大鼠藍斑核之持續性鈉離子電流

Abstract

藍斑核 (locus coeruleus；LC) 神經元廣泛投射於許多腦區。它是大腦正腎上腺素 (norepinephrine；NE) 的主要來源，並且扮演許多認知和生理功能的角色，如：睡眠醒覺循環、注意力、學習與記憶、疼痛和大腦新陳代謝…等。因此，探索藍斑核細胞放電的調控可以瞭解此藍斑核正腎上腺素系統的運作與認知功能的關係。在此篇報告探討的是持續性鈉離子電流 (persistent Na+ current；INaP) 有可能成為藍斑核細胞自發性放電 (spontaneous firing) 的可能原因。以30mV 到 -100mV (20 mV/s) 的斜波電位法 (ramp) 引發電流，加入河豚毒素 (tetrodotoxin；TTX) 前後的電流紀錄線 (current trace) 差異即為INaP。INaP開啟的電位大約在-50到-60 mV，電流為-103 +- 16 pA (n = 12)。INaP的活化曲線 (activation curve) 可被二狀態一級波茲曼方程式 (two-state first-ordered Boltzmann equation) 描述，得到k為6.8 +- 0.8和Vh為-24 +- 1.5 mV。以非侵入性法量測17顆細胞膜電位，得知膜電位平均為-51 +- 1.5 mV。其中10顆為非自發性放電 (non-spontaenous firing) 的細胞，其膜電位 (membrane potential；Vm) 為-54 +- 1.3 mV，而7顆會自發性放電的細胞膜電位為-47 +- 1.7 mV。平均上，細胞膜電位和INaP的開啟電位相近，若膜電位達其閾值 (threshold) 則可穩定地推動膜電位提升達到自發性放電，反之則否。在電位箝制模式 (voltage clamp mode) 或電流箝制模式 (current clamp mode) 紀錄下，可以看到藍斑核細胞膜電位在動作電位 (action potential；AP) threshold以下仍然產生規律的振盪 (oscillation)，此振盪可被卡貝索酮 (carbenoxolone；CBX)抑制，表示是經connexin調控之相鄰細胞傳入的動作電流。這個振盪可以使藍斑核細胞受正電流 (positive current) 興奮後，產生相位式放電 (phasic firing)。使用力如太 (riluzole) 抑制INaP，可以完全或部分地抑制藍斑核細胞的自發性放電，並且改變動作電位波型，造成動作電位頂點 (peak) 電位下降與動作電位的去極化 (depolarize) 速度降低，甚至可讓部分細胞產生適應性放電 (adaptive firing)，可見INaP對於藍斑核細胞自發性放電，動作電位完整性有很重要的地位。

Locus coeruleus (LC) neurons have widely projections to various areas of brain; they provide the forebrain the major supply of norepinephrine which is well known to play important roles in many cognitive functions, including sleep-awake cycle, alertness attention, learning and memory, pain, and brain metabolism, etc. Accordingly, investigating the regulation of LC neurons is essential to understand how LC-NE system associated brain functions are regulated. Here we reported the persistent voltage-gated sodium current (INaP) could be a major player for generating spontaneous firing in LC neurons. We recorded membrane currents responded to a voltage-ramp from 30mV to -100mV (slope = 20 mV/s) in control and in the presence of 1 μM tetrodotoxin (TTX) in LC neurons; subtracting recording in TTX from that in control condition yielded INaP, the range of activation threshold and peak amplitude of which were about -50 to -60 mV and -103 +- 16 pA (n = 12 cells), respectively. The activation curve of INaP was fitted with two-state fisrt-ordered Boltzmann equation with estimated k-value and half-activation voltage (Vh) being 6.8 +- 0.8 and -24 +- 1.5 mV, respectively. By using noninvasive membrane potential measurement to estimate resting membrane potential (Vm) without interrupting cytosol compositions of the recorded neurons and misjudging the seal quality in whole cell recording, the averaged Vm from 17 LC neurons was -51 +- 1.5 mV, 10 of them whose Vm is -54 +- 1.3 mV showed no spontaneous firing and the 7 of them with estimated Vm = -47 +- 1.7 mV could fire spontaneously. These results show that Vm and activation threshold of INaP in LC neurons are about the same value. This feature together with the high k-value of INaP could allow the self-generation of AP in LC neurons. In current clamp recording, most LC neurons showed sub-threshold voltage-oscillation, which was blocked by connexin blocker, carbenoxolone, showing the activity was generated by action currents of discharging neighboring cells flowing through gap junctions. This voltage-oscillation enable the generation of phasic firing in recorded LC neuron. Moreover, selective blocker of INaP─riluzole can fully or partially inhibit the spontaneous firing in LC neurons, abolish the repetitive firing pattern in some neurons, and change the AP waveform when injecting 100 or 200 pA from Vm at I = 0. The peak amplitude and rate of rising phase are both decreased. Also, INaP probably can enhance the oscillation amplitude due to the similar range between threshold of INaP and Vm at I = 0. In conclusion, INaP is an important factor that completes the intact firing ability in LC neurons.