A comparison of benzodiazepine, serotonin, and dopamine agents in the taste-reactivity paradigm

Previous studies have shown that rats' positive, palatability-dependent consummatory reactions to infused tastes are selectively facilitated by a benzodiazepine agonist (chlordiazepoxide), and that this effect can be blocked by the coadministration of benzodiazepine antagonists (e.g., Ro 15-1788). The purpose of the present study was to determine whether agents acting at other receptor sites (dopaminergic, serotonergic), which have been shown to modulate food consumption, might also modify rats' palatability-dependent reactivity to infused tastes. In this experiment, the benzodiazepine agonist, diazepam, facilitated positive palatability reactions, while dopaminergic agents (haloperidol, apomorphine, amphetamine) had no significant effects on either positive or aversive reactions. The putative 5-HT1A agonists, buspirone and gepirone, had a general inhibitory action on both positive and aversive palatability reactions. These results are surprising in view of the effects of serotonergic and dopaminergic agents on food and fluid intake. Our results suggest that the benzodiazepine receptor system may play a special role in the neural control of appetite through its enhancement of the positive palatability of tastes. Dopamine systems, by contrast, appear to control food intake by modulating processes that are independent of food affect evaluation.     

Chlordiazepoxide directly enhances positive ingestive reactions in rats

Benzodiazepines such as chlordiazepoxide (CDP) promote feeding in a number of species. This effect has been interpreted generally to be an indirect consequence of benzodiazepine anti-anxiety action, although some have questioned whether it might not reflect instead a direct action upon the reinforcing properties of foods. The present study employed a behavioral measure that can discriminate between these possibilities: palatability-dependent consummatory actions elicited in rats by tastes. The results suggest that chlordiazepoxide enhances the positive palatability of tastes selectively while having little or no effect on aversive palatability. The net effect is to make tastes more reinforcing following CDP administration.     

The sedative effects of CL 218,872, like those of chlordiazepoxide,are reversed by benzodiazepine antagonists

The effects of CL 218,872, initially classified as a non-sedative anxiolytic, were investigated and compared with those of chlordiazepoxide in the holeboard. The ability of two drugs that antagonise the effects of benzodiazepines, CGS 8216 and Ro 15-1788, to reverse the effects of CL 218,872 and chlordiazepoxide were also investigated, to see whether their effects might be mediated via benzodiazepine receptors. CL 218,872 (10 mg/kg) was found to be significantly sedative in both mice and rats (i.e., both locomotor activity and head-dipping were significantly decreased). In mice, the effects of CL 218,872 and of chlordiazepoxide were very similar over a range of doses, except that the stimulatory effect seen with low doses of chlordiazepoxide on head-dipping just failed to reach significance with CL 218,872. This study is in agreement with recently published results from different tests showing that sedative effects can be obtained with doses of CL 218,872 that are low and not much higher than those leading to anxiolysis. The sedative effects of both CL 218,872 (10 mg/kg) and chlordiazepoxide (20 mg/kg) were significantly reversed by RO 15-1788 (10 and 20 mg/kg) and CGS 8216 (10 mg/kg), suggesting that their effects are mediated via benzodiazepine receptors. The increase in head-dipping seen with chlordiazepoxide (2.5 mg/kg) was also reversed by RO 15-1788 and CGS 8216.     

Similar effects of ethanol and flumazenil on acquisition of a shuttle-box avoidance response during withdrawal from chronic ethanol treatment.

1. Acquisition of a two-way shuttle-box avoidance response is facilitated by ethanol. This facilitated acquisition of an avoidance response to ethanol was attenuated during withdrawal from chronic-ethanol diet intake (i.e. tolerance developed by ethanol). The deficit in the avoidance task after chronic ethanol treatment could be overcome by increasing the dose of ethanol. 2. Flumazenil, a benzodiazepine antagonist, also facilitated acquisition of the avoidance response in control rats. This response to flumazenil was significantly reduced during withdrawal from chronic-ethanol treatment. This reduced avoidance responding during withdrawal also could be overcome by increasing the dose of flumazenil. 3. The benzodiazepine-inverse agonist, RO 15-4513, produced a deficit in avoidance responding that was antagonized by both ethanol and flumazenil in a dose-related manner. 4. To determine whether flumazenil has the properties of a benzodiazepine agonist, it was established that, unlike the benzodiazepine chlordiazepoxide, flumazenil did not enhance the ethanol-induced deficit in the aerial righting reflex. Additionally, flumazenil blocked the action of chlordiazepoxide in this procedure, consistent with the benzodiazepine antagonist action of flumazenil. 5. Data collected are consistent with the hypothesis that an endogenous substance with the properties of a benzodiazepine-inverse agonist antagonizes the anticonflict actions of acutely administered ethanol during withdrawal from chronic-ethanol exposure.     

