Sex differences in lower urinary tract function in mice with or without spinal cord injury.

OBJECTIVES: We examined sex differences of lower urinary tract function and molecular mechanisms in mice with and without spinal cord injury (SCI). METHODS: SCI was induced by Th8-9 spinal cord transection in male and female mice. We evaluated cystometrograms (CMG) and electromyography (EMG) of external urethral sphincter (EUS) at 6 weeks after SCI in spinal intact (SI) and SCI mice. The mRNA levels of Piezo2 and TRPV1 were measured in L6-S1 dorsal root ganglia (DRG). Protein levels of nerve growth factor (NGF) in the bladder mucosa was evaluated using an enzyme-linked immunosorbent assay. RESULTS: Sex differences were found in the EUS behavior during voiding as voiding events in female mice with or without SCI occurred during EUS relaxation periods without EUS bursting activity whereas male mice with or without SCI urinated during EUS bursting activity in EMG recordings. In both sexes, SCI decreased voiding efficiency along with increased tonic EUS activities evident as reduced EUS relaxation time in females and longer active periods of EUS bursting activity in males. mRNA levels of Piezo2 and TRPV1 of DRG in male and female SCI mice were significantly upregulated compared with SI mice. NGF in the bladder mucosa showed a significant increase in male and female SCI mice compared with SI mice. However, there were no significant differences in Piezo2 or TRPV1 levels in DRG or NGF protein levels in the bladder mucosa between male and female SCI mice. CONCLUSIONS: We demonstrated that female and male mice voided during EUS relaxation and EUS bursting activity, respectively. Also, upregulation of TRPV1 and Piezo2 in L6-S1 DRG and NGF in the bladder could be involved in SCI-induced lower urinary tract dysfunction in both sexes of mice.

Role of opioid and ß-adrenergic receptors in bladder underactivity induced by prolonged pudendal nerve stimulation in cats.

AIMS: To determine the role of opioid and ß-adrenergic receptors in bladder underactivity induced by prolonged pudendal nerve stimulation (PNS). METHODS: In α-chloralose anesthetized cats, 30-min PNS was applied repeatedly for 3-9 times to induce poststimulation or persistent bladder underactivity. Then, naloxone (opioid receptor antagonist, 1 mg/kg, IV) or propranolol (ß-adrenergic receptor antagonist, 3 mg/kg, IV) was given to reverse the bladder underactivity. After the drug treatment, an additional 30-min PNS was applied to counteract the drug effect. Repeated cystometrograms were performed by slowly (1-2 mL/min) infusing the bladder with saline via a urethral catheter to determine the bladder underactivity and the treatment effects. RESULTS: Prolonged (2-4.5 h) PNS induced bladder underactivity evident as a large bladder capacity (169 ± 49% of control) and a reduced amplitude of bladder contraction (59 ± 17% of control). Naloxone fully reversed the bladder underactivity by reducing bladder capacity to 113 ± 58% and increasing the amplitude of bladder contraction to 104 ± 34%. After administration of naloxone an additional 30-min PNS temporarily increased the bladder capacity to the underactive bladder level (193 ± 74%) without changing the amplitude of the bladder contraction. Propranolol had no effect on bladder underactivity. CONCLUSIONS: A tonic enkephalinergic inhibitory mechanism in the CNS plays a critical role in the bladder underactivity induced by prolonged PNS, while the peripheral ß-adrenergic receptor mechanism in the detrusor is not involved. This study provides basic science evidence consistent with the clinical observation that comorbid opioid usage may contribute to voiding dysfunction in patients with Fowler's syndrome.

Pudendal Nerve Block by Adaptively Stepwise Increasing the Intensity of High-Frequency (10 kHz) Biphasic Stimulation.

