The Study Of Pre-synaptic And Post-synaptic NMDA Receptor Activity-dependent Changes In The Spinal Dorsal Horn Nociceptive First Synapse

In recent years, the mechanism of chronic pain has made considerable progress, a largenumber of receptors, channels, kinases system in chronic pain occurs, the development of therole is gradually revealed. The first stage of neuronal synaptic functional changesglutamatergic synapses played a key role in the development and maintenance of chronicpain after nerve injury[1]. Chronic pain in the process of central sensitization, NMDAreceptors plays an important role. Immunohistochemical studies showed that NMDAreceptors are widely expressed in the central nervous system, while a large number of studieshave shown that, NMDA receptors are also expressed in the presynaptic terminal and DRGneurons[2-5]. DRG as part of peripheral nervous system, its small and middle neurons,including cell body and the central projection and peripheral projections A and C fibers areprimarily responsible for the transmission of pain information, has been also known as the nociceptors (nociceptor). Since these NMDA receptors in nociceptors are activated byglutamate release itself, it is also known as the nociceptors itself receptor (autoreceptor).Glutamate release from primary afferent terminals in the spinal dorsal horn can be regulatedby many receptors located presynaptically on the primary afferent terminals. For example,On the other hand, activation of group I metabotropic glutamate receptors[11], P2X3increasesglutamate release from the central terminals of primary afferents, on the other hand,glutamate release from the primary afferents in the spinal dorsal horn is reduced uponactivation of μ-opioid receptors[6], α2adrenoreceptors[7], CB1cannabinoid receptors[8],GABABreceptors[9]as well as group II and group III metabotropic glutamate receptors[10].The function of glutamatergic synapses is governed by three key factors including theamount of glutamate release from presynaptic terminals, the rate at which glutamate is takenup by glutamate transporters, and the number and function of postsynaptic glutamatereceptors[12]. In other regions of the CNS, such as hippocampus, cerebellum, and enthorinalcortex, immunocytochemical and functional studies have documented presynaptic NMDAreceptor expression and their involvement in modulating transmitter release[13-18]. Bardonietet.al3found that NMDA caused a depression of peak amplitude and an increase in synapticlatency and failures of evoked monosynaptic AMPA EPSCs in most lamina II neurons,suggest that this synaptic inhibition is attributable to a presynaptic mechanism, directlymediated by NMDA receptors. The results suggest that presynaptic NMDA receptor itselfmay play an negative feedback regulation of afferent nociceptive information[3]. Therefore,peripheral presynaptic glutamate release can act directly on presynaptic NMDA receptors,resulting in complex and diverse role. Conventional wisdom is that its main role is as anegative feedback regulation mechanism of inhibition of presynaptic neurotransmitterrelease, thereby preventing excessive neurotransmitter concentration in the synaptic cleft.However, activation of presynaptic NMDA receptors in morphine tolerant rats canenhance presynaptic glutamate release[5]. In animal models of epilepsy, activating forebrainpresynaptic NMDA receptors also result in the enhancement of glutamate release bypresynaptic terminals[19,20]. Immunohistochemistry and western blot results found in injury or inflammation-induced chronic pain condition, nociceptor NMDA receptor itself[4,21-23]expression levels were significantly increased. Functional studies showed that nociceptor intheir functional activity of NMDA receptors in chronic pain states showed significantenhancements, such as nociceptor in experimental colitis induced by NMDA receptorautophosphorylation levels were significantly increased, NMDA-induced inward currentamplitude and intracellular calcium concentration was also significantly increased[4]. Thesefindings strongly suggest that in chronic pain condition, the function of nociceptor NMDAautoreceptor was significantly enhanced. Intrathecal injection of NMDA to activatepresynaptic NMDA receptors