201.1 -46.0 133.6
INT/NonEXP -3.33 134.8 -16.1 176.9
CHR/NonEXP -3.69 145.3 -16.2 111.1

 

 

36
-;, ... . . : . , ., ;, . ., 1,. g;,. . .u.. ;. s

The group means for hypothermia, ataxia, and
pentobarbital brain concentrations are shown in Table 3.

The pharmacokinetic tolerance component was determined
by comparing the intermittently-treated animals to the
chronically-treated rats after a 40 mg/kg test dose (see
Figure 42) . INT/EXP had a significantly greater pentobarbital
brain concentration compared to CHR/EXP at all time points
(F=14.068, p<0.002) . Likewise, INT/NonEXP had a significantly
greater brain pentobarbital concentration than CHR/NonEXP at
all time points (F=14.238, p<0.002). There was obviously a
large amount of pharmacokinetic tolerance present in the CHR
animals. The pharmacokinetic tolerance observed in the CHR
groups is due to increased metabolism and can not be
attributed to a change in blood:brain distribution (Okamoto
and Boisse, 1975).

.A determination of the presence of cellular tolerance
requires use of a statistical treatment (analysis of
covariance) to statistically control brain pentobarbital
concentrations, essentially removing the difference in these
concentrations between the CHR and INT animals. By using
analysis of covariance, a comparison of the pentobarbital-
induced hypothermia produced in INT/EXP and CHR/EXP (F=4.618,
p<0.05), as well as that.produced.in.INT/NonEXP and CHR/NonEXP
(F=7.571, p<0.01) demonstrates the existence of significant

cellular tolerance for hypothermia as a result of chronic

37

TAQLE 3

Pentobarbital (40 mg/kg) Hypothermia, Ataxia
and Brain Concentration Group Means 1 SEN
Duplication of the Postchronic Testing Procedure

 

 

 

 

 

 

 

 

 

 

 

Body Motor
Group n Temperature Performance [Brain]
('C) (seconds) (HQ/g)
15migute
INT/EXP -0.85 i .16 0 i O 40.1 i
INT/NonEXP -l.26 i .08 0 i O 40.7 _
CHR/EXP -0.38 i .30 15 i 14 27.7 :
CHR/NonEXP -0.42 i .29 19 i 18 25.0 1
32212222
INT/EXP -1.05 i .16 3 i 3 34.8 :
INT/NonEXP -2.00 1 .22 o i o 40.9 :
CHR/EXP -0.13 i .45 70 i 15 19.0 :
CHR/NonEXP -0.62 i .44 53': 18 22.8 :
60minute
INT/EXP -1.43 i .42 23 i 23 27.4 :
INT/NonEXP -2.07 i .30 0 i 0 26.7 :
CHR/EXP -0.06 i .13 90 i O 14.7 :
CHR/NonEXP 0.10 i .17 90 i 0 9.9 :
120minute
INT/EXP -O.50 : .64 69 i 21 16.0 :
INT/NonEXP -0.44 i .37 90 i O 14.5 :
CHR/EXP 0.83 i .15 90 i 0 7.7 :
CHR/NonEXP 0.18 i .20 90 i 0 8.8 :

 

 

38
pentobarbital treatment (see Figure 43). Perhaps a simpler
representation of the cellular tolerance difference between
INT and CHR groups is displayed in Figure 44. This figure
depicts the regression lines produced from the mean
hypothermia produced at the mean brain concentration of each
time point for each group. Unfortunately, there is no non-
parametric equivalent to analysis of covariance for similar

treatment of the ataxia data.

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The postchronic body temperature data from the INT
animals indicated that behavioral tolerance, separable from
metabolic and cellular tolerances, developed. INT/EXP
demonstrated significant tolerance, as compared to the
prechronic measures, that INT/NonEXP lacked (see Figures 1
through 3 and 7 through 9). However, there was no difference
between the EXP and NonEXP groups.

The slight tolerance development to the hypothermic
effect demonstrated by the NonEXP animals may have been due
to an inability to totally eliminate drug-induced.hypothermia
using the BTW procedure or to some generalization of various
tolerance phenomena. Due to the large number of animals being
maintained, at times the body temperatures may have exceeded
the control range for a short time, providing some experience
in the NonEXP group to the loss of temperature control. Even
though subjects were monitored for up to 20 minutes after
turning off the heat lamp (sometimes up to three hours), some
animals may also have experienced some prolonged hypothermia
that was undetected after being returned to their home cages.
An automated temperature control system for each animal could,
have eliminated this problem.

84

85

The lack of dissipation of tolerance to hypothermia after
withdrawal in the INT animals was expected since there should
have been negligible cellular or metabolic tolerance produced
with this dosing schedule. Tolerance loss after extinction
trials was expected for the INT/EXP animals but was not
observed.

It is possible that the extinction trials were
unsuccessful in the INT/EXP animals due to their previous
behavioral experiences in the absence of the drug effect.
The drug-dosing cues (i.p. administration) were partially
reinforced since these animals received saline injections
during the chronic drug treatment period. Siegel (1983)_
demonstrated that partial reinforcement inhibits the
development of an association or extinguishes the association
of the CS (conditioned stimulus, cues for drug administration)
with the UCS (unconditioned stimulus, drug effect). Since the
extinction trials in the current study utilized i.p.
administration of saline as the conditioned stimulus, the
previous partial reinforcement of this stimulus may have
caused a failure for the extinction trials to disassociate the
drug administration from the drug effect. The stimulus cues
for drug effect (or other influence) which developed to become
the CS in these animals may have been weakened or may have
been something other than the drug administration.

