Calcium homeostasis in rat septal neurons in tissue culture

Septal neutons from embryonic rats were grown in tissue culture. Microfluorimetric and electrophysiological techniques were used to study Ca²⁺ homeostasis in these neurons. The estimated basal intracellular free ionized calcium concentration ([Ca²⁺]_i) in the neurons was low (50–100 nM). Depolarization of the neurons with 50 mM K⁺ resulted in rapid elevation of [Ca²⁺]_i to 500–1,000 nM showing recovery to baseline [Ca²⁺]_i over several minutes. The increases in [Ca²⁺]_i caused by K⁺ depolarization were completely abolished by the removal of extracellular [Ca²⁺], and were reduced by 80% by the ‘L-type’ Ca²⁺ channel blocker, nimodipine (1 μM). [Ca²⁺]_i was also increased by the excitatory amino andl-glutamate, quisqualate, AMPA and kainate. Responses to AMPA and kainate were blocked by CNOX and DNOX. In the absence of extracellular Mg²⁺, large fluctuations in [Ca²⁺]_i were observed that were blocked by removal of extracellular Ca²⁺, by tetrodotoxin (TTX), or by antagonists ofN-methyld-aspartate (NMDA) such as 2-amino 5-phosphonovalerate (APV). In zero Mg²⁺ and TTX, NMDA caused dose-dependent increases in [Ca²⁺]_i that were blocked by APV. Caffeine (10 mM) caused transient increases in [Ca²⁺]_i in the absence of extracellular Ca²⁺, which were prevented by thapsigargin, suggesting the existence of caffeine-sensitive ATP-dependent intracellular Ca²⁺ stores. Thapsigargin (2 μM) had little effect on [Ca²⁺]_i, or on the recovery from K⁺ depolarization. Removal of extracellular Na⁺ had little effect on basal [Ca²⁺]_i or on responses to high K⁺, suggesting that Na⁺/Ca²⁺ exchange mechanisms do not play a significant role in the short-term control of [Ca²⁺]_i in septal neurons. The mitochondrial uncoupler, CCCP, caused a slowly developing increase in basal [Ca²⁺]_i; however, [Ca²⁺]_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 [Ca²⁺]_i. In simultaneous electrophysiological and microfluorimetric recordings, the increase in [Ca²⁺]_i associated with action potential activity was measured. The amplitude of the [Ca²⁺]_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 [Ca²⁺]_i from the modest Ca²⁺ loads imposed on the neuron by action potential trains follows a simple exponential decay (τ = 3–5s).