OBJECTIVE: The purpose of this study is to determine whether adaptively stepwise increasing the intensity of a high-frequency (10 kHz) biphasic stimulation (HFBS) can produce nerve conduction block without generating a large initial response. MATERIALS AND METHODS: In anesthetized cats, three cuff electrodes were implanted on the left pudendal nerve for stimulation or block. The urethral pressure increase induced by pudendal nerve stimulation was used to measure the pudendal nerve block induced by HFBS. RESULTS: HFBS applied suddenly with a large step increase in intensity induced a large (86 ± 16 cmH2O) urethral pressure increase before it blocked pudendal nerve conduction. However, HFBS applied by adaptively stepwise increasing the intensity every 10 to 60 seconds over a long period (33-301 minutes; average 108 ± 35 minutes) with many small intensity increases (0.005-0.1 mA) induced no response or low-amplitude high-frequency urethral pressure changes before it blocked pudendal nerve conduction. The minimal HFBS intensities required by the two different methods to block pudendal nerve conduction are similar. CONCLUSION: This study is important for better understanding the possible mechanisms underlying the HFBS-induced nerve block and provides the possibility of developing a new nerve block method for clinical applications in which an initial large response is a concern.

Molecular Mechanisms of Neurogenic Lower Urinary Tract Dysfunction after Spinal Cord Injury.

This article provides a synopsis of current progress made in fundamental studies of lower urinary tract dysfunction (LUTD) after spinal cord injury (SCI) above the sacral level. Animal models of SCI allowed us to examine the effects of SCI on the micturition control and the underlying neurophysiological processes of SCI-induced LUTD. Urine storage and elimination are the two primary functions of the LUT, which are governed by complicated regulatory mechanisms in the central and peripheral nervous systems. These neural systems control the action of two functional units in the LUT: the urinary bladder and an outlet consisting of the bladder neck, urethral sphincters, and pelvic-floor striated muscles. During the storage phase, the outlet is closed, and the bladder is inactive to maintain a low intravenous pressure and continence. In contrast, during the voiding phase, the outlet relaxes, and the bladder contracts to facilitate adequate urine flow and bladder emptying. SCI disrupts the normal reflex circuits that regulate co-ordinated bladder and urethral sphincter function, leading to involuntary and inefficient voiding. Following SCI, a spinal micturition reflex pathway develops to induce an overactive bladder condition following the initial areflexic phase. In addition, without proper bladder-urethral-sphincter coordination after SCI, the bladder is not emptied as effectively as in the normal condition. Previous studies using animal models of SCI have shown that hyperexcitability of C-fiber bladder afferent pathways is a fundamental pathophysiological mechanism, inducing neurogenic LUTD, especially detrusor overactivity during the storage phase. SCI also induces neurogenic LUTD during the voiding phase, known as detrusor sphincter dyssynergia, likely due to hyperexcitability of Aδ-fiber bladder afferent pathways rather than C-fiber afferents. The molecular mechanisms underlying SCI-induced LUTD are multifactorial; previous studies have identified significant changes in the expression of various molecules in the peripheral organs and afferent nerves projecting to the spinal cord, including growth factors, ion channels, receptors and neurotransmitters. These findings in animal models of SCI and neurogenic LUTD should increase our understanding of pathophysiological mechanisms of LUTD after SCI for the future development of novel therapies for SCI patients with LUTD.

Model Analysis of Post-Stimulation Block of a Myelinated Axon by Direct Current.

OBJECTIVE: To determine the role of ion concentrations and ion pump activity in conduction block of myelinated axon induced by a long-duration direct current (DC). METHODS: A new axonal conduction model for myelinated axons based on the classical Frankenhaeuser-Huxley (FH) equations is developed that includes ion pump activity and allows the intracellular and extracellular Na+ and K+ concentrations to change with axonal activity. RESULTS: Action potential generation, propagation, and acute DC block occurring within a short period (milliseconds) that do not significantly change the ion concentrations or trigger ion pump activity are successfully simulated by the new model in a similar way as the classical FH model. Different from the classical model, the new model also successfully simulates the post-stimulation block phenomenon, i.e., the axonal conduction block occurring after terminating a long-duration (30 seconds) DC stimulation as observed recently in animal studies. The model reveals a significant K+ accumulation outside the axonal node as the possible mechanism underlying the post-DC block that is slowly reversed by ion pump activity during the post-stimulation period. CONCLUSION: Changes in ion concentrations and ion pump activity play an important role in post-stimulation block induced by long-duration DC stimulation. SIGNIFICANCE: Long-duration stimulation is used clinically for many neuromodulation therapies, but the effects on axonal conduction/block are poorly understood. This new model will be useful for better understanding of the mechanisms underlying long-duration stimulation that changes ion concentrations and triggers ion pump activity.

