Postnatal Development Of Segmental Synaptic Connections In The Lumbar Spinal Cord Of Rat

Peripheral somatosensory information produced in peripheric receptor, andconveyed from central axons of dorsal root ganglion which control periphericreceptor of primary sensory neurons to dorsal horn. Present knowledgeconsiders that Aβ fibers which dominate somatosensory ascend to posteriorfunicular nucleus of brainstem via dorsal column, and they processnon-nociceptive information like proprioceptive sensation, pressure sensation,fine touch sensation and so on.A and C fibers mainly project to dorsal horn,and process nociceptive information. Many neuroanatomical andelectrophysiology experimental evidences prove that some of Aβ-fiber centralaxons ascend2-3segment in dorsal column to invade to dorsal column, andpossibly involve procession of nociceptive information. For example, spinalcord stimulation (SCS), a popular used in clinic pain treatment,control pain though electrode positioned in epidural dorsal column (50Hz,0.2mspulse,30min). Although we have been know that Aβ fibers distribute in dorsal horn,little is known about functions of those fibers due to limitation of technique.This academic dissertation investigate postnatal development of synapticnetwork of nociceptive and non-nociceptive primary afferents in lumbar spinalcord of rat by using neurochemistry method to observe primary afferents,transmission electron microscope to observe myelin sheath and multi-electrodearray for electrophysiology recording. The aim of this study is to establish newmethods and models to investigate mechanism of Aβ roles in involvement ofnociceptive information in segmental spinal cord.The results were shown as following:1. Postnatal development of cytoarchitecture in rat lumbar spinal cord andestablishment of experimental paradgims for studying synapticnetworkFirst, NeuNstaining by using immunohistochemistrywas used to identifycytoarchitectonics of coronal section of lumbar spinal cord at postnatal day7(P7), P14and P28. We selected suitable multi-electrode dish (MED) probe (88,64channels) to match dorsal horn size in different postnatalage.Multi-electrode array technique (Alpha Med Scientific, Japan) and itsspecial probe (MED-64Probe) were used in this experiment. Base oncytoarchitectonics of lumbar spinal cord and Rexed laminaeof rat, we selectedMED-P210A (probe area:0.7x0.7mm， size:20x20μ m， distance of twoelectrodes:100μ m， chamberheight:10mm) for P7,and MED-P515A (probearea:1x1mm， size:50x50μ m， distance of two electrodes:150μ m，chamberheight:10mm) for P14and P28. The thickness of spinal cord slice is450-500μ m. The technology difficulty of this experiment was to prepare spinalcord slice with attached dorsal root.The first, to make sure field excitatory postsynaptic potential (fEPSP) of spinal cord induced by dorsal root and dorsalcolumn electric stimulation (ES) at the same slice, so this experiment canprogress all right.2. Postnatal development of primary afferent neurochemistry and myelinin rat lumbar spinal cordTo observe postnatal changes and structural featureof nociceptive primaryafferents of dorsal horn conveyed through dorsal root and non-nociceptiveprimary afferents of dorsal horn conveyed through dorsal column,immunohistochemistry and immunofluorescence were used at this part. We usedanti-substance P (SP) antibody and IsolectinB4(IB4)-binding to identifypeptidergic and non-peptidergic Cfibers respectively, and anti-neurofilament200(NF200) to identify A fibers.In addition, we used anti-myelinoligodendroglia glycoprotein (MOG) and anti-myelin basic protein (MBP) tostain myelin sheath of myelinated fibers in postnatal life.Distribution ofpeptidergic and non-peptidergic C fibers in dorsal horn at postnatal life wereshown as following:(1) distribution of peptidergic fibers terminals was widelyin superficial laminae (I-II) and deep laminae (III-VI) of spinal dorsal horn at P7and P14, and was limited in superficial laminae of dorsal horn at P28.