Somatic GABA Release In DRG And Nerve Injury Effect On Gaba Current In DRG

Somatosensory neuron,with its somata residing in dorsal root ganglion(DRG),is important part of sensory nervous system.Peripheral processes of DRG neurons transform the versatile environmental stimuli to nerve impulses,and convey them to central nervous system to form variable sensation such as temperature,tactile and pain sensation etc.According to our previous study,we believe that DRG neurons also involve in modulating of pain signal transmission as well.We have demonstrated previously that DRG has a local GABA circuitry with the function of GABA release under multiple excitatory stimuli and can reduce acute pain behavior induced by Bradykinin.Therefore,we assumed that this GABAergic system may be a potential therapeutic target for pain.Chronic neuropathic pain is a common and notoriously incurable disease in clinic and its pathogenesis is not clear yet.In this study,we focus on the role of DRG GABAergic system in neuropathic pain.Firstly,a new established experimental paradigm,"Sniffer Patch",was used to investigate GABA release from DRG neurons.This will be another direct evidence of functional GABAergic system in DRG.Secondly,we tested GABA induced current density in DRG neurons from rats with chronic constrictive injury(CCI)of sciatic nerve for evaluating involvement of GABAergic system during neuropathic pain development.Finally,a DRG targeted mini-pump drug delivery system was used to testify the effect of focal GABA application on neuropathic pain behavior for assessing the potency of the GABAergic system as therapeutic target for neuropathic pain.Part 1 Testify somatic GABA release from DRG through "Sniffer patch" recordingObjective:Establishing a new patch recording method,"Sniffer Patch",to testify the somatic GABA release from DRG neurons,t-test method.Methods:Perforated "Sniffer Patch" recording;VGA-ChR2-YFP genotyping through semi-quantitative RT-PCR;Immunofluorescence visualization of DRG section through confocal microscopy.Results:(1)Chemical stimulation in "Sniffer patch" recording:we co-cultured DRG neurons with HEK293 cells were transiently transfected with GABAA receptor and GFP.Then ’sniffing’ patch-clamp recordings were performed from HEKGABAA cells juxtaposed to small-diameter DRG neurons.In such HEKGABAA cells we could record robust inward currents in response to 200 μM GABA;application of TRPV1 agonist capsaicin(1 μM)produced inward currents very similar in kinetics(although smaller in amplitude)to GABA currents.HEKGABAA cells in monoculture(without DRG co-culture)never responded to capsaicin,likewise,we never observed a response to capsaicin when recording from non-transfected HEK cells(HEKcontrol)juxtaposed to a small-diameter DRG neurons in HEKcontrol-DRG co-culture.(2)Mechanical stimulation in "Sniffer patch" recording:We were unable to elicit a response in HEKGABAA cells when we stimulated juxtaposed DRG neurons(of any size)mechanically.(3)Optogenetic stimulation in“Sniffer Patch" recording of DRG neurons from VGAT-ChR2-YFP mouse:’Sniffing’patch-clamp recordings were performed form HEKGABAA cells juxtaposed to DRG neurons of VGAT-ChR2-YFP mouse.In such HEKGABAA cells we could record small inward currents.We observed very subtle responses when recording from HEKGABAA cells in monoculture andHEKGABAA cells juxtaposed to DRG neurons of wild type mouse.Conclusions:These results suggested that some DRG neurons such as TRPV1+ and VGAT+ had function of somatic GABA release during their excitation.Part 2 Role of GABAergic system within Dorsal Root Ganglion in neuropathic painObjective:(1)Evaluating changes of GABA current density in different sized DRG neurons during process of neuropathic pain.(2)Investigating effect of focal GABA application in DRG on neuropathic pain behavior.Methods:Sciatic nerve chronic contractive injury(CCI)model of rats;Radiate heat test and Von Frey filament test for thermal and mechanical pain threshold measurement;Established DRG targeted mini-pump drug delivery method;Whole cell patch clamp recording;Two independent samples nonparametric tests the Mann-Whitney Test.Results:(1)CCI pain model:Rats with CCI surgery showed the typical behavior of neuropathic pain compared with the control group.The mechanical and thermal pain threshold in the CCI groups decreased in the post-operation 1-14 days(P<0.05).(2)GABA induced currents Patch recording:Among large and small diameter DRG neurons,there are no significant changes in GABA currents density and the proportion of GABA-response neurons between contralateral and ipsilateral DRG neurons on the 5th and the 14th post-operation day.Among medium diameter DRG neurons,DRG neurons from operated side showed larger GABA currents density compared with contralateral control on the 5th post-operation day(P<0.05).At the meantime,the proportion of GABA-response neurons also increased(P<0.05)in medium sized neurons.(3)Behavioral study:mini-punmp delivery of GABA in neuropathic pain rats:One week of GABA(200 μM)application through new established DRG targeted mini-pump drug delivery system increased the thermal thresholds of CCI groups on the 1t and the 5th post-operation day of CCI(P<0.05,compared with the NS.groups).The mechanical pain thresholds were unaffected by GABA application.Concliusions:These results suggested DRG GABAergic system may involve in the some process of neuropathic pain by increasing its GABA sensitivity,especially in medium sized DRG neurons.The GABA system activation could be a potent target for alleviate neuropathic pain.