Effects of pregnanolone and flunitrazepam on the retention of response sequences in rats.

Neuroactive steroids produce effects similar to other GABA(A) modulators (e.g., benzodiazepines and barbiturates) and have a large therapeutic potential; however, a greater understanding of the effects of these substances on learning and memory is needed. To specifically assess the effects of a neurosteroid on memory, pregnanolone (1-18 mg/kg) was administered to male Long-Evans rats responding under a repeated acquisition and delayed-performance procedure in which different 4-response sequences were acquired and then retested after varying delays. Responding was maintained under a second-order fixed-ratio (FR) 2 schedule of food reinforcement, and incorrect responses (errors) produced a 5-sec timeout. For comparison purposes, both a high (flunitrazepam) and low efficacy agonist/antagonist (flumazenil) of the GABA(A) receptor complex were also administered both alone and in combination. Retention of each sequence was quantified as percent savings in errors-to-criterion and this dependent measure was shown to be sensitive to increases in delay. When administered 15 min prior to the end of either a 30- or 180-minute delay, pregnanolone produced both dose- and delay-dependent decreases in percent savings, response rate and accuracy; this effect was selective in that decreases in retention occurred at doses lower than those that disrupted response rate or accuracy. Flunitrazepam (0.056-1mg/kg) produced similar disruptions in retention and these disruptions were antagonized by 5.6 mg/kg of flumazenil. Both an ineffective (0.056 mg/kg) and an effective (0.18 mg/kg) dose of flunitrazepam also potentiated the dose- and delay-dependent disruptions in retention produced by pregnanolone. These data indicate that the neurosteroid pregnanolone disrupts retention in a manner similar to the benzodiazepine flunitrazepam, and suggests that the interaction of flunitrazepam and pregnanolone on retention may be mediated by the GABA(A) receptor complex.

Tolerance to chronic delta-9-tetrahydrocannabinol (Δ9-THC) in rhesus macaques infected with simian immunodeficiency virus.

Although Δ9-THC has been approved to treat anorexia and weight loss associated with AIDS, it may also reduce well-being by disrupting complex behavioral processes or enhancing HIV replication. To investigate these possibilities, four groups of male rhesus macaques were trained to respond under an operant acquisition and performance procedure, and administered vehicle or Δ9-THC before and after inoculation with simian immunodeficiency virus (SIV(mac251), 100 TCID50/ml, i.v.). Prior to chronic Δ9-THC and SIV inoculation, 0.032-0.32 mg/kg of Δ9-THC produced dose-dependent rate-decreasing effects and small, sporadic error-increasing effects in the acquisition and performance components in each subject. Following 28 days of chronic Δ9-THC (0.32 mg/kg, i.m.) or vehicle twice daily, delta-9-THC-treated subjects developed tolerance to the rate-decreasing effects, and this tolerance was maintained during the initial 7-12 months irrespective of SIV infection (i.e., +THC/-SIV, +THC/+SIV). Full necropsy was performed on all SIV subjects an average of 329 days post-SIV inoculation, with postmortem histopathology suggestive of a reduced frequency of CNS pathology as well as opportunistic infections in delta-9-THC-treated subjects. Chronic Δ9-THC also significantly reduced CB-1 and CB-2 receptor levels in the hippocampus, attenuated the expression of a proinflammatory cytokine (MCP-1), and did not increase viral load in plasma, cerebrospinal fluid, or brain tissue compared to vehicle-treated subjects with SIV. Together, these data indicate that chronic Δ9-THC produces tolerance to its behaviorally disruptive effects on complex tasks while not adversely affecting viral load or other markers of disease progression during the early stages of infection.

Relative potency and effectiveness of flunitrazepam, ethanol, and beta-CCE for disrupting the acquisition and retention of response sequences in rats.

