Possible interaction between central cholinergic muscarinic and opioid peptidergic systems during memory consolidation in mice

BEHAVIORAL AND NEURAL BIOLOGY40, 155-169 (1984)

Possible Interaction between Central Cholinergic Muscarinic and Opioid Peptidergic Systems during Memory Consolidation in Mice CARLOS M. BARATTI,1 INES B . INTROINI, AND PATRICIA HUYGENS 2 Cdtedra de Farmacologfa, Facultad de Farmacia y Bioqulmica, Universidad de Buenos Aires, Junln 956, Buenos Aires 1113, Argentina Naloxone (0.01-1.00 mg/kg, ip) facilitated retention of a one-trial inhibitory avoidance task, when administered to male Rockland mice immediately after training, as indicated by performance on a retention test 48 hr later. The doseresponse curve was an inverted U in this range of dose. In these conditions naloxone did not lengthen latencies to step-through during the retest of unshocked mice. Higher doses of naloxone (3.00 and 10.00 mg/kg, ip) tended to increase latencies to step-through of both shocked and unshocked mice. These facts rule out an aversive effect of naloxone for low and moderate doses but not for high doses. The influence of naloxone (0.10 mg/kg, ip) on retention was time dependent, which suggests that naloxone facilitated memory consolidation processes. The effects of naloxone were prevented by morphine in both an amnesic and a nonamnesic dose (1.0 and 0 5 mg/kg, ip, respectively). Therefore, naloxone probably facilitated retention as a function of its opiate antagonist properties. The memory facilitation induced by naloxone (0.10 mg/kg, ip) was antagonized by atropine (0.5 mg/kg, ip) but not by methylatropine (0.5 mg/kg, ip), mecamilamine (5 mg/ kg, ip), or hexametonium (5 mg/kg, ip). Further, there was a mutual potentiation for both naloxone (0.01 mg/kg, ip) and the muscarinic agonist oxotremorine (6.25 and 12.5 /zg/kg, ip) administered simultaneously, in doses which had no effect on their own. Moreover, an amnesic dose of atropine (10.00 mg/kg, ip) prevented the enhancement of retention induced by naloxone, while an amnesic dose of morphine (I.00 mg/kg, ip) did not modify the facilitatory effect of oxotremorine (50/zg/kg, ip) on retention. An inhibitory modulatory role for endogenous opioid systems on the activity of central cholinergic muscarinic systems during memory consolidation is suggested.

Evidence is accumulating that the opioid brain peptides may participate in the regulation of learning and memory processes (Izquierdo et al., 1981; Izquierdo, 1982a; Kastin, Scollan, King, Schally, & Coy, 1976; Member of the Carrera del Investigador Cientffico (CON1CET). z The authors wish to express their thanks to Dra. Maria A. Enero for her kind supply of naloxone and for her generous advice, Send requests for reprints to Dr. Carlos M. Baratti. 155 0163-1047/84 $3.00 Copyright © 1984by AcademmPress, lnc All rightsof reproductionIn any form reserved