Therapeutic effects of p38 mitogen-activated protein kinase inhibition on hyperexcitability of capsaicin sensitive bladder afferent neurons in mice with spinal cord injury.

AIMS: Nerve growth factor (NGF) has been implicated as a key molecule of pathology-induced changes in C-fiber afferent nerve excitability, which contributes to the emergence of neurogenic detrusor overactivity due to spinal cord injury (SCI). It is also known that the second messenger signaling pathways activated by NGF utilize p38 Mitogen-Activated Protein Kinase (MAPK). We examined the roles of p38 MAPK on electrophysiological properties of capsaicin sensitive bladder afferent neurons with SCI mice. MAIN METHODS: We used female C57BL/6 mice and transected their spinal cord at the Th8/9 level. Two weeks later, continuous administration of p38 MAPK inhibitor (0.51 µg/h, i.t. for two weeks) was started. Bladder afferent neurons were labelled with a fluorescent retrograde tracer, Fast-Blue (FB), injected into the bladder wall three weeks after SCI. Four weeks after SCI, freshly dissociated L6-S1 dorsal root ganglion neurons were prepared and whole cell patch clamp recordings were performed in FB-labelled neurons. After recording action potentials or voltage-gated K+ currents, the sensitivity of each neuron to capsaicin was evaluated. KEY FINDINGS: In capsaicin-sensitive FB-labelled neurons, SCI significantly reduced the spike threshold and increased the number of action potentials during 800 ms membrane depolarization. Densities of slow-decaying A-type K+ (KA) and sustained delayed rectifier-type K+ (KDR) currents were significantly reduced by SCI. The reduction of KA, but not KDR, current density was reversed by the treatment with p38 MAPK inhibitor. SIGNIFICANCE: P38 MAPK plays an important role in hyperexcitability of capsaicin-sensitive bladder afferent neurons due to the reduction in KA channel activity in SCI mice.

Effect of high-frequency membrane potential alternation between depolarization and hyperpolarization on dorsal root ganglion neurons of rats.

The purpose of this study was to determine how sensory neurons respond to high-frequency membrane potential alternation between depolarization and hyperpolarization. Membrane currents were recorded from dissociated dorsal root ganglion (DRG) neurons of adult rats using the whole cell patch clamp technique in voltage clamp mode. Stepwise depolarization of the membrane was applied first to determine the threshold membrane potential for inducing an action potential (AP) current. Then, membrane potential alternation between depolarization (to +20 mV) and hyperpolarization (to -110 mV) was applied to the neuron for 10 s at different frequencies (10 Hz to 1 kHz). The tested DRG neurons had APs of either a long duration (>10 ms) or a short duration (<10 ms). Membrane potential alternation at ≥500 Hz completely disrupted the AP generation, disabled the ion channel gating function, and produced membrane current alternating symmetrically across zero. Replacing extracellular sodium with potassium increased the amplitude of the membrane current response and caused the membrane current to be larger during hyperpolarization than during depolarization. These results support the hypothesis that high-frequency biphasic stimulation blocks axonal conduction by driving the potassium channel open constantly. Understanding neural membrane response to high-frequency membrane potential alternation is important to reveal the possible mechanisms underlying axonal conduction block induced by high-frequency biphasic stimulation.

Temperature Effect on Nerve Conduction Block Induced by High-Frequency (kHz) Biphasic Stimulation.

