J.R. Roppolo

Neural Control of the Urethra

Coordination between the urinary bladder and the urethra is mediated by multiple reflex pathways organized in the brain and spinal cord. Some reflexes promote urine storage; whereas other reflexes facilitate voiding. During bladder filling, activation of mechanoreceptor afferent nerves in the bladder wall triggers firing in the cholinergic efferent pathways to the external urethral sphincter (EUS) and in sympathetic adrenergic pathways to the urethral smooth muscle. These storage reflexes are dependent upon interneuronal circuitry in the spinal cord. During voiding the spinal storage reflexes are inhibited by supraspinal mechanisms which originate in the pontine micturition center. Glutamatergic, serotonergic and alpha 1 adrenergic excitatory transmission as well as GABAergic/glycinergic inhibitory transmission have been implicated in the central control of sphincter reflexes. During voiding, a parasympathetic nitrergic inhibitory input to the urethral smooth is activated. This reflex mechanism which is triggered by bladder afferents persists in paraplegic rats and therefore must be mediated at least in part by spinal interneuronal circuitry. In female rats, the parasympathetic nitrergic pathway is prominent; but in male rats it is obscured by a dominant parasympathetic cholinergic excitatory input to the urethral smooth muscle. The function of the cholinergic pathway in voiding is uncertain. Stimulation of urethral afferents can also influence bladder activity. Contraction of the external urethral sphincter activates afferents that inhibit reflex bladder contractions; whereas infusion of fluid through the urethra facilitates bladder contractions. These reflexes are also organized in the spinal cord and presumably play a role in urine storage and elimination. Alterations in primitive bladder-to-urethra and urethra-to-bladder reflex mechanisms may contribute to neurogenic bladder dysfunction.Coordination between the urinary bladder and the urethra is mediated by multiple reflex pathways organized in the brain and spinal cord. Some reflexes promote urine storage; whereas other reflexes facilitate voiding. During bladder filling, activation of mechanoreceptor afferent nerves in the bladder wall triggers firing in the cholinergic efferent pathways to the external urethral sphincter (EUS) and in sympathetic adrenergic pathways to the urethral smooth muscle. These storage reflexes are dependent upon interneuronal circuitry in the spinal cord. During voiding the spinal storage reflexes are inhibited by supraspinal mechanisms which originate in the pontine micturition center. Glutamatergic, serotonergic and alpha, adrenergic excitatory transmission as well as GABAergic/glycinergic inhibitory transmission have been implicated in the central control of sphincter reflexes. During voiding, a parasympathetic nitrergic inhibitory input to the urethral smooth is activated. This reflex mechanism which is triggered by bladder afferents persists in paraplegic rats and therefore must be mediated at least in part by spinal interneuronal circuitry. In female rats, the parasympathetic nitrergic pathway is prominent; but in male rats it is obscured by a dominant parasympathetic cholinergic excitatory input to the urethral smooth muscle. The function of the cholinergic pathway in voiding is uncertain. Stimulation of urethral afferents can also influence bladder activity. Contraction of the external urethral sphincter activates afferents that inhibit reflex bladder contractions; whereas infusion of fluid through the urethra facilitates bladder contractions. These reflexes are also organized in the spinal cord and presumably play a role in urine storage and elimination. Alterations in primitive bladder-to-urethra and urethra-to-bladder reflex mechanisms may contribute to neurogenic bladder dysfunction.

Simulation analysis of conduction block in unmyelinated axons induced by high-frequency biphasic electrical currents

Nerve conduction block induced by high-frequency biphasic electrical currents is analyzed using a lumped circuit model of the unmyelinated axon based on Hodgkin-Huxley equations. Axons of different diameters (5-20 /spl mu/m) can not be blocked completely when the stimulation frequency is between 2 kHz and 4 kHz. However, when the stimulation frequency is above 4 kHz, all axons can be blocked. At high-frequency a higher stimulation intensity is needed to block nerve conduction. The larger diameter axon has a lower threshold intensity for conduction block. The stimulation waveform in which the pulsewidth changes with frequency is more effective in blocking nerve conduction than the waveform in which the pulsewidth is fixed. The activation of potassium channels, rather than inactivation of sodium channels, is the possible mechanism underlying the nerve conduction block of the unmyelinated axon. This simulation study further increases our understanding of axonal conduction block induced by high-frequency biphasic currents, and can guide future animal experiments as well as optimize stimulation waveforms that might be used for electrical nerve block in clinical applications.

