THE ABILITY OF PROGLUMIDE TO MODULATE CHANGES IN MORPHINE’S
AVERS IVE PROPERTIES DURING CHRONIC MORPHINE EXPOSURE
by
Meredith A. Fox
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: -- f t Anthony L. Riley
Laura
Dean df College of Arts and Sciences Scott Parker
C.j s 'ha, Date ^ Cora Lee Wetherington^
2003
American University
Washington, DC 20016
AMERICAN UNIVERSITY LIBRARY
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. THE ABILITY OF PROGLUMIDE TO MODULATE CHANGES IN MORPINE’S
AVERSIVE PROPERTIES DURING CHRONIC MORPHINE EXPOSURE
by
Meredith A. Fox
ABSTRACT
Just as chronic exposure to morphine alters its antinociceptive properties, chronic
exposure to morphine has been shown to alter the strength of its aversive effects. As
cholecystokinin (CCK) antagonists have been shown to prevent or reverse tolerance to
morphine’s analgesic effects, such compounds could potentially modulate morphine’s
aversive properties. The current experiment assessed the ability of the CCK antagonist
proglumide to block the development of tolerance to the aversive properties of morphine
in rats utilizing a conditioned taste aversion (CTA) design. Specifically, animals were
preexposed to vehicle, morphine (5 mg/kg), proglumide (5 mg/kg) or a combination of
proglumide administered either immediately or 15 min before morphine. On subsequent
conditioning days, saccharin was presented followed immediately by administration of
either morphine (10 mg/kg) or vehicle. Over conditioning, control animals increased
saccharin consumption, regardless of preexposure condition. Animals preexposed to
vehicle or proglumide and injected with morphine during conditioning acquired a
morphine-induced saccharin aversion. Animals preexposed to morphine and conditioned
with morphine acquired an attenuated morphine-induced saccharin aversion. Animals ii
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. preexposed to the combination of proglumide and morphine and injected with morphine
during conditioning also acquired an attenuated morphine-induced saccharin aversion.
Importantly, saccharin consumption in animals preexposed to either proglumide-
morphine combination was not different from animals preexposed to morphine,
indicating that proglumide had no effect on the development of tolerance to the aversive
properties of morphine in a CTA design. In order to assess whether the inability of
proglumide to block the development of tolerance was due to the specific parameters of
Experiment 1, Experiment 2 examined the effects of 5 mg/kg proglumide administered
either immediately, 5 or 15 min before CCK (3 or 10 ptg/kg) on CCK-induced
suppression of feeding. When proglumide was administered immediately before 3 pg/kg
CCK, it attenuated the CCK-induced suppression of feeding. The findings from the
current study suggest that CCK may not be involved in the aversive properties of
morphine.
iii
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS
ABSTRACT...... ii
LIST OF ILLUSTRATIONS...... vi
Chapter
1. INTRODUCTION...... 1
2. PROCEDURE: EXPERIM ENT!...... 12
Subjects
Drugs and Solutions
Phase 1: Habituation
Phase 2: Preexposure
Phase 3: Conditioning
Test for Saccharin Aversion
Statistical Analysis
3. RESULTS: EXPERIMENT 1...... 16
Preexposure
Conditioning
4. DISCUSSION: EXPERIMENT 1...... 22
5. PROCEDURE: EXPERIMENT 2 ...... 27
Subjects
iv
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Drugs
Phase 1: Habituation
Phase 2: Drug Effects on Feeding
Statistical Analysis
6. RESULTS: EXPERIMENT 2 ...... 29
7. DISCUSSION...... 33
BIBLIOGRAPHY...... 46
v
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF ILLUSTRATIONS
1. Mean (± S.E.M.) consumption of water over repeated preexposure days for subjects in Preexposure Groups PM, PiM, PV, VM and VV. Water was available 5 h before injections...... 16
2. Mean (± S.E.M.) consumption of saccharin over repeated conditioning trials for control subjects and subjects in Groups VM-M and VV-M. * Significantly different from baseline...... 18
3. Mean (± S.E.M.) consumption of saccharin over repeated conditioning trials for control subjects and subjects in Groups PM-M and PV-M. * Significantly different from baseline...... 19
4. Mean (± S.E.M.) consumption of saccharin over repeated conditioning trials for control subjects and subjects in Groups PiM-M and PV-M. * Significantly different from baseline...... 20
5. Mean (± S.E.M.) consumption of food for food-deprived subjects over repeated 60-min experimental sessions following saline, 10 pg/kg CCK, 3 pg/kg CCK or 5 mg/kg proglumide. * Significantly different from baseline (saline)...... 29
6. Mean (± S.E.M.) consumption of food for food-deprived subjects over repeated 60-min experimental sessions following saline, 10 pg/kg CCK or 5 mg/kg proglumide (P) followed either 15 or 5 min later by 10 pg/kg CCK. * Significantly different from baseline (saline)...... 30
7. Mean (± S.E.M.) consumption of food for food-deprived subjects over repeated 60-min experimental sessions following saline, 3 pg/kg CCK or 5 mg/kg proglumide (P) followed either 15 min later or immediately by 3 pg/kg CCK. * Significantly different from baseline (saline). **Significantly different from 3 pg/kg CCK alone. ***Significantly different from proglumide followed 15 min later by 3 pg/kg CCK...... 32
vi
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 1
INTRODUCTION
Cholecystokinin (CCK) is a gastrointestinal hormone and one of the most widely
distributed neuropeptides in the brain (Fink, Rex, Voits & Voigt, 1998), where it acts as a
neurotransmitter (Mutt, 1994). CCK has been shown to be involved in many biological
processes, including the reduction of food intake, the induction of anxiety-related
behavior, and may be involved in learning and memory processes (for a review, see
Crawley & Corwin, 1994; de Tullio, Delarge & Pirotte, 2000; Fink et al., 1998;
Lindefors, Linden, Brene, Sedvall & Persson, 1993). CCK has also been shown to be
involved in pain and antinociception/analgesia (for a review, see Wiesenfeld-Hallin &
Xu, 1996), where it acts as an anti-opioid peptide (Wiesenfeld-Hallin, de Arauja, Alster,
Xu & Hokfelt, 1999).
In relation to its involvement in antinociception, the exogenous administration of
CCK produces a physiological antagonism of morphine-induced antinociception (Faris,
Komisaruk, Watkins & Mayer, 1983). For example, Faris et al. (1983) reported that rats
injected with CCK 20 min prior to morphine displayed significantly attenuated morphine-
induced analgesia, as assessed on the tail-flick test. CCK has also been found to reduce
analgesia induced by endogenous opiates. Specifically, CCK administered 30 min before
the delivery of foot shock to the front paws, which results in opiate-induced analgesia,
reduced foot-shock induced analgesia (Faris et al., 1983). Nonopiate hind paw foot- 1
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shock analgesia was not reduced by CCK, suggesting that CCK specifically inhibits
opiate analgesia (Faris et al., 1983). Thus, on an acute basis, CCK attenuates analgesia
induced by both morphine and endogenous opiates, suggesting that CCK, as an
endogenous opiate antagonist (Faris et al., 1983), may be released in response to opiate
administration, returning the organism to its basal level of pain sensitivity (Watkins,
Kinscheck & Mayer, 1985).
Chronic morphine administration results in the development of tolerance to its
antinociceptive effects (Zhou, Sun, Zhang & Han, 1992), and CCK is involved in this
development. For example, Dourish et al. (1990) induced tolerance to morphine
analgesia by a 6-day schedule of twice daily injections of increasing doses of morphine.
Animals were then challenged with morphine, and analgesia was assessed with the tail-
flick test. Animals treated chronically with morphine became tolerant and exhibited
only a small analgesic response when challenged with morphine (Dourish et al., 1990).
CCK antiserum has been reported to reverse this tolerance to morphine analgesia by
approximately 50% (Ding, Fan, Zhou & Han, 1986).
It has been suggested that chronic opiate administration might induce a
compensatory increase in the synthesis or release of CCK, which could result in a
progressive antagonism of opiate analgesia (Watkins, Kinscheck & Mayer, 1984). For
example, Zhou et al. (1992) found a marked increase in preproCCK mRNA in the
brains of rats receiving chronic morphine treatment for 1, 3 and 6 days, with morphine-
treated rats showing an increase of 52%, 62% and 92%, respectively, over control
levels. This increase paralleled the time course of the development of morphine
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3
tolerance. These findings suggest that an acceleration of CCK gene expression during
chronic morphine treatment might comprise one of the mechanisms for morphine
tolerance, i.e., the CCK system might be activated during chronic morphine treatment,
producing a negative feedback mechanism that leads to the development of tolerance to
opioids (Zhou et al., 1992).
