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In considering characteristics of action of anxiolytic drugs and the mechanism of drug action in the brain, it may be necessary to study not only the behavioral pharmacology but also the brain site. In the present study, anxiolytic drugs have been examined in various kinds of behaviors induced by stimulating the brain areas with regard to emotional expression such as reward (pleasure) or aversion in rats. First, the low rate responding on lateral hypothalamic self-stimulation behavior was induced by schedules of low current brain stimulation, variable interval (VI) and differential reinforcement of low rate (DRL). Anxiolytic drugs such as benzodiazepines facilitated these low rate responses. The drug susceptibility was high in the order of the low current stimulation > VI > DRL schedules. Furthermore, it was found by the auto-titration method on intracranial self-stimulation behavior that anxiolytic drugs decreased the threshold of stimulation reward. Second, it was recognized using the decremental lever pressing (DLP) paradigm that anxiolytic drugs increased the threshold of aversive stimulation of mesencephalic dorsal central gray (DCG), and this increasing effect of the drug was antagonized by GABA receptor blockers such as biccuculline. Third, it has been examined whether the conflict situation is established by combining with brain stimulation reward and aversion such as foot-shock or DCG stimulation. As a result, the conflict behavior was established by combining with not only the brain stimulation reward and foot shock aversion, but also the brain stimulation reward and DCG stimulation aversion. Further anxiolytic drugs exhibited the anti-conflict action to both situations. The susceptibility of anxiolytic drugs to the conflict behavior by intracranial reward and aversion was higher than the conventional method based on milk reward and foot shock aversion. Indeed, in the present brain stimulation behavioral study, anxiolytic drugs such as benzodiazepines increased the stimulation threshold in lateral hypothalamic self-stimulation and inhibited the DCG aversive stimulation, i.e. resulting in an anticonflict action of the drugs. Recently we have preliminarily established a new model for evaluating the drug which may facilitate the motivation contributing to result in various behaviors, using the priming stimulation paradigm in intracranial self-stimulation behavior. Diazepam, benzodiazepine, and nomifensine, an dopamine uptake inhibitor, exhibited a delay of the extinction process which included non-reinforcing stimulation and pretrial electric stimulation (priming stimulation) in the self-stimulation behavior. This action may be related to the brain-mechanism of motivation.

In considering the characteristics of the action of anxiolytic drugs and their mechanism in the brain, it may be necessary not only to study the behavioral pharmacology but also to perform brain site research. In the present study, the action of anxiolytic drugs was examined with respect to various behaviors that were induced by stimulating the brain areas related to emotions such as reward (pleasure) or aversion in rats. First, the low rate of response in lateral hypothalamic self-stimulation behavior was induced by schedules of low current brain stimulation, variable interval (VI) and differential reinforcement of low rate (DRL). Anxiolytic drugs such as benzodiazepines facilitated these low-rate responses. The drug susceptibility was highest in the low current stimulation, lower in the VI stimulation, and lowest in the DRL stimulation schedules. Furthermore, it was found by the auto-titration method in intracranial self-stimulation behavior that anxiolytic drugs decreased the threshold of stimulation reward. Second, it was recognized using the decremental lever pressing (DLP) paradigm that anxiolytic drugs increased the threshold of aversive stimulation of mesencephalic dorsal central gray (DCG), and this increasing effect of the drug was antagonized by GABA receptor blockers such as biccuculline. Finally, it was examined whether or not the conflict situation is established by combining brain stimulation reward and aversion, such as foot-shock or DCG stimulation. As a result, the conflict behavior was established by combining not only the brain stimulation reward and foot-shock aversion, but also the brain stimulation reward and DCG stimulation aversion. Further anxiolytic drugs exhibited anti-conflict action in both situations. The susceptibility of anxiolytic drugs was higher with respect to the conflict behavior induced by intracranial reward and aversion than to that induced by the conventional method based on milk reward and foot-shock aversion. These results suggest that behavioral methods using brain stimulation can examine the mechanisms of direct drug action at the brain stimulation site. Indeed, in the present brain stimulation behavioral study, anxiolytic drugs such as benzodiazepines increased the stimulation threshold in lateral hypothalamic self-stimulation and inhibited the DCG aversive stimulation, thus resulting in an anticonflict action of the drugs.

