Involvement of glutamate receptors within the central nucleus of the amygdala in naloxone-precipitated morphine withdrawal-induced conditioned place aversion in rats

Chronic use of morphine leads to physical and psychological dependence. The amygdala is known to be involved in the expression of emotion such as anxiety and fear, and several studies have shown that the central nucleus of the amygdala (CeA) is involved in morphine dependence. In the present study, we investigated the role of glutamate receptors within the CeA in the negative affective component of morphine abstinence by evaluating naloxone-precipitated withdrawal-induced conditioned place aversion (CPA) in morphine-dependent rats. We found that microinjection of the AMPA/kainate-glutamate-receptor antagonist CNQX (30 nmol/side) into the bilateral CeA significantly attenuated the naloxone-precipitated withdrawal-induced CPA, as well as several somatic signs, in morphine-dependent rats, without preference or aversive effects by itself in non-dependent rats. Furthermore, microinjection of the non-competitive NMDA-receptor antagonist MK-801 (30 nmol/side) or competitive NMDA-receptor antagonist D-CPPene (0.01 and 0.1 nmol/side) into the CeA significantly attenuated the naloxone-precipitated morphine withdrawal-induced CPA, but not somatic withdrawal signs. These results suggest that the activation of AMPA /kainate and NMDA receptors within the CeA play a crucial role in the negative affective component of morphine abstinence.     

Involvement of noradrenergic system within the central nucleus of the amygdala in naloxone-precipitated morphine withdrawal-induced conditioned place aversion in rats

Rationale We previously reported that the central nucleus of the amygdala (CeA) plays a crucial role in the negative affective, rather than somatic, component of morphine withdrawal. However, numerous studies have reported that the central ascending noradrenergic system is implicated in morphine withdrawal syndrome, although the roles of the noradrenergic system within the CeA in the negative affective component remains less clear. Objectives The possible role of the noradrenergic system within the CeA in the negative affective component of morphine withdrawal was investigated. Methods The extracellular noradrenaline level within the CeA during naloxone-precipitated morphine withdrawal was measured using an in vivo microdialysis experiment on unanesthetized and freely moving rats. The effects of microinjection of β-adrenoceptor antagonists into the bilateral CeA on the naloxone-precipitated morphine withdrawal-induced conditioned place aversion (CPA) and somatic signs were examined. Results The extracellular noradrenaline level within the CeA was transiently elevated during morphine withdrawal. Intra-CeA injections of β-adrenoceptor antagonists propranolol (30 nmol per side) and timolol (10 nmol per side) significantly attenuated the morphine withdrawal-induced CPA. Similarly, β₁-antagonist atenolol (30 nmol per side) or β₂-antagonist butoxamine (30 nmol per side) significantly attenuated the CPA. In contrast, they did not affect morphine withdrawal-induced somatic signs, except for propranolol. Conclusion These results suggest that the activation of the noradrenergic system within the CeA contributes to naloxone-precipitated morphine withdrawal-induced CPA, rather than somatic signs, through β₁- and β₂-adrenoceptors.     

Neurosteroids and reward: allopregnanolone produces a conditioned place aversion in rats

The neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone) has been reported to have rewarding properties in mice tested for place conditioning. Another study found that allopregnanolone reduced dopamine (DA) output in the nucleus accumbens (NAc) of rats. As many rewarding stimuli increase accumbens DA, these results may appear contradictory. Thus, the present study examined the rewarding properties of allopregnanolone in rats tested for place conditioning using an unbiased conditioning procedure. In control studies, a place preference was observed following conditioning with intraperitoneal (2.0 mg/kg) or intracerebroventricular (i.c.v.) (100 microg/0.5 microl) amphetamine. Conditioning with i.c.v. allopregnanolone produced a significant aversion at a dose of 5.0 microg (in 5.0 microl) and a near aversion at 25.0 microg (in 8.3 microl); doses of 0 microg (i.e., vehicle alone, in 10 microl) or 30.0 microg (in 10 microl) produced little effect on place preference. During conditioning, locomotor activity was stimulated by amphetamine using either route of administration, but allopregnanolone had no significant main effect on locomotor activity. Thus, there was a dissociation between the effects of drugs on locomotor activity vs. place conditioning. Results show that i.c.v. amphetamine produces a place preference, whereas allopregnanolone produces either no effect or an aversion, depending on the dose.     

