Effects of cocaine on conflict behavior in the rat

The present studies examined the effects of acute cocaine administration, chronic cocaine administration and cocaine withdrawal on behavior in the Conditioned Suppression of Drinking (CSD) conflict paradigm, an animal model for the study of anxiety. In daily 10-minute sessions, water deprived rats were trained to drink from a tube that was occasionally electrified (0.25 mA), electrification being signalled by a tone. Within 3-4 weeks, control (i.e., non-drug) CSD behavior stabilized (30-50 shocks and 10-12 ml/session) and drug studies were initiated. Acute administration of cocaine (30-min pretreatment) produced a selective pro-conflict effect only at a dose of 10 mg/kg cocaine, with lower doses (2.5, 5 mg/kg) exerting no effect on CSD behavior and a higher dose (20 mg/kg) depressing both punished and unpunished responding. In a second experiment, cocaine (10 mg/kg, IP, 2/day) or saline was administered to separate groups of subjects for 7 weeks. In this chronic treatment study, CSD testing was conducted 12 hours after each evening cocaine administration. Although it had no effect on CSD behavior during the first week of treatment, this chronic cocaine administration produced a significant and selective pro-conflict effect which was stable during the period from Weeks 2-7. In a final experiment, a high dose of cocaine (20 mg/kg, 3/day) or saline was given to separate groups of subjects for 2 weeks and the behavioral effects of these treatments and their subsequent termination were examined. In this study, CSD testing was conducted 8 hours after each evening cocaine treatment. During the first week of high dose cocaine treatment, a decrease in punished responding was observed; this parameter returned to baseline levels by Week 2. Discontinuation of this high dose chronic cocaine treatment resulted in a selective decrease in punished responding. This pro-conflict effect was greatest at 3 days, and lasted for 6 days after the last cocaine dose. These data are consistent with clinical findings demonstrating the anxiogenic effects of both acute and chronic cocaine treatment as well as cocaine withdrawal and suggest that conflict paradigms such as the CSD may be useful for the study of cocaine-induced anxiety states.     

Do benzodiazepine receptors mediate the anticonflict action of pentobarbital?

The effects of the benzodiazepine antagonist CGS 8216 (2-phenylpyrazolo[4,3-c]quinoline-3(5H)-one) were examined in a thirsty rat conflict test in the presence and absence of pentobarbital. CGS 8216 (2.5-10 mg/kg i.p.) did not affect nonpunished responding, but doses of 5 and 10 mg/kg significantly reduced the rate of punished responding (i.e., the number of 3 second drinking episodes in a "shock" contingency). However, a dose of CGS 8216 which did not significantly alter punished responding (2.5 mg/kg) antagonized the anticonflict actions of pentobarbital. These observations suggest that while high doses of CGS 8216 may elicit an "anxiogenic" response in rodents, lower doses of CGS 8216 antagonize the anticonflict actions of a compound which has been shown to enhance benzodiazepine affinity in vitro. These data imply that the anticonflict actions of pentobarbital may be mediated through benzodiazepine receptors.     

Antipunishment effects of acute and repeated administration of phencyclidine and NPC 12626 in rats.

The effects of phencyclidine (PCP) and NPC 12626 on punished responding were examined using a modified Geller-Seifter procedure in rats. Both drugs are known to antagonize N-methyl-D-aspartate (NMDA) receptor mediated neurotransmission, albeit at different sites on the NMDA receptor complex. Rats were trained to lever press for food reinforcement under a multiple schedule, with responding in one component reinforced under a fixed-interval 60-sec schedule, while each response in the other component resulted in both food and brief electric shock. Both PCP and NPC 12626 produced selective increases in punished responding, although the effects were not as large as those produced by chlordiazepoxide. Repeated daily administration of each of these drugs for 6 days resulted in increases in punished responding during different portions of the treatment. A 5 mg/kg dose of chlordiazepoxide produced increases over the last 2 days of administration. PCP (2 mg/kg) produced an increase only during the second session, whereas NPC 12626 (30 mg/kg) produced increases for all but the first and fifth days of the 6-day regimen. Both competitive and noncompetitive NMDA antagonists can have antipunishment effects in this model.     