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Martinez et al., 1981; McGaugh, 1983). Evidence has been obtained using opiate receptor antagonists (Fulginiti & Cancela, 1983; Gallagher & Kapp, 1978; Gallagher, 1982; Izquierdo, 1979; Messing et al., 1979), as well as exogenous opiod alkaloids (Castellano, 1975; Izquierdo, 1979; St~iubli & Huston, 1980) and peptides such as endorphins and enkephalins (Izquierdo, 1980; Izquierdo et al., 1981; Martinez et al., 1981; McGaugh, 1983). Several reports indicated that post-training administration of naloxone, an opiate antagonist, increases retention of both passive and active avoidance conditioning (Gallagher & Kapp, 1978; Gallagher, 1982; Izquierdo, 1979; Messing et al., 1979; Zimmerman, Gorelick, & Colbern, 1980). The effects of naloxone on memory processes are independent of the response requirements of the tasks and of the presence of pain during the training session (Izquierdo, 1979). In addition, the effects of naloxone are blocked by morphine (Izquierdo, 1979; Messing et al., 1979). It has been suggested that naloxone facilitates memory consolidation through release of catecholaminergic systems from a probably opiate-mediated inhibition (Izquierdo & Graudenz, 1980) which is the result of the release of endogenous opioid peptides during or immediately after the training (Izquierdo et al., 1980; Izquierdo et al., 1981). On the other hand, it has been reported that morphine and related drugs inhibit the release of brain acetylcholine in vivo, through a specific action on opioid receptors (Jhamandas & Sutak, 1974; Jhamandas, Hron, & Sutak, 1975). Endogenous opioid peptides methionine-enkephalin, leucine-enkephalin (Hughes et al., 1975), and beta-endorphin (Guillemin, Ling, & Burgus, 1976) also reduce the release of acetylcholine or its turnover in several brain regions through a naloxone- or naltrexonesensitive mechanism (Botticelli & Wurtman, 1979; Jhamandas & Sutak, 1974; Jhamandas et al., 1975; Morony, Cheney, & Costa, 1978; Versteeg, 1980; Wood & Stotland, 1980). Further, Jhamandas and Sutak (1983) showed that naloxone and naltrexone stereospecifically enhance the evoked release of brain acetylcholine. These studies with opioid agonists suggest the possibility that the endogenous opioid agonists may act as inhibitory modulators of cholinergic neurons. If this is true, pharmacological antagonism of the endogenous opioids would enhance the activity of cholinergic neurons. Taking into account the already known participation of the central cholinergic system in memory processes (Baratti, Huygens, Mifio, Merlo, & Gardella, 1979; Baratti, Introini, Huygens, & Gusovsky, 1983; Izquierdo, Baratti, Torrelio, Ar6valo, & McGaugh, 1973; Karczmar & Dun, 1978; Russell, 1982; Squire & Davis, 1981; Zornetzer, 1978) it seems interesting to study a possible opioid-cholinergic interaction on these processes. The present study investigates the effect of the post-training injection of naloxone and morphine on the retention of an inhibitory avoidance response in mice and the possible interaction on this form of memory of morphine

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and naloxone with drugs which are known to affect the cholinergic system both centrally and/or peripherally.

METHODS

Animals. Male Rockland mice (Squibb Laboratories, Argentina) weighing 22-25 g were maintained on a light-dark cycle of 12 hr (lights on 7:00 AM--7:00 PM). All mice were experimentally naive and were only tested once. There were acclimatized to laboratory conditions for at least 3 days before being used for experimental purposes. Food and water were provided ad libitum except during the experimental testing. Apparatus. The step-through inhibitory avoidance apparatus employed was a dark wooden box (35-cm long, 35-cm wide, and 20-cm deep) with a grid floor consisting of bronze bars of 0.3-cm diameter, spaced 0.5 cm apart. There was a platform (5 x 5 cm) in front of the box which was illuminated by a 25-W incandescent light positioned 15 cm above the platform. It was divided from the dark compartment by a wall containing a 5 x 5-cm hole. Training and testing procedure. Mice were given one training trial as follows: each mouse was placed on the lighted platform facing away from the dark compartment. When the mouse stepped through the hole off the platform and onto the metallic grid, it immediately received a footshock (0.8 mA, 50 Hz, 1-sec duration) (Baratti et al., 1979: Baratti, Introini, Huygens, et al., 1983). Forty-eight hours later, each mouse was placed on the lighted platform again and its step-through latency was recorded as a measure of retention of the training experience. If a mouse failed to step into the dark compartment within 300 sec, the retention test was terminated, and the mouse was assigned a score of 300. Drugs and experimental groups. The drugs used in these experiments were the opiate antagonist naloxone hydrochloride (Endo Laboratories), the opiate agonist morphine sulfate (Merck), the cholinergic muscarinic agonist oxotremofine sesquifumarate (Aldrich), the cholinergic muscarinic antagonists atropine sulfate and atropine methyl bromide (Merck), and the cholinergic nicotinic antagonists mecamilamine hydrochloride (May & Baker) and hexametonium hydrochloride (Sigma). All doses were calculated as the base. All drugs were dissolved in saline immediately before being used and were given intraperitoneally (10 ml/kg). Controls received the same volume of saline. In the first experiment we examined the effects of the immediate posttraining injection of saline or naloxone (0.01, 0.03, 0.10, 0.30, 1.00, 3.00, and 10.00 mg/kg) to mice that had or had not received a footshock during the training. In another experiment we examined the effect of delayed injections of saline or naloxone (0.10 mg/kg) on retention performance. The purpose of the third experiment was to determine if the behavioral effects of naloxone are mediated by opiate receptors. Thus, different

