Regulation of Action Potential Frequency and Amplitude by T-type Ca²⁺ Channel During Spontaneous Synchronous Activity of Hippocampal Neurons

In this paper, the changes in the frequency and amplitude of action potentials (AP) were investigated depending on the depolarization caused by the Ca²⁺ channels activity during the spontaneous synchronous activity (SSA) of hippocampal neurons in culture. It is known that the pacemaker neuronal electrical activity can be both tonic and bursting. Using the image analysis to measure [Са²⁺]_i and patch-clamp to register the membrane potential we show that depolarization caused by the GABA(A) receptor inhibitor results in a pattern of SSA in which tonic APs frequency of 2−3 Hz are generated by a neuron without any changes in cytosolic free Ca²⁺ concentration, ([Ca²⁺]_i). The tonic mode is interrupted by bursts activity, which is accompanied by slow depolarization and calcium pulses. The inhibitor of T-type calcium channels, ML218, suppresses this process. The frequency and amplitude of the AP are regulated by slow depolarization pulses as follows: on the depolarization front, the APs frequency increases. At the same time, the amplitude decreases due to Na⁺ channels inactivation. The higher is the depolarization rate, the higher is the APs frequency. If slow depolarization amplitude exceeds Na⁺ channels reactivation potential, the neuronal impulse activity stops. As the [Ca²⁺]_i increases and Ca²⁺-dependent K⁺ channels are activated, depolarization amplitude decreases slowly, and Na⁺ channels are reactivated, which leads to a gradual increase in the amplitude of APs against the background of depolarization decrease. The frequency of APs on the backside of depolarization pulse slows down to 3–4 Hz due to the occurrence of the faster Ca²⁺-potential oscillations (micro bursts of AP). Under these conditions, only one AP can be generated due to rapid depolarization at the leading edge. After that, APs generation is suppressed due to Na⁺ channel inactivation. The frequency of APs in this case coincides with the Ca²⁺ channel activation frequency (3−4 Hz). The burst firing is terminated due to [Ca²⁺]_i increase, Ca²⁺-dependent K⁺ channels activation, and voltage-gated Ca²⁺ channels (VGCC) inactivation. As a result, the membrane is hyperpolarized even more (10 mV lower than the critical potential), that suppress AP generation, activate HCN-like channels and reactivate Na⁺ and VGCC. The activity of HCN-like channels increases, the membrane slowly depolarizes and reaches the threshold potential. The generation of tonic APs begins, and then, the Ca²⁺ channels opens, and Ca²⁺ potential and Ca²⁺ signal induced again. Thus, the Ca²⁺-channels, is determining the pulse of slow depolarization, control the frequency and the amplitude of APs during SSA, regulating the activation and inactivation conditions of Na⁺ channels. The T-type channels inhibitors reduce the duration of burst firing and Ca²⁺ impulse in neurons in vitro, stimulating their survival during hyperexcitation and ischemia. Thus, reducing the Ca²⁺ pulse duration caused by the inhibitors of T-type Ca²⁺-channels, can be one of the reasons for the known neuroprotective effect of these compounds.

Keywords: spontaneous synchronous activity of neurons, calcium signal, T-type calcium channels, voltage-gated calcium channels, action potential, genesis of bursting activity, action potential bursts, depolarization shift, critical potential