Effect Of Optogentic Activation Of DRG GABAergic Circuit On Peripheral Pain Transduction

Pain is a kind of sensory signal contributing to avoidance of injury from noxious stimuli for organism.Classic gate control theory suggests that GABA system reside in the dorsal horn of the spinal cord forms a "gate" for the integration of pain signals.Our previous study found that rat DRG expresses GABA receptors,GABA synthetase(GAD),GABA vesicular transporter(VGAT)and membrane GABA transporter(GAT),and DRG neurons may have function of GABA release under excitatory stimulation.In addition,we also proved that administration of GABA in DRG could relieve acute inflammatory pain caused by bradykinin(BK)and neuropathic pain induced by sciatic nerve compression.To further confirm this hypothesis,we expanded the pain model in the experiment.We verified that the pain behavior caused by formalin and capsaicin can be relieved by GABA application in DRG.It showed that the activation of GABA receptor in DRG neurons could inhibit the transmission of many kinds of pain signals.At the same time,by observing the change of c-fos expression in the dorsal horn of spinal cord in rats,it was confirmed that the local administration of GABA in DRG indeed reduced the transduction of the pain signals induced by inflammation to the spinal cord.This is suggesting that the activation of GABA receptors in DRG neurons leads to a certain extent of integration of pain signals within DRG.Furthermore,through a new established optogenetic experiment targeted to DRG in freely moving mice,it was shown that both basic pain threshold and inflammatory pain of mice were significantly inhibited by activating GABAergic neurons in DRG.This affirms that activation of endogenous GABA circuit within the DRG plays an important role of gating control on pain signal transmission similar to dorsal horn of spinal cord.Objective:1 To investigate effect of focal GABA application in DRG through casing on pain behavior induced by multiple pain factors and pain signals transmission;2 To observe the effect of in vivo activation of endogenous GABA circuit in DRG on basic pain threshold of animals and inflammatory pain behavior.Methods:Focal drug delivery though retained cannula targeted to DRG;Rat inflammatory pain models by subcutaneous injection of algogenic substances(bradykinin,formalin and capsaicin)in paw;Immunofluorescence staining of frozen sections of DRG and spinal cord;Genotype identification of VGAT-ChR2-EYFP transgenic mice;Measurement of mechanical pain threshold of mice through Frey Von fibers;Measurement thermal pain threshold of mice through Hargreaves test;Optogenetic activation of DRG in moving mice.Results:1 Focal application of GABA on DRG reduced inflammatory pain induced by formalin pain in rats.Nocifensive behavior of rat right hind paw in FM +GABA group(Phase ?:41.5±1.64 s;Phase ?:199.5 ± 23.96 s)was significant inhibited compared with FM group(Phase ?:71.8±9.20 s,P<0.05 vs FM +GABA group;Phase ?:429.2 ± 19.55 s,P<0.05 vs FM + GABA group).2 Focal application of GABA on DRG suppresses peripheral pain transduction.(1)Expression of c-fos in the ipsilateral spinal cord dorsal horn after subcutaneous injection of BK in right hind paw of rats(11±0.58,P<0.05 vs control)was significantly higher than control group(0.3 ± 0.33).While the application of GABA to DRG remarkably reduced the expression of c fos compared with control group(3 ± 0.58,P<0.05 vs saline control).(2)Expression of c-fos in the ipsilateral spinal cord dorsal horn after subcutaneous injection of capsaicin in right hind paw of rats was significantly increased(14.3 ± 2.33,P<0.05 vs control);Likewise,application of GABA to DRG remarkably reduced the expression of c fos induced by capsaicin injection compared with control group(5.7 ± 1.45,P<0.05 vs saline control).3 Establishment of in vivo optogenetic experiment of DRG in V GAT-ChR2-EYFP transgenic mice.Genotype identification of VGAT-ChR2-EYFP mice was used for optogentic experiment.Immunofluorescence staining showed some proportion of GFP-positive DRG neurons in frozen section from VGAT-ChR2-EYFP transgenic mice.The implantation of optical fiber was performed similarly to the DRG cannula implant with modifications.A stainless steel cannula guide was forced into the hole in the transverse process over the L4 DRG and the optical fiber was inserted through the guide.The incision was closed with sutures and the fiber was firmly fixed in place with dental cement;the rest of the procedure was similar to the cannula implantation.Laser stimulation(473 nm,3 mW,30 Hz,with a 10 s interval every 20 s)were elicited using MLL-FN-473-50 unit controlled by pulsing set.4 Optogentic stimulation of GABAergic DRG neurons increased basic pain thresholds of VGAT-ChR2-EYFP transgenic mice.Optical stimulation caused increases of mechanical(1.05 ± 0.12 g,P<0.05 vs absent of laser)and thermal pain threshold(16.09 ± 1.65 s,P<0.05 vs absent of laser)of ipsilateral paw of VGAT-ChR2-EYFP transgenic mice.The mechanical and thermal pain threshold of contralateral paw of VGAT-ChR2-EYFP transgenic mice under laser stimulation had no significant change.5 Optogentic stimulation of GABAergic DRG neurons inhibited acute pain induced by BK in VGAT-ChR2-EYFP transgenic mice.BK jnjection induced similar response of nocifensive behavior in both VGAT-ChR2-EYFP transgenic mice and WT mice.Under optical stimulation,VGAT-ChR2-EYFP transgenic mice showed significant decrease of the nocifensive time(11.83 ± 1.74 s vs.WT 28.5 ±7.91 s)compared with wild type mice.There are no differences on left paw pain responses after BK injection between transgenic mice and wild type mice with optical stimulation.Conclusion:1 Behavioral results showed that focal administration of GABA in DRG could alleviate inflammatory pain not only induced by bradykinin but also formalin.It indicates that activation of GABA receptor expressed on DRG neurons has a general inhibitory effect on many kinds of inflammatory pain signal transduction.2 Immunofluorence staining of C-fos protein in the spinal cord dorsal horn proved that application of GABA in DRG could reduce pain signals induced by bradykinin and capsaicin transduction from the peripheral to the spinal cord.This suggested that DRG functionally integrated pain signals by activation of GABA receptor expressed on DRG neurons.3 Optogenetic experiment demonstrated that activation of endogenous GABAergic circuit involved in inhibition of physiological pain responses and poathological pain process as well,which provide convincing evidence for DRG as a "gate" of pain signal transduction.