Although much of the research described has focused on CCK and its effect on
behavior, other research in this area has focused on the mechanism of CCK’s effects. In
so doing, two subtypes of CCK receptors have been revealed, specifically the CCKA
receptor, located in both peripheral tissues and in some brain areas, and the CCKB
receptor, located throughout the central nervous system (for a review, see de Tullio et
al., 2000). While mediating the effects of CCK, these receptors also have affinity for a
variety of compounds with no intrinsic activity, but which block the effects of CCK (for
a review, see Lindefors et al., 1993). These CCK antagonists differ in their relative
affinity for each of the receptor subtypes (for a review, see Woodruff & Hughes,
1991; Dunlop, 1998). There are nonspecific CCK antagonists, including proglumide,
lorglumide and benzotript, which have affinity for both CCKA and CCKB receptors,
CCKA-selective antagonists including L-365,071, PD 140548 and devazepide (also
known as L-364,718 and MK-329) and CCKB-selective antagonists including CI-988
and L-365,260. CCK antagonists have been shown to block CCK-induced
suppression of feeding as well as the anxiogenic effects of CCK (for a review, see
Woodruff & Hughes, 1991). As mentioned above, administration of exogenous CCK
produces a physiological antagonism of morphine-induced antinociception (Faris et
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al., 1983), and this antagonism of morphine's antinociceptive effects is blocked by the
nonselective CCK antagonist proglumide (Tang, Chou, Iadarola, Yang & Costa, 1984).
In addition to their ability to block various effects of CCK, CCK antagonists
have been shown to induce effects on their own. For example, on an acute basis,
CCK antagonists potentiate morphine's analgesic effects on a variety of analgesia
assays (Dourish, Hawley & Iverson, 1988; Dourish et al., 1990; Kellstein & Mayer,
1991; Ossipov, Kovelowski, Vanderah & Porreca, 1994; Singh et al., 1996; Tang et
al., 1984; Watkins, Kinscheck & Mayer, 1985). This effect has been shown with
nonspecific CCK antagonists such as proglumide (Kellstein & Mayer, 1990; Tang et
al., 1984; Watkins, Kinscheck & Mayer, 1994; 1995), as well as the CCKA-selective
antagonists L-365,718 (Dourish et al., 1988) and L-365,031 (Dourish et al, 1990) and
the CCKB-selective antagonists L-365,260 (Dourish et al., 1990; Ossipov et al., 1994)
and CI-988 (Singh et al., 1996). However, it has also been reported that the CCKB-
selective antagonist CI-988 fails to potentiate the antinociceptive effects of morphine
in morphine-naive animals (Hoffman & Wiesenfeld-Hallin, 1994).
CCK antagonists have also been shown to potentiate analgesia induced by
endogenous opiates. For example, proglumide produces a marked potentiation of
front paw foot-shock induced analgesia, analgesia mediated by the release of
endogenous opiates (Watkins, Kinscheck, Kaufman et al., 1985). Further, this effect is
specific to opiate systems, as proglumide attenuates or has no effect on various forms of
non-opiate analgesia (Watkins, Kinscheck, Kaufman et al., 1985). This potentiation of
morphine analgesia by CCK antagonists appears to be mediated by an interaction
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between CCK and opiate systems, as naltrindole, an opioid 5 receptor antagonist, has
been shown to block the enhancement of the antinociceptive actions of morphine
induced by CCK antagonists (Ossipov et al., 1994).
Interestingly, when administered alone CCK antagonists do not alter baseline
pain thresholds in several assessments of analgesia, including the hot-plate (Dourish et
al., 1990; Hoffmann & Wiesenfeld-Hallin, 1994), tail-flick (Dourish et al., 1990;
Kellstein & Mayer, 1991; Ossipov et al., 1994; Singh et al., 1996; Watkins, Kinscheck
& Mayer, 1985) and rat paw formalin (Dourish et al., 1990) tests. This lack of effect of
CCK antagonists on baseline pain thresholds has been shown with nonspecific CCK
antagonists such as proglumide (Kellstein & Mayer, 1991; Panerai, Rovati, Cocco,
Sacerdote & Mantegazza, 1987; Watkins Kinscheck & Mayer, 1985; Watkins et al,
1984), lorglumide (Kellstein & Mayer, 1991) and benzotript (Panerai et al., 1987), the
CCKA-selective antagonists L-365,071 (Dourish et al., 1990) and PD 140548 (Singh et
al., 1996) and the CCKB-selective antagonists L-365,260 (Dourish et al., 1990; Ossipov
et al., 1994) and CI-988 (Hoffmann & Wiesenfeld-Hallin, 1984; Singh et al., 1996).
Given that CCK antagonists exert no analgesic effects on their own and potentiate the
analgesic effects of opiates on an acute basis, they appear to exert their analgesic
effects via their interactions with the opiate system (Watkins, Kinscheck & Mayer,
1985). These findings may suggest that CCK systems are not tonically active, but
rather are activated in response to either the administration or release of opiates
(Watkins et al., 1984).
As mentioned above, chronic exposure to morphine results in the development
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of tolerance to its analgesic effect (Zhou et al., 1992). Interestingly, CCK antagonists
have been shown to affect opiate tolerance in morphine-tolerant animals. For
example, the nonselective CCK antagonist proglumide (Watkins et al., 1984; Tang et
al., 1984) and the CCKB-selective antagonist CI-988 reverse tolerance to morphine
on the hot-plate test (Hoffman & Wiesenfeld-Hallin, 1994). CCK antagonists have
also been shown to block or reduce the development of tolerance to morphine-induced
analgesia in several assessments of analgesia, including the hot-plate (Xu,
Wiesenfeld-Hallin, Hughes, Horwell & Hokfelt, 1992; Hoffman & Wiesenfeld-Hallin,
1994) and tail-flick (Dourish et al., 1988; Kellstein & Mayer, 1991) tests. This effect
has been shown with nonspecific CCK antagonists such as proglumide (Panerai et al.,
1987; Kellstein & Mayer, 1991; Tang et al., 1984), lorglumide (Kellstein & Mayer,
1991) and benzotript (Panerai et al., 1987), as well as the CCKA-selective antagonists
devazepide (Dourish et al., 1988) and L-365,031 (Dourish et al., 1990) and the
CCKB-selective antagonists L-365,260 (Dourish et al., 1990) and CI-988 (Singh et
al., 1996; Xu et al., 1992).
Thus, CCK antagonists potentiate morphine analgesia (acute) and block or
reverse tolerance to morphine’s analgesic effects (chronic). These findings suggest
that endogenous CCK systems affect the action of opiates (Watkins et al., 1984;
Wiesenfeld-Hallin et al., 1999), possibly as a physiological opiate antagonist (Panerai
et al., 1987; Watkins, Kinscheck & Mayer, 1985). Acutely, CCK’s effects on opiate
analgesia can be blocked by CCK antagonists. Tolerance to the exogenous opioids
may be associated with an up-regulation of endogenous CCK, which induces greater
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blockade of opiate analgesia, resulting in tolerance (Wiesenfeld-Hallin & Xu, 1996).
CCK antagonists block the action of this up-regulated CCK system, which restores some
of the analgesic effect of the opioid, resulting in the reversal of morphine tolerance
(Wiesenfeld-Hallin & Xu, 1996). Further, upregulation of the CCK system may require
chronic stimulation of CCK receptors by repeated opiate administration, as CCK
antagonists also prevent the development of morphine tolerance (Wiesenfeld-Hallin &
Xu, 1996).
Although most of the research to date on the interactions of CCK and the opiates
has focused on morphine-induced analgesia, a few studies have investigated the role of
CCK and CCK antagonists in the rewarding motivational properties of morphine. Drugs
with abuse potential, such as morphine, possess opposing motivational properties in that
they concurrently produce both rewarding and aversive effects (Wise, Yokel & de Witt,
1976; Bechara & van der Kooy, 1985). Specifically, animals will self-administer a
compound as well as avoid tastes paired with that same compound, indicating that the
compound is rewarding and aversive, respectively (Wise et al., 1976). For example,
White, Sklar and Amit (1977) trained rats to run towards a goal box to obtain food.