Despite the knowledge that gamma-aminobutyric acid(A) modulators can affect learning and memory, their capacity for disrupting each of these complex processes is rarely compared, and often mistakenly assumed to occur with identical potency. For these reasons, the effects of flunitrazepam (0.056-3.2 mg/kg), ethanol (0.25-1.5 g/kg), and ethyl-beta-carboline-3-carboxylate (beta-CCE; 1-17.8 mg/kg) were compared in groups of rats responding under baselines that assessed learning and memory separately. The first baseline was a multiple schedule of repeated acquisition and performance of tandem response sequences, whereas the second baseline was a retention or memory procedure where a tandem response sequence was acquired and then retested after a 30-min delay. Under both procedures, responding was maintained under a second-order fixed-ratio-2 schedule of food reinforcement, and incorrect responding (errors) produced a 5-s timeout. With regard to the effects of the three drugs on sequence acquisition (learning), all three drugs dose dependently decreased the overall response rate and increased the percentage of errors. Both flunitrazepam and beta-CCE affected accuracy more potently than response rate, whereas ethanol was equipotent in affecting these two dependent measures. With regard to the effects of these drugs on sequence retention (memory), both flunitrazepam and ethanol dose dependently decreased retention at doses that had little or no effect on sequence acquisition under the multiple schedule, whereas beta-CCE decreased retention and sequence acquisition similarly at the doses tested. Together, these data show that drugs with differing capacities for altering the function of gamma-aminobutyric acid(A) receptors differ in their capacity for disrupting the acquisition and retention of response sequences and that positive modulation of this receptor complex may be more predictive of disruptions in memory than disruptions in learning.

Estradiol replacement in gonadectomized male rats alters scopolamine-induced disruptions of nonspatial learning.

Recent evidence indicates that testosterone can modulate learning in males through an interaction with the cholinergic system. However, the mechanism for this interaction between testosterone and the cholinergic system on learning remains uncharacterized and may involve several of testosterone's active metabolites. In the present study, two of the active metabolites of testosterone, 5alpha-dihydrotestosterone and estradiol, were administered in combination with the muscarinic receptor antagonist scopolamine (0.1-1 mg/kg, i.p.) to adult gonadectomized male rats that were trained to respond under a multiple schedule of repeated acquisition and performance of response sequences. In the acquisition component, subjects acquired a different three-response sequence each session, whereas in the performance component they responded on the same three-response sequence each session. When scopolamine was administered, it produced greater rate-decreasing and error-increasing effects in gonadally intact subjects than in gonadectomized subjects, even though gonadectomy had little or no effect on these measures under control conditions. In gonadectomized rats receiving 5alpha-dihydrotestosterone replacement, the disruptions produced by scopolamine were also smaller than those produced in gonadally intact subjects. In contrast, gonadectomized rats receiving estradiol replacement were as sensitive, or more sensitive, to scopolamine-induced disruptions of response rate and accuracy than those under the gonadally intact condition. These results suggest that testosterone's interactive effects with the cholinergic system on learning in gonadectomized male rats may not be mediated directly via androgen receptors, but rather by estrogen receptors following the aromatization of testosterone to estradiol.

Differential antagonism of cocaine self-administration and cocaine-induced disruptions of learning by haloperidol in rhesus monkeys.

Six rhesus monkeys responding under a three-component multiple schedule were administered haloperidol to determine its effects on cocaine self-administration and on cocaine's disruptive effects on the repeated acquisition and performance of response chains. In the absence of haloperidol, 0.0032-0.032 mg/kg/infusion of cocaine increased response rate and the number of infusions in the self-administration component when compared to saline administration, whereas 0.1-0.32 mg/kg/infusion decreased response rate and the number of infusions. When compared to saline administration, the two lowest infusion doses of cocaine had little or no effect on responding in the acquisition and performance components; however, higher infusion doses of cocaine dose-dependently decreased response rate in these components. In addition, the higher doses of cocaine also increased the percentage of errors in the acquisition and performance components. Pretreatment with haloperidol (0.0032 or 0.01 mg/kg, i.m.) antagonized the effects of low doses of cocaine on the number of infusions in the self-administration component, whereas only the 0.01-mg/kg dose antagonized the effects of high doses of cocaine on the number of infusions. Neither dose of haloperidol antagonized the rate-decreasing effects of cocaine on responding in the acquisition and performance components significantly; the highest dose of haloperidol alone decreased rates of responding in each component. Antagonism of cocaine's error-increasing effects by haloperidol was only evident at one dose of cocaine (0.032 mg/kg/infusion), and was more complete in the performance components than in the acquisition components. Together, these data show the limited suitability of haloperidol for selectively antagonizing cocaine self-administration in the context of a multiple schedule involving transition behavior, and show the lack of uniform antagonism across operant behaviors.

