Influence Of Peripheral Nerve Injury To Girk Family Proteins Expression In DRG And Spinal Cord Neuronal Cells

Increased nociceptive neuronal excitability underlies chronic pain conditions.Various ion channels, including sodium, calcium and potassium channels have pivotal roles in the control of neuronal excitability. Members of G protein-gated inwardly rectifying potassium(GIRK) channels family subunits GIRK1~4 have been implicated in modulating neuronal excitability. Previous studies have indicated that GIRK1~4 m RNAs are expressed in the dorsal root ganglion(DRG) of rats and human beings. Functional GIRK channels are homo- or hetero-tetramer composed of one or two GIRK channels subunits. GIRK channels are activated and opened through interacting with inhibitory G protein-coupled receptors, resulting in outflow of potassium, which makes the membrane potential more “negative”, thus,decreasing the excitability of neurons. This thesis aims to study the neurochemical characteristics of GIRK channels subunits in normal rat DRG and spinal cord, and their regulation by peripheral nerve injury using neuropathic pain model. Furtherly,we will analyze the role of GIRK channels on neuropathic pain based on our data.In this study, animals were perfused, and then, lumbar 4-5(L4-5) DRG and L5 spinal cord were dissected for frozen tissue sections preparation.Immunohistochemistry(IHC) assay and quantification analysis results showed that~70% and <10% neurons expressed GIRK1 and GIRK2 in normal rat DRG,respectively. We used CGRP, IB4 and NF200 to differentiate small unmyelinated peptidergic neurons, small unmyelinated non-peptidergic neurons and medium-large myelinated neurons, respectively. Our data indicated that GIRK1 and GIRK3 were extensively expressed in these three neuronal subpopulations, however, GIRK2 was expressed only in CGRP- and NF200-positive(+) neurons. To further characterize the distribution of GIRK channels subunits, four calcium-binding proteins(Ca BPs)CB, CR, PV and Scgn were used to label different DRG neuronal subpopulations.We found that GIRK1 co-localized with all these four Ca BPs to varying degrees.However, GIRK2 was only expressed in CB+ neurons. To characterize the distribution of GIRK channels subunits in spinal dorsal horn, we used different markers to label different lamina. GIRK1+and GIRK2+terminals were observed mainly in limina II, GIRK3+ and GIRK4+ terminals mainly in lamina I and outer lamina II. In addition, co-localization study of GIRK1~4 with VGLUT1 or VGLUT2 in spinal dorsal horn indicated a presynaptic location. Neu N was used as a neuronal marker for labelling the local neurons in spinal dorsal horn, we found a large amount of GIRK1+ and GIRK2+, but not GIRK3+ or GIRK4+ local neurons.We studied the regulation of GIRK channels subunits by peripheral nerve injury using a successfully established rat neuropathic pain model of sciatic nerve axotomy. The expressions of GIRK1~4 were analyzed at m RNA and protein levels by IHC test, in situ hybridization test, real-time quantitative PCR, western blot test and quantification analysis. Our results indicated that GIRK1,-2, and-4 were downregulated in the ipsilateral DRG and spinal dorsal horn compared with contralateral side. In contrast, GIRK3 was upregulated after peripheral nerve injury.Combining the previous studies, we concluded that the upregulation of GIRK3 might decrease functional GIRK1/2 hetero-tetramer and GIRK2/2 homo-tetramer on the surface of neuronal membrane.In order to detect the axonal transport of GIRK channels subunits, we successfully established rat sciatic nerve ligation model and spinal dorsal roots rhizotomy model for centrifugal transport study, both peripherally and centrally.Our results showed that GIRK1~4 conducted both peripheral and central transport in DRG neurons. Moreover, retrograde transport of GIRK1~3 were found from periphery to the DRG cell bodies. Using IHC test, we further detected the expression of GIRK1~4 in normal rat sciatic nerve and glabrous skin of hind paw.The results indicated an extensive expression of GIRK1~4 in different nerve fibers of sciatic nerve, including sensory fibers, and expression in the nerve end of dermis layer in the glabrous skin of hind paw.In conclusion, we first systematically illustrated the neurochemical characteristics of GIRK channel subunits GIRK1~4 in normal rat DRG and spinal cord in the present thesis. We have revealed the axonal transport of GIRK1~4 in DRG neurons using various nerve injury models and molecular biological methods.The downregulation of GIRK channels in axotomized rat DRG may underlie the generation and maintenance of neuropathic pain.