OBJECTIVES: This study aims to determine temperature effect on nerve conduction block induced by high-frequency (kHz) biphasic stimulation (HFBS). MATERIALS AND METHODS: Frog sciatic nerve-muscle preparation was immersed in Ringer's solution at a temperature of 15 or 20 °C. To induce muscle contractions, a bipolar cuff electrode delivered low-frequency (0.25 Hz) stimulation to the nerve. To induce nerve block, a tripolar cuff electrode was placed distal to the bipolar cuff electrode to deliver HFBS (2 or 10 kHz). A bipolar hook electrode distal to the blocking electrode was used to confirm that the nerve block occurred locally at the site of HFBS. A thread tied onto the foot was attached to a force transducer to measure the muscle contraction force. RESULTS: At 15 °C, both 2- and 10-kHz HFBSs elicited an initial transient muscle contraction and then produced nerve block during the stimulation (ie, acute block), with the 10 kHz having a significantly (p < 0.001) higher acute block threshold (5.9 ± 0.8 mA peak amplitude) than the 2 kHz (1.9 ± 0.3 mA). When the temperature was increased to 20 °C, the acute block threshold for the 10-kHz HFBS was significantly (p < 0.0001) decreased from 5.2 ± 0.3 to 4.4 ± 0.2 mA, whereas the 2-kHz HFBS induced a tonic muscle contraction during the stimulation but elicited nerve block after terminating the 2-kHz HFBS (ie, poststimulation block) with an increased block duration at a higher stimulation intensity. CONCLUSION: Temperature has an important influence on HFBS-induced nerve block. The blocking mechanisms underlying acute and poststimulation nerve blocks are likely to be very different.

Penile Erection Induced by Stimulation of Sacral S1/S2 Spinal Root in Cats.

OBJECTIVE: This study aimed at determining whether stimulation of sacral spinal roots can induce penile erection in cats. MATERIALS AND METHODS: In anesthetized cats, a 20-gauge catheter was inserted into the corpus cavernosum to measure the penile pressure. Stimulus pulses (5-80 Hz, 0.2 ms) were applied through bipolar hook electrodes to sacral ventral roots alone or to combined ventral and dorsal roots of a single S1-S3 segment to induce penile pressure increases and penile erection. RESULTS: Stimulation of the S1 or S2 ventral root at 30 to 40 Hz induced observable penile erection with rigidity and the largest increase (169 ± 11 cmH2O) in penile pressure. Continuous stimulation (10 minutes) of afferent and efferent axons by simultaneous stimulation of the S1 or S2 dorsal and ventral roots at 30 Hz also produced a large increase (190 ± 8 cmH2O) in penile pressure that was sustainable during the entire stimulation period. After a complete spinal cord transection at the T9-T10 level, simultaneous stimulation of the S1 or S2 dorsal and ventral roots induced large (186 ± 9 cmH2O) and sustainable increases in penile pressure. CONCLUSION: This study indicates the possibility to develop a novel neuromodulation device to restore penile erection after spinal cord injury using a minimally invasive surgical approach to insert a lead electrode through the sacral foramen to stimulate a sacral spinal root.

Mechanisms Underlying Poststimulation Block Induced by High-Frequency Biphasic Stimulation.

OBJECTIVE: To reveal the possible mechanisms underlying poststimulation block induced by high-frequency biphasic stimulation (HFBS). MATERIALS AND METHODS: A new axonal conduction model is developed for unmyelinated axons. This new model is different from the classical axonal conduction model by including both ion concentrations and membrane ion pumps to allow analysis of axonal responses to long-duration stimulation. Using the new model, the post-HFBS block phenomenon reported in animal studies is simulated and analyzed for a wide range of stimulation frequencies (100 Hz-10 kHz). RESULTS: HFBS can significantly change the Na+ and K+ concentrations inside and outside the axon to produce a post-HFBS block of either short-duration (<500 msec) or long-duration (>3 sec) depending on the duration of HFBS. The short-duration block is due to the fast recovery of the Na+ and K+ concentrations outside the axon in periaxonal space by diffusion of ions into and from the large extracellular space, while the long-duration block is due to the slow restoration of the normal Na+ concentration inside the axon by membrane ion pumps. The 100 Hz HFBS requires the minimal electrical energy to achieve the post-HFBS block, while the 10 kHz stimulation is the least effective frequency requiring high intensity and long duration to achieve the block. CONCLUSION: This study reveals two possible ionic mechanisms underlying post-HFBS block of axonal conduction. Understanding these mechanisms is important for improving clinical applications of HFBS block and for developing new nerve block methods employing HFBS.

Current Knowledge and Novel Frontiers in Lower Urinary Tract Dysfunction after Spinal Cord Injury: Basic Research Perspectives.