Stimulation of serotonin1B receptors induces conditioned place aversion and facilitates cocaine place conditioning in rats

RATIONALE: Changes in serotonin(1B) (5-HT(1B)) receptor function appear to modify the reinforcing properties of cocaine, but the direction of this effect is not completely clear. Pharmacological stimulation of 5-HT(1B) enhanced the rewarding properties of self-administered cocaine while attenuating the threshold-reducing effect of cocaine in the intracerebral brain stimulation procedure. OBJECTIVE: The present study investigates how pharmacological modification of 5-HT(1B) receptor-mediated neurotransmission influence cocaine motivational properties in the conditioned place preference paradigm in rats. METHODS: In separate groups of rats the motivational properties of CP 94,253, a selective 5-HT(1B) agonist, or GR 127935, a 5-HT(1B/D) receptor partial agonist, given alone or in combination, were determined. To evaluate their influence on cocaine-induced place conditioning, CP 94,253, that was found to be aversive, was given every day before each conditioning session, while GR 127935, which given alone had no effect, was administered only before cocaine conditioning sessions. RESULTS: CP 94,253, injected IP at 2.5 and 10 (but not 0.5) mg/kg produced place aversion in the place conditioning paradigm. The aversive effect of 2.5 mg/kg CP 94,253 was completely reversed by 10 mg/kg SC GR 127935. Given before every conditioning session, CP 94,253 did not modify place conditioning by four injections of 10 mg/kg cocaine but at 2.5 mg/kg it potentiated a sub-threshold dose of cocaine. The place preference caused by these two drugs was completely reversed by 10 mg/kg GR 127935. The antagonism by GR 127935 of CP 94,253's effects was shown not to be due to the induction of state-dependent effects. CONCLUSION: The results suggest that stimulation of 5-HT(1B) receptors causes place aversion, and enhances the effect of low doses of cocaine in the conditioned place preference paradigm.     

Reinstatement of both a conditioned place preference and a conditioned place aversion with drug primes

In two experiments, we report that the place-conditioning paradigm can be used to demonstrate reinstatement of place preference/aversion by a drug prime following extinction training. In Experiment 1, rats were trained to prefer a chamber paired with morphine. Following extinction training, a morphine drug prime reinstated the morphine place preference. In Experiment 2, a lithium-induced conditioned place aversion was reinstated following extinction training by a lithium prime prior to testing. These results indicate that not only do rewarding drug primes produce reinstatement of learned responses (as demonstrated in the drug self-administration paradigm), but also aversive drug primes reinstate aversive learned responses.     

The dopamine antagonist, alpha-flupenthixol, interferes with naloxone-induced place aversion learning, but not with acute opiate dependence in rats.

Pretreatment with the dopamine antagonist alpha-flupenthixol (alpha-flu) interfered with the establishment of a naloxone-induced place aversion in two experiments. In Experiment 1, the potential of pretreatment with alpha-flu to interfere with the establishment of a naloxone-induced place aversion was evaluated in rats administered morphine (Group MN) or saline (Group SN) 24 h prior to the naloxone conditioning trial. Naloxone-precipitated withdrawal from morphine administered 24 h prior to the trial produced a stronger place aversion than produced by naloxone alone. The neuroleptic, alpha-flu, attenuated the naloxone-induced place aversion, but did not selectively interfere with the place aversion produced by acute opiate dependence. Experiment 2 replicated demonstration of interference with naloxone-place aversion learning by neuroleptic pretreatment with the inclusion of saline controls. These results suggest that dopamine modulates either the aversive motivational properties of naloxone or learning, even in opiate na?ve rats.     