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groups of mice were injected with saline, naloxone (0.10 mg/kg), morphine (0.5 or 1.00 mg/kg), or naloxone (0.10 mg/kg) plus morphine (either 0.5 or 1.00 mg/kg) as a cocktail immediately after training. In the next experiment different groups of mice were injected with saline, naloxone (0.10 mg/kg), any of the cholinergic blocking agents atropine (0.5 mg/kg), methylatropine (0.5 mg/kg), mecamilamine (5 mg/ kg) or hexamethonium (5 mg/kg), or each of the cholinergic blocking agents plus naloxone (0.10 mg/kg) as a cocktail immediately after training. For a better characterization of the possible interaction between cholinergic and endogenous opioid mechanisms on the behavioral effects of naloxone, we studied the resulting effect of the simultaneous post-training injection of naloxone (0.01 mg/kg) and oxotremorine (6.25 or 12.50/xg/ kg) in doses which had no effect on their own. Further analysis of the possible interaction between cholinergic and opioid peptidergic systems was performed. For this purpose, different groups of mice were injected with saline, atropine (10.00 mg/kg), naloxone (0.10 mg/kg), atropine (10.00 mg/kg) plus naloxone (0.10 mg/kg), morphine (1.00 mg/kg), oxotremorine (50 /xg/kg), or morphine (1.00 mg/kg) plus oxotremorine (50 ~g/kg), immediately after training. Statistical analysis. Data were submitted to a one- or two-way analysis of variance according to the experimental design (Scheff6, 1959; Winer, 1971). Post hoc analysis with the Newman-Keuls' test was made. In all cases p values less than .05 were considered significant (Winer, 1971). RESULTS

Effects of the Immediate Post-training Injection of Naloxone The retention test data obtained in the first experiment are presented in the upper part of Fig. 1. An analysis of variance revealed that naloxone treatment markedly influenced the latencies to step-through during the retention test (F(7, 312) = 2.992, p < .01). Newman-Keuls' multiple comparisons indicated that retention performances varied in a non-monotonic fashion as a function of the dosage of naloxone. The doses of 0.01, 0.30, and 1.00 mg/kg had a small nonsignificant effect on retention performance whereas the doses of 0.03 and 0.10 mg/kg significantly increased retention latencies when compared with the saline control group (p < .05 and < .01, respectively). Thus the dose-retention response curve for naloxone shows an inverted U-shaped form in the range of doses from 0.01 to 1.00 mg/kg; i.e., it facilitated retention at median doses and had no effect at low and high doses. Such curves are typical of drugs that affect learning and memory processes (Gold & McGaugh, 1975). On the other hand, higher doses of naloxone (3.00 and 10.00 mg/ kg) tended to enhance retention. The retention latencies of those mice that were injected with naloxone in a dose of 10.00 mg/kg were significantly higher than saline controls' (p < .05).

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Effect of Delayed Post-training Naloxone Injections Figure 2 shows that the actions of naloxone (0.10 mg/kg) on inhibitory avoidance conditioning in mice are time dependent. A 2 (saline-naloxone)

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× 4 (times) A N O V A s h o w s significant interaction o f t r e a t m e n t x time (F(3, 312) = 2.613, p < .05). N a l o x o n e administered immediately or 10 min after training significantly increased retention latencies (p < .01 and < .05, r e s p e c t i v e l y ) . H o w e v e r , if the injections were given either 30 o r 180 min after training, retention latencies did not significantly differ as c o m p a r e d with their c o n t r o l groups.

Effects of the Immediate Post-training Morphine or Morphine plus Naloxone Injections I n o u r e x p e r i m e n t a l conditions, the post-training injection o f m o r p h i n e (0.5 a n d 1.00 mg/kg) a n t a g o n i z e d the e n h a n c e m e n t o f retention i n d u c e d

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by naloxone (0.10 mg/kg) when administered after training (F(2, 234)e= 5.902, p < .01). Moreover, the dose of 1.00 mg/kg of morphine impaired retention on its own (p < .05, as compared with saline control group). The dose of 0.10 mg/kg of naloxone was unable to antagonize the amnesic effect of morphine (1.00 mg/kg) (Fig. 3).