Behavioral effects and benzodiazepine antagonist activity of Ro 15-1788 (flumazepil) in pigeons

The selective benzodiazepine receptor antagonist, Ro 15-1788, produced behavioral effects in pigeons at doses at least 100 times lower than those previously reported to possess intrinsic pharmacological activity in mammals. In contrast to its effects in mammalian species, in pigeons, Ro 15-1788 does not exhibit partial agonist activity. Key-peck responses of pigeons were studied under a multiple fixed-interval 3-min, fixed-interval 3-min schedule in which the first response after 3-min produced food in the presence of red or white keylights. In addition, every 30th response during the red keylight produced a brief electric shock (punishment). Under control conditions, punished responding was suppressed to 30% of unpunished response levels. Ro 15-1788 (0.01 mg/kg, i.m.) increased unpunished response rates by 33% without affecting rates of punished responding. Doses of 0.1 to 1.0 mg/kg Ro 15-1788 produced dose-related decreases in both punished and unpunished responding. As is characteristic of other benzodiazepines, midazolam (0.1 and 0.3 mg/kg, i.m.) markedly increased punished responding but had little effect on rates of unpunished responding. Ro 15-1788 antagonized the increases in punished responding and also reversed the rate-decreasing effects of higher doses of midazolam. However, the effectiveness of Ro 15-1788 as a benzodiazepine antagonist was limited by its intrinsic activity: rate-decreasing doses of Ro 15-1788 were unable to completely reverse behavioral effects of midazolam. Midazolam was an effective antagonist of the behavioral effects of Ro 15-1788 (up to 0.1 mg/kg) but midazolam did not influence the rate-decreasing effects of 1.0 mg/kg Ro 15-1788 across a 100-fold dose range. In the pigeon, the behavioral effects of relatively low doses of Ro 15-1788 (0.01-0.1 mg/kg) appear to be related to benzodiazepine receptor mechanisms, whereas other systems appear to be involved in the effects of higher doses.     

Potentiation of the behavioral effects of pentobarbital, chlordiazepoxide and ethanol by thyrotropin-releasing hormone

Effects of pentobarbital, chlordiazepoxide and ethanol were studied alone and in combination with thyrotropin-releasing hormone (TRH), IM, on punished behavior. Key-peck responses of pigeons were maintained by food presentation under a fixed-interval 3-min schedule in which every 30th response produced shock. Moderate doses of pentobarbital, chlordiazepoxide and ethanol increased punished responding to 150-200% of control values while the higher doses of these drugs almost completely eliminated responding. TRH (0.01-1 mg/kg) had little effect on punished responding and 3 mg/kg produced 50% decreases. Although the lower doses of TRH were without effect when given alone, doses of 0.03 mg/kg and greater markedly potentiated the rate-increasing effects of pentobarbital, chlordiazepoxide and ethanol. Increases in punished responding of 350% were obtained with combinations of TRH and these drugs. The rate-decreasing effects of the sedative-hypnotic and anxiolytic compounds were not reversed by TRH. Potentiation of the behavioral effects of sedative-hypnotic and anxiolytic drugs by TRH suggests that TRH may play an important role in modulating the behavioral effects of these compounds and that combinations of neuroactive peptides with certain psychotherapeutic agents may be of some therapeutic value.     

Opposite effects of two ligands for peripheral type benzodiazepine binding sites, PK 11195 and RO5-4864, in a conflict situation in the rat

The effects of two drugs acting at the peripheral type benzodiazepine binding sites, PK 11195 and RO5-4864, were examined in shock-induced suppression of drinking in rats. These two compounds have opposite effects : RO5-4864 (3.1-1205 mg/kg i.p.) enhanced whereas PK 11195 (25-50 mg/kg i.p.) decreased the punished responding, and PK 11195 (6.25 mg/kg, a dose which did not alter the punished responding) blocked the proconflict action of RO5-4864 (6.25 and 12.5 mg/kg). The effects of RO5-4864 and PK 11195 were not antagonized by RO15-1788, a selective antagonist of the central benzodiazepine site. In addition, PK 11195 (6.25 mg/kg) did not reverse the proconflict effect of two beta-carbolines : beta-CEE and FG 7142. AS picrotoxin did not change the punished responding, these data imply that the effects of RO5-4864 and PK 11195 on the one hand and those of chlordiazepoxide and beta-carbolines on the other hand are differentially mediated and suggest that the peripheral type benzodiazepine binding sites are involved in this conflict model.     