Extinction of ethanol-induced conditioned place preference and conditioned place aversion: effects of naloxone

 Four experiments examined the effect of naloxone pretreatment on the expression and extinction of ethanol-induced conditioned place preference (experiments 1, 2, 4) or conditioned place aversion (experiments 1, 3). DBA/2 J mice received four pairings of a distinctive tactile (floor) stimulus (CS) with injection of ethanol (2 g/kg) given either immediately before or after 5-min exposure to the CS. A different stimulus was paired with injection of saline. Pre-CS injection of ethanol produced conditioned place preference, whereas post-CS injection of ethanol produced conditioned place aversion. Both behaviors extinguished partially during repeated choice testing after vehicle injection. Naloxone (10 mg/kg) had little effect on the initial expression of conditioned place preference, but facilitated its extinction. Moreover, repeated naloxone testing resulted in the expression of a weak conditioned place aversion to the CS that initially elicited a place preference. In contrast, naloxone (1.5 or 10 mg/kg) enhanced expression of conditioned place aversion, thereby increasing its resistance to extinction. A control experiment (experiment 4) indicated that repeated testing with a different aversive drug, lithium chloride, did not affect rate of extinction or produce an aversion to the CS previously paired with ethanol. These findings do not support the suggestion that naloxone facilitates the general processes that underlie extinction of associative learning. Also, these data are not readily explained by the conditioning of place aversion at the time of testing. Rather, naloxone’s effects appear to reflect a selective influence on maintenance of ethanol’s conditioned rewarding effect, an effect that may be mediated by release of endogenous opioids. Overall, these findings encourage further consideration of the use of opiate antagonists in the treatment of alcoholism. Received: 4 December 1997 / Final version: 16 February 1998     

D1 receptor blockade stereospecifically impairs the acquisition of drug-conditioned place preference and place aversion

The motivational effects of dopamine (DA) D1 receptor blockade and its influence on the motivational effects of amphetamine (1.0mg/kg s.c.), morphine (1.0mg/kg s.c.) and lithium (40mg/kg s.c.) were studied in a place-conditioning paradigm. Drugs tested were two potent D1 receptor antagonists, SCH 23390 and SCH 39166, that differ in the poor affinity of the latter for 5-HT(2) receptors, and SCH 23388, the inactive enantiomer of SCH 23390. SCH 23390 and SCH 39166, at low doses (12.5 and 25μg/kg s.c.), paired for 30min with one compartment, elicited place aversion. Higher doses of the D1 antagonists or pairing for 60min with one compartment failed to elicit place aversion. SCH 39166 (50μg/kg s.c.) paired with both compartments completely prevented the place-aversion elicited by SCH 23390 (12.5μg/kg s.c.). SCH 23390 and SCH 39166 at low doses (12.5 and 25μg/kg s.c. respectively), paired with both compartments, abolished amphetamine-induced place preference. The D1 antagonists also impaired the acquisition of morphine-induced place preference and lithium-induced place aversion but only at higher doses (50 and 100μg/kg s.c.). These effects were stereospecific as the inactive enantiomer SCH 23388, up to a dose of 500μg/kg s.c. failed to impair the acquisition of amphetamine and morphine-induced place preference. It is concluded that DA plays a dual role in motivation: one role is that of assigning motivational valence to stimuli in relation to changes in DA transmission; another role of DA relates to the learning process involved in the acquisition of positive as well as negative incentive properties by otherwise neutral stimuli (incentive learning).     

Central administration of nociceptin/orphanin FQ blocks the acquisition of conditioned place preference to morphine and cocaine, but not conditioned place aversion to naloxone in mice

Rationale Previous studies have suggested that nociceptin (known also as orphanin FQ) suppresses the rewarding potential of morphine and alcohol in the rat. However, little is known of the effect of nociceptin on the rewarding properties of these and other drugs in the mouse.Objective To determine the effect of nociceptin on opiate or psychostimulant-induced conditioned place preference, or naloxone-induced conditioned place aversion in mice.Methods C57BL6 mice were implanted with chronically indwelling intracranial cannulae targeted at the lateral cerebroventricle through which nociceptin (0.06, 0.6, or 6 nmol) could be administered. Animals were conditioned in an unbiased balanced paradigm to study the effect of nociceptin administration alone, or the effect of nociceptin on the acquisition of place conditioning to morphine, cocaine, or naloxone (all 7.6 mg/kg subcutaneous).Results Administration of 0.06 nmol nociceptin alone stimulated locomotion during conditioning sessions, but had no hedonic effects. In contrast, administration of 6 nmol nociceptin alone markedly reduced basal locomotion during the conditioning sessions and induced a mild place aversion. Both morphine and cocaine induced robust place preferences, the acquisition of which was dose dependently suppressed by administration of nociceptin at doses of 0.6 nmol and above. Conditioning with naloxone produced a robust place aversion that was only weakly blocked by the maximum dose of nociceptin tested.Conclusion Nociceptin blocks the rewarding properties of drugs in both narcotic analgesic and psychostimulant classes in the mouse. In contrast, nociceptin has only a minor effect on the negative affective state experienced following naloxone administration.     