Other possible conditioned stimuli are the testing
procedure, environment, altered response pattern and low-level

drug effects (Schuster et al., 1966; Demellweek and Goudie,

 

86

1983). INT/EXP animals had experienced the testing procedure
without pentobarbital during the training period. They had
also never’ experienced. the drug’ outside of“ the testing
procedure. The animals were all in the same room
(environment) for all treatments, there was no change in
environment to allow cues for conditioning drug effects. The
design of this study may have forced these INT/EXP subjects
to utilize low level drug cues as the only consistent CS for
the drug effect. Evidence that low level drug cues were
involved as the CS can be observed from the postchronic dose-
response effects on all the animals. For all measures the
greater tolerance observed in the experienced subjects as
compared to the non-experienced subjects was diminished with
increasing dose, and was often reversed at the highest dose.
As drug dosing increases the low-level drug cues were altered
or' missing, which. may’ have resulted in a loss of the
conditioned response (i.e., a loss of behavioral tolerance).
An introduction of different cues for drug testing days and
the chronic treatment (towel wrap) days such as different
environments involving changes in visual, auditory, and/or
olfactory stimuli may have allowed for extinguishable cues for
drug effect.

It is possible that this conditioned behavior was
difficult to extinguish using extinction trials that did not
employ the drug experience outside of the testing procedure,
particularly when the subjects had previous nondrugged testing

experience. These hypotheses gain support by the observance

87

of successful extinction trials in CHR/EXP for both
hypothermic and ataxic tolerance. This group had drug
experience outside the testing procedure (in the towel wrap)
and did not have partial reinforcement of drug administration
cues during the chronic drug treatment period.

A difference between the EXP and NonEXP groups of the
INT animals was also demonstrated in the amount of
pentobarbital-induced ataxia produced. There was an increased
sensitivity to the ataxic effect of pentobarbital in the
NonEXP group after the chronic treatment period. Furthermore,
no apparent tolerance development was measured for the EXP
group, as compared to the prechronic dose-response
measurements (see Figs 19 through 21 and 25 through 27).

Behavioral tolerance to rotarod-measured ataxia has been
demonstrated previously in this laboratory (Commissaris and
Rech, 1981). A comparison of the methods employed for the
current study and that of Commissaris and Rech (1981) reveals
some differences. These differences are in the sex of the
subjects, the dose of pentobarbital, and the other behavioral
experiences of the subjects (mainly the towel wrap). It is
possible that the male subjects and the larger doses of
pentobarbital used in the current study may not be as optimum
for behavioral tolerance development to ataxia as the previous
study's. female subjects and the 15 mg/kg dose of
pentobarbital. In the current study the higher doses of
pentobarbital did not allow for much intermediate drug effect

practice. The animals generally went from a rotarod

88

performance score of 0 seconds to that of 90 seconds within
one or two trials. The device used in this study was a single
speed rotarod. Ataxia changes could be measured more
sensitively with the use of a variable speed rotarod or an
inclined plane. Additional senitivity would also be achieve
by the use of parametric statistics applicable to data
obtained from a variable speed rotarod or an inclined plane.

The apparent lack of behavioral tolerance to ataxia in
INT/EXP of the current study may also be a reflection of a
non-contiguous drug treatment effect relating to the
behavioral experience other than the rotarod training (the
towel wrap) that was offset by drug-effect experience (i.e.,
behavioral tolerance). A comparison of the first three days
to the last three days of the initial (prechronic) dose-
response determination indicates there was a trend for less
ataxia in the last three days as compared to the first three
days (see Figure 45) at the middle dose, which becomes
significant at the high dose. This was probably due to a
rapidly developing behavioral tolerance during this prechronic
testing. (Due to these results the prechronic scores reported
were only of the first three days of the prechronic dose-
response determination for the ataxia. The hypothermia data
did not demonstrate a significant difference between the first
three days and the last three days of the initial dose-
response determination probably due to a more slowly
developing behavioral tolerance, and thus all six days of

measurements were reported.) At the postchronic test of the

 

89

40 mg/kg dose in the INT/EXP animals, the level of ataxia was
no longer significantly less than the score for the prechronic
days 1-3 determination. Therefore, it appears that INT/EXP
actually lost tolerance over the chronic treatment period.

This discrepancy may relate to a conditioned behavior
similar to learned helplessness (Maier et a1. , 1983) produced
by the animals' repeated non-drug experiences of inability to
ambulate in the towel wrap. It has been demonstrated by
Carder (1978) that behavioral experiences that do not involve
exposure to the drug or the specific drug-effect testing
procedure can alter the drug effect in the specific test.
This learned helplessness may explain the increased
sensitivity to the drug effect in INT/NonEXP as well as the
apparent lack of tolerance in INT/EXP. Although a slight
trend (non-significant) for an increase in sensitivity was
observed at the 40 mg/kg dose in INT/EXP postchronically as
compared to the last three days (4-6) of the prechronic
testing, there was no difference as compared to the first
three days of prechronic testing (Figure 45). It is likely
that the increased sensitivity also expected to occur in
INT/EXP by a learned helplessness type of effect was offset
by the behavioral tolerance produced in these subjects. Thus,
the behavioral tolerance in INT/EXP may be masked by a non-
drug behavioral experience that was not anticipated in the
design of this study that enhanced the drug effect to impair
rotarod performance. Furthermore, a learned helplessness-like

effect may have been extinguished during the extinction

 

90
training period, resulting then in a decreased drug effect.
A trend for this type of decreased impairment was observed for
both the pentobarbital-induced hypothermia and ataxia (see
Figures 12 through 15, 30, 31 and 32) for INT/EXP.

Restraint stress may also play a role in the unexpected
results observed in this study. Restraint stress alters
hormone levels (Rupe et al., 1963), drug metabolism (Rupe et
al., 1963: Stitzel and Furner, 1967), the regulation of many
neurotransmitters (Hendley et al., 1977) and the drug
responses (Chung and Brown, 1976; Martin.and Papp, 1979). Any
one of these alterations could affect tolerance development
in these animals. Since the amount of restraint stress is
equivalent in the groups being compared, these factors do not
play a role in the differences between EXP and NonEXP animals.