Interactions between the benzodiazepine receptor antagonist Ro 15-1788 (flumazepil) and the inverse agonist beta-CCE: behavioral studies with squirrel monkeys

The effects of Ro 15-1788 and ethyl-beta-carboline-3-carboxylate (beta-CCE) were studied alone and in combination on the behavioral performances of squirrel monkeys. Under one procedure, performances maintained by food were suppressed by electric shock presentation (punishment or "conflict" procedure). Under a second procedure, responding was maintained either by food or electric shock delivery under a 5-min fixed-interval schedule. Doses of beta-CCE between 0.1 and 3.0 mg/kg, i.m., produced graded decreases in punished responding which were reversed by pretreatment with Ro 15-1788 (1.0 - 10.0 mg/kg, i.m.). Low doses of beta-CCE (0.03 - 0.3 mg/kg, i.m.) increased responding of monkeys maintained by shock presentation, but did not affect food-maintained responding; higher doses of beta-CCE decreased responding under both schedules. These effects of beta-CCE are opposite those produced by the benzodiazepines under this procedure. Ro 15-1788 (1.0 mg/kg i.m.) antagonized the effects of beta-CCE, producing a shift to the right in the dose-response curves. These findings provide further support for the view that beta-CCE and Ro 15-1788 produce effects mediated by the same benzodiazepine receptor recognition site.     

Withdrawal from chronic phencyclidine produces a pentylenetetrazol-like discriminative stimulus.

Rats trained to discriminate the anxiogenic compound pentylenetetrazol (PTZ) from water were implanted with osmotic mini-pumps containing 7 mg/kg/day phencyclidine (PCP) or water. Rats were tested for generalization to PTZ 24 hours prior to pump removal, and 4 to 96 hours after pump removal. While the pumps were in place, rats did not generalize to PTZ. When the pumps were removed on day 10, rats in the water group did not generalize to PTZ, but 69% of the rats in the chronic PCP group responded on the PTZ lever at 4 and/or 24 hours after pump removal, suggesting that the PCP withdrawal state mimics the interoceptive cue produced by PTZ. This withdrawal phenomenon was repeatable, in that rats that generalized once to PTZ during PCP withdrawal, generalized a second time when the procedure was repeated. In addition, the phenomenon was dose-dependent, as rats that did not generalize to PTZ after 7 mg/kg/day PCP did generalize when the chronic dose of PCP was increased to 10 mg/kg/day. These findings suggest that there is an anxiogenic component of PCP withdrawal and that tolerance does not develop to this effect.     

RO 15-1788 selectively reverses antagonism of pentylenetetrazol-induced discriminative stimuli by benzodiazepines but not by barbiturates

The specificity of ethyl 8-fluro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo (1,5-a) (1,4) benzodiazepine-3-carboxylate (RO 15-1788) in reversing the effectiveness of diazepam and des-methylclobazam, but not of pentobarbital, in antagonizing discriminative stimuli produced by pentylenetetrazol is described. Male hooded rats were trained to discriminate pentylenetetrazol-induced interoceptive discriminative-stimuli (IDS) in a two-lever choice paradigm on an FR10 schedule of food reinforcement. These IDS pharmacologically model verbal report of anxiogenic activity in humans. Diazepam (1,4 benzodiazepine), des-methylclobazam (1,5 benzo-diazepine), and pentobarbital antagonized pentylenetetrazol-IDS. RO 15-1788 neither generalized to nor antagonized pentylenetetrazol-IDS. It also did not cause convulsions in pentylenetetrazol sensitized rats at doses up to 40 mg/kg. It did, however, antagonize the action of diazepam (10 mg/kg) as well as that of des-methylclobazam (160 mg/kg) but not that of pentobarbital. These data suggest that RO 15-1788 is not an anxiomimetic, anxiolytic or a convulsant drug, but it is a specific and effective antagonist of anxiolytic action of benzodiazepines.     

beta-Carboline-3-carboxylate-t-butyl ester: a selective BZ1 benzodiazepine receptor antagonist

