Membrane properties underlying patterns of GABA-dependent action potentials in developing mouse hypothalamic neurons

Spikes may play an important role in modulating a number of aspects of brai n development. In early hypothalamic development, GABA can either evoke act ion potentials, or it can shunt other excitatory activity. In both slices a nd cultures of the mouse, hypothalamus, we observed a heterogeneity of spik e patterns and frequency in response to GABA. To examine the mechanisms und erlying patterns and frequency of GABA-evoked spikes, we used conventional whole cell and gramicidin perforation recordings of neurons (n = 282) in sl ices and cultures of developing mouse hypothalamus. Recorded with gramicidi n pipettes, GABA application evoked action potentials in hypothalamic neuro ns in brain slices of postnatal day 2-9 (P2-9) mice. With conventional patc h pipettes (containing 29 mM Cl-), action potentials were also elicited by GABA from neurons of 2-13 days in vitro (2-13 DIV) embryonic hypothalamic c ultures. Depolarizing responses to GABA could be generally classified into three types: depolarization with no spike, a single spike, or complex patte rns of multiple spikes. In parallel experiments in slices, electrical stimu lation of GABAergic mediobasal hypothalamic neurons in the presence of glut amate receptor antagonists [10 muM 6-cyano-7-nitroquinoxaline-2,3-dione (CN QX), 100 muM 2-amino-5-phosphonopentanoic acid (A-P5)] resulted in the occu rrence of spikes that were blocked by bicuculline (20 muM). Blocking ionotr opic glutamate receptors with AP5 and CNQX did not block GABA-mediated mult iple spikes. Similarly, when synaptic transmission was blocked with Cd2+ (2 00 muM) and Ni2+ (300 muM), GABA still induced multiple spikes, suggesting that the multiple spikes can be an intrinsic membrane property of GABA exci tation and were not based on local interneurons. When the pipette [Cl-] was 29 or 45 mM, GABA evoked multiple spikes. In contrast, spikes were not det ected with 2 or 10 mM intracellular [Cl-1]. With gramicidin pipettes, we fo und that the mean reversal potential of GABA-evoked current (E-GABA) was po sitive to the resting membrane potential, suggesting a high intracellular [ Cl-1] in developing mouse neurons. Varying the holding potential from -80 t o 0 mV revealed an inverted U-shaped effect on spike probability. Blocking voltage-dependent Na+ channels with tetrodotoxin eliminated GABA-evoked spi kes, but not the GA-BA-evoked depolarization. Removing Ca2+ from the extrac ellular solution did not block spikes, indicating GABA-evoked Na+-based spi kes. Although EGABA was more positive within 2-5 days in culture, the proba bility of GABA-evoked spikes was greater in 6- to 9-day cells. Mechanistica lly, this appears to be due to a greater Na+ current found in the older cel ls during a period when the E-GABA is still positive to the resting membran e potential. GABA evoked similar spike patterns in HEPES and bicarbonate bu ffers, suggesting that Cl-, not bicarbonate, was primarily responsible for generating-multiple spikes. GABA evoked either single or multiple spikes; n eurons with multiple spikes had a greater Na+ current, a lower conductance, a more negative spike threshold, and a greater difference between the peak of depolarization and the spike threshold. Taken together, the present res ults indicate that the patterns of multiple action potentials evoked by GAB A are an inherent property of them developing hypothalamic neuron.