Immediately upon eating the food, animals were injected with either saline or morphine
and were then returned to the goal box for 50 min. Over five conditioning trials, rats
injected with morphine increased their running speed to the goal box by approximately
400%, indicating that the drug has rewarding effects, while at the same time decreasing
the amount of food consumed in the goal box to approximately 70% of baseline
consumption, indicating that the drug also has aversive effects. Animals injected with
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saline showed no change in either running speed or food consumption. It has been
suggested that it is the relative strength of each of these motivational properties that
affects whether or not a drug will be taken (Ettenberg, Sgro & White, 1982; Rademacher,
Anders, Thompson & Steinpreis, 2000; Riley & Simpson, 2000; Stolerman, 1989). For
example, a drug high in aversive properties and low in rewarding properties is less likely
to be used than a drag that is low in aversive properties and high in rewarding properties
(Stolerman, 1989).
Although no research has directly examined CCK antagonists and the aversive
properties of morphine, results from research investigating CCK antagonists and the
rewarding properties of morphine have been variable. For example, the CCKA-selective
antagonists PD 140548 (Singh et al., 1996) and devazepide (Higgins, Nguyen & Sellers,
1992) have been shown to dose-dependently antagonize or attenuate the development of a
conditioned place preference (CPP) to morphine, indicating that CCK antagonists may
affect the rewarding properties of morphine. However, these same effects were not seen
with the CCKB-selective antagonists CI-988 (Singh et al., 1996) nor L-365,260 (Higgins
et al, 1992). Conversely, Lu and colleagues (Lu, Huang, Liu & Ma, 2000; Lu, Huang,
Ma & Li, 2001) found that morphine CPP was significantly attenuated by the CCKB-
selective antagonist L-365,260, whereas this effect was not observed with the CCKA-
selective antagonist MK-329. In this same study, following a 28-day extinction period,
the morphine CPP disappeared and was reactivated by a single morphine injection.
However, pretreatment with the CCKB-selective antagonist L-365,260 blocked the
reactivation of the morphine CPP, whereas the CCKA-selective antagonist MK-329
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failed to produce these same results (Lu et al., 2001).
In other studies, CCKB-selective antagonists have been shown to potentiate
morphine place preferences. For example, in animals pretreated with L-365,260, a
subthreshold dose of morphine induced a significant CPP (Higgins et al., 1992),
indicating that CCK antagonists may increase the rewarding motivational properties of
morphine. Further, although the CCKB-selective antagonist PD-134,308 elicited neither
a place preference nor a place aversion, when given with a subthreshold dose of
morphine, a CPP was observed (Valverde, Foumie-Zaluski, Roques & Maldonado,
1996). This same effect was also seen when this CCKB-selective antagonist was
administered with a subthreshold dose of RB 101, a complete inhibitor of enkephalin
catabolism (Valverde et al., 1996).
Just as chronic morphine exposure results in tolerance to its analgesic effects,
chronic exposure to morphine has also been shown to alter the strength of its aversive
effects. Specifically, tolerance to the aversive properties of morphine develops following
chronic morphine exposure (e.g., see Dacanay & Riley, 1982; Domjan & Siegel, 1983;
Riley, Dacanay & Mastropaolo, 1984; Stewart & Eikelboom, 1978). As CCK antagonists
prevent the development of tolerance to morphine's analgesic effects, CCK antagonists
could potentially modulate the aversive motivational properties of morphine. Of interest
to the current research is whether or not CCK antagonists are also effective in blocking
tolerance to the aversive properties of morphine. To this end, the current research
investigated the effects of the nonselective CCK antagonist proglumide on changes in the
strength of the aversive motivational properties of morphine during chronic morphine
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exposure.
One way that the aversive properties of morphine can be assessed is by the
conditioned taste aversion (CTA) design (e.g., see Hutchinson et al., 2000; Mucha &
Herz, 1985; Riley, Jacobs & LoLordo, 1978, Switzman, Hunt & Amit, 1981; for an
alternate interpretation, see Grigson, 1997), a behavioral index of toxicity (Riley & Tuck,
1985). Specifically, in this design water-deprived animals are given access to a novel
solution, such as saccharin, which is subsequently followed by administration of a drug,
such as morphine (e.g., see Hutchinson et al., 2000). Saccharin consumption is measured
over conditioning trials, and animals administered drug typically decrease saccharin
consumption, whereas control animals administered vehicle typically increase saccharin
consumption over conditioning trials. For example, Switzman et al. (1981) paired saccharin
and either 4, 8 or 12 mg/kg morphine. Following only one such pairing, there were
significant decreases in saccharin consumption in the groups of animals exposed to either 8
or 12 mg/kg morphine, whereas there was an increase in saccharin consumption in animals
administered vehicle on the conditioning day. There was no significant change in the
animals administered 4 mg/kg morphine. Thus, at the two higher doses tested, morphine
induced a CTA evidenced by a significant decrease in saccharin consumption on the test day.
This effect has been seen with other opioids (Gaiardi, Gubellini & Bartoletti, 1998; Grigson,
Twinning & Carelli, 2000; Hutchinson et al., 2000), in other rat strains (Lancelotti, Bayer,
Glowa, Houghtling & Riley, 2000), in both females (Jacobs, Zellner, LoLordo & Riley,
1981) and males (Bardo & Valone, 1994) and with different routes of administration
(Bechara, Martin, Pridgar & van der Kooy, 1993; Hutchinson et al., 2000).
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Although morphine is effective in inducing a CTA, it is less effective in doing so
following preexposure to morphine (e.g., Cappell & Le Blanc, 1977). For example, Capped
& Le Blanc (1977) found that after six morphine-saccharin pairings animals displayed a
significant aversion to saccharin, i.e., a significant decrease in saccharin consumption.
However, animals exposed to morphine prior to the pairing of saccharin and morphine
showed an attenuated aversion to saccharin. Thus, chronic exposure to morphine attenuates
subsequent morphine-induced taste aversions, presumably due to the development of
tolerance to its aversive properties over repeated exposure (Capped & Le Blanc, 1977;
Randich & LoLordo, 1979; Riley & Simpson, 2001). This change in the relative strengths of
the aversive motivational properties of morphine may lead to an increase in future use and/or
abuse of the drug (for a discussion, see Riley & Simpson, 2001).
The current research assessed the ability of the CCK antagonist proglumide to
block the development of tolerance to the aversive properties of morphine in rats
utilizing a CTA design. Specifically, animals were preexposed to either vehicle alone,
morphine alone or a combination of either proglumide and morphine or proglumide and
vehicle. Subsequently, saccharin consumption was measured within a CTA design,
where saccharin was paired with either morphine or vehicle. If the effects of morphine
preexposure, which generally result in an attenuation of morphine-induced taste
aversions, are blocked by this CCK antagonist, it may suggest that CCK antagonists
block tolerance to the aversive properties of morphine, further implicating the role of
CCK in morphine tolerance.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 2
PROCEDURE: EXPERIMENT 1
Subjects
Subjects were 84 experimentally-naive male Sprague Dawley rats, approximately
60-90 days in age and approximately 300-450g in weight at the beginning of the
experiment. Animals were housed individually in stainless-steel, wire-mesh cages and
maintained on a 12 light: 12 dark cycle (lights on at 0800h) and at an ambient temperature
of 23° C for the duration of the experiments. Standard rat chow and water (except where
noted) were availablead libitum. Animals were housed for approximately two weeks
prior to the commencement of the experiments to allow for habituation to their new
environment.
Druss and Solutions
Morphine sulfate was generously provided by the National Institute on Drug
Abuse (NIDA) and was dissolved in distilled water. Proglumide was obtained from
Panos Therapeutics and was suspended in a solution of 1.2% DMSO and 98.8% 7.0 pH
buffer solution. All drugs were administered by intraperitoneal (i.p.) injection. For
vehicle injections (see below), the solution in which the drug was dissolved is defined as
that drug’s vehicle. Saccharin (0.1% sodium saccharin, Sigma Chemical Company) was
prepared as a 1 g/1 solution in tap water.
12
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Phase 1: Habituation
Following 23-h water deprivation, animals were given 20-min access to water.
This procedure was repeated each day until all subjects drank within 2 s of water
presentation.