The effectiveness of beta-carboline-3-carboxylate-t-butyl ester (beta CCtB) in antagonizing the anticonvulsant, ataxic and antipunishment effects of diazepam were evaluated. In mice, beta CCtB at doses of 3 and 10 mg/kg produced a dose-related antagonism of the anticonvulsant effects of diazepam against pentylenetetrazole (80 mg/kg). A dose of 30 mg/kg of beta CCtB did not produce a further shift in the diazepam dose-effect curve, apparently because beta CCtB failed to block the muscle-relaxant effects of diazepam. Further, beta CCtB (30 mg/kg) failed to antagonize the ataxic effects of diazepam in an inverted screen test. Rats responded under a multiple schedule where in one component every twentieth response (FR20) resulted in water presentation (unpunished component) and in another component every twentieth response (FR20) resulted in both shock and water presentation (punished component). Diazepam p.o. (0.1 to 10 mg/kg) first increased and then decreased rates in the punished component but only decreased rates in the unpunished component. beta CCtB had no effect on response rates when administered alone, but antagonized the rate-increasing effects of diazepam in the punished component. beta CCtB did not alter the rate-decreasing effects of diazepam in either component. Thus, beta CCtB selectively antagonized the effects of diazepam on punished behavior as well as the anticonvulsant effects of diazepam, but beta CCtB failed to antagonize the rate-decreasing and ataxic effects of diazepam. These results are consistent with the interpretation that beta CCtB is a selective BZ1 benzodiazepine receptor antagonist.     

Alcohol reverses the proconflict effect of corticotropin-releasing factor

Alcohol has tension reducing properties in man that are reflected in a release of punished responding in a rat operant conflict test. In contrast, corticotropin releasing factor (CRF), injected centrally produces a suppression of punished and non-punished responding in the conflict test consistent with its hypothesized role in mediating behavioral responses to stress. Alcohol in a dose of 0.75 g/kg reversed the suppressive effects of 0.5 microgram CRF injected intracerebroventricularly on punished responding but augmented the suppression of unpunished responding by CRF. Results suggest that one mechanism for the tension reducing properties of acute alcohol intoxication may involve a suppression of brain CRF systems.     

Anxiolytic-like effect of neuropeptide S in the rat defensive burying

Neuropeptide S (NPS) has been recently identified as the endogenous ligand of a previously orphan G-protein-coupled receptor now named NPSR. Both NPS and its receptor are expressed in the brain, where they modulate different functions. In particular, it has been demonstrated that intracerebroventricular (i.c.v.) injection of NPS in rodents increases wakefulness and promotes anxiolytic-like effects. In the present study we used the defensive burying (DB) test in rats to further investigate the action of human NPS (0.1–10 nmol, i.c.v.) on anxiety-related behaviors. Diazepam (1.5 mg/kg, i.p.) and caffeine (20 mg/kg, i.p.) were used in parallel experiments as standard anxiolytic and anxiogenic drugs, respectively. None of the tested drugs produced statistical differences in the latency to contact the probe, burying behavior latency, number of shocks received or immobility/freezing duration. Caffeine increased cumulative burying behavior and the buried bedding height in a statistically significant manner thus promoting anxiogenic like effects. Opposite results were obtained with diazepam that significantly reduced these behavioral parameters. The anxiolytic-like action of diazepam was mimicked by NPS that reduced cumulative burying behavior in a dose dependent manner. Collectively, robust anxiolytic-like effects were recorded in response to NPS in the DB test. These results are of particular interest since the outcome of this assay is marginally influenced by drug effects on locomotor activity. In conclusion, we provide further evidence that NPS evokes genuine anxiolytic-like effects in the rat; therefore NPSR selective agonists are worthy of development as innovative drugs for the treatment of anxiety disorders.     

Naloxone blocks 'anxiolytic' effects of neuropeptide Y

Intracerebroventricular injection of neuropeptide Y (NPY) produces potent 'anxiolytic' effects in animal models of anxiety. Administration of opioid receptor antagonists suppresses NPY-induced food intake and thermogenesis. The present study examined whether the opiate antagonist naloxone would also suppress the 'anxiolytic' effects of neuropeptide Y. Following training and stabilization of responding in an operant conflict model of anxiety, rats were injected with either NPY or diazepam. Both NPY (veh., 2, 4, 6 microg, i.c.v.) and chlordiazepoxide (veh., 2, 4, 6 mg/kg, i.p.) produced a dose-dependent increase in punished responding in the conflict test. The 'anxiolytic' effects of NPY were not blocked by the administration of flumazenil (3, 6, 12 mg/kg, i.p.). The administration of naloxone (0.25-2.0 mg/kg, s.c.) antagonized the effects of NPY. Central administration of the selective mu opiate antagonist CTAP (1 microg, i.c.v.) partially blocked NPY-induced conflict responding. These results support the hypothesis that NPY may play an important role in experimental anxiety independent of the benzodiazepine receptor and further implicate the opioid system in the behavioral expression of anxiety.     

