| Dorsal root ganglion(DRG)neurons,as primary somatosensory afferent neurons,represent one of important components in sensory signal transmitting pathway.A variety of functional molecules were expressed in DRG neurons and play important roles in sensory signal formation and transmission.The first part of this paper aims to explore the the effect of DWSA7 protein in DRG neurons on peripheral mechanical transmission;the second part focuses on the alteration and contribution of alpha5 subunit of GABAA receptors in DRG neurons in chronic neuropathic pain.Part one Effect of DWSA7 protein in DRG neurons on peripheral mechanical sensory transmissionDWSA protein belongs to a family of transmembrane ion channel-like genes,including eight members DWSA1-8,which can be divided into three subfamilies based on sequence homology.The DWSA protein subfamily A consists of three proteins,DWSA1,DWSA2 and DWSA3;subfamily B consists of DWSA5 and DWSA6;subfamily C consists of DWSA4,DWSA7 and DWSA8.The function of DWSA protein has not been well understood because the special characrtistic of DWSA family,which makes the research about DWSA protein a hot spot in scientific research.DWSA1 protein is the most widely studied member of the family.Some studies have shown that DWSA1 may function as an ion channel.However rare studies on the functions of other members of the DWSA family are published.Our preliminary experiments found that DWSA7 protein was highly expressed in peripheral DRG neurons.Therefore,it was speculated that DWSA7 protein may be involved in the perception of sensory neurons to the stimulation of the external environment.This part aims to demonstrate this speculation through experiments.And this part of study will add new concept of the function of the DWSA family and improve the understanding of regulation of peripheral sensation.Objective:To investigate the effect of DWSA7 knockout on behavior in rats;to explore the effect of DWSA7 protein knockout on mechanically sensitive currents(RA,IA,SA)in rat DRG neurons and investigate the regulation of DWSA7 protein on Piezo2 channel.Methods:1.Behavior tests:heat test and Von Frey filament test for thermal and mechanical threshold measurement;2.Knocking down or overexpressing DWSA7 protein in DRG neurons through AAV/BLV virus infection;3.Immunohistochemical and qPCR techniques;4."Mechano-clamp' electrophysiological patch clamp recording;5 Western blot and immunoprecipitation methods.Results:1 The effect of DWSA7 on peripheral perception of rats1.1 The phenotype of DWSA7-KO rats in perception:The mechanical withdrawal thresholds of DWSA7-KO male Rats(11.18 ± 1.20 g)significantly decreased compared with the male littermate/control group(14.50±0.50 g,two samples t test,*P<0.05),while no significant difference in the thermal withdrawal latencies.Likewise,DWSA7-KO female rats showed significantly lower mechanical thresholds(10.56±1.02 g)compared with the female littermate/control group(14.22±0.53 g,two samples t test,*P<0.05),but there was no significant increase in the thermal withdrawal latency.1.2 Behavioral results of rats with DRG infected with AAV virus carrying DWSA7shRNA/mRNA:The mechanical withdrawal thresholds showed significantly reduced after the injection of AAV5-DWSA7shRNA-GFP virus within DRG(3.13±0.75 g,two samples t test,P<0.05),compared with scramble virus group(7.75±1.93 g);but there was no significant change in the thermal withdrawal latency.On the contrary,the mechanical withdrawal thresholds were significantly increased after overexpresing DWSA7 in DRG through injection of AAV5-DWSA7mRNA-GFP virus into DRG(13.42±1.86 g,two samples t test,P<0.05)compared with scramble virus group(7.75±1.93 g),no significant change in the thermal withdrawal latency was observed.2 The effects of DWSA7 protein on the electrophysiopgical characteristics of DRG neurons2.1 The effect of DWSA7 protein on the the excitability of DRG neurons:No changes of the resting membrane potential,action potential and excitability of the membrane was detected within DRG neurons of DWSA7-KO rat and the littermats rats by patch calmp recording.So such tresults don't affect the peripheral nociceptive transmission.2.2 The "mechano-clamp' recording on DRG neurons dissociated from DWSA7-KO rat and the littermates:The proportion of DRG neurons with RA current in DWSA7-KO rats(32/54,59.26%)showed significantly increase compared with that of the littermate control group(45/91,49.45%).But there was no significant difference between the two groups(Pearson Chi-Square,x2=2.09,P>0.05).Otherwise,the inactivation constant of RA current in DRG neurons was significantly reduced(Mann-Whitney test,*P<0.05).2.3 The mechanically sensitive current recording in DRG neurons infected with DWSA knocking down:The RA proportion of DRG neurons infected with BLV-DWSA7shRNA-GFP(31/48,64.58%)was increased compared with that of DRG neurons infected with scramble virus(27/52,51.92%).But there was no significant difference between the two groups(Pearson Chi-Square,x 2=4.731,P>0.05).2.4 The RA proportion of DWSA7-KO DRG neurons infected with BLV-DWSA7mRNA-GFP(8/36,22.22%)was significantly decreased compared with that of DWSA7-KO DRG neurons(32/54,59.62%,Pearson