Morphological Study Of Connections Between Glutamatergic, Serotoninergic Or Substance Pergic Axon Terminals And Cell Bodies Of The Mesencephalic Trigeminal Nucleus Neurons In The GAD67-GFP Knock-in Mouse

Some clinical diseases which are functional disturbance in trigeminal nerve field such as disturbances syndrome of temporomandibular joint, anarthria literalis and acataposis have close relationship with the transmission and regulation of the trigeminal proprioceptive sensory signals. The mesencephalic trigeminal nucleus(Vme) are the collection of transferring the orofacial proprioceptive sensory signals, meanwhile the important place of regulating these signals. Vme is a special nuclei of neurons, the nervous cells spreading nonconcentrately from the superior colliculus to between locus ceruleus and the parabrachial nucleus. Vme composes of the large pseudounipolar neurons and the small multipolar neurons. It is now generally contained that the former have excitatory neurotransmitter like glutamate, the latter are the interneurons containing inhibitory neurotransmitter like GABA. As we know, glutamatergic Vme neurons play important roles in the transmission of orofacial proprioceptive sensory signals, but GABAergic Vme neurons play inhibitory roles in the regulation of the proprioceptive sensory signals through part circuit loop. GABA is an important inhibitory neurotransmitter in central nervous system(CNS), which combining with GABA receptors can play inhibitory roles in the regulation. Our topic group have observed 5-HT-, VGluT1/VGluT2- or substance P-like immunoreactive(-LI) axon terminals made close contact with neuronal cell bodies of the glutamatergic Vme in the rat. However, those researches can't mark the GABAergic interneurons in Vme, connections between 5-HT-, VGluT1/VGluT2- or substance P-LI axon terminals and the cell bodies of the GABAergic interneurons in Vme are still obscure now.GAD67-GFP knock-in mouse is a kind of transgenic animal widespread using for study and research in recent years. These transgenic mice are coincubated by Department of Morphological Brain Science of Medicine in Kyoto University and Department of Anatomy in our university. In the brain of this kind mouse, the GABAergic neurons expressing green fluorescent protein(GFP) are labeled exactly. Under the control of GAD67 promoter, the GABAergic neurons could be revealed with GFP expression. It is confirmed that GFP-positive neurons are almost GABAergic neurons.Therefore, in the present study, an attempt was made to examine the possible relationships of 5-HT-, VGluT1/VGluT2- or substance P-LI axon terminals or the cell bodies of large pseudounipolar neurons (glutamatergic) and small multipolar neurons (GABAergic) by using techniques of triple-immunofluorescence histochemistry and double-labeled electron microscopic immunohistochemistry in GAD67-GFP knock-in mouse.The results were observed under confocal laser-scanning microscope and electron microscope and showed:(1) Many cell bodies showed PV-LI and the majority of them were large or medium pseudounipolar neurons(diameter: 18~40μm); some GFP-positive neurons were observed in the Vme and these neurons were small multipolar neurons(diameter: 8～15μm);(2) A considerable number of VGluT1- and VGluT2-LI terminals were widely distributed in Vme, some of them surrounded the cell bodies or the dendrites of Vme neurons showing PV- or GFP-LI,respectively. The quantitative estimation showed that 71.8% and 28.2% VGluT1-LI terminals made close contact with PV- and GFP-positive Vme neurons, respectively; whereas 74.5% and 25.5% VGluT2-LI terminals made close contact with PV- and GFP-positive Vme neurons, respectively;(3) Some 5-HT- or SP-LI terminals were distributed in the Vme, some of them surrounded the cell bodies of Vme neurons showing PV- or GFP-LI respectively. The quantitative estimation showed that 77.7 % of 5-HT-LI terminals made close contact with the PV-LI Vme neuronal soma, whereas approximately 22.3 % 5-HT-LI terminals made close contact with the GFP-positive neuronal soma or the dendrites. On the other hand, 78.2% SP-LI terminals made close contact with the PV-LI Vme neurons, 21.8% SP-LI terminals made close contact with the GFP-positive neurons;(4)Under electron microscope, some 5-HT-LI terminals were observed to make synaptic contact with GFP-positive neuronal soma or the dendrites. The majority of axon-dendritic or axon-somatic synapses were a symmetric.The above results suggest that in the transmission of the proprioceptive sensory information from the orofacial region to the higher centre, glutamatergic, 5-HTergic or SPergic terminals may exert some modulatory effects directly and indirectly through regulating the activity of the GABAergic Vme interneurons.