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Origin and modulation of action potential evoked calcium signals in hippocampal CA1 pyramidal neurons Sandler, Vladislav Michael

Abstract

Calcium is an important second messenger that participates in triggering and regulating numerous neuronal processes. Action potentials (APs or AP) initiate rapid changes of intracellular Ca2+-concentration ([Ca2+];) in both soma and dendrites of central neurons. We addressed two major questions about the origin and modulation of these changes in [Ca2 +]i. Firstly, how does a neurotransmitter, serotonin (5-HT), modulate the backpropagation of APs and associated changes in the [Ca2+]; in CA1 hippocampal pyramidal neurons? Secondly, do APs trigger Ca2+-induced Ca2+-release (CICR) from internal stores in these neurons? We used whole-cell somatic or dendritic patch-clamp recordings combined with high-speed imaging of [Ca2+]; and analyses of the responses to applications of pharmacological agents, studying the changes in electrical membrane properties and [Ca2+]i. The experiments were conducted in the CA1 pyramidal neurons of in vitro slices from the rat hippocampus (11 day- to 5 week-old). Changes in [Ca2+]; were measured in neurons filled with bisfura- 2, Calcium Green-1 or fura-2-AM. Bath applications of 5-HT increased membrane conductance and hyperpolarized both soma and apical dendrites. They also lowered peak potentials of antidromically-activated, backpropagating APs in the dendrites. In the soma, 5-HT applications increased the absolute AP-amplitude while slightly decreasing peak potentials. 5-HT reduced the amplitude of the AP-evoked changes in [Ca2+]i at all locations along the apical dendrites and soma. The application of 5-HT and antidromically evoked APs generated, through synergistic actions, increases in [Ca2+]j that propagated along dendrites (Ca2 + waves). Such waves originated in the proximal or middle apical dendrites and were not accompanied by a significant change in somatic membrane potential. A minimum of five APs was required to evoke the waves. According to these new observations and supporting literature, the waves are a likely consequence of Ca2+-induced Ca2+-release (CICR) from internal stores through (inositol-1,4,5- triphosphate) IP3-sensitive channels. In the absence of 5-HT, APs evoked CICR. Caffeine application increased the amplitude of AP-induced changes in [Ca2+]i. During simultaneous calcium imaging, the whole-cell recordings showed that caffeine application did not significantly change either the resting membrane potential or amplitude and shape of APs. The enhancement of AP-evoked Ca2+-transients due to caffeine application could not be attributed to protein phosphorylation or modulation of high-threshold Ca2+-channels. Applications of IBMX, a non-specific inhibitor of phosphodiesterases, forskolin, an activator of adenylyl cyclase, H-89, an inhibitor of PKA and PKG, or nifedipine, a blocker of high-threshold Ca2 + channels, did not mimic or prevent the caffeine effect. Pretreatment of neurons with thapsigargin or cyclopiazonic acid (CPA) -- substances that facilitate depletion of intracellular Ca2+-stores by blocking endoplasmic reticulum specific Ca2+-ATPases -- precluded this effect. Similar pretreatment with ryanodine, a blocker of 'ryanodine-sensitive' channels, also precluded the caffeine effect. Despite a presence or absence of caffeine, applications of thapsigargin, ryanodine or CPA reduced the AP-evoked changes in [Ca2+]i. From these new experimental observations, we can conclude that the CICR through ryanodine-sensitive channels contributes to the AP-induced changes of [Ca2+]; in hippocampal CA1 pyramidal neurons. [Scientific formulae used in this abstract could not be reproduced.]

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