A Preliminary Study On The Formation And Characteristic Of Synapse Between Spinal Motoneuron And Superior Cervical Ganglion Neuron In Vitro

Patients with spinal cord injury can achieve controllable micturition by surgically establishing an artificial somatic-central nervous system-autonomic reflex pathway. However, little is known about the mechanism underlying the synaptogenesis during the formation of the artificial somatic-central nervous system-autonomic reflex arc, though it is obvious that somatic motor neurons somehow manage to form synapses with autonomic neurons. In recent years, in vivo studies have been undertaken to observe the characteristic of this newly formed synapse, and have demonstrated that acetylcholine (ACh) is the major neurotransmitter for the relay of neural signals. Nevertheless, the many intervening factors inherent to anastomosis such as adhesion, inflammation, and complex components in concerned tissue have prevented surgeons from removing noise and making a complete, thorough and systematic investigation on the characteristics of such new type synapse. So far, the only form of the new type synapse that has been successfully established is the one that takes shape between somatic motor neurons and parasympathetic postganglionic neurons. As is known, the major neurotransimitter used by spinal motor neuron and parasympathetic presynaptic neurons under physiological condition is acetylcholine, which has also been reported by in vivo studies to be the main neurotransmitter in the newly formed synapse in the somatic-central nervous system-autonomic reflex pathway. It is thus rational to speculate that the same receptors in the somatic and autonomic postsynaptic membrane for acetylcholine may serve to facilitate the formation of this new type synapse. It is also tempting to investigate whether neurons synthesizing the same neurotransmitters are prone to forming functional synapse with neurons expressing corresponding receptors, and whether motor neurons from spinal cord can establish synapses with sympathetic postganglionic neurons given that sympathetic preganglionic neurons share the same neurotransmitter (ACh) with motor neurons. In this study, we first dissected out and cultured spinal motor neuron(SMN) and superior cervical ganglion neuron(SCGN) in conditional culture medium( Neurobasal medium, B27), which was supplemented with GDNF and NGF respectively that are indispensable for the their survival and growth. Primary culture of SMN and SCGN were successfully established and cell types were confirmed by double-immunofluorescent staining of NF200 (for neurons), ChAT (for SMN) or GFAP (for astrocytes), as well as by Hoechst (for cell nuclei), followed by quantitative study. We next established in vitro SMN-SCGN synapse model. Cover-slips were microlandized by agarose and PDL, planted with low-density SCGNs which were subsequently labeled by DiI. Cell suspensions made by different adhesion time from ventral spinal cord of rat E16 (SMN) were implanted to the prepared SCGN culture. Co-culture of SCGN and SMN were double-immunofluorescently labeled by p38 antibody and ChAT antibody to illustrate the synaptogenesis in the presynaptic neurons, followed by quantitative study. Ultrastructural studies under transmission electronic microscope, western blot and RT-PCR were also performed to confirm the synaptogenesis in the co-culture of SMN-SCGN. Miniature spontaneous postsynaptic current (mSPC) was recorded in SCGN so as to observe the influence of co-culture and verify the formation of functional synapses. We then investigated the neurotransmitter and corresponding receptors used by SMN-SCGN synapse by introducing AChR blocker (HEX), AChE antagonist (THA), and glutamate non-NMDA receptor blocker (CNQX). Finally, astrocyte conditioned medium(ACM) was exogenously added to the co-culture medium, and the changes in the synaptogenesis of SMN-SCGN interaction was observed.Main results:1. Primary culture of SMN and SCGN achieved satisfactory growth states. For SMN culture, on the 14th day in vitro, outgrowth disappeared, and tiny black granules and bubble-like structure were observed in the cytoplasm. Most neurites were broken and the majority of neurons became spherical and dim. Cell debris were scattered around. In contrast, SCGN still kept excellent growth state on the 16th day, as indicated by clear outgrowth. Double-fluorescent staining of NF200 , ChAT or GFAP and Hoechst for nucli revealed that neuronal purity was up to 87.8%, with the purity of SMN being 70.9% and purity of SCGN being 95%. 2. In SCGN culture group, neurons could be labelled by DiI on day 1, and the red fluororescence could last many days without obvious negative influence on the survival of SCGN.3. SMN and SCGN could be co-cultured on the cover-slips that have been microlandized; quantitative studies showed that co-culture groups significantly outnumbered either single culture group in the number of synapse.4. Results of western blot indicated that the content of p38 was greatly increased in co-culture group compared with SMN culture group.5. RT-PCR experiment showed that the level of p38 mRNA was significantly higher in co-culture group than either single culture group or the summation of the two groups.6. Synapse-like structures were observed under electronic microscope in co-culture group along the interface between SMN and SCGN.7. On day 4 in vitro, mSPC was detected in SCGN in the co-culture group; while no similar postsynaptic current was detected in mere SCGN culture group.8. The mSPC detected in SCGN could be blocked by HEX, and could be enhanced by THA; while CNQX did not have obvious influence on recorded mSPC.9. Frequency and amplitude of mSPC detected in SCGN could be fastened and augmented by ACM in co-culture group.Main conclusions:1. SMN and SCGN could be cultured by serum-free culture medium, with the former surviving up to 13 days and the latter even longer.2. Cover-slips could be microlandized so as to provide an ideal base for the establishment of SMN-SCGN synapse model.3. SMN-SCGN synapse functions through the release of ACh, serving as the neurotransmitter, and the activation of nAChR.4. ACM can promote the formation of SMN-SCGN.5. Different types of neurons (somatic motor neuron and visceral neuron) secreting the same neurotransmitter and distributed with corresponding receptors are able to establish functional synapses. The types of neurotransmitter and receptor do not change much in the synapses, whose formation involves contribution from glial cells. Collectively, we dissected out and cultured SMN and SCGN, and harnessed microlandized cover-slips to establish SMN-SCGN synapse model. Various methods such as double-fluorescence, western blot, RT-PCR, TEM and patch clamp were utilized to confirm the formation of functional synapse. It was found that this type of synapse functions through the release of ACh and activation of nAChR. We also found that ACM could promote the synaptogenesis between SMN and SCGN, though its molecular regulation needed to be elucidated. Our study provides experimental evidence for the understanding of mechanism underlying the establishment of artificial somatic-central nervous system-autonomic reflex pathway, and serves as a preliminary investigation of the synaptogenesis under pathological conditions. The establishment of in vitro SMN-SCGN synapse model paves the way for further studies about the mechanism responsible for the formation of synapse between different types of neurons and provokes new perspectives and ideas that may be of great use in the treatment of patients with spinal cord injury.