Chronic pentobarbital treatment produced profound
cellular/metabolic tolerance, as expressed by decreased
hypothermia and ataxia. Behavioral tolerance for ataxia was
only suggested in.CHR/EXPIat‘the‘postchronic tests by'a slight
trend (non-significant) for increased tolerance to ataxia due
to EXP in the low and middle dose measures (Figures 22, 23,
25, 26 and 27) . However, a conditioned component to the
tolerance in the CHR/EXP animals became apparent at the
postwithdrawal and.postextinction testing phases for both the
tolerances to hypothermia and ataxia (Figures 30, 31 and 34).
Withdrawal either failed to or only partially reversed
tolerance to hypothermia and ataxia in CHR/EXP, suggesting

that a masked behavioral tolerance was uncovered by withdrawal

91

which. then. maintained. significant tolerance at the
postwithdrawal test. This unmasked behavioral tolerance was
then diminished, as would be expected with a conditioned-
tolerance component, by extinction trials, as evidenced by the
significant tolerance loss at the postextinction test in the
CHR/EXP animals. This conditioned component was absent in the
CHR/NonEXP animals, which displayed significant tolerance loss
only after withdrawal and not from extinction training
(Figures 30, 31 and 35).

The slight trend for additional loss of tolerance after
extinction trials observed in CHR/NonEXP could be explained
if all of the metabolic tolerance was not lost after the ten
day withdrawal period. However, previous studies have
indicated that metabolic tolerance to barbiturates dissipates
within 6—10 days (Aston, 1965; Stevenson and Turnbull, 1968).
Since it has also been established that cellular tolerance
dissipates ‘within 7-10 days (Okamoto et al, 1976), the
significant tolerance remaining within the CHR/EXP animals at
the postwithdrawal test, that is lost after extinction trials,
must be attributed to a conditioned behavioral tolerance
component.

The appearance of a large conditioned component of
tolerance in CHR/EXP revealed by the postwithdrawal and
postextinction tests introduces a discrepancy with regard to
the postchronic test results. The postchronic tests failed
to Ireveal a major behavioral tolerance component when

comparing CHR/EXP tolerance to that of CHR/NonEXP. It is

92
possible that a behavioral component was present but was
overshadowed or masked by the prominent cellular/metabolic
tolerance developed by the chronic drug treatment. The total
tolerance produced after chronic dosing may have a
ceiling/floor effect imposed by this experimental design.
Even though an accelerated dosing regimen was used during the
chronic treatment period, the acceleration may not have been
enough to produce maximal functional tolerance development.

Okamoto et al (1978) demonstrated that chronic dosing
failed to produce significant functional tolerance when the
dose did not challenge the function being tested
(anesthesia/consciousness) . Almost all of the animals in
both CHR groups achieved the criteria for increasing the dose
on every test day. Perhaps if the dosing increase was greater
for these animals and thus more challenging to the functional
tolerance development, a larger amount of total functional
tolerance could have been achieved and a difference between
the CHR/EXP and CHR/NonEXP groups would have been observed at
the postchronic testing.

Alternatively, if CHR/EXP had received initial training
to develop behavioral tolerance first and then been treated
with chronic drug, perhaps an initial behavioral tolerance may
then have augmented a cellular/metabolic tolerance. Another
possible explanation for a functional tolerance ceiling is
that development of behavioral tolerance produces less of a

demand for cellular tolerance development.

 

93

The approach for demonstrating behavioral tolerance after
chronic drug treatment in this study is unconventional. The
conventional approach is an additive one, illustrating
behavioral tolerance by demonstrating a significant increase
in tolerance for animals with task-practice after drug
administration as compared to animals receiving drug after
task-practice following the chronic treatment period. The
unconventional approach exhibited in the current study is a
subtractive approach. After subtracting the cellular and
metabolic components of drug tolerance, the behavioral
tolerance is uncovered and its conditioning nature is
confirmed by subtracting it with extinction trials.

Changes in the peak effect would traditionally be
attributed to cellular tolerance, whereas changes in duration
of effect would be attributed to metabolic tolerance.
However, these distinctions are far from absolute. Behavioral
tolerance can cause changes in both the peak effect and
duration of effect (Commissaris and.Rech, 1981: San-Marina et
al., 1989) . The decrease in duration may be due to an
increase in acute behavioral tolerance which may be
strengthened by conditioning (Tabakoff et al, 1986) . The
decrease in peak effect may be due to conditioning from the
drug exposure combined with behavioral testing experience.
Small decreases in peak effect and duration of effect were
observed for the INT/EXP animals with regard to hypothermia.
The behavioral experience of the towel wrap combined with drug

exposure in INT/NonEXP appeared to produce an increase in both

94

the peak ataxia and duration of ataxia after the pentobarbital
test doses. The potential mechanism(s) for this increased
sensitivity remain unresolved, though a development of
"learned helplessness" was postulated above. It would be
possible to test whether the BTW actually produces a "learned
helplessness" on its own. Naive animals would be exposed to
five weeks of daily BTW, then a dose-effect test performed for
pentobarbital ataxia and compared to animals maintained in
home cages without BTW experience.

Thus, behavioral experiences during intermittent
barbiturate administration may have an influence on the amount
of behavioral deficit produced by the drug. This did not
appear to be the case with chronic administration in this
model. Although there were large decreases in the peak
effects and duration of effects after chronic administration
there was no appreciable difference between the CHR/EXP and
CHR/NonEXP animals for the total tolerance level to
hypothermia. These effects were undoubtedly largely due to
cellular and metabolic tolerance. There was a slight trend
for behavioral tolerance development to ataxia in the lower
test.doses for the peak effect, duration and total effect (see
Figures 25, 26 and 27). It is not possible to determine from
these data whether this was due to (a) an increase in
tolerance in CHR/EXP due to drug exposure combined with RR or
(b) to an enhanced deficit in CHR/NonEXP due to drug exposure
combined with BTW. Therefore, analysis of the postchronic log

dose-response curves was performed. Even though there was a

95

lack of significant differences in the regression analyses,
there was a consistent trend through all six graphs. This
trend suggests that exposure of the towel wrap in combination
with the pentobarbital produces an increase in drug effect.
From this analysis it appears that the towel-wrap behavioral
experience combined with drug exposure did influence the peak
ataxia and may have also influenced the hypothermia peak
effect, duration of effect and total effect to some extent
(Figures 36, 37, 38, and 39). This suggests that the learned
helplessness type of behavior was increased by the combination
of the drug and the towel wrap (both being incapacitating and
leading to a ”learned helplessness") and may not have been
limited to the ataxia.