(2)Terminals of IB4binding non-peptidergic fiber distributed superficial laminae ofdorsal horn from P7to P28. At P7-P14, fibers marked by NF200mainlydistributed in substantia alba medullae spinalis (dorsal column, lateral columnand anterior column) and deep lamiae of dorsal horn, but some of them also insuperficial of dorsal horn. At P28, NF200labeledstaining was disappear fromsuperficial laminae. By using transmission electron microscope andimmunohistochemistry of myelin sheathassociated proteintechnology, we found myelin sheath of myelinated fibers have a remarkable postnatal changes. Indorsal column at P7, about20-30%fibers wrapped with myelin sheath and nothick myelin sheath observed. Diameter of these fibers was smaller than5m.In P14, myelinated fibers increased to50-60%, fibers which diameter wasbigger than5m were not observed. In P28, almost all of myelinated fiberswrapped with myelin sheath, and fibers which diameter was bigger than5mmwere observed. In dorsal column, immuno-positivematerial of MBP and MOGwas weakly expressed at P7, notable increased at P14, and reach top expressionat P28. In dorsal horn, immuno-positivematerial of MBP and MOG wasobserved at descending or ascendingfasciculus and dorsal column, little wasobserved at the other part.3. Application of multi-electrode array recordings in studying postnataldevelopment of segmental synaptic network in rat lumbar spinal cordSimultaneous recording of fEPSP induced by dorsal root ES and dorsal columnES by Using64-channels (8x8) multi-electrode array technique (Med64System) was used to observe postnatal changes of function and synaptic networkwhich formed by nociceptive primary afferents and non-nociceptive primaryafferents in the dorsal horn. When ES was in dorsal root, fEPSP recorded fromsuperficial laminae of dorsal horn at P28, and also in deep laminae at P7andP14. Waves of fEPSP in superficial laminae were negative-going responses at P7and P14and P28. The amplitude was decreased with age. Waves of fEPSP indeep laminae were positive-going responses with two peaks at all of three ages,and the amplitude was decreased with age. Distribution of fEPSP was decreasedwith age, and limited to superficial laminae. The peak latency was decreasedwith age When ES was in dorsal column, fEPSP recorded mainly from deep laminae of dorsal horn, and in superficial laminae at P7and P14. Waves offEPSP in superficial laminae were two-way-going responses, and amplitude wasdecreased with age. Waves of fEPSP in deep laminae were negative-goingresponses at all of three ages, and amplitude was decreased with age too. Thepeak latency was also decreased with age.Spinal cord slice were perfused withionotropic glutamate non-NMDA receptor antagonistCNQX and NMDAreceptor antagonistAP5and sodium channel blockerTTX, the results showedthat the field potentials (FPs) whatever induced by dorsal root ES or dorsalcolumn ESwasmainly mediated by non-NMDA (AMPA/KA) receptor, andNMDA receptor worked a little.Conclusion:synaptic network of nociceptive and non-nociceptive primary afferents insegment of rat lumbar spinal cord have a postnatal and fictionalchangecharacteristics.(1) Primary afferents from dorsal column have remarkablypostnatal changes. Diameter of dorsal column fibers from small (<5m) to big(>5m), and from some fibers wrapped with myelin sheath (70-80%isunmylinated fibers) to all fibers wrapped with myelin sheath. Myelin sheathchange was from thin myelin sheath to thick myelin sheath. This postnataldevelopment affects formation of synaptic network and function in the dorsalhorn.(2) In all primary afferent fibers though dorsal root to dorsal horn,peptidergic fiber have significantly postnatal changes with age.This kind offibers distribute in superficial laminar and deep laminae of dorsal horn, and limitin superficial laminae when maturation. Non-peptidergic fibers have no changein postnatal life, and limit in lamina Ⅱ. So fEPSP induced by dorsal root ES indeep laminae may be mediated bypeptidergic fibers, and in superficial laminae may be mediated by peptidergic and non-peptidergic fibers.(3) Results frompreliminary experiment showed that dorsal column stimulation could inhibitfEPSP which ES on dorsal root in superficial laminae of adult rat, suggestingthat primary afferent from dorsal column form a segmental regulation circuitbesides dcsccnding pain inhibitory systcm. This circuit may be one ofmechanism underlying pain relieving effect of SCS.(4)64-channels (8x8)multi-electrode array technique which used to study synaptic network of dorsalroot and dorsal column afferents is a good model tostudy mechanism ofnon-nociceptive primary afferents regulate nociceptive afferents in segmentalspinal dorsal horn.