Further investigation of the stimulus properties of chlordiazepoxide and zolpidem. Agonism and antagonism by two novel benzodiazepines

The effects of Ro 16-6028 and Ro 17-1812, two novel benzodiazepines with mixed agonist and antagonist properties, were studied in rats trained to discriminate either chlordiazepoxide or the benzodiazepine receptor ligand zolpidem. In rats discriminating 5 mg/kg chlordiazepoxide from saline both Ro 16-6028 and Ro 17-1812 produced responding on the drug lever. At a dose of 10 mg/kg both compounds gave rise to 100% responding on the lever associated with chlordiazepoxide. The dose-response curve produced by Ro 16-6028 was flatter than that for Ro 17-1812, however. The discriminative cue produced by 2 mg/kg zolpidem did not generalise to either Ro 16-6028 or Ro 17-1812. In contrast, both of those compounds antagonised the zolpidem cue and also antagonised the reduction in response rates produced by zolpidem. These results are consistent with previous findings that Ro 16-6028 and Ro 17-1812 may exert anxiolytic-like effects typical of benzodiazepines while antagonising the depressant actions of benzodiazepine receptor ligands. The results are also consistent with the suggestion that the discriminative cues produced by chlordiazepoxide and zolpidem may be mediated, at least partially, by activity at different sub-types of benzodiazepine receptors.     

Drug alterations of punished responding after chlordiazepoxide: possible screen for agents useful in minimal brain dysfunction

In the present study, the effect of various stimulant drugs on the action of chlordiazepoxide to increase punished responding was studied. Drugs such as d-amphetamine, methylphenidate and imipramine that are effective in attentional deficit disorder (MBD) were found to reverse this benzodiazepine-induced increase in responding. Phenobarbital which worsens this condition enhanced the benzodiazepine effect. Since the impairment caused by chlordiazepoxide may be analogous to the lack of impulse control noted in MBD, the bupropion antagonism of this action of chlordiazepoxide suggests that bupropion may be useful in MBD.     

Intrahippocampal injections of benzodiazepine and muscimol impair working memory but not reference memory of rats in the three-panel runway task.

In a three-panel runway task, the benzodiazepine chlordiazepoxide at 3.2 and 10 mg/kg i.p. significantly increased the number of errors (attempts to pass through two incorrect panels of the three panel-gates at four choice points) in a test of working memory, but it had no effect on errors in a test of reference memory. This effect of 10 mg/kg chlordiazepoxide on working memory was blocked by the benzodiazepine receptor antagonist flumazenil at 10 mg/kg. Intrahippocampal injection of chlordiazepoxide at 10 and 32 micrograms/side significantly increased the number of working memory errors. This effect of intrahippocampal chlordiazepoxide (32 micrograms/side) was attenuated not only by flumazenil at 10 mg/kg but also by the gamma-aminobutyric acid (GABA)A receptor antagonist bicuculline at 3.2 mg/kg. Intrahippocampal injection of the GABAA receptor agonist muscimol at 100 and 320 ng/side also significantly increased working memory errors. Neither chlordiazepoxide nor muscimol affected the number of reference memory errors when injected into the hippocampus at doses up to 32 micrograms/side or 320 ng/side, respectively. These results suggest that activation of the GABAA/benzodiazepine receptor complex in the hippocampus impairs working memory, but does not affect reference memory.     