Mechanism of Nerve Conduction Block Induced by High-Frequency Biphasic Electrical Currents

The mechanisms of nerve conduction block induced by high-frequency biphasic electrical currents were investigated using a lumped circuit model of the myelinated axon based on Frankenhaeuser-Huxley (FH) model or Chiu-Ritchie-Rogart-Stagg-Sweeney (CRRSS) model. The FH model revealed that the constant activation of potassium channels at the node under the block electrode, rather than inactivation of sodium channels, is the likely mechanism underlying conduction block of myelinated axons induced by high-frequency biphasic stimulation. However, the CRRSS model revealed a different blocking mechanism where the complete inactivation of sodium channels at the nodes next to the block electrode caused the nerve conduction block. The stimulation frequencies to observe conduction block in FH model agree with the observations from animal experiments (greater than 6 kHz), but much higher frequencies are required in CRRSS model (greater than 15 kHz). This frequency difference indicated that the constant activation of potassium channels might be the underlying mechanism of conduction block observed in animal experiments. Using the FH model, this study also showed that the axons could recover from conduction block within 1 ms after termination of the blocking stimulation, which also agrees very well with the animal experiments where nerve block could be reversed immediately once the blocking stimulation was removed. This simulation study, which revealed two possible mechanisms of nerve conduction block in myelinated axons induced by high-frequency biphasic stimulation, can guide future animal experiments as well as optimize stimulation waveforms for electrical nerve block in clinical applications

Simulation analysis of conduction block in myelinated axons induced by high-frequency biphasic rectangular pulses

Nerve conduction block induced by high-frequency biphasic rectangular pulses was analyzed using a lumped circuit model of the myelinated axon based on Frankenhaeuser-Huxley (FH) equations. At the temperature of 37 /spl deg/C, axons of different diameters (2-20 /spl mu/m) can be blocked completely at supra-threshold intensities when the stimulation frequency is above 10 kHz. However, at stimulation frequencies between 6 kHz and 9 kHz, both nerve block and repetitive firing of action potentials can be observed at different stimulation intensities. When the stimulation frequency is below 6 kHz, nerve block does not occur regardless of stimulation intensity. Larger diameter axons have a lower threshold intensity to induce conduction block. When temperature is reduced from 37 /spl deg/C to 20 /spl deg/C, the lowest frequency to completely block large axons (diameters 10-20 /spl mu/m) decreased from 8 kHz to 4 kHz. This simulation study can guide future animal experiments as well as optimize stimulation waveforms for electrical nerve block in clinical applications.

Localization of P2X and P2Y Receptors in Dorsal Root Ganglia of the Cat

The distribution of P2X and P2Y receptor subtypes in upper lumbosacral cat dorsal root ganglia (DRG) has been investigated using immunohistochemistry. Intensity of immunoreactivity for six P2X receptors (P2X5 receptors were immuno-negative) and the three P2Y receptors examined in cat DRG was in the order of P2Y2 = P2Y4>P2X3>P2X2 = P2X7>P2X6>P2X1 = P2X4>P2Y1. P2X3, P2Y2, and P2Y4 receptor polyclonal antibodies stained 33.8%, 35.3%, and 47.6% of DRG neurons, respectively. Most P2Y2, P2X1, P2X3, P2X4, and P2X6 receptor staining was detected in small- and medium-diameter neurons. However, P2Y4, P2X2, and P2X7 staining was present in large- and small-diameter neurons. Double-labeling immunohistochemistry showed that 90.8%, 32.1%, and 2.4% of P2X3 receptor-positive neurons coexpressed IB4, CGRP, and NF200, respectively; whereas 67.4%, 41.3%, and 39.1% of P2Y4 receptor-positive neurons coexpressed IB4, CGRP, and NF200, respectively. A total of 18.8%, 16.6%, and 63.5% of P2Y2 receptor-positive neurons also stained for IB4, CGRP, and NF200, respectively. Only 30% of DRG neurons in cat were P2X3-immunoreactive compared with 90% in rat and in mouse. A further difference was the low expression of P2Y1 receptors in cat DRG neurons compared with more than 80% of the neurons in rat. Many small-diameter neurons were NF200-positive in cat, again differing from rat and mouse.

