1. A combination of confocal microscopy, whole-cell patch-clamp recording, intracellular dialysis and pharmacological techniques have been employed to study Ca2+ signalling in CA1 pyramidal neurones, within rat hippocampal slices. 2. In the soma of CA1 neurones, depolarizing steps 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 the Ca2+ currents recorded through the pipette. Both the somatic fluorescence transients and the voltage-activated Ca2+ currents ran down in parallel over a period of between approximately 15-45 min. The fluorescence transients were considered, therefore, to be caused by increases in cytosolic free Ca2+. 3. Under current-clamp conditions, high-frequency (tetanic) stimulation (100 Hz, 1 s) of the Schaffer collateral-commissural pathway led to compound excitatory postsynaptic potentials (EPSPs) and somatic Ca2+ transients. The somatic Ca2+ transients were sensitive to the N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-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+ transients could not be elicited under voltage-clamp conditions. 4. Fluorescence images were obtained from the dendrites of CA1 pyramidal neurones starting at least 30 min after obtaining whole-cell access to the neurone. Measurements were obtained only after voltage-gated Ca2+ channel activity had run down completely. 5. Tetanic stimulation of the Schaffer collateral-commissural pathway resulted in compound EPSPs and excitatory postsynaptic currents (EPSCs), under current- and voltage-clamp, respectively. In both cases, these were invariably associated with dendritic Ca2+ transients. In cells voltage-clamped at -35 MV, the fluorescent signal increased on average 2-fold during the tetanus and decayed to baseline values with a half-time (t1/2) of approximately 5 s. 6. The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 muM) partially reduced the tetanus-induced EPSC without affecting the Ca2+ transients. 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 displayed the typical region of negative slope conductance in the peak I-V relationship (between -90 and -35 mV). The dendritic tetanus-induced Ca2+ transients also displayed a similar anomalous voltage dependence, decreasing in size from -35 to -90 mV. 8. In slices perfused for at least 1 h with medium which was nominally free of Mg2+, the voltage dependence of the dendritic tetanus-induced Ca2+ transients and EPSCs was linear. At membrane potentials slightly more positive than 0 mV, small dendritic tetanus-induced Ca2+ transients, associated with outward synaptic currents, were recorded. 9. The L-type Ca2+ channel antagonist nitrendipine (10 muM) reversibly abolished the voltage-gated Ca2+ currents evoked within minutes of obtaining whole-cell access, by voltage steps between -35 and 0 mV. It had no effect, however, on tetanus-induced dendritic Ca2+ transients, evoked from -35 mV (or -70 mV). 10. Analysis of small (circa 4 muM) lengths of dendrite revealed oscillations in the decaying phase of the tetanus-induced Ca2+ transients, particularly where the decay phase was relatively slow (t1/2 > 5 s). Neighbouring small segments displayed markedly heterogeneous behaviour, indicating the independence of Ca2+ signalling within these regions. The oscillations were substantially reduced by AP5 (100 muM). 11. Ryanodine (10 muM) or thapsigargin (10 muM), which interfere with the release of Ca2+ from intracellular stores, reduced the peak tetanus-induced Ca2+ transient by approximately 65 12. Analysis within spine-like structures also revealed Ca2+ transients. The Ca2+ transients within 'spines' decayed rapidly. Sustained Ca2+ elevations were not seen. 13. We conclude that tetanic stimulation of the Schaffer collateral-commissural pathway, through the synaptic activation of NMDA receptors, elevates Ca2+ by at least three mechanisms. There is entry into the soma through voltage-gated Ca2+ channels, entry into the dendrites by permeation through NMDA receptor-operated channels and release from intracellular stores. The significance of these distinct Ca2+ signals for synaptic plasticity is discussed.
