The Distribution Of Ca_v3 T-type Channel Isoforms In Rat Spinal Dorsal Horn

Objective:According to its coding gene,T-type calcium channel(Ca_v3)can be divided into three subtypes:Ca_v3.1,Ca_v3.2,and Ca_v3.3,the expression and electrophysiological characteristics of which play an important role in the occurrence and development of neuropathic pain(NP).Spinal dorsal horn(SDH)is the primary center of pain signal transduction and local processing and it is also the key site of NP central sensitization.The differences in the distribution of Ca_v3 channel subtypes in the cell body and dendrites affects the contribution of Ca_v3 channel-mediated calcium current to cell output.Although immunohistochemical studies have demonstrated that the Ca_v3 three subtypes are differentially distributed in the adult rodent brain and dorsal root ganglion,this problem has not been elucidated in SDH.Therefore,the purpose of this study is to clarify cellular and subcellular localizations of Ca_v3 channel subtypes in SDH.These data may provide new theoretical evidence for the study of pathological pain mechanism and the development of novel analgesic drug.Methods:Sprague-Dawley(SD)rats(5-8 w)of either sex were anesthetized with cold ices.After transcardial perfusion and extraction of the lumbosacral spinal cords(L1-S3),the pia-arachnoid membrane as well as all dorsal and ventral roots were removed with fine forceps.The spinal cord segments were removed and post-fixed in 4%paraformaldehyde,then transferred to 30%sucrose.The head and tail of the spinal cord were removed and the lumbosacral enlargement of L4-L6 was left behind,then embedded by OCT-medium before frozen section.A serial of 30-?m-thick spinal cord slices in transverse or parasagittal planes were cut with a freezing microtome.The SDH marker was incubated with Ca_v3.1-3.3 primary antibodies by immunofluorescence histochemistry method and then incubated with the corresponding second antibodies overnight at 4°C.The general distribution of Ca_v3.1-3.3-immunolabeling in spinal cord and the cellular and subcellular localizations of Ca_v3.1-3.3-immunolabeling in SDH were observed and analyzed under confocal microscope.Results:1.Ca_v3.1,Ca_v3.2 and Ca_v3.3 channel proteins are widely distributed throughout the gray matter of the spinal cord,with the most expressed in the laminae I-II of SDH,followed by the laminae III-IV,lamina IX,and the least in the laminae V-VI.In addition,Ca_v3.1-3.3 also have weak expression in the white matter of the spinal cord;2.Ca_v3.1 shows a punctate staining pattern in SDH,while Ca_v3.2 and Ca_v3.3showed a neuron-like on SDH.And Ca_v3.2 exhibited the strongest intensity in medial region compared to the lateral and central region of SDH.All three subtypes of Ca_v3channel express in the motor neurons of the ventral spinal horn;3.Ca_v3.1 and Ca_v3.3 exhibit a moderate degree distribution in dendrites,with less Ca_v3.1-3.3 distribution in axons;4.Ca_v3.1 is distributed at primary afferent terminals and the afferent terminals of interneurons,and mainly located in peptidergic nerve terminals and axon terminals of inhibitory interneurons.Ca_v3.3 also has a small distribution at the axon terminals of inhibitory interneurons.Conclusions:In SDH,Ca_v3.1 is mainly distributed in the nerve endings,while Ca_v3.2 and Ca_v3.3 are mainly expressed in neurons,and Ca_v3.1 and Ca_v3.3 are the major subtypes distributed in dendrites,indicating that the distribution of Ca_v3 channel subtypes in SDH is different in cellular and subcellular structures.It is suggested that Ca_v3 channel isoforms may play different roles in participating in electrical activity of SDH neuronal circuits.