Additional evidence for pharmacokinetic tolerance was
demonstrated by the brain concentration time courses. The CHR
animals have lower pentobarbital brain concentrations at all
time points as compared to the INT animals. Since there was
such a large difference between the CHR and INT brain levels,
even at 15 minutes, the brain/serum.ratios were determined at
15 minutes and were found to show no significant difference
between any of the groups. This indicates that there was no
change in the ability of pentobarbital to cross the blood-
brain barrier. The pharmacokinetic tolerance in the CHR
animals was most likely due to changes in metabolism as
indicated by Okamoto et al. (1975) and Commissaris et a1.

(1982).

96

Additionally, cellular tolerance was demonstrated by an
analysis of covariance of the brain pentobarbital
concentrations and their corresponding hypothermic deficits.
The depiction of this analysis, Figure 43, reveals not only
a difference in amount of effect between the chronic and
intermittent treatment groups, but also a change in shape
between the experienced and non-experienced groups. Whether
this change in shape was due to the drug-effect experience or
to the drug-towel wrap experience is not discernible without
naive animal data for comparison.

The cellular tolerance expressed to the ataxia could not
be determined due to the non-parametric nature of the data.
Again, the solution to this problem would be to employ a
variable speed rotarod or an inclined plane to produce data
that can be analyzed with parametric statistical tests

(Analysis of Covariance).

1)

2)

EEHELBX_BED_SQE£LDEIQE§

The aims of this study were achieved as follows:
Significant tolerance to the pentobarbital-induced
hypothermia was observed after both chronic and
intermittent treatment. Significant tolerance to the
pentobarbital-induced ataxia was observed after chronic
treatment but not after intermittent treatment.

INT/EXP animals expressed a tolerance to pentobarbital-
induced hypothermia that was attributed to learned
adaptation based on drug exposure combined with
behavioral testing experience.

INT/NonEXP animals expressed an enhanced sensitivity to
pentobarbital-induced ataxia that. was attributed to
learned adaptation (learned helplessness; Maier et al.,
1983) based on drug exposure combined with the towel wrap
experience.

Development of behavioral tolerance to pentobarbital-
induced ataxia may have been masked in the INT/EXP by a
non-drug related 'learned helplessness' type of effect
(Carder, 1978).

CHR/EXP animals expressed a tolerance to pentobarbital-

induced hypothermia and ataxia which was attributed to

97

 

3)

4)

98

components of learned adaptation, unmasked after
Postwithdrawal and Postextinction testing, and
cellular/metabolic tolerance.

CHR/NonEXPIanimals expressed tolerance to pentobarbital-
induced hypothermia and ataxia which was attributable to
cellular/metabolic tolerance.

Analysis of brain pentobarbital concentrations
determined that pharmacokinetic tolerance was present
after chronic pentobarbital treatment. Analysis of
pentobarbital-induced.hypothermia.as correlated with.the.
brain pentobarbital concentrations (by analysis of
covariance) determined that cellular tolerance was also
present after chronic pentobarbital treatment.

Cellular and metabolic tolerances can occur without
requiring experience to specific drug deficits.
Behavioral tolerance (or sensitivity) occurs as a
consequence of behavioral experiences, without requiring
chronic drug treatment or the development of cellular

tolerance.

The following conclusions can be drawn. with respect to

pentobarbital-induced hypothermia and ataxia:

1)

2)

Behavioral experiences (whether drug-related or not) can
alter drug effects by learned adaptations.
These learned adaptations are responsible for the drug-

tolerance or drug-sensitivity observed after intermittent

 

3)

4)

5)

99
drug treatment without requiring the production of
cellular or metabolic drug tolerance.
These learned adaptations are also a component of the
total drug tolerance observed after chronic drug
treatment which includes the presence of cellular and
metabolic tolerances. Behavioral tolerance was uncovered
in the chronic treatment group after a withdrawal period
and confirmed after extinction trials, using a
subtractive approach.
Behavioral tolerance is separable from both cellular and
metabolic types of tolerance.
Cellular or metabolic tolerance development does not

require specific experience to drug effects.

 

LIST 01' REFERENCES

LI£I_QI_B£IEBBEQES

Adams, W. J., S. Y. Yeh, L. A. Woods and C. L. Mitchell.
Drug-test interaction as a factor in the development of
tolerance to the analgesic effect of morphine. J.
Pharmacol. Exp. Ther. 168: 251-257, 1969.

Akera, T., R. H. Rech, W. J. Marquis, T. Tobin, and T. M.
Brody. Lack of relationship between brain (Na+ + K“)
activated adenosine triphosphatase and the development
of tolerance to ethanol in rats. J. Phanacol. Exp.
Ther. 185: 594-601, 1973.

Alkana, R. L., D. A. Finn and R. D. Malcolm. The importance
of experience in the development of tolerance to ethanol
hypothermia. Life Sciences 32: 2685-2692, 1983.

Annear, W. C. and M. Vogel-Sprott. Mental rehearsal and
classical conditioning contribute to ethanol tolerance
in humans. Psychopharnacol. 87: 90-93, 1985.

Aston, R. Quantitative aspects of tolerance and posttolerance
hypersensitivity to pentobarbital in the rat. J.
Pharmacol. Exp. Ther. 150: 253-258, 1965.

Balster, R. L. Behavioral studies of tolerance and
dependence. In: Behavioral Pharmacology: The Current
status, edited by L. S. Seiden. New York, Alan R. Liss,
Inc., 1985, 403-418.

Barnes , C. D. and L. G. Eltherington. Drug Dosage in
Laboratory Animals. Univ. of California Press, Berkeley,
1973, p.181.

Baum, D., J. B. Halter, G. J. Taborsky, Jr. and D. Porte, Jr.
Pentobarbital effects on plasma catecholamines:
temperature, heart rate, and blood pressure. Am. J.
Physiol. 248: E95-E100, 1985.

