Release and sequestration of calcium by ryanodine‐sensitive stores in rat hippocampal neurones

Abstract

1. The properties of ryanodine-sensitive Ca¥ stores in CA1 pyramidal cells were investigated in rat hippocampal slices by using whole-cell patch-clamp recordings combined with fura_2-based fluorometric digital imaging of cytoplasmic Ca¥ concentration ([Ca¥]é). 2. Brief pressure applications of caffeine onto the somata of pyramidal cells caused large transient increases in [Ca¥]é (Ca¥ transients) of 50-600 nÒ above baseline. 3. The Ca¥ transients evoked by caffeine at −60 mV were not associated with an inward current, persisted after blocking voltage-activated Ca¥ currents and were completely blocked by bath-applied ryanodine. Similar transients were also evoked at +60 mV. Thus, these transients reflect Ca¥ release from intracellular ryanodine-sensitive Ca¥ stores. 4. The Ca¥ transients evoked by closely spaced caffeine pulses rapidly decreased in amplitude, indicating progressive depletion of the Ca¥ stores. The amplitude of the Ca¥ transients recovered spontaneously with an exponential time constant of 59 s. Recovery was accelerated by depolarization-induced elevations in [Ca¥]é and blocked by cyclopiazonic acid (CPA) and thapsigargin, indicating that store refilling is mediated by endoplasmic reticulum Ca¥-ATPases. 5. Even without prior store depletion the caffeine-induced Ca¥ transients disappeared after 6 min exposure to CPA, suggesting that ryanodine-sensitive Ca¥ stores are maintained at rest by continuous Ca¥ sequestration. 6. Caffeine-depleted Ca¥ stores did not refill in Ca¥-free saline, suggesting that the refilling of the stores depends upon Ca¥ influx through a 'capacitative-like' transmembrane influx pathway operating at resting membrane potential. The refilling of the stores was also blocked by Ni¥ and gallopamil (D600). 7. Elevations of basal [Ca¥]é produced by bath-applied KCl markedly potentiated (up to 6-fold) the caffeine-induced Ca¥ transients. The degree of potentiation was positively related to the increase in basal [Ca¥]é. The Ca¥ transients remained potentiated up to 9 min after reversing the KCl-induced [Ca¥]é increase. Thus, the ryanodine-sensitive Ca¥ stores can 'overcharge' when challenged with an increase in [Ca¥]é and slowly discharge excess Ca¥ after basal [Ca¥]é returns to its resting level. 8. Pressure applications of caffeine onto pyramidal cell dendrites evoked local Ca¥ transients similar to those separately evoked in the respective somata. Thus, dendritic ryanodinesensitive Ca¥ stores are also loaded at rest and can function as independent compartments. 9. In conclusion, the ryanodine-sensitive Ca¥ stores in hippocampal pyramidal neurones contain a releasable pool of Ca¥ that is maintained by a Ca¥ entry pathway active at subthreshold membrane potentials. Ca¥ entry through voltage-gated Ca¥ channels transiently overcharges the stores. Thus, by acting as powerful buffers at rest and as regulated sources during activity, Ca¥ stores may control the waveform of physiological Ca¥ signals in CA1 hippocampal pyramidal neurones.

A characterization of muscarinic receptor‐mediated intracellular Ca2+ mobilization in cultured rat hippocampal neurones

“…The magnitude of the agonist-evoked responses was inversely related to the time between membrane depolarization and exposure to agonist ( Fig. 2A), which is consistent with the spontaneous discharging of intracellular Ca¥ stores following removal of a depolarizing stimulus (Shmigol et al 1994;Garaschuk et al 1997). The rate of run-down varied between cells: in 6Ï12 cells studied responses to carbachol could only be obtained within 6 min of removal of the high K¤-containing medium, whereas in the remaining cells responses persisted for up to 12 min following the depolarizing episode.…”

Synergistic Control of Protein Kinase Cγ Activity by Ionotropic and Metabotropic Glutamate Receptor Inputs in Hippocampal Neurons

“…This was not unexpected because Ca 2ϩ stores in neurons have been reported to be empty at rest and to replenish only when Ca 2ϩ stores are filled by electrical activity (Shmigol et al, 1994;Rae et al, 2000). This condition is not specific of primary cultures, because also in unstimulated slices of hippocampus the intracellular stores were reported to act as powerful buffers, rather than regulated sources of Ca 2ϩ (Garaschuk et al 1997) or to provide a significant contribution only after they were loaded by a preceding exposure to high [Ca 2ϩ ] i (Rae and Irving, 2004). It was more surprising that Ca 2ϩ influx, mediated either by NMDARs or VOCCs, did not only induce PKC␥ translocation but also triggered the production of DAG.…”

Activity-Dependent Calcium Sequestration in Dendrites of Hippocampal Neurons in Brain Slices

“…Since then, a related but distinct Ca 2ϩ -regulating function of the ER, namely, that of a releasable Ca 2ϩ store, has been well characterized (Alford et al, 1993;Llano et al, 1994;Seymour-Laurent and Barish, 1995;Pozzo-Miller et al, 1996;Garaschuk et al, 1997;Golovina and Blaustein, 1997). This store, because it is engaged in cycles of Ca 2ϩ uptake and release, plays an important role in neuronal Ca 2ϩ buffering (Andrews et al, 1988;Markram et al, 1995).…”