The Contribution Of LTD In A Feed-forward Spinal Inhibitory Circuit To The Development Of Neuropathic Allodynia

Melzak and Wall[2] proposed a well-known hypothesis, the gate control theory ofpain, which is highly influential for nearly50years. However, the theory remains aconjecture because of insufficient evidence to substantiate the neural circuitry underlyingpain signaling and modulation in the spinal dorsal horn. The author’s supervisor has beenengaged in the study of gate control theory proposed neuronal circuit since the year of2000. Our recent study [1]found that the spinal dorsal horn contains a feed-forwardinhibitory circuit that can gate incoming low-threshold A input to nociceptive pathway.This study identified a novel “gate” with definite morphological and functional details inthe spinal dorsal horn, in which activation of low-threshold innocuous A fibers inneuropathic pain condition “opens” the gate to elicit mechanical allodynia. This “allodyniagate” may contribute to the development of neuropathic pain.Based upon our previous findings, the present study utilized patch-clamp whole-cellrecordings, morphological and behavior techniques examined the contribution of LTD inthe “allodynia gate” circuits to the development of neuropathic allodynia induced byperipheral nerve injury in SD rat. We found that the peripheral nerve injury induced the endocytosis of postsynaptic glutamate AMPA receptors of glycinergic neurons, andinvolved in the development of the circuit-specific LTD in synapses betweenlow-threshold innocuous A fibers and glycinergic neurons, and finally, open the“allodynia gate” and contribute to the development of neuropathic allodynia.Experiment one: The long-term synaptic plasticity in the spinal gatecontrol circuits.Objective: To examine the long-term synaptic changes of the spinal gate control circuitsinduced by high frequency electric stimulation (HFS).Methods：Parasagittal spinal cord slices with dorsal root were made from the lumbosacralspinal cord of4~5week-old Sprague-Dawley rats. Whole-cell patch clamp recordingswere made from lamimae Ⅱand Ⅲ neurons of the Prasagittal spinal cord slices. HFS（100Hz for1s, repeated three times at10-s intervals, stimulus strength at20V）wasapplied to the dorsal root to induce the long-term changes of the recorded neurons.TAT-GluR2was superfused to test its effect on the LTD induction.Results： After HFS, the amplitude of A-EPSP recorded from Gly neurons wassignificantly reduced for at least40min (n=6, P <0.001, one-way AVOVA); the amplitudeof unitary IPSP recorded from PKC neurons evoked by Gly cell action potentials wasalso significantly reduced for at least40min (n=6, P <0.001, one-way AVOVA).Pre-superfusion of TAT-GluR2for20min before HFS significantly alleviated theinduction of both EPSP-LTD in Gly neurons (n=6, P <0.001, one-way AVOVA) andIPSP-LTD in PKC neurons (n=6, P <0.001, one-way AVOVA). However,Pre-superfusion of TAT-GluR2s has no effect on the LTD induction (n=5，P>0.05,one-way AVOVA).Experiment two: The effect of Tat-GluR2on the allodynia induced bySNL.Object: To study the effect of intrathecal-applied TAT-GluR2on the mechanical allodyniainduced by SNL. Methods: SNL model was made by tight ligation of L5spinal nerve of SD rats.TAT-GluR2was injected through an intrathecal catheter (PE-10polyethylene tubes).Mechanical Withdraw Thresholds (MWT) were measured using von Frey hairs before andafter TAT-GluR2injection.Results：(1) One single intrathecal injection of TAT-GluR2before SNL significantly slowdown the decline of MWT (n=6, P <0.05, one-way AVOVA).(2) Sustained injection ofTAT-GluR2for7days also slow down the decline of MWT (n=6, P <0.01, one-wayAVOVA).(3) Intrathecal injection of TAT-GluR23days after SNL can reverse the MWT(n=10, P <0.01, one-way AVOVA).Experiment three: The location changes of GluR2and GlyRα3onneurons of the inhibitory circuits after SNLObject: To observe the location changes of GluR2and GlyR3on neurons of theinhibitory circuits after SNL.Methods: Male adult SD rats,180~220g, were divided into2groups (SNL and control).Seven days after SNL, the lumbosacral spinal cord was cut into30μm thick sections anddouble-labeling immunofluorescence was performed (PKC and GluR2, PKC andGlyR3, GlyT2and GluR2).Results：(1) GluR2receptors were mainly expressed on the cell membrane of a smallamount of PKCγ+neurons in normal adult rats; GluR2endocytosis occurred after SNL.(2)GlyR3receptors were mainly expressed on the cell membrane of PKCγ+neurons innormal adult rats; the membrane location of GlyR3receptors remained unchanged afterSNL.(3) GluR2receptors were mainly expressed on the cell membrane of glycinergicneurons in normal adult rats; GluR2endocytosis occurred after SNL.Summary：1. The electrophysiological data indicated that the excitatory synapses between primaryA fibers and Gly neurons underwent circuit-specific LTD after HFS to the dorsal root.The inhibitory synapses from Gly neurons to PKC neurons also underwent LTD afterHFS to the dorsal root. The endocytosis of GluR2on Gly neurons may contributed to the induction of LTD. The LTD induced in the specific inhibitory circuit afterperipheral nerve injury may open the “allodynia gate” to elicit mechanical allodynia.2. The behavior studies suggested that pharmacological suppression of the endocytosis ofGluR2may prevent or alleviate mechanical allodynia induced by peripheral nerveinjury.3. The morphological observations demonstrated that the GluR2receptors on themembrane of Gly neurons do underwent endocytosis after peripheral nerve injury andcontributed to the LTD induction. The present study failed to observe the locationchanges of GlyR3receptors suggesting that the GlyR3may be not involved in theinduction of IPSP-LTD in PKC neurons.Conclusion：This preliminary study proposed that the GluR2endocytosis of glycinergic neurons isrequired for the LTD induction in a feed-forward spinal inhibitory circuit, thiscircuit-specific LTD may contribute to the development of neuropathic allodyniainduced by peripheral nerve injury.