Phase 2: Preexposure
On Day 1 of this phase, animals received their regular 20-min access to water. At
the outset of this phase, animals were assigned to a drug preexposure group based on the
average amount of water consumed over the last three days of water habituation.
Specifically, animals were rank ordered according to water consumption and were then
alternately assigned to one of five preexposure groups. Five hours later, subjects
received injections of their preexposure drug(s). Specifically, animals received i.p.
injections of either proglumide (5 mg/kg) or its vehicle (see above) followed 15 min later
by an i.p. injection of either morphine (5 mg/kg) or its vehicle. One group of animals
received an injection of proglumide followed immediately by morphine. This resulted in
five groups (PM, PV, VM, VV and PiM). The first letter refers to the first compound to
be administered [proglumide (P) or vehicle (V)], and the last letter refers to the second
compound to be administered [morphine (M) or vehicle (V)]. PiM refers to the group
that was injected with proglumide immediately followed by morphine. These
preexposure injections were administered every other day for a total of five preexposure
days. Animals received 20-min access to water on the recovery day between preexposure
injections, as well as on the day following the last drug preexposure day.
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Phase 3: Conditionins
On the second day after the last drug preexposure injections in Phase 2, all
animals received 20-min access to a novel saccharin solution. Immediately following
access to saccharin, each preexposure group was divided in half based on saccharin
consumption such that saccharin consumption was comparable between groups.
Specifically, animals in each Preexposure Group were ranked according to saccharin
consumption and were then alternately assigned to one of two groups. Each animal then
immediately received an i.p. injection of either morphine (10 mg/kg) or vehicle. This
resulted in a total often groups (PM-M, PM-V, PV-M, PV-V, VM-M, VM-V, VV-M,
VV-V, PiM-M and PiM-V). Names for these groups are the same as above, with the
letter after the hyphen representing whether that group was injected with morphine (M)
or vehicle (V) during this phase. For example, Group PM-M was preexposed to
proglumide and morphine and was injected with morphine during this phase. On the
following three water-recovery days, all animals received 20-min access to water. No
injections followed water access on those days. This alternating procedure of
conditioning followed by three water-recovery days was repeated for four cycles.
Test for Saccharin Aversion
On the day after the final water-recovery session of Phase 3, animals received 20-
min access to saccharin in a final test of the aversion to saccharin. No injections
followed saccharin access on the test day.
Statistical Analysis
A 5 x 5 repeated measures ANOVA with one within-group factor (Preexposure
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 15
Day) and one between-group factor (Preexposure Drug) was performed on mean water
consumption during preexposure. Post-hoc analyses were conducted using Tukey HSD
pairwise comparisons. Significance was based on p < 0.05. Within-subject differences in
water consumption from baseline (Preexposure Day 1) were assessed using paired sample
t-tests with a Bonferroni correction (g = 0.0125).
A 5 x 2 x 5 repeated measures ANOVA with one within-group factor (Trial) and
two between-group factors (Preexposure Drug and Conditioning Drug) was performed to
study saccharin consumption during conditioning. Post-hoc analyses were conducted
using Tukey HSD pairwise comparisons. Significance was based on g < 0.05. Within-
subject differences in saccharin consumption from baseline (Trial 1) were assessed using
paired sample t-tests with a Bonferroni correction (g = 0.0125).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 3
RESULTS: EXPERIMENT I
Preexposure
Figure 1 illustrates the mean (± standard error of the mean (S.E.M.)) consumption
of water for subjects during preexposure. A 5 x 5 repeated-measures ANOVA revealed a
significant effect of Preexposure Day (F(4,268) = 9.075, p < 0.001) and of Preexposure
Drug (F(4,67) = 3.881, p = 0.007). There was no significant interaction for Preexposure
Day x Preexposure Drug (F(l6,268) = 1.283, p = 0.207), indicating that water
consumption was comparable among preexposure groups during preexposure. On the
initial drug preexposure day, animals consumed approximately 15 ml and this level was
maintained over multiple exposures.
PM PiM PV ■VM ■VV
03 ° £ 4-
2- 0-1
i------1------1------1------1— 12 3 4 5 Preexposure Day Figure 1 Mean (± S.E.M.) consumption o f water over repeated preexposure days for subjects in Preexposure Groups PM, PiM, PV, VM and VV. Water was available 5 h before injections. 16
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 17
Conditioning
A 5 x 5 (Group x Trial) repeated measures ANOVA was performed to assess
differences in saccharin consumption among control groups (groups not conditioned with
morphine) across conditioning trials. There was a significant main effect for Trial
(F(4,140) = 75.296, p < 0.001). However, there was no significant effect for Group
(F(4,35) = 1.491, p = 0.226), nor was there a significant Group x Trial interaction
(F(16,140) = 1.212, g = 0.266). Consequently, all control groups were combined for all
further analyses.
A 5 x 2 x 5 repeated measures ANOVA revealed significant main effects for
Preexposure Drug (F(4,73) = 5.439, g = 0.001), Conditioning Drug (F(l,73) = 138.573, g
< 0.001) and Trial (F(4,292) = 34.973, g < 0.001). There were also significant two-way
interactions for Trial x Preexposure Drug (F(16,292) = 7.365, g < 0.001), Trial x
Conditioning Drug (F(4,292) = 53.697, g < 0.001) and Preexposure Drug x Conditioning
Drug (F(4,73)= 10.353, g < 0.001), as well as a significant three-way interaction for
Preexposure Drug x Conditioning Drug x Trial (F(16,292) = 3.607, g <0.001).
Figure 2 illustrates the mean (± S.E.M.) consumption of saccharin over repeated
conditioning trials and on the test day for Controls and animals in Groups VM-M and
VV-M. Post-hoc Tukey HSD analyses revealed that there were no significant differences
among groups on the first trial (all g’s > 0.05), indicating that all groups drank similar
amounts of saccharin on this initial conditioning day (approximately 11.8 ml). Within-
group paired sample t-tests revealed significant differences between each trial and each
group’s respective baseline (Trial 1). Specifically, Controls significantly increased
saccharin consumption relative to baseline on all trials (all g’s < 0.0125). Group VV-M,
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 18
subjects preexposed to the vehicle and conditioned with morphine, decreased saccharin
consumption significantly from its baseline on all trials (all p’s < 0.0125). Group VM-M,
subjects preexposed to morphine and conditioned with morphine, increased consumption
significantly relative to its baseline on Trial 2 (p = 0.011). Saccharin consumption on
Trials 3 and 4 as well as on the Test Day did not differ from baseline (all p’s > 0.0125).
<=> 22 "i s, 20- 8 —*— Controls K. 16- •-■’•-VM-M | 14- C/2 4 § 12‘ O 10- 8
Test Conditioning Trial Figure 2 Mean (± S.E.M.) consumption of saccharin over repeated conditioning trials for control subjects and subjects in Groups VM-M and W -M . * Significantly different from baseline.
Post-hoc Tukey HSD analyses also revealed significant differences among
groups. Specifically, Group VV-M consumed significantly less saccharin than Controls
on Trials 2, 3 and 4 as well as on the Test Day (all p’s < 0.05). Consumption for Group
VM-M was also significantly less than that of Controls on Trials 2, 3, 4 and on the Test
Day (all p’s < 0.05), but it was significantly greater than that of nonpreexposed animals
in Group VV-M on Trials 2, 3 and 4 as well as on the Test Day (all p’s < 0.05), indicating
that preexposure to morphine significantly attenuated the aversion to morphine.
Figure 3 illustrates the mean (± S.E.M.) consumption of saccharin over repeated
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 19
conditioning trials and on the test day for Controls and animals in Groups PM-M and PV-
M. As previously described, Controls significantly increased saccharin consumption
relative to baseline on all trials. Group PV-M, subjects preexposed to proglumide and
vehicle and conditioned with morphine, decreased saccharin consumption significantly
from its baseline on Trials 3 and 4 as well as on the Test Day (all p’s < 0.0125). Group
PM-M, subjects preexposed to proglumide followed 15 min later by morphine and
conditioned with morphine, significantly increased saccharin consumption relative to its
baseline on Trial 2 only (p = 0.001).
C, 22-1 S 20- —•— Controls I 18_ 'S, 16- -o-PM-M | 14- § 12- PV-M O 10- 8
Test Conditioning Trial Figure 3 Mean (± S.E.M.) consumption of saccharin over repeated conditioning trials for control subjects and subjects in Groups PM-M and PV-M. * Significantly different from baseline.
