A novel pyrazoloquinoline that interacts with brain benzodiazepine receptors: characterization of some in vitro and in vivo properties of CGS 9896

The novel pyrazoloquinoline, CGS, 9896, was a potent inhibitor of specific [3H]-flunitrazepam binding in several brain regions with subnanomolar KI values. The inhibition of [3H] propyl beta-carboline-3-carboxylate ([3H]-PCC-) binding by CGS 9896 was enhanced by gamma-aminobutyric acid (GABA) but not by chloride ion. GABA enhancement of CGS 9896 inhibition of [3H]-PCC binding predicts this compound has benzodiazepine (BZD) agonist-type activity. Behavioral studies support this prediction. CGS 9896 was found to protect mice against bicuculline and metrazol induced seizure at doses that did not induce ataxia or sedation. CGS 9896 may represent a class of compounds with potential therapeutic value. The high affinity of this non-BZD compound suggests that CGS 9896 may also be of value as a high affinity ligand for the continued study of BZD receptors.     

A novel pyrazoloquinoline that interacts with brain benzodiazepine receptors: Characterization of some and properties of CGS 9896.

The novel pyrazoloquinoline, CGS, 9896, was a potent inhibitor of specific [³H]-flunitrazepam binding in several brain regions with subnanomolar K_I values. The inhibition of [³H] propyl beta-carboline-3-carboxylate ([³H]-PCC) binding by CGS 9896 was enhanced by gamma-aminobutyric acid (GABA) but not by chloride ion. GABA enhancement of CGS 9896 inhibition of [³H]-PCC binding predicts this compound has benzodiazepine (BZD) agonist-type activity. Behavioral studies support this prediction. CGS 9896 was found to protect mice against bicuculline and metrazol induced seizures at doses that did not induce ataxia or sedation. CGS 9896 may represent a class of compounds with potential therapeutic value. The high affinity of this non-BZD compound suggests that CGS 9896 may also be of value as a high affinity ligand for the continued study of BZD receptors.     

Differential properties between TRK-820 and U-50,488H on the discriminative stimulus effects in rats

It has been demonstrated that the newly synthesized kappa-opioid receptor agonist TRK-820, which has a unique structure that is different from those of other prototypical kappa-opioid receptor agonists such as U-50,488H, exert some behavioral effects that differ from those induced by U-50,488H. Therefore, the present study was designed to examine the possible difference between the discriminative stimulus effects of TRK-820 and U-50,488H in rats. Substitution tests with several kappa-opioid receptor agonists were initiated in rats trained to discriminate between TRK-820 (40 microg/kg) or U-50,488H (3.0 mg/kg) and saline. In the cross-substitution tests, U-50,488H substituted for the discriminative stimulus effects of TRK-820, whereas TRK-820 did not substitute completely for those of U-50,488H, indicating that the discriminative stimulus effects of TRK-820 and U-50,488H were somewhat different. In the substitution tests, E-2078, but not R-84760, substituted for the discriminative stimulus effects of both TRK-820 and U-50,488H. KT-90, CI-977 and ICI-199441 each substituted for the discriminative stimulus effects of U-50,488H, but not to those of TRK-820. These results imply that these kappa-opioid receptor agonists possess U-50,488H-like discriminative stimulus effects. Furthermore, that U-50,488H and the other kappa-opioid receptor agonists substituted for the discriminative stimulus effects of U-50,488H, produced aversive effects in rats. These findings suggest the possibility that unlike those of TRK-820, the cue of the discriminative stimulus effects of U-50,488H may be, at least in part, associated with its aversive effects.     

Strategy of auditory discrimination of scale in Java sparrows: they use both "imagery" and specific cues

We examined whether Java sparrows use imagery of auditory stimuli (imagery is a subject's mental representation of a stimulus by which the subject's behaviour may be governed under stimulus control even in the absence of the physical stimulus). Three types of ascending tone sequences were used. In the intact scale, sequence tones were played in ascending order. In the intact-masked scale, part of the sequence was masked by noise but the remaining scale was identical with the intact scale, whereas in the violated scale, the sequence could be heard as if tones were played slowly (Experiment 1) or quickly (Experiment 2). Subjects were divided into two groups: one group was trained to respond to the intact and intact-masked scales and to suppress response to the violation scale (imagery-positive group). The contingency was reversed for the other (violation-positive) group. In Experiment 1, all the birds acquired discrimination, but successful transfer to novel stimuli was observed only in the imagery-positive group, suggesting that the imagery of the tone sequence was used as a discriminative cue. Experiment 2 confirmed that the stimulus duration was a discriminative cue for both groups, suggesting that the birds also acquired discrimination using only specific cues.     