Augmentation of the NO-cGMP cascade induces anxiogenic-like effect in mice.

Several studies have reported the anxiolytic-like effects of various nitric oxide synthase inhibitors in distinct animal models. However, in the context of anxiety, the possible involvement of cyclic GMP, believed to be one of the main targets of NO, remains obscure. Cyclic GMP is degraded by the specific phosphodiesterases in the brain. Therefore, we studied the effect of the selective phosphodiesterase type 5 inhibitor sildenafil in the mouse elevated plus-maze test of anxiety and in the open field test of locomotion. We found that sildenafil (0.05-10 mg/kg i.p.) alone did not affect the behavior of animals in the plus-maze or open field tests, but the anxiogenic beta-carboline DMCM given in a subconvulsive dose (2 mg/kg i.p.) decreased the time spent on open arms in the elevated plus-maze. Treatment with the NO precursor L-arginine (200 mg/kg i.p.) did not modify the behavior of animals in the plus-maze, however, when sildenafil (1 mg/kg i.p.) was administered in combination with L-arginine (200 mg/kg i.p.), both the time spent on the open arms and the percentage of open arm visits were significantly decreased. We conclude that augmentation of the NO-cGMP cascade induces anxiogenic-like effect in mice.     

Effect of amino acid derivatives of beta-carboline-3-carboxylate on rat behavior

It was shown, that administration of methyl ether N-(beta-carboline-3-carbonyl)-glycine (GA) at dose level of 1-10 mg/kg markedly reduced exploratory behavior and motor activity in the open field test and facilitated manifestation of different rats defend reaction types. Methyl ether N-(beta-carboline-3-carbonyl)-leucine (LA) at the same doses was less effective. Besides GA (10 mg/kg) like earlier described anxiogenic compound FG 7142 suppressed isolation induced muricide behavior of rats. The results obtained combined with literary data allow us to conclude, that GA possesses expressed anxiogenic activity.     

Mechanisms involved in the control of punished responding in mother rats

In a previous study we found that mother rats show more drinking responses than virgins in the punished drinking paradigm, an animal model for anxiety. The present investigation was carried out to determine the possible mechanisms underlying this naturally occurring anticonflict effect. In Experiment 1, we investigated whether the induction of maternal behavior in virgin females (by long-term estrogen and progestin treatment in combination with pup exposure) enhances punished drinking. However, no release from shock-induced (0.25 mA) suppression of drinking was observed in maternally responsive virgins deprived of water for 24 hr. Unlike natural mothers, then, no anticonflict effect is seen in maternal virgins. A considerable body of evidence suggests that facilitation of gamma-aminobutyric acid (GABA) activity in the brain increases punished responding in rats. In the second experiment, therefore, lactating females were injected with pentylenetetrazol, a GABA antagonist, before being monitored for punished drinking. The drug attenuated the enhanced acceptance of shock in mothers, the effect being observed at a dose level that did not reliably affect unpunished responding. Experiment 3 addressed the possible influence of ovarian and adrenal hormones on punished drinking in lactating females. No significant behavioral effects were observed in mothers subjected to adrenalectomy or ovariectomy 4 days before testing.     

Anxiolytic-like effect of neuropeptide Y (NPY), but not other peptides in an operant conflict test.