Post-hoc Tukey HSD analyses revealed significant between-group differences.
Specifically, Group PV-M consumed significantly less saccharin than Controls on Trials
2, 3 and 4 as well as on the Test Day (all p’s < 0.05). Consumption for Group PM-M was
also significantly less than that of Controls on Trials 3 and 4 and on the Test Day (all p’s
< 0.05), but it was significantly greater than that of nonpreexposed animals in Group PV-
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 20
M on Trials 2, 3 and 4 as well as on the Test Day (all p’s < 0.05), indicating that
preexposure to morphine attenuated the aversion to morphine, even in subjects receiving
proglumide 15 min prior to morphine drug preexposure.
Figure 4 illustrates the mean (± S.E.M.) consumption of saccharin over repeated
conditioning trials and on the test day for Controls and animals in Groups PiM-M and
PV-M. As described above, controls significantly increased saccharin consumption
relative to baseline on all trials and animals in Group PV-M decreased saccharin
consumption significantly from its baseline on Trials 3 and 4 as well as on the Test Day.
Group PiM-M, subjects preexposed to proglumide immediately prior to morphine and
conditioned with morphine, did not differ in saccharin consumption from its baseline on
any trials (all p ’s > 0.0125).
— Controls PiM-M pv-M
8 CO /- j s 6 I 4 2 3 4 Test Conditioning Trial Figure 4 Mean (± S.E.M.) consumption of saccharin over repeated conditioning trials for control subjects and subjects in Groups PiM-M and PV-M. * Significantly different from baseline. Post-hoc Tukey FISD analyses revealed significant between-group differences. As previously described, Group PV-M consumed significantly less saccharin than Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 21 Controls on Trials 2, 3 and 4 as well as on the Test Day. Group PiM-M consumed significantly less saccharin than Controls on Trials 2, 3 and 4 as well as on the Test Day (all p’s < 0.05), but consumed significantly more saccharin than Group PV-M on Trials 3 and 4 as well as on the Test Day, indicating that morphine preexposure significantly attenuated the aversion to saccharin, even in subjects receiving proglumide immediately prior to morphine drug preexposure. There were no significant differences in saccharin consumption between Groups PV-M and VV-M, subjects not preexposed to morphine and conditioned with morphine, on any trial (all p’s > 0.05). Further, there were no differences in saccharin consumption on any trial among Groups VM-M, PM-M and PiM-M, subjects preexposed to morphine or a combination of proglumide and morphine and conditioned with morphine (all p’s > 0.05), indicating that proglumide had no effect on tolerance to the aversive properties of morphine. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 4 DISCUSSION: EXPERIMENT 1 Chronic exposure to morphine results in tolerance to its analgesic effects, and CCK antagonists block and/or reverse this development. Chronic exposure to morphine has also been shown to alter the strength of its aversive effects (e.g., see Riley et al., 1984), and, thus, of interest to the current research was whether or not CCK antagonists would also be effective in affecting tolerance to these properties. Specifically, the current research assessed the ability of the nonselective CCK antagonist proglumide to block the development of tolerance to the aversive properties of morphine in rats utilizing a UCS preexposure design. In such a design, exposure to a drug prior to taste aversion conditioning with that drug results in an attenuated taste aversion (see Cappell & Le Blanc, 1977), presumably due to the development of tolerance to its aversive properties over repeated exposure (e.g., see Dacanay & Riley, 1982; Domjan & Siegel, 1983; Riley, Dacanay & Mastropaolo, 1984; Stewart & Eikelboom, 1978; for a review, see Riley & Simpson, 2001). In the current study, animals were preexposed to either vehicle alone, proglumide alone, morphine alone or a combination of proglumide and morphine. Subsequently, saccharin was paired with either morphine or vehicle. As reported, animals preexposed to either proglumide or vehicle and conditioned with morphine significantly decreased saccharin consumption. This decrease presumably reflects the association of saccharin with the aversive effects of morphine (Riley & Tuck, 22 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 23 1985) and not to the unconditioned suppressant effects of morphine, as chronic exposure to morphine (e.g., 40 mg/kg; Riley, West, Sipel & Woods, 1983) results in an increase in the size and duration of feeding and frequency of drinking. This aversion was significantly attenuated when animals were preexposed to morphine and conditioned with morphine, indicating that preexposure to morphine attenuated the aversion to morphine (for other examples of UCS with morphine, see Cappell & Le Blanc, 1977; Martin, Bechara & van der Kooy, 1988; Riley et al., 1984). In animals preexposed to proglumide administered either immediately or 15 min prior to morphine and then conditioned with morphine, the aversions were also attenuated. This attenuated aversion was similar to that seen in animals preexposed to morphine (in the absence of proglumide), indicating that proglumide had no effect on the development of tolerance to the aversive properties of morphine. Although proglumide has been shown to be effective in blocking the development of tolerance to the analgesic effects of morphine and has also been shown to reverse such tolerance, proglumide is ineffective in preventing the development of tolerance to the aversive effects of morphine within the current preparation. Whether this is a function of proglumide or the specific conditions under which proglumide was tested, however, remains unknown. There are several parametric issues that might have affected the ability of proglumide to block tolerance to morphine’s aversive effects, e.g., the dose of proglumide (5 mg/kg) and the amount of time between the administration of proglumide and morphine (immediate or 15 min). In the current study, 5 mg/kg proglumide was administered prior to 5 mg/kg Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 24 morphine during drug preexposures. Although tolerance to morphine’s aversive effects did develop, 5 mg/kg proglumide did not block this development. In other reports, proglumide at lower doses does modulate morphine’s effects (Ben-Horin, Ben-Horin & Frenk, 1984; Watkins, Kinscheck & Mayer, 1985). However, these lower doses potentiate, rather than block, morphine’s effects, including morphine-induced analgesia and hypokinesia. There is only one study assessing the ability of proglumide to block morphine’s chronic effects (Tang et al., 1984). In this study, animals were administered either 4 mg/kg morphine subcutaneously (s.c.) alone or morphine and 15 mg/kg proglumide i.p. every 2 hours for a total of seven times, and the development of tolerance to morphine’s analgesic effects was assessed over time. Under these conditions, proglumide partially blocked the development of tolerance to morphine’s analgesic effects (Tang et al., 1984). In other behavioral assessments, 50 mg/kg proglumide reverses CCK-induced satiety (Willis, Hansky & Smith, 1986) and 150 mg/kg proglumide blocks CCK’s suppression of food intake (Collins, Walker, Forsyth & Belbeck, 1983). Lower doses of proglumide have also been shown to be effective in blocking CCK’s effects. For example, 2 mg/kg s.c. blocks the effect of CCK-induced suppression of locomotion scores (Katsuura, Hsiao & Itoh, 1984). Further, 5 mg/kg proglumide blocks CCK-induced reduction of passive avoidance latency (Deupree & Hsaio, 1984). Thus, although ineffective in the current study, 5 mg/kg proglumide has been shown to be effective in blocking CCK’s effects within another behavioral paradigm and is certainly within the range of effective doses. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 25 Another possible reason that proglumide may not have blocked the development of tolerance to the aversive effects of morphine is the timing between the injections of proglumide and morphine. In the current study, proglumide was administered either immediately or 15 min before the administration of morphine. A range in the amount of time between injections has been shown to be effective in various behavioral assessments. Although the single study investigating the ability of proglumide to block the development of morphine tolerance did not describe the amount of time between injections (Tang et al., 1984), proglumide administered immediately before morphine is effective in blocking the effects of other drugs, such as CCK. For example, Deupree and Hsaio (1984) found that 5 mg/kg proglumide, the dose used in the current study, administered concurrently with CCK blocked CCK’s reduction of passive avoidance latency. Proglumide followed immediately by CCK also blocks CCK-induced suppression of feeding (Collins et al., 1983; Willis et al., 1986). Thus, the dose and amount of time between injections used in the current study have been shown to be within the range of doses and within the proglumide-morphine interval that have been effective in other studies. In order to assess whether this dose and these intervals between proglumide and morphine injections would be effective under the conditions in the first experiment (e.g., strain of rats, gender, route of administration) utilizing another behavioral assessment, Experiment 2 investigated the ability of 5 mg/kg proglumide, administered either immediately, 5 or 15 min before CCK, to block or attenuate CCK-induced suppression of feeding. Specifically, following 23 h of food deprivation, animals were injected with either saline, proglumide, CCK or a combination Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 26 of proglumide followed either immediately, 5 or 15 min later by CCK. Animals were then allowed access to food for 60 min. The amount of food eaten at the end of the 60- min period was measured. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 5 PROCEDURE: EXPERIMENT 2 Subjects Subjects were 16 male Sprague Dawley rats, approximately 350-500g in weight at the beginning of the experiment. Animals were housed individually in stainless-steel, wire-mesh cages and maintained on a 12 light: 12 dark cycle (lights on at 0800h) and at an ambient temperature of 23° C for the duration of the experiment. Standard rat chow and water (except where noted) were availablead libitum. Drugs Proglumide was obtained from Panos Therapeutics and was suspended in a solution of 1.2% DMSO and 98.8% 7.0 pH buffer solution. CCK octapeptide (generously supplied by Bristol-Myers Squibb) was prepared in distilled water. All drugs were administered by i.p. injection. Phase 1: Habituation Animals were deprived of food in their home cages for 23 h prior to each training or experimental session. The rats received three habituation sessions in which they were injected with distilled water and were then placed for 60 min in experimental cages that contained a weighed amount of food. The amount of food eaten at the end of the 60-min period was measured. 27 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 28 Phase 2: Drus Effects on Feedins During these sessions, the rats were injected i.p. with either saline, proglumide (5 mg/kg), CCK (3 or 10 pg/kg) or a combination of proglumide followed either immediately, 5 min or 15 min later by CCK. Five min after the animals were injected with one of the drugs or drug combinations mentioned above, they were placed for 60 min into experimental cages that contained a weighed amount of food. The amount of food eaten at the end of the 60-min period was measured. Animals were run in two separate groups, and each group received the vehicle and each drug or drug combination in a different order. There was at least one recovery day between sessions, during which animals hadad-libitum access to food and water. Water was availablead libitum throughout all sessions. Statistical Analysis A repeated measures ANOVA with one within-subjects factor (Drug) was performed on the amount of food consumed during each 60-min period of Phase 2. Significance was based on p < 0.05. Within-subject differences in food consumption were assessed using planned paired sample t-tests with a Bonferroni correction (p = 0.0038). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 6 RESULTS: EXPERIMENT 2 Figure 5 illustrates the mean (± S.E.M.) consumption of food for subjects injected with saline, 3 pg/kg CCK, 10 pg/kg CCK or 5 mg/kg proglumide. A repeated-measures ANOVA revealed a significant effect for Drug (F(7,105) = 30.314, g < 0.001). Following saline injections, mean consumption was approximately 8.1 g. Both 3 pg/kg and 10 pg/kg CCK significantly decreased feeding relative to this saline baseline (g’s < 0.0038), with animals consuming approximately 3.7 g and 2.6 g, respectively. Proglumide, alone, did not significantly alter feeding compared to baseline (g - 0.133), with subjects eating approximately 7.2 g. Kh w _o53 tx os O T3o o P3 Saline 10 CCK 3 CCK Proglumide (P) Condition Figure 5 Mean (± S.E.M.) consumption of food for food-deprived subjects over repeated 60-min experimental sessions following saline, 10 pg/kg CCK, 3 pg/kg CCK or 5 mg/kg proglumide. * Significantly different from baseline (saline). 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 30 Figure 6 illustrates the mean (± S.E.M.) consumption of food for subjects injected with saline, 10 pg/kg CCK or proglumide followed either 5 or 15 min later by 10 pg/kg CCK. As described, animals consumed approximately 8.1 g of food following saline injections and this consumption was decreased significantly by 10 pg/kg CCK (to 2.6 g). Proglumide did not affect this CCK-induced suppression of feeding. Specifically, food consumption was significantly suppressed relative to baseline following the combination of proglumide and 10 pg/kg CCK, administered either 5 or 15 min apart (p’s < 0.001), when animals consumed approximately 3.4 g and 2.6 g, respectively. The amount of food consumed following either of the proglumide and 10 pg/kg CCK combinations was not significantly different from the amount consumed following 10 pg/kg CCK alone, indicating that proglumide did not block the decrease in feeding induced by this dose of CCK (p’s > 0.0038). Further, food consumption following proglumide and 10 pg/kg CCK administered 15 min apart did not differ from food consumption following this proglumide and CCK combination administered 5 min apart (p = 0.217). ^ 10-i HD .2 8 ' 4—»a, 6H cnI C o O 4 -a O O tu 2- Saline 10 CCK P-15min-10 CCK P-5min-10 CCK Condition Figure 6 Mean (± S.E.M.) consumption of food for food-deprived subjects over repeated 60-min experimental sessions following saline, 10 pg/kg CCK or 5 mg/kg proglumide (P) followed either 1 5 or 5 min later by 1 0 pg/kg CCK. * Significantly different from baseline (saline). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 31 Figure 7 illustrates the mean (± S.E.M.) consumption of food for subjects injected with saline, 3 pg/kg CCK or proglumide followed either immediately or 15 min later by 3 pg/kg CCK. As described, animals consumed approximately 8.1 g of food following saline injections, and this consumption was decreased significantly by 3 pg/kg CCK (to 3.7 g). Proglumide administered 15 min before 3 pg/kg CCK did not affect this CCK- induced suppression of feeding. Specifically, consumption following proglumide administered 15 min before 3 pg/kg was significantly decreased from baseline (p < 0.001) to approximately 4.0 g. Further, consumption following proglumide and 3 pg/kg 15 min later did not did not differ from feeding following 3 pg/kg alone (p = 0.615). However, proglumide administered immediately before 3 pg/kg CCK did block its suppression of feeding. Specifically, the amount of food consumed following proglumide immediately before 3 pg/kg CCK, when consumption was approximately 6.2 g, did not differ relative to baseline consumption (p > 0.0038). Further, following this proglumide and CCK combination, animals consumed significantly more food than that consumed by animals administered 3 pg/kg alone (p < 0.001). Further, food consumption following proglumide given immediately prior to 3 pg/kg CCK was significantly higher than that consumed when proglumide was administered 15 min before 3 pg/kg CCK (p = 0.003). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Saline 3 CCK P-15min-3 CCK P-immediate-3 CCK Condition Figure 7 Mean (± S.E.M.) consumption of food for food-deprived subjects over repeated 60-min experimental sessions following saline, 3 pg/kg CCK or 5 mg/kg proglumide (P) followed either 15 min later or immediately by 3 pg/kg CCK. * Significantly different from baseline (saline). * * Significantly different from 3 pg/kg CCK alone. * * * Significantly different from proglumide followed 15 min later by 3 pg/kg CCK. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 7 DISCUSSION Chronic exposure to morphine results in tolerance to its analgesic effects, and CCK antagonists block and/or reverse this development. Chronic exposure to morphine has also been shown to alter the strength of its aversive effects (e.g., see Cappell & Le Blanc, 1977; Martin et al., 1988; Riley et al., 1984), and, thus, of interest to the current research was whether or not CCK antagonists would also be effective in affecting tolerance to these properties. Specifically, the current research assessed the ability of the nonselective CCK antagonist proglumide to block the development of tolerance to the aversive properties of morphine in rats utilizing a UCS preexposure design. In such a design, exposure to a drug prior to taste aversion conditioning with that drug results in an attenuated taste aversion (see Cappell & Le Blanc, 1977), presumably due to the development of tolerance to its aversive properties over repeated exposure (e.g., see Dacanay & Riley, 1982; Domjan & Siegel, 1983; Riley, Dacanay & Mastropaolo, 1984; Stewart & Eikelboom, 1978; for a review, see Riley & Simpson, 2001). In the current study, animals were preexposed to either vehicle alone, proglumide alone, morphine alone or a combination of proglumide and morphine. Subsequently, saccharin was paired with either morphine or vehicle. As described, in Experiment 1 a significant morphine-induced taste aversion was obtained in animals preexposed to either proglumide or vehicle and conditioned with 33 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 34 morphine. Further, this aversion was significantly attenuated when animals were preexposed to, and conditioned with, morphine, indicating that preexposure to morphine attenuated the morphine-induced taste aversion (for other examples of UCS with morphine, see Cappell & Le Blanc, 1977; Martin et al., 1988; Riley et al., 1984). In animals preexposed to proglumide administered either immediately or 15 min prior to morphine and then conditioned with morphine, the aversions were also attenuated. This attenuated aversion was similar to that seen in animals preexposed to morphine (in the absence of proglumide), indicating that proglumide had no effect on the development of tolerance to the aversive properties of morphine. Although proglumide has been shown to be effective in blocking the development of tolerance to the analgesic effects of morphine and has been shown to reverse such tolerance, proglumide is ineffective in preventing the development of tolerance to the aversive effects of morphine within the current preparation. Whether this is a function of parametric issues employed in the current study, or other nonparametric issues such as the UCS preexposure design or the role of CCK antagonists in the motivational properties of morphine, however, remains unknown. In relation to specific parameters used in the current assessment, there are several issues that might have affected the ability of proglumide to block tolerance to morphine’s aversive effects. For example, it is possible that proglumide might have been effective had a different dose of morphine, rather than 5 mg/kg, been utilized during