Belknap, J. K., G. Ondrusek, J. Berg and S. Waddingham.
Barbiturate dependence in mice: Effects of continuous
vs discontinuous drug administration. Psychopharnacol.
51: 195-198, 1977.

Bird, D. C. and F. A. Holloway. Development and loss of
tolerance to the effects of ethanol on DRL performance
of rats. Psychopharaaool. 97: 45-50, 1989.

100

101

Cappell, H. and A. E. LeBlanc. Tolerance to, and physical
dependence on, ethanol: Why do we study them? Drug
Alcohol Depend. 4: 15-31, 1979.

Cappell, H. and A. E. LeBlanc. Tolerance and physical
dependence: Do they play a role in alcohol and drug
self-administration? In: Research Advances in Alcohol
and Drug Dependence, vol. 6, edited by Y. Israel, F. B.
Glaser, H. Kalant, R. E. Popham, W. Schmidt, and R. G.
Smart. New York, Plenum Press, 1981, 159-196.

Cappell, H., C. Roach and C. X. Poulos. Pavlovian control of
cross-tolerance between pentobarbital and ethanol.
Psychopharmacol. 74: 54-57, 1981.

Carder, B. Environmental influences on marihuana tolerance.
In: Behavioral Tolerance: Research and Treatment
Implications. NIDA Research Monograph Series Vol. 18,
edited by N. A. Krasnegor. Rockville, MD, DHEW Pub. #
(ADM) 78-551, 1973, pp. 90-102.

Chen, C. S. A further note on studies of acquired behavioural
tolerance to alcohol. Psychopharmacol. 27: 265-274,
1972.

Chen, C. 8. Acquisition of behavioral tolerance to ethanol
as a function of reinforced practice in rats.
Psychopharmacol. 63: 285-288, 1979.

Chung, H and D. R. Brown. Alcohol-hexobarbital interaction
in rats under acute stress. Life Sci. 18: 123-128,
1976.

Commissaris, R. L. and R. H. Rech. Tolerance to pentobarbital
and ethanol following chronic pentobarbital
administration in the rat. Substance and Alcohol
Actions/Misuse 2: 331-339, 1981.

Commissaris, R. L. and R. H. Rech. Tolerance and cross-
tolerance to central nervous system depressants after

chronic pentobarbital or chronic methaqualone
administration . Pharmacol . Biochem. Behav. 18 : 327-3 3 1 ,
1983 .

Commissaris, R. L., D. R. Semeyn and R. H. Rech.
Dispositional without functional tolerance to the
hypothermic effects of pentobarbital in the rat. J.
Pharmacol. Exp. Ther. 220: 536-539, 1982.

Crowell, C. R., R. E. Hinson and S. Siegel. The role of
conditional drug responses in tolerance to the
hypothermic effects of ethanol. Psychoparmacol. 73: 51-
54, 1981.

102

Dafters, R. and G. Anderson. Conditioned tolerance to the
tachycardia effect of ethanol in humans.
Psychopharmaccl. 78: 365-367, 1982.

Demellweek, C. and A. J. Goudie. Behavioural tolerance to
amphetamine and other psychostimulants: the case for
considering behavioural mechanisms. Psychopharmacol. 80 :
287-307, 1983.

Dews, P. B. Behavioral tolerance. In: Behavioral Tolerance:
Research and Treatment Implications. NIDA Research
Monograph Series Vol. 18, edited by N. A. Krasnegor.
Rockville, MD, DHEW Pub. # (ADM) 78-551, 1973, pp. 18-
26.

Emmett-Oglesby, M. W., D. G. Spencer, Jr., D. M. Wood and H.
Lal. Task-specific tolerance to d-amphetamine.
Neuropharmacol. 23: 563-568, 1984.

Fraser, H. F., H. Isbell, A. J. Eisenman, A. Wikler and F. T.
Pescor. Chronic barbiturate intoxication: Further
Stfldies. Aexele Itch. Inte HOG. 8 34-41, 195 e

Ferraro, D. P. Behavioral tolerance to marihuana. In:
Behavioral Tolerance: Research and Treatment
Implications. NIDA Research Monograph Series Vol. 18,
edited by N. A. Krasnegor. Rockville, MD, DHEW Pub. #
(ADM) 78-551, 1978, pp. 103-117.

Goldstein, D. B. Physical dependence on ethanol: Its
relation to tolerance. Drug Alcohol Depend. 4:33-42,

1979.

Goudie, A. J. and J. W. Griffiths. Behavioural factors in
drug tolerance. Trends Pharmacol. Sci. : 192-196,
1986.

Greeley, R. H. New approach to derivatization and gas-
chromatographic analysis of barbitruates. Clin. Chem.
20: 192-193, 1974.

Hendley E. D., G. H. Burrows, E. S. Robinson, K. A.
Heidenreich.and.c. A. Bulman. .Acute stress and the brain
norepinephrine uptake mechanism in the rat. Pharmacol.
Biochem. Behav. 6: 197-202, 1977.

Herberg, L. J. and A. M. J. Montgomery. Learnt tolerance to
sedative effects of chlordiazepoxide on self-stimulation
performance, but no tolerance to facilitatory effects
after 80 days. Psychopharmacol. 93: 214-217, 1987.

Hinson, R. E. and S. Siegel. The contribution of Pavlovian
conditioning to ethanol tolerance and dependence. In:
Alcohol Tolerance and Dependence, edited by Rigter and
Crabbe. N. Holland: Elsevier, 1980, pp. 181-199.

103

Isbell, H., S. Altschul, S. H. Kornetsky, A. J. Eisenman, H.
G. Flanary and. H. F. Fraser. Chronic barbiturate
intoxication: An experimental study. Arch. Neurol.
Psychiat. 64: 1-28, 1950.

Jones, R. T. Behavioral tolerance: lessons learned from
cannibis research. In: Behavioral Tolerance: Research
and Treatment Implications. NIDA Research Monograph
Series Vol. 18, edited by N. A. Krasnegor. Rockville,
MD, DHEW Pub. # (ADM) 78-551, 1978, pp. 118-126.