Chi-Square,x2=19.695,***P<0.001).The inactivation constant of RA current in DWSA7-KO DRG neurons infected with BLV-DWSA7mRNA-GFP(medium=14.15,n=30)was significantly increased compared with that of DRG neurons infected with scramble virus(medium=14.15,n=30,Mann-Whitney test,**P<0.01).These results suggest that DWSA7 protein may play a role in the regulation of RA current in DRG neurons.It is known that Piezo2 is an important channel for mediating RA in DRG neurons.Therefore,the subsequent experiments in this part mainly study the modulation of Piezo2 channels by DWSA7.3 The modulation of the Piezo2 channel by DWSA7 protein.3.1 The effect of DWSA7 protein on the expression of Piezo2 channel:1)The expression of Piezo2 in the DRG neurons of DWSA7-KO rats:No obvious changes of Piezo2 expression were observed within DRG of DWSA7-KO rats and littermate rats by using immunohistochemical staining,western blot and real-time qPCR analysis.These results indicated that DWSA7 protein didn't affect the expression of Piezo2 channel in rat DRG neurons.2)The effect of DWSA7 on expression of Piezo2 in the exogenous HEK293T cells:Real-time qPCR analysis showed compared with HEK293T cells transfected with Piezo2,there was no significantly different in expression of Piezo2 in HEK293T cells cotransfected with DWSA7+Piezo2.Western blot analysis showed both whole cell protein and membrane protein expression of Piezo2 had no significant difference in HEK293T cells cotranfected with DWSA7+Piezo2,DWSA1+Piezo2 and Piezo2 single-transfected HEK293T cells.Notably,our western blot results showed relatively abundant Piezo2 protein expressed in blank HEK293T cells.Unfortunately we can not exclude the possibility of the unspecificity of antibody in current stage.3.2 The effect of DWSA7 protein on the function of Piezo2 channel.The"mechano-clamp" recording in HEK293A cells transfected with Piezo2 channel,RA MA(mechano-acivated)currents can be recorded in all HEK293A transiently transfected with Piezo2(n=8,100%).We co-transfected Piezo2+DWSA7 into HEK293A cells,and HEK293A cells transfected with Piezo2+DWSA1 was taken as control,to see the effect of DWSA7 protein on the function of Piezo2 channel.The results showed as follows:1)RA currents still can be recorded in all HEK293A transiently transfected with Piezo2+DWSA1(n=10,100%).But ratio of RA in HEK293A transfected with Piezo2+DWSA7 was significantly decreased.Among all 18 recorded cells,the proportion of cells elicited RA,IA and SA currents were 2/18,4/18 and 4/18 respectively.And 8/18 cells showed no response to the mechano-stimuatation.2)In the above responsive HEK293A cells,the amplitude of MA from HEK293A cells transfected by piezo2+DWSA7 showed significantly decreased compared with HEK293A cells transfected by piezo2+DWSA1.(Repeated measures ANOVA,F(1,14)=10.938,,##P<0.01).3)The inactivation constant of MA current mediated by HEK293A cells transfected with Piezo2+DWSA7(median=23.27)significantly increased compared with that of HEK293A cells transfected with Piezo2+DWSA1(median=4.96,Mann-Whitney test,***P<0.001).The results suggest that DWSA7 protein may inhibit the mechanosensitivity of Piezo2 channel and may also convert its currents from RA to IA or SA mechanically sensitive currents.4 The interaction of DWSA7 protein with Piezo2 channelCo-transfected Piezo2+DWSA7-MYC-DDK into HEK293A cells and immunoprecipitated Piezo2 protein using anti-DDK antibody.No positive Piezo2 band was detected in this experiment.This result suggests that there may be no direct interaction between DWSA7 protein and piezo2.Conclusion:1.DWSA7 knockout significantly increase the peripheral mechaosens-itivity of rats but not affect resting membrane potential,action potential and excitability of DRG neurons.2.DWSA7 protein does not affect expression of Piezo2 channel but inhibits its mechaosensitivity and convert its RA current to IA or SA currents.3.There may be no direct interaction between DWSA7 protein and piezo2 channel.Part two Effect of alpha5 subtype of GABAA receptors in DRG neurons on chronic pain of ratsGABA is an important inhibitory neurotransmitter in adult mammalian central nervous system(CNS).Its inhibitory effect is principally mediated via anion-selective ionotropic GABAA receptors.In pain pathway,it is well known that spinal dorsal horn is the first location of integrating and gating pain signals during which transmitting from peripheral to central nervous system.GABAergic interneurons play critically important roles in this gating process.We have demonstrated that dorsal root ganglia,similar to spinal dorsal horn,contain local GABAergic circuits,including multiple subunits of the glutamate decarboxylase isoforms Gad65 and Gad67(which decarboxy-late glutamate to produce GABA),the plasma membrane GABA transporters Gat1-3(which remove released GABA from the extracellular space),GABA receptors(GABAA receptor and GABAB receptor).This system activation inhibits the transmission of pain signals from the peripheral to the central nervous system,which results in reducing pain behavior.This finding makes us recognize that DRG not only plays a role in the transmission of pain signals,but also has a role in the regulation of pain signals.So we proposed