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Rationale: Administration of flumazenil, a benzodiazepine (BZD) antagonist, has therapeutic efficacy against some anxiogenic effects of ethanol withdrawal. This observation has led to the suggestion that anxiety associated with ethanol withdrawal is related to release in brain of an endogenous BZD inverse agonist., Objective: The present studies further tested this hypothesis by assessing the effect of flumazenil on withdrawal-induced changes in a behavioral task and on the expression of the neuronal protein, Fos., Methods: Male Sprague-Dawley rats were withdrawn from a chronic ethanol regimen and tested, with or without flumazenil pretreatment, for either ultrasonic vocalization in response to air puff or for the induction of Fos protein-like immunoreactivity (Fos-LI) in brain. In addition, flumazenil effects on Fos-LI were measured in a group of animals treated with the BZD inverse agonist DMCM (0.75 and 1.0 mg/kg)., Results: Flumazenil (5.0 mg/kg) significantly reduced the number of ultrasonic vocalizations observed following withdrawal from chronic ethanol. In contrast, flumazenil (5.0 mg/kg), given either 14 h before withdrawal from chronic ethanol, or during hours 3 and 5 following withdrawal, did not attenuate the effects of withdrawal on Fos-LI. Subsequent testing with DMCM confirmed that a benzodiazepine inverse agonist can induce Fos-LI in most of the same brain regions as observed following ethanol withdrawal, and that this change in Fos protein can be attenuated by pretreatment with flumazenil (5.0 mg/kg)., Conclusions: Overall, these results demonstrate that specific behavioral indices of anxiety, but not measures of Fos-LI, support the contribution of an endogenous BZD inverse agonist in the ethanol withdrawal syndrome.

The purpose of this study was to determine: 1) whether 6-hydroxydopamine (6-OHDA), previously shown to deplete brain catecholamines (CA) in rodents, depletes brain CA in rhesus monkeys; 2) whether depletion of brain CA produces changes in behavior; and, 3) whether urinary output of 3-methoxy-4-hydroxyphenylglycol (MHPG) reflects brain norepinephrine (NE) depletions. Repeated intracerebroventricular (ICV) injection of 6-OHDA (N = 20; 15.5-73.3 mg/subject) produced chronic changes in social behavior and, at higher dosages, reduced output of urinary MHPG. However, 4 weeks after the last ICV 6-OHDA injection, urinary MHPG excretion returned to baseline values and whole brain CA content was not reliably different from control. A single treatment with 6-OHDA microinjected into the substantia nigra (SN) (N = 12; 120-240 microgram/subject) produced chronic whole brain depletions of brain CA without depleting serotonin. Reductions in brain CA were associated with a specific set of motor behaviors, aphagia, and adipsia. SN 6-OHDA produced greater brain NE depletions than ICV 6-OHDA, but urinary MHPG output was not reduced. SN 6-OHDA treated subjects showed chronic changes in social behavior and were more sensitive to the operant response rate decreasing effects of alpha-methyl-para-tyrosine (AMPT) than control subjects. Subjects with the largest depletions of brain dopamine (DA) (greater than 90%) were hypokinetic, rigid, and had a distal limb tremor. These results show that SN but not ICV injection of 6-OHDA can deplete brain CA in the rhesus monkey. The most prominent behavioral changes were characterized by disturbances in motor function. Urinary MPHG output does not reflect depletions of brain NE in this species.

The selectivity and specificity of fenmetozole (DH-524) [2(3,4-dichlorophenoxy-methyl)2-imidazole HCl] as an antagonist of the actions of ethanol were examined. Fenmetozole (15--30 g/kg) reduced ethanol-induced impairment of the aerial righting reflex without changing blood or brain ethanol content, indicating that the antagonistic actions of fenmetozole were not de to change in the pharmacokinetics of ethanol. Since fenmetozole also reduced aerial righting reflex impairment due to phenobarbital, chlordiazepoxide, and halothane, this action of fenmetozole was not specific to ethanol. In mice, both the ethanol-induced increase in locomotor activity at 2.0 g/kg and the decrease caused by 4.0 g/kg were antagonized by fenmetozole. In addition, fenmetozole attenuated the ethanol-induced reduction in cerebellar cyclic guanosine monophosphate (cGMP) content, but the drug also significantly elevated cGMP levels in this tissue when given alone. Fenmetozole did not alter ethanol-induced increases in punished drinking in a conflict test, except at a high dose which alone decreased both punished and unpunished responding. Fenmetozole also failed to precipitate ethanol withdrawal-like reactions when given to physically-dependent, intoxicated rats. Thus, the antagonistic action of fenmetozole against ethanol would not seem to be related to a specific receptor interaction but rather may be the result of a physiological antagonism.