Naltrexone-induced conditioned place aversion following a single dose of morphine in the rat

Limited preclinical research has been conducted investigating the motivational or "affective" properties of withdrawal from acute opioid dependence following a single morphine exposure. Therefore, the purpose of the present study was to pharmacologically characterize the motivational properties associated with naltrexone-precipitated withdrawal after a single injection of morphine using place conditioning. Conditioned place aversion was assessed using a biased two-compartment apparatus and procedure. Adult male Sprague-Dawley rats were given 15 min free access to the entire apparatus on day one to determine initial preferences. Beginning on the second day, combinations of either saline or morphine (1.0-10 mg/kg, s.c.) followed by naltrexone (0.003-3.0 mg/kg, s.c.) 3.75 h later were administered. Rats were then immediately confined to one compartment for 30 min. The next day, rats received the alternative treatment and were confined to the opposite compartment. Twenty-four hours later animals were tested again for 15 min while they had access to the entire apparatus. Morphine followed by naltrexone conditioned significant place aversion (CPA) with just one pairing. This effect was a function of the naltrexone and morphine doses. CPA was also dependent on morphine pretreatment time, with significant aversion only occurring 4 h after morphine pretreatment. Finally continuous morphine administration followed by a single injection of naltrexone resulted in CPA. These data extend the range of behavioral effects associated with antagonist-precipitated withdrawal from acutely administered morphine and suggest that place conditioning is an effective model in assessing motivational aspects of withdrawal from acute opioid dependence in rats.     

Strain differences in the acquisition of nicotine-induced conditioned taste aversion

Lewis (LEW) and Fischer (F344) rat strains differ on a variety of physiological and behavioral endpoints, including reactivity to drugs of abuse. Although they differ in drug reactivity, such assessments are generally limited to morphine and cocaine. To determine if these differences generalize to other drugs, the present study examined these strains for their reactivity to the affective properties of nicotine, specifically their sensitivity to nicotine in the conditioned taste aversion preparation. For four or five conditioning cycles given every other day, rats from both strains were allowed access to saccharin and injected with nicotine (0.1, 0.4, 0.8 mg/kg) or vehicle. On intervening days, all rats were given access to water and injected with vehicle. Under this one-bottle training and testing procedure, neither strain displayed aversions at the lowest dose of nicotine (0.1 mg/kg). Aversions were evident for both strains at 0.4 and 0.8 mg/kg, although the F344 rats acquired the aversions at 0.4 mg/kg faster and displayed a significantly greater aversion at 0.8 mg/kg than subjects from the LEW strain. For both strains, aversions were evident at all doses (and in a dose-dependent manner) when subjects were given access to saccharin and water in a two-bottle test. There were, however, no strain differences on this test. Differences between the two strains in their acquisition of nicotine-induced taste aversions were discussed in the context of aversion assessments with other compounds as well as in relation to differences in the self-administration of nicotine in the two strains.     

Modulation of multiple ethanol withdrawal-induced anxiety-like behavior by CRF and CRF1 receptors

Previous work demonstrated that rats subjected to multiple withdrawals from chronic ethanol exhibit a sensitization of anxiety-like behavior compared to animals withdrawn from treatment with an equal but continuous amount of ethanol. This study sought to examine whether corticotropin-releasing factor (CRF) could modulate this ethanol-withdrawal-induced anxiety-like behavior. Initially, rats were administered with CRF (1 microg) or vehicle intraventricularly on two occasions 5 days apart while on control diet (CD) followed by exposure to 7% ethanol diet (ED) for 5 days, with social interaction assessed 5 h into withdrawal. Social interaction was significantly reduced in the CRF-treated animals compared to vehicle-treated rats and vehicle- and CRF-treated rats maintained on CD, indicative that CRF given before ethanol exposure was capable of inducing an adaptive change that sensitized withdrawal-induced anxiety-like behavior. Next, the CRF(1) receptor antagonist CRA1000 (3 mg/kg, systemically), the CRF(2) receptor antagonist antisauvagine-30 (20 microg intraventricularly), or vehicle was injected 4 h after the ethanol was removed following the first and second cycles of chronic ethanol exposure and the effect on the multiple-withdrawal-induced anxiety-like behavior determined after the third withdrawal cycle. The CRF(1) receptor antagonist blocked the reduced social interaction behavior, whereas the CRF(2) receptor antagonist was without effect. Similar pretreatment with another CRF(1) receptor antagonist CP-154,526 (10 mg/kg systemically) during the first and second withdrawals also counteracted anxiety-like behavior. These findings indicate that the CRF system and CRF(1) receptors play key roles in the adaptive change responsible for the anxiety-like behavior induced by repeated withdrawals from chronic ethanol.     

