Roadmap for the expression of canonical and extended endocannabinoid system receptors and metabolic enzymes in peripheral organs of preclinical animal models.

The endocannabinoid system is widely expressed throughout the body and is comprised of receptors, ligands, and enzymes that maintain metabolic, immune, and reproductive homeostasis. Increasing interest in the endocannabinoid system has arisen due to these physiologic roles, policy changes leading to more widespread recreational use, and the therapeutic potential of Cannabis and phytocannabinoids. Rodents have been the primary preclinical model of focus due to their relative low cost, short gestational period, genetic manipulation strategies, and gold-standard behavioral tests. However, the potential for lack of clinical translation to non-human primates and humans is high as cross-species comparisons of the endocannabinoid system have not been evaluated. To bridge this gap in knowledge, we evaluate the relative gene expression of 14 canonical and extended endocannabinoid receptors in seven peripheral organs of C57/BL6 mice, Sprague-Dawley rats, and non-human primate rhesus macaques. Notably, we identify species- and organ-specific heterogeneity in endocannabinoid receptor distribution where there is surprisingly limited overlap among the preclinical models. Importantly, we determined there were no receptors with identical expression patterns among mice (three males and two females), rats (six females), and rhesus macaques (four males). Our findings demonstrate a critical, yet previously unappreciated, contributor to challenges of rigor and reproducibility in the cannabinoid field, which has implications in hampering progress in understanding the complexity of the endocannabinoid system and development of cannabinoid-based therapies.

Roadmap For The Expression Of Canonical and Extended Endocannabinoid System Receptors and Proteins in Peripheral Organs of Preclinical Animal Models.

The endocannabinoid system is widely expressed throughout the body and is comprised of receptors, ligands, and enzymes that maintain metabolic, immune, and reproductive homeostasis. Increasing interest in the endocannabinoid system has arisen due to these physiologic roles, policy changes leading to more widespread recreational use, and the therapeutic potential of Cannabis and phytocannabinoids. Rodents have been the primary preclinical model of focus due to their relative low cost, short gestational period, genetic manipulation strategies, and gold-standard behavioral tests. However, the potential for lack of clinical translation to non-human primates and humans is high as cross-species comparisons of the endocannabinoid system has not been evaluated. To bridge this gap in knowledge, we evaluate the relative gene expression of 14 canonical and extended endocannabinoid receptors in seven peripheral organs of C57/BL6 mice, Sprague-Dawley rats, and non-human primate rhesus macaques. Notably, we identify species- and organ-specific heterogeneity in endocannabinoid receptor distribution where there is surprisingly limited overlap among the preclinical models. Importantly, we determined there were only five receptors (CB2, GPR18, GPR55, TRPV2, and FAAH) that had identical expression patterns in mice, rats, and rhesus macaques. Our findings demonstrate a critical, yet previously unappreciated, contributor to challenges of rigor and reproducibility in the cannabinoid field, which has profound implications in hampering progress in understanding the complexity of the endocannabinoid system and development of cannabinoid-based therapies.

Behavioral effects and pharmacokinetics of gamma-hydroxybutyrate (GHB) precursors gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) in baboons.

RATIONALE: Gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) are prodrugs for gamma-hydroxybutyrate (GHB). Like GHB, GBL and 1,4-BD are drugs of abuse, but their behavioral effects may differ from GHB under some conditions. OBJECTIVES: The first study compared the behavioral effects of GBL (32-240 mg/kg) and 1,4-BD (32-240 mg/kg) with each other and to effects previously reported for GHB (32-420 mg/kg). A second study determined GHB pharmacokinetics following intragastric administration of GHB, GBL, and 1,4-BD. METHODS: Operant responding for food, observed behavioral effects, and a fine-motor task occurred at multiple time intervals after administration of drug or vehicle. In a separate pharmacokinetics study, blood samples were collected across multiple time points after administration of GHB, GBL, and 1,4-BD. RESULTS: Like GHB, GBL, and 1,4-BD impaired performance on the fine-motor task, but the onset of motor impairment differed across drugs. GBL and 1,4-BD dose dependently decreased the number of food pellets earned, but at lower doses than previously observed for GHB. Similar to GHB, both GBL and 1,4-BD produced sedation, muscle relaxation, gastrointestinal symptoms, and tremors/jerks. Administration of GBL and 1,4-BD produced higher maximum concentrations of GHB with shorter times to maximum concentrations of GHB in plasma when compared to GHB administration. CONCLUSIONS: GBL and 1,4-BD produced behavioral effects similar to those previously reported with GHB and the time course of effects were related to blood levels of GHB. Given their higher potency and faster onset of effects, the abuse liability of GBL and 1,4-BD may be greater than GHB.