Investigations on drug produced and subjectively experienced discriminative stimuli: 1. The fentanyl cue,a tool to investigate subjectively experienced narcotic drug actions.

Fentanyl is reported to produce a discriminative stimulus that can control food-reinforced lever pressing in rats. Other narcotics (i.e. dextromoramide, morphine, phenoperidine and piritramide) are found to be generalized with fentanyl injection, whereas the neuroleptic haloperidol is not. The subjectively experienced narcotic cue is effectively controlled by the experimental procedures described.     

CGS8216 noncompetitively antagonizes the discriminative effects of diazepam in rats

The benzodiazepine antagonist properties of CGS8216 were evaluated in rats trained to discriminate between saline and 1.0 mg/kg of diazepam in a two-choice, stimulus-shock termination procedure. CGS8216 (0.3 to 100 mg/kg) administered alone, either s.c., p.o. or i.p., occasioned only saline-appropriate responding. When administered concomitantly with a constant 1.0 mg/kg dose of diazepam, CGS8216 produced dose-related decreases in drug-appropriate responding. CGS8216 was most potent by the i.p. route, and approximately tenfold less potent by the oral route. CGS8216 was dermatotoxic after s.c. administration. CGS8216 i.p. had a long duration of action. A dose of 30 mg/kg completely antagonized the discriminative effects of the 1.0 mg/kg training dose of diazepam when the antagonist was administered 8 hr before the start of the test session. In order to determine the type of antagonism by CGS8216, the dose-effect curve for diazepam was redetermined in the presence of varying doses of CGS8216 (0.3 to 3.0 mg/kg, i.p.). CGS8216 produced a dose-related rightward shift in the diazepam dose-effect curve, but also decreased the slope and appeared to decrease the maximal effect. These results are consistent with the interpretation that CGS8216 antagonizes diazepam in a noncompetitive manner. It may do so because either it interacts with a subpopulation of benzodiazepine receptors, it functions as a pseudo-irreversible antagonist due to its high affinity, or because it is an antagonist with agonist properties.     

Measuring naloxone antagonism of discriminative opioid stimulus

The numerous studies of opioids as discriminative stimuli, beginning in 1971, have shown specificity, similarity of several opioids, differences in potency (fentanyl greater than heroin greater methadone greater than morphine), and antagonism by naloxone and naltrexone. The discriminative opioid stimulus is differentiated from those of other classes of drugs, such as sedatives and anxiolytics. Greater potency of the opioid stimulus has been found in rats after subcutaneous (s.c.) than intraperitoneal administration. The discriminative opioid stimulus and its antagonism by naloxone or naltrexone have been demonstrated in rats, squirrel monkeys, gerbils, and pigeons. A few studies have quantified the competitive agonist-antagonist interaction at the receptor by calculating the pA2, which reflects the dose of the antagonist that requires doubling the agonist dose to obtain the original agonist response. The pA2 for naloxone is the same in groups of rats trained to discriminate different doses of morphine (1, 2, or 4 mg/kg s.c.) from saline. Higher pA2 values in tests after fentanyl and methadone than after heroin and morphine in rats trained to discriminate fentanyl (0.04 mg/kg s.c.) from saline reflect greater susceptibility of the synthetic than the natural exogenous opioids to antagonism by naloxone. Different pA2 values are usually interpreted as indicating differences among populations of receptors.     

Benzodiazepine receptor mediated discriminative cues: Effects of GABA-ergic drugs and inverse agonists

Rats were exposed to a two-layer drug discrimination procedure using the benzodiazepine (BZ) receptor inverse agonists N′-methyl-β-carboline-3-carboxamide (FG 7142) or methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM). FG 7142 (30 mg/kg) failed to acquire discriminative stimulus control, although it did suppress responding. The same group of animals was trained successfully to discriminate diazepam (DZP, 2.5 mg/kg) from vehicle. The DZP cue was potentiated by the GABA agonist 4,5,6,7-tetrahydro-isoxazolo [5, 4-c] pyridin-3-ol (THIP, 1–3 mg/kg); THIP alone produced vehicle-appropriate responding. In addition, clonazepam (0.2 mg/kg) and chlordiazepoxide (5 mg/kg) substituted for DZP (with potencies of 7.5 and 0.25 times that of DZP, respectively). In antagonism tests, FG 7142 (5–17.5 mg/kg), methyl-β-carboline-3-carboxylate (β-CCM, 2.5 mg/kg), nicotine (0.3 mg/kg), harmaline (5 mg/kg) and naltrexone (10 mg/kg) did not effect, bicuculine (2 mg/kg) and DMCM (1 mg/kg) partially blocked, and the BZ receptor antagonist Ro 15–1788 (40 mg/kg) completely blocked the discriminative stimulus effects of DZP. In animals trained to discriminate DMCM (0.2 mg/kg) from vehicle, 95% substitution occured with bicuculline (2 mg/kg); DZP (1–5 mg/kg) completely antagonized DMCM. These results indicate that the DZP cue is mediated by GABA-coupled BZ receptors and that GABA may modulate the efficacy of a BZ at its receptor site. However, since inverse BZ receptor agonists (FG 7142, DMCM and β-CCM) were, at best, only marginally effective in antagonizing DZP, the DZP cue may be mediated by a distinct subclass of BZ receptors.     

