| Backgrounds and Objectives: This team did systematic study in the lumbosacral nerveinjury. In the study of mechanism ofcauda equina syndrome, professor Shi Jiangangpresented horsetail nerve damage "two-way feedback" theory, the reverse feed change leadto damage of spinal cord anterior horn and posterior horn neurons. Also found in thestudy of peripheral nerve and sciatic nerve injury secondary to produce the anterior horn ofspinal cord after a large number of motor neuron apoptosis. The most of spine injurypatients found that dominated muscle dysfunction, which nerve roots damage occurred，even have nerve decompression surgery, functional recovery is not ideal, for example: footdrop, etc. Nerve root damage source of spinal cord anterior horn motor neurons damageoccurs, is the concern of the spine and neurologists. In order to further explore thepathological mechanism of the secondary damage in spinal cord anterior horn cells，looking for ways to restrain the mechanism occurred, this topic mainly through BDNFglycine can normal spinal motor neurons of rat neurotransmitter regulation mechanismresearch, understand the BDNF conduction function change for spinal cord anterior horncells, the influence of the objective is to study the pathological mechanism of neurons ofBDNF in spinal cord anterior horn motor neurons of the repair mechanism of damage anddysfunction.The research status: The role of BDNF include promoting the survival、differentiation of neurons、synapses control, promote myelination and regulating thegrowth of axons and dendrites. Yet the BDNF in the spinal cord motor neurons, which is amajor role in motor output structure still poorly understood. Boyce, etc by exprementstudy, electrophysiology, and immunohistochemical results found that slowly gives thebrain derived neurotrophic factor can increase spinal cord excitability of motor neuroncells, improve the motor function of lower limbs. By inference, BDNF have a regulatingeffect on motor neuron excitability. The excitability of neurons in the regulation is dividedinto two inhibitory and excitatory effect, it is because of the two work together, to make motor coordination, precision by the line.renshew cells as inhibitory interneurons,recurrent inhibition play very important role, especially the release of neurotransmittersglycine. Glycine is itself a inhibitory neurotransmitter. With the literature When thepostsynaptic inhibition occurs, BDNF can directly enhance NMDA presynaptic excited.Tothis end, we in the study of BDNF on adjustment of spinal motor neuron excitability,whether brain derived neurotrophic factor involved in the glycine regulation of spinalmotor neuron excitability, no similar literature reported so far. We will further examine theeffect of BDNF on spinal motor neurons, due to the limited subject, mainly usingwhole-cell patch clamp technique in vitro slices, auxiliary immunohistochemicaltechniques, such as observation of BDNF is regulation function of spinal motor neuronexcitability. A preliminary study of BDNF is regulation function of spinal motor neuronexcitability. With glycine participation mechanism? The pattern of synergism andantagonism effect? The relationship with TrKB receptor mediated?objective:This study,we will use in vitro slices of whole cell patch clamp technique, real timequantitative PCR and immunofluorescence histochemical double marking technology toassess the BDNF in spinal motor neuron activity and its regulation of glycine toneurotransmitter and its receptor mechanism. Observation of BDNF, glycine inducedchanges the current size of motor neurons, and receptors are involved in. Furtherunderstanding of BDNF in the central nervous system, especially the role of signal outputstructure.The research content and methods:1、Take of lumbosacral spinal cord of rats spinal cord slices. Using14to24days afterbirth the SD rats. Animals after anesthesia depth of ground water freezing rapid beheaded,scissors to cut off the thoracic lumbar cone plate, step by step a separate exposure andspinal cord, free cutting spinal nerve, quickly remove spinal cord into the ready access tosaturated95%95%O2/CO2mixture of2°C artificial cerebrospinal fluid, then lumbarspinal cord adhesion in AGAR block, fixed on the vibration slicing machine, start the slicerfor spinal cord coronal slices number (thickness is300-400microns), the production ofspinal cord slices.2、Identifying and selecting of spinal motor neurons. From40only12to24days old ratlumbosacral spinal cord) section of the brain slice of the ventral horn， we obtained40selection standards of neurons. Through morphology and electrophysiological method, We further determine these neurons are spinal motor neurons.3、Selecting the spinal cord anterior horn motor neurons for A, B, C, D four groups, Indifferent perfusion reagent (BDNF, glycine), perfusion concentration, length of time and soon, Voltage clamp down record spinal cord neurons in different ways to stimulate the casesinduced by the outgoing current size, order to observe the relationship between the BDNFand glycine, which appeared at the time of the outgoing neurons induced current.4、Dividing two groups, blocking presynaptic neuron’s action potential distributionrespectively group（Perfusion artificial cerebrospinal fluid containing0.3μM TTX toblock fast Na+channel caused by action potentials15minutes)，No intervention group.Glycine stimulus to generate neurons inhibitory reaction,we record the size of the differentgroups of the membrane potential. We can determine that glycine is acting on thepresynaptic or postsynaptic.5、Dividing four groups under the condition of different interventions, We recordedneurons induced by the outgoing current size, observed the the interaction relationsbetween BDNF, glycine and TrkB.6、Using real-time quantitative PCR and immunofluorescence double standard techniqueto observe the BDNF TrkB and its receptor in rat spinal cord in the expression anddistribution of motor neurons.results:(1) BDNF did not produce a direct excitatory or inhibitory effect on themotoneurons;(2) BDNF dose-dependently increased the glycinergic transmission in themotoneurons;(3)The glycinergic transmission motor neurons was a direct postsynapticeffect;(4) the BDNF-induced enhancement of the glycinergic transmission was mediatedby the activation of TrkB receptors.(5) BDNF and its receptors TrkB had an extensiveexpression in the motoneurons.Conclusion: These results suggest that BDNF is directly involved in the regulation ofglycinergic transmission on the motoneurons through postsynaptic TrkB receptors.Considering that the glycinergic synaptic transmission of motoneurons mainly comes fromRenshaw cells, an importent inhibitory interneurons of the spinal cord, we speculate thatBDNF may play an important role in the information integration in the spinal cord andparticipate in the reaction sensitivity of motoneurons. |
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