Physical dependence in baboons chronically treated with low and high doses of diazepam.

Physical dependence on diazepam was evaluated in male baboons chronically treated with either low or high doses of diazepam. Baboons received either a single oral daily administration of a low dose (0.5 mg/kg per day) of diazepam (n=4) or continuous intragastric infusion of a high dose (20 mg/kg per day) of diazepam (n=7). Development of physical dependence during chronic dosing with 0.5 mg/kg per day diazepam was assessed at 2 and 4 weeks and then monthly, during 1-h behavioral observations, following injections of the benzodiazepine competitive antagonist flumazenil. After 3-24 months of diazepam treatment, dosing was discontinued and physical dependence assessed via observation and responding for food pellets. In baboons that received 0.5 mg/kg per day diazepam, flumazenil precipitated a mild- to intermediate-intensity benzodiazepine withdrawal syndrome, which included decreases in the number of food pellets earned per day and increases in withdrawal postures, self-directed behaviors, aggressive behaviors and retching/vomiting. Three of four baboons showed signs of precipitated withdrawal after only 2 weeks of chronic low-dose treatment. Flumazenil continued to precipitate withdrawal signs, but with no systematic increase in severity, throughout the 6-10 months of 0.5 mg/kg diazepam administration. When 0.5 mg/kg per day diazepam dosing was discontinued, the number of food pellets earned per day decreased in two of the four baboons, but no systematic changes in behavioral signs were observed. In contrast, within 7-10 days of termination of 20 mg/kg per day diazepam dosing, withdrawal signs of intermediate intensity and a decrease in the number of food pellets earned per day occurred in all baboons. In the present study, physical dependence developed after 2 weeks of a chronic low dose of diazepam administration but did not increase further over long-term exposure to diazepam.

Zaleplon and triazolam physical dependence assessed across increasing doses under a once-daily dosing regimen in baboons.

The ability of the GABA(A)-receptor-subtype-selective hypnotic zaleplon to produce physical dependence was compared to the nonselective benzodiazepine triazolam. Progressively increasing doses of zaleplon and triazolam were given to baboons by intragastric infusion once each day, with doses increasing every 17 days. Next, the highest dose was given for 10-34 additional days by continuous infusion. Both drugs produced increases in food-maintained lever pressing, ataxia, and time to complete a fine motor task. Plasma levels increased dose-dependently; drug was detectable 24 h after higher doses. Flumazenil produced a mild or intermediate precipitated-withdrawal syndrome on day 14 of all dosing conditions. When drug delivery ended after 85-100 days, a benzodiazepine-type withdrawal syndrome occurred. Physical dependence potential of zaleplon and triazolam appear similar.

Evaluation of the intravenous reinforcing effects of clonidine in baboons.

Clonidine HCl is an antihypertensive that is also sometimes used to alleviate symptoms of withdrawal during narcotics detoxification. Recently, there have been reports abuse of clonidine by methadone patients and opioid abusers. The present study evaluated the intravenous self-administration of clonidine in four baboons. Self-injections were available 24 h/day under a fixed-ratio (FR 120 or 160) schedule of injection with a 3-h timeout after each injection. Doses of clonidine (0.0001-0.056 mg/kg per injection) or its vehicle (saline) were substituted for cocaine (0.32 mg/kg) for at least 15 days. Food pellets were available continuously under a concurrent FR 30 schedule of pellet delivery. Clonidine maintained self-injection greater than its saline vehicle in all four baboons. Although self-injection of clonidine increased as a function of dose within each baboon, there were differences between baboons in the range of doses of clonidine that maintained self-injection. Doses of 0.032 or 0.056 mg/kg maintained peak mean levels of clonidine self-injection which ranged from low (3.2 injections/day) to high (> 6 injections/day) across baboons. Levels of self-injection were similar to vehicle at 0.01 mg/kg clonidine in two of four baboons. However, in the other two baboons, very low doses of clonidine (0.0001-0.001 mg/kg) maintained low to moderate levels of self-injection. Acute administration of clonidine produced signs of sedation including lip droop, drooling and sitting with eyes closed. At high doses, some toxicity was apparent: Baboons were pale and not responsive. Food intake was generally increased in a dose dependent manner. The present study indicates that clonidine functions as a positive reinforcer.

