UDC 612.825.5:612.833 The discovery of benzodiazepine receptors in the brain and the important role postulated for the benzodiazepine system of the brain, including an as yet unidentified endogenous figand, in the regulation of its functional activity makes the study of the role of this system in learning and memory a problem for urgent consideration. The urgency of this problem is also determined by the fact that among the many pharmacological effects of benzodiazepine tranquilizers [i-3], memory disturbances of the anterograde amnesia type have been described [5, 7, 8, ii, 14-16]. The ability of benzodiazepine receptors of synaptic membranes to bind exogenous benzodiazepines, can serve as a parameter of the functional state of the benzodiazepine system of the brain, for it varies under the influence of functional loads [6, 12]. It has been suggested that changes in binding of exogenous benzodiazepines by synaptic membranes are due to changes in the number and affinity of the receptors [6] and competition between the endogenous ligand secreted during appropriate functional loads with exogenous benzodiazepines for the receptor [12]. The effect of conditioning on binding of 3H-diazepam ~n v~t~o by synaptic membranes isolated from the cerebral cortex was investigated. EXPERIMENTAL METHOD Noninbred male albino rats weighing 130-150 g were used. Two-way defensive conditioned avoidance reflexes were formed in a shuttle box. The conditioned stimuli were flashes at the 6th second of action of which an electric shock was applied to the animals through the floor. Conditioning was continued until five out of six correct responses were obtained. Animals receiving the same number of photic and electrodermal stimulations as the corresponding experimental animals, but not as combinations, served as the active control (AC). Animals not exposed to any specific procedures served as the passive control. Each group consisted of 5-7 rats. The animals were decapitated immediately after the experiments, the brain removed, and the separated cortex was placed in liquid nitrogen. After thawing, the tissue was homogenized in a Potter's homogenizer in 25 volumes of Tris-HCl buffer solution consisting of 0.32M sucrose, O.001 EDTA, and 0.05M Tris-HCl, pH 7.4, at 4~ The suspension thus obtained was centrifuged for 15 min at 200g, the residue was discarded, and the supernatant was centrifuged for 20 min at 20,000g. The residue thus obtained (R2) was suspended in 25 volumes (of the initial tissue) of 0.05M Tris-HCl solution (pH 7.4) and the suspension was frozen at --20~ for keeping not more than i0 days. Binding of 3H-diazepam (specific activity 87 Ci/mmole, Amersham, England) with synaptic membranes of R2 was carried out in a total volume of 0.5 ml. The reaction mixture consisted of 50 ~i of a solution of 3H-diazepam of appropriate concentration (from 0.37 to 70 nM), 50 ~l water or 2"10 -4 M diazepam (to determine nonspecific binding). The reaction was started by addition of 400 ~i of membrane suspension from R2. Incubation continued for 30 min at 0.5-I~ The mixture was treated with 4 ml of 0.05M Tris-HCl buffer, pH 7.4, and the suspension was filtered on a water-jet pump through GF/B filters (from Whatman, England), which were then washed twice with 4 ml of the same buffer. After drying overnight, the radioactivity of the filters was Laboratory of Neurochemical Mechanisms of Conditioned Reflexes, Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. S. Rusinov.) Translated from Byulleten' Eksperimental'noi