Opioid Drug Discrimination: Characterization of the Morphine Stimulus

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission ofof the the copyright copyright owner. owner. Further Further reproduction reproduction prohibited prohibited without without permission. permission. OPIOID DRUG DISCRIMINATION: CHARACTERIZATION OF THE MORPHINE STIMULUS. by Glenn William Stevenson submitted to the Faculty of the College of Arts and Sciences of American University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Psychology Chair: Anthonvltnonv L. Riley, Ph.D. UP

Glowa, Ph.D.

Dean “of the College o f Arts and Sciences

3 * Date

2002

American University

Washington, D C. 20016

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by GLENN STEVENSON

2002

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. OPIOID DRUG DISCRIMINATION:

CHARACTERIZATION OF THE MORPHINE STIMULUS

Glenn William Stevenson

ABSTRACT

There have been few assessments of delta receptor activity in morphine

drug discrimination learning (DDL) and these assessments have been limited to

nonselective compounds or selective delta peptides administered i.c.v.

Experiment 1 assessed the role of the delta receptor in the mediation of

morphine drug discrimination by examining the ability of the systemicaily-

administered, delta opioid agonist SNC80 to substitute for (and the delta opioid

antagonist naltrindole to antagonize) morphine stimulus effects in rats trained to

discriminate morphine from its vehicle in the conditioned taste aversion baseline

of drug discrimination learning. Although morphine and methadone produced

dose-related substitution for morphine (10 mg/kg), there was no evidence of

substitution for morphine by SNC80 at any dose tested. Further, although

naloxone (3.2 mg/kg) completely blocked the discriminative effects of morphine,

naltrindole (3.2 -1 0 mg/kg) did not significantly affect the morphine stimulus.

These data suggest that the discriminative control established to morphine is

mediated by its activity at the mu, but not the delta, receptor.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The failure of SNC80 to substitute for morphine may be due to the general

inability of activity at the delta receptor to support discrimination learning. To test

this, Experiment 2 assessed the establishment of discriminative control by the

selective delta agonist SNC80 in rats and its generalization to and antagonism by

compounds relatively selective to the delta and mu receptor subtypes using the

conditioned taste aversion baseline of DDL. Following acquisition, SNC80 and

the delta agonist SNC162 produced SNC80-appropriate responding at a dose of

18 mg/kg. Conversely, the mu agonist morphine produced vehicle-appropriate

responding at all doses tested. The discriminative effects of SNC80 were

maximal at 20 min, partial at 120 min and lost at 240 min. These selective

generalization patterns with SNC162 and morphine suggest that the

discriminative effects of SNC80 were mediated at the delta, but not the mu,

receptor, a conclusion supported by the fact that SNC80’s discriminative control

was completely blocked by the delta-selective antagonist naltrindole (NTI), but

not by the mu-selective antagonist naltrexone. The present findings indicate that

the discriminative effects of morphine are mediated at the mu, but not the delta,

receptor.

iii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS

ABSTRACT...... ii

TABLE OF CONTENTS...... iv

LIST OF ILLUSTRATIONS...... vi

Chapter

1. EXPERIMENT 1...... 1

2. METHOD...... 9

Subjects and Apparatus

Drugs

Procedure

Data Analyses

3. RESULTS...... 14

4. DISCUSSION...... 27

5. EXPERIMENT 2...... 30

6. METHOD ...... 35

Subjects and Apparatus

Drugs

Procedure

Data Analyses

7. RESULTS...... 40

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1. DISCUSSION...... 56

2. GENERAL DISCUSSION...... 59

BIBLIOGRAPHY...... 71

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES

1. Mean amount (±S.E.M.) of saccharin consumption for subjects in Groups ML and MW during Water Baseline (W) and Saccharin Habituation (S) and throughout the first seven conditioning cycles. * Significantly different between Groups ML and MW. ** For Group ML, significantly different from Trials 1 and 2. 16

2. Mean amount of saccharin consumption (±S.E.M) for subjects in Groups ML and MW during recovery (R) and conditioning (C) and following various doses of morphine (1.8 -10 mg/kg). * Significantly different between Groups ML and MW. ** For Group ML, significantly greater than consumption at 10 mg/kg. *** For Group ML, significantly greater than consumption at 5.6 mg/kg 18

3. Mean amount of saccharin consumption (±S.E.M) for subjects in Groups ML and MW during recovery (R) and conditioning (C) and following various doses of methadone (3.2 - 7.5 mg/kg. * Significantly different between Groups ML and MW. ** For Group ML, significantly different from consumption at 6.5 and 7.5 mg/kg. *** For Group MW, significantly different from consumption at 7.5 mg/kg...... 20

4. Mean amount of saccharin consumption (±S.E.M) for subjects in Groups ML and MW during recovery (R) and conditioning (C) and following various doses of SNC80 (3.2 - 24 mg/kg). *** For Group ML, significantly different from consumption at 24 mg/kg ...... 22

5. Mean amount of saccharin consumption (±S.E.M) for subjects in Groups ML and MW following the naloxone/morphine combination. * Significantly different between Groups ML and MW. ** For Group ML, significantly different from consumption at 0 mg/kg. *** For Group MW, significantly different from consumption at 3.2 mg/kg ...... 24

6. Mean amount of saccharin consumption (±S.E.M) for subjects in Groups ML and MW following the naltrindole/morphine combination. * Significantly different between Groups ML and MW. ** For Group MW, significantly different from consumption at 0, 3.2 and 5.6 mg/kg ...... 26

7. Mean amount (± S.E.M) of saccharin consumption for subjects in Groups SL and SW during Water Baseline (W) and Saccharin Habituation (S) and throughout the first seven conditioning cycles. * Significantly different between Groups SL and SW. ** For Group SL, significantly different from Trial 1...... 42

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 8. Mean amount (± S.E.M) of saccharin consumption for subjects in Groups SL and SW during recovery (R) and conditioning (C) in this phase and following various doses of SNC80 (0.56 - 5.6 mg/kg). * Significantly different between Groups SL and SW. ** For Group SL, significantly different from consumption at 3.2 and 5.6 mg/kg ...... 44

9. Mean amount (± S.E.M) of saccharin consumption for subjects in Groups SL and SW during recovery (R) and conditioning (C) in this phase and following various doses of SNC162 (1.8 - 32 mg/kg). * Significantly different between Groups SL and SW. For Group SL, significantly different from consumption at 18 and 32 mg/kg. *** For Group SW, significantly different from consumption at 1.8 - 18 mg/kg...... 46

10. Mean amount (± S.E.M) of saccharin consumption for subjects in Groups SL and SW during recovery (R) and conditioning (C) in this phase and following various doses of morphine (1.8-10 mg/kg). ** For Group SL, significantly different from consumption at conditioning. *** For Group SW, significantly different from consumption at 1.8, 3.2 and 5.6 mg/kg ...... 48

11. Mean amount (± S.E.M) o f saccharin consumption fo r Groups SL and SW following 5.6 mg/kg SNC80 given 20,60,120 and 240 min prior to saccharin access. * Significantly different between Groups SL and SW. ** For Group SL, significantly different from consumption at 120 and 240 min ...... 50

12. Mean amount (± S.E.M) of saccharin consumption for subjects in Groups SL and SW during recovery (R) and conditioning (C) in this phase and following NTI, SNC80 alone as well as the NTI/SNC80 combination. * Significantly different between Groups SL and SW. ** For Group SL, significantly different from consumption at 1 mg/kg ...... 53

13. Mean amount (± S.E.M) of saccharin consumption for subjects in Groups SL and SW during recovery (R) and conditioning (C) in this phase and following naloxone, SNC80 alone as well as the naloxone/SNC80 combination. * Significantly different between Groups SL and SW. ** For Group SW, significantly different from consumption at 0.18 -1 mg/kg ...... 55

14. Hypothetical intrinsic activities generated at the mu, delta and kappa receptor subtypes by 10 mg/kg morphine across time in the DDL procedure 67

15. Hypothetical intrinsic activities generated at the mu, delta and kappa receptor subtypes by 5.6 mg/kg SNC80 across time in the DDL procedure...... 69

vii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 1

EXPERIMENT 1

Although exteroceptive cues (e.g., lights and tones) have been

traditionally used as a means to establish discriminative control of behavior,

drugs (by acting as interoceptive cues) have been shown to have discriminative

effects as well (Overton, 1971). Drug discrimination training usually employs a

design in which an animal is reinforced for making a specific response following

administration of a specific drug and a different response following administration

of the drug's vehicle (though see Stolerman, 1993 for more complex designs).

Ultimately, the animal acquires the drug discrimination (i.e., behavior comes

under the control of the drug) such that the animal performs drug- and vehicle-

appropriate responding following administration of the drug or its vehicle,

respectively. Although the initial demonstration of DDL was with alcohol (Conger,

1951), later reports revealed that many different classes of compounds were also

capable of producing discriminative control of behavior (Overton, 1971; Colpaert,

1978) and that this stimulus control was evident using a variety of preparations

and a variety of species (Stolerman & Shine, 1985).

Although DDL procedures initially examined the ability of drugs to produce

discriminative cues, subsequent reports assessed the nature of such cues. Such

assessments included studies of generalization, time course, antagonism,

stereospecificity and sites of action (Bertalmio & Woods, 1987; Gianutsos & Lai,

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1974; Jaeger & van der Kooy, 1993; Shannon & Holtzman, 1976,1977a, 1977b;

Shoaib & Spanagel, 1994; Winter, 1975). One class of compounds widely

examined within the DDL literature has been the opioids, with particular focus on

the prototypical opioid morphine. Some of the earlier work examining the nature

of the morphine discriminative stimulus assessed the ability of this cue to

generalize to other drugs (Gianutsos & Lai, 1975; Shannon & Holtzman, 1976a,

1977a, 1977b). For example, Shannon and Holtzman (1976a, 1977b)

demonstrated that the opioids oxymorphone, levorphanol, methadone and

meperidine all produced dose-related, morphine-appropriate responding in

animals trained to discriminate morphine from saline, whereas the non-opioid

psychoactive drugs d-amphetamine, pentobarbital, chlorpromazine, mescaline,

ketamine, physostigmine and scopolamine all failed to substitute for the

morphine discriminative cue. In another study, Gianutsos and Lai (1975)

demonstrated that the opioids morphine and methadone dose-dependently

substituted for the morphine cue, whereas, the non-opioids loperamide and

haloperidol produced saline-appropriate responding. It is thought that compounds

that substitute for the training drug belong to the same pharmacological class.

Given that the opioid, but not the non-opioid, drugs generalized to the morphine

stimulus suggested that the discriminative effects of morphine were due to its

opioidergic actions. Similar patterns of opioids, but not non-opioids, substituting

for morphine have been generated in studies examining analgesia and reward

(Easterling & Holtzman, 1998; Mamoon, Barnes, Ho, & Hoskins, 1995; Morgan,

Cook, & Picker, 1999; Mucha & Herz, 1985; Switzman, Hunt, & Amit, 1981).