The anticonflict activities of buspirone and gepirone were examined in the Vogel's water licking test in rats. Acute treatment with buspirone induced a significant increase in water licking response, but gepirone showed slightly more marked effect than buspirone. The anticonflict activities of these compounds were potentiated by chronic administration. Especially, gepirone exhibited a dramatically remarkable anticonflict effect. These results suggest that gepirone has a great possibility of promising drug for anxiety.

Each animal was chronically implanted with bipolar electrodes in dorsal central gray matter (DCG) and was trained to press a lever to decrease the DCG-stimulation current. Chlordiazepoxide (5-20 mg/kg, PO), diazepam (2-10 mg/kg, PO) and bromazepam (1-5 mg/kg, PO) produced dose-dependent increases in the DCG-stimulation threshold 1-4 h after administration without affecting motor performance. Meprobamate (200 mg/kg, PO) and pentobarbital (10 mg/kg, PO) also slightly increased the stimulation threshold. Their potency was in the order of bromazepam greater than diazepam greater than chlordiazepoxide greater than pentobarbital greater than meprobamate. The increase in the threshold induced by diazepam (10 mg/kg, PO) was inhibited by the GABA antagonists, bicuculline (1 mg/kg, IP) and picrotoxin (0.1 mg/kg, IP). These results suggest that decreased susceptibility to brain stimulation is involved in suppressing effects of anxiolytic drugs on the escape behavior, and also that the antiaversive action of benzodiazepines may be related to a GABAergic mechanism.

Rats were chronically implanted with electrodes aimed at the lateral hypothalamus (LH) and the dorsal central gray (DCG) and trained to press a lever that delivered rewarding stimulation of the LH and punishing stimulation of the DCG. In this situation, both diazepam (5-20 mg/kg, PO) and bromazepam (2-10 mg/kg, PO) caused a marked dose-dependent increase of the lever pressing response in the punished period. In addition, the facilitation of lever pressing in unpunished period was also seen in diazepam (5 and 10 mg/kg). These results show that behavioral suppression on lever pressing maintained self-stimulation reward is inducible following DCG stimulation, and that benzodiazepines exhibit an anti-behavioral suppression effect in this situation.

Formation of gastric lesions in response to conditioned emotional stimulus (CES) and effects of benzodiazepines were studied in mice. The CES was introduced in the form of affective communication through a communication box. The "senders" were exposed to electric foot shock and the "responders" were able to receive affective cues such as visual, auditory and olfactory from "senders". The "senders" and "responders" exhibited significantly greater gastric lesions than the controls. Diazepam at doses of 2 X 1 - 2 mg/kg (p.o.) and oxazolam at a dose of 2 X 2 mg/kg (p.o.), reduced the formation of gastric lesions of the "responders" induced non-physically by CES. The present results indicate that "responders" showed bodily changes under CES treatment, particularly in the development of gastric lesions, and that gastric lesions produced by CES were protected by diazepam and oxazolam.

Effects of gamma-oryzanol were studied on gastric lesions induced by both conditioned emotional stimuli (CES) and REM sleep deprivations and on the facilitation of small intestinal propulsive activity by CES in mice. The CES were given by the communication box method, and the REM sleep deprivations were performed by a modified flower pot method. The incidence of gastric lesions in responder mice induced by CES was reduced by twice p.o. administrations at 6 hr interval of gamma-oryzanol at 200 and 500 mg/kg, oxazolam at 2 mg/kg and atropine at 1-10 mg/kg. The incidence in sender mice was also reduced by gamma-oryzanol at 200 and 500 mg/kg. In addition, the incidence of gastric lesions induced by REM sleep deprivation was also reduced by single administration of gamma-oryzanol at 100 and 200 mg/kg and oxazolam at 5 mg/kg. The facilitation of small intestinal propulsive activity in responder mice induced by CES was suppressed by gamma-oryzanol at 100 and 200 mg/kg and atropine at 10 mg/kg. These results indicate that gamma-oryzanol has an antiulcerative action on gastric lesions induced by CES and REM sleep deprivation, and it has a suppressive action on the facilitation of intestinal propulsion induced by CES.

The present study was designed to examine the anticonflict action of benzodiazepines injected into the mammillary body (MB) using the rat conflict-punishment procedure. Diazepam 20 micrograms/microliters, chlordiazepoxide 60 micrograms/microliters and midazolam 30 micrograms/microliters, bilaterally injected into the MB, produced a significant increase in the punished responses without changes in the unpunished responses. These findings have demonstrated for the first time that the MB could be a potential site of antianxiety action of benzodiazepines.

