Effects Of An Artificial Somatic-Autonomic Reflex Arc Procedure On Voltage-gated Sodium Channels In Bladder Major Pelvic Ganglion Neurons

PART I: Electrophysiological Properties of Voltage-gated Sodium Channels in acutely dissociated bladder Major Pelvic Ganglion Neurons of the Adult Male Rat[OBJECTIVE] To study on the mechanisms underlying the micturition reflex, we examined the electrophysiological properties of the voltage-gated sodium channel (VGSC) in acutely dissociated neurons from the major pelvic ganglion (MPG) which innervates the urinary bladder.[METHODS] The MPG neurons innervating the urinary bladder of the adult male rat were identified by retrograde axonal tracing methods using Fluorogold (FG) . Whole-cell patch-clamp recording techniques were performed to analyze the kinetics of sodium channel steady-state activation, inactivation and recovery.[RESULTS]1. The bladder MPG neurons had a spherical shape or similar to spherical, a mean diameter of 24.28 ± 3.28μm (n = 46), range from 16 to 38μm. It had an average membrane capacitance of 24.05±3.31 pF at holding potential of -80 mV.2. The sodium current (I_Na) had a mean peak amplitude of -165.13 ± 13.51 pA/pF (n=29) at -20 mV when activated from the holding potential of -80 mV in the external solution containing 120 mM Na⁺ and was reversibly blocked by 1 μM tetrodotoxin (TTX). The threshold for steady-state activation of I_Na was approximately -35mV (mean -33.79 ± 1.82 mV, n=29) and I_Na reached a peak amplitude at approximately -20 mV (mean -19.65 ± 0.78 mV, n=29). The reversal potential (V_rev) was approximately +55 mV. The conductance reached half-maximal activation at a membrane potential of -25.70 ± 0.95 mV mV with a slope factor of 2.55 ± 0.42 mV.3. The steady-state inactivation of I_Na occurred at membrane potentials more depolarized than -80 mV and the half-maximal inactivation potential (V_1/2) was -39.92 ± 0.79 mV with a slope factor (V_C) of 6.93 ± 0.46 mV(n = 31).4. The time constant of recovery was 2.85 ± 0.12 ms (n=23).[CONCLUSIONS] This work has demonstrated the feasibility of combiningpatch-clamp recording and retrograde axonal tracing techniques to study the electrophysiological properties of VGSCs in morphologically identified autonomic ganglion neurons of bladder. The results indicated that the MPG neurons may have special electrophysiological properties different from sympathetic and parasympathetic neurons. These findings may be useful to further define and regulate the role of VGSCs of MPG neurons in micturition reflex, especially in disease states.PART II: The isoforms of Voltage-gated Sodium Channels in MajorPelvic Ganglion Neurons of the Adult Male Rat[OBJECTIVE] We investigate the isoforms of voltage-gated sodium channels in major pelvic ganglion neurons of the adults male rat, in order to explain the mechanism underlying the micturition, and for future treatment of associated clinical diseases.[METHODS] The isoforms of VGSCs in MPG neurons were identified with Reverse transcriptase-Polymerase chain reaction and DNA sequencing.[RESULTS] Only the isoforms Na_V1.1, Na_V1.6, Na_V1.7 were expressed in MPG neurons, and all of them are TTX-sensitiveVGSCs. We did not found the expression of the isforms of Na_V1.2, Na_V1.3, Na_V1.8, Na_V1.9.[CONCLUSIONS] Three isoforms of major pelvic ganglion, Na_V 1.1, Na_V 1.6, Na_V 1.7 may involve the mechanism underlying the normal micturition and control the urinary bladder detrusor.PART III: Effects in electrophysiology properties of an Artificial Somatic-Autonomic Reflex Arc Procedure on Voltage-gated Sodium Channels in Bladder Major Pelvic Ganglion Neurons[OBJECTIVE] To investigate the changes in electrophysiology properties of Voltage-gated Sodium Channels in bladder major pelvic ganglion neurons following the artificial somatic-autonomic reflex arc procedure. And establish the basis for elucidating the mechanisms underlying the new reflex.