Comparison of the intravenous reinforcing effects of propofol and methohexital in baboons.

Propofol is a widely used intravenous anesthetic that can directly activate and positively modulate the GABA(A)-receptor. Propofol is not currently regulated under the USA Controlled Substances Act. The present study evaluated the intravenous reinforcing effects of propofol compared to the intravenous barbiturate anesthetic methohexital in baboons using a procedure in which doses of the test drug were substituted for a standard cocaine dose. Drug or vehicle was available for self-injection during daily 5.5-h sessions under a fixed-ratio 120 or 160 schedule of reinforcement. A 40-min timeout after each injection limited the maximum of injections per session to eight. Food pellets were available continuously during the session under a fixed ratio 10 schedule of reinforcement. Self-injection of cocaine (0.001-0.32 mg/kg/injection) and vehicle was characterized first. Cocaine maintained self-injection in a dose-dependent manner, with peak injections maintained by 0.32 mg/kg. Vehicle and each dose of propofol (0.1-1.0 mg/kg/injection) and methohexital (0.01-1.0 mg/kg/injection) were substituted for 0.32 mg/kg cocaine for at least 10 sessions. Propofol and methohexital maintained self-injection greater than vehicle in all three baboons, and these effects were dose dependent. Methohexital maintained peak mean levels of self-injection that were >6 injections/day at doses of 0.56 and 1.0 mg/kg, and did not alter food intake systematically. Propofol maintained peak mean levels of self-injection at 1.0 mg/kg that ranged from 2.2 to >6 injections/day across the baboons. Food intake was increased slightly above vehicle levels by propofol self-injection in two baboons, and was decreased in the third baboon. These data indicate that propofol, like methohexital, can function as a positive reinforcer.

Evaluation of limited and unlimited food intake during withdrawal in triazolam-dependent baboons.

Chronic administration of benzodiazepine (BZ) agonists in baboons typically increases food intake, in a dose-dependent manner, during drug administration and suppresses food intake after termination of drug dosing. To determine if suppressed food intake after termination of chronic BZ administration (i.e. withdrawal) was related to increased food consumption during drug administration, the effects of chronic triazolam (1.0 mg/kg/day, intragastrically, for 30-34 days) and subsequent triazolam withdrawal on food intake was studied under two conditions in each of four baboons: (1) when the number of pellets was unlimited; and (2) when the number of pellets was limited so that pellet intake could not increase above the mean number of pellets per day obtained during a preceding vehicle condition. Pellets were available during daily 20-h sessions under a fixed-ratio 10 schedule of reinforcement. All baboons completed both pellet conditions, and the order of exposure was counterbalanced across subjects. During the unlimited pellet condition, pellets per day were increased during triazolam administration and then were suppressed in a time-limited manner when triazolam was discontinued in all four baboons. When pellet intake was limited during triazolam administration, pellet intake after triazolam discontinuation was suppressed in three of four baboons, and the magnitude and duration of suppression was generally less than during the unlimited pellet condition. Other behavioral signs of withdrawal (e.g., tremor/jerk, vomit/retch) were observed in all four baboons under both pellet conditions. These data suggest that the hyperphagic effects of triazolam appear to contribute to the subsequent suppression of food intake during triazolam withdrawal. However, these hyperphagic effects do not account for the entire phenomenon of suppressed food intake during BZ withdrawal.

Alcohol and heightened aggression in individual mice.

