视网膜节细胞与视交叉上核AVP神经元联系的研究Ⅱ.假狂犬病毒顺行追踪与免疫荧光双重标记

为进一步揭示视网膜节细胞与视交叉上校精氨酸血管加压素神经元间有无直接的联系，本文将假狂犬病毒注入大鼠眼球内通过顺行追踪结合免疫荧光双重标记法观察到：（1）视交叉上核神经元被病毒感染的时间始于病毒注入后56h，并随存活时间的延长而增多；（2）呈绿色荧光的病毒感染神经元见于双侧视交叉上核，注射对侧优于同侧，主要位于视交叉上核的腹外侧部和嘴侧份，个别散在于二者之外;（3）视交叉上核内个别病毒感染的神经元可同时呈精氨酸血管加压素的红色荧光，此类双标神经元系病毒由视网膜节细胞顺行运输并跨突触传给视交叉上核的精氨酸血管加压素神经元所致。表明视网膜节细胞与视交叉上核的精氨酸血管加压素神经元间有直接的突触性联系。这一结果为视交叉上核节律机制和精氨酸血管加压素神经元的机能调控提供了进一步研究的线索。

A STUDY ON THE CONNECTION BETWEEN RETINAL GANGLION CELL AND ARGININE VASOPRESSIN NEURON IN SUPRACHIASMATIC NUCLEUS Ⅱ.PSEUDORABIES VIRUS ANTEROGRADE TRACING AND IMMUNOFLUORESCENCE DOUBLE-LABELLING TECHNIQUES

The retinohypothalamic tract (RHT) is a direct monosynaptic projection from the retinal ganglion cells (RGC) to the hypothalamic suprachiasmatic nucleus (SCN) which is the oscillator of circadian rhythm in mammal. Our previous study found that RGC terminals contact closely with AVP neuron's soma in the SCN. To prove these facts further, the present study applied 20 SD rats receiving uniocular injection of pseudorabies virus (PRV, Bartha strain, 4 μl,5 × 108 pfu/ml). After 48 h, 56 h, 72h, 96 h(5 rats in each group), animals were perfused with fixative containing 4% para formaldehyde, 1. 37 % lysine and 0. 21 %sodium-periode. A block including SCN from animal was removed and cut on cryostat in coronal plane. 40μm sections were incubated in an antiserum mixture of Goat anti-PRV (1: 200) and Rabbit anti-AVP (1:2000) for 24 h, then transferred to a solution containing 1: 100 FITC-conjugated Donkey anti-Goat IgG and 1: 200 Texas Red-conjugated Donkey anti-Rabbit IgG (Jacson), for 1 h. Results showed (1) The first signs of PRV infected neurons in the SCN occurred 56 h following intraocular injection of the virus, and the number of infected cells increased progressively with longer survival intervals. (2) Green fluorescentPRV infected neurgns, which distributed bilaterally and much more contralaterally than ipsilabrally, were numerously displayedin ventrolateral and rostral portion of the SCN. (3) A few PRV-infected neurons in the SCN showed red fluorescence of AVP simultaneously in each group, and the number of PRV/AVP double-labelled neurons increased with longer survival time. Theseresults indicate that there is a direct synaptic junction between RGC terminals and AVP neurons of the SCN. Regarding to immunoelectron microscopic evidence and the functional significance of the projection of RGC to AVP neuron in the SCN will be explored latter.