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In addition to demonstrations of generalization of the discriminative effects

of the opioids, the temporal parameters of these effects have also been

determined. Specifically, using a discrete trial operant procedure, Bertalmio and

Woods (1987) reported that the stimulus effects of morphine were greatest at 30

min postinjection and still evident at 56 min, while stimulus control for the opiate

agonist alfentanil peaked in the range of 3 to 10 min postinjection and faded at

18 to 30 min. The temporal parameters o f morphine antinociception are

comparable in peak effect and duration to morphine’s discriminative effects (see

Shannon & Holtzman, 1976a). For example, Gades and colleagues (2000) have

shown that the analgesic effects of morphine peaked at 30 min and lasted for 2

to 3 hrs.

The stimulus effects of the opioids can be blocked by pharmacological

antagonists with known activity at opioid receptors. For example, Shannon and

Holtzman (1976a) using a discrete trial avoidance task demonstrated that

naloxone blocked the discriminative effects of morphine in a dose-dependent

manner (see also Stevenson, Poumaghash & Riley, 1992). This antagonism was

reversible in that, in the presence of naloxone, a dose of 56 mg/kg morphine was

able to produce morphine-appropriate responding. Opioid antagonists have also

been shown to block morphine-induced place preference, self-adminstration,

antinociception, delayed gastrointestinal transit and immunosuppression within a

dose range comparable to that used to block the discriminative effects of

morphine (Carr, Gerak, & France, 1994, Culpepper-Morgan, Holt, Laroche, &

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Kreek, 1995; Hoiaday, Pasternak, & Faden, 1983; Lysle, Coussons, Watts,

Bennett, & Dykstra, 1993).

Stereospedfidty has also been exhibited with the opioids. For example,

the /-isomers of morphine and fentanyl produce discriminative effects, whereas

the d-isomers generally do not (Shannon & Holtzman, 1977; Winter, 1975).

Specifically, Winter (1975) showed that in rats trained to discriminate morphine

from saline, the morphine congener ievorphanol substituted for morphine at

doses up to 200-fold less than it’s optical isomer dextrorphan. In another report,

Shannon and Holtzman (1977) showed that Ievorphanol, but not dextrorphan,

substituted for morphine stimulus control. It was thought that because these

opioids had optically active isomers that showed differential activity, a precise

structure was most likely required to activate the receptor, further suggesting that

receptor mediation was likely. Along with the demonstration of antagonism of the

morphine cue, studies demonstrating stereospecificity of this cue lent further

support to the notion that the morphine discriminative stimulus was contingent

upon its binding to opiate receptors (Colpaert, 1978). Comparable findings of

stereospecificty for the opioids (i.e., /-isomers are active) have also been

demonstrated in analgesic and place conditioning procedures (Ling, Spiegel,

Lockhart, Pasternak, 1985; Mucha & Herz, 1985).

Various DDL reports have demonstrated that the discriminative effects of

opioids are mediated at specific sites in the brain (Jaeger & van der Kooy, 1993;

Shoaib & Spanagel, 1994). For example, using the conditioned taste aversion

baseline of DDL, Jaeger and van der Kooy (1993) have shown that the

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discriminative effects of morphine are produced in the parabrachial nucleus.

More specifically, infusions of morphine into the parabrachial nucleus dose-

dependently substituted for subcutaneously-administered morphine in the rat.

Morphine infusions into the medial prefirontal cortex, anterior dorsolateral

striatum, medial thalamus, amygdaloid nucleus, dorsal hippocampus and

periaqueductal grey generalized to systemic saline. In a subsequent report,

Shoaib and Spanagel (1994) reported that microinjections of morphine into the

ventral tegmental area and nucleus accumbens completely and partially

substituted for, respectively, the systemic administration of morphine. These data

suggest that mesolimbic structures may mediate the discriminative stimulus

effects of morphine. These results are interesting in light of the overwhelming

evidence that the rewarding effects of the opiates are mediated by some of the

same anatomical substrates that mediate drug discrimination, (Koob & Bloom,

1988; Koob, 1992). Interestingly, drugs that have weak discriminative cues are

rarely abused (Overton, 1971), suggesting that the discriminative effects of drugs

may be related to their abuse potential (Holtzman, 1990; though see Preston &

Bigelow, 1991). The finding that central administration of opioids (at doses 1000-

fold less than systemic doses required to produce morphine stimulus control)

substituted for systemically administered cues, strongly suggested that the

receptor-driven discriminative effects of morphine were centrally mediated.

Although the above findings suggested that morphine is acting on opiate

receptors to effect its discriminative control, the specific opiate receptor subtypes

mediating this stimulus control are not known. More recent reports have

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assessed the specific receptor mediation of this cue. Both biochemical and

behavioral investigations have implicated three distinct opiate receptors,

specifically, mu, delta and kappa (Gilbert & Martin, 1976; Lord, Waterfield,

Hughes & Kostertitz, 1977; Magnan, Paterson, Tavani, & Kostertitz, 1982; Martin,

Eades, Thompson, Huppler & Gilbert, 1976; Paterson, Robson & Kostertitz,

1983). Although morphine is relatively selective for the mu receptor, depending

on the dose, it binds to and has effects at all three (Akil, Meng, Devine & Watson,

1997; Chen, Smith, Cahill, Cohen & Fishman, 1993; Magnan et al., 1982). In one

of the initial assessments of the receptor mediation of morphine’s discriminative

effects, Bertalmio and Woods (1987) demonstrated that the highly mu-selective

antagonist quadazocine blocked the discriminative stimulus effects of the mu

agonists, codeine, Ievorphanol and alfentanil, with a dose 100-fold less than the

dose required to block stimulus effects of the kappa agonist, ethylketazocine.

Similarly, Negus and his colleagues (Negus, Picker & Dykstra, 1990) reported

that rats trained to discriminate morphine from its vehicle selectively generalized

morphine control to opioid agonists with relative selectivity for the mu receptor,

but not to those selective for the kappa receptor. Specifically, the mu agonist

fentanyl and the partial mu agonist buprenorphine substituted for morphine,

whereas the relatively selective kappa agonists U50488H and bremazocine failed

to do so. It was not the case that these latter compounds lacked discriminative

effects. Animals could be trained to discriminate U50488H from its vehicle, and

bremazocine (but not morphine) substituted for U50488H in subsequent

generalization assessments. Thus, similar to a variety of other opioid-induced

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effects, e.g., analgesia, respiratory depression, inhibition of gastrointestinal

transit, morphine discriminative control appears to be based on its activity at the

mu, but not the kappa, receptor (Moerschbaecher, Devia, & Brocklehurst, 1988;

Reisine & Pasternak, 1996; Shannon & Holtzman, 1976; Young, Woods, Herling

& Hein, 1983).

To date, there have been relatively few reports assessing delta receptor

activity in morphine drug discrimination learning. Further, these assessments

have been limited to nonselective opioid compounds (e.g., BW373U86) or

selective delta peptides (Locke & Holtzman, 1986; Ukai & Holtzman, 1988)

administered intracerebroventricularly (icv). Given that BW373U86 has low

selectivity for mu and delta receptor subtypes, this precludes any definitive

assessments of delta mediation of morphine control with this compound. Further,

it is not known if findings with the centrally administered peptides generalize to

systemically active compounds. For example, in rats trained to discriminate

morphine from saline the delta peptide metkephamid produces morphine-

appropriate responding when injected i.c.v. and saline-appropriate responding

when injected systemically (Locke & Holtzman, 1986). Recently a systemically

active delta agonist (+)-4-[(alphaft)-aipha-((2S,5R)-4-allyl-2,5-dimethyl-1-

piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide) (SNC80), the methyl ether

of one enantiomer of BW373U86 (Calderon, Rothman, Porreca, Flippen-

Anderson, McNutt, Xu, Smith, Bilsky, Davis & Rice 1994), has been synthesized

that demonstrates high selectivity for the delta receptor and at the highest doses

tested shows no signs of toxicity (Bilsky, Calderon, Wang, Bernstein, Davis,

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Hruby, Mcnutt, Rothman, Rice, & Porreca, 1995; Negus, Gatch, Mello, Zhang &

Rice, 1998). Such a compound allows for an assessment of the role of the delta

receptor in the mediation of morphine drug discrimination. Accordingly, in

Experiment 1 animals were trained to discriminate 10 mg/kg morphine from

vehicle using the conditioned taste aversion baseline of drug discrimination

learning (Grabus, Smurthwaite & Riley, 1999; Kautz, Geter, McBride,

Mastropaolo & Riley, 1989; Mastropaolo, Moskowitz, Dacanay, & Riley, 1989;

Riley, 1997; Riley & Poumaghash, 1995; Riley & Melton, 1997; Stevenson et al.,

1992). Once discriminative control was established, a range of doses of SNC80

was administered to assess the ability of this compound to substitute for the

morphine discriminative cue. A range of doses of the opioid agonist morphine

and the mu agonist methadone (Payte, 1991) were also assessed for their ability

to engender morphine-appropriate responding. In a further assessment of the

receptor mediation of the discriminative effects of morphine, both the opioid

receptor antagonist naloxone (Akil et al., 1997; Magnan et al., 1982), as well as

the delta receptor antagonist naltrindole (Portoghese, Sultana & Takemori,

1988), were administered prior to morphine to determine if the stimulus

properties of morphine could be differentially blocked.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 2

METHOD

Experiment 1

Subjects and Apparatus

The subjects were 12 experimentally naive, Long-Evans female rats

approximately 200-290 g at the beginning of the experiment. They were housed

in individual wire-mesh cages and maintained on a 12:12 L:D cycle and at an

ambient temperature of 23° C for the duration of the experiment. Rat chow

(Prolab Rat, Mouse, Hamster 3000) was available ad libitum.

Druos

Morphine sulfate, methadone hydrochloride, naltrexone hydrochloride,

naltrindole (all generously supplied by the National Institute on Drug Abuse,

NIDA) and naloxone hydrochloride (generously supplied by DuPont

Pharmaceuticals) were dissolved in distilled water. SNC80 (generously supplied

by the National Institute of Diabetes and Digestive and Kidney Diseases, NIDDK)

was prepared as a base dissolved in distilled water and 6M HCI. All drugs were

injected intraperitoneally (ip) and prepared at the following concentrations:

morphine (10 mg/ml), methadone (10 mg/ml), SNC80 (2 mg/ml), naloxone

(1 mg/ml) and naltrindole (2 mg/ml).