The objective of the current research was to study the large individual differences in alcohol effects on aggressive behavior under systematically varied conditions in experimental protocols with mice. Three experiments were conducted with outbred Swiss-Webster derived mice that identified those individuals whose aggressive behavior was reliably heightened by low acute alcohol doses. In all experimental protocols, low alcohol doses were orally administered to a "resident" male mouse that subsequently confronted an "intruder" opponent for 5 min while all salient elements of aggressive behavior and motor activities were quantified. In all three experiments, alcohol (1.0 g/kg) heightened aggressive behavior by at least two standard deviations of the individual's water vehicle control mean in 27% of the mice. In 64% of mice, no reliable change in aggressive behavior was detected after the identical alcohol treatment, and in 9% of the mice alcohol decreased aggressive behavior. Experiments differed in protocol indicating that these aggression-heightening effects were evident in resident mice that were either maintained at restricted or unlimited amounts of food, housed singly or in breeding pairs with a female partner, and conditioned to perform daily a food-reinforced task or remained undisturbed. The first experiment found the aggression-heightening effects to persist during weekly challenges for at least 2 months (n = 8 of 30). The second experiment showed these effects at intervals from 5 to 60 min after alcohol administration. Blood alcohol concentrations reached peak level within 5 to 10 min after oral administration in mice that had confronted an intruder. Those mice in whom alcohol heightened aggressive behavior (n = 21) did not differ from those that showed suppressed levels (n = 9) in terms of blood alcohol concentrations (79.6 vs. 82.4 mg%), suggesting that the intensity and frequency of aggressive behavior after alcohol were not directly dependent on the amount of alcohol in the circulation. The third experiment revealed that alcohol's (0.1 to 5.6 g/kg) effects on heightened aggressive behavior (n = 11) are dissociated from those on concurrently measured high- or low-rate operant performance as engendered by a multiple FR 30-FI 600 sec schedule of reinforcement. Current results indicate that this alcohol effect is relatively specific to aggressive behavior in individual animals, offering the opportunity for neuropharmacological and molecular characterization.

Zolpidem self-injection with concurrent physical dependence under conditions of long-term continuous availability in baboons.

Intravenous zolpidem self-injection, and the concurrent development of physical dependence under conditions of continuous drug availability, was characterized in three baboons. Previously, under similar conditions, 1.0 mg/kg midazolam maintained low, but stable, daily rates of self-injection (e.g. less than 20 injections/day) over 30 or more days and resulted in the development of physical dependence in baboons. In the current experiments, saline and zolpidem (1.0 mg/kg) were available for self-injection under a fixed-ratio (FR-30) schedule of lever-pull responses with a 5 min time-out after each injection. Saline maintained only low levels of responding (i.e. less than five injections per day). Zolpidem maintained an orderly spaced within-day pattern of injections and daily rates of self-injection were higher than saline (i.e. 10 or more injections per day). Daily rates of zolpidem self-injection were relatively stable in two baboons, and increased over time in the third baboon. Substitution of saline for zolpidem produced a rapid decrease in responding that remained low (i.e. less than five injections per day) in all three baboons. Chronic self-injection of zolpidem produced an increase in responding maintained by food pellet delivery and an increase in body weights. Administration of flumazenil (0.1-1.0 mg/kg, i.v.) after at least 35 days of zolpidem self-injection produced postures and behavioral signs typical of a classic flumazenil-precipitated benzodiazepine withdrawal syndrome. Substitution of saline after chronic zolpidem self-injection produced a time-limited spontaneous withdrawal syndrome. Behavioral signs and postures were similar to those observed during flumazenil-precipitated withdrawal and were most prominent during the first 8 days after zolpidem was discontinued. Therefore, like midazolam, zolpidem maintained self-injection and physical dependence developed under conditions of long-term continuous availability.

Dissociation of consummatory and vocal components of feeding in squirrel monkeys treated with benzodiazepines and alcohol.