Effects of Cholinergic Blockers on Naloxone Memory Facilitation The effect of naloxone (0.10 mg/kg) on retention was prevented by atropine (0.5 mg/kg) (F(1, 156) = 5.667, p < .05) but not by methylatropine (F(1, 156) = .171, p > .05) in the same experimental conditions (Fig. 4). The Newman-Keuls' test showed significant differences as compared 30G

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with its control for the groups that received either naloxone (p < .01) or methylatropine plus naloxone (p < .01) but not atropine plus naloxone. Furthermore, the enhancement of retention induced by naloxone was prevented neither by mecamilamine (5 mg/kg) (F(1, 156) = .075, p > .05) nor hexamethonium (5 mg/kg (F(1, 156) = .011, p > .05) (Fig. 4). Mutual Potentiation between Naloxone and Oxotremorine

Low doses of naloxone (0.01 mg/kg) or oxotremorine (6.25 and 12.50 /xg/kg) did not influence retention on their own (Fig. 5), but when they were injected together after training they each potentiated the facilitatory effects on retention of the other (F(2, 234) = 3.079, p < .05). Post hoc analysis with the Newman-Keuls' test showed that naloxone (0.01 mg/ kg) plus oxotremorine (6.25 /z/kg) significantly increased the retention latencies to step-through (p < .05) while naloxone (0.01 mg/kg) plus oxotremorine (12.50/x/kg) tended to increase retention latencies although this increase was not significant (Fig. 5).

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Interaction between Morphine and Naloxone with Drugs which Affect the Cholinergic Muscarinic System Finally we analyzed the mutual interaction between muscarinic and endogenous opioid systems on retention. Figure 6 shows that an amnesic dose of atropine (10.0 mg/kg) prevented the enhancement of retention induced by naloxone (0.10 mg/kg) (F(1, 156) = 8.067, p < .01). On the other hand, an amnesic dose of morphine (1.00 mg/kg) was unable to reverse the enhancement of retention induced by oxotrernorine (50/xg/kg) (F(1, 156) = 4.938, p < .05). DISCUSSION

The effects of post-training injection of naloxone on retention of an inhibitory avoidance response in mice reported in the present work are

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FIG. 6. Effects of the simultaneous administration of naloxone and atropine or morphine and oxotremorine on the retention latencies to step-through. (**) p < .01, as compared with its control group: (00) p < .01, as compared with saline-injected control group; (~r'l') p < .01, as compared with naloxone-injected group; n = 40 mice per group. in agreement with the results obtained by other authors in other experimental situations (Gallagher & Kapp, 1978; Gallagher, 1982; Izquierdo, 1979; Izquierdo, 1980; Izquierdo & Graudenz, 1980; Messing et al., 1979; Zimmerman et al., 1980). N a l o x o n e increased retention in a dose-dependent manner. The d o s e - r e s p o n s e curve exhibits an inverted U-shaped form in the d o s e range from 0.01 to 1.00 mg/kg. T h e s e results are in a c c o r d a n c e with those o f Messing et al. (1979) which were obtained using an inhibitory a v o i d a n c e task in rats. In this dose range the post-training administration o f n a l o x o n e to u n s h o c k e d mice did not modify the latencies to stepthrough 48 hr later. This fact rules out a punishing effect of the drug as an explanation for the o b s e r v e d m e m o r y e n h a n c e m e n t in mice w h i c h