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Procedure

Phase I: Acquisition. A l the outset of training, subjects were given 20-min

access to water once a day for 14 consecutive days in their home cages until all

subjects consistently drank levels greater than 10 ml. On Days 15-17 (Saccharin

Habituation), a novel saccharin solution (0.1 w/v Sodium Saccharin, Sigma

Pharmaceuticals) replaced water during the daily 20-min fluid-access period and

was preceded on the last day of Saccharin Habituation by an ip injection of

distilled water (1 ml/kg).

On Day 18, conditioning began. All subjects were injected with 10 mg/kg

of morphine 30 min prior to 20-min access to saccharin. Immediately following

saccharin access, subjects were ranked according to saccharin consumption

(i.e., from lowest to highest) and assigned to one of two groups (Group ML, i.e.,

morphine-lithium chloride, n_= 6, and Group MW, i.e., morphine-water, n = 6).

Subjects in Group ML were then injected with 1.8 milliequivalents (mEq), 0.15 M

LiCI (76.8 mg/kg), while subjects in Group MW were given an equivolume

injection of distilled water (i.e., the LiCI vehicle). On the following three recovery

days, subjects in both groups were injected with distilled water 30 min prior to 20-

min access to the same saccharin solution. No injections followed saccharin on

these recovery days. This alternating procedure of conditioning and recovery was

repeated until discriminative control had been established for all experimental

subjects (i.e., each subject in Group ML had consumed at least 50% less than

the mean of Group MW on two consecutive conditioning trials).

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Phase II: Generalization. The procedure during this phase was identical to

that of Phase I with the following exception. On the second day following

conditioning (the second recovery day within Phase I, but a probe day in this

phase), subjects were administered one of a range of doses of morphine (1.8 -

10 mg/kg), methadone (3.2 - 7.5 mg/kg) or SNC80 (3.2 - 24 mg/kg) 30 min prior

to saccharin access. On any specific probe day, subjects in Group ML were

given an injection only if they had consumed at least 50% less than the mean of

the control subjects on the two preceding conditioning trials. Doses were

administered in a mixed pattern. No injections followed saccharin access on

these probe sessions.

Phase III: Naloxone challenge. The procedure for this phase was identical

to that of Phase I with the exception that on the second recovery day following

each conditioning trial, animals were given 3.2 mg/kg of naloxone 60 min prior to

the training dose of morphine (i.e., 10 mg/kg). This time course of 60 min (for

naloxone preexposure) was based on previous research in our laboratory

(Stevenson et al., 1992) demonstrating complete antagonism of the

discriminative effects of morphine when naloxone was administered 60 min prior

to morphine. Thirty min following the injection of morphine, all subjects were

given 20-min access to saccharin. To assess the effects of naloxone alone on

saccharin consumption, all subjects were given naloxone 60 min prior to an

injection of distilled water and then 30 min later given access to saccharin. No

injections followed saccharin access on these test days.

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Phase IV: Naltrindole challenge. The procedure for this phase was

identical to that of Phase III with the exception that animals were given one of a

range of doses of naltrindole (3.2 -10 mg/kg) 60 min prior to the injection of

morphine. Thirty min following the injection of morphine, all subjects were given

20-min access to saccharin. To assess the effects of naltrindole alone on

saccharin consumption, all subjects were given naltrindole 60 min prior to an

injection of distilled water and then 30 min later given access to saccharin. No

injections followed saccharin access on these test days.

Data Analyses

Phase I: Acquisition. Statements of statistical significance are based on a

repeated measures Analysis of Variance (ANOVA) with between-subjects

variable of Group (ML and MW) and within-subjects variables of Trial (1 - 8). The

repeated measures ANOVA was followed by one-way ANOVAs for each trial.

Within-group changes in consumption across trials were evaluated using paired

sample t-tests. The accepted level of significance for all tests was p < 0.05.

Phase II: Generalization. For each drug, statements of statistical

significance are based on individual repeated measures ANOVA with between-

subjects variable of Group (ML and MW) and within-subjects variables of Dose

(1 .8 -1 0 mg/kg, morphine; 3.2 - 7.5 mg/kg, methadone; 3.2 - 24 mg/kg SNC80).

The repeated measures ANOVA was followed by one-way ANOVAs for each

dose. Within-group changes in consumption across doses were evaluated using

paired sample t-tests. The accepted level of significance for all tests was p <

0.05.

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Phase III: Antagonism. For each combination of drugs, statements of statistical

significance are based on individual repeated measures ANOVA with between-

subjects variable of Group (ML and MW) and within-subjects variables of dose (0

- 3.2 mg/kg, naloxone; 0 -1 0 mg/kg, naltrindole). The repeated measures

ANOVA was followed by one-way ANOVAs for each dose. Within-group changes

in consumption across doses were evaluated using paired sample t-tests. T-tests

were used for between-groups comparisons in single-dose studies. The accepted

level of significance for all tests was p < 0.05.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 3

RESULTS

Experiment 1

P/rase I: Acquisition

Figure 1 presents the mean amount (± SEM) of saccharin consumption for

Groups ML and MW during Water Baseline (W) and Saccharin Habituation (S)

and throughout the first seven conditioning cycles during this phase. As

illustrated, there were no significant differences in fluid consumption between

groups during Water Baseline (t (io> = -.258; g = 0.802) or Saccharin Habituation

(t (io) = 0.380; c = 0.551). A repeated measures ANOVA revealed significant

main effBcts of Group (F(i,io r 29.589; g £ 0.05) and Trial (F ^jo r 7 37°; g < 0.05)

and a significant Group X Trial interaction (F p jor 6.530; g < 0.05) during

acquisition of the drug discrimination. There were no significant differences in

saccharin consumption between groups during Saccharin Habituation or over the

first two conditioning trials (all g’s > 0.05). On the third conditioning trial, subjects

in Group ML drank significantly less saccharin than subjects in Group MW

(F(i,io)B10.53; g £ 0.05). This difference was maintained for the remainder of

conditioning. For subjects in Group ML, there were no changes in consumption

from the first to the third conditioning trial (all g’s £ 0.369). By Trial 4, however,

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 15

consumption significantly decreased below the level on the initial trial (p < 0.05).

Consumption remained significantly reduced throughout this phase (all p’s <

0.05). For subjects in Group MW, there were no changes in saccharin

consumption over conditioning (all p’s £ 0.066), with consumption approximating

habituation levels on each trial. On the final conditioning trial of this phase,

subjects in Groups ML and MW drank 2.5 and 9.83 ml of saccharin, respectively.

During recovery sessions, consumption for both groups remained high,

approximating habituation levels (data not shown).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 16

Figure I A ML

o MW

15-i

I 1 - s 1 s a

a sh **

T" T T T ~ r T T W S 2 3 4 5 6

T ria l

Figure 1. Mean amount (±S.E.M.) of saccharin consumption for subjects in Groups ML and MW during Water Baseline (W) and Saccharin Habituation (S) and throughout the first seven conditioning cycles.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 17

Phase II: Generalization

Figure 2 presents the mean amount (± SEM) of saccharin consumption for

Groups ML and MW during conditioning (C) and recovery (R) and following

various doses of morphine. Subjects in Group ML drank significantly less

following conditioning than recovery (g < 0.05), whereas subjects in Group MW

displayed no significant differences in consumption between conditioning and

recovery (g = 0.204), indicating maintenance of discriminative control in this

phase. A repeated measures ANOVA revealed that there were significant main

effects of Group (F(1i10)= 25.822; g < 0.05) and Dose (F(3,30)s 28.820; g < 0.05)

and a significant Group X Dose interaction (F( 3 ,30)s 11 810; g < 0.05). For

subjects in Group ML, there was an inverse relationship between the dose

of morphine and the amount of saccharin consumed (i.e., as the dose of

morphine increased, the amount of saccharin consumed decreased).

Consumption at 1.8, 3.2 and 5.6 mg/kg was significantly greater than

consumption during conditioning (all g’s < 0.05). There were no significant

differences in consumption between 10 mg/kg and conditioning. For subjects in

Group MW, there were no consistent changes in saccharin consumption over the

increasing doses of morphine. Further, consumption at these doses

approximated consumption during conditioning. At 3.2, 5.6 and 10 mg/kg,

subjects in Group ML drank significantly less than subjects in Group MW (all g’s

j£ 0.05).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2 a m l O MW

l5 -i

1 10 - § I E

5 -

T" t T i “T” “T ~ R C 1.8 3.2 5.6 10

Morphine Dose (mg/kg) Figure 2. Mean amount of saccharin consumption (±S.E.M) for subjects Groups ML and MW during recovery (R) and conditioning (C) and following various doses of morphine (1.8 -1 0 mg/kg).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 19

Figure 3 presents the mean amount (± SEM) of saccharin consumption for

Groups ML and MW during conditioning (C) and recovery (R) and following

various doses of methadone. During this probe, subjects in Group ML drank

significantly less following conditioning than recovery (p < 0.05), whereas

subjects in Group MW drank significantly more during conditioning than recovery

(p < 0.05). There were significant main effects of Group (F(i,«»= 20.154; p < 0.05)

and Dose (F(3, 30)= 44.650; p < 0.05) and a significant Group X Dose interaction

(F(3,30)= 5.795; p < 0.05). As illustrated, for subjects in Groups ML and MW there

was an inverse relationship between the dose of methadone and the amount of

saccharin consumed. For subjects in Group ML, consumption at 3.2 mg/kg was

significantly greater than consumption during conditioning (p < 0.05). There were

no significant differences in consumption between 6.5 and 7.5 mg/kg and

conditioning (all p’s > 0.088). For subjects in Group MW, consumption at 5.6,6.5

and 7.5 mg/kg was significantly less than consumption during conditioning (ail p’s

< 0.05). There was no significant difference in consumption between conditioning

and the lower dose of 3.2 mg/kg (p = 0.697). At 5.6 and 6.5 mg/kg, subjects in

Group ML drank significantly less than subjects in Group MW (all p’s < 0.05).