Overlapping, but not identical, discriminative stimulus effects of the neuroactive steroid pregnanolone and ethanol.

Many behavioral effects of neuroactive steroids are mediated by GABA(A) receptors; however, other receptors might be involved. Ethanol has a complex mechanism of action, and many of the same receptors have been implicated in the effects of neuroactive steroids and ethanol. The goal of this study was to determine whether actions of neuroactive steroids and ethanol at multiple receptors result in similar discriminative stimulus effects. Rats discriminated 5.6 mg/kg of pregnanolone while responding under a fixed-ratio 20 schedule of food presentation. Pregnanolone, flunitrazepam and pentobarbital produced >80% pregnanolone-lever responding. In contrast, neither morphine nor the negative GABA(A) modulator beta-CCE substituted for pregnanolone up to doses that markedly decreased response rates. Ethanol substituted only in some rats; in other rats, ethanol produced <20% pregnanolone-lever responding up to rate-decreasing doses. Thus, substitution of positive GABA(A) modulators, and not morphine or beta-CCE, for pregnanolone in all rats suggests that positive modulation of GABA(A) receptors is important in the discriminative stimulus effects of pregnanolone. Although pregnanolone might have actions at other receptors, in addition to actions at GABA(A) receptors, substitution of ethanol for pregnanolone only in some rats suggests that the mechanisms of action of pregnanolone and ethanol overlap, but are not identical.

Testosterone potentiates scopolamine-induced disruptions of nonspatial learning in gonadectomized male rats.

Whereas research into the effects of the gonadal hormones on learning and memory has primarily focused on estrogen in females, recent evidence suggests that testosterone can also modulate learning in males through an interaction with the cholinergic system. In the present study, the interactive effects of testosterone and scopolamine (0.1- 0.32 mg/kg), a muscarinic receptor antagonist, on complex behavioral processes were investigated in male rats trained to respond under a multiple schedule of repeated acquisition and performance. In the acquisition component, subjects acquired a different 3-response sequence each session, whereas in the performance component, they responded on the same 3-response sequence each session. Although gonadectomy did not disrupt responding in either component, gonadectomized rats were less sensitive to the disruptive effects of scopolamine on both response rate and accuracy. In contrast, after receiving exogenous testosterone replacement, these gonadectomized males were more sensitive to the behavioral disruptions produced by scopolamine (i.e., the effects of scopolamine were similar to those obtained in gonadally intact males). These results suggest that testosterone replacement can enhance scopolamine-induced behavioral effects in gonadectomized male rats responding under a multiple schedule of repeated acquisition and performance, a finding that is in contrast to those previously found for certain spatial tasks. Furthermore, the present findings suggest that testosterone may decrease the activity of the cholinergic system during nonspatial tasks and thereby work in concert with the antagonism produced by scopolamine.

Effects of gamma-hydroxybutyric acid and flunitrazepam on ethanol intake in male rats.