Based on lateral hypothalamic self-stimulation behavior of the rat in a Skinner box, a "conflict" situation was established by combining foot shock punishment with brain stimulation. Diazepam (10-20 mg/kg, PO) caused a marked increase in the lever pressing response in the punished period without affecting the unpunished response. Bromazepam (10--20 mg/kg PO) also caused an increase in the lever pressing response in the punished period and a decrease of the punished response. These results indicate that a "conflict" situation based on self-stimulation behavior is useful for the evaluation of antianxiety action.

Satiated rats could be trained to give stable rates of responding for rewarding stimulation of the lateral hypothalamus delivered on differential reinforcement of low rate (DRL) schedule requiring 2 to 8 sec interresponse intervals for reinforcement (DRL-2 to 8). The performance on a DRL-8 schedule was tested 30 min after the oral administration of benzodiazepines. Diazepam (5 and 10 mg/kg) and meprobamate (200 mg/kg) caused significant increases in response rates during the first 5 min of a session, but not thereafter. Bromazepam (1 and 5 mg/kg) also caused a significant increase in the rates during the first and second 5 min. On the other hand, chlorpromazine (20 mg/kg) caused no effect in the first 5 min but decrease in second and third 5 min. These results indicate that DRL schedules with a brain stimulation reward provided a useful tool for evaluation of antianxiety drugs. The advantage of the brain stimulation reward over food reward is that the possible effects of the drugs on hunger motivation need not be considered.

Differences between the pharmacological effects of bromazepam given by oral and rectal administration were investigated in mice and rats. 1) Bromazepam dose-dependently prolonged the sleeping time induced by thiopental-Na, ethanol and ether by both administration routes. 2) The analgesic action of bromazepam was recognized by the hot-plate method and the algolytic test. In the hot-plate test, analgesic actions of morphine and pentazocine were potentiated by bromazepam in a dose of 0.5 mg/kg by both routes. 3) The muscle relaxant effect of bromazepam administered rectally was more potent than that administered orally in the inclined screen test and the rotarod test. This effect of bromazepam by rectal administration was approximately 2 times as potent as that by oral administration. 4) Bromazepam inhibited the convulsion induced by maximum electric shock, pentylenetetrazol and picrotoxin. In pentylenetetrazol-induced convulsion, the inhibitory effect of bromazepam administered rectally was 2 times as potent as that administered orally. In the other convulsion test, no significant differences between oral and rectal administration could be recognized. 5) Hyperemotionality and muricide (mouse-killing behaviour) of rats with bilateral olfactory bulb ablations (OB rat) were reduced by oral and rectal administrations of bromazepam in a dose-dependent manner. The effects by rectal administration were more potent than that by oral administration. Bromazepam was approximately 20 times as potent as diazepam administered by the same route. Fighting behaviour in mice subjected to footshock was suppressed by rectal administration of bromazepam, and this effect was as same as that by oral administration. 6) The rate of lever pressing response in the lateral hypothalamic self-stimulation test in the Skinner box was markedly increased by rectal administration of 0.2 mg/kg bromazepam. 7) Methamphetamine-induced hyperactivity of mice was significantly suppressed only by bromazepam administered rectally in a dose of 5 mg/kg. 8) The falling effect of bromazepam on body temperature in normal rats was the same in both administration routes and was dose-dependent. From these data, significant differences of the pharmacological effects between oral and rectal administration of bromazepam were recognized in the duration of action and, in part, potencies; and therefore, rectal administration of bromazepam may be a useful dosage form for clinical use.

In order to study effects of antianxiety drugs on the threshold of intracranial self-stimulation (ICSS), the auto-titration technique was used in the two lever Skinner box. This procedure consisted of conventional ICSS except the brain stimulation current intensity was decreased after every 20 lever press for ICSS, and the animal could press the another reset lever (at any time) to reset the stimulation current intensity to the preseted level. The current intensity at which the animal pressed the reset lever (reset current) was defined as the threshold of ICSS. Since reset currents that the animals showed were very stable during this experiment, the effects of chlordiazepoxide, diazepam and meprobamate were studied. The reset current was significantly lowered by 5.0 mg/kg chlordiazepoxide, p.o., at 1 hr after administration accompanied by the significant increase of ICSS, and the reset current was significantly lowered by 20.0 mg/kg of chlordiazepoxide. On the other hand, diazepam did not lower the reset current, although a significant increase of ICSS was observed at 1.0 mg/kg, p.o. The reset current was lowered by meprobamate at 100 mg/kg, p.o., accompanied by the increase of ICSS, and a significant increase of ICSS was observed at 200 mg/kg but not accompanied by lowered reset current. From these results, increased susceptibility to the brain stimulation current may be involved in the facilitating effects of chlordiazepoxide and meprobamate on ICSS.