can lead to the release of substance P in the periphery of theterminal[24]. Yan et.al[25]found that in case of damage to the periphery, the enhancement ofactivity of the presynaptic NMDA receptors can increase the release of presynapticglutamate. Under pathological conditions, behavioral studies have revealed that localinjection on the NMDA receptor antagonists on inflammation or injury induced hyperalgesiacan inhibit neuronal discharge, rather than enhancement[26-28]. This suggests that presynapticNMDA receptor may undergoes activity-dependent changes after inury or inflammation.Although these results suggest that the peripheral NMDA receptors may have inducedhyperalgesia in the pathological state, but these results were derived from local injection pfthe peripheral NMDA receptor antagonists, thus specificity of these antagonists, the timecourse of their action and the exact targets are difficult to determine, therefore, these resultscan not reveal the exact role and function of specific nociceptor NMDA receptor itself inpathological states. Taken together, in order to find out the effect of nociceptor NMDAreceptor itself plays a role in chronic pain, Mice lacking NR1specifically in a primarynociceptor-specific manner (SNS-NR1-/-) were generated via Cre/loxP-mediatedrecombination by mating mice carrying the floxed prkg1allele (NR1fl/fl) with a mouse lineexpressing Cre recombinase under control of the Nav1.8promoter (SNS-Cre). We havepreviously demonstrated that SNS-Cre mice enable gene recombination commencing at birthselectively in nociceptive (Nav1.8-expressing) sensory neurons, without affecting geneexpression in the spinal cord, brain, or any other organs in the body.Therefore, we intend to establish a conditional knockout transgenic animal model of NMDA receptor itself, furtherresearch presynaptic NMDA receptors in the first synapse role in physiological andpathological conditions state noxious, in order to study activity-dependent changes in thepathological process of presynaptic NMDA receptors, to lay the foundation for the furtherunderstanding of the role and function of NMDA receptors.Meanwhile, changes in the spinal dorsal horn ion channels synaptic neurons play animportant role in chronic pain central sensitization. Dynamic changes in neuronalpostsynaptic Ca2+concentration can encode the timing of action potentials before and afterthe postsynaptic neurons, thereby start causing changes in the efficiency of synaptictransmission cellular processes[29-31]. Generally, after repeated stimulation of postsynapticneurons EPSP generate action potentials can be highly activated after synaptic NMDAreceptors, resulting in substantial Ca2+influx, activating calcium/calmodulin-dependentprotein kinase II (CaMKII) and leading to the generation of LTP. Thus, postsynaptic NMDAreceptor function changes produce central sensitization and the development of its key role.CCL2, also known as monocytes chemoattractant protein1(MCP-1), can specificallyrecruit monocytes to sites of inflammation, infection, trauma, toxin exposure, and ischemia.Electrophysiological studies indicate that application of CCL2increases Ca2+in culturedeonatal DRG neurons[36]. DRG neurons isolated from animals exhibiting neuropathic painbehavior are strongly depolarized by CCL2[35,37]. CCL2-induced sensitization of nociceptorsmay be caused by activation of TRP channels and inhibition of K+conductance[37]. CCL2inhibits a voltage-dependent, non-inactivating outward current, presumably a delayedrectifier type K+conductance, which is known to regulate neuronal excitability[40]. CCL2inDRG neurons should be transported to peripheral terminals in the skin, as intradermalinjection of MCP-1directly induces mechanical hyperalgesia[41]. While a large number ofstudies have shown that after peripheral nerve injury, CCL2may act as a nerve modulation,has an activity-dependent release characteristic from peripheral nerves to the spinal corddorsal horn[34,42,43]. Studies have shown that, CCR2is also expressed in the spinal dorsal horn neuronal cellbodies[43,44]. But how does CCL2/CCR2work on neuronal cell bodies is still unknown. Ourstudy further found that, CCL2can combine spinal dorsal horn neurons CCR2receptors tofurther activate ERK signaling