the preexposure phase of Experiment 1. However, the doses used were known to produce the UCS preexposure effect, i.e., block the development of tolerance to the aversive effects of morphine (Stevenson & Riley, 2002). Further, this Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 35 preexposure dose, in conjunction with a conditioning dose of 10 mg/kg morphine, results in a moderate, but significant, attenuation of the morphine-induced taste aversion. This was important, as proglumide could have altered the UCS preexposure effect in either direction, i.e., potentiate or attenuate. Thus, these doses of morphine were necessary in order to assess the effects of proglumide on the UCS preexposure effect. Other parameters that may have affected the results of the current study include the dose of proglumide (5 mg/kg) and the amount of time between the administration of proglumide and morphine (immediate or 15 min). As reported in Experiment 2, however, this combination of parameters was pharmacologically effective. As reported, CCK (3 and 10 pg/kg) significantly decreased feeding relative to baseline. Proglumide did not block the suppression of feeding induced by 10 pg/kg when administered either 5 or 15 min before CCK. Further, when proglumide was administered 15 min before 3 pg/kg, the suppression of feeding induced by CCK was not affected. However, when 5 mg/kg proglumide was administered immediately before 3 pg/kg CCK, proglumide attenuated the CCK-induced suppression of feeding. Thus, although these injection parameters of proglumide were ineffective in antagonizing the development of tolerance to the aversive effects of morphine in Experiment 1, under similar conditions as in Experiment 1, e.g., strain of rats, gender, route of administration, these dose and injection parameters of proglumide produced antagonism in another behavioral preparation. It remains possible, however, that other doses or injection parameters might have been effective in blocking the development of tolerance to the aversive effects of morphine, given that antagonism has been parametric-dependent in previous studies (see also Experiment 2). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 36 Another parametric issue concerns the role of CCKA versus CCKB receptors in the motivational properties of morphine. Although the nonspecific CCK antagonist proglumide used in the current study has a relatively low affinity for CCK receptor subtypes (Wiesenfeld-Hallin & Xu, 1996), it does have activity at both CCKA and CCKB receptors (Wiesenfeld-Hallin & Xu, 1996). Thus, if there were an interaction between CCK antagonists and the development of tolerance to the aversive effects of morphine, regardless of whether the this effect is mediated by CCKA versus CCKB receptors, proglumide would have been effective. Thus, employing a CCKA- or CCKB- selective antagonist may not have altered the results of the current study. However, it remains possible that a CCK antagonist with a higher affinity for either CCKA or CCKB receptors may have been effective in the current study. There are also other issues related to the preparation utilized in Experiment 1 that might account for the findings of the current study, including whether or not the UCS preexposure design is susceptible to antagonism. The UCS preexposure effect in CTA learning is well established (for a review, see Riley & Simpson, 2001). This effect has been shown with many compounds, including drugs of abuse, and can be altered by a variety of parametric conditions, e.g., the number of preexposures, the specific compound used during preexposure and conditioning, the length of time separating drug preexposure and conditioning, the dose of both the preexposure drug and the conditioning drug. However, few studies have examined whether or not this effect can be blocked. Previous research has found that various compounds have failed to block the development of tolerance to a drug’s aversive effects within a UCS preexposure design when examining Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 37 the effects of preexposure to a drug on an aversion to that same drug. For example, Stevenson & Riley (2002) found that the NMDA receptor antagonist MK-801, which inhibits the development of tolerance to analgesia induced by chronic morphine administration, did not block the development of tolerance to the aversive properties of morphine under similar conditions as in Experiment 1. Specifically, MK-801 was administered with morphine prior to conditioning with morphine in a CTA design. Animals preexposed to vehicle and conditioned with morphine consumed significantly less saccharin than animals not conditioned with morphine, and animals preexposed to morphine and conditioned with morphine displayed an attenuated morphine aversion. MK-801 had no effect on this attenuation, indicating that MK-801 does not block the development of tolerance to the aversive effects of morphine. This has also been seen with ethanol. Specifically, June et al. (1992) administered the inverse benzodiazepine agonist Rol5-4513 to animals immediately before each of five daily consecutive ethanol exposures prior to conditioning with ethanol. Animals preexposed to saline and conditioned with ethanol displayed a significant ethanol-induced saccharin aversion compared to animals not conditioned with ethanol. Further, animals preexposed to ethanol and conditioned with ethanol displayed an attenuated aversion, and drank at levels similar to controls. However, administration of Ro 15-4513 with ethanol during preexposure did not block the effects of ethanol preexposure on ethanol-induced conditioned taste aversions. Although in these studies the UCS preexposure effect was not blocked, two studies examining cross-drug preexposure effects, where the effects of preexposure to Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 38 drug A on conditioning with drug B in a taste aversion design are assessed, did block the UCS preexposure effect. For example, Kunin and colleagues (Kunin, Bloch, Smith & Amit, 2001) examined whether nicotine preexposure would attenuate a caffeine-induced taste aversion and whether or not this attenuation could be blocked by concurrent administration of mecamylamine, a nicotine receptor antagonist. The lowest dose (but not higher doses) of nicotine attenuated the caffeine-induced CTA, and this effect was reversed by mecamylamine. In another study, the opiate antagonist naloxazone injected 4.5 h before the preexposure drug, either morphine or ethanol, significantly attenuated the preexposure effect of morphine on ethanol CTA as well as the effect of ethanol on morphine CTA (Ng Cheong Ton & Amit, 1987). Given the results with cross-drug preexposure, it appears that the UCS preexposure effect of a drug can be affected pharmaco logical ly. The current study was based on the premise that the UCS preexposure effect is due to the development of tolerance to the aversive effects of the drug. Accordingly, the design should allow an assessment of the ability of proglumide to affect this development. It should be noted, however, that a variety of mechanisms have been proposed to account for the attenuating effects of drug preexposure, of which tolerance is only one. These mechanisms include other nonassociative processes, such as habituation to the novelty of the drug, habituation to the drug-induced illness, creation of an unnatural need state, sensitization to reward, activation of an opponent B process, as well as associative processes such as learned helplessness and associative blocking, i.e., the acquisition of stimulus associations during preexposure that interfere with taste aversion Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 39 learning (for a review, see Randich & LoLordo, 1979; Riley & Simpson, 2001). Importantly, it is not necessarily the case that there is a single mechanism underlying the UCS preexposure effect for all drugs. Of interest to the current study is morphine, and whether or not tolerance is the mechanism underlying the preexposure effect seen with this drug of abuse. In one of the first studies to examine the UCS preexposure effect with morphine, Dacanay and Riley (1982) preexposed rats to morphine or water in a novel environment and later conditioned them with morphine in either the same novel environment or the home cage. If an associative mechanism were responsible for any effects of morphine preexposure, it would be expected that the preexposure effects would only be evident when preexposure and conditioning occurred in the same environment. Only under such conditions would stimuli associated with morphine during preexposure affect aversion conditioning with morphine. On the other hand, if a nonassociative mechanism mediated the effects of morphine preexposure, it would be expected that the preexposure effect would occur independent of any similarity between the preexposure and conditioning environments (though see Siegel & MacRae, 1984). In the Dacanay and Riley (1982) report, animals preexposed to morphine displayed attenuated aversions, independent of the similarity of the preexposure and conditioning environments, supporting a nonassociative basis for the effects of morphine preexposure on morphine- induced taste aversion learning (see also Domjan & Siegel, 1983; Riley, Dacanay & Mastropaolo, 1984; Stewart & Eikelboom, 1978). This effect with morphine is in contrast to that of LiCl. Specifically, animals administered LiCl preexposure and conditioning in a distinct environment display significantly attenuated aversions, while Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 40 animals preexposed to LiCl in the distinct environment and conditioned with LiCl in the home cage do not. Such findings with LiCl are consistent with an associative (blocking) explanation (see Dacanay & Riley, 1982; Willner, 1978). Although the effect of morphine preexposure appears nonassociative in nature, the specific nonassociative mechanism underlying these effects remains unknown. As described above, several nonassociative processes have been proposed