Kalant, H. Comparative aspects of tolerance to, and
dependence on, alcohol, barbiturates and opiates. In:
Alcohol, Intoxication and Withdrawal, vol. 38, edited.by
M. M. Gross. New York, Plenum Press, 1977, pp. 169-186.

Kalant, H., A. E. LeBlanc and R. J. Gibbins. Tolerance to,
and dependence on, some non-opiate psychotropic drugs.
Pharmacol. Rev. 23: 135-191, 1971.

Krasnegor, N. A. Introduction. In: Behavioral Tolerance:
Research and Treatment Implications. NIDA Research
Monograph Series Vol. 18 , edited by N. A. Krasnegor.
Rockville, MD, DHEW Pub. # (ADM) 78-551, 1978, pp. 1-3.

Laties, V. G. and B. Weiss. Effects of alcohol on timing
behavior. J. Comp. Physiol. Psychol. 55: 85-91, 1962.

Le, A. D., C. X. Poulos and.H. Cappell. Conditioned tolerance
to the hypothermic effect of ethyl alcohol. Science 206:
1109-1110, 1979.

Le, A. D., J. M. Khanna and H. Kalant. Role of Pavlovian
conditioning in the development of tolerance and cross-
tolerance to the Jhypothermic effect. of ethanol and
hydralazine. Psychopharmacol. 92: 210-214, 1987.

LeBlanc, A. E. and H. Cappell. Tolerance as adaptation:
Interactions with behavior and parallels to other
adaptive processes. In: Alcohol and Opiates.
Neurochemical and Behavioral Mechanisms, edited by K.
Blum. New York, Academic Press, 1977, pp. 65-77.

LeBlanc, A. E., R. J. Gibbins and H. Kalant. Behavioral
augmentation of tolerance to ethanol in the rat.
Psychopharmacol. 30: 117-122, 1973.

LeBlanc, A. E., H. Kalant and R. J. Gibbins. Acquisition and
loss of behaviorally augmented tolerance to ethanol in
the rat. Psychopharmacol. 48: 153-158, 1976.

LeBlanc, A. E., C. X. Poulos and H. D. Cappell. Tolerance as
a behavioral phenomenon: evidence from.two experimental
paradigms . In: Behavioral Tolerance : Research and

 

104

Treatment Implications. NIDA Research Monograph Series
vol. 18, edited by N. A. Krasnegor. Rockville, MD, DHEW
Pub. # (ADM) 78-551, 1978, pp. 72-89.

Maier, S. F., R. Drugan, J. W. Grau, R. Hyson, A. J.
MacLennan, T. Moye, J. Madden IV and J. D. Barchas.
Learned helplessness, pain inhibition, and the endogenous
opiates. In: Advances in Analysis of Behavior, vol. 3,
edited by M. D. Zeiler and P. Harzem. New York, N.Y.,
John Wiley and Sons, 1983, pp. 275-320.

Mansfield, J. G. and C. L. Cunningham. Conditioning and
extinction of tolerance to the hypothermic effect of
ethanol in rats. J. Compar. Physiol. Psychol. 94: 962-
969, 1980.

Mansfield, J. G., R. S. Benedict and S. C. Woods. Response
specificity of behaviorally augmented tolerance to
ethanol supports a learning interpretation.
Psychopharmacol. 79: 94-98, 1983.

Martin, G. E. and N. L. Papp. Effect on core temperature of
Restraint after’ peripherally and centrally injected
morphine in the Sprague-Dawley rat. Phamraccl. Biochem.
Behav. 10: 313-315, 1979.

Melchior, C. L. and B. Tabakoff. Modification of
environmentally cued tolerance to ethanol in mice. J.
Pharmacol. Exp. Ther. 219: 175-180, 1981.

Melchior, C. L. and B. Tabakoff. Features of environment-
dependent tolerance to ethanol. Psychopharmacol. 87:
94-100, 1985.

O'Brien, C. P., Testa, T., Ternes, J. and Greenstein, R.
Conditioning effect of narcotics in humans. In:
Behavioral Tolerance: Research and Treatment
Implications. NIDA Research Monograph Series vol. 18,
edited by N. A. Krasnegor. Rockville, MD, DHEW Pub. #
(ADM) 78-551, 1978, pp. 67-71.

O'Brien, C. P., R. N. Ehrman and J. W. Ternes. Classical
conditining in human opioid dependence. In: Behavioral
Analysis of Drug Dependence. edited by S. R. Goldberg and
I. P. Stolerman. Orlando, Academic Press, Inc., 1986,
pp. 329-356.

Okamoto, M. and N. R. Boisse. IEffect of chronic pentobarbital
treatment on blood-cerebrospinal fluid kinetics. Eur.
J. Pharmacol. 33: 205-209, 1975.

Okamoto, M. and N. R. Boisse. Sedative-hypnotic tolerance
and physical dependence. Trends in Pharmacol. Sci. 2:
9-13, 1981.

105

Okamoto, M., H. C. Rosenberg and N. R. Boisse. Tolerance
characteristics produced during the maximally tolerable
chronic pentobarbital dosing in the cat. J. Pharmacol.
Exp. Ther. 192: 555-569, 1975.

Okamoto, M., H. C. Rosenberg and N. R. Boisse. Withdrawal
characteristics following chronic pentobarbital dosing
in cat. Eur. J. Pharmacol. 40: 107-119, 1976.

Okamoto, M., N. R. Boisse, H. C. Rosenberg and R. Rosen.
Characteristics of functional tolerance during
barbiturate physical dependency production. J.
Pharmacol. Exp. Ther. 207: 906-915, 1978.

Okamoto, M., D. J. Hinman and L. M. Aaronson. Comparison of
ethanol and barbiturate physical dependence. J.
Pharmacol. Exp. Ther. 218: 701-708, 1981.

Okamoto, M., S. Rao and J. L. Walewski. Effect of dosing
frequency on the development of physical dependence and
tolerance to pentobarbital. J. Pharmacol. Exp. Ther.
238: 1004-1008, 1986.

Paletta, M. S. and A. R. Wagner. Development of context-
specific tolerance to morphine: Support for a dual-
process interpretation. Behav. Neurosci. 100: 611-623,
1986.