Effects of cue information on response production and inhibition measured by event-related potentials

The aim of this study was to investigate how information carried by a cue stimulus modulate event-related potentials (ERPs) to a subsequent target stimulus which either calls for an overt response (Go stimulus) or no response (Nogo stimulus). One of the cues predicted the likely appearance of the Go stimulus (Go cue) whereas the other cue predicted the likely appearance of the Nogo stimulus (Nogo cue). Our results showed that unpredicted Nogo stimuli elicited enlarged N200 component. This finding supports the notion that Nogo N200 reflects response inhibition processes, i.e., the amplitude of the N200 is a function of the difficulty of response inhibition. In other words, increased N200 to Nogo stimuli following Go cues might be related to increased efforts in activating the response inhibition system thereby interrupting preparations to respond.     

Discriminative stimulus properties of methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), an inverse agonist at benzodiazepine receptors

Rats (N = 8) were trained to discriminate the stimulus properties of the potent benzodiazepine (BZ) receptor inverse agonist methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) from saline in a two-lever operant task. The initial training dose of DMCM was 0.4 mg/kg at which the discrimination developed slowly; increasing the dose to 0.8 mg/kg resulted in rapid acquisition. However, since convulsions eventually developed during further training (sensitization), the training dose was finally individualized below the convulsive threshold (0.4-0.7 mg/kg). The DMCM cue was mimicked by FG 7142 (10 mg/kg), a non-convulsant anxiogenic beta-carboline, by pentylenetrazol (20-30 mg/kg), and by the GABA antagonist bicuculline (2 mg/kg). The DMCM cue was not, or marginally, blocked by diazepam (2.5 mg/kg) or pentobarbital (10-15 mg/kg). Furthermore, the BZ receptor antagonists CGS 8216 (2.5 mg/kg), ZK 93426 (20 mg/kg), and Ro 15-1788 (20-80 mg/kg) also did not, or only marginally, block the DMCM cue. However, the receptor antagonists (alone) substituted for DMCM although Ro 15-1788 was less effective. The partial BZ receptor agonist ZK 91296 (25 mg/kg), which is structurally similar to DMCM, blocked completely the DMCM stimulus effect. THIP (4 mg/kg) did not block the DMCM cue. To explain these results, we suggest that the repeated DMCM treatment, necessary for maintaining the discrimination, shifts the balancing point ("set-point") for positive (i.e., BZ-like) agonist efficacy versus inverse agonist efficacy, towards inverse action. This hypothesis was supported by the finding of an enhanced ability of GABA to reduce 3H-DMCM binding to cortical neuronal membranes of animals treated chronically with DMCM in a regimen similar to that used to maintain the DMCM discrimination. Furthermore, this treatment did not affect baseline 3H-DMCM binding, baseline or GABA stimulated 3H-diazepam binding, or 35S-TBPS binding (to chloride channels).     

Object discrimination by pigeons: effects of object color and shape

Can nonhuman animals attend to visual stimuli as whole, coherent objects? We investigated this question by adapting for use with pigeons a task in which human participants must report whether two visual attributes belong to the same object (one-object trial) or to different objects (two-object trial). We trained pigeons to discriminate a pair of differently colored shapes that had two targets either on a single object or on two different objects. Each target equally often appeared on the one-object and two-object stimuli; therefore, a specific target location could not serve as a discriminative cue. The pigeons learned to report whether the two target dots were located on a single object or on two different objects; follow-up tests demonstrated that this ability was not entirely based on memorization of the dot patterns and locations. Additional tests disclosed predominate stimulus control by the color, but not by the shape of the two objects. These findings suggest that human psychophysical methods are readily applicable to the study of object discrimination by nonhuman animals.     

