Electrophysiological studies of luteinizing hormone releasing hormone (LHRH) neurons in the hypothalamus were done by culturing explants of olfactory pit regions of embryonic mice (E-11.5) where LHRH neurons are abundant. LHRH neurons were maintained up to 3 weeks in these explant cultures and their electrical and synaptic properties were studied using patch-pipette, whole-cell current and voltage-clamp techniques. Cells were marked intracellularly with Lucifer yellow (LY) after recordings were made, and their phenotypes were subsequently identified immunocytochemically. Sixty-two cells were unequivocally identified as LHRH-immunoreactive. Morphologically, these cells were unipolar or bipolar, and resembled LHRH neurons found in vivo. Cultured LHRH-neurons had resting potentials of about -50 mV and exhibited tonic to partially phasic spontaneous discharges generated by both endogenous and synaptic mechanisms. Voltage-clamp analysis of the somatic membranes of the LHRH neurons revealed the following voltage-sensitive and insensitive ionic currents: at least two types of K currents - a transient current (I/A) and a delayed rectifier current (I/K), a tetrodotoxin (TTX)-sensitive Na current, and low threshold and high threshold Ca currents. Direct application of GABA-induced depolarizations at the resting membrane potential, due to Cl-conductance increases which were inhibited by picrotoxin or bicuculline. Spontaneous depolarizing synaptic potentials (in >10-day cultures), which exhibited the same reversal potentials as GABA-induced potentials, were also abolished by picrotoxin or bicuculline. These observations indicate the presence of functional GABA/A synapses on these neurons. The cultured LHRH neurons were also depolarized by glutamate. GABAergic neurons which were multipolar, and possessed similar voltage-dependent ion channels and receptors as those of LHRH neurons, were also present in the cultures. Neurons of the nucleus of the diagonal band (DBN), supraoptic nucleus (SON) and paraventricular nucleus (PVN) of PN 5-10 rats were dissociated and cultured on the monolayer of the brain astrocytes for about 1 month. Following electrophysiological analyses by patch pipettes, the phenotypes of the recorded neurons were identified by double-labelling immunocytochemical methods. Both phasically and tonically firing spontaneous discharges were recorded from clearly visible neurons on the inverted microscope. Using these preparations, Ca-sensitive dyes Fura-2 (5K) or Ca green were introduced intracellularly and the dynamic states of intracellular Ca ions in these peptidergic neurons were analyzed by the Ca imaging/photometry set-up, which was coupled to the voltage-clamp system.
