成年蜜蜂脑神经细胞的培养和电生理特征

为了研究杀虫剂等对蜜蜂毒性作用的神经机制,需在体外建立成年蜜蜂脑神经细胞的分离培养和电生理记录技术并研究其正常电生理特征,而对成年蜜蜂脑神经细胞的分离培养和电生理特性的研究报道甚少。我们采用酶解和机械吹打相结合的方法获得了数量较多且活力较好的成年意大利蜜蜂Apis mellifera脑神经细胞,并用全细胞膜片钳技术研究了成年意大利蜜蜂脑神经细胞对电流和电压刺激的反应,获得了成年意蜂脑神经细胞的基本电生理特征以及钠电流和钾电流的特性。全细胞电流钳的记录结果表明,在体外培养条件下,细胞无自发放电发生,注射电流后仅引起细胞单次放电,引起细胞放电的阈电流平均为60.8±63 pA; 细胞动作电位产生的阈电位平均为−27.4±2.3 mV。用全细胞电压钳记录了神经细胞的钠电流和钾电流。钠电流的分离是在电压刺激下通过阻断钾通道和钙通道实现。细胞的内向钠电流在指令电压为−40～−30 mV左右激活,−10 mV达峰值,钠通道的稳态失活电压V_1/2为−58.4 mV; 外向钾电流成份至少包括较小的快速失活钾电流和和较大的缓慢失活钾电流（占总钾电流的80%）,其半激活膜电位V_1/2为3.86 mV,无明显的稳态失活。结果提示缓慢失活钾电流的特征可能是细胞单次放电的机制之一。

Primary culture and electrophysiological properties of brain neurons of adult Apis mellifera (Hymenoptera: Apidae)

To explore the neuronal mechanisms underlying pesticides intoxication in adult honeybees, the primary culture of brain neurons of adult honeybees and electrophysiological recordings are required. However, little is known of the electrophysiological properties and sodium currents or potassium currents in normal adult honeybee brain neurons. We first dissociated neurons from brains of adult Apis mellifera by gentle trituration after a 15-min incubation with papain. This preparation resulted in a large number of isolated viable neurons. Using the whole-cell configuration of the patch-clamp technique, we investigated the neuronal responses of the isolated neurons to the current or voltage stimulation. Current-clamp data showed that the tested neurons showed no spontaneous discharges but were capable of firing a single action potential in response to depolarizing current injection. The mean current threshold for action potential was 60.8±6.3 pA and the mean voltage threshold −27.4±2.3 mV. Under voltage clamp, sodium currents were isolated by blockade of potassium channel and calcium channel. The sodium current was activated at command potentials more positive than −40 mV with a maximum around −10 mV and a half maximal voltage for inactivation of −58.4 mV. The neurons expressed at least two distinct K⁺ currents, a small transient current and a large sustained current (80% of total K⁺ current) with a half maximal voltage for activation of 3.86 mV. The large sustained potassium current showed little or no inactivation. The results suggest that the prominent sustained potassium current might account for the a single spiking in the recorded adult honeybee neurons.