that DRG serves as a new "gate' for sensory transmission and represents as a novel target for pain therapeutics.Chronic pain is notorious in therapeutic in clinic.Our previous experiments have shown that activation of the local GABAergic circuits system can alleviate the symptoms of chronic inflammatory pain and chronic neuropathic pain in animals.To further investigate the role of the local GABAergic circuits system in the development of chronic pain and its potential as a novel target for pain therapeutics.We examined the the alteration of various components of the local DRG GABAergic circuits in chronic inflammatory pain and chronic neuropathic pain.The preliminary results showed that the ?5-GABAA receptor subunit showed the most obvious expression alteration during chronic pain.So this section will focus on studying the role of ?5-GABAA receptor subunit within DRG in the development and treatment of chronic pain.Objective:To investigate the expression pattern of ?5-GABAAreceptor subtype in DRG neurons;To explore the role of ?5-GABAA receptor subtype in the development of pain and the potential of ?5-GAB AA receptor as a novel target for pain therapeutics.Methods:1.Real time qPCR;2.Immunohistochemical staining;3.CCI,SNI and CFA model of rats;4.Von Frey filament test and heat test for thermal and mechanical pain threshold measurements;5.DRG targeted AAV2/5-?5GABAA shRNA-GFP virus injection.Results:1 Expression pattern of ?5-GABAA receptor subtype in DRG neurons?5-GABAA receptors were expressed in most DRG neurons and partially overlaped with such markers of sensory neuron subtypes as CGRP(peptidergic nociceptors),IB4(nonpeptidergic nociceptors)and NF200(myelinated fibers).2 The alteration of ?5-GABAA subtype mRNA within DRG in the develop-ment of chronic pain of rats The expression of ?5-GABAA mRNA was tested significantly up-regulated in L4-6 DRGs of rats with CCI or SNI surgery on both the 5th and the 14th post-operation days(Mann-Whitney test,***P<0.001).The up-regulation of ?5-GABAA protein was further verified through western blotting detection on the both days after CCI surgery.3 The role of ?5-GABAA subtype in the development of chronic pain3.1 Effect of kocking down of ?5-GABAA subtype in DRG neurons on basal pain thresholds:The mechanical withdrawal threshold significantly increased(10.63±0.93 g,)at four weeks after the injection of AAV5-?5 GABAA shRNA-GFP compared with the injection of scramble virus(7.88±0.86 g,Two-sample t test,n=16,*P<0.05),but there was no significant change in the thermal withdrawal latency with ?5-GABAA knocking down.3.2 The involvement of ?5 GABAAin development of chronic neuropathic pain:CCI surgery was performed four weeks after AAV5-?5 GABAA shRNA-GFP injection to ipsilateral L5 DRG of rats.The withdrawal threshold to mechanical stimulation and latency to thermal stimulation of rats with DRG were measured in 1st,3rd,5th,7th,10th and 14th days after CCI surgery.The withdrawal threshold to mechanical stimulation and latency to thermal stimulation were both significantly increased compared with scramble group(repeated measures ANOVA,#P<0.05).The thermal threshold on the 5th and 14th days after CCI injury were significantly increased compared with the same days of control rats(post hoc t-test comparison,*P<0.05).3.3 The involvement of ?5-GABAA in development of chronic inflamm-atory pain:CFA injection to rat hind paw was performed four weeks after AAV5-?5GABAA shRNA-GFP ipsilateral injection to L5 DRG of rats.The withdrawal threshold to mechanical stimulation and latency to thermal stimulation of rats with DRG targeted AAV2/5-?5GABAA shRNA-GFP virus were measured in 1st,3rd,5th,7th,10th and 14th days after CFA injection.Compared with rats injected with virus carrying scramble virus,the thermal thresholds of the rats significantly increased in the AAV2/5-?5GABAA shRNA-GFP virus injection group(repeated measures ANOVA,#P<0.05),especially on the 7th and 14th day after CFA injection(post hoc t-test comparison,*P<0.05).No significant difference in mechanical threshold was observed between the two groups.4 ?5-GABAA receptor as a target for pain therapeutics.Two weeks of ?5-GABAA receptor inhibitor L655,701 application through DRG targeted mini-pump drug delivery system significantly increased the thermal threshold and mechanical threshold of CCI rats,(compared with the saline group,repeated measures ANOVA,#P<0.05,##P<0.01).Post hoc t-test comparison showed L655,701 application increased mechanical thresholds on the 7th,10th and 14th day,and thermal thresholds on 7th and 10th after in rats suffered from CCI surgery compared with those of rats in same time points respectively in saline group(*P<0.05).Conclusion:1.?5-GABAA subtype located in most DRG neurons;2.The expression of ?5-GABAA receptor increased significantly in the process of neuronpathic pain development;3.?5-GABAA subtype was involved in regulating the basal mechanical withdrawal threshold of rats;4.?5-GABAA subtype plays an important role in the development of chronic neuropathic pain and chronic inflammatory pain.5.?5-GABAA subtype can be an effective target for chronic neuropa-thic pain therapeutics. |
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