This review article aims to summarize the recent advancement in basic research on lower urinary tract dysfunction (LUTD) following spinal cord injury (SCI) above the sacral level. We particularly focused on the neurophysiologic mechanisms controlling the lower urinary tract (LUT) function and the SCI-induced changes in micturition control in animal models of SCI. The LUT has two main functions, the storage and voiding of urine, that are regulated by a complex neural control system. This neural system coordinates the activity of two functional units in the LUT: the urinary bladder and an outlet including bladder neck, urethra, and striated muscles of the pelvic floor. During the storage phase, the outlet is closed and the bladder is quiescent to maintain a low intravesical pressure and continence, and during the voiding phase, the outlet relaxes and the bladder contracts to promote efficient release of urine. SCI impairs voluntary control of voiding as well as the normal reflex pathways that coordinate bladder and sphincter function. Following SCI, the bladder is initially areflexic but then becomes hyperreflexic due to the emergence of a spinal micturition reflex pathway. However, the bladder does not empty efficiently because coordination between the bladder and urethral sphincter is lost. In animal models of SCI, hyperexcitability of silent C-fiber bladder afferents is a major pathophysiological basis of neurogenic LUTD, especially detrusor overactivity. Reflex plasticity is associated with changes in the properties of neuropeptides, neurotrophic factors, or chemical receptors of afferent neurons. Not only C-fiber but also Aδ-fiber could be involved in the emergence of neurogenic LUTD such as detrusor sphincter dyssynergia following SCI. Animal research using disease models helps us to detect the different contributing factors for LUTD due to SCI and to find potential targets for new treatments.

Intracellular sodium concentration and membrane potential oscillation in axonal conduction block induced by high-frequency biphasic stimulation.

Objective.A new axonal conduction model was used to analyze the interaction between intracellular sodium concentration and membrane potential oscillation in axonal conduction block induced by high-frequency (kHz) biphasic stimulation (HFBS).Approach.The model includes intracellular and extracellular sodium and potassium concentrations and ion pumps. First, the HFBS (1 kHz, 5.4 mA) was applied for a duration (59.4 s) long enough to produce an axonal conduction block after terminating the stimulation, i.e. a post-stimulation block. Then, the intensity of HFBS was reduced to a lower level for 4 s to determine if the axonal conduction block could be maintained.Main results.The block duration was shortened from 1363 ms to 5 ms as the reduced HFBS intensity was increased from 0 mA to 4.1 mA. The block was maintained for the entire tested period (4000 ms) if the reduced intensity was above 4.2 mA. At the low intensity (<4.2 mA) the membrane potential oscillation disrupted the post-stimulation block caused by the increased intracellular sodium concentration, while at the high intensity (>4.2 mA) the membrane potential oscillation was strong enough to maintain the block and further increased the intracellular sodium concentration.Significance.This study indicates a possibility to develop a new nerve block method to reduce the HFBS intensity, which can extend the battery life for an implantable nerve stimulator in clinical applications to block pain of peripheral origin.

Sacral neuromodulation of bladder underactivity induced by prolonged pudendal afferent firing in cats.

This study examined the effect of sacral neuromodulation on persistent bladder underactivity induced by prolonged pudendal nerve stimulation (PudNS). In 10 α-chloralose-anesthetized cats, repetitive application of 30-min PudNS induced bladder underactivity evident as an increase in bladder capacity during a cystometrogram (CMG). S1 or S2 dorsal root stimulation (15 or 30 Hz) at 1 or 1.5 times threshold intensity (T) for inducing reflex hindlimb movement (S1) or anal sphincter twitch (S2) was applied during a CMG to determine if the stimulation can reverse the bladder underactivity. Persistent (>3 h) bladder underactivity consisting of a significant increase in bladder capacity to 163.1 ± 11.3% of control was induced after repetitive (1-10 times) application of 30-min PudNS. S2 but not S1 dorsal root stimulation at 15 Hz and 1 T intensity reversed the PudNS-induced bladder underactivity by significantly reducing the large bladder capacity to 124.3 ± 12.9% of control. Other stimulation parameters were not effective. After the induction of persistent underactivity, recordings of reflex bladder activity under isovolumetric conditions revealed that S2 dorsal root stimulation consistently induced the largest bladder contraction at 15 Hz and 1 T when compared with other frequencies (5-40 Hz) or intensities (0.25-1.5 T). This study provides basic science evidence consistent with the hypothesis that abnormal pudendal afferent activity contributes to the bladder underactivity in Fowler's syndrome and that sacral neuromodulation treats this disorder by reversing the bladder inhibition induced by pudendal nerve afferent activity.

