Endocannabinoid Contributed To The Synaptic Disinhibition In Both Innocuous Mechanoreceptive Pathway And Nociceptive Pathway Of Rat Spinal Dorsal Horn

Neuropathic pain has always been a difficult problem to be tackled in the field of medicine.Compared with physical pain,neuropathic pain is not only reflected in the quantitative change of stimulus and feeling but also in qualitative change.The occurrence and persistence of chronic process of neuropathic pain may be involved in the plasticity of structure and function of nervous system entirely.Therefore,to clarify the mechanism of the pathological pain of neural circuits,looking for potential drug targets has vital significance for the treatment of neuropathic pain.The "gate control theory" has been published for more than a half century,and the composition and function of the putative gate circuit have not been fully elucidated.A recent study found that the "feedforward inhibition” in spinal dorsal horn afferent pathway may play a role of "allodynia gate".After nerve injury,the “gate” can be opened,and the tactile information may lead to pain by nociceptive pathways(J Clin Invest.2013).In recent years,the role of endocannabinoid system(endocannabinoids,e CBs)in the pathogenesis of neuropathic pain has become an intense focus.Although most studies on the e CBs system were concentrated in the mechanism of analgesia,it is noticeable that,however,the e CBs does not produce cannabinoid analgesic effect in some cases,or even plays a role in pain production.In 2009,Christie et al proposed a new hypothesis: “e CBs can open the gate of pain”,but it has not been confirmed.CB1 receptors are widely expressed in the dorsal horn of the spinal cord,and it is not clear that the influence of activation of CB1 receptors on innocuous mechanoreceptive pathway and nociceptive pathway in the spinal dorsal horn.This study puts forward a hypothesis on mechanisms of "allodynia gate”: the endocannabinoid(e CBs)has a specific inhibitive effect on feedforward inhibition in spinal innocuous mechanoreceptive pathway and nociceptive pathway,and contributes to the development of allodynia and hyperalgesia.Whole cell patch clamp recordings were used in this study.Exogenous cannabinoid ligands,2-AG and AEA were applied in the spinal cord slices with a dorsal root attached,to observe its effect on Ab,Ad and C fiber mediated excitatory or inhibitory postsynaptic potential and thus,to provide electrophysiological basis for further clarification of the role of the endocannabinoid system in the pathogenesis and development of neuropathic pain.Part 1: The effects of endocannabinoid on innocuous mechanoreceptive pathway in spinal dorsal hornObjective: To observe the effect of increased endocannabinoid to the synaptic transmission of mechanoreceptive pathway in spinal dorsal horn.Methods: Four to six weeks old male SD rats were used in this study.After anesthesia,the rats were perfused with artificial cerebrospinal fluid from the heart,and the spinal cord lumbar enlargement was removed rapidly to prepare the parasagittal spinal cord slices with a long dorsal root attached.Whole cell patch clamp recordings were used to search for and record from neurons that receive monosynaptic,Ab fiber input in the deep layers(IIi andIII)of superficial dorsal horn.Excitatory(e EPSPs)or inhibitory(e IPSPs)postsynaptic potentials were evoked by electrical stimulation of the dorsal root.2-AG,AEA,and JZL195/184 were perfused onto spinal cord slices to investigate their effects on Aβ fiber mediated e EPSPs and e IPSPs.Results: Both 2-AG and AEA presynaptically inhibited the amplitude of e EPSPs recorded from putative glycinergic neurons mediated by activation of primary Ab fibers(P<0.01,paired,t-test).2-AG,AEA and hydrolase inhibitor JZL195/184 significantly inhibited the amplitude of e IPSPs recorded from putative PKCg positive neurons which receives feed-forward inhibition from putative glycinergic neurons(P<0.01,paired,t-test).Part 2: The effects of endocannabinoid on nociceptive pathway in spinal dorsal hornObjective: To observe the effect of increased endocannabinoid to the synaptic transmission of nociceptive pathway in superficial layers of the spinal dorsal horn.Methods: Whole cell patch clamp recordings were used to search for and record from neurons that receive monosynaptic Ad or C fiber input and feedforward inhibition in the superficial layer(IIo)of superficial dorsal horn.Excitatory(e EPSPs)or inhibitory(e IPSPs)postsynaptic potentials were evoked by electrical stimulation of the dorsal root.2-AG was perfused onto spinal cord slices to investigate their effects on C or Ad fiber mediated e EPSPs and e IPSPs.Results:(1)2-AG had little effect on e EPSPs recorded from Lamina IIo neurons activated by Aδ or C fiber stimulation.(P > 0.05,paired t-test).(2)2-AG significantly inhibited the amplitude of e IPSPs recorded from Lamina IIo neurons(putative nociceptive)which receives C fiber or Ad fiber inputs and feed-forward inhibition(P<0.01,paired,t-test).Summary:1.Endocannabinoid directly acted on the Ab fiber nerve endings innervating the lamina III glycinergic neurons,and inhibited the release of excitatory neurotransmitters.In other words,the endocannabinoid attenuated the excitability of glycinergic neurons.2.Endocannabinoid directly acted on lamina III inhibitory glycinergic interneurons receiving Ab fiber input,and inhibited the release of glycine neurotransmitter.Namely,Endocannabinoid may play disinhibitory effect on innocuous mechanoreceptive pathway and contributed to the development of mechanical allodynia.3 Endocannabinoid also may play disinhibitory effect on nociceptive pathway and may contribute to the development of hyperalgesia.Conclusions: The increased endocannabinoid in the pathological condition produced disinhibition in both innocuous mechanoreceptive pathway and nociceptive pathway of rat spinal dorsal horn.The present study provided clues to explore the mechanisms underlying the neuropathic allodynia and hyperalgesia.