Handling blocks expression of conditioned place aversion but not conditioned place preference produced by ethanol in mice

Previous findings implicate opioid receptors in the expression of the conditioned rewarding and aversive properties of ethanol. We have recently reported that the conditioned rewarding effect of ethanol is mediated by opioid receptors in the ventral tegmental area (VTA). We attempted to determine whether VTA opioid receptors also mediate the expression of the conditioned aversive properties of ethanol. However, the magnitude of conditioned place aversion (CPA) was not consistent with our previous findings and prevented us from making definitive conclusions. We hypothesized that the handling required to make intracranial infusions in mice alters the expression of CPA, but not conditioned place preference (CPP). Therefore, non-operated animals underwent a Pavlovian conditioning procedure for either ethanol CPA or CPP. Just before testing, half of the animals were held by the scruff of the neck to mimic intracranial infusion handling. Animals conditioned for CPA did not express CPA if they were handled. However, animals conditioned for CPP exhibited robust CPP, regardless of handling. These findings provide additional evidence that the conditioned rewarding and aversive effects of ethanol are mediated by different neural mechanisms.     

Pentylenetetrazol produces a state-dependent conditioned place aversion to alcohol withdrawal in mice

The purpose of this study was to determine if aversive effects of alcohol withdrawal could be detected in mice using the place conditioning procedure and whether the GABA_A receptor antagonist, pentylenetetrazol (PTZ), would increase the aversive effects of alcohol withdrawal and increase the probability of detecting conditioned place aversion. Subjects were alcohol-naïve mice from a specific line selectively bred for low alcohol preference (LAP1; n = 91) and were assigned to three groups: alcohol withdrawal, PTZ alone, and PTZ + alcohol withdrawal. On four trials, mice received either a 4.0 g/kg intraperitoneal (i.p.) injection of alcohol (alcohol withdrawal, PTZ + alcohol withdrawal groups) or saline (PTZ group) 8 h prior to being placed on a distinctive floor texture for a 30-min conditioning session. Five minutes before these sessions, mice in the PTZ and PTZ + alcohol withdrawal groups received PTZ (5.0 mg/kg; i.p.) and the alcohol withdrawal group received saline. On intervening days mice received two saline injections at the same time points prior to being placed on a different floor texture. Post-conditioning floor preference was assessed in two 60-min tests; the first test was drug-free and the second test was state-dependent. Neither alcohol withdrawal nor PTZ produced significant place conditioning. The PTZ + alcohol withdrawal group showed a significant place aversion during the state-dependent test. These data suggest that the combined stimulus properties of PTZ and alcohol withdrawal facilitated the expression of conditioned place aversion to alcohol withdrawal.     

Inhibition of 5-HT neurotransmission increases clonidine protective effects on naloxone-induced conditioned place aversion in morphine-dependent rats.

Previous pharmacological studies have implicated serotonergic brain systems in opiate-withdrawal-precipitated conditioned place aversion. To assess this hypothesis, we tested the effects of either (i). a near-total 5,7-dihydroxytryptamine-induced lesion (90% depletion) or (ii). an acute serotonin (5-HT) inhibition induced by the specific stimulation of 5-HT1A autoreceptors (8-OHDPAT 5-100 microg/kg), on naloxone-induced conditioned place aversion in morphine-dependent rats. Morphine dependence was induced by the implantation of morphine slow-release pellets. The protective properties of clonidine (an alpha-2 adrenergic agonist classically given for opiate detoxification) were also tested after inhibition of 5-HT transmission. Serotonergic lesions in morphine-dependent rats failed to alter naloxone-induced conditioned place aversion but increased the sensitivity to the protective effects of clonidine. Acute neuropharmacological blockade of serotonin transmission also potentiated the clonidine effects on naloxone-induced conditioned place aversion. When combined with the 5-HT1A agonist 8-OHDPAT, clonidine was also found to be more potent. Further understanding of this serotonin/noradrenaline interaction might help devise new therapeutic treatments for the acute opiate withdrawal syndrome.     