Both gamma-hydroxybutyric acid (GHB) and flunitrazepam are often used illicitly in combination with ethanol. Nevertheless, the effects that these and other drugs of abuse have on the reinforcing effects of ethanol remain inconclusive. To test the effects of GHB and flunitrazepam on contingent ethanol intake, twelve male Long-Evans rats were trained to orally consume ethanol using a saccharin-fading procedure. After training, all animals preferentially consumed ethanol instead of water at each of five ethanol concentrations (0-32%) when tested with a two-bottle preference test in the homecage. Animals then received a noncontingent dose of ethanol (0.32, 0.56, 1, and 1.33 g/kg), flunitrazepam (0.032, 0.1, and 0.32 mg/kg), or GHB (100, 180, 320, and 560 mg/kg) prior to each subject's daily access to ethanol (18% v/v). Noncontingent doses of ethanol decreased ethanol intake, however, the subjects consumed enough ethanol to maintain a consistent total ethanol dose in g/kg. Flunitrazepam did not affect ethanol intake at any dose tested, whereas GHB only affected intake at the highest dose (560 mg/kg), a dose that also produced sedation. These data suggest that there are perceptible or qualitative differences between GHB, flunitrazepam, and ethanol in terms of their capacity for modulating oral ethanol intake in outbred rats.

Effects of GABA modulators on the repeated acquisition of response sequences in squirrel monkeys.

The present study investigated the effects of positive and negative GABA(A) modulators under three different baselines of repeated acquisition in squirrel monkeys in which the monkeys acquired a three-response sequence on three keys under a second-order fixed-ratio (FR) schedule of food reinforcement. In two of these baselines, the second-order FR schedule and the discriminative stimuli for the response sequence were manipulated ("chain-strained" and "tandem-strained"). In the third baseline condition, response-independent tail shock was presented during acquisition of the response sequence. All of these baselines maintained high error levels and produced slow rates of acquisition. Under both the chain-strained and tandem-strained conditions, the positive GABA(A) modulator triazolam (0.0032-0.1 mg/kg) and the negative GABA(A) modulators beta-CCE (ethyl-beta-carboline-3-carboxylate; 0.01-1 mg/kg), beta-CCM (methyl-beta-carboline-3-carboxylate; 0.0032-0.1 mg/kg), and FG-7142 (methyl-beta-carboline-3-carboxamide; 0.18-10 mg/kg) dose-dependently decreased overall response rate compared to administration of saline (control). Under the same two conditions, triazolam and the negative GABA(A) modulators also increased the percentage of errors; however, the effects on accuracy frequently depended on the baseline condition and the particular modulator. In contrast, triazolam only decreased errors and enhanced acquisition in the presence of concurrent response-independent tail shock when compared to saline administration under this condition. The neutral GABA(A) modulator, flumazenil (1 mg/kg), had no effect on rate or accuracy of responding when administered alone, but antagonized the rate-decreasing and error-increasing effects produced by the negative GABA(A) modulators. Together, these data suggest that the effects of both the positive and negative GABAA modulators on acquisition can be similar in squirrel monkeys (i.e., both types of modulator may produce rate-decreasing and error-increasing effects) and that their effects on acquisition depend, in part, on the environmental conditions maintaining acquisition.

Effects of pregnanolone alone and in combination with other positive GABAA modulators on complex behavior in rats.

RATIONALE: Although positive modulators of gamma-aminobutyric acid(A) (GABA(A)) receptors generally produce similar behavioral effects, regardless of which modulatory site on the GABA(A) receptor complex mediates these effects, some differences have been observed between the effects of neuroactive steroids and those of other positive GABA(A) modulators. OBJECTIVE: The current study was designed to compare the behavioral effects of a neuroactive steroid to those of other positive GABA(A) modulators. METHODS: Rats responded under a multiple schedule of repeated acquisition and performance of response chains, with responding maintained under a second-order fixed-ratio 2 schedule of food presentation. RESULTS: Pregnanolone, flunitrazepam, pentobarbital and ketamine, an antagonist at NMDA receptors, dose-dependently decreased response rates and increased the percentage of errors in both components of the multiple schedule. Although the rate-decreasing and error-increasing effects of pregnanolone, pentobarbital and ketamine were quantitatively similar to each other, flunitrazepam was less effective in decreasing response rates and more effective in increasing errors than the other three drugs. A dose of 3.2 mg/kg pregnanolone potentiated the effects of flunitrazepam and pentobarbital, producing 2- to 3-fold shifts to the left in the dose-effect curves. In contrast, pregnanolone did not alter the ketamine dose-effect curves. CONCLUSIONS: The disruptive effects of the neuroactive steroid pregnanolone are qualitatively similar to those of other positive GABA(A) modulators as well as ketamine; however, the potentiation of the effects of flunitrazepam and pentobarbital, and not ketamine, emphasizes the importance of GABA(A) receptors in the behavioral effects of pregnanolone.

