Gary T. Shearman

Evidence that GABA mechanisms mediate the anxiolytic action of benzodiazepines: a study with valproic acid.

Abstract Valproic acid and diazepam were tested for “anti-anxiety” activity in two animal tests known to predict the anxiolytic action of drugs. In one test, male hooded rats were trained to discriminate the “anxiomimetic” action of pentylenetetrazol from saline by responding on one of two levers for food reinforcement after pentylenetetrazol (1450 μmol/kg) injection and the other lever after saline injection. Once the pentylenetetrazol-saline discrimination was acquired, pretreatment with either diazepam (4.4–35.0 μmol/kg) or valproic acid (278–2220 μmol/kg) produced a dose-dependent antagonism of the “anxiomimetic” stimulus in this test. In the other test, male Wistar rats were trained to respond for milk reinforcement and to suppress those responses when the reinforcement was accompanied by the simultaneous delivery of foot shock. Treatment of these rats with diazepam (7–28 μmol/kg) or valproic acid (1110–2220 μmol/kg) antagonized the suppression of responding induced by the foot shock in a dose-dependent manner. In both tests, diazepam and valproic acid showed anxiolytic activity at doses which did not result in an overall suppression of responding. The demonstration of anxiolytic activity by the GABAmimetic drug, valproic acid, suggests that GABA mechanisms may mediate the anxiolytic action of benzodiazepines.

Discriminative stimulus properties of pentylenetetrazol and bemegride: Some generalization and antagonism tests

In an operant procedure of lever pressing on a FR 10 schedule of food reinforcement, male hooded rats were trained to respond with a lever on one side of a food cup following a drug injection, and to respond with a lever on the alternate side following a 1ml/kg saline injection. All of 14 subjects learned to discriminate reliably between the effects of 20 mg/kg pentylene-tetrazol (PTZ) and saline. Seven of eight rats learned to discriminate between the effects of bemegride (5 mg/kg) and saline. None of 14 rats learned to discriminate between 5 mg/kg PTZ and saline. The bemegride discriminative stimulus generalized to PTZ (20 mg/kg) and was antagonized by chlordiazepoxide (10 mg/kg). Chlordiazepoxide, diazepam, flurazepam, clobazam, and meprobamate were all effective antagonists of PTZ in a dose-dependent manner. Bemegride and cocaine generalized to the PTZ discriminative stimulus in a dose-dependent manner, but d-amphetamine, methylphenidate, and nicotine did not. Since bemegride and PTZ are convulsants at higher doses, the discriminative stimulus properties of these drugs might be based on a subtle convulsive brain state. The anxiolytic properties of benzodiazepines and meprobamate suggest that the discriminative stimulus produced by these convulsants is related to an “anxiety-inducing” action.

Generalization and antagonism studies with convulsant, GABAergic and anticonvulsant drugs in rats trained to discriminate pentylenetetrazol from saline☆

Abstract Male hooded rats were trained on an FR 10 schedule of food reinforcement to discriminate the interoceptive stimulus produced by a subconvulsive dose of pentylenetetrazol from that produced by saline, by responding on a lever on one side of a food cup following a 20 mg/kg pentylenetetrazol injection and responding on a lever on the alternate side following a 1 ml/kg saline injection. All of the subjects learned this discrimination reliably. Picrotoxin and strychnine, but not bicuculline nor gamma-hydroxybutyrate, partially generalized to the pentylenetetrazol discriminative stimulus. Valproic acid, sodium pentobarbital and sodium phenobarbital antagonized the pentylenetetrazol discriminative stimulus but etomidate, gamma-hydroxybutyrate, gamma-acetylenic GABA, gamma-vinyl GABA, trimethadione, ethosuximide and diphenylhydantoin did not. These data suggest that the pentylenetetrazol discriminative stimulus is based upon neither a subconvulsant brain state produced by this drug nor a deficiency of GABA neuronal function. Drugs which antagonize the pentylenetetrazol stimulus are all anxiolytic drugs that show no other pharmacological property common to all of them.

Chapter 6. Interoceptive Discriminative Stimuli in the Development of CNS Drugs and a Case of an Animal Model of Anxiety

Publisher Summary This chapter identifies the new animal procedures to quantitatively measure interoceptive discriminative stimuli (IDS) produced by many psychoactive and other drugs. These economical and reliable procedures lend themselves too many problems of drug development including, but not limited to, the identification of new drugs. This is illustrated by describing IDS produced by pentylenetetrazol, and its applications in identifying anxiolytic drugs as well as anxiogenic side effects of new chemicals. It is interesting to note that the anxiogenic property of cocaine is not the dominant IDS produced by this drug, and is thus not recognized in the animals that are not specifically trained to attend to the anxiomimetic effect. Animals trained only to discriminate cocaine from saline learn to perceive more predominant IDS that is related to euphoria and that can be readily blocked by dopamine antagonist drugs. In contrast, cocaine generalization to pentylenetetrazol in animals trained to discriminate pentylenetetrazol from saline is not blocked by haloperidol as is the case with human anxiety, but instead the anxiogenic IDS of cocaine is antagonized by diazepam. To develop new drugs, novel animal models that are reliable, efficacious, and rapid in predicting the clinical usefulness of new chemicals are continuously needed. It is because of the availability of this wealth of recent evidence that the discriminative stimulus properties of drugs have become the focus of interest in developing new bioassays for drug development. It is because of the availability of this wealth of recent evidence that the discriminative stimulus properties of drugs have become the focus of interest in developing new bioassays for drug development.