Superficial peroneal neuromodulation of nonobstructive urinary retention induced by prolonged pudendal afferent activity in cats.

The purpose of this study is to determine whether superficial peroneal nerve stimulation (SPNS) can improve nonobstructive urinary retention (NOUR) induced by prolonged pudendal nerve stimulation (PNS). In this exploratory acute study using eight cats under anesthesia, PNS and SPNS were applied by nerve cuff electrodes. Skin surface electrodes were also used for SPNS. A double lumen catheter was inserted via the bladder dome for bladder infusion and pressure measurement and to allow voiding without a physical urethral outlet obstruction. The voided and postvoid residual (PVR) volumes were also recorded. NOUR induced by repetitive (4-13 times) application of 30-min PNS significantly (P < 0.05) reduced voiding efficiency by 49.5 ± 16.8% of control (78.3 ± 7.9%), with a large PVR volume at 208.2 ± 82.6% of control bladder capacity. SPNS (1 Hz, 0.2 ms) at 1.5-2 times threshold intensity (T) for inducing posterior thigh muscle contractions was applied either continuously (SPNSc) or intermittently (SPNSi) during cystometrograms to improve the PNS-induced NOUR. SPNSc and SPNSi applied by nerve cuff electrodes significantly (P < 0.05) increased voiding efficiency to 74.5 ± 18.9% and 67.0 ± 15.3%, respectively, and reduced PVR volume to 54.5 ± 39.0% and 88.3 ± 56.0%, respectively. SPNSc and SPNSi applied noninvasively by skin surface electrodes also improved NOUR similar to the stimulation applied by a cuff electrode. This study indicates that abnormal pudendal afferent activity could be a pathophysiological cause for the NOUR occurring in Fowler's syndrome and a noninvasive superficial peroneal neuromodulation therapy might be developed to treat NOUR in patients with Fowler's syndrome.

High-frequency stimulation induces axonal conduction block without generating initial action potentials.

The purpose of this modeling study is to develop a novel method to block nerve conduction by high frequency biphasic stimulation (HFBS) without generating initial action potentials. An axonal conduction model including both ion concentrations and membrane ion pumps is used to analyze the axonal response to 1 kHz HFBS. The intensity of HFBS is increased in multiple steps while maintaining the intensity at a sub-threshold level to avoid generating an action potential. Axonal conduction block by HFBS is defined as the failure of action potential propagation at the site of HFBS. The simulation analysis shows that step-increases in sub-threshold intensity during HFBS can successfully block axonal conduction without generating an initial response because the excitation threshold of the axon can be gradually increased by the sub-threshold HFBS. The mechanisms underlying the increase in excitation threshold involve changes in intracellular and extracellular sodium and potassium concentration, change in the resting potential, partial inactivation of the sodium channel and partial activation of the potassium channel by HFBS. When the excitation threshold reaches a sufficient level, an acute block occurs first and after additional sub-threshold HFBS it is followed by a post-stimulation block. This study indicates that step-increases in sub-threshold HFBS intensity induces a gradual increase in axonal excitation threshold that may allow HFBS to block nerve conduction without generating an initial response. If this finding is proven to be true in human, it will significantly impact clinical applications of HFBS to treat chronic pain.

Defecation induced by stimulation of sacral S2 spinal root in cats.