Piascik, M. T. and L. F. McNicholas. Effect of pentobarbital
dependence on adenylate cyclase activity and calmodulin
levels in rat cerebral cortex. Life Sci. 37: 55-62,
1985.

Rech, R. H., H. K. Borys and K. E. Moore. Alterations in
behavior and brain catecholamine levels in rats treated
with a-methyltyrosine. J. Pharmacol. Exp. Ther. 153:
412-419, 1966.

Ritzmann, R. F. and B. Tabakoff. Body temperature in mice:
A quantitative measure of alcohol tolerance and physical
dependence. J. Pharmacol. Exp. Ther. 199: 158-170,
1976.

Roffman, M. and H. Lal. Stimulus control of hexobarbital
narcosis and metabolism in mice. J. Pharmacol. Exp.
Thor. 191: 358-369, 1974.

Ruddle, H. V., M. J. Morley, C. M. Bradshaw and E. Szabadi.
The effect of pentobarbitone on variable-interval
performance: Analysis in terms of Herrnstein's equation.
Psychopharmacol. 84: 520-525, 1984.

Rupe, B. D., W. F. Bousquet and T. S. Miya. Stress
modification of drug response. Science 141: 1186-1187,
1963.

 

 

106

San-Marina, A., J. M. Khanna and H. Kalant. Relationship
between initial sensitivity, acute tolerance and chronic
tolerance to ethanol in a heterogeneous population of
Swiss mice. Psychopharmacol. 99: 450-457, 1989.

Schuster, C. R. Theoretical basis of behavioral tolerance:
implications of the phenomenon for problems of drug
abuse. In: Behavioral Tolerance: Research and
Treatment Implications. MIDA Research Monograph Series
va1. 18, edited by N. A. Krasnegor. Rockville, MD, DHEW
Pub. # (ADM) 78-551, 1978, pp. 4-17.

Schuster, C. R., W. S. Dockens and J. H. Woods. Behavioral
variables affecting the development of amphetamine
tolerance. Psychopharmacol. 9: 170-182, 1966.

Schuster, C. R. and T. Thompson. Self administration of and
behavioral dependence on drugs. .Ann. Rev. Pharmacol. 9:
483-502, 1969.

Shapiro, A. P. and P. E. Nathan. Human tolerance to alcohol:
The role of Pavlovian conditiong processes.
Psychopharmacol. 88: 90-95, 1986.

Siegel, S. Evidence from rats that morphine tolerance is a
learned response. J. Compar. Physiol. Psychol. 89: 498-
506, 1975.

Siegel, S. A pavlovian conditioning analysis of morphine
tolerance. In: Behavioral Tolerance: Research and
Treatment Implications. NIDA Research Monograph Series
vol. 18, edited by N. A. Krasnegor. Rockville, MD, DHEW
Pub. # (ADM) 78-551, 1978, pp. 27-53.

Siegel, S. Classical conditioning, drug tolerance, and drug
dependence. In: Research Advances in Alcohol and Drug
Problems, vol. 7, edited by Y. Israel, F. B. Glaser, H.
Kalant, R. E. Popham, W. Schmidt and R. E. Smart. New
York, Plenum Press, 1983, pp. 207-246.

Siegel, S. and J. MacRae. Environmental specificity of
tolerance. Trends MeuroSci. 7: 140-143, 1984.

Siew, C. and D. B. Goldstein. Osmotic minipumps for
administration of barbital to mice: Demonstration of
functional tolerance and physical dependence. J.
Pharmacol. Exp. Ther. 204: 541-546, 1978.

Stevenson, I. H. and M. J. Trunbull. Hepatic drug-
metabolising enzyme activity and duration of
hexobarbitone anaesthesia in. barbitone-dependent and
withdrawn rats . Biochem. Pharmacol . 17 : 2 297-2 3 05 ,
1968.

107

Stitzel R. E. and R. L. Furner. Stress-induced alterations
in microsomal drug metabolism in the rat. Biochem.
Pharmacol. 16: 1489-1494, 1967.

Tabakoff, B., N. Cornell and P. L. Hoffman. Alcohol
Tolerance. Ann. Emerg. Med. 15: 1005-1012, 1986.

Tang, Maisy and J. L. Falk. Behavioral and pharmacological
components of phenobarbital tolerance. In: Behavioral
Tolerance: Research and Treatment Implications. NIDA
Research Monograph Series Vol. 18, edited by N. A.
Krasnegor. Rockville, MD, DHEW Pub. # (ADM) 78-551,
1978, pp. 142-148.

Tiffany, S. T., K. J. McCal and P.M. Maude-Griffin. The
contribution of classical conditioning to tolerance to
the antinociceptive effects of ethanol. Psychopharmacol.
92: 524-528, 1987.

Vincent, F., C. Feuerstein, M. Gavend and J. Faure. Routine
identification and determination of 11 barbiturates in
biological samples. Clin. Chim. Acta 93: 391-399, 1979.

Vogel-Sprott, M. and K. Sdao-Jarvie. Learning alcohol
tolerance: The contribution of response expectancies.
Psychopharmacol. 98: 289-296, 1989.

Wenger, J. R., V. Berlin and S. C. Woods. Learned tolerance
to the behaviorally disruptive effects of ethanol.
Behav. Neural Biol. 28: 418-430, 1980.

Wenger, J. R., T. M Tiffany, C. Bombardier, K. Nicholls and
S. C. Woods. Ethanol tolerance in the rat is learned.
Science 213: 575-577, 1981.

Wigell, A. H. and D. H. Overstreet. Acquisition of
behaviorally augmented tolerance to ethanol and its
relationship to muscarinic receptors. Psychopharmacol.
83: 88-92, 1984.

Williams, A. J., T. W. G. Jones and J. D. H. Cooper. A rapid
method for the determination of therapeutic barbiturate
levels in serum using gas-liquid chromatography. Clin.
Chim.Acta 43: 327-332, 1973.

