WHOLE-CELL NMDA-EVOKED CURRENT IN SUPRACHIASMATIC NEURONS OF THE RAT - MODULATION BY EXTRACELLULAR CALCIUM-IONS

The action of N-methyl-D-aspartic acid (NMDA) on,suprachiasmatic neuro nes was studied using whole-cell recordings in coronal hypothalamic sl ices of the rat. The location of the recorded neurones within the supr achiasmatic nucleus was ascertained by intracellular labelling with bi ocytin, followed by histological processing of the slice. Suprachiasma tic neurones had an input resistance of 780+/-20 M Omega (mean+/-S.E.M .; n=106). They were voltage-clamped at or near their resting membrane potential and their responsiveness to NMDA was tested by adding this compound to the perfusion solution. NMDA generated an inward current i n about 85% of the neurones. At 50 mu M, the average induced peak curr ent was 30+/-10 pA (n=32); at 100 mu M, it was 50+/-10 pA (n=12). The NMDA-induced current was reduced by D-2-amino-5-phosphopentanoic acid (D-AP5), an NMDA receptor antagonist, and was suppressed by MK-801, an NMDA channel blocker. Reducing the extracellular magnesium concentrat ion from 1 to 0.01 mM caused a 2- to 3-fold increase in the amplitude of this current. Thus, suprachiasmatic neurones are endowed with funct ional NMDA receptor-channels, which may play a role in glutaminergic t ransmission in this nucleus. Decreasing the extracellular calcium conc entration from 2 to 0.01 mM caused a 1.3- to 4.5-fold enhancement in t he whole-cell NMDA current. This effect was probably nor mediated by a change in the intracellular free calcium concentration. Indeed, loadi ng suprachiasmatic neurones with 11 or 20 mM of the calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N'-N'-tetracetic acid (BAPTA) suppre ssed a calcium-dependent slowly decaying outward aftercurrent but did not affect the low-calcium-induced facilitation of the NMDA response. NMDA current-voltage relations were established in normal and low-calc ium perfusion solutions. In the normal solution, the net current gener ated by NMDA contained a region of negative slope conductance and reve rsed in polarity at 7+/-2 mV. In the low-calcium solution, this curren t increased in amplitude in the region of negative slope conductance, whereas at more depolarized potentials it was not altered. The NMDA-in duced current was fitted using the Boltzmann equation. The effect of l ow-calcium solution could be modelled by shifting the activation of th e NMDA-sensitive conductance in the negative direction, by about 17 mV . We conjecture that lowering external calcium can unmask negative sur face charges located on or near the NMDA channel and that this, in tur n, weakens the voltage-dependent block of the channel by magnesium. A voltage-dependent blockade of the NMDA channel by calcium, however, ma y also contribute to this effect. This low-calcium-induced facilitatio n of the NMDA response could play a regulatory role by enhancing calci um influx through the NMDA channel in case of calcium depletion in its vicinity.