Electrical properties of oxytocin neurons in organotypic cultures from postnatal rat hypothalamus

Abstract

1. Intracellular recordings were performed on immunocytochemically identified oxytocin (OT) neurons (n = 101) maintained for 2-7 wk in hypothalamic organotypic cultures derived from 4-to 6-day-old rat neonates. The neurons displayed a resting potential of -58.9 +/- 6.8 mV (mean +/- SD, n = 74), an input resistance of 114 +/- 26.8 M omega (n = 66), and a time constant of 9.6 +/- 1.4 ms (n = 57). Voltage-current (V-I) relations, linear at resting potential, showed a pronounced outward rectification when depolarized from hyperpolarized membrane potentials. At these hyperpolarized potentials, depolarizing current pulses induced a delayed action potential. 2. Action potentials had an amplitude of 73.4 +/- 9.7 mV and a duration of 1.9 +/- 0.2 ms. Each action potential was followed by an afterhyperpolarization of 7.9 +/- 2.0 mV in amplitude lasting 61.7 +/- 11.3 ms. The depolarizing phase of action potentials was both Na+ and Ca2+ dependent, whereas repolarization was due to a K+ conductance increase. 3. When Ba2+ was substituted for Ca2+ in the medium, OT neurons displayed prolonged sustained depolarizations. In the presence of tetrodotoxin (TTX), these depolarizations were triggered by depolarizing current pulses and arrested by hyperpolarizing current pulses or by local application of Ca2+, Co2+, Cd2+, No sustained depolarization was obtained when nifedipine was added to the medium. These data suggest that OT cells in organotypic culture possess L-type Ca2+ channels. 4. All OT neurons generated spontaneous action potentials at resting potential. Of 59 neurons, 29 showed a slow, irregular firing pattern (< or = 2.5 spikes/s), 24 generated a fast continuous firing pattern (> or = 2.5 spikes/s), and 6 cells displayed a bursting pattern of activity consisting of alternating periods of spike discharge and quiescence. None of the bursting cells exhibited regenerative endogenous potentials (plateau potentials). On the contrary, in four of these cells, the bursting activity was clearly due to patterned synaptic activity. 5. The cultured OT cells responded to exogenous gamma-aminobutyric acid (GABA) and muscimol with a hyperpolarization and an increase in membrane conductance. These effects still were observed in the presence of TTX, indicating that they were due to direct activation of GABA receptors in the cells. The GABA-induced response was mediated by GABAA receptors because it was blocked by bicuculline, but not by GABAB receptors, because baclofen and hydroxysaclofen had no effect on membrane potential and input resistance. 6. OT neurons responded to exogenous glutamate, quisqualate, and kainate with a depolarization concomitant with an increase in membrane conductance. N-methyl-D-aspartate depolarized the cells in Mg(2+)-free medium. These effects were observed in the presence of TTX, suggesting that OT cells expressed ionotropic glutamate receptors. Trans-(1S,3R)-1-amino-1,3-cyclopentane-dicarboxylic acid and (+/-)-alpha-amino-4-carboxymethylphenylglycine had no effect on OT cells, thus excluding the presence of metabotropic glutamate receptors. 7. Taken together, our observations demonstrate that hypothalamic slice cultures from 4- to 6-day-old rat neonates contain well-differentiated OT neurons that display electrical properties similar to those shown by adult neurons in vitro. Such cultures provide a reliable model to investigate membrane properties of adult OT neurons and a useful means to study the long-term modulation of their electrical behaviour by various agents known to affect OT cells in vivo.