Witkin, J. M. and J. E. Barrett. Effects of pentobarbital on
punished behavior: Persistent increases with chronic
administration. Psychopharmacol. 75: 324-325, 1981.

Woods, J. H. and J. Carney. Narcotic tolerance and operant
behavior. In: Behavioral Tolerance: Research and
Treatment Implications. NIDA Research Monograph Series
vol. 18, edited by N. A. Krasnegor. Rockville, MD, DHEW
Pub. # (ADM) 78-551, 1978, pp. 54-66.

108

Woolverton, W. L. and C. R. Schuster. Behavioral tolerance
to cocaine. In: Behavioral Tolerance: Research and
Treatment Implications. NIDA Research Monograph Series
Vol. 18, edited by N. A. Krasnegor. Rockville, MD, DHEW
Pub. # (ADM) 78-551, 1978, pp. 127-141.

 

APPENDICES

APPENDIX A

m4
NUNBER OP SUBJECTS POR EACH TEST (N) AND GROUP (n)

 

 

 

 

WWW W

TEST N n n n n
PreCHR 50 12 -- 13 --
PostCHR 46 12 11 12 11
PostCHR

vs.
PostWITH 46 12 11 12 11

vs.
PostEXT .

 

 

 

109

 

 

110

APPENDIX A (continued)

IABLE.§
PENTOBARBITAL CHRONIC TREATNENT PERIOD DOSING PAIRS

IHILEKB IHILHQDEXB Dose QEBLEIB QHBAHQDEXB Dose
Deter- Dependent Achieved Deter- Dependent Achieved

 

mining Animal (mg/kg) mining Animal (mg/kg)
Animal Animal
76 138 36 79 139 44
78 150 40 80 140 46
82 94 36 89 141 46
84 128 36 95 146 46
85 81 38 96 75 46
92 137 32 97 107 46
93 118 36 102 112 46
101 77 38 105 135 46
111 90 38 114 120 46
115 106 36 117 86 46
125 104 30 121 103 46
126 100 38 122 88 46
127 109 42 123 91 46
129 98 36 124 142 46
130 119 38 131 110 46
136 83 34 132 116 46
145 139 36 133 87 46
154 144 36 134 108 46
143 --- 32 149 152 46
--- 147 42 153 151 46
--- 148 38
_ x = 45.9
X = 36.5

111

APPENDIX A (continued)

TABLE_§

PENTOBARBITAL TEST DOSES
for Postchronic vs. Postwithdrawal vs. Postextinction Tests

 

IEILEKB IEILHQDEXB QHBLEXR QEBLNQDEXB
rat dose rat dose rat dose rat dose
26 28 31 28 37 40 27 40
28 28 39 20 40 80 29 80
33 20 41 20 42 40 30 28
34 40 47 20 48 40 32 28
36 28 49 20 53 40 35 40
38 20 51 20 54 80 44 40
43 20 52 20 56 40 45 40
55 28 62 20 59 80 50 80
66 20 63 20 60 40 58 80
67 20 65 28 58 40 61 40
70 20 71 28 59 28 72 40

74 28 73 80

x = 25.011.S 22.211.1 52.316.0 48.716.2

112

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APPENDIX B (continued)

TABLI_§

PENTOBARBITAL HYPOTRERIIA
Postchronic vs. Postwithdrawal vs. Postextinction Tests
F-values and Probability Levels

 

 

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tht 0.97 .420 5.25 .003 2.03 .131 5.42 .005
16551 1.10 .350 10.95 .002 1.95 .159 7.05 .005
sinuses ~
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20 1.55 .219 2.52 .075 4.75 .009 2.49 .079
30 1.00 .405 3.91 .019 4.27 .013 2.52 .077
40 2.08 .121 3.39 .030 2.55 .057 4.09 .015
50 1.07 .375 5.42 .005 1.23 .315 4.55 .015
50 0.70 .558 9.52 .001 2.27 .101 5.45 .003
70 0.50 .683 12.20 .001 1.59 .190 9.27 .001
80 0.87 .458 14.93 .001 2.05 .128 9.53 .001
90 1.29 .293 14.05 .001 1.59 .191 7.71 .001
100 2.05 .125 14.09 .001 1.94 .144 9.29 .001
110 1.55 .221 12.24 .001 1.11 .352 9.89 .001
120 2.02 .130 12.79 .001 2.34 .094 9.94 .001

114

 

 

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116

Ilflhl_11

PBNTOBARBIILL 331113
Postchronic EXP vs NonEXP Time Course
Mann-Whitney U Test Probability Levels

 

TIME 20 28 40 28 40 80
“Q/kg mg/kg “Q/kg mg/kg mg/kg mg/kg

0 .317 .900 .338 .926 .228 .696
15 .328 .376 .296 .184 .539 .296
30 .312 .305 .296 .366 .625 .131
45 .070 .293 .563 .495 .465 .284
60 .113 .130 .563 .296 .121 .442
75 .068 .180 .926 1.000 .856 .224
90 .442 .115 .078 1.000 .277 .788
105 .317 .011 .055 1.000 .277 .677
120 1.000 .013 .066 .296 1.000 .852

117

APPSNDIX a (continued)

IABLI_11

PENTOBARBITAL POSTCBRONIC LOG DOSE-RESPONSE
RBGRBBBIOH LIN! PARALLBLIBI
T-test Values for
Hypothermia Duration and Peak,
Ataxia Duration and Peak

 

Groups Peak Duration Peak Duration
Prechr. vs.

INT/EXP 0.239 1.426 0.689 0.810
Prechr. vs.

CHR/EXP 0.861 0.659 0.405 1.141
Prechr. vs.

INT/NonEXP 1.641 0.734 1.339 0.240
Prechr. vs.

CHR/NonEXP 1.954 0.786 1.775 1.812
INT/EXP vs.

CHR/EXP 0.341 1.151 0.650 1.907
INT/NonEXP vs.

CHR/NonEXP 0.220 0.165 0.005 1.616
INT/EXP vs.

INT/NonEXP 1.062 1.682 1.505 0.488
CHR/EXP vs.

CHR/NonEXP 1.186 1.463 1.009 0.812

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