The primary aim of the current experiments was to develop methods that engender vocalizations associated with positive social situations comprising affiliative behavior and feeding that could be quantified under controlled laboratory conditions and were sensitive to anxiolytic drugs. Classical conditioning procedures were used to elicit vocalizations during presentation of stimulus lights (i.e., CS condition) previously paired with either preferred foods (e.g., grapes, peanuts, bananas) or standard foods (e.g., monkey chow) as well as during presentation of both food types (i.e., UCS condition). When compared to the period before stimulus light presentation (i.e., Pre-CS condition), the rate, duration and number of elemental units of food-related "twitter" vocalizations were increased during the CS conditions regardless of food type. Monkeys spent significantly more time oriented toward the food box during the light stimulus that preceded preferred food than for the light stimulus that preceded standard food. However, twitter vocalizations were higher for standard food regardless of the stimulus conditions (i.e., Pre-CS, CS and UCS). Administration of the benzodiazepine full agonist chlordiazepoxide (CDP, 1-10 mg/kg), the partial agonist bretazenil (BRZ, 1-10 mg/kg), the antagonist flumazenil (FLZ, 1-10 mg/kg) and ethyl alcohol (EtOH, 0.1-1.0 g/kg) differentially altered vocalizations. Although CDP and BRZ increased feeding of standard food, twitters were reduced across stimulus conditions. CDP and BRZ did not alter other social contact calls (i.e., "peeps"). FLZ also reduced twitters without altering peeps, but did not increase feeding. In contrast, EtOH did not increase feeding or peeps, but did increase food-related twitters. These results indicate that there is a dissociation between food-related behaviors, such as food consumption and orientation towards the food source, and vocal behaviors associated with group communication during feeding.

Zolpidem physical dependence assessed across increasing doses under a once-daily dosing regimen in baboons.

The current study examined behavioral effects and possible development of physical dependence after once-daily doses of zolpidem (0, 1.0, 3.2, 10.0, 32.0 mg/kg intragastrically [i.g.]) in three baboons. Each dose was administered for 17 days and then the dose was increased; the 32.0 mg/kg dose was administered for 27 days. Baboons had access to food pellets for 20 hr/day beginning 15 min after dosing. Each day, baboons were presented with a fine motor task. Observation sessions were conducted 1 hr after dosing on days 1, 10, 12 and 14 of each dose condition and after termination of drug dosing. On days 10 and 14 of each dose condition, vehicle and flumazenil (5 mg/kg i.m.) were administered, respectively. Zolpidem increased the number of pellets obtained by two of three baboons. Vomit and/or retch and grimace (signs believed to be indicative of abdominal discomfort) were observed in one or two baboons during all zolpidem dose conditions (1.0-32.0 mg/kg). Time to complete the fine motor task increased dose-dependently in all three baboons, and incoordination was observed during the task in two baboons at 10.0 and 32.0 mg/kg. Analysis of blood plasma showed that measurable levels of zolpidem were present 24 hr after dosing in all drug conditions. The signs of flumazenil-precipitated withdrawal were summarized on a 9-point scale. Scores ranged from 1 to 5 in the 1.0 mg/kg condition, from 2 to 5 in the 3.2 and 10.0 mg/kg conditions and from 4 to 6 in the 32.0 mg/kg condition. Signs that were considered intermediate in severity were observed. Specifically, tremor, jerk and/or rigidly braced posture was observed in one baboon at 1.0 mg/kg, two baboons at the next two doses and all three baboons at 32.0 mg/kg. Vomit and/or retch also occurred in two baboons at dose conditions above 1.0 mg/kg. Discontinuation of zolpidem dosing after 78 to 79 days resulted in mild withdrawal signs (e.g., number of pellets obtained were lower and number of 1-min intervals increased in which eyes were closed, or in which lying down, head lower than torso posture and/or withdrawn posture were observed) on the first day in two baboons. The peak withdrawal scores were 4 or 5 on days 5 to 10; two baboons vomited and/or retched and all three baboons showed tremor, jerk and/or rigidly braced posture. Thus, zolpidem produced physical dependence under once-daily dosing conditions, and the severity of the withdrawal syndrome can be characterized as intermediate.

Stable low-rate midazolam self-injection with concurrent physical dependence under conditions of long-term continuous availability in baboons.