Chlordiazepoxide alters intravenous cocaine self-administration in rats

This investigation was designed to examine the effects of benzodiazepines on intravenous cocaine self-administration in rats. Pretreatment with low doses of the benzodiazepine receptor agonist, chlordiazepoxide (0.3 to 1.0 mg/kg, IP), resulted in small but nonsignificant increases in drug intake with 0.5 mg/kg cocaine, while higher doses (10 mg/kg, IP) significantly decreased drug intake in all rats tested. The effects of chlordiazepoxide on self-administration were attenuated when the concentration of cocaine was increased to 1.0 mg/kg, suggesting that chlordiazepoxide was opposing rather than augmenting the pharmacological actions of cocaine. Pretreatment with the benzodiazepine receptor antagonist, Ro 15-1788 (1.0 to 10 mg/kg, IP), had no effect on self-administration, suggesting that the reinforcing properties of cocaine do not result from direct interactions with benzodiazepine receptors. The result of this investigation demonstrate that chlordiazepoxide alters intravenous cocaine self-administration in rats. Although additional research will be necessary to confirm these data, the results of this investigation suggest that chlordiazepoxide may decrease the reinforcing efficacy of cocaine through indirect actions on dopaminergic neuronal activity potentially mediated through GABAergic mechanisms via benzodiazepine receptor activation.     

A new sensitive method which unlike the Vogel test detects the anxiolytic effect of tofisopam

A new, more sensitive than previously used anxiolytic test is described. The test consists in measuring of inhibition by punishment of drinking water necessary to swallow dry food by very hungry rats. This test reveals the anxiolytic properties of tofisopam, a clinically effective benzodiazepine anxiolytic, and of very low doses of chlordiazepoxide, both ineffective in the Vogel test, as well as anxiolytic properties of high doses of chlordiazepoxide and other anxiolytics, and confirms the lack of anxiolytic effects of major tranquilizers. The results suggest that chlordiazepoxide (and possibly other benzodiazepines) acts on two subgroups of benzodiazepine receptors, named BRI (high affinity) and BR2 (low affinity), while tofisopam acts specifically on BRI receptors. The new test is proposed as a tool for a search for drugs specifically acting on high affinity benzodiazepine BRI receptors.     

Endogenous opioids are necessary for benzodiazepine palatability enhancement: naltrexone blocks diazepam-induced increase of sucrose-'liking'

Opioid agonists and benzodiazepine agonists each increase food intake. Both also increase hedonic 'liking' reactions to sweet tastes in rats. Do opioids and benzodiazepines share overlapping mechanisms of hedonic impact? Or are benzodiazepine and opioid effects on hedonic impact mediated by independent mechanisms? The present study examined whether blockade of opioid receptors prevents benzodiazepine-induced enhancement of taste palatability, as assessed by the affective taste reactivity test. Rats were implanted with oral cannulae, and prior to an oral infusion of bittersweet quinine-sucrose solution, all received i.p. injections of either vehicle, or diazepam alone (5 mg/kg diazepam+0 mg/kg naltrexone), naltrexone alone (1 mg/kg naltrexone+0 mg diazepam), or both diazepam plus naltrexone (5 mg/kg diazepam+1mg/kg naltrexone). Videotaped hedonic ('liking') and aversive ('disliking') orofacial reactions elicited by sucrose/quinine taste were compared across drug conditions. Diazepam administration alone more than doubled hedonic 'liking' reactions to the bittersweet taste, while reducing 'disliking' in half, compared to vehicle levels. Naltrexone by itself had little effect on taste-elicited affective reactions, and only marginally increased aversive gapes. However, naltrexone completely blocked diazepam's enhancement of positive hedonic 'liking' reactions, and naltrexone similarly disrupted diazepam-reduction of aversive 'disliking' taste reactions. These results indicate that endogenous opioid neurotransmission may be crucial to benzodiazepine enhancement of hedonic 'liking' for natural taste reward.     

Reduction by two benzodiazepines and pentobarbitone of the multiunit activity in substantia nigra, hippocampus, nucleus locus coeruleus and nucleus raphé dorsalis of encéphale isolé rats

Multiunit activity was recorded simultaneously in the substantia nigra pars compacta, the pyramidal layer of the hippocampus dorsalis CA 1 area, the nucleus locus coeruleus and the nucleus raphé dorsalis of encéphale isolé rats; drugs were injected intravenously in increasing doses. Chlordiazepoxide decreased multiunit activity dose-dependently to a similar extent in substantia nigra, hippocampus and dorsal raphé, but was less effective in the locus coeruleus. Midazolam reduced multiunit activity in all 4 nuclei to a similar degree and was more potent than chlordiazepoxide. Specific benzodiazepine antagonists completely reversed the effects of the two benzodiazepines. Pentobarbitone was a less potent depressant than chlordiazepoxide, and its effect was not reversed by the benzodiazepine antagonist Ro 15-1788.     

