The Functional Expression Of GABAergic Circuitry In Dorsal Root Ganglion

Dorsal root ganglia(DRG)are part of the peripheral nerve system,which convey a variety of information to the central nervous system(such as spinal cord and brain).In many chronic pain conditions,aberrant sensory ganglia excitability contributes to pathological peripheral nerve over-excitation.y-Amino butyric acid(GABA)is a major inhibitory neurotransmitter in the adult mammalian central nervous system and participates in the transmission and regulation of pain.However,the structure and the role of the peripheral DRG GABA system are not well established.The common conception is that there are no sources of GABA interfacing peripheral sensory fibers or ganglia.Yet,there is evidence for a relatively high level of expression of multiple GABAA receptor subunits in the majority of DRG neurons,and either cultured or acutely isolated DRG neuron cell bodies express very large GABAA Cl-currents.These results indicate that there exists a local GABA release in DRG which form part of the GABA circuit operating locally in DRG rather than through spinal cord.Here in this study,we describe a fully functional local GABAergic circuitry in rat DRG.Except GABAA receptors,the expression in DRG of other key components of GABAergic transmission is not well documented.Thus,we investigated expression in DRG of glutamate decarboxylase(GAD65,GAD67),GABA transports such as vesicular GABA transporter(VGAT,GAT 1-3),GABAB(B1,B2)as well as various GABAA receptor subunits.We also measured the release of GABA from DRG.Furthermore,two models of the neuropathic pain(CCI,SNT)were established and mRNA expression level of components in GABAergic circuit in DRG neurons relating to the neuropathic pain were studied,which provide information for a new approach for the study and treatment of chronic pain in clinic.Part 1 Dorsal root ganglia express key components of functional GABAergic circuitry and release somatic GABAObjective:(1)To identify the expression in rat DRG of glutamate decarboxylase(GAD),GABA transporters such as vesicular GABA transporter(VGAT)and GAT(1-3),GABAB(B1,B2)as well as various GABAA receptor subunits.(2)To determine the GABA release and the mechanism in the acutely-extracted rat DRG.Methods:mRNA was quantified using real time PCR technique(qPCR);Protein expression was visualized using immunofluorescence technique in the frozen section of rat DRG;GABA release from rat DRG was measured using high performance liquid chromatography(HPLC).Results:(1)mRNA expression detected using qPCR technique demonstrated that multiple ?,? and y subunits of GABAA receptors presented in rat DRG,with particularly strong expression of ?1-3 and ?2 subunits.Moreover,mRNA of GAD67,GAT1-3,VGAT,GABAB1 and GABAB2 were also detected in the DRG.Among GAT subunits GAT1 showed strongest expression;GAD65 was not detected in the DRG.(2)We observed high levels of immune reactivity for ?1,?4 and ?2 subunits of GABAA receptors,and GABA transporters(GAT1,VGAT)were also found expressed in many DRG neurons of various sizes.At the same time,the high level of GAT1 immune reactivity was also found in the satellite glia cells.(3)High concentration of potassium solution(high-K)induced significant GABA release from rat DRG.GAT1 inhibitor N0711(200 ?M)in the normal K+solution produced significant increase of tonic GABA release but partially reduced the high-K-induced release of GABA.Inhibitors of VGAT concanamycinA(0.5 ?M)and tetanus toxin(100 ?g/ml)both inhibited high-K-induced GABA release almost completely.Conclusions:These data suggest that all major components of a GABA circuit are present in rat DRG and the machinery for production,release and recycling of GABA in DRG is functional.In DRG neurons GABA is released via a classical vesicular mechanism but also via a reversed GAT1 transportation mechanism.Part 2 The expression changes of mRNA of the key components of DRG GABAergic circuitry after neuropathic pain-causing nerve injuryObjective:To compare the mRNA expression of key components of DRG GABAergic circuitry and to understand the role of GABAergic in the development of neuropathic pain.Methods:Two models of neuropathic pain(CCI and SNT)were established.Thermal hyperalgesia and mechanical allodynia of neuropathic pain were assessed using the radiate heat test and Von Frey filament test,respectively.Real time PCR technique(qPCR)was used to quantify mRNA.Results:(1)Rats with CCI and SNT surgery showed the typical behavior of neuropathic pain compared with the Sham group.(2)The mechanical threshold in the CCI and SNT groups decreased in the post-operation 1-14 days(P<0.05).On the other hand,the thermal pain thresholds decreased only in the CCI group(P<0.05).(3)In the post-operation 5 days,mRNA of ?1,?2,GAD67 in CCI group were significantly decreased(P<0.05),and ?2,?5,?1,GAT2 were increased(P<0.05);mRNA of ?1,?2 in SNT group,were significantly decreased(P<0.05),and ?5,?1,?3,GAT2,GAT3 were increased(P<0.05).In the post-operation 14 days,mRNA of ?1,?2,?2,?2,?3,GAT2,GAD67 in CCI group were significantly decreased(P<0.05),and ?5 was increased(P<0.05);mRNA of ?1,?2,?2,?3,GAT1,GAT3,GAD67,Bl,B2 in SNT group were significantly decreased(P<0.05).Conclusions:Two models(CCI and SNT)of neuropathic pain were successfully established with behavior of thermal hyperalgesia and mechanical allodynia.mRNA expression of many key components of DRG GABA circuit was altered compared with the sham control,and mRNA level of GABAA receptor ?1 and ?2 subunits were most consistently and significantly reduced in the DRG of the two neuropathic models.The results suggest that the DRG circuit may play an important role in the development of neuropathic pain.