Penile erection produced by microstimulation of the sacral spinal cord of the cat

The sacral neural pathways mediating penile erection in the cat were studied by measuring the change in cavernous sinus pressure (CSP) elicited by stimulation of the sacral ventral roots or by microstimulation of the sacral spinal cord. Ventral root stimulation revealed that the S1 segment rather than S2 and S3 spinal segments could evoke the largest CSP responses. Microstimulation in the S1 spinal cord elicited large CSP responses but small or no bladder contractions. Maximal CSP responses were evoked by microstimulation in the middle of the S1 ventral horn, 1.6-2.8 mm below the cord surface and midway between the midline and the lateral edge of the gray matter. The area was 200-400 microm wide (medial to lateral) and extended 1-2 mm in the rostrocaudal direction. Maximal CSP responses to spinal cord microstimulation were elicited by stimulus intensities of 50-150 microA, at a pulse width of 0.2 ms and at frequencies of 3040 Hz and occurred after delay of 8-40 s. This study suggests that focal microstimulation of the sacral spinal cord might be useful in eliciting penile erectile activity in patients with spinal cord injury.

Isometric torque about the knee joint generated by microstimulation of the cat L6 spinal cord

Isometric torque was generated about the knee joint by microstimulation of the cat L6 spinal cord using a single microelectrode. The torque responses varied with microstimulation location. Appreciable extension torque was generated by microstimulation in ventrolateral locations of the L6 spinal cord. Stimulation parameters (intensity, frequency and pulse-width) also influenced the extension torque. Specific stimulation parameters (100 microA intensity, 40 Hz frequency and 0.20 ms pulse-width) appear best suited for mapping the spinal cord based on knee joint torque responses. Low levels of cocontraction of the extensor and flexor could be achieved when extension torque was produced, but also varied with the stimulation locations. There are locations in the L6 ventral horn where microstimulation could evoke sustained extension for at least 4 min with only a slight change in torque. This study suggests the possibility of restoring lower limb function in patients with spinal cord injury above the lumbar level.

Multimicroelectrode stimulation within the cat L6 spinal cord: influences of electrode combinations and stimulus interleave time on knee joint extension torque

During multimicroelectrode stimulation within the cat L6 spinal cord, the number of electrodes activated, their separation distance, and the stimulus interleave time all influenced isometric knee joint extension torque. The torque evoked by stimulation with a three electrode combination could be enhanced or suppressed when compared with that evoked by single or paired electrode stimulation. A similar difference was noted when comparing two electrode combination versus single electrode stimulation. Relative fatigue was not improved significantly by interleaving the stimuli from two or three microelectrodes. Compared with the extension torque response evoked by noninterleaved stimulation, torque evoked by interleaved stimulation with the two microelectrode combination was decreased when the electrode distance was 2.0 mm or less and increased when the electrode distance was 3.0 mm. Designing an optimal stimulation strategy for multimicroelectrode spinal cord stimulation will be challenging and complex if a suppression effect among these electrodes is to be avoided. To reduce muscle fatigue, an asynchronous, interleaved strategy of stimulation may be required.

Corticotropin-releasing factor family peptide signaling in feline bladder urothelial cells

Corticotropin-releasing factor (CRF) plays a central role in the orchestration