Attenuation of defensive threat and attack in wild rats (Rattus rattus) by benzodiazepines

A battery of tests designed to elicit reactions to a variety of non-painful threat stimuli was used to study the effects of chlordiazepoxide (5–20 mg/kg), diazepam (1–5 mg/kg) and midazolam (1–10 mg/kg) on the defensive repertoire of wild Rattus rattus. The most consistent effect of benzodiazepine treatment, across compounds and tests, was a marked reduction in defensive threat and attack behaviors, with midazolam effective over a wider range of situations. In contrast, effects on freezing and flight reactions were more variable, differing substantially as a function of stimulus context. The general profile of observed changes in defense cannot be explained in terms of either non-specific behavioral suppression or a global reduction in defensiveness. Rather, our findings suggest that benzodiazepines may primarily induce a shift within the defense repertoire.     

Differential EEG effects of the anxiolytic drugs, deramciclane (EGIS-3886), ritanserin and chlordiazepoxide in rats

The influence of serotonergic and benzodiazepine type anxiolytic drugs on the cortical activation and sleep-wakefulness cycle were compared by evaluating the effects of ritanserin and deramciclane (EGIS-3886), two 5-HT₂ receptor antagonists, and chlordiazepoxide on the electroencephalogram (EEG) in freely moving rats. Following drug administration (1, 3, and 10 mg/kg, PO for all drugs), EEG was continuously sampled for 6 h and power spectra were calculated for every 5 s to assess changes in slow wave activity and sleep phases. In a separate test, anticonvulsant effects of the drugs were examined in mice. Both deramciclane and ritanserin slightly increased total time spent in deep sleep (DS) and lengthened sleep episodes. In contrast, chlordiazepoxide had a strong inhibitory action on DS, sleep time being shifted to more superficial light sleep (LS). The incidence and length of the high voltage spindle (HVS) episodes characteristic for the motionless, awake rat were increased at the highest dose of both deramciclane and ritanserin, while it was decreased by chlordiazepoxide. In mice, chlordiazepoxide had a marked anticonvulsant effect, while deramciclane was moderately effective and ritanserin ineffective. In conclusion, the 5-HT₂ receptor antagonist anxiolytic drugs seem to be superior compared to the benzodiazepine type anxiolytic drug, chlordiazepoxide, as ritanserin and deramciclane improved sleep quality by increasing sleep episode length and time spent in DS, while chlordiazepoxide enhanced sleep fragmentation and decreased DS. Received: 1 July 1998/Final version: 4 September 1998     

Role of the periaqueductal gray substance in the antianxiety action of benzodiazepines

In order to study the interactions between serotonergic mechanism and electrical stimulation of the mesencephalic central gray substance, rats were trained to lever-press for terminating aversive electric stimuli applied at the Periaqueductal gray and adjoining tectum of the mesencephalon. Experimental sessions consisted of 40 discrete escape trials of a maximum of 30 sec duration, separated by 30 sec intervals. Dose-effect curves of two tryptamine antagonists, cyproheptadine and methysergide, as well as of the benzodiazepine minor tranquilizer, chlordiazepoxide, on average escape latencies and on frequency distribution of individual latencies were determined. Doses of 3 to 10 mg/kg of cyproheptadine decreased average latencies of escape responding in six of eight rats studied. Doses of 10 and 30 mg/kg of methysergide also facilitated escape responding in one of three rats. In contrast, doses from 1 to 10 mg/kg of chlordiazepoxide, that cause little sedation or ataxia, produced dose-dependent increases in escape latencies. Furthermore, doses of 5.6 and 10 mg/kg of chlordiazepoxide partially blocked escape responding. The facilitatory effects of the tryptamine antagonists suggest that escape behavior is inhibited by brain tryptaminergic mechanisms, whereas the specific depressant effect of chlordiazepoxide on escape from Periaqueductal gray electrical stimulation suggest that this region may be involved in the antianxiety action of benzodiazepines.     

