The Expression Of Two Isotypes Of Vesicular Glutamate Transporters(VGLUT) In The Orofacial Sensory Information Transmission Pathway And Related Functional Research

As one kind of neurotransmitters which is most widely distributed in the nervoussystem, glutamate was involved in regulation of varieties of neurological functions.Through complicated transmission pathways, the organic bodies receive, transmit andintegrate the sensory information from outside so as to feel the surroundings, and thenmake the proper response which is important for maintaining the normal physicalfunctions. Abundant studies have indicated that, glutamate, as a classical excitatoryneurotransmitter, also carries the sensory information transmission. Spinal and trigeminalpathways are two major somatic sensory information systems, which are responsible forthe transmission of trunk and orofacial sensory information to the upper brain centralstructures, respectively. The previous research indicated both of these two pathways were mainly consist of glutamatergic neurons. Among them, the trigeminal pathway includedthe following parts:(1) the peripheral terminals of the pseudounipolar neurons intrigeminal ganglion (TG) and mesencephalic trigeminal nucleus (Vme) receive the variouskinds of peripheral sensory information from orofacial regions, and the central terminalsof these pseudounipolar neurons send the information to the trigeminal sensory nucleuscomplex (TSNC, from the caudal to the oral included caudal subnucleus Vc, interpolarsubnucleus Vi, and oral subnucleus Vo of the trigeminal spinal nucleus as well asprincipal sensory trigeminal nucleus Vp), TSNC then project to the sensory thalamus(mainly included ventral posteromedial thalamic nucleus VPM and posterior thalamicnucleus Po), and the sensory information was finally transmitted to the cerebral sensorycortex after the integration. By immunohistochemistry and pharmacologicalelectrophysiology, researchers have showed several components of the trigeminal systemwere all made of glutamatergic neurons, then therefore, the synaptic releasing of glutamatethrough these components, were very important for the normal transmission of theorofacial sensory information, and may also underlie the pathogenesis of various orofacialsensory transmission disorders, such as trigeminal neuralgia.Classical models indicated that the neurotransmitters were released into the synapticgap by the function of synaptic vesicular. The membrane proteins constituting the synapticvesicular which is responsible for the glutamate releasing, were cloned just until the end oflast century, and these proteins were therefore named as vesicular glutamate transporters(VGLUTs). To date, there were three different kinds of VGLUTs cloned, which wereVGLUT1,VGLUT2and VGLUT3. Among them, VGLUT1and VGLUT2werespecifically expressed in the glutamatergic neurons, and are commonly used as markerproteins for the glutamatergic neurons. With the deeper probing for the study of these twoproteins, the researchers found a interesting phenomenon, that is VGLUT1and VGLUT2showed a complementary distribution pattern in the nervous system, VGLUT1wasexpressed mainly in cortex and telencephalon, while VGLUT2was expressed mainly inthalamus and brainstem. In addition, the co-expression of VGLUT1and VGLUT2in single neuron is hardly found in the whole nervous system either. Then one question arose,why the two different kinds of proteins were recruited for conducting the same function,and why their locating were exclusive to each other, the most likely explaination may be,VGLUT1and VGLUT2must have some functional differences though they both mediatethe vesicular releasing of the glutamate.In fact, there have been some studies suggesting that VGLUT1was more related to thesynapses which showed activity dependent synaptic plasticity (e.g., LTP, long termpotentiation), while the synapses expressing VGLUT2featured the high fidelity forinformation transmission. In addition, the experiments conducted on the gene knock-outanimals also found, the animals with VGLUT1knocked out and those with VGLUT2knocked out showed the different behavioral defective changes. More and more studies bymorphological methods testified that two isotypes of the VGLUT showed high level ofcomplementary distribution patterns in the central nervous systems. Series of studiesindicated that, although VGLUT1and VGLUT2showed82%of similarities for aminoacid constitution, although they both act as the membrane protein of the vesicularmediating the synaptic releasing of glutamate, the two isotypes of VGLUT must still havesome functional differences.As abovementioned, the transmission of orofacial sensory information was completedby the glutamatergic neurons which constituting the whole trigeminal pathway, thenwhether VGLUT1and VGLUT2, the two isotypes of VGLUT which were specificallyexpressed in the glutamatergic neurons, showed the similar complementary distributionpatterns in the trigeminal system is a possible question. If so, whether these two VGLUTshave some functional difference, may also underlie the transmission and integration ofdifferent types of orofacial sensory information by the trigeminal pathway depending onrecruiting different isoforms of VGLUTs. To unveil these questions may help us to clarifythe normal physical functions of excitatory trigeminal pathway, as well as to know moreabout the pathogenesis of some orofacial sensation defection, especially the orofacialabnormal pain (e.g. trigeminal neuralgia). Experimental