CHARACTERIZATION OF FUNCTIONAL GABAERGIC SYNAPSES FORMED BETWEEN RAT HYPOTHALAMIC NEURONS AND PITUITARY INTERMEDIATE LOBE CELLS IN COCULTURE - CA2+ DEPENDENCE OF SPONTANEOUS IPSCS

Rat hypothalamic neurons and endocrine cells from the intermediate lob e of the pituitary were grown in dissociated coculture. Neurons positi vely stained with an antibody against glutamate decarboxylase establis hed apparent contacts with the ar-melanocyte-sti mutating hormone-posi tive endocrine cells. These sites of contact were intensely labeled wi th an antibody against the synaptic protein synapsin 1 and displayed u ltrastructural features characteristic of synapses. Using patch-clamp recordings, we have demonstrated that these contacts correspond to fun ctional GABAergic synapses. The synaptic currents were blocked reversi bly by bicuculline (5 mu M) and SR95531 (5 mu M), two competitive anta gonists of the GABA(A) receptor. At a holding potential of -60 mV, spo ntaneously occurring IPSCs (s-IPSCs) had small amplitudes (10-100 pA), whereas electrically evoked IPSCs (ee-IPSCs) had amplitudes up to 1 n A. The rise times of both types of IPSCs were fast (less than or equal to 1 msec), and their decaying phases were fitted in most cases with a single exponential function (time constant, 50 msec). The amplitude distribution of s-IPSCs did not reveal clear, equally spaced peaks and was little affected by tetrodotoxin, suggesting that most s-IPSCs wer e miniature IPSCs. Reduction of extracellular calcium concentration to 0.3 mM induced a marked decrease in s-IPSC frequency and revealed a s ingle amplitude peak at 10 pA, suggesting that a single quantum of GAB A activates 8-10 GABA(A) channels. Thus, our preparation might be an i nteresting model to study different aspects of synapse formation betwe en a central neuron and its target as well as the fundamental mechanis ms of synaptic transmission at central synapses.