The current research was undertaken to characterize intravenous midazolam self-injection and the concurrent development of physical dependence under conditions of continuous drug availability. Baboons (n=6) i.v. self-injected midazolam under conditions of continuous availability under a fixed-ratio 30 schedule of lever-pull responses with a 5-min time-out after each injection. Midazolam (1.0 mg/kg) maintained an orderly spaced within-day pattern of injections and low, but stable, daily rates of self-injection over 30 or more days (e.g. <20 injections/day). Sequential substitution of saline and then midazolam produced rapid extinction and then reinstatement of responding at the same stable rate. In subsequent manipulations, a range of lower doses of midazolam (0.0156-0.25 mg/kg) were also shown to reinstate self-injection responding after extinction on saline; however, both chronic and acute dose manipulations indicated that dose-regulation was poor. Chronic self-injection of the high dose (1.0 mg/kg) but not lower doses produced a suppression in responding maintained by food pellet delivery. Chronic self-injection of 1.0 and 0.25 mg/kg midazolam produced physical dependence as reflected in classic benzodiazepine spontaneous and flumazenil-precipitated withdrawal syndromes, including tremor, vomiting and, in one instance, seizure. The stable, low-rate self-injection of midazolam, with concurrent development of physical dependence, demonstrated in the present study may provide a useful model system for investigating factors which contribute to long-term inappropriate use of benzodiazepines by physically dependent patients.

Benzodiazepine self-administration in humans and laboratory animals--implications for problems of long-term use and abuse.

Drug reinforcement may represent the primary behavioral-pharmacological mechanism underlying two types of problematic use of benzodiazepines--recreational abuse by polydrug abusers and inappropriate chronic use by patients. High dose polydrug abuse for the purpose of getting high is readily recognized as a significant social problem. Inappropriate chronic benzodiazepine use is more subtle but relatively common: for anxiolytics, 36% of past-year users (3% of the adult population in the US) report using these drugs for 4 consecutive months or longer. The risks of such long-term use are much better documented than the benefits. This paper provides a current review of various problems that have been identified with the long-term use and the recreational abuse of benzodiazepines, including memory impairment, risk of accidents, falls and hip fractures in the elderly, a withdrawal syndrome, brain damage, overuse in the elderly, overuse by chronic pain patients, overuse by alcoholics and recreational abuse among alcoholics and polydrug abusers. A comprehensive review of the literature on benzodiazepine reinforcing effects in humans and laboratory animals is also provided. Drug self-administration studies in humans and laboratory animals provide models of both types of problematic benzodiazepine use. Recreational abuse of benzodiazepines has been modeled in human research with polydrug abusers and in laboratory animal studies, which show that the reinforcing effect of benzodiazepines is intermediate relative to other sedative compounds and is increased in subjects with histories of previous sedative drug self-administration. The problem of inappropriate long-term use of benzodiazepines by people without histories of drug abuse has been partially modeled in human studies showing that benzodiazepines function as reinforcers in subjects with anxiety, insomnia, and histories of moderate alcohol consumption, and in preclinical studies showing stable, low-rate benzodiazepine self-injection with concurrent physical dependence under conditions of continuous availability. Both human and animal research suggests that the drug history and current behavioral context may be important in the establishment of benzodiazepines as reinforcers. Limited human and animal research provides little support for the common belief that physical dependence enhances benzodiazepine reinforcement.

Primate vocalizations during social separation and aggression: effects of alcohol and benzodiazepines.

The most common group of squirrel monkey vocalizations, peeps, are emitted during different social situations including social separation, affiliative interactions, feeding and aggressive confrontations. The present experiments investigated whether peeps and other vocalizations emitted during different social contexts are pharmacologically altered in a similar manner. First, vocalizations were characterized during (1) social separation in juveniles, and (2) "resident-intruder" aggressive confrontations between dominant monkeys from different social groups. Then, the effects of alcohol (EtOH) and the benzodiazepine chlordiazepoxide (CDP) on vocalizations during social separation and during aggression were examined. Isolated juveniles emitted only one type of call, the isolation peep. Resident monkeys primarily emitted peeps, but also emitted cackles, chucks, noisy calls and pulsed calls. Aggressive peeps were similar in structure and frequency (kHz) to isolation peeps, but were shorter in duration. At the same doses, both CDP (0.3-3 mg/kg) and EtOH (0.1-1.0 g/kg) reduced explosive motor behaviors and isolation peeps in juvenile monkeys during social separation and increased threat displays and aggression peeps in resident monkeys during confrontations with an intruder monkey from a different social group. Thus, similarly structured vocalizations that were emitted during social separation and aggression were very sensitive to EtOH and CDP, but the social context determined the direction and magnitude of effects.