The locomotor activity of groups of three CD-1 female mice was increased by 1.0 and 2.0 g/kg ethanol, IP, was decreased during the first hour and increased during the second hour by 3.0 and 4.0 g/kg, and was decreased by 5.0 g/kg. The dose (2.0 g/kg) that caused the greatest increase in locomotor activity did not impair motor coordination, measured by the height of aerial righting in mice. Tests after oral administration of ethanol showed that the increase in locomotor activity of mice was not due to peritoneal irritation. The same dose (2.0 g/kg) did not increase the locomotor activity of C57BL/6J mice. Ethanol (0.1 to 3.0 g/kg) had no effect or decreased the locomotor activity of individual male Sprague-Dawley rats. These findings suggest that biological differences in strains and species of laboratory rodents contribute to the apparent variability of locomotor stimulation caused by ethanol. The presence or absence of an ethanol-induced increase in locomotor activity was not dependent on the sex or number of mice or rats tested. Intertrial-interval crossing by rats acquiring or performing an active avoidance task in a shuttle box was increased by ethanol. This action was dependent on the presentation of electric foot shock. Apomorphine (0.25 and 2.5 mg/kg) and fenmetozole (7.5 and 15.0 mg/kg) failed to inhibit the ethanol-induced increase in intertrial-interval crossing by rats, although these drugs have been shown previously to antagonize the ethanol-induced increase in the activity of mice ethanol treatment. The ethanol-induced increases in the spontaneous locomotor activity of CD-1 mice in photocell activity monitors and in intertrial-interval crosses in rats in a shuttle box task thus do not appear to share a common mechanism.

The intracisternal administration of 5,7-dihydroxytryptamine (5,7-DHT) to rats resulted in a potentiated response to 5-hydroxytryptophan (5-HTP) when the animals were tested 30 days later. The 5-HTP-induced changes include elevation of serum prolactin, decrease in operant responding, and the magnitude of the "serotonin behavioral syndrome" observed after 5-HTP administration. The serotonin concentration in brains of 5,7-DHT-treated animals reached maximum earlier and remained elevated longer than that of controls following administration of 5-HTP. Brain norepinephrine and dopamine concentration were not affected by 5-HTP in either group of animals. The increase in serum prolactin concentration elicited by administration of the serotonergic agonists quipazine or 5-methoxy-N,N-dimethyltryptamine and by the serotonin uptake inhibitor fenfluramine also was potentiated by pretreating rats with 5,7-DHT. These data suggest that both serotonergic receptor supersensitivity and the absence of presynaptic uptake sites contribute to the enhanced responses to 5-HTP occurring in rats previously treated with 5,7-DHT. The findings further demonstrate that both behavioral and hormonal measures can be used to assess the sensitivity of serotonergic receptors and indicate that 5,7-DHT may be useful in evaluating the role of serotonergic neurons in neuroendocrine function.

Scopolamine reversed the reduction in avoidance responding caused by spiperone and antagonized the inhibitory effects of spiperone on the behavioral actions of d-amphetamine or apomorphine. Scopolamine-induced locomotor activity was greater in 6-hydroxydopamine (6-OHDA)-treated animals than in controls. This increase was prevented by administration of alpha-methyltyrosine, but not by inhibition of dopamine-beta-hydroxylase, indicating that this action of scopolamine was associated with presynaptic dopaminergic fibers. Therefore, the possibility that pre-synaptic dopaminergic function was the locus of the antagonism of spiperone by scopolamine was examined using drug interaction studies in 6-OHDA-treated rats. However, when 6-OHDA-treated rats were given alpha-methyltyrosine, scopolamine still reversed the spiperone blockade of apomorphine-induced locomotion. Although these data provided evidence for a post-synaptic action for this cholinergic blocking agent, scopolamine affected neither dopamine-stimulated adenylate cyclase activity nor 3H-spiperone binding in vitro. Furthermore, scopolamine did not alter the level of specific 3H-spiperone binding found in brain after in vivo administration. This suggests that the post-synaptic mechanism affected by scopolamine is different from the site affected by spiperone. Thus, it is concluded that scopolamine can affect both pre- or post-synaptic events associated with dopaminergic function and that both may contribute to the reversal of the actions of spiperone.

The latency, duration, hindlimb kick frequency, and total activity components of the post-decapitation reflex (PDR) were measured in the rat using a movement-sensitive transducer. Reduction of brain and spinal cord norepinephrine (NE) caused by neonatal administration of 6-hydroxydopamine (6-OHDA) or 5,7-dihydroxytryptamine, which also reduced brain serotonin, decreased all components of the PDR. Depletion of serotonin or dopamine alone reduced the vigor of the reflex, suggesting that these pathways can influence the PDR but are not essential for the response. Lesions of neurons in the Locus coeruleus, made electrolytically or with 6-OHDA, decreased the intensity of the PDR, with the 6-OHDA-induced lesion being more effective. Depletion of forebrain NE terminals with 6-OHDA did not alter the PDR, consistent with a critical involvement of spinal noradrenergic fibers. The PDR was also decreased by phentolamine and prazosin, but not by propanolol, suggesting an involvement of alpha-adrenergic receptors in the response. This hypothesis was further supported by the finding that the efficacy of a variety of drugs (such as tricyclic antidepressants, phenothiazines, and anti-hypertensive compounds) for blocking the reflex was apparently related to their affinity for alpha-adrenergic receptors. Thus, the PDR is dependent on noradrenergic fibers in the spinal cord and may provide a simple screen for drugs with suspected alpha-adrenergic blocking properties or for agents that disrupt the function of central noradrenergic fibers.