contents are as follows: Part1Expression of two isotypes of VGLUT in the orofacial primary sensoryinformation transmission pathwayObjective: To analyze the expression patterns and origins of VGLUT in trigeminalprimary sensory terminals, and to clarify the possible functions of two kinds of VGLUT asfor the orofacial primary sensory information transmission. Methods: Observe theexpression of VGLUT1-and VGLUT2-immunopositive axonal terminals in subnuclei ofTSNC and the co-expression of two VGLUTs in single terminals in TSNC, by dualimmunofluorescence; Detect the change of density VGLUT1-and VGLUT2-immunopositive axonal terminals in subnuclei of TSNC after the rhizotomy of sensoryroot of trigeminal nerve; by trigeminal trans-ganglionic tract tracing methods combiningdual immunofluorescence, to check the expression patterns of VGLUT in peripheraloriginated primary sensory fibers in TSNC. Results: With the exception that superficiallayer of Vc expressed high density of only VGLUT2, in all subnuclei of TSNC, theVGLUT1and VGLUT2were evenly distributed, while co-expression of these twoVGLUTs was hardly detected; after the unilateral rhizotomy of sensory root of trigeminalnerve, the density of VGLUT1positive terminals significantly decreased in Vp, Vo, Viand Vc on the ipsilateral side, while the density of VGLUT2positive terminals did notshow obvious changes but only decreased in superficial layer of Vc; after the peripheralinjection of trans-ganglionic tract tracer CTB which label the myelinated nerve fibers,abundant CTB labeled terminals were distributed in the Vp, Vo, Vi and deep layer of theVc, and these CTB labeled terminals only expressed VGLUT1but not VGLUT2; after theperipheral injection of trans-ganglionic tract tracer WGA-HRP which label theunmyelinated nerve fibers, small amount of WGA-HRP labeled terminals can only bedetected in superficial layer of the Vc, and these tracer labeled terminals only expressedVGLUT2but not VGLUT1. Conclusion: The orofacial peripheral primary sensoryinformation transmission nerve fibers mainly express VGLUT1, which is the main originsof VGLUT1immunopositive terminals in TSNC, and the VGLUT2immunopositiveterminals in TSNC mainly take central origins, with the exception that the VGLUT2immunopositive unmyelinated nerve fibers in superficial layer of Vc take the peripheral origins as well. These results indicated that VGLUT1and VGLUT2were respectivelyrelated to the transmission of oroficial primary general and nociceptive sensoryinformation from peripheral.Part2The expression of two isotypes of VGLUTs in the TSNC output projections-orofacial secondary sensory information transmission pathwayObjective: To investigate the expression patterns of VGLUT in output projectionterminals to different upper relay brain structures from TSNC, and to clarify the possiblefunctions of two kinds of VGLUTs as for the orofacial secondary sensory informationtransmission. Methods: Detect the expression of VGLUT1mRNA and VGLUT2mRNAin subnuclei of TSNC, and their co-expression in single neuron by dual fluorescence insitu hybridization (dual FISH); by anterograde tract tracing combining dualimmunofluorescence as well as dual immunoelectron microscopy, to check the expressionpattern of VGLUT in output projection terminals originated from subnuclei of TSNC; byretrograde tract tracing combining FISH, to check the expression pattern of VGLUTmRNA in thalamic-and cerebellar-projecting neurons in subnuclei of TSNC. Results:Co-existing of VGLUT1mRNA and VGLUT2mRNA in single cells can hardly beenfound in Vo, Vi and Vc, in Vp there are some small to medium size neurons co-expressingVGLUT1mRNA and VGLUT2mRNA; the projection terminals from Vo, Vi and Vc tothe thalamus only express VGLUT2, while projection terminals from Vp, Vo and Vi to thecerebellum only express VGLUT1, projection terminals from Vp to the VPM co-expressVGLUT1and VGLUT2; the study by the retrograde tract tracing experiment show thesame results. Conclusion: In TSNC, the thalamus projecting neurons are mainly VGLUT2mRNA positive, the cerebellar projecting neurons are mainly VGLUT1mRNA positive,the VPM projecting neurons in Vp express both VGLUT1mRNA and VGLUT2mRNA.Part3The effect on orofacial pain information transmission by down-regulation theVGLUT2in medullary dorsal hornObjective: To observe the influence on rats’ pain related behavioral by targetdown-regulation the VGLUT2in medullary dorsal horn of the animals. Methods: Co-transfect HEK293cells by use of VGLUT2shRNA expressing plasmid and VGLUT2expressing plasmid with reporter genes, to select the effective VGLUT2shRNA sequenceform designed ones; package the VGLUT2shRNA expressing lentivirus and then infectthe primary cultured cortical neurons of rats, to testify the down-regulation effect onVGLUT2; inject this lentivirus into the medullary dorsal horn of the rats and thenobserved the influence on the animals’ pain behavior. Results: The effective shRNAsequence which can down-regulate the expression of VGLUT2was successfully selectedby the luciferase reporting value, the lentivirus expressing this sequence can effectivelyinfected the rats’ primary cultured cortical neurons and down-regulated the VGLUT2expression in them; Injection of this lentivirus into the medullary dorsal horn caneffectively down-regulated the VGLUT2expression in it, and meanwhile be able toweaken the hyperalgesia of oro-facial pain model rats. Conclusion: Targetingdown-regulation of VGLUT2in medullary dorsal horn of rats can effectively interferetheir pain behavior.