CHARACTERIZATION OF CA2+ SIGNALS INDUCED IN HIPPOCAMPAL CA1 NEURONS BY THE SYNAPTIC ACTIVATION OF NMDA RECEPTORS

1. A combination of confocal microscopy, whole-cell patch-clamp record ing, intracellular dialysis and pharmacological techniques have been e mployed to study Ca2+ signalling in CA1 pyramidal neurones, within rat hippocampal slices. 2. In the soma of CA1 neurones, depolarizing step s applied through the patch-pipette resulted in transient increases in the fluorescence emitted by the Ca2+ indicator fluo-3. The intensity of the fluorescence transients was proportional to the magnitude of th e Ca2+ currents recorded through the pipette. Both the somatic fluores cence transients and the voltage-activated Ca2+ currents ran down in p arallel over a period of between approximately 15-45 min. The fluoresc ence transients were considered, therefore, to be caused by increases in cytosolic free Ca2+. 3. Under current-clamp conditions, high-freque ncy (tetanic) stimulation (100 Hz, 1 s) of the Schaffer collateral-com missural pathway led to compound excitatory postsynaptic potentials (E PSPs) and somatic Ca2+ transients. The somatic Ca2+ transients were se nsitive to the N-methyl-D-aspartate (NMDA) receptor antagonist D-2-ami no-5-phosphonopentanoate (AP5; 100 muM). These transients, but not the EPSPs, disappeared with a time course similar to that of the run-down of voltage-gated Ca2+ currents. Tetanus-induced somatic Ca2+ transien ts could not be elicited under voltage-clamp conditions. 4. Fluorescen ce images were obtained from the dendrites of CA1 pyramidal neurones s tarting at least 30 min after obtaining whole-cell access to the neuro ne. Measurements were obtained only after voltage-gated Ca2+ channel a ctivity had run down completely. 5. Tetanic stimulation of the Schaffe r collateral-commissural pathway resulted in compound EPSPs and excita tory postsynaptic currents (EPSCs), under current- and voltage-clamp, respectively. In both cases, these were invariably associated with den dritic Ca2+ transients. In cells voltage-clamped at -35 MV, the fluore scent signal increased on average 2-fold during the tetanus and decaye d to baseline values with a half-time (t1/2) of approximately 5 s. 6. The pha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 muM) parti ally reduced the tetanus-induced EPSC without affecting the Ca2+ trans ients. In contrast, AP5, which also depressed the EPSC, substantially reduced or eliminated the Ca2+ transients. 7. In normal (i.e. 1 mM Mg2 +-containing) medium, NMDA receptor-mediated synaptic currents display ed the typical region of negative slope conductance in the peak I-V re lationship (between -90 and -35 mV). The dendritic tetanus-induced Ca2 + transients also displayed a similar anomalous voltage dependence, de creasing in size from -35 to -90 mV. 8. In slices perfused for at leas t 1 h with medium which was nominally free of Mg2+, the voltage depend ence of the dendritic tetanus-induced Ca2+ transients and EPSCs was li near. At membrane potentials slightly more positive than 0 mV, small d endritic tetanus-induced Ca2+ transients, associated with outward syna ptic currents, were recorded. 9. The L-type Ca2+ channel antagonist ni trendipine (10 muM) reversibly abolished the voltage-gated Ca2+ curren ts evoked within minutes of obtaining whole-cell access, by voltage st eps between -35 and 0 mV. It had no effect, however, on tetanus-induce d dendritic Ca2+ transients, evoked from -35 mV (or -70 mV). 10. Analy sis of small (circa 4 muM) lengths of dendrite revealed oscillations i n the decaying phase of the tetanus-induced Ca2+ transients, particula rly where the decay phase was relatively slow (t1/2 > 5 s). Neighbouri ng small segments displayed markedly heterogeneous behaviour, indicati ng the independence of Ca2+ signalling within these regions. The oscil lations were substantially reduced by AP5 (100 muM). 11. Ryanodine (10 muM) or thapsigargin (10 muM), which interfere with the release of Ca 2+ from intracellular stores, reduced the peak tetanus-induced Ca2+ tr ansient by approximately 65 12. Analysis within spine-like structures also revealed Ca2+ transients. The Ca2+ transients within 'spines' dec ayed rapidly. Sustained Ca2+ elevations were not seen. 13. We conclude that tetanic stimulation of the Schaffer collateral-commissural pathw ay, through the synaptic activation of NMDA receptors, elevates Ca2+ b y at least three mechanisms. There is entry into the soma through volt age-gated Ca2+ channels, entry into the dendrites by permeation throug h NMDA receptor-operated channels and release from intracellular store s. The significance of these distinct Ca2+ signals for synaptic plasti city is discussed.