Discriminative stimulus effects of m-chlorophenylpiperazine as a model of the role of serotonin receptors in anxiety

Serotonin is known to play a role in anxiety. The roles of serotonin reuptake and 5-HT1A receptors have been well characterized, but the contribution of other serotonin receptor subtypes is not as clear. 1-(3-Chlorophenyl)-piperazine (mCPP), which binds non-selectively to a wide range of serotonin receptors, has often been used to produce anxiety in humans and in animal models. Because functional assays indicate that mCPP is significantly more potent at 5-HT2C receptors, it may serve as a tool to investigate the contribution of 5-HT2C receptors to anxiety. This paper reviews the results of behavioral tests using mCPP, including the drug discrimination assay, to model anxiety. Although the discriminative stimulus effects of mCPP do not seem to be a useful screen for general anxiolytics, they do seem to be useful for characterization of the contribution of 5-HT1B and 5-HT2C receptors to the mediation of anxiety-like behaviors.     

GABA and the behavioral effects of anxiolytic drugs

Much recent research has shown that benzodiazepine binding sites in the central nervous system are associated with GABA receptors. It is therefore possible that the pharmacological and therapeutic effects of benzodiazepines and drugs with similar profiles are mediated through GABAergic mechanisms. In this paper the evidence is considered for a possible involvement of GABA in the behavioral effects of anxiolytic drugs. There are a number of reports that the behavioral actions of anxiolytics can be antagonised by GABA antagonists such as bicuculline or picrotoxin but there are many contradictory findings and these drugs are difficult to use effectively in behavioral studies. In general, GABA agonists do not exert anxiolytic-like behavioral effects after systemic injection but intracerebral administration of muscimol has been shown to produce benzodiazepine-like actions. Although a number of questions remain unanswered, current evidence does not provide strong support for a role for GABA in the behavioral effects of anxiolytic drugs.     

Breakfast in the nook and dinner in the dining room: time-of-day discrimination in rats

Four studies were conducted which demonstrate that most (63%) male Sprague-Dawley rats can attain criterion, nine correct choices over ten consecutive trials, on a time-of-day discrimination in an elevated T-maze, but that the task is relatively difficult. The discrimination required that the rats go to one goal arm during a morning session and the other in an afternoon session. The sessions always began at the same time and were at least 6 h apart. A larger proportion of rats attained criterion and required fewer trials when the discriminative cue was a maze insert providing visual and tactile stimulation (0.72), orientation and position of the maze in the room (0.88), or the rats were required to always make the same left or right turn (0.94). Also, once criterion was attained, rats trained on time-of-day discrimination only made about 70% correct choices with continued training. Housing the rats with continuous light, all laboratory noises masked with white noise, and a random feeding schedule did not prevent them from acquiring the time-place discrimination. Testing the rats with a random number of trials during morning and afternoon sessions and with added or omitted sessions revealed that the rats did not use response or session alternation strategies to perform the discrimination. Also, the particular experimenter administering the morning or afternoon sessions did not serve as a cue for the discrimination. The relative difficulty of the task suggests that time of day does not normally function as a discriminative stimulus for choices, but probably as a contextual stimulus. Further, performance of the task in the absence of time-of-day cues suggests that the discrimination is based on event memory combined with an internal timing mechanism.     

The acquisition in dogs of temporal regulation by differentiated reinforcement of the response duration using an exteroceptive component and the pharmacological modulation of this reflex (diazepam, clozapine)]

Transformation of expectation phenomenon into the phenomenon of temporal regulation is usually achieved by the suppression of preliminary factors or by means of their physical modification. Our studies show that such transformation can be obtained in dogs using Kupalov paradigm with the presentation of additional stimuli. These stimuli strictly identical, from the physical point of view, to the signals which interrupt expectation are randomly introduced into the temporal limit. Absence of the reinforcement in response to the additional stimulus impels the animal to include temporal regulation in its behaviour, and an additional negative discriminative stimulus promotes an expression of active character of inhibition. These circumstances make our pattern closer to DRRD (differential reinforcement of response duration). In order to evaluate the merits of this procedure the influence was studied of anxiolytic (diazepam) and neuroleptic (closepine) on the stabilized reaction of experimental animals. The increase of responses duration by closepine and their shortening by diazepam as well as the influence of these pharmacological substances on the frequency of responses in dependence of dose, confirm the results of the previous studies of DRRD and DRL (differential reinforcement of low rate of responses) and prove differential sensitivity of our procedure to pharmacological substances.     