as mechanisms underlying the US preexposure effect. For drugs of abuse in general, it appears that tolerance may be responsible. Support for this position comes from several sources, indirectly, by investigating the development of tolerance to other drug effects occurring along with attenuated aversions in the same animals, and, more directly, by varying the parameters of drug preexposure known to affect the development of tolerance (for a review, see Riley & Simpson, 2001) and assessing their effects on UCS preexposure in aversion learning. In relation to studies examining the issue of tolerance indirectly, Cannon, Baker and Berman (1977) assessed the effects of ethanol preexposure on aversion learning with ethanol and on the ability of ethanol to affect motor activity. They found that ethanol preexposure attenuated ethanol-induced taste aversions and reduced the effects of ethanol on rotor-rod performance (indicating tolerance to ethanol’s effects on motor behavior). The parallel between the development of tolerance to the motor suppressant effects of ethanol and the development of the UCS preexposure effect with ethanol provides indirect support for the role of tolerance in the UCS preexposure effect. Other studies have examined tolerance to the aversive effects of a drug more directly by examining the effects of variations in parameters known to affect tolerance, e.g., the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 41 number of preexposures, the time between preexposure and conditioning, etc. (for a review, see Riley & Simpson, 2001) on the UCS preexposure effect. For example, Cappell and Le Blanc (1975) examined the effects of varying the amount of time between preexposure to and conditioning with amphetamine. Specifically, animals were conditioned with amphetamine 0, 1, 7 or 14 days following amphetamine preexposure. Animals conditioned either 0 or 1 day after preexposure displayed attenuated aversions. Animals conditioned 7 or 14 days after preexposure also displayed attenuated aversions; however, this attenuation was weaker than that seen in animals conditioned with the shorter intervals, indicating that the degree of attenuation was affected by the amount of time between preexposure and conditioning, an effect consistent with tolerance. As with other drugs of abuse, research has examined the issue of tolerance with morphine preexposure. For example, Cappell and Le Blanc (1977) examined the effects of the spacing of morphine preexposures, a manipulation known to affect the development of tolerance to behavioral effects of the opioids (see Goldstein, Aronov & Kalman, 1974). Specifically, different groups of animals were given morphine exposures every 24 (massed) or 120 (spaced) h for a total of 15 exposures prior to taste aversion conditioning with morphine. Interestingly the massed preexposures to morphine were no more effective in attenuating morphine-induced taste aversions than were the spaced preexposures, an effect inconsistent with what might be expected if the preexposure effect with morphine was a function of tolerance. Although apparently inconsistent, it is possible that the intervals assessed by Cappell and LeBlanc were too long to see differential effects on the development of tolerance. This suggestion is supported by Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 42 earlier work by Goldstein et al. (1974) who reported that greater tolerance was evident with shorter intervals between repeated drug injections (8 h). As the inter-injection interval exceeded 24 h, there was little evidence of differential tolerance (or of tolerance itself), suggesting a direct effect of such injection schedules on tolerance development. It remains unknown if variations in the interval of morphine preexposure would affect morphine-induced aversion learning if intervals less than 24 h were used. More direct assessments of tolerance to morphine preexposure come from other work by Cappell & Le Blanc (1977) who examined the effects of varying the amount of time between preexposure to and conditioning with morphine, another parameter known to affect tolerance. Specifically, animals were preexposed to morphine1,7, 14 or 28 days before six conditioning trials with morphine. Although the preexposure effect was evident as long as 28 days following morphine preexposure, on the third through the last trials, the degree of aversion was ordered according to the number of days since the last preexposure day, i.e., the degree of aversion following 28 days was less than the degree of attenuation observed following fewer days, consistent with tolerance. It is interesting to note in this context that morphine-induced taste aversions have been reported to weaken with extended conditioning (Siegel, Parker & Morez, 1995). This weakening is suggestive of the development of tolerance over conditioning and is consistent with the reduction of the aversive effects of morphine with repeated administration. Although limited, the work with morphine preexposure points to a nonassociative mechanism underlying the effects of UCS preexposure. Further, manipulations assessing variations in the parameters known to affect tolerance are consistent with tolerance as the specific Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 43 nonassociative mechanism. Thus, the failure of proglumide to block the UCS preexposure effect is not likely due to the fact that tolerance was not assayed in this design, although more research is required before conclusively determining that tolerance is responsible for the US preexposure effect with morphine. The issues discussed to this point are parametric and procedural in nature. However, the results of the current study might be due to the role of CCK in the motivational properties of morphine. In other words, perhaps CCK is not involved in the motivational effects of morphine. Only a few studies have examined the interaction between CCK antagonists and the motivational properties of morphine. In relation to the rewarding effects of morphine, the CCKA-selective antagonists PD140548 (Singh et al., 1996) and devazepide (Higgins et al., 1992) and the CCKB-selective antagonist L- 365,260 (Lu et al., 2000; 2001) attenuate or block morphine-induced CPP. Further, following extinction, the CCKB-selective antagonist L-365,260 blocks the reactivation of morphine place preference by morphine (Lu et al., 2001). Other studies, however, have found that CCK antagonists have the opposite effect, as the CCKB-selective antagonists L-365,260 (Higgins et al., 1992) and PD-134,308 (Valverde et al., 1996) potentiate morphine CPP. Although this would indicate that CCK has a role in the rewarding effects of morphine, several studies have found that CCK antagonists have no effect. For example, the CCKA-selective antagonist MK-329 (Lu et al., 2000; 2001) and the CCKB-selective antagonist CI-988 (Singh et al., 1996), administered with morphine during the development of morphine place preference, do not affect morphine-induced place preferences. Further, following extinction of a morphine-induced place preference, Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 44 the CCKA-selective antagonist MK-329 does not block the reactivation of morphine place preference induced by the administration of morphine (Lu et al., 2001). Given the current findings, it appears that CCK may play a limited role (if any) in the aversive effects of morphine. Drugs with abuse potential, such as morphine, possess opposing motivational properties in that they concurrently produce both rewarding and aversive effects (Wise et al., 1976; Bechara & van der Kooy, 1985). It has been suggested that the relative strength of each of these motivational properties affects whether or not the drug will be taken (Stolerman, 1989). For example, a drug that is both high in rewarding properties and low in aversive properties is more likely to be used than a drug that is both low in rewarding properties and high in aversive properties (Stolerman, 1989). Any manipulation that could alter these properties may affect the drug’s abuse liability. One such manipulation that may alter the relative strength of these properties, and, thus, drug acceptability, is drug history. In relation to the aversive properties, decreases with repeated exposure would result in an increased acceptability of the drug, which may lead to increased use. If the effects of drug history on drug acceptability could be altered, subsequent drug use might be affected. In other words, manipulating the changes in the motivational properties of a drug of abuse with exposure could have therapeutic value. The current study was the first to examine directly the effects of a CCK antagonist, proglumide, on the development of tolerance to the aversive effects of morphine. The interest in CCK antagonists lies in their potential therapeutic value to chronic pain patients treated with morphine. Recent studies have investigated the utility of CCK Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 45 antagonists for such patients, specifically assessing the ability of CCK antagonists to increase the analgesic effects of morphine (e.g., see McCleane, 2002; 2003; Price, von der Gruen, Miller, Rafii & Price, 1985). CCK antagonists could also be useful in preventing the development of tolerance to the analgesic effects of morphine, thus, decreasing the need for higher doses of morphine for continued effective pain relief (Price et al., 1985). Had CCK antagonists also been effective in blocking the development of tolerance to the aversive effects of morphine, it could have potentially blocked any increase in drug acceptability following chronic exposure. Given that drug history has also been reported to increase the rewarding properties of morphine, i.e., sensitization, resulting in an increase in drug acceptability (e.g., Gaiardi, Bartoletti, Bacchi, Gubellini, Costa & Babbini, 1991), future research should further examine the effects of proglumide and other CCK antagonists on these properties of morphine. 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