Electrophysiological And Morphological Properties Of Ascending Commissural Interneurons And NMDA Modulation In The Neonatal Rodent Spinal Cord

Locomotion is the basic exercise of vertebrates, such as:walking, swimming, flying and so on. CPGs that controlling the lower or hind limb which responsible for locomotion are located in thoracolumbar spinal cord. The interneurons responsible for the alternating movement of the limbs in the CPG are called commissural interneurons (CINs) which project their axons to the opposite side through the ventral midline of the spinal cord. In cats and rodents, the CINs can be divided into short-range projection, upward projection, downward projection and the two-way projection four categories depending on the projecting direction of the axons. We have known the functions and characteristics of the four types CINs and the Acin show rhythmic excitement during fictive locomotion induced by special drugs, suggesting that these IVuciferlVrons are involved in the coordination between left and right limbs during locomotion. However we know little about the relevance between the morphology and distribution of aCIN or how does NMDA regulate the excitation of aCIN. In view of this, we decided to figure out the morphology, distribution and the regulation of membrane properties by NMDA of aCIN.Objective:In the current study, we use the retrograde labeling and patch clamp to figure out the morphology and electrophysiology characteristics of the aCINs; the intracellular staining technology and whole cell patch recording were used to determine the membrane properties and the location of the aCINs in the lumbar spinal cord; we want to find out the neurochemical mechanisms that how does NMDA regulate the excitability of the aCINs during the fictive locomotion.Methods:In this study, experiments were carried out on neonatal(P1-P10) wild type rats. After decapitation, torso was placed in a supine position and then pinned down via the limbs onto the base of the Sylgard-lined metaldish, which was filled with DaCSF and bubbling with carbogen(95% O2-5% CO2). The spinal cord was released from the column by the vertebrectomy then one hemicord was isolated from rostral end to T12 segments prepare for Retrograde labeling.(1 Distribution:After retrograde labeled aCINs using the water-soluble fluoresecent tracer-tetramethylrhodamine dextran amine (RDA), the distribution of the aCINs in the spinal cord transection could be figured out.(2)Electrophysiology:Membrane properties observed in the current study include rest membrane potential(Em), input resistance(Rin), voltage threshold(Vth), rheobase, amplitude and half width of the action potential, afterhyperpolarization(AHP) half decay time and AHP half width in the whole cell patch.(3)Morphology:All the cells recorded in this study were labeled with Vucifer yellow in the recording pipette, then the slides were scanned and photographed with Zeiss microscope. The diameter and superficial area were calculated tuo analysis the cell morphology.(4)NMDA regulation:In order to examine the effect of the NMDA on the aCINs, measurements of the rest membrane potential(Em), input resistance(Rin), voltage threshold(Vth), rheobase, amplitude and half width of the action potential, afterhyperpolarization(AHP) half decay time and AHP half width were compared before and after the bath application of 10μ MNMDA.Results:(1) Most of the aCINs are located in lamina Ⅶ and lamina Ⅷ however we can also find aCINs in lamina X.(2) Membrane properties obtained in current study included rest membrane potential (-60.7 ± 6.5mV)、input resistance (1508.1±683.lmΩ)、voltage threshold (-35.6±5.8mV). rheobase (13.1±8.3pA)、action potential amplitude (51.8±14.5mV)、action potential half-width (2.9±0.7 ms)、afterhyperpolarization amplitude (15.0 ± 9.0 mV) and afterhyperpolarization half decay (211.0±66.lms)(3) The aCINs can be classified into fusiform, triangle and multipolar basing on the shape of the soma. Morphological indexes measured in this study include diameter(16.2±2,9μm) and area(850.5±326.5μm2). The results suggest that there is no significant difference between the neurons distributed in lamina Ⅶ, and X on the diameter (Ⅶ 15.8±2.0μm, Ⅷ 16.5± 3.1μm, Ⅹ 16.2+4.9μm) and superficial area(Ⅶ 799.5±202.9μm2,Ⅷ 882.1±351.5μ m2,Ⅹ881.7±326.5μm2).(4) There is no statistic significant correlation between the morphology and the lumbar distribution of the aCINs.(5) All the cells recorded in this study were classified into three types basing on their response to the injected depolarizing current. Type I has significantly depolarized rheobase and voltage threshold than type Ⅱ and typeⅢ however there is not any statistic difference between the type II and typeⅢ.(6) Depolarized sag was found in 24% aCINs and some of the cells had postinhibitory rebound.(7) Bath application of 10 μ-MNMDA can depolarize the rest membrane potential of the aCINs.(8) NMDA hyperpolarized the voltage threshold and rheobase in all aCINs.Conclusion(1) In the electrophysiological properties of cell membrane, three types of aCINs showed differences, without indifference on the morphology and the spinal distribution, indicating that aCINs may provide the diversity of motion control mechanism through the properties of membrane,while their morphology and distribution characteristics may not play an important role in locomotion.(2) NMDA,as an excitatory neurotransmitter, plays a crucial role for production of locomotion.The results of this paper showed that NMDA can increase excitatory of aCINS,demonstrating that aCINs can accept regulation of NMDA in locomotion and exert a role in the coordination of body movement.