The aim of this study was to determine whether stimulation of sacral spinal nerve roots can induce defecation in cats. In anesthetized cats, bipolar hook electrodes were placed on the S1-S3 dorsal and/or ventral roots. Stimulus pulses (1-50 Hz, 0.2 ms) were applied to an individual S1-S3 root to induce proximal/distal colon contractions and defecation. Balloon catheters were inserted into the proximal and distal colon to measure contraction pressure. Glass marbles were inserted into the rectum to demonstrate defecation by videotaping the elimination of marbles. Stimulation of the S2 ventral root at 7 Hz induced significantly (P < 0.05) larger contractions (32 ± 9 cmH2O) in both proximal and distal colon than stimulation of the S1 or S3 ventral root. Intermittent (5 times) stimulation (1 min on and 1 min off) of both dorsal and ventral S2 roots at 7 Hz produced reproducible colon contractions without fatigue, whereas continuous stimulation of 5-min duration caused significant fatigue in colon contractions. Stimulation (7 Hz) of both dorsal and ventral S2 roots together successfully induced defecation that eliminated 1 or 2 marbles from the rectum. This study indicates the possibility to develop a novel neuromodulation device to restore defecation function after spinal cord injury using a minimally invasive surgical approach to insert a lead electrode via the sacral foramen to stimulate a sacral spinal root.NEW & NOTEWORTHY This study in cats determined the optimal stimulation parameters and the spinal segment for sacral spinal root stimulation to induce colon contraction. The results have significant implications for design of a novel neuromodulation device to restore defecation function after spinal cord injury (SCI) and for optimizing sacral neuromodulation parameters to treat non-SCI people with chronic constipation.

Downstream projection of Barrington's nucleus to the spinal cord in mice.

Barrington's nucleus (Bar), which controls micturition behavior through downstream projections to the spinal cord, contains two types of projection neurons, BarCRH and BarESR1, that have different functions and target different spinal circuitry. Both types of neurons project to the L6-S1 spinal intermediolateral (IML) nucleus, whereas BarESR1 neurons also project to the dorsal commissural nucleus (DCN). To obtain more information about the spinal circuits targeted by Bar, we used patch-clamp recording in spinal slices from adult mice in combination with optogenetic stimulation of Bar terminals. Recording of opto-evoked excitatory postsynaptic currents (oEPSCs) in 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine, 4-chlorobenzenesulfonate (DiI)-labeled lumbosacral preganglionic neurons (LS-PGNs) revealed that both Bar neuronal populations make strong glutamatergic monosynaptic connections with LS-PGNs, whereas BarESR1 neurons also elicited smaller-amplitude glutamatergic polysynaptic oEPSCs or polysynaptic opto-evoked inhibitory postsynaptic currents (oIPSCs) in some LS-PGNs. Optical stimulation of BarCRH and BarESR1 terminals also elicited monosynaptic oEPSCs and polysynaptic oIPSCs in sacral DCN neurons, some of which must include interneurons projecting to either the IML or ventral horn. Application of capsaicin increased opto-evoked firing during repetitive stimulation of Bar terminals through the modulation of spontaneous postsynaptic currents in LS-PGNs. In conclusion, our experiments have provided insights into the synaptic mechanisms underlying the integration of inputs from Bar to autonomic circuitry in the lumbosacral spinal cord that may control micturition.NEW & NOTEWORTHY Photostimulation of BarCRH or BarESR1 axons in the adult mouse spinal cord elicits excitatory or inhibitory postsynaptic responses in multiple cell types related to the autonomic nervous system including preganglionic neurons (PGNs) in the lumbosacral intermediolateral nucleus and interneurons in the lumbosacral dorsal commissure nucleus. Integration of excitatory inputs from Bar and from visceral primary afferents in PGNs may be important in the regulation of micturition behavior.

Low pressure voiding induced by stimulation and 1 kHz post-stimulation block of the pudendal nerves in cats.

The goal of this study is to induce low-pressure voiding by stimulation and bilateral 1 kHz post-stimulation block of the pudendal nerves. In anesthetized cats, wire hook electrodes were placed on the left and/or right pudendal nerves. Stimulus pulses (30 Hz, 0.2 ms) were applied to one pudendal nerve to induce a reflex bladder contraction and to produce contractions of the external urethral sphincter (EUS). High frequency (1 kHz) biphasic stimulation was applied to block axonal conduction in both pudendal nerves and block EUS activity. In 4 cats, a catheter was inserted into the distal urethra to perfuse and measure the back pressure caused by the EUS contraction. In another 5 cats, a catheter was inserted into the bladder dome and the urethra was left open to allow voiding. The 1 kHz stimulation (30-60 s, 0.5-5 mA) delivered via a wire hook electrode completely blocked pudendal nerve conduction for ≥2 min after terminating the stimulation, i.e., a post-stimulation block. The block gradually disappeared in 6-18 min. The block duration increased with increasing amplitude or duration of the 1 kHz stimulation. Without the 1 kHz block, 30 Hz stimulation alone induced high-pressure (90 cmH2O) voiding. When combined with the 1 kHz block, the 30 Hz stimulation induced low-pressure (≤50 cmH2O) voiding with a high voiding efficiency (80%). In summary, a minimally invasive surgical approach might be developed to restore voiding function after spinal cord injury by stimulation and block of the pudendal nerves using lead electrodes.

