Synapse Formation Between Rat Spinal Motor Neurons And Major Pelvic Ganglion Neurons In Vitro

PARTⅠ: Experimental study on co-culture of ventral spinalcord explants and major pelvic ganglion explants【Objective】In the present study, we investigated the interaction between explantsof ventral spinal cord containing motor neurons from newborn rat and explants of majorpelvic ganglion from adult rat.【Methods】We have developed a system for long-term co-culturing of ventralspinal cord explants and major pelvic ganglion explants. Morphology of cells wereobserved by microscopy.【Results】Explanted tissues of both types survived well in co-culture. Spinalventral root neurons emitted numerous outgrowing processes(axons)some of which cameinto association with neurons in the explanted major pelvic ganglion.【Conclusions】Some apparently specific motor neuron-major pelvic ganglianeuron contacts were observed, suggesting that a functional interaction may developbetween spinal motor neurons axon and major pelvic ganglion neurons in vitro. PARTⅡ: co-culture of spinal motor neurons from newborn ratsand major pelvic ganglion neurons from adult ratsPaper1: Isolation and culture of motor neurons from the newborn ratspinal cord【Objective】To explore the method of isolation, purification and culture of motorneurons from the newborn rat spinal cord.【Methods】Spinal motor neurons (SMNs) were dissociated from ventral spinal cordof postnatal day 1 rats. The culture system for SMNs was established by density gradientcentrifugation, differential adhesion, serum free conditions and in defined media. SMNswere identified by immunocytochemistry with a specific antibody, ChAT.【Results】After 72 h in culture, the cultured neurons continued to exhibit extensiveneuritic processes and displayed the characteristic morphology of motor neurons. Theseneurons have been shown to survive at least 2 weeks. SMNs expressed cholineacetyltransferase (ChAT) and were immunoreactive to the antibody against ChAT.【Conclusions】We have now demonstrated a reliable protocol for the isolation andculture of motor neurons from the postnatal spinal cord. It can be useful for further study ofSMNs.Paper2: Culture of major pelvic ganglion neurons from adult rat【Objective】The aim of the present study was to establish a method for the cultureof major pelvic ganglia (MPG) neurons from adult rat, which would permit further studiesfor establishing the SMNs-MPGN co-culture system.【Methods】Studies were conducted in adult male rats.The MPG tissues werechopped in a number of slices and treated enzymatically in two steps, then neurons were dissociated by sequential mechanical trituration in a small volume of defined culturemedium. MPG neurons were identified by immunostained with a neuron specific antibody,neurofilament-200 (NF-200).【Results】We reported here the development of a novel method for the dissociationand the culture of MPG neurons from adult rat. Neurons dissociated from adult rat MPGreadily adapted to cell culture and survived for more than 2 weeks. MPG neurons wereimmunoreactive to the antibody against NF-200.【Conclusions】The results demonstrated that the protocol we used was able toobtain a relatively high yield of MPG neurons. Furthermore, the new protocol iscost-effective and improved the speed and simplicity of neuronal isolation.Paper3: Synapse formation between rat spinal motor neurons andmajor pe lvic ganglion neurons in vitro【Objective】To find out whether synapses would form between spina motorneurons from newborn rats and major pelvic ganglion neurons from adult rats, and furtherto investigate the mechanisms of an artificial somatic-autonomic reflex arc.【Methods】Major pelvic ganglion neurons were transfected with Ad -GFP vectorto stably express enhanced green flourescent protein(GFP), a tracing marker in thefollowing experiment. These GFP major pelvic ganglion cells were cultured in adefined medium together with spinal motor neurons. In these co-cultures, establishmentof synaptic contacts between spinal motor neurons and major pelvic ganglionneurons,was studied by morphology and immunocytochemistry approaches.【Results】Spinal motor neurons and major pelvic ganglion neurons survived welland developed into mature neurons in co-cultures. Phase-contrast observation ofco-cultures showed apparent spinal motor neurons-major pelvic ganglion neurons contacts after only DIV 3- 4d. Using immunostaining with antibody directed againstNeurofilament -200, we demonstrated synaptic contacts between spinal motor neuronsand major pelvic ganglion neurons in vitro.【Conclusions】The results presented here described the establishment ofSMNs-MPGN synapses in vitro, which were thought to play an essential role in themachanisms of regenerated bladder following an artificial somatic-autonimic reflex arc.