Castration in rats impairs performance during acquisition of a working memory task and exacerbates deficits in working memory produced by scopolamine and mecamylamine.

RATIONALE: Although much research has focused on the effects of ovarian hormones on learning and memory in females, less information is available regarding the effects of testicular hormones on learning and memory in males. Additionally, despite evidence of an interaction of testicular hormones and the cholinergic system in areas of the brain implicated in learning and memory, no information is available regarding the behavioral consequences of that interaction. OBJECTIVES: We assessed the effect of castration in male rats on working memory during acquisition of a radial maze. We also assessed the interactive effects of castration and scopolamine, a muscarinic receptor antagonist, as well as mecamylamine, a nicotinic receptor antagonist, on behavior. METHODS: Young adult male rats were castrated or underwent sham surgeries. Beginning 10 days after surgeries, performance on a task of working memory was assessed across 24 days of acquisition in an eight-arm radial maze. Following acquisition, scopolamine and mecamylamine dose-effect curves were established. RESULTS: Castration of male rats significantly decreased arm-choice accuracy during acquisition. Castration significantly exacerbated impairments in arm-choice accuracy produced by scopolamine as well as mecamylamine, without altering the disruptive effects of the drugs on the rate at which rats entered the arms of the maze. CONCLUSIONS: These results indicate that castration in male rats impairs working memory during acquisition of a spatial maze task. Additionally, these results suggest that the absence of testicular hormones increases the sensitivity of male rats to the impairing effects of scopolamine as well as mecamylamine on working memory.

Effects of triazolam, 8-OH-DPAT, and buspirone on repeated acquisition in squirrel monkeys.

The nonserotonergic benzodiazepine, triazolam, was compared with two 5-HT1A receptor agonists, 8-OH-DPAT and buspirone, in squirrel monkeys responding under a repeated-acquisition procedure. In each session, subjects acquired a 4-response sequence by responding sequentially on 3 keys in the presence of 4 discriminative stimuli (colors). Response sequences for each session were maintained by food presentation under a second-order fixed-ratio schedule. Errors produced a brief time-out but did not reset the sequence. In general, all of the drugs produced dose-dependent decreases in overall response rate and increases in the percentage of errors as the cumulative dose was increased. Together, these results indicate that 5-HT1A receptor agonists disrupt learning in squirrel monkeys by producing rate-decreasing and error-increasing effects in a manner comparable with the nonserotonergic benzodiazepine triazolam.

Alcohol unmasks simian immunodeficiency virus-induced cognitive impairments in rhesus monkeys.