Aggression, anxiety and vocalizations in animals: GABAA and 5-HT anxiolytics.

A continuing challenge for preclinical research on anxiolytic drugs is to capture the affective dimension that characterizes anxiety and aggression, either in their adaptive forms or when they become of clinical concern. Experimental protocols for the preclinical study of anxiolytic drugs typically involve the suppression of conditioned or unconditioned social and exploratory behavior (e.g., punished drinking or social interactions) and demonstrate the reversal of this behavioral suppression by drugs acting on the benzodiazepine-GABAA complex. Less frequently, aversive events engender increases in conditioned or unconditioned behavior that are reversed by anxiolytic drugs (e.g., fear-potentiated startle). More recently, putative anxiolytics which target 5-HT receptor subtypes produced effects in these traditional protocols that often are not systematic and robust. We propose ethological studies of vocal expressions in rodents and primates during social confrontations, separation from social companions, or exposure to aversive environmental events as promising sources of information on the affective features of behavior. This approach focuses on vocal and other display behavior with clear functional validity and homology. Drugs with anxiolytic effects that act on the benzodiazepine-GABAA receptor complex and on 5-HT1A receptors systematically and potently alter specific vocalizations in rodents and primates in a pharmacologically reversible manner; the specificity of these effects on vocalizations is evident due to the effectiveness of low doses that do not compromise other physiological and behavioral processes. Antagonists at the benzodiazepine receptor reverse the effects of full agonists on vocalizations, particularly when these occur in threatening, startling and distressing contexts. With the development of antagonists at 5-HT receptor subtypes, it can be anticipated that similar receptor-specificity can be established for the effects of 5-HT anxiolytics.

Defeat engenders pentylenetetrazole-appropriate responding in rats: antagonism by midazolam.

Defeat and the threat of defeat by an aggressive conspecific is stressful and may engender an anxiety- or fear-like state in animals; the present experiment investigated whether defeat generalized to the discriminative stimulus properties of PTZ and how benzodiazepine receptors were involved in this generalization. Separate groups of male Long-Evans rats (Rattus norvegicus) were trained to discriminate 20 mg/kg pentylenetetrazole (PTZ) or 0.4 mg/kg midazolam (MDZ) from saline in a two-choice drug-discrimination task. After establishing stimulus control, PTZ- and MDZ-trained rats were exposed to an aggressive conspecific which resulted in defeat, as defined by the display of defensive and submissive postures as well as audible and ultrasonic vocalizations. Administration of saline after defeat resulted in greater than 80% PTZ lever selection in 15 out of 25 PTZ-trained rats; this effect was attenuated through pretreatment with MDZ (1 mg/kg). Furthermore, short-term defeat substitution for the PTZ discriminative stimulus was not accompanied by long-term changes in the post-defeat generalization curves for PTZ and MDZ when compared to pre-defeat generalization curves. Nor did defeat alter the antagonism of PTZ by diazepam (2.5 mg/kg) or MDZ by flumazenil (10 mg/kg). In order further to characterize the necessary features for defeat substitution for the PTZ discriminative stimulus, exposure to a threatening conspecific was also attempted by PTZ-trained rats protected from physical contact with a wire mesh cage. In these tests, saline continued to engender greater than 50% PTZ lever responding in 15 of 25 rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol, benzodiazepine-GABAA receptor complex and aggression: ethological analysis of individual differences in rodents and primates.