Discriminative stimulus properties of the benzodiazepine receptor partial agonist beta-carbolines abecarnil and ZK 95962: a comparison with chlordiazepoxide

Separate groups of rats were trained to discriminate one of three benzodiazepine receptor ligands from vehicle. The three ligands used, the benzodiazepine chlordiazepoxide, and the beta-carboline partial agonists ZK 95962 and abecarnil, have been reported to have different agonistic profiles. All three ligands formed specific benzodiazepine-receptor mediated discriminative stimuli antagonizable by at least one benzodiazepine antagonist. Different patterns of generalization were observed for each cue. As reported previously full and partial agonists substituted for chlordiazepoxide, whereas generalization to ZK 95962 was obtained more readily with partial agonists and antagonists with weak partial agonist activity. In contrast to the other two cues, the abecarnil discriminative stimulus was difficult to train and was unstable over time. Additionally, the abecarnil cue showed commonalities only with sedative or BZ1 receptor agonists. These results demonstrate qualitative differences between different benzodiazepine receptor ligands dependent on the intrinsic activity of the compound used.     

Benzodiazepine receptor mediation of behavioral effects of nitrous oxide in mice

Nitrous oxide produces behavioral effects, the underlying mechanism of which is not known. In the mouse staircase test, exposure to nitrous oxide caused a reduction in rearing activity, an effect similar to that produced by benzodiazepines in this paradigm, when its opioid action on locomotion is blocked by naloxone. In this study, we tested whether effects of nitrous oxide might be mediated by benzodiazepine receptors, using chlordiazepoxide as a control. The abilities of nitrous oxide and chlordiazepoxide to reduce rearing were significantly attenuated in mice pretreated with the benzodiazepine receptor blocker flumazenil or rendered tolerant to benzodiazepines. These findings suggest an involvement of benzodiazepine receptors in mediation of certain behavioral effects of nitrous oxide.     

Synthesis and pharmacological evaluation of a series of 4-piperazinylpyrazolo[3,4-b]- and -[4,3-b][1,5]benzodiazepines as potential anxiolytics

The synthesis and pharmacological evaluation of a series of pyrazolo[b][1,5]benzodiazepines are described. Some of the 4-piperazinyl-2,10-dihydropyrazolo[3,4-b][1,5]benzodiazepine derivatives demonstrated potent anxiolytic activity in the three-part operant anticonflict test in rats. Compounds 21 and 30 were more active than the clinically effective anxiolytic chlordiazepoxide in releasing conflict-suppressed behavior. This study shows a dissociation of the anxiolytic and antidopaminergic activities found in the thieno- and dibenzodiazepine derivatives flumezapine and clozapine, respectively. Examples of the three other dihydropyrazolo[b][1,5]benzodiazepine ring systems are described and evaluated for comparison and were found to be less active.     

The effects of prenatal chlordiazepoxide administration on avoidance behavior and benzodiazepine receptor density in adult albino rats

Adult male and female offspring of rats injected daily with 10 mg/kg chlordiazepoxide during their pregnancy exhibited a facilitated acquisition of a two-way active avoidance response. Benzodiazepine receptor assays carried out on cortex and cerebral samples taken from experimental and control female rats showed a significant decrease in the density of benzodiazepine receptors, without changes in receptor affinity.     

Differential antagonism of the anticonflict effects of typical and atypical anxiolytics

Selected benzodiazepine and non-benzodiazepine agents were studied alone or in the presence of benzodiazepine antagonists in the shock-induced suppression of drinking (SSD) procedure in rats. The disinhibitory activity of chlordiazepoxide, CL218,872, zopiclone and CGS 9896 was antagonized by two benzodiazepine antagonists, RO-15-1788 and CGS 8216. In contrast, the disinhibitory activity of fenobam, meprobamate, phenobarbital and tracazolate was not antagonized by either RO 15-1788 and CGS 8216. From these data it is apparent that the anticonflict activity of agents that bind to benzodiazepine receptors is blocked by benzodiazepine antagonists. In contrast, the activity of anxiolytics that are not displacers are unaffected even at higher doses.     

Facilitation of benzodiazepine binding by sodium chloride and GABA

An endogenous substance was found which facilitated benzodiazepine binding and was subsequently identified as GABA. The facilitation of benzodiazepine binding produced by GABA obeyed Michaelis — Menten Kinetics and Scatchard analysis revealed that this facilitation was due to an increased affinity of the benzodiazepine receptor. Sodium chloride itself increased benzodiazepine binding and also markedly enhanced the facilitation caused by GABA, by a further increase in the affinity of the benzodiazepine receptor. The enhancement of the GABA effect produced by sodium chloride showed sigmoidal kinetics indicating cooperativity, no evidence could be found for the involvement of sodium-dependent GABA binding in this interaction.