[METHODS] MPG neurons were identified by retrograde axonal tracing with Fluorogold and whole-cell patch-clamp recording were performed on the MPG neurons to detect the electrophysiology properties of VGSCs in 3 group of control, 7 day and 90 day postoperatively.[RESULTS]1. The bladder MPG neurons had a mean diameter of 24.58 ± 2.08, 25.87 ± 1.38, 24.07 ± 1.42μm and an average membrane capacitance of 25.21 ± 0.47, 26.12 ± 0.43 and 23.90 ± 0.47 pF for control, 7 day group and model, respectively. No significant differenece was found among the three groups.2. The threshold for steady-state activation of I_Na was -33.79 ± 1.82 mV, -34.5 ± 2.04 mV and -34.78 ± 2.50 mV, the membrane potential which I_Na reached a peak amplitude was -19.65 ± 0.78 mV, -21.05 ± 1.03 mV,-19.13 ± 0.87 mV, and the mean peak amplitude of I_Na was -165.13 ± 13.51 pA/pF, -127.34 ± 4.81 pA/pF, -152.13 ± 14.38 pA/pF for group control, 7 day and model, respectively. Compared with control, the mean peak of group 7 day decreased significantly (P< 0.05), but no significance were found in threshold and membrane potential for peak amplitude among three groups.3. The half-maximal activation potential was -25.70 ± 0.95 mV, -25.19 ± 0.72 mV, -24.29 ± 0.68 mV with slope 2.55 ± 0.42 mV, 2.35 ± 0.33 mV, 1.91 ± 0.23 mV, respectively for the three groups and no significance found.4. The half-maximal inactivation potential was -39.92 ± 0.79 mV, -37.06 ± 0.82, -37.74 ± 0.89 mV with slope 6.93 ± 0.46 mV, 6.55 ± 0.72 mV, 6.86 ± 0.78 mV, respectively for the three groups and no siginificance found.5. The time constant of recovery was 3.06 ± 0.83 ms, 3.10 ± 0.53 ms, 3.17 ± 0.49ms for the 3 groups respectively. No significance was found among three groups.[CONCLUSIONS] This study has demonstrated electrophysiologically the feasibility of the somatic nerve fibre regenerating into autonomic preganglionic nerves. The steady kinetics of VGSCs of the MPG neurons innervating the bladder may contribute to it's function after the establishment of the artificial reflex arc. The dynamic alteration of VGSCs current density was associated with the mechanism of artificial reflex and may be involved in the nerve plasticity.PART IV: Semiquatitative of Voltage-gated Sodium Channels protein in Major Pelvic Ganglion Neurons following the ArtificialSomatic-Autonomic Reflex Arc Procedure[OBJECTIVE] To investigate the quantitative of VGSCs protein in MPG neurons of the adults male rat with an artificial somatic-autonomic reflex arc, to try to clarify the mechanism underlying the reflex micturition.[METHODS] Semiquantitative-PCR and photograph analysis were performed to detect the expression in MPG of VGSCs in rats divided to groups of control, 7 day or 90-day after the procedure for an artificial somatic-autonomic reflex arc.[RESULTS]The expression of isoforms Na_V 1.1 had a mean integrated optical density (IOD) 0.7760 ± 0.1030, 0.4584 ± 0.0335 and 0.7298 ±0.1156 for group control, 7 day and 90-day prosoperatively, respectively. The mean IOD for Nay 1.6 were 0.7170 ± 0.0697, 0.7820 ± 0.0663 and 0.7951 ± 0.0810, and for Na_V 1.7 were 1.2780 ± 0.1876, 0.6168 ± 0.09211 and 1318 ± 0.1055, for the three groups, respectively. The quantum of Na_V1.1 and Na_V 1.7 expressed in MPG decreased markedly at 7 days after anastomosis and rescovery in group 90-day obviously (P<0.05). Compared with control, no change was detected in Na_V1 .6 expression in the process.[CONCLUSIONS] Results suggest that the two isoforms in MPG neurons, Na_V1.1 and Na_V 1.7, may be involved in plasticity of regeneration and rehabilitation following the artificial somatic-autonomic reflex arc procedure. The dynamic alteration of Na_V 1.1 and Na_V 1.7 may be associated with the mechanism underlying new reflex voiding.