Research in animals has only recently been successful in reliably mimicking the long-established link between alcohol and heightened aggressive behavior. The present review highlights the large individual differences in the effects of acute low alcohol doses on aggressive behavior in rodent and primate species, paralleling the human condition. Subpopulations of both species show reliable and repeatable enhancement of aggressive behavior when administered low, acute alcohol doses. Statistical analysis of the temporal patterns of aggressive behavior indicate that alcohol prolongs aggressive bouts or "bursts" and increases the number of aggressive behaviors within each burst. However, the latency to initiate attack and the time between aggressive bursts are relatively unaltered by alcohol. These alcohol-induced increases in aggression can be potentiated by benzodiazepine agonists and prevented by antagonists. In addition, highly aggressive animals can be differentiated from nonaggressive ones at the GABAA-benzodiazepine receptor complex. These data suggest an important link between alcohol, aggression and the GABAA-benzodiazepine receptor complex.

"Anxiolytic" and "anxiogenic" benzodiazepines and beta-carbolines: effects on aggressive and social behavior in rats and squirrel monkeys.

Ethopharmacological studies on the behavior of socially housed rats and squirrel monkeys were conducted to explore the role of the benzodiazepine GABAA-coupled ionophore receptor complex in aggressive and social interactions. Benzodiazepine receptor (BZR) antagonists, ZK 93426 (1-10 mg/kg) and flumazenil (3-10 mg/kg), the partial agonist, ZK 91296 (1-10 mg/kg) and the partial inverse agonists Ro 15-4513 (0.3-10 mg/kg), were administered to (1) squirrel monkeys prior to 1 h focal observations within established social groups or to (2) resident male rats before confrontations with a naive male intruder in their home cage for 5 min. Aggression was modified in a similar manner in both species, although squirrel monkeys were more sensitive to BZR challenges. Specifically, resident male rats showed dose dependent reductions in attack bites directed at intruder males that were significant at the highest dose of ZK 93426 (10 mg/kg). In squirrel monkeys, ZK 93426 (3 and 10 mg/kg) reduced aggressive grasps, threats and displays, as well as reducing the duration of being the target of aggression from untreated group members (1-10 mg/kg). The BZR partial agonist, ZK 91296 and the antagonist, flumazenil produced few effects on social behavior, low and high intensity aggression and motor activity in both species. Flumazenil (10-30 mg/kg) and ZK 91296 (10 mg/kg), but not ZK 93426, produced significant increases in foraging and feeding behaviors in squirrel monkeys. The hyperphagic effects of ZK 91296 and flumazenil, that are typical of BZR agonists compounds, were not observed in rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of the pro-aggressive effects of alcohol in rats and squirrel monkeys by benzodiazepine receptor antagonists.

Pharmacological manipulations at the benzodiazepine-GABAA-chloride ionophore receptor complex modify some of the behavioral and physiological actions of alcohol (ethanol). The interactions between alcohol, benzodiazepines and aggression were examined in similar ethopharmacological studies in squirrel monkeys and in rats in confrontations with conspecifics. Dominant male squirrel monkeys were tested (1) within their social groups, and (2) in dyadic confrontations with "rival" males from a different social group, and resident male rats were tested in their home cage in confrontations with an inexperienced male intruder. Low doses of alcohol (0.1-0.3 g/kg) increased aggressive behaviors in dominant squirrel monkeys and a subgroup of resident rats, whereas high doses of alcohol (1-3 g/kg) decreased aggression and produced marked motor incoordination. Individuals that showed alcohol-enhanced aggression were selected, and pretreated with benzodiazepine antagonists (flumazenil, ZK 93426) prior to alcohol administration. Both ZK 93426 (3 mg/kg) and flumazenil (10 mg/kg) blocked the aggression-enhancing effects of alcohol in dominant squirrel monkeys and resident rats in confrontations with conspecifics. Neither compound altered the reductions in aggression and increases in inactivity produced by high doses of alcohol. Interestingly, agonist-like increased feeding and inverse agonist-like reductions in social behaviors were observed simultaneously at the same dose of flumazenil, in the same individual and testing situation. ZK 93426 did not alter feeding but also reduced social behaviors. The two antagonists were also not equipotent in their interactions with alcohol. ZK 93426 reduced alcohol-induced motor incoordination in squirrel monkeys, whereas flumazenil did not. In fact, flumazenil potentiated the effects of low doses of alcohol.(ABSTRACT TRUNCATED AT 250 WORDS)