中国对虾(Fenneropenaeus chinensis)视神经节神经内分泌细胞电生理特征

ELECTROPHYSIOLOGICAL STUDY OF NEUROSECRETORY NEURONS IN EYESTALK OF SHRIMP FENNEROPENAEUS CHINENSIS

利用膜片钳技术对体外培养 2 4— 48h中国对虾的视神经节端髓A型和B型神经内分泌细胞进行电生理特征研究。电流钳的结果表明 ,A型细胞具有自发和诱发放电活动 ;B型细胞没有记录到明显放电活动。在电压钳模式下 ,中国对虾视神经节神经内分泌细胞表达TTX敏感Na+通道、高电压激活L型Ca2 +通道、TEA敏感晚钾通道、早钾通道。Ca2 +通道电流在- 40— - 30mV被激活 ,在 - 1 0— 0mV时达到峰值 ;Ca电流受钳制电压的影响。一定电压范围内 ,钳制电压越负 ,Ca电流就越大。外向的钾通道电流均在 - 40mV左右被激活。晚钾通道电流对TEA敏感 ,但TEA不能完全阻断晚钾通道电流。

X-organ-sinus gland (XO-SG) complex is an important neurosecretory organ in nervous system of Crustaceans. This complex produces and secretis neuropeptidergic hormones, including crustacean hyperglycemic hormone (CHH), molting-inhibiting hormone (MIH), gonad-inhibiting hormone (GIH), mandibular organ-inhibiting hormone (MOIH), pigment concentrating hormone (PCH) and pigment dispersing hormone (PDH). These neuropeptides play a key role in the regulation of a variety of important physiological processes, such as molting, reproduction, carbohydrate metabolism, pigment migration and osmoregulation. Due to importance of this complex, it has become a model structure in crustacean endocrinology research. Due to limited knowledge on this system, especially no single neuron, studies on this complex have still stopped at tissue level, many basic problems of this system have yet to be clarified. Chinese shrimp, Fenneropenaeus chinensis, with great commercial value, is a major aquaculture species in China. It also provides a source of cell and tissue types for basic research and biotechnological application. In this study the medulla terminalis neurons in Chinese shrimp eyestalk were used to carry out the culture. The neurons showed immediate outgrowth in the culture medium. The gain of single neurons makes it possible to carry out research on secretion mechanisms and hormone regulation at cellular and molecular level. The patch clamp recording technique developed by Neher and Sakmann in 1976. This technique is an invaluable tool for determination of currents biophysical properties. and can be easily combined with other techniques such as fluorescence microscopy and flash photolysis. In recent years, whole cell patch pipette has been used to introduce into the cell ion selective fluorophores for quantitative measurements of calcium, magnesium, sodium and proton concentrations. Similarly, various caged compounds have been introduced into the cell to study the role of second messengers in the cell by flash photolysis. In this paper, the whole-cell patch clamp technique was used to study properties of voltage-gated ion channels expressed by the cultured in vitro secretory neurons (24—48h after plating) of medulla terminalis in the eyestalks of F. chinensis. The study focused on the type A and B cells. Under current clamp, spontaneous and inducing excitability and secretion were observed in type A neurons, no excitability was observed in type B cells. Overshooting action potential could be recorded in type A neurons when given a 30pA stimulus. Under voltage clamp, net current consisted of inward and outward currents. When solutions that suppressed the outward currents were used, a tetrodotoxin (TTX)-sensitive Na + current (I Na) and Cd 2+-sensitive Ca 2+ current (I Ca) were resolved in two cell types. When holding potential (V h) was -50mV, I Ca was activated at potential -40—-30mV and maximal at -10—*$0mV. I Ca was altered by change of holding potential, the peak value increased about 15%—20% when V h changed from -40mV to -70mV. In the presence of 1μmol/L TTX and 0.5mmol/L Cd 2+, two cell types expressed a slower-rising, TEA-sensitive current and a 4-AP-insensitive transient current. Two kinds of outward current were activated at the same potential of -40mV.