pathway, which leads to increase in the spinal dorsal hornneuronal synaptic NMDA receptor channel function, and thus play a regulatory role inchronic pain, this role is mainly through NR2B subunit.Under inflammatory conditions, arachidonic acid can produce prostaglandins (PG) byCox hydrolysis, wherein the prostaglandin E2(PGE2) involved in the inflammatory response,and lead to hyperalgesia. The expression of Cox-2in inflammation significantly increased.Cox-2inhibition is possible to suppress PGE2. Studies have shown that in hippocampus thePGE2can act on presynaptic EP2receptor resulting in presynaptic glutamate release[45]. Alsostudies have shown that, PGE47]2can induce CCL2/MCP-1release[46,. However, in ourexperiments, the dorsal horn neurons postsynaptic NMDA current enhancement effect cannot be completely blocked by Cox-2inhibitor.Part I: Pre-synaptic NMDA receptors induced hyperalgesia and its mechanismIn the process of chronic pain central sensitization, NMDA receptor plays an importantrole. Numbers of results suggest that the periphery NMDA receptors in pathological statesmay cause hyperalgesia, but the application antagonists study has some limitations, so theseresults can not exactly reveal their nociceptor NMDA receptors specific roles and functionsin pathological states. We intend to use the conditional knock transgenic animal models tofurther study presynaptic NMDA receptors in the first synapse role in physiological andpathological states on the injury status, thus before the study activity-dependent changes inthe pathological process of synaptic NMDA receptors. Results：1. Create SNS-NR1-/-miceWe used Cre-loxP technology for establishment of conditional knock transgenicanimals except NR1subunit in the nociceptor. Fluorescein-labeled488IB4(IB4-Fluor488)in nociceptive DRG neuron was recording by whole-cell patch clamp. Perfusion of NMDAin NR1fl/flmice can induce a significant inward current which was sensitive with the NMDAreceptor antagonist AP5; whereas NMDA-induced inward current in SNS-NR1-/-mice wasalmost completely blocked. Described in DRG neurons, nociceptive neurons NMDAreceptor function was completely knockouted. And other non-nociceptive neurons NMDAreceptor function was unaffected.2. Behavioral phenotypesSNS-NR1-/-mice showed reduced mechanical hypersensitivity and thermalhyperalgesia in chronic inflammatory pain states. There was no significant difference inNR1fl/flmice and SNS-NR1-/-mice in mechanical threshold and thermal hyperalgesiaincubation period. NR1fl/flmice and SNS-NR1-/-mice also display no significant differencein reaction time in Capsaicin induced acute pain. The second phase persistent spontaneousreaction was significantly lower than the wild-type mice in Formalin induced pain inSNS-NR1-/-mice. In SNS-NR1-/-mice injected hind foot CFA induced mechanical allodyniaand thermal hyperalgesia were significantly weaker than wild-type mice.3. MechanismArtificial action potentials induce an inward current in a neuron in whole-mount DRG,which is blocked by AP5(50M). In normal condition, NR1fl/fland SNS-NR1-/-DRGneurons’ resting membrane potential, the rheobase and the threshold were not significantlydifferent. In CFA animal models, resting membrane potential, rheobase and the thresholdof NR1fl/flneurons were all dereased, while the SNS-NR1-/-nociceptive DRG neurons’resting membrane potential, the threshold value were significantly reduced, but the strengthof the rheobase did not change. DRG neuronal excitability changes after CFA-induced inflammation. To further studyexcitability of DRG neurons with injection ramp current in NR1fl/fland SNS-NR1-/-nociceptive neurons, depending on number of spikes with different the slope of the injectioncurrent ramp. In NR1fl/flmice, IB4-Fluor488-positive nociceptive DRG neurons in responseto ramp current injection with increasing slope displayed a significant increase in spike firingfrequency at24h after CFA injection as compared to basal state. In SNS-NR1-/-mice, thespike firing frequency induced by ramp current injection in IB4-Fluor488-positivenociceptive DRG neurons was not altered after CFA injection.SNS-NR1-/-mice showed diminished LTP in spino-PAG projection neurons. Afterlow-frequency stimulation of the dorsal root, we