Latency to enter a mirrored chamber: a novel behavioral assay for anxiolytic agents.

Many animal species exhibit approach-avoidance responses upon the novel placement of a mirror into an individual animal's environment. With a view toward identifying new behavioral measures with qualitatively or quantitatively different responses to anxiolytic agents, we developed a mirrored chamber apparatus for which adult male BALB/cByJ mice showed an extended latency to enter. Administration of diazepam significantly reduced this latency to enter a mirrored chamber in a dosage-dependent manner. The psychomotor stimulant, methylphenidate, had no effect on latency to enter the mirrored chamber at a dose which stimulated locomotor activity to the same extent as diazepam. Thus, the decreased latency to enter the mirrored chamber brought about by diazepam seems unlikely to reflect the motor effects of this benzodiazepine. The potency of diazepam was significantly lower in the mirrored chamber assay than it was on three other measures of exploratory activity--"head-dipping" performance, plus-maze performance and locomotor activity stimulation. The findings of our study indicate that the mirrored chamber method is simple to carry out, nonpunishing, rapid and quantitative and that it possesses pharmacological attributes which distinguish its response to anxiolytics from other assays of exploratory behavior.

3,7-Dimethyl-1-propargylxanthine: a potent and selective in vivo antagonist of adenosine analogs.

3,7-Dimethyl-1-propargylxanthine (DMPX), a caffeine analog that exhibits in vitro selectivity for A2-adenosine receptors, compared to A1-adenosine receptors, has now been investigated with respect to in vivo potency and selectivity. DMPX potently and selectively blocked the actions of the potent A2 adenosine agonist, 5'-N-ethylcarboxamidoadenosine (NECA), in DBA/2 mice, compared to blockade of the same responses elicited by the selective A1-adenosine agonist, N6-cyclohexyladenosine (CHA). DMPX was 57-fold more potent versus NECA-induced hypothermia than versus CHA-induced hypothermia and 11-fold more potent versus NECA-induced behavioral depression than versus CHA-induced behavioral depression. The hypothermia is mediated by peripheral receptors, based on blockade by 8-(p-sulfophenyl)theophylline (PSPT), while the behavioral depression is centrally mediated, based on lack of blockade by PSPT. DMPX was 28- and 15-fold more potent than caffeine in blocking peripheral and central NECA-responses, respectively. DMPX was equipotent with caffeine versus CHA-induced hypothermia and 2.5-fold more potent than caffeine versus CHA-induced behavioral depression. The motor stimulating potency of DMPX (ED50 10 mumol/kg) was slightly greater than caffeine.

Different genes specify hyporesponsiveness to seizures induced by caffeine and the benzodiazepine inverse agonist, DMCM.

Two strains of inbred mice differed significantly in their susceptibility to tonic seizures induced by caffeine and the benzodiazepine inverse agonist, methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM). The hyporesponsive strain, SWR, was not less susceptible to the convulsant action of other chemical convulsants, an observation which indicated that the response differences between the strains were pharmacologically specific. These observations and drug interaction studies suggested that caffeine-induced seizures might be mediated through an "inverse" agonist-like action of caffeine on benzodiazepine receptors associated with GABA receptor-benzodiazepine receptor-chloride ionophore complex. To determine whether the coincident alteration in susceptibility to DMCM and caffeine resulted from a single mutational change or was the result of two different genetic changes occurring coincidentally between these two strains of mice, progeny from conventional Mendelian crosses (F1, F2 and reciprocal backcrosses) were analyzed for the co-segregation of susceptibility to DMCM and caffeine. The inheritance of DMCM sensitivity was consistent with a single autosomal gene determinant in which the allele specifying increased responsiveness was dominant to the allele determining hyporesponsiveness. The frequent occurrence of recombinant phenotypes (e.g., caffeine hyporesponsive but DMCM sensitive mice) among F2 and backcross progeny established that different genetic determinants encode DMCM susceptibility and caffeine susceptibility in these two strains of mice. Thus, while these data establish a simply inherited difference in benzodiazepine responsiveness between the two mouse strains, they also indicate that this pair of strains is inappropriate for a genetic analysis aimed at probing the relationship between caffeine-induced seizures and the benzodiazepine receptor.

Inherent hyporesponsiveness to methylxanthine-induced behavioral changes associated with supersensitivity to 5'-N-ethylcarboxamidoadenosine (NECA).

Two inbred mouse strains, SWR and CBA, differed significantly in their susceptibility to acute dose dependent theophylline- and caffeine-induced stimulation of locomotor activity. The efficacy of both methylxanthines was reduced in the SWR strain compared to the CBA strain. When brain levels of theophylline were determined at a dose (32 mg/kg IP) which gave maximal behavioral separation of the two strains, no significant differences were found between them (SWR levels 12.5 +/- 1.9, CBA levels 14.3 +/- 1.7 micrograms/g wet weight brain). The dose dependent ability of several adenosine agonists (N6-cyclohexyladenosine, (-)-N6-phenylisopropyladenosine, 5'-N-ethylcarboxamidoadenosine) to depress locomotor activity was investigated. SWR mice were found to be significantly more sensitive to NECA-induced depression of locomotor activity and the NECA-induced hypothermia than were CBA mice (respective ED50 values for inhibition of activity, 11.6 and 30.5 nmoles/kg IP). No differences were found in brain [3H]-NECA levels at doses which produced marked differences in behavioral effects between the two strains. The differences in adenosine agonist sensitivity between the strains were both agonist- and behavior-specific. These data indicate that an inherited alteration in behavioral responsiveness to methylxanthine administration can be inversely associated with inherent alterations in susceptibility to the action of specific adenosine analogs. An adenosine A-2 receptor sub-class may be involved in these changes in in vivo pharmacological susceptibility to the action of both methylxanthines and adenosine agonists on locomotor activity.