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received footshock and naloxone post-training. On the other hand, higher doses of naloxone tended to increase retention, too. In this case unshocked mice showed a significant increase in their latencies to step-through during the retest 48 hr later. Therefore, an aversive effect of the opioid antagonist may exist which represents an extra punishment given shortly after training, adding to the motivational component of the task (Izquierdo, 1982). We are not aware that this punitive effect of naloxone had been previously reported. The effects of naloxone on retention were time dependent, since the enhancement of retention decreased as the training-treatment interval was increased. These results are in accordance with other authors' (Gallagher & Kapp, 1978; Gallagher, 1982; Messing et al., 1979) and suggest an action of naloxone on mechanisms involved in memory consolidation processes (Gold & McGaugh, 1975). In addition, the time-dependent actions of naloxone rule out a proactive pharmacological effect of the drug at the time of the retention test. Both the lower dose of morphine, which has no effect on retention, and the higher one, which significantly impaired retention, prevented the facilitatory effect of naloxone. Thus, our results suggest that the effects of naloxone on retention are due to an action of naloxone on opioid peptides receptors. It has been reported that morphine releases betaendorphin from the brain (Carrasco et al., 1982), which is also released during the training (Izquierdo et al., 1980). On the other hand, naloxone does not prevent the release of beta-endorphin induced by morphine, but antagonizes the effects of both morphine and beta-endorphin on retention (Izquierdo, 1982). We cannot now say whether, in our experimental conditions, the facilitatory effect of naloxone on retention is directly prevented by morphine or by the beta-endorphin probably released by morphine. The enhancement of retention induced by naloxone was prevented by the simultaneous administration of atropine but not by methylatropine, mecamilamine, or hexamethonium. Previously we observed that these cholinergic blocking agents administered in the doses employed in these experiments had no effect of their own on retention (Baratti et al., 1979; Baratti, 1982). Further, atropine but not methylatropine, mecamilamine, or hexamethonium, prevents the facilitatory effects of both the muscarinic agonist oxotremorine (Choi, Roch, & Jenden, 1973) and the anticholinesterase physostigmine on the retention of the inhibitory avoidance response (Baratti et al., 1979; Baratti, Introini, Gusovsky, & Huygens, 1983). So, the present results suggest that central cholinergic muscarinic systems may participate in the enhancement of retention induced by naloxone. If opioid peptidergic systems and central cholinergic muscarinic systems interact during the memory consolidation process, low doses of naloxone and of the central muscarinic agonist oxotremorine, without

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effects on their own, administered simultaneously, should mutually potentiate each other, and thereby facilitate memory of the inhibitory avoidance task. This hypothesis was fully corroborated in the present paper. There is evidence that endogenous opioids exert an inhibitory influence on acetylcholine release or its turnover in different brain regions (see introduction of this work). Thus, it is possible that the effect of naloxone on retention may be through a blockade of the inhibitory effect on acetylcholine release of endogenous opioids released during or immediately after training. Since, muscarinic agonists which have about the same affinity to pre- and postsynaptic receptors (Yamamura & Snyder, 1976), inhibit acetylcholine release through an interaction with presynaptic receptors (Szerb, 1979), the lack of potentiation between the higher dose of oxotremorine and naloxone observed in this paper may be explained through a mutual compensation between the desinhibition of acetylcholine release induced by naloxone and its inhibition elicited by oxotremorine. More studies are necessary to support this assumption. On the other hand, the muscarinic agonist tended to induce a small but not significant enhancement on retention by itself through an interaction with postsynaptic muscarinic receptors. Further, preliminary evidence indicates that atrophine administered in a nonamnesic dose prevents the effect of the concurrent administration of oxotremorine plus naloxone. Another indication of opioid modulation of central muscarinic influences on memory processes is that atropine, in an amnesic dose, prevented the enhancement of retention induced by naloxone; however, an amnesic dose of morphine did not modify the facilitatory effect of the cholinergic agonist oxotremorine, which directly acts on central muscarinic receptors. In summary, we suggest that in addition to the possible inhibitory modulatory role of endogenous opioids on catecholaminergic systems during memory consolidation (Izquierdo, 1982), endogenous opioid peptides may exert an inhibitory influence on cholinergic systems involved in these processes. In both cases, the post-training administration of naloxone facilitates memory consolidation through an antagonism of the endogenous peptides released during and/or after training, and then enhances the activity of catecholaminergic and/or cholinergic brain neurons at levels compatible with memory consolidation. If true, these suggestions raise interesting possibilities concerning the interaction among peptidergic, cholinergic, and catecholaminergic systems during memory consolidation processes. REFERENCES Baratti, C. M., Huygens, P., Mifio, J. H., Merlo, A. B.. & Gardella. J. L. (1979). Memory facilitation with post-trial injection of oxotremorine and physostigmine in mice. Psychopharmacology, 64, 85-88. Baratti, C. M. (1982). ParticipaciOn de Mecanismos Colindrgieos en Procesos de Aprendizaje y Memoria Animal. Su Posible Interrelaci6n con Mecanismos Monoamingrgicos. Doctoral thesis, Universidad de Buenos Aires, Facultad de Farmacia y Bioqufmica.

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