Calcium homeostasis in rat septal neurons in tissue culture

Abstract

Septal neutons from embryonic rats were grown in tissue culture. Microfluorimetric and electrophysiological techniques were used to study Ca 2+ homeostasis in these neurons. The estimated basal intracellular free ionized calcium concentration ([Ca 2+] i) in the neurons was low (50–100 nM). Depolarization of the neurons with 50 mM K + resulted in rapid elevation of [Ca 2+] i to 500–1,000 nM showing recovery to baseline [Ca 2+] i over several minutes. The increases in [Ca 2+] i caused by K + depolarization were completely abolished by the removal of extracellular [Ca 2+], and were reduced by ∼80% by the ‘L-type’ Ca 2+ channel blocker, nimodipine (1 μM). [Ca 2+] i was also increased by the excitatory amino and l-glutamate, quisqualate, AMPA and kainate. Responses to AMPA and kainate were blocked by CNOX and DNOX. In the absence of extracellular Mg 2+, large fluctuations in [Ca 2+] i were observed that were blocked by removal of extracellular Ca 2+, by tetrodotoxin (TTX), or by antagonists of N-methyl d-aspartate (NMDA) such as 2-amino 5-phosphonovalerate (APV). In zero Mg 2+ and TTX, NMDA caused dose-dependent increases in [Ca 2+] i that were blocked by APV. Caffeine (10 mM) caused transient increases in [Ca 2+] i in the absence of extracellular Ca 2+, which were prevented by thapsigargin, suggesting the existence of caffeine-sensitive ATP-dependent intracellular Ca 2+ stores. Thapsigargin (2 μM) had little effect on [Ca 2+] i, or on the recovery from K + depolarization. Removal of extracellular Na + had little effect on basal [Ca 2+] i or on responses to high K +, suggesting that Na +/Ca 2+ exchange mechanisms do not play a significant role in the short-term control of [Ca 2+] i in septal neurons. The mitochondrial uncoupler, CCCP, caused a slowly developing increase in basal [Ca 2+] i; however, [Ca 2+] i recovered as normal from high K + stimulation in the presence of CCCP, which suggests that the mitochondria are not involved in the rapid buffering of moderate increases in [Ca 2+] i. In simultaneous electrophysiological and microfluorimetric recordings, the increase in [Ca 2+] i associated with action potential activity was measured. The amplitude of the [Ca 2+] i increase induced by a train of action potentials increased with the duration of the train, and with the frequency of firing, over a range of frequencies between 5 and 200 Hz. Recovery of [Ca 2+] i from the modest Ca 2+ loads imposed on the neuron by action potential trains follows a simple exponential decay ( τ = 3–5s).