Deciphering Spinal Endogenous Dopaminergic Mechanisms That Modulate Micturition Reflexes in Rats with Spinal Cord Injury.

Spinal neuronal mechanisms regulate recovered involuntary micturition after spinal cord injury (SCI). It was recently discovered that dopamine (DA) is synthesized in the rat injured spinal cord and is involved in lower urinary tract (LUT) activity. To fully understand the role of spinal DAergic machinery in micturition, we examined urodynamic responses in female rats during pharmacological modulation of the DA pathway. Three to four weeks after complete thoracic SCI, the DA precursor L-DOPA administered intravenously during bladder cystometrogram (CMG) and external urethral sphincter (EUS) electromyography (EMG) reduced bladder overactivity and increased the duration of EUS bursting, leading to remarkably improved voiding efficiency. Apomorphine (APO), a non-selective DA receptor (DR) agonist, or quinpirole, a selective DR2 agonist, induced similar responses, whereas a specific DR2 antagonist remoxipride alone had only minimal effects. Meanwhile, administration of SCH 23390, a DR1 antagonist, reduced voiding efficiency by increasing tonic EUS activity and shortening the EUS bursting period. Unexpectedly, SKF 38393, a selective DR1 agonist, increased EUS tonic activity, implying a complicated role of DR1 in LUT function. In metabolic cage assays, subcutaneous administration of quinpirole decreased spontaneous voiding frequency and increased voiding volume; L-DOPA and APO were inactive possibly because of slow entry into the CNS. Collectively, tonically active DR1 in SCI rats inhibit urine storage and enhance voiding by differentially modulating EUS tonic and bursting patterns, respectively, while pharmacologic activation of DR2, which are normally silent, improves voiding by enhancing EUS bursting. Thus, enhancing DA signaling achieves better detrusor-sphincter coordination to facilitate micturition function in SCI rats.

TRP Channel Agonists Activate Different Afferent Neuromodulatory Mechanisms in Guinea Pig Urinary Bladder.

Activation of TRP channels expressed in urinary bladder afferent nerves and urothelium releases neurotransmitters that influence bladder function. Experiments were undertaken to examine the mechanisms underlying effects of TRPA1 (allyl isothiocyanate, AITC), TRPV1 (capsaicin, CAPS), and TRPC (oleoyl-2-acetyl-sn-glycerol, OAG) agonists on guinea pig bladder activity. Effects of these agonists were compared with effects of nitro-oleic acid (OA-NO2), an electrophilic nitro-fatty acid, known to activate TRPV1, TRPA1 or TRPC channels in sensory neurons. AITC (100 µM) increased (231%) area of spontaneous bladder contractions (SBCs) an effect reduced by a TRPA1 antagonist (HC3-03001, HC3, 10 µM) and reversed to inhibition by indomethacin (INDO, 500 nM) a cyclooxygenase inhibitor. The post-INDO inhibitory effect of AITC was mimicked (39% depression) by calcitonin gene-related peptide (CGRP, 100 nM) and blocked by a CGRP antagonist (BIBN, 25 µM). CAPS (1 µM) suppressed SBCs by 30% in 81% of strips, an effect blocked by a TRPV1 antagonist (diarylpiperazine, 1 µM) or BIBN. SBCs were suppressed by OA-NO2 (30 µM, 21% in 77% of strips) or by OAG (50 µM, 30%) an effect blocked by BIBN. OA-NO2 effects were not altered by HC3 or diarylpiperazine. OA-NO2 also induced excitation in 23% of bladder strips. These observations raise the possibility that guinea pig bladder is innervated by at least two types of afferent nerves: [1] Type A express TRPA1 receptors that induce the release of prostaglandins and excite the detrusor, [2] Type B express TRPV1, TRPA1 and TRPC receptors and release CGRP that inhibits the detrusor.