BACKGROUND: Alcohol and human immunodeficiency virus (HIV) produce similar neuropathological profiles, including loss of neurons in the frontal cortex. Additionally, HIV-positive patients with a history of alcohol abuse have greater neurologic deficits, and chronic alcohol abuse produces electrophysiological deficits earlier in the HIV disease process. Few studies, preclinical or clinical, have examined whether alcohol administration exacerbates the neuropsychological deficits in subjects with lentiviruses such as HIV. METHODS: To examine the combined effects of alcohol and immunodeficiency viruses (IVs) on neuropsychological functioning, four groups of young adult rhesus monkeys trained to respond under two complex behavioral tasks were administered ethanol 4 days per week via an intragastric catheter for 3 months and then infected with simian immunodeficiency virus (SIV). Behavioral testing after SIV inoculation was conducted in each group (-ethanol [EtOH]/-SIV, -EtOH/+SIV, +EtOH/-SIV, and +EtOH/+SIV) on days when alcohol was not administered to avoid a direct confound and on several occasions when ethanol or sucrose was administered as a probe of the effect of alcohol alone and the effect of caloric supplementation on the food-maintained tasks, respectively. RESULTS: During the days of the week when ethanol was not administered, little or no disruption was observed in either response rate or the percentage of errors (accuracy) across the different treatment groups. In contrast, behavioral testing during alcohol administration revealed that subjects in the various treatment groups had different susceptibilities to ethanol administration. As expected, a two-way ANOVA (ethanol condition, SIV condition) indicated there were significant main effects of ethanol on both response rate and percent errors in both behavioral tasks, but it also indicated there was a significant interaction between ethanol administration and SIV infection on the accuracy of responding in the acquisition (learning) task. In addition, the main effect of SIV on percent errors was in the performance task. CONCLUSIONS: The fact that alcohol administration in SIV-infected monkeys produced greater behavioral deficits than either alcohol or SIV alone further strengthens the supposition that IVs adversely affect neural substrates involved in cognition and that the adverse effects of many central nervous system drugs may be enhanced in IV-infected individuals.

Estrogen improves response accuracy and attenuates the disruptive effects of delta9-THC in ovariectomized rats responding under a multiple schedule of repeated acquisition and performance.

Despite evidence of an interaction between cannabinoids and estrogen in the brain, little information is available regarding the consequences of this interaction on behavior. A within-subjects design was used to examine the effects of estrogen and delta9-tetrahydrocannabinol (delta9-THC) on learning and memory in ovariectomized rats responding under a multiple schedule of repeated acquisition and performance. Treatment with low physiological levels of estrogen, delivered in Silastic capsules, improved response accuracy without affecting response rate during acquisition. Estrogen also attenuated the ability of delta9-THC (0.56- 3.2 mg/kg) to decrease response accuracy and rate during acquisition and response accuracy during performance. Results indicate that estrogen can improve accuracy during acquisition of a nonspatial operant task and can attenuate delta9-THC- induced behavioral deficits.

Effects of fenfluramine, m-CPP and triazolam on repeated-acquisition in squirrel monkeys before and after neurotoxic MDMA administration.

RATIONALE: Establishing functional deficits as a result of neurotoxic dosing regimens of MDMA has been difficult. However, moderate success has been achieved when sensitive animal models and drug challenge have been used together. OBJECTIVE: The present study used a repeated-acquisition technique and dose-effect determinations before, during and after neurotoxic MDMA exposure to characterize the effects of serotonergic drugs on learning, and to determine if MDMA-induced serotonin (5-HT) neurotoxicity is associated with learning deficits as measured by changes in response rate or the percentage of errors. METHOD: The effects of various serotonergic drugs were characterized in six squirrel monkeys responding under a repeated-acquisition procedure before and after neurotoxic dose regimens of MDMA. Specifically, cumulative dose-effect curves for m-CPP (0.032-1 mg/kg), fenfluramine (0.1-3.2 mg/kg) and triazolam (0.0032-0.1 mg/kg) were obtained prior to MDMA administration, with the latter drug serving as a non-5-HT control. RESULTS: In general, all of the drugs tested decreased overall response rate as the cumulative dose increased, whereas only triazolam markedly increased the percentage of errors. MDMA treatment produced significant (80-99%) decreases in brain 5-HT and 5-HIAA axonal markers, but did not lead to changes in either dependent measure of responding or shifts in the dose-effect curves obtained during pharmacological challenges with m-CPP, fenfluramine or triazolam. CONCLUSIONS: Taken together, these results demonstrate that serotonergic drugs can disrupt learning in monkeys, but indicate that MDMA-induced 5-HT neurotoxicity does not lead to disruptions in this particular type of serial learning task.