Morphological Study For Expression Of VGluT1 In The Spinal Cord, Dorsal Root Ganglion And Peripheral Nerve Endings Of The Rat

As an important amino acid of mammalian, glutamate widely distributed in the central nervous system(CNS). Functionally, glutamate involve the process of development, differentiation and synaptic transmission. With the development of molecular biological techniques, special glutamate transporters have been cloned in the brain. They specially located on the membrane of glutamate vesicles. With the change of H+ concentration and existence of Cl-, they are responsible for the transmission of glutamate from plasma into the vesicles. So they were renamed as vesicular glutamate transporters (VGluT1, VGluT2 and VGluT3). The recent studies found that VGluTl and VGluT2 specially distributed in the glutamatergic axonal terminals in the CNS. They are regarded as the markers of glutamatergic terminals. In the spinal cord (SC), VGluT1-like immunoreactivity (-LI) was mainly distributed in the medial part of laminae II-VI, while VGluT2-LI was dense in the laminae I-II. VGluT3 was specially distributed not only in glutamatergic terminals, but in the somata, dendrites or axon terminals ofnon-glutamatergic neurons. In the dorsal root ganglion (DRG) and trigeminal ganglion (TG), VGluT1 mRNA or protein was mainly observed in the large or medial diameter somata of neurons. Moreover, other studies showed that the VGluT1-LI could be found in the muscle-spindles of the triceps surae muscle. The above research demonstrated that VGluTl may be involved in the non-nociceptive information transmission to the supraspinal center. However, there are some questions still unknown: (1) Whether the axon terminals showing VGluT1-LI in the spinal cord originated from the peripheral DRG neurons and/or from the supraspinal structures; (2) Whether VGluTl was transfered peripherally and centrally by the axonal flow; (3) Whether the ligation of peripheral nerve affected synthesize of VGluTl in the DRG neurons. It is not yet reported whether VGluTl was expressed in the neuronal endings of mechanoreceptors except the muscle spindle.On the basis of above questions, the present study examined: (1) The distribution of VGluT-LI in DRG and the change of VGluT1-LI in L4 segment of DRG after the sciatic nerve (SN) ligation; (2) The origination of the axon terminals of VGluTl in the lumbar spinal cord; (3) The effects of complete ligation of the unilateral sciatic nerve (SN) on transmission of VGluT1-LI by axonal flow peripherally and centrally through axons of DRG neurons; (4) Expression of VGluTl and VGluT2 in the mechanoreceptors of the hind palm skin.Part oneChange of expression of VGluTl in the L4 segment of DRG neurons after the complete SN ligationThe rats were divided into 2 groups in the present study (normal group and operated group). Immunohistochemical method and counterstained with eosin were used to examine the distribution and expression of VGluT1-LI in the different diameter neurons of fourth lumbar segment of DRG. And change of the density of VGluT1-LI in fourth lumbar segment of DRG after the complete SC ligation of the rat with different survival time. The results were as follows:(1) VGluT1-immunoreativity weas seen as punctate immunostaining in the neuronal perikarya of the L4 segmentof DRG in the normal rats. There were a lot of DRG neurons expressing VGluT1-LI. In these neurons, medium- diameter neurons (20~40μm) is predominance and the ratio was about 65.5%.(2)The number of VGluT1-positive neurons in L4 DRG was not detected the obvious change after 1, 2 days of complete SN ligation. But from 4 days after complete SN ligation, the number of VGluT1-positive neurons decreased transiently with the survival time. From 1 to 4 weeks after complete SN ligation, the number of VGluT1-positive neurons decreased significantly than that of control side of same rat (P<0.05 or P<0.01). On the other hand, comparing the operation side in the ligation 1 to 4 weeks with in the ligation 1 to 3 days, the significant difference in the reduction of number of VGluT1-positive neurons between them wasfound(P<0.05 or P< 0.07).The results indicated that VGluTl was synthesized in the cell bodies of DRG neurons and was transported peripherally by axonal flow. Therefore, VGluTl in the DRGs was vulnearable to the damage of the peripheral nerves.Part twoThe expression and origination of VGLuT1 in the lumbar spinal cord and peripheral nerve stump of the ratThe rats were divided into three groups in present experiment: the twelfth thoracic cord hemisection group; dorsal rhizotomy from the first lumbar nerve(L1) to the first sacral nerve (S1) group; unilateral ligation of SN and kept survival for 1, 2, 4 days and 1, 2, 4 weeks goup. Immunoreactivity method was used to observe: (1) the change of expression of VGluTl in the L4 spinal cord segment after hemisection thoracic cord or the ligation of SN; (2) the density of VGluT1-LI in L4 spinal cord segment and nerve stump distal or proximal to the ligation site. The result were as follows:(1) In the rats survived 7 days after the twelfth thoracic cord hemisection, no obvious change of VGluT1-positive density of L4 spinal cord segments on the operation side compared with contralateral side.(2) In the rats survived 7 days after dorsal rhizotomy from L1 to S1, the obvious decrease of VGluT1-LI could be detected in the all lamina of L4 spinal cord segment, especially in the lamina II to IV on the operation side compared with contralateral side.(3) In the rats that were unilaterally ligated of SN and allowed to survive for 1, 2, 4 days and 1, 2, 4 weeks, respectively, there is no apparent change of VGluT1-LI in the L4 spinal cord in the first 2 days. But from 4d, the intensity of VGluT1-LI in the inner part of lamina II, lamina III, the medial part of laminae IV-VI and laminae VIII-IX of the spinal cord on the side ipsilateral to ligation decreased persistently, and the VGluT1-LI has been showed sparsely after ligation 4 weeks.(4) In group 3, VGluT1-LI was observed to increase in the nerve stump proximal to ligation 1d after unilateral ligation of the SN, but to decrease persistently in the nerve stump proximal to ligation from 4d. While VGluT1-LI decreased persistently in the nerve stump distal to ligation from 1d and disappeared from 4d after unilateral ligation of the SN.The present results indicate that: (1) Part of VGluT1-positive axonal terminals in the lumbar spinal cord originated from DRG neurons, and the rest part might originate from the interneurons of the spinal cord; (2) VGluTl was transported peripherally by axonal flow from DRG neurons.Part threeThe expression of VGluT1-like immunoreactivity in the nerve endings in the skin of ratIn this study, immunohistochemical method was used to detect the expression of VGluTl or VGluT2 in the various receptors of the dermal papillae of hind paw. The results showed that: VGluT1-LI was detected...