found that evoked EPSC in Lamina Ineurons was significantly enhanced in NR1fl/flmouse, and this enhancement can bemaintained more than30min (LTP). In the SNS-NR1-/-mice which specifically knockoutpre-synaptic NMDA receptors of Lamina I neurons, low-frequency stimulation-induced LTPwas nearly blocked.Part II: The direct effect of CCL2on the spinal cord dorsal horn Lamina IIpostsynaptic neurons NMDA receptorsThis paper fouce on the spinal cord dorsal horn Lamina II neurons NMDA receptor,observation the effect of monocyte chemotactic factor-1(MCP-1) on the spinal horn LaminaII neurons NMDA receptors, to further explain the inflammatory process inducedhyperalgesia, the activation of microglia and astrocytes release of cytokines, the chemokinescan be applied to not only act on the glial cells themselves and increasing microglialreactivity for enhancement of postsynaptic neurons, but also directly effect on thepostsynaptic neuron cell body, thus directly regulating the expression of postsynapticNMDA receptors on the cell body to regulate the response properties of neurons,activity-dependent changes in postsynaptic NMDA receptors may lead to further centralsensitization, cause inflammatory damage. Results：1. CCL2induced pain behavioral responses of animalsAgainst118w C57BL/6animal behavior can be detected CCL2induced pain behaviorenhancement. Intrathecal injection of100μl of undiluted CCL2induced a hindpaw of ratsproduces marked inflammation and inflammatory pain. Application CCL2natural receptorCCR2antagonist RS504393, can inhibit CCL2-induced inflammatory pain sensitizationreactions.C57Bl6mice exhibited a persistent mechanical allodynia and heat hyperalgesiafollowing CFA inflammation. CFA-induced mechanical allodynia and heat hyperalgesia waspartially but significantly attenuated by RS504393in ipsilateral. Meanwhile behavioral testsshowed, after CFA24hours, the animal’s mechanical allodynia and thermal hyperalgesia canpartially reverses Ifenprodil.2. Inflammatory injury induced upregulation of CCL2and CCR2Real-time PCR revealed distinct regulation of CCL2and CCR2mRNA expression inthe spinal cord following inflammation. Western blot shows that, CCL2can lead to asignificant increase in NR2B.3. Cellular and molecular mechanisms of CCL2-mediated hyperalgesiaWe conducted single-cell PCR to characterize the colocalization of NMDAR and CCR2receptors in spinal cord lamina II neurons. Among5lamina II neurons we collected,4ofthem expressed NMDA receptors, but2of them expressed CCR2. We postulate that theseCCR2terminals in lamina IIo form synapses with vGluT2excitatory neurons, but notVIAAT inhibit neurons.CCL2may directly increase neuronal pERK levels, and this effect was blocked byintracellular adding PD98059(pERK blocker) and extracellular adding RS504393(CCR2inhibitors). Superfusion of CCL2at a concentration (100ng/ml) that caused in significant increasesin NMDAinduced currents and able to be abolished by CCR2antagonist RS504393.CCL2two minute didn’t increase AMPAR induced evoked EPSC but significantincrease NMDAR induced evoked EPSC. That suggested that CCL2has directly orindirectly regulation effect on postsynaptic NMDAR-induced currents.We superfuse CCR2antagonist RS504393on CFA inflammatory pain model spinalcord lamina II neurons, application of RS504393could directly reduce NMDA enducedcurrents in2min. Meanwhile Ifenprodil, NR2B specific blocker, partially inhibit NMDAcurrents in lamina II neurosn and can abolish CCL2enhancment role for NMDAcurrent.In anesthetized mice, spinal LTP was induced by tetanic stimulation (100Hz,1s,4trains) in all C57/B6WT mice, lasting for>2h, and LTP can be reversal by RS504393.Conclusion:1. Spinal dorsal horn first synapses presynaptic NMDA receptors inhibitory synaptictransmission under physiological conditions。2. Under pathological conditions of chronic inflammation,pre-synaptic NMDA receptorchanges activity-dependently，enhance synaptic transmission of nociceptive signals,exacerbate the formation of central hyperalgesia at spinal dorsal horn first synapses.3. The direct effect of CCL2/MCP-1on CCR2receptor in spinal dorsal horn postsynapticneurons.4. CCR2can result in increased phosphorylation of ERK, and furtherenhance postsynapticNMDA activity by NR2B receptor.