Glutamate receptors in the dorsal hippocampus mediate the acquisition,but not the expression,of conditioned place aversion induced by acute morphine withdrawal in rats

Aim:

To evaluate the role of glutamate receptors in the dorsal hippocampus (DH) in the motivational component of morphine withdrawal.

Methods:

NMDA receptor antagonist D-AP5 (5 μg/0.5 μL per side) or AMPA receptor antagonist NBQX (2 μg/0.5 μL per side) was microinjected into DH of rats. Conditioned place aversion (CPA) induced by naloxone-precipitated morphine withdrawal were assessed.

Results:

Preconditioning microinjection of D-AP5 or NBQX into the DH impaired the acquisition of CPA in acute morphine-dependent rats. However, intra-DH microinjection of D-AP5 or NBQX after conditioning but before the testing session had no effect on the expression of CPA.

Conclusion:

Our results suggest that NMDA and AMPA receptors in the dorsal hippocampus are involved in the acquisition, but not in the expression, of the negative motivational components of acute morphine withdrawal in rats.     

Cannabinoid CB(1) receptor antagonist rimonabant attenuates reinstatement of ketamine conditioned place preference in rats

Recent evidence suggests that cannabinoid CB(1) receptors may represent effective targets for therapeutic agents used to treat cocaine and heroin relapse. However, the role of cannabinoid CB(1) receptors in the potential treatment for other drugs of abuse is still largely unknown. The present study was conducted to determine whether cannabinoid CB(1) receptors play a similar role in relapse to ketamine abuse. To establish a ketamine reinstatement model in the conditioned place preference paradigm, rats were trained to develop place preference conditioned by ketamine, which was subsequently extinguished through daily exposure to the test chambers in the absence of ketamine. On the day following the last extinction session, four groups of rats were injected with ketamine (1, 5, 10 and 15 mg/kg, i.p.) to reinstate previously extinguished conditioned place preference. To investigate the effects of rimonabant, a cannabinoid CB(1) receptor antagonist, on reinstatement of ketamine-induced place preference, different doses of rimonabant (0.1, 0.5 and 3 mg/kg, i.p) were injected 30 min prior to the ketamine (5 and 15 mg/kg, i.p.) priming injection in a separate group of rats. To determine whether rimonabant itself produces conditioned place preference or conditioned place aversion, rats were trained for conditioned place preference or place aversion with rimonabant (0, 0.1, 0.5, 3.0 mg/kg, i.p.). While ketamine priming injections reinstated extinguished place preference, rimonabant administration significantly attenuated the reinstatement of ketamine-induced place preference in a dose-dependent manner. Importantly, rimonabant itself did not produce conditioned place preference or place aversion. Since the reinstatement effects of ketamine administration were inhibited by rimonabant, these findings suggest that a cannabinoid CB(1) receptor antagonist may be useful in preventing relapse to ketamine abuse.     

Glutamate receptors in the dorsal hippocampus mediate the acquisition,but not the expression,of conditioned place aversion induced by acute morphine withdrawal in rats

Aim： To evaluate the role of glutamate receptors in the dorsal hippocampus （DH） in the motivational component of morphine withdrawal. Methods： NMDA receptor antagonist D-AP5 （5 pg/0.5 pL per side） or AMPA receptor antagonist NBQX （2 pg/0.5 pL per side） was micro- injected into DH of rats. Conditioned place aversion （CPA） induced by naloxone-precipitated morphine withdrawal were assessed. Results： Preconditioning microinjection of D-AP5 or NBQX into the DH impaired the acquisition of CPA in acute morphine-dependent rats. However, intra-DH microinjection of D-AP5 or NBQX after conditioning but before the testing session had no effect on the expres- sion of CPA. Conclusion： Our results suggest that NMDA and AMPA receptors in the dorsal hippocampus are involved in the acquisition, but not in the expression, of the negative motivational components of acute morphine withdrawal in rats.