The anxiolytic activity of calcium-channel blockers in tests of anxious behavior in mice and rats]

The evidence obtained from the studies of the anxiolytic activity of calcium channel blockers in the tests for anxiety: dark-light chamber, elevated plus-maze, social interaction, incomplete sensory deprivation, and change in the excitability of the CNS is analyzed. Methodological aspects of studying the anxiolytic activity of drugs in these tests are discussed. The problems are considered related with simulation of the normal situational anxiety and pathological anxiety as well as the prospects of using the calcium channels blockers as anxiolytics. In view of these aspects, the problem of search for an "ideal" anxiolytic and possible results of revealing its activity in the tests for the normal and pathological anxiety are discussed.     

Loop diuretics have anxiolytic effects in rat models of conditioned anxiety

A number of antiepileptic medications that modulate GABA(A) mediated synaptic transmission are anxiolytic. The loop diuretics furosemide (Lasix) and bumetanide (Bumex) are thought to have antiepileptic properties. These drugs also modulate GABA(A) mediated signalling through their antagonism of cation-chloride cotransporters. Given that loop diuretics may act as antiepileptic drugs that modulate GABAergic signalling, we sought to investigate whether they also mediate anxiolytic effects. Here we report the first investigation of the anxiolytic effects of these drugs in rat models of anxiety. Furosemide and bumetanide were tested in adult rats for their anxiolytic effects using four standard anxiety models: 1) contextual fear conditioning; 2) fear-potentiated startle; 3) elevated plus maze, and 4) open-field test. Furosemide and bumetanide significantly reduced conditioned anxiety in the contextual fear-conditioning and fear-potentiated startle models. At the tested doses, neither compound had significant anxiolytic effects on unconditioned anxiety in the elevated plus maze and open-field test models. These observations suggest that loop diuretics elicit significant anxiolytic effects in rat models of conditioned anxiety. Since loop diuretics are antagonists of the NKCC1 and KCC2 cotransporters, these results implicate the cation-chloride cotransport system as possible molecular mechanism involved in anxiety, and as novel pharmacological target for the development of anxiolytics. In view of these findings, and since furosemide and bumetanide are safe and well tolerated drugs, the clinical potential of loop diuretics for treating some types of anxiety disorders deserves further investigation.     

Hippocampal State-Dependent Behavioral Reflex to an Identical Sensory Input in Rats

We examined the local field potential of the hippocampus to monitor brain states during a conditional discrimination task, in order to elucidate the relationship between ongoing brain states and a conditioned motor reflex. Five 10-week-old Wistar/ST male rats underwent a serial feature positive conditional discrimination task in eyeblink conditioning using a preceding light stimulus as a conditional cue for reinforced trials. In this task, a 2-s light stimulus signaled that the following 350-ms tone (conditioned stimulus) was reinforced with a co-terminating 100-ms periorbital electrical shock. The interval between the end of conditional cue and the onset of the conditioned stimulus was 4±1 s. The conditioned stimulus was not reinforced when the light was not presented. Animals successfully utilized the light stimulus as a conditional cue to drive differential responses to the identical conditioned stimulus. We found that presentation of the conditional cue elicited hippocampal theta oscillations, which persisted during the interval of conditional cue and the conditioned stimulus. Moreover, expression of the conditioned response to the tone (conditioned stimulus) was correlated with the appearance of theta oscillations immediately before the conditioned stimulus. These data support hippocampal involvement in the network underlying a conditional discrimination task in eyeblink conditioning. They also suggest that the preceding hippocampal activity can determine information processing of the tone stimulus in the cerebellum and its associated circuits.     

Gender and Facial Dominance in Gaze Cuing: Emotional Context Matters in the Eyes That We Follow

Gaze following is a socio-cognitive process that provides adaptive information about potential threats and opportunities in the individual’s environment. The aim of the present study was to investigate the potential interaction between emotional context and facial dominance in gaze following. We used the gaze cue task to induce attention to or away from the location of a target stimulus. In the experiment, the gaze cue either belonged to a (dominant looking) male face or a (non-dominant looking) female face. Critically, prior to the task, individuals were primed with pictures of threat or no threat to induce either a dangerous or safe environment. Findings revealed that the primed emotional context critically influenced the gaze cuing effect. While a gaze cue of the dominant male face influenced performance in both the threat and no-threat conditions, the gaze cue of the non-dominant female face only influenced performance in the no-threat condition. This research suggests an implicit, context-dependent follower bias, which carries implications for research on visual attention, social cognition, and leadership.