Brain Research, 289 (1983) 385-390 385 Elsevier Behavioral analysis of benzodiazepine-medlated inhibition in the early chick embryo JEROME L. MADERDRUT*, RONALD W. OPPENHEIM* and JOHN L. REITZEL Neuroembryology Laboratory, North Carolina Division of Mental Health, Research Section, Anderson Hall, Dorothea Dix Hospital, Raleigh, NC 27611 (U.S.A.) (Accepted August 30th, 1983) Key words: benzodiazepines - - GABA - - embryology - - receptors - - inhibition - - motility - - synaptic transmission Diazepam, a depressant benzodiazepine, produced a dose-dependent decrease in the spontaneous motility of 5-day embryos while Ro 5-3663, a convulsant benzodiazepine, had no apparent effect. Diazepam and 4 other benzodiazepines inhibited motility in 5-day embryos with a potency that paralleled their effectiveness in ligand-binding studies. Ro 11-5073/Ro 11-5231, depressant benzodiaze- pine enanti0mers, stereospecifically inhibited motility in 4- and 5-day embryos. Ro 15-1788, a benzodiazepine antagonist, reversed the decrease in the motility of 4- and 5-day embryos caused by a depressant benzodiazepine, clonazepam. Ro 5-6945, a potent agonist for the non-neuronal (but not the neuronal) benzodiazepine receptor, had no apparent effect on motility in 5-day embryos. Benzodia- zepine receptors appear at least as early as day 5 (and perhaps as early as day 4) in the chick embryo. ~-Amino-N-butyric acid (GABA) receptors 35 and GABA-mediated synaptic transmission (J. L. Reit- zel, J. L. Maderdrut and R. W. Oppenheim, unpub- lished observations) controlling overt behavior ap- pear in the chick embryo at least as early as the 4th and 5th day of embryogenesis, respectively. Benzo- diazepine receptors3, 23 are an integral part of the ma- ture GABA postsynaptic membrane receptor-iono- phore complex10As,24,26 and benzodiazepines appar- ently modulate behaviour by modulating GABA-me- diated synaptic transmission7, 40. Radioligand-bind- ing and autoradiographic techniques have detected benzodiazepine receptors in the rat brain beginning on the 14th day of gestation4,19, 38. Electrophysiolog- ical techniques have detected benzodiazepine recep- tors on spinal cord and dorsal root ganglion neurons dissociated from 7-day chick embryos and cultured for 2-3 days 7. However, it is unknown whether the receptors detected by either radioligand-binding, au- toradiographic or electrophysiological techniques (vide supra) control overt behaviour or are only func- tionally silent receptors7, 8. It is also unknown wheth- er benzodiazepine receptors appear simultaneously with GABA receptors during embryonic devel- opment. The absence of a blood-brain barrier]4,33, the presence of spontaneous, neurogenic, motor ac- tivity (motility) throughout the last 85% of the incu- bation period1,27, as well as the ability to quantify be- havior under relatively undisturbed conditions make the chick embryo suitable for the functional analysis of drug effects on the central nervous system in situ. The present experiments indicate that behaviorally relevant benzodiazepine receptors (like behaviorally relevant GABA receptors) appear at least as early as the 4th day of embryogenesis in the chick. White Leghorn chicken embryos were used. Em- bryos (4- and 5-day) were exposed to view by the lat- eral window technique 29 and the eggs were sealed with Parafilm. The eggs were then placed in an obser- vation chamber with a standard environment of 37 °C and 60% relative humidity. The Parafilm was removed within 1 h and a pre-drug recording of embry- onic movements was made by observing the embryo through a dissecting microscope 30. Only embryonic somatic movements (motility) as opposed to amniot- ic contractions were recorded. Drugs were applied directly onto the vascularized chorioallantoic mem- brane. Drug doses were expressed as absolute * Present address: Department of Anatomy, Bowman Gray School of Medicine, Winston-Salem, NC 27103, U.S.A. Correspondence: J. L, Maderdrut, Department of Anatomy, Bowman Gray School of Medicine, Winston-Salem, NC 27103, U.S.A. 0006-8993/83/$03.00 ~) 1983 Elsevier Science Publishers B.V.