There were no significant differences in consumption between Groups ML and

MW at the remaining doses (all p’s > 0.652).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 20

Figure 3

15- B, •+ ¥

E 10 - m9 uS

5 -

0J 1 1 1 1 1 1------1“ R C 3.2 5.6 10

Methadoae Dose (mg/kg)

Figure 3. Mean amount of saccharin consumption (±S.E.M) for subjects in Groups ML and MW during recovery (R) and conditioning (C) and following various doses of methadone (3.2 - 7.5 mg/kg).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 21

Figure 4 presents the mean amount (± SEM) of saccharin consumption for

subjects in Groups ML and MW during conditioning (C) and recovery (R) and

following various doses of SNC80. During these probes, subjects in Group ML

drank significantly less following conditioning than recovery (g < 0.05), whereas

subjects in Group MW displayed no significant differences in consumption

between conditioning and recovery (g = 0.936). A repeated measures ANOVA

revealed a significant main effect of Dose (F(4,4 0 )s 8.732; g < 0.05), but not of

Group (F(i,io)s 0.953; g = 0.352), and no significant Group X Dose interaction

(F(4,40)= 1.809; g - 0.146). For subjects in both Groups ML and MW, there was an

inverse relationship between the dose of SNC80 and the amount of saccharin

consumed. For subjects in Group ML, consumption at all doses was significantly

greater than consumption during conditioning (all g’s £ 0.05). Consumption at 3.2

and 5.6 mg/kg was significantly greater than consumption at 24 mg/kg (all g’s <

0.05). For subjects in Group MW, consumption at 10,18 and 24 mg/kg was

significantly less than consumption during conditioning (all g’s £ 0.05). There

were no significant differences between Groups ML and MW at any dose of

SNC80 tested (all g’s >0.091).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 22

Figure 4

I 10 - 8 * e

£ 5-

~r~ ” i------1— — i— "T“ T R C 3.2 5.6 10 18 32

SNC80 Dote (mg/kg)

Figure 4. Mean amount of saccharin consumption (±S.E.M) for subjects in Groups ML and MW during recovery (R) and conditioning (C) and following various doses of SNC80 (3.2 - 24 mg/kg).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 23

Phase III: Antagonism

Figure 5 presents the mean amount (± SEM) of saccharin consumption for

subjects in Groups ML and MW during conditioning (C) and recovery (R), as well

as following the combination of various doses of naloxone (0-3.2 mg/kg)

administered 60 min prior to the training dose of 10 mg/kg morphine (see

Stevenson et al., 1992). During this phase, subjects in Group ML drank

significantly less following conditioning than recovery (q£ 0.05), whereas

subjects in Group MW drank significantly more following conditioning than

recovery (g =0.036). A repeated measures ANOVA revealed significant main

effects of Group (F(1i10)= 26.467; g < 0.05) and Dose (F(i,i0)= 63.241; g < 0.05)

and a significant Group X Dose interaction (F(i,i0>= 48.419; g < 0.05) when

naloxone was injected prior to morphine. For subjects in Group ML, consumption

following the combination of 3.2 mg/kg naloxone and morphine (10.92 ml) was

significantly greater than consumption following the combination of distilled water

vehicle and morphine (0.40 ml), reflective of the blocking of morphine’s stimulus

effects (g < 0.05). Conversely, subjects in Group MW displayed no significant

differences in consumption between the combination of 3.2 mg/kg naloxone and

morphine (10.95 ml) and the combination of distilled water vehicle and morphine

(14.70 ml) (g = 0.852). There were no significant differences in consumption

between Groups ML and MW at 3.2 mg/kg, indicative of complete antagonism of

morphine's discriminative effects at this dose.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5

▲ ML 20 —| o MW *** * 15- I I

8 I

10 - I9

J ** 5- I

T" ~ r T —T~ R c 0 3.2

Naloxone Dose (m g/kg)

Figure 5. Mean amount of saccharin consumption (±S. E. M) for subjects Groups ML and MW following the naloxone/morphine combination.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 25

Figure 6 presents the mean amount (± SEM) of saccharin consumption for

subjects in Groups ML and MW during conditioning (C) and recovery (R) in this

phase, as well as following the combination of various doses of naltrindole (0.0 -

10 mg/kg) administered 60 min prior to the training dose of morphine (10 mg/kg).

As above, subjects in Group ML drank significantly less following conditioning

than recovery (g < 0.05), whereas subjects in Group MW displayed no significant

differences in consumption between conditioning and recovery (g = 0.211). There

were significant main effects of Group (F(i,io)s 223.677; g < 0.05) and Dose

(F(3.30)= 8.993; g < 0.05) but no significant Group X Dose interaction (F(3i30)=

0.195; g = 0.899) when naltrindole was injected prior to morphine. For subjects in

Group ML, consumption following morphine alone (C) and in combination with all

doses of naltrindole did not differ (all g’s > 0.073), indicating that naltrindole did

not block the discriminative effects of morphine. For subjects in Group MW,

consumption following the combination of 10 mg/kg naltrindole and morphine

was significantly greater than consumption following the combination of all lower

doses of naltrindole and morphine (all g’s < 0.05). Subjects in Group ML drank

significantly less than subjects in Group MW following all combinations of

naltrindole and morphine (all g’s < 0.05).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced Groups ML and MW following the naltrindole/morphine combination. naltrindole/morphine the following MW and ML Groups Figure 6. Mean amount o f saccharin consumption (±S.E.M) for subjects in in subjects for (±S.E.M) consumption saccharin f o amount Mean 6. Figure Consumption (m l) lrnoeDs mg/kg) (m Dose altrindole N Figure 6 Figure o ▲ ML MW 26 CHAPTER 4

DISCUSSION

EXPERIMENT 1

Both biochemical and behavioral studies have implicated three distinct

opiate receptors: mu, delta and kappa (Gilbert & Martin, 1976; Lord et al., 1977;

Magnan et at., 1982; Martin et al., 1976; Paterson et al., 1983). Although

research suggests that the discriminative effects of morphine are mediated at the

mu, but not the kappa, receptor (Bertalmio & Woods, 1987; Negus et al., 1990;

Picker, Doty, Negus, Mattox, & Dykstra, 1990), there have been relatively few

reports assessing delta receptor activity in morphine drug discrimination learning

and they have been limited generally to the peptides (Locke & Holtzman, 1986;

Ukai & Holtzman, 1988). Because it is not dear to what extent findings with delta

peptides generalize to systemically active compounds (interestingly, some delta

peptides produce morphine- or vehide-appropriate responding depending on the

route of administration; see Locke & Holtzman, 1986), in the present experiment

the systemically active delta selective compound SNC80 was assessed for its

ability to substitute for morphine in animals trained to discriminate morphine from

distilled water using the conditioned taste aversion baseline of drug

discrimination learning (Grabus et al., 1999; Kautz et al.. 1989; Riley &

Poumaghash, 1995; Riley & Melton, 1997; Stevenson et al., 1992). A range of

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 28

doses of the opioid agonist morphine and the mu agonist methadone were also

assessed for their ability to engender morphine-appropriate responding.

Subsequently, both the opioid receptor antagonist naloxone and the delta opioid

receptor antagonist naltrindole were administered prior to morphine to determine

if the stimulus properties of morphine could be diiferentially blocked.

As described, animals injected with morphine prior to a saccharin and LiCI

pairing acquired the drug discrimination, consuming significantly less saccharin

following morphine than following the morphine vehicle. The discrimination was

dose-dependent in that as the dose of morphine increased, the amount of

saccharin consumed decreased. In subsequent generalization tests, the mu

agonist methadone produced morphine-appropriate responding in a dose-

dependent manner. That both morphine and methadone substituted for the

discriminative effects of the training dose of 10 mg/kg morphine is consistent with

other research that demonstrates that morphine’s discriminative effects are

mediated at the mu opioid receptor (Gianutsos & Lai, 1975; Shannon &

Holtzman, 1976a). Conversely, the highly selective, systemically active delta

agonist SNC80 produced vehicle-appropriate responding. These selective

generalization patterns with methadone and SNC80 suggest that the

discriminative effects of morphine are mediated at the mu, but not the delta,

receptor.

The conclusion that morphine’s stimulus effects are mediated at the mu

receptor is further supported by the feet that morphine’s discriminative control

was completely blocked by the opioid antagonist naloxone, but not by the delta

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 29

antagonist naltrindole. That naloxone completely antagonized morphine’s

discriminative effects is in agreement with a variety of other reports (Colpaert,

1978; Shannon & Holtzman, 1976a; Shannon & Holtzman, 1976b; Stevenson et

al., 1992) demonstrating complete antagonism of morphine stimulus control by

naloxone. Although naltrindole failed to block the discriminative effects of

morphine, it should be noted that there was a slight increase in saccharin

consumption in subjects given naltrindole prior to morphine, suggesting a partial

antagonism of the morphine cue. However, this increase in consumption was not

significant (relative to consumption when the vehicle was given prior to

morphine). Given that the delta agonist SNC80 did not substitute at any dose for

morphine, the slight increase in saccharin consumption following the

naltrindole/morphine combination unlikely reflects a partial antagonism of the

morphine cue or any delta mediation of morphine’s discriminative effects. The

fact that control subjects drank significantly more saccharin following the highest

dose of naltrindole than following vehicle suggests that naltrindole alone may

have an unconditioned stimulant effect on drinking.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 5

EXPERIMENT 2

The failure of morphine to generalize to the delta agonist SNC80 (or to be

antagonized by the delta antagonist naltrindole) suggests that morphine’s

stimulus control is based on its activity at the mu (but not the delta) receptor

subtype. Although the basis of this selectivity is not known, one possibility is the

general inability of activity at the delta receptor to support discimination learning.

That is, although most drugs produce effects sufficient to serve as discriminative

cues, some do not (Overton, 1971). Drugs acting on the delta receptor may be

one such class of drugs. If this is the case, the failure to see any delta mediation

of morphine’s stimulus effects may be due to this general inability of delta activity

to support such learning. Experiment 2 assessed whether or not activity at the

delta receptor would support DDL on its own.

Although the discriminative stimulus effects of mu and kappa compounds

have been well characterized (Bertalmio and Woods, 1987; France, Jacobson, &

Woods, 1984; Gianutsos and Lai, 1976; Grabus et al., 1999; Heriing, Valentino,

Solomon, Woods, 1984; Hill, Jones, Bell, & 1971; Jarbe, 1978; Locke and

Holtzman, 1986; Morgan and Picker, 1998; Negus, Morgan, Cook, & Picker,

1996; Negus et al., 1990; Picker, Benyas, Horwitz, Thompson, Mathewson,

Smith, 1996; Picker, 1994; Picker etal., 1990; Picker and Dystra, 1989; Picker

and Dykstra, 1987; Poumaghash and Riley, 1993; Schaefer and Holtzman, 1977;

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 31

Schaefer and Holtzman, 1978; Shannon and Holtzman, 1977a; Shannon and

Holtzman, 1977b; Shannon and Holtzman, 1976a; Smurthwaite and Riley, 1994;

Stevenson et al., 1992; Suzuki, Mori, Tsuji, Misawa, Nagase, 1995; Ukai and

Holtzman, 1988; Winter, 1975), work assessing the discriminative stimulus

effects of delta agonists, on the other hand, has been quite limited (Brandt,

Negus, Mello, Furness, Zhang, Rice, 1999; Comer, Mcnutt, Chang, DeCosta,

Mosberg, & Woods, 1993; Jewett, Mosberg, & Woods, 1996; Negus e tal., 1998;

Negus, Butelman, Chang, DeCosta, Winger, Woods, 1994; Picker and Cook,

1998).