The peptide messengers neuropeptide Y (NPY), growth hormone-releasing hormone (GHRH), atrial natriuretic peptide (ANP) and beta-endorphin (BEND) were tested in an animal model of anxiety, the Geller-Seifter conflict test. Rats were subjected to a multiple schedule consisting of three components: in the first component, lever-pressing produced food-reward ('unpunished responding'). The second component was a time-out period, during which lever-pressing had no consequences. During the third component, lever-pressing produced food-reward, but was also punished by an incremental foot-shock ('punished responding'). After establishing a stable baseline of both unpunished and punished responding, animals were injected with various doses of NPY, GHRH, ANP, BEND, or with saline into the lateral cerebral ventricle, and testing was repeated. While changes in unpunished responding can reflect alterations in performance factors or motivational strength, increases in punished responding have previously been shown to be highly specific for anxiety-reducing drugs, such as the benzodiazepines. NPY markedly and dose-dependently increased punished responding. A smaller increase of unpunished responding was also seen. These results add further support to the hypothesis that NPY may be an endogenous anxiolytic. GHRH, ANP and END did not affect punished responding.     

Differential response of rat brain polyamines to convulsant agents.

The polyamines putrescine (PUT), spermidine (SD) and spermine (SM) have been studied in rat brain after treatment with several convulsant agents. Kainic acid (10 mg/kg), picrotoxinin (1.5 mg/kg), pentylenetetrazol (60 mg/kg) and lindane (gamma-hexachlorocyclohexane) (60 mg/kg) were given to male Wistar rats. Twenty-four hours later, the animals were sacrificed and their brains removed. Cortical polyamines were analyzed by HPLC with fluorimetric detection of their respective dansyl derivatives, using 1,6-diaminohexane as internal standard for the measurements. Polyamine levels are not affected by short periods of time (30 min) of brain exposure to room temperature before freezing the samples, as compared to a quick procedure (less than 40 s from animal death). Kainic acid induced a 14-fold increase of cortical PUT with respect to control values, leaving unchanged the other polyamines. Lindane also increased cortical PUT (4-fold) without affecting SM or SD. Neither picrotoxinin, nor pentylenetetrazol groups were different from controls for any of the polyamines assayed. The results are discussed in relation to the possible mechanism of action of these convulsant agents and the role of the polyamines in cell injury.     

Effects of acute and chronic nicotine on elevated plus maze in mice: involvement of calcium channels

The current experiments examined the anxiety-related effects of acute and repeated nicotine administration using the elevated plus maze test in mice. Nicotine (0.1 mg/kg s.c., 5 and 30 min after injection; 0.5 mg/kg, s.c., 5 min after injection) had an anxiogenic effect, shown by specific decreases in the percentage of time spent on the open arms and in the percentage of open arm entries. Tolerance developed to this anxiogenic action after 6 days of daily nicotine administration (0.1 mg/kg, s.c.). Five minutes after the seventh injection, an anxiolytic effect was observed, i.e., specific increases in the percentage of time spent on the open arms and in the percentage of open arm entries. L-type voltage-dependent calcium channel antagonists nimodipine (5 and 10 mg/kg, i.p.), flunarizine (5 and 10 mg/kg, i.p.), verapamil (5, 10, 20 mg/kg) and diltiazem (5, 10, 20 mg/kg, i.p.) were also injected prior to an acute low dose of nicotine or to each injection of chronic nicotine. Our results revealed that calcium channel blockers dose-dependently attenuated both an anxiogenic effect of nicotine as well as the development of tolerance to this effect. Our results suggest that neural calcium-dependent mechanisms are involved in the anxiety-related responses to acute and chronic nicotine injection that may ultimately lead to addiction and smoking relapse in human smokers.     

A discriminative two-lever test of dizocilpine's ability to reinstate ethanol-seeking behavior

Because ethanol has N-methyl-D-aspartate (NMDA) antagonist effects, we tested whether dizocilpine, an NMDA antagonist, reinstates ethanol-seeking behavior. Rats were trained to lever-press for a 10% ethanol/2% sucrose (EtOH) or a 3% sucrose (Suc) solution using a two-lever (one lever active) procedure (FR2). After extinction, rats were injected with ethanol (0.5 g/kg). The EtOH group emitted more active than inactive lever presses and the Suc group showed minimal responding. Thus, ethanol reinstated ethanol-seeking behavior in a specific manner. In contrast, dizocilpine (0.175 mg/kg) increased responding on both levers in both groups suggesting a loss of discriminative control. Dizocilpine fails to reinstate ethanol-seeking behavior. These data also demonstrate the necessity of using a discriminative, two-lever test for drug reinstatement.