Effectiveness of lofexidine in blocking morphine-withdrawal signs in the rat

Discontinuation of chronic morphine infusion in rats resulted in the reliable occurrence of withdrawal body shakes. This sign of narcotic withdrawal was dose-dependently reduced by lofexidine (0.04-0.64 mg/kg) and clonidine (0.01-0.16 mg/kg). As with clonidine, the activity of lofexidine was not prevented by naloxone (5 mg/kg). In addition, diarrhea induced by naloxone (5 mg/kg) in morphine dependent rats was also prevented by lofexidine or clonidine pretreatment. These data suggest that lofexidine, like clonidine, may reduce the narcotic withdrawal syndrome in humans.

Nonnarcotic Antidiarrheal Action of Clonidine and Lofexidine in the Rat

Abstract: Clonidine (0.01 to 0.16 mg/kg) and lofexidine (0.01 to 0.64 mg/kg) produced a dose‐dependent inhibition of diarrhea induced by castor oil treatment in the rat. Both drugs were more potent and longer acting than diphenoxylate. Pre‐ and posttreatment with naloxone (5 mg/kg) failed to prevent or antagonize the antidiarrheal effect of clonidine and lofexidine. These data suggest that clonidine and lofexidine may provide potent antidiarrheal activity of a nonnarcotic nature.

Discriminative stimulus properties of desipramine

In an operant procedure of lever pressing on a FR10 schedule of food reinforcement, male hooded rats were trained to respond on a lever on one side of a food cup following a desipramine (10 mg/kg) injection and to respond on a lever on the other side following a saline injection. Sixty percent of the rats learned to reliably discriminate desipramine from saline. The rate of lever pressing at this dose of desipramine was 49% of the saline response rate. No tolerance to this depressant effect was observed after 20 injections. Subjects selected the appropriate desipramine lever 91, 29 and 18% of the time following 10, 2.5 and 0 (saline) mg/kg of desipramine. Imipramine and protryptyline each produced 50% selection of the desipramine lever at a dose of 40 mg/kg. Lower doses were less effective. d-Amphetamine produced 20 and 67% desipramine lever selection at 0.64 and 1.25 mg/kg respectively.

Effect of acute and chronic pentylenetetrazol treatment on benzodiazepine and cholinergic receptor binding in rat brain

The binding of [3H] diazepam and [3H] flunitrazepam in rat cerebral cortex was not altered by either acute or chronic administration of pentylenetetrazol except in rats made to convulse 30 min before sacrifice. Rats treated for up to 6 months with doses of pentylenetetrazol which are below seizure threshold in naive rats, became increasingly sensitive to the CNS stimulant effect of pentylenetetrazol as demonstrated by the development of myoclonus and convulsions during treatment periods. These effects were not correlated with any changes in benzodiazepine binding in cerebral cortex or cerebellum and [3H] quinuclidinyl benzilate binding in cerebral cortex. Acute convulsant doses of pentylenetetrazol increased benzodiazepine binding in cerebral cortex, but only in those rats which actively convulsed. Benzodiazepine and cholinergic receptors of the cortex, and benzodiazepine receptors of the cerebellum, therefore, do not appear to change with either the acute or chronic subconvulsive administration of pentylenetetrazol.

Lack of tolerance development to benzodiazepines in antagonism of the pentylenetetrazol discriminative stimulus

In an operant procedure of lever pressing on FR 10 schedule of food reinforcement male hooded rats were trained to respond on a lever on one side of a food cup following a 20 mg/kg pentylenetetrazol (PTZ) injection and to respond on a lever on the alternate side following a 1 ml/kg saline injection. Upon acquisition of the PTZ-saline discrimination, diazepam and chlordiazepoxide were tested and found to antagonize the PTZ discriminative stimulus. The animals were then injected with 10 mg/kg diazepam or chlordiazepoxide for ten consecutive days. New dose-response curves obtained following this treatment indicated that tolerance did not develop to the antagonism of the PTZ discriminative stimulus by these benzodiazepines.

Effect of valproic acid on anxiety related behaviors in the rat

Abstract Valproic acid was tested for anxiolytic activity in two behavioral tests most often used to predict anxiolytic activity in man. In one test, male hooded rats were trained to discriminate the anxiomimetic action of pentylenetetrazol by responding on one of two levers for food reinforcement after pentylenetetrazol injection and on the other lever after saline injection. Pretreatment of these rats with valproic acid (320 mg/kg) antagonized the discriminative stimulus produced by pentylenetetrazol. In the other test, male Wistar rats were trained to respond for milk reinforcement in a conflict procedure where some of the reinforced responses resulted in the simultaneous delivery of footshock. Treatment of these rats with valproic acid (320 mg/kg) antagonized the suppression of responding induced by the footshock. In both tests, valproic acid showed anxiolytic activity comparable to diazepam. These data suggest that valproic acid may be useful in treating clinical anxiety.