In the first study assessing delta agonists as training drugs within the drug

discrimination procedure (Comer et al., 1993), pigeons were trained to

discriminate the systemically active, delta agonist BW373U86 (Chang, Rigdon,

Howard, & Mcnutt, 1993) from sterile water in a two key, food-reinforced

procedure. Animals acquired the discrimination in approximately 100 training

sessions and were subsequently tested for the generalization of BW373U86

control to a variety of compounds with varying degrees of selectivity for mu, delta

and kappa receptor subtypes. BW373U86 partially generalized to several other

systemically active, delta agonists, e.g., oxymorphindole and LY 123502, but

failed to generalize to either the delta peptides [D-Pen^D-Pen^enkephalin

(DPDPE) or [D-Sef^-L-Leu^enkephalyl-Thr (DSLET) icv. BW373U86 also

generalized partially to morphine, alfentanil and ethylketocyclazocine. These

generalization patterns are consistent with the general low selectivity of

BW373U86 for the mu and delta receptor subtypes (Negus et al., 1996; Picker

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 32

and Cook, 1998). Although nonselective, BW373U86's stimulus effects were fully

blocked by the delta antagonist naltrindole at doses 1000 fold less than those

needed to block morphine's stimulus effects, an effect suggestive of delta

mediation of its discriminative properties.

Subsequent to these findings, Jewett and his colleagues (1996) trained

pigeons to discriminate the delta agonist DPDPE (icv administration) from saline

in a two-key, food-reinforced procedure. Animals acquired the discrimination

relatively rapidly and subsequently generalized DPDPE stimulus control to the

highly selective peptidergic delta agonists, DSLET and deltorphin II (but not to

the mu selective peptide, D-Ala2-NmePhe4- Gly5(o!)enkephalin, DAMGO).

Interestingly, BW373U86 substituted only partially for DPDPE, a finding

consistent with the aforementioned work in which BW373U86 was the training

drug (Comer et al,, 1993). Morphine and U69.593 (mu- and kappa-agonists,

respectively) produced only saline-appropriate responding. The finding that

DPDPE generalized to DSLET and deltorphin II is consistent with the feet that

DPDPE’s stimulus control was based on its agonist activity at the delta receptor.

The failure of BW373U86 to substitute for DPDPE again may be a function of the

general low selectivity of BW373U86 for the mu and delta receptor subtypes

(Negus et al., 1996; Picker and Cook, 1998).

One problem with the assessment of stimulus control by delta agonists

has been the general unavailability of systemically active, highly selective delta

compounds. Such an unavailability has resulted in the assessment primarily of

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 33

icv-administered delta peptides (e.g., OPDPE) or nonselective. systemic

alkaloids such as BW373U86. In this context, Brandt and his colleagues (Brandt

et al., 1999) have reported the acquisition of discriminative control in monkeys

trained to discriminate the systemically active delta agonist SNC80 from saline in

a food-reinforced procedure. Following acquisition of the discrimination, SNC80

generalized to other systemically active piperazinyl benzamide delta agonists,

e.g., SNC 86, SNC162 and SNC 243A, but failed to generalize to the (-)-

enantiomer of SNC80, opioids selective for mu and kappa receptor subtypes or

several nonopioid compounds, i.e., cocaine and ketamine. That these

generalization patterns reflected the delta mediation of SNC80 control were

further supported by the finding that the delta antagonist naltrindole competitively

blocked SNC80’s stimulus effects while the mu antagonist quadazocine was

without effect.

Together, the work with DPDPE, BW383U86 and SNC80 all point to the

ability of delta agonists to serve as stimuli in drug discrimination learning. To

date, however, the work with these delta agonists has been limited to pigeons

and monkeys, i.e., there have been no assessments of delta stimulus control in

rodents, although rats have been used to assess the ability of delta agonists to

substitute for morphine when morphine was used as the training drug (Locke and

Holtzman, 1986; Takita, Herienius, Lindahl, & Yamamoto, 1997). Given that

much of the work in the literature about the discriminative stimulus properties of

opioids has been done in rats (Broqua, Wettstein, Rocher, Riviere, & Dahl, 1998;

Gianutsos and Lai, 1976; Gianutsos and Lai, 1975; Grabus e t al., 1999; Hill et al.,

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 34

1971; Locke, Gomey, Comfeldt, & Fielding, 1989; Negus e tal., 1990; Picker et

al., 1990; Poumaghash and Riley, 1993; Riley and Poumaghash, 1995; Shannon

and Holtzman, 1979; Shannon and Holtzman, 1977a; Shannon and Holtzman,

1977b; Shannon and Holtzman, 1976a; Smurthwaite and Riley, 1994; Stevenson

et al., 1992; Winter, 1975; Young, Masaki, & Geula, 1992) and that species

differences in the discriminative properties (and other effects) of opioids with

relative selectivity for mu and kappa receptors (Herling, Coale, Valentino, Hein, &

Woods, 1980; Jarbe, 1978; Picker and Dykstra, 1987), as well as those with

mixed action at these receptor subtypes (Grabus et al., 1999; Picker, 1994), have

been reported, Experiment 2 assessed the establishment of discriminative

control with SNC80 in rats. Specifically, rats were trained to discriminate 5.6

mg/kg ip SNC80 from distilled water within the taste aversion baseline of drug

discrimination learning. Following acquisition of the discrimination, the ability of

various compounds with selectivity for mu (morphine) and delta (SNC 162)

receptors to substitute for SNC80 was assessed. Subsequently, the

determination of SNCSO’s temporal characteristics was determined. Finally,

animals were administered naltrexone and naltrindole concurrent with SNC80 to

assess their ability to block the stimulus properties of SNC80.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 6

METHOD

Experiment 2

Subjects and Apparatus

The subjects were 12 experimentally naive, female rats of Long-Evans

descent, weighing approximately 200-250 g at the start of the experiment. They

were housed in individual wire-mesh cages and were maintained on a 12-h

light/12-h dark cycle and at an ambient temperature of 23°C. Subjects received

restricted access to fluid for the duration of the study, but they were maintained

on ad-lib access to food (Prolab Rat, Mouse, Hamster 3000).

Druas

SNC80 and SNC162 (generously supplied by NIDDK) were prepared as a

base dissolved in distilled water and 6M HCI. Morphine sulfate (generously

supplied by NIDA), naloxone hydrochloride (generously supplied by DuPont

Pharmaceuticals) and naltrindole (generously supplied by NIDDK) were

dissolved in distilled water. All drugs were injected ip and prepared at the

following concentrations: SNC80 (2 mg/ml), morphine (4 mg/ml), SNC162 (2

mg/ml), naltrindole (2 mg/ml) and naloxone (1 mg/ml).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 36

Procedure

Phase I: Conditioning. The procedure for the first 15 days in this phase

was identical to that of Phase I of Experiment 1 (see above). On Day 16,

conditioning began. All subjects were injected with 5.6 mg/kg of SNC80 20 min

prior to 20-min access to saccharin. Immediately following saccharin access,

subjects were ranked according to saccharin consumption (i.e., from lowest to

highest) and assigned to one of two groups (Group SL, n = 6 and Group SW, n =

6). Subjects in Group SL were then injected with 1.8 mEq, 0.15 M LiCI (76.8

mg/kg), while subjects in Group SW were given an equivolume injection of

distilled water (i.e., the LiCI vehicle). On the following three recovery days,

subjects in both groups were injected with distilled water 20 min prior to 20-min

saccharin access. No injections followed saccharin on these recovery days. This

alternating procedure of a single conditioning day followed by 3 recovery

sessions was repeated until discriminative control had been established for all

experimental subjects (i.e., each subject in Group SL had consumed at least

50% less than the mean of Group SW on two consecutive conditioning trials).

Phase II: Generalization. The procedure during this phase was identical to

that of Phase I with the following exception. On the second day following

conditioning (the second recovery day within Phase I, but a probe day in this

phase), subjects in Groups SL and SW were administered one of a range of

doses of SNC80 (0.56 - 5.6 mg/kg), SNC162 (1.8 - 32 mg/kg) or morphine (1.8 -

10 mg/kg) 20 min prior to saccharin access. On any specific probe day, subjects

in Group SL were given an injection only if they had consumed at least 50% less

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 37

than the mean of the control subjects on the two preceding conditioning trials.

Doses were administered in a mixed pattern. No injections followed saccharin

access on these probe days.

Phase III: SNC80 time course. The procedure in this phase was identical

to that of Phase I with the following exception. During each probe day (the

second recovery day following conditioning), subjects in Groups SL and SW were

administered the training dose of SNC80 (5.6 mg/kg) 20,60,120 or 240 min prior

to saccharin access. Subjects in Group SL were probed only if they had

consumed at least 50% less than the mean of Group SW on the two preceding

conditioning trials. No injections followed saccharin access on these probe days.

Phase IV: Antegonism. The procedure during this phase was identical to

that of Phase I with the exception that on the second recovery day following each

conditioning trial (probe day) animals were given a range of doses of naltrindole

(1 - 5.6 mg/kg) or naltrexone (0.18 -1.8 mg/kg) 40 min and 10 min, respectively,

(injection times based on Spina et al., 1998 and Locke & Holtzman, 1986) prior to

the training dose injection of SNC80 (i.e., 5.6 mg/kg). Twenty min following the

injection of SNC80, all subjects were given 20-min access to saccharin. No

injections followed saccharin access on these probe days.

Date Analyses

Phase I: Acquisition. Statements of statistical significance are based on a

repeated measures ANOVA with between-subjects variables of Group (SL and

SW) and within-subjects variables of Trial (1 - 7). The repeated measures

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 38

ANOVA was followed by one-way ANOVAs for each trial. Within-group changes

in consumption across trials were evaluated using paired sample t-tests. The

accepted level of significance for all tests was p < 0.05.

Phase II: Generalization. For each drug, statements of statistical

significance are based on individual repeated measures ANOVA with between-

subjects variables of Group (SL and SW) and within-subjects variables of Dose

(0.56 - 5.6 mg/kg, SNC80; 1 .8 -3 2 mg/kg, SNC162; 1 .8 -1 0 mg/kg, morphine).

The repeated measures ANOVA was followed by one-way ANOVAs for each

dose. Within-group changes in consumption across doses were evaluated using

paired sample t-tests. The accepted level of significance for all tests was p <

0.05.

Phase III: SNC80 Time course. Statements of statistical significance are

based on a repeated measures ANOVA with between-subjects variables of

Group (SL and SW) and within-subjects variables Time (20 - 240 min). The

repeated measures ANOVA was followed by one-way ANOVAs for each time.

Within-group changes in consumption across times were evaluated using paired

sample t-tests. The accepted level of significance for ail tests was p < 0.05.