Effects of triazolam on various types of conditioned behavior were investigated and compared mainly with diaepam in rats. The active conditioned avoidance response of the rat in a Shuttle box was inhibited by triazolam and diazepam only at large doses. The passive avoidance response in a step-down method was not affected by either triazolam or diazepam, but was markedly suppressed by chlorpromazine. The low rate response of hypothalamic self-stimulation behavior was markedly increased by triazolam at doses ranging from 2 to 40 mg/kg p.o., but was suppressed at doses over 80 mg/kg p.o. The high rate response was unaffected by triazolam even at doses of 40 approximately 180 mg/kg p.o. The low rate response was increased by diazepam at doses of 1 approximately 10 mg/kg p.o. and was suppressed at 80 mg/kg p.o. The high rate response was reduced by diazepam at 180 mg/kg p.o. In the conflict situation of the rat subjected to food reward and foot-shock punishment, the lever press response in the unpunished period was reduced by triazolam at doses of 1 approximately 5 mg/kg p.o., whereas that in the punished period was markedly increased. Similar effects were observed with diazepam at doses of 15 approximately 20 mg/kg p.o. Triazolam appeared to be 10 approximately 15 times more potent than diazepam in this anticonfluct effect. Thus, triazolam appears to be a potent antianxiety agent.

Behavioral and electroencephalographic effects of clobazam (CBZ), a 1, 5 benzodiazepine, were investigated in mice, rats and rabbits and compared with the effects of diazepam (DZP) and chlordiazepoxide (CDP). In EEG studies of rabbits, CBZ, DZP and CDP at doses of 2-10 mg/kg, i.v., caused a drowsy pattern, i.e., high voltage slow waves in the frontal cortex and the desynchronization of hippocampal theta wave. EEG arousal responses induced not only by auditory stimulation but also by electric stimulation of the mesencephalic reticular formation were inhibited by CBZ; CBZ was less potent than DZP, but more potent than CDP. On hypothalamic self-stimulation behavior of rats, low rate responses induced by low current brain stimulation, VI or DRL procedure were increased by oral administration of the three drugs. CBZ was less potent than DZP in the above respondings, but the same potency as CDP in VI or DRL responding and more potent in low rate responding induced by low current stimulation. The preventive effect of CBZ on MES convulsion in mice was less potent than DZP, but 2.5 times as potent as CDP. The preventive effect of CBZ was 1.3 times as potent as DZP and 2.5 times as potent as CDP. CBZ reduced the hyperemotionality of olfactory bulbectomized rats, and this effect was less than DZP in suppressing muricide. The muscle relaxant effect of CBZ in inclined screen and rotarod tests of mice was less than that of DZP. CBZ was 2.2 times as potent as CDP in potentiating thiopental sleep in mice, but less than DZP. These results indicate that CBZ is qualitatively similar to 1, 4 benzodiazepines, DZP and CDP, and is more potent than CDP, but less potent than DZP.

The effect of clobazam, a 1,5-benzodiazepine, on kindling seizures was compared with the effects of 1,4-benzodiazepines, diazepam and bromazepam, in amygdaloid and hippocampal kindled rats. After kindled seizures were established (stage 5 seizure), the test drugs were administered intraperitoneally. In amygdaloid kindled rats, clobazam significantly suppressed the motor seizures (MS) at doses of 20 and 50 mg/kg and significantly shortened the duration of after-discharge (AD) at doses of 10 to 50 mg/kg. Diazepam at doses of 2 to 10 mg/kg and bromazepam at 1 to 5 mg/kg also significantly suppressed the MS and significantly shortened the duration of AD. Similar suppressive effects by these three benzodiazepines were observed in hippocampal kindled rats. From these results, clobazam was found to have a qualitatively similar but weaker anticonvulsive effect than those of 1,4-benzodiazepines.