Phase IV: Antagonism. For each drug combination, statements of

statistical significance are based on individual repeated measures ANOVA with

between-subjects variables of Group (SL and SW) and within-subjects variables

of Dose (1 - 5.6 mg/kg, naltrindole; 0.18 -1 .8 mg/kg, naltrexone). The repeated

measures ANOVA was followed by one-way ANOVAs for each dose. Within-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 39

group changes in consumption across doses were evaluated using paired

sample t-tests. The accepted level of significance for all tests wasp < 0.05.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 7

RESULTS

Experiment 2

Phase I: Acquisition

Figure 7 presents the mean amount (± SEM) of saccharin consumption for

Groups SL and SW during Water Baseline (W) and Saccharin Habituation (S)

and throughout the first seven conditioning cycles during this phase. As

illustrated, there were no significant differences in fluid consumption between

groups during Water Baseline (tio = 1.059; g = 0.321) or Saccharin Habituation

(tio = 0.496; g = 0.631). A repeated measures ANOVA revealed significant main

effects of Group (F(i,i0)= 123.894: g < 0.05) and Conditioning (F(6 ,60 )s 39.429 g <

0.05) and a significant Group X Conditioning interaction (F<6,6or 46.305; g < 0.05)

during acquisition of the drug discrimination. For subjects in Group SL, there

were no changes in consumption from the first to the third conditioning trial (all

g’s > 0.616). By Trial 4, however, consumption significantly decreased below the

level on the initial trial (g < 0.05). Consumption remained significantly reduced

throughout this phase (all g’s < 0.05). For subjects in Group SW, consumption at

Trial 6 was significantly greater than consumption during saccharin habituation (g

< 0.05). Consumption approximated habituation levels for all remaining trials (all

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 41

g’s > 0.250). Although subjects in Groups SL and SW did not differ in saccharin

consumption on Conditioning Trials 1 and 2 (all g’s > 0.559), subjects in Group

SL significantly reduced consumption of saccharin relative to that in Group SW

by the third conditioning trial (g < 0.05). This difference was maintained over

conditioning. On the final conditioning trial of this phase, subjects in Groups SL

and SW drank 0.16 and 11.08 ml, respectively. During recovery sessions,

consumption for both groups remained high, approximating habituation levels

(data not shown).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 42

Figure 7 A SL O SW

15-i

0^ 1 10 - 8 I ma 8 u

** ** O-1 T~ T T i "T “T w S 2 3 4 5 6

T ria l

Figure 7. Mean amount (± S.E.M) of saccharin consumption for subjects in Groups SL and SW during Water Baseline (W) and Saccharin Habituation (S) and throughout the first seven conditioning cycles.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 43

Phase II: Generalization

Figure 8 presents the mean amount (± SEM) o f saccharin consumption for

Groups SL and SW during conditioning (C) and recovery (R) and following

various doses of SNC80. Subjects in Group SL drank significantly less following

conditioning than recovery (g < 0.05), whereas subjects in Group SW displayed

no significant differences in consumption between conditioning and recovery (g =

0.305), indicating maintenance of discriminative control in this phase. A repeated

measures ANOVA revealed that there were significant main effects o f Group

(F(i,io)s 48.26, g < 0.05) and Dose (F( 4 .40)s 11.12, g < 0.05) and a significant

Group X Dose interaction (F(4>40)s 9.149; g < 0.05). For subjects in Group SL,

there was an inverse relationship between the dose of SNC80 and the amount of

saccharin consumed (i.e., as the dose of SNC80 increased, the amount of

saccharin consumed decreased). Consumption at 0.56,1 and 1.8 mg/kg was

significantly greater than consumption during conditioning (all g’s < 0.05). There

were no significant differences in consumption between 3.2 and 5.6 mg/kg and

conditioning (all g’s > 0.350). For subjects in Group SW, there were no consistent

changes in saccharin consumption over the increasing doses of SNC80 (all g's >

0.415). Further, consumption at these doses approximated consumption during

conditioning. At 1,1.8, 3.2 and 5.6 mg/kg, subjects in Group SL drank

significantly less than subjects in Group SW (all g's < 0.05).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 44

Figure 8

4 SL 20i O SW

* * * 15-

10 -

0.56 1.8 3.2 5.6

SNC80 Dose (mg/kg)

Figure 8. Mean amount (± S.E.M) o f saccharin consumption for subjects in Groups SL and SW during recovery (R) and conditioning (C) in this phase and following various doses of SNC80 (0.56 - 5.6 mg/kg).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 45

Figure 9 presents the mean amount (± SEM) of saccharin consumption for

Groups SL and SW during conditioning (C) and recovery (R) and following

various closes o f SNC162. During these probes, subjects in Group SL drank

significantly less following conditioning than recovery (p < 0.05), whereas

subjects in Group SW displayed no significant differences in consumption

between conditioning and recovery (p = 0.067). There were significant main

effects of Group (F fljp 5.696; p < 0.05) and Dose ( F ^ r 9.75; p < 0.05) and a

significant Group X Dose interaction (F(5,40)s 2.911; p < 0.05). As illustrated, for

subjects in Groups SL and SW there was an inverse relationship between the

dose of SNC162 and the amount of saccharin consumed. For subjects in Group

SL, consumption at 1.8,3.2, 5.6 and 10 mg/kg was significantly greater than

consumption during conditioning (ail p’s < 0.05). There were no significant

differences in consumption between 18 mg/kg and conditioning (p = 0.194).

Consumption at 32 mg/kg was significant greater than consumption at

conditioning (p < 0.05). For subjects in Group SW, consumption at 32 mg/kg was

significantly less than consumption during conditioning. There were no significant

differences in consumption between conditioning and the other five lower doses

(all p’s > 0.066). At 18 mg/kg, subjects in Group SL drank significantly less than

subjects in Group SW (p < 0.05). There were no significant differences in

consumption between Groups SL and SW at the remaining doses (all p’s >

0.072).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 46

Figure 9

▲ SL 20i o SW

15-

IB *** 10 a u

~ i ------1------1 r ■ i ------1------1------1— R C 1.8 3.2 5.6 10 18 32

SNC162 Dose (mg/kg)

Figure 9. Mean amount (± S.E.M) of saccharin consumption for subjects in Groups SL and SW during recovery (R) and conditioning (C) in this phase and following various doses of SNC162 (1.8 - 32 mg/kg).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 47

Figure 10 presents the mean amount (± SEM) of saccharin consumption

for subjects in Groups SL and SW during conditioning (C) and recovery (R) and

following various doses of morphine. During these probes, subjects in Group SL

drank significantly less following conditioning than recovery (g < 0.05), whereas

subjects in Group SW displayed no significant differences in consumption

between conditioning and recovery (g = 0.509). A repeated measures ANOVA

revealed a significant main effect of Dose (Fp aop 9.989; g < 0.05) but not of

Group (F(i,i0)= 0.487; g = 0.501) and no significant Group X Dose interaction

(F(3,3oj= 2.812; g = 0.056). For subjects in Group SL, there were no consistent

changes in saccharin consumption over the increasing doses of morphine.

Consumption at all doses was significantly greater than consumption during

conditioning (all p's < 0.05). For subjects in Group SW, there was an inverse

relationship between the dose of morphine and the amount of saccharin

consumed. Consumption at 10 mg/kg was significantly less than consumption

during conditioning (g < 0.05). There were no significant differences between

Groups SL and SW at any dose of morphine tested (all g’s > 0.094).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 48

Figure 10

A SL 20- 0 SW

15- f

10 -

ea U

***

— i— — i— R C 1.8 3.2 5.6 10

Morphine Dose (mg/kg)

Figure 10. Mean amount (± S.E.M) of saccharin consumption for subjects in Groups SL and SW during recovery (R) and conditioning (C) in this phase and following various doses of morphine (1.8-10 mg/kg).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 49

Phase III: SNC80 Time course

Figure 11 presents the mean amount (± SEM) of saccharin consumption

for Groups SL and SW following 5.6 mg/kg SNC80 given 20 (baseline interval),

60,120 and 240 min prior to saccharin access. As illustrated, there were

significant main effects of Group (F(1,8)= 65.979; g < 0.05) and Time (F p ^ r

13.332; g < 0.05) and a significant Group X Time interaction (F(3>2 4)= 6.627; g £

0.05). For Group SL, there was a direct relationship between the preexposure

interval of the training dose of SNC80 and the amount of saccharin consumed

(i.e., as the preexposure interval increased, the amount of saccharin consumed

increased). For subjects in Group SL, consumption at 20 and 60 min was

significantly less than consumption at 240 min (all g’s < 0.05). For subjects in

Group SW, there were no consistent changes in saccharin consumption over the

increasing preexposure intervals. Subjects in Group SL drank significantly less

saccharin than subjects in Group SW at 20,60 and 120 min (all g's < 0.05). At

240 min, Groups SL and SW did not differ in saccharin consumption (g = 0.744),

indicating loss of discriminative control at this preexposure interval.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 50

Figure 11

2 0 -i

® 15-

B 10 - «a a £ ** 5 - **

1 1------1— 20 m in 60 min 120 min 240 min

lim e (m in )

Figure 11. Mean amount (± S.E.M) of saccharin consumption for Groups SL and SW following 5.6 mg/kg SNC80 given 20,60,120 and 240 min prior to saccharin access.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 51

Phase IV: Antagonism

Figure 12 presents the mean amount (± SEM) o f saccharin consumption

for subjects in Groups SL and SW during conditioning (C) and recovery (R), as

well as following the combination of various doses of naltrindole (1-5.6 mg/kg)

administered 40 min prior to the training dose of 5.6 mg/kg SNC80 (a pilot study

in our laboratory demonstrated that antagonism of SNC80 by naltrindole was

greatest at 40 min preexposure; administration at 10 min was ineffective while

pretreatment at 20 min produced partial antagonism - data not shown). During

this phase, subjects in Group SL drank significantly less following conditioning

than recovery 02 ^ 0.05), whereas subjects in Group SW displayed no significant

differences in consumption between conditioning and recovery (g = 0.397). A

repeated measures ANOVA revealed significant main effects of Group (F(1|8p

19.167; g < 0.05) and Dose (F(3^ 4)= 20.887; g < 0.05) and a significant Group X

Dose interaction (Fp^p 22.562; g < 0.05) when naltrindole was injected prior to

SNC80. For subjects in Group SL, consumption following the combination of 1

mg/kg naltrindole and SNC80 was not significantly different (g = 0.084) than

consumption following SNC80 alone (C) but was significantly different (g < 0.05)

from consumption during recovery (R). For these same subjects, consumption

following the combination of 1.8, 3.2 and 5.6 mg/kg naltrindole and SNC80 was

significantly greater than consumption following SNC80 alone and the

combination of 1 mg/kg naltrindole and SNC80 (all g’s < 0.05). For subjects in

Group SW, there were no consistent changes in saccharin consumption over the

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 52

increasing doses of naltrindole (all g’s > 0.083). Subjects in Group SL drank

significantly less than subjects in Group SW at 1 mg/kg (g < 0.05). There were no

significant differences in consumption between Groups SL and SW at the three

remaining doses (1.8, 3.2 and 5.6 mg/kg), indicative of complete antagonism of

SNC80’s discriminative effects at these three doses (all g's > 0.167).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 53

Figure 12 ▲ SL o SW

2 0 -i

I 15- I 8

1 0 - ** **

I o-J ~r T —r~ “T" ~ T “ R C 1.8 3.2 5.6

Naltrindole Dose (mg/kg)

Figure 12. Mean amount (± S.E.M) of saccharin consumption for subjects in Groups SL and SW during recovery (R) and conditioning (C) in this phase and following naltrindole, SNC80 alone as well as the naltrindole/SNC80 combination.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 54

Figure 13 presents the mean amount (± SEM) of saccharin consumption

for subjects in Groups SL and SW during conditioning (C) and recovery (R) in

this phase, as well as following the combination of various doses of naltrexone

(0.18-1.8 mg/kg) administered 10 min prior to the training dose of SNC80 (5.6

mg/kg) (see Walker et al., 1994). As above, subjects in Group SL drank

significantly less following conditioning than recovery (g < 0.05), whereas

subjects in Group SW displayed no significant differences in consumption

between conditioning and recovery (g = 0.499). There were significant main

effects of Group (F(i,sr 246.117; g < 0.05) and Dose (F(4,32)= 4.095; g = 0.05)

and a significant Group X Dose interaction ( F ^ p 6.436; g < 0.05) when

naltrexone was injected prior to SNC80. For subjects in Group SL, consumption

following SNC80 alone (C) and in combination with all doses of naltrexone did

not differ (all g’s > 0.095). Consumption following recovery (R) was significantly

greater than that following all doses of naltrexone in combination with SNC80 (all

g’s £ 0.05). For subjects in Group SW, consumption following SNC80 alone was

significantly greater than consumption following the combination of all doses of

naltrexone and SNC80 (all g’s < 0.05). Consumption at 1.8 mg/kg naltrexone was

significantly less than consumption at the other lower doses (all g’s < 0.05),

indicating the unconditioned suppressive effects of naltrexone on fluid

consumption. Subjects in Group SL drank significantly less than subjects in

Group SW following all combinations of naltrexone and SNC80 (all g’s < 0.05).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 55

Fig u re 13 A SL o SW

is-

£ 10- 5 £ E «aa

i 5 -

T 0.18 0J2 0^6 1.0 1 J

Naltrexone Dose (mg/kg)

Figure 13. Mean amount (± S.E.M) of saccharin consumption for subjects in Groups SL and SW during recovery (R) and conditioning (C) in this phase and following naltrexone, SNC80 alone as well as the naltrexone/SNC80 combination.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 8

DISCUSSION

EXPERIMENT 2

The failure of morphine to generalize to the delta agonist SNC80 (or to be

antagonized by the delta antagonist naltrindole) in Experiment 1 suggests that

morphine’s stimulus control is based on its activity at the mu (but not the delta)

receptor subtype. Although morphine binds to (and has agonist activity at) the

delta receptor, the failure to observe any evidence of delta stimulus control may

be due to the general inability of delta activity to support drug discrimination

learning. Experiment 2 tested the latter possibility, namely, whether or not

activity at the delta receptor would support DDL on its own.

As noted, prior assessments of delta stimulus control have not only been

limited to nonselective compounds and centrally administered peptides (see

Introduction, Experiment 2) but also to the monkey (Brandt et al., 1999; Negus et

al., 1998; Negus et al., 1994) and pigeon (Comer et al., 1993; Jewett et al., 1996;

Negus et al., 1996; Picker and Cook, 1998). No assessment of delta stimulus

control has been examined in rodents. To that end, the present experiment

examined discriminative control by the selective, systemically-administered delta

agonist SNC80 in rats and its generalization to and antagonism by compounds

relatively selective for the delta and mu receptor subtypes. As described, animals

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acquired the discrimination between 5.6 mg/kg SNC80 and distilled water within

approximately seven conditioning cycles (the same number of cycles required to

produce discrimination between morphine and vehicle in Experiment 1), avoiding

saccharin consumption when it was preceded by an injection of SNC80 and

consuming the same saccharin solution when it was preceded by vehicle. The

discriminative effects of SNC80 were maximal at 20 min, partial at 120 min and

lost at 240 min. The discrimination was dose dependent in that as the dose of

SNC80 increased, the amount o f saccharin consumed decreased. In subsequent

generalization tests, the delta agonist SNC162 produced SNC80-appropriate

responding at a dose of 18 mg/kg. Although generalization of SNC80 stimulus

control was evident at 18 mg/kg of SNC162, mean consumption at this dose was

higher than the mean consumption following the training dose of SNC80, i.e., 10

mg/kg, an effect consistent with at least one other report demonstrating that the

effects of SNC162 are sometimes smaller and more variable than those of

SNC80 (Negus et al., 1998). Assessments with higher doses o f SNC162 could

result in full generalization of SNC80’s stimulus effects; however, as described,

with higher doses there was marked unconditioned suppression of consumption

in control animals (see Experiment 1 for similar dose-response suppression with

methadone). Thus, it is unknown if complete generalization could be produced

with SNC162 (although see Brandt et al., 1999 for complete generalization in

monkeys). While SNC162 partially substituted for SNC80, the mu agonist

morphine produced vehicle-appropriate responding at all doses tested. Although

these selective generalization patterns with SNC162 and morphine suggest that

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 58

the discriminative effects of SNC80 were mediated at the delta, but not the mu,

receptor, assessments with other delta and mu agonists within this preparation

are needed before general conclusions can be made about the mediation of

SNC80’s stimulus effects. As noted, SNC80’s discriminative control was

completely blocked by the delta-selective antagonist naltrindole, but not by the

mu-selective antagonist naltrexone, again suggestive of delta mediation of

SNC80’s stimulus effects. However, it is possible that had higher doses of

naltrexone been given, antagonism of SNC80’s discriminative effects might have

been produced. Although possible, the dose range examined is highly effective in

antagonizing mu stimulus control (Locke and Holtzman, 1986). Further, with the

highest dose assessed (i.e., 1.8 mg/kg), generalized behavioral suppression was

evident in the control subjects, precluding interpretation of any possible

antagonism of SNC80 discriminative control. The dose range examined for

naltrindole was also within the range of doses effective in antagonizing delta-

mediated effects (see Spina et al., 1998). As described, in the present

experiment antagonism was evident at a dose as low as 1.8 mg/kg.

Thus, in summary, rats can discriminate compounds selective for the delta

opioid receptor, which suggests that the inability of morphine stimulus control to

generalize to SNC80 (Experiment 1) is not due to the failure of delta compounds

to support discrimination learning.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 9

GENERAL DISCUSSION

Experiment 1 demonstrated that morphine discriminative control is

mediated at the mu, but not delta, opioid receptor. Specifically, although

morphine and methadone produced dose-dependent substitution for morphine,

there was no evidence of substitution for morphine by SNC80 at any dose tested.

Further, although naloxone completely blocked the discriminative effects of

morphine, naltrindole did not significantly affect the morphine stimulus. Although

data on the ability of systemically active delta agonists to substitute for morphine

stimulus control are relatively scarce (see Introduction), the present findings are

consistent with previous work reporting that [o-Pen2L-Pen5]enkephalin (DPLPE)

fails to substitute for morphine in a discrete-trial avoidance procedure, while the

mu selective compounds (DAMGO and FK33824) produce morphine-appropriate

responding (Ukai & Holtzman, 1988). These findings are also similar to those in a

recently published report with monkeys (Brandt et al., 1999) demonstrating

failure of the discriminative effects of morphine to generalize to SNC80, a

similarity that is consistent with the position that rats and monkeys share similar

sensitivities for the discriminative effects of opiates (Woods, Bertalmio, Young,

Essman & Winger, 1988). That naloxone, but not naltrindole, completely blocked

the discriminative effects of morphine is in agreement with several other reports

(Brandt et al., 1999; Colpaert, 1978; Jewett et al., 1996; Shannon & Holtzman,

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Misawa, & Nagse 1993; 1976a; Shannon & Holtzman, 1976b; Stevenson et al.,

1992; Suzuki, Kishimoto, Ozaki, & Narita, 2001) demonstrating antagonism of

mu-mediated effects by naloxone and the failure o f such antagonism by

naltrindole (but see Bilsky et al., 1995; Narita et al., 1993; Suzuki, Yoxhiike,

Mizoguchi, Hamei, Misawa, Nagase, 1994).

As noted, the failure to see any delta mediation of morphine s stimulus

effects in Experiment 1 may be due to a general inability of delta activity to

support drug discrimination learning. Experiment 2 tested this possibility, namely,

the ability of the delta receptor to support discrimination learning. Although

previous reports assessing delta mediation of morphine stimulus control

assessed the ability of either nonselective compounds (e.g., BW373U86) or

centrally-administered peptides to substitute for morphine in monkeys or pigeons

(see above), the present study assessed delta mediation of morphine stimulus

control with the systemically-administered delta selective agonist SNC80 in rats.

As described, following acquisition of discriminative control, subsequent

generalization tests established that both SNC80 and the delta agonist SNC162,

but not morphine, produced SNC80-appropriate responding. These selective

generalization patterns and the finding that the discriminative effects of SNC80

were completely blocked by the delta-selective antagonist naltrindole, but not the

mu-selective antagonist, naltrexone, strongly suggest that the SNC80

discrimination was delta mediated. That SNC80 serves a discriminative function

is consistent with previous work demonstrating delta agonist stimulus control

(Brandt et al., 1999; Jewett et al., 1996). For example, Jewett et al. (1996)

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demonstrated that pigeons trained to discriminate 100 jig of DPDPE from saline

generalized this control to a variety of delta agonists, e.g., DSLET and

deltorphine II, but not to the mu agonists morphine and DAMGO. More relevant

to the present findings, Brandt et al. (1999) demonstrated that monkeys trained

to discriminate 0.32 mg/kg of the systemically active SNC80 from saline

generalized SNC80 control to the delta agonists SNC80, SNC162, SNC86, and

SNC243A, but not the the mu agonists morphine and fentanyt. That the delta

antagonist naltrindole, but not the mu antagonist naltrexone, completely blocked

SNC80’s stimlulus effects in rats is in agreement with other reports

demonstrating delta mediation of the discriminative effects of SNC80, DPDPE

and BW373U86 (Brandt et al., 1999; Comer et al., 1993; Jewett et al., 1996).

The results of Experiment 2 suggest that the failure of SNC80 to substitute

for morphine in Experiment 1 is not due to the general inability of delta

compounds to support DDL. These results with compounds selective for the delta

receptor are similar to reports assessing the ability of kappa compounds to

substitute for morphine control. Specifically, although activity at the kappa

receptor is capable of supporting DDL on its own (Bertalmio and Woods, 1987;

Picker et al., 1990; Negus et al., 1996; 1998), activity at this receptor subtype

does not mediate morphine stimulus control (see Introduction to Experiment 1).

Thus, although morphine binds to all three receptor subtypes (mu, delta and

kappa), the present study and others (Bertalmio and Woods, 1987; France et al.,

1984; Gianutsos and Lai, 1976; Grabus et al., 1999; Heriing et al., 1984; Hill et

al., 1971; Jarbe, 1978; Locke and Holtzman, 1986; Morgan and Picker, 1998;

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 62

Negus e ta l., 1996; Negus etal., 1990; Picker e ta l., 1996; Picker, 1994; Picker et

al., 1990; Picker and Dystra, 1989; Picker and Dykstra, 1987; Poumaghash and

Riley, 1993; Schaefer and Holtzman, 1977; Schaefer and Holtzman, 1978;

Shannon and Holtzman, 1977a; Shannon and Holtzman, 1977b; Shannon and

Holtzman, 1976; Smurthwaite and Riley, 1994; Stevenson e tal., 1992; Suzuki et

al., 1995; Ukai and Holtzman, 1988; Winter, 1975) suggest that only the mu

receptor is responsible for producing the discriminative effects of this compound.

Given that morphine binds to mu, delta and kappa receptors, the issue

arises as to why delta and kappa receptors do not contribute to morphine's

stimulus effects. One possible explanation may be perceptual masking (Gauvin,

Peirce & Holloway, 1994; Gauvin & Young, 1989; Overton, 1983). Masking refers

to a situation in which one stimulus “masks” or reduces the perception of a

concurrently-administered second stimulus (Zwislocki, 1978). Although

perceptual masking was first demonstrated with exteroceptive stimuli such as

tones and visual cues, Gauvin and colleagues (1989,1993) were among the first

to demonstrate this phenomenon with drug stimuli. For example, Gauvin and

Young (1989) reported that the stimulus effects of morphine were blocked by the

opioid antagonist naltrexone and the psychostimulant amphetamine.

Amphetamine blocked the perceptual response measure only (i.e., the

percentage of morphine-appropriate responding), whereas, naltrexone produced

right-ward shifts in both the discriminative and rate-decreasing functions of

morphine - an expected outcome given that pharmacological antagonists

typically block all behavioral response measures of the agonist. Thus, it appears

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 63

that amphetamine perceptually masked (as opposed to antagonized) the

discriminative effects of morphine. Perceptual masking may also explain the data

in the present experiment. For example, given that morphine binds to mu, delta

and kappa receptors, morphine may produce subjective effects at all three

receptor subtypes. However, morphine’s effects at the mu receptor may be

sufficiently salient to mask or block its effects at the delta and kappa receptors.

To test this, it might be useful to functionally inactivate the mu receptor thereby

terminating any potential masking properties of morphine at this receptor

subtype. Functional inactivation of this receptor could be achieved by any of the

following methods: 1) use of a non-competitive antagonist or alkylating agent

(e.g., PFNA, see Adams, Paronis & Holtzman, 1990), 2) use of antisense (which

would inactivate the mRNA transcript for the mu receptor subtype, see McMahon

et al., 2001), 3) knocking out the MOR gene itself (e.g., using a MOR-knockout

mouse, see Matthes, Maldonado, Simonen, Valverde, Slowe, Kitch, Dierich,

LeMeur, Dolle, Tzauara, Hanoune, Roques, & Kieffer, 1996) or 4) inducing

tolerance to the discriminative effects of mu activity with a highly selective mu

agonist such as etorphine or sufentanil (Easterling & Holtzman, 1999). These

techniques have been used to evaluate the receptor mediation of morphine-

induced antinociception (McMahon et al., 2001), drug discrimination (Riley &

Poumaghash, 1995), locomotion (Sora, Elmer, Funada, Pieper, Li, Hall & Uhl,

2001), reward (Becker, Grecksch, Brodemann, Kraus, Peters, Schroeder,

Thiemann, Loh, & Holft, 2000), immunosuppression (Lysle et al., 1993),

gastrointestinal transit (Pol, Valle, Sanchez-Blazquez, Garzon, & Puig, 1999) and

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 64

respiratory depression (Holaday et al., 1983). In order to demonstrate that activity

at the mu receptor is perceptually masking the subjective effects produced at the

delta and kappa receptors, it would be necessary to show that functional

inactivation of the mu receptor subtype (by any of the above proposed methods)

results in delta and kappa mediation of the discriminative effects of morphine.

Another method to test for perceptual masking might be to assess delta and

kappa substitution in animals trained on lower doses of morphine (if the notion

that masking strength at mu decreases with decreasing doses of morphine is

accepted). However, the subjective effects of delta and kappa activity may also

decrease with decreasing doses of morphine, thereby, precluding any empirical

assessment of masking with this method. Although perceptual masking may be

one possible explanation for why the delta or kappa receptors do not mediate

morphine stimulus control, one should be cautious when extrapolating from the

Gauvin and Young report to the present experiment, since procedural differences

exist between the two studies. Specifically, Gauvin and Young (1989, see also

Gauvin, Pierce & Holloway, 1994) used drug combinations (morphine and

amphetamine), whereas in the present study, one drug with multiple effects (mu,

delta and kappa activity) was assessed.

A more parsimonious explanation for the present findings may be that of

intrinsic activity (Colpaert, 1988). Colpaert’s theory on intrinsic activity adheres to

basic principles of molecular pharmacology. One such principle is that the

magnitude of pharmacological activity generated by a drug is due to the dose or

concentration of the drug as well as the intrinsic activity of that compound

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 65

(Kenakin, 1997). The intrinsic adtivity of a drug imparts the biological signal at

the drug receptor, which ultimately results in a biological response. Intrinsic

activity is a property of the drug and not the tissue, is graded in nature and is an

important factor in the general characterization of drugs (Kenakin, 1997).

Colpaert (1988) incorporated these principles of molecular pharmacology to

explain differential patterns of responding in animals to the same drug across

different tasks. Specifically, different response systems (e.g., analgesia,

conditioned place preference, DDL, self-administration) require different levels of

intrinsic activity by a given drug at a given receptor. For example, the level of

intrinsic activity required for morphine to produce a behavioral response may be

self-administration > CPP > analgesia > DDL. Thus, a given dose of morphine

may have sufficient intrinsic activity to produce DDL or analgesia, but not enough

for place conditioning or self-administration. Although Colpaert applied the

principle of intrinsic activity to drugs acting at a single receptor, it is possible to

extend this analysis to drugs acting at multiple receptors. For example, let us

assume that morphine generates different intrinsic activities at the mu, delta and

kappa receptors such that the intrinsic activity at mu is greater than the intrinsic

activity at either delta or kappa. Figure 14 depicts a set of hypothetical intrinsic

activities generated at each receptor subtype by the training dose of 10 mg/kg

morphine across time in the DDL procedure. As shown in the figure, at this dose

of morphine the intrinsic activity at the mu receptor is well above the threshold of

discriminability (i.e., the point at which subjective effects are discriminable,

denoted by the dotted line), whereas the intrinsic acitvity at delta and kappa

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receptors is below this threshold (i.e., below the clotted line). Therefore, although

morphine may produce subjective effects at all three receptor subtypes, only the

subjective effects produced at the mu receptor are discriminable. More

specifically, at 10 mg/kg, morphine’s intrinsic activity at the mu receptor is

sufficient for generating a discriminative cue, whereas activity at delta or kappa

receptors is not sufficient to mediate any portion of the morphine discriminative

stimulus. However, it is not the case that delta activity cannot produce

discriminative effects. Thus, a possible explanation for the failure of SNC80 to

substitute for morphine may be that delta stimulus control was never conditioned

or trained in Experiment 1.

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Morphine Training Intrinsic Activity

Threshold/or discriminative effects

Time

Figure 14. Hypothetical intrinsic activities generated at the mu, delta and kappa receptor subtypes by 10 mg/kg morphine across time in the DDL procedure.

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Figure 15 illustrates that activity at the delta receptor can support

discrimination learning when animals are trained with 5.6 mg/kg SNC80

(Experiment 2) and depicts a set of hypothetical intrinsic activities generated at

each receptor subtype by SNC80 across time, also in a DDL task. As shown in

the figure, at this dose of SNC80 the intrinsic activity at the delta receptor is well

above the threshold of discriminabiltiy (i.e., the point at which subjective effects

are discriminable, denoted by the dotted line), whereas the intrinsic acitvity at mu

and kappa receptors is below the threshold of discriminabiltiy (below the dotted

line). Therefore, although SNC80 may produce subjective effects at all three

receptor subtypes (although to a lesser degree than morphine, given that SNC80

is more selective for delta receptors than morphine is for mu receptors), only the

subjective effects produced at the delta receptor are discriminable. More

specifically, at 5.6 mg/kg, SNC80’s intrinsic activity at the delta receptor is

sufficient for generating a discriminative cue, whereas activity at mu or kappa

receptors is not sufficient to mediate any portion of the SNC80 discriminative

stimulus. As above, a possible explanation for the inability of morphine to

substitute for SNC80 in Experiment 2 may be that activity at mu was never

conditioned or trained.

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SNC80 Training Intrinsic Activity

Threshold for discriminative effects

Time

Figure 15. Hypothetical intrinsic activities generated at the mu, delta and kappa receptor subtypes by 5.6 mg/kg SNC80 across time in the DDL procedure.

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The above conceptual suggestions for why the mu, but not the delta or

kappa, receptors mediate morphine’s stimulus effects are strongly theoretical in

nature. It is dear that although the mu receptor mediates the discriminative

effects of morphine, the reason for the lack of mediation by delta or kappa

receptors remains unknown. Continued research will be necessary to eluddate

further this mechanism of drug discrimination to morphine.

in summary, the present experiment demonstrates that the discriminative

effects of morphine are mediated at the mu, but not the delta, receptor. Further,

rats can discriminate compounds selective for the delta receptor, thereby,

suggesting that the failure of morphine stimulus control to generalize to the delta

selective agonist SNC80 is not due to the failure of delta compounds to support

discrimination learning, but rather to the fact that morphine’s discriminative

stimulus effects are not delta mediated. Finally, it is possible that perceptual

masking and/or relative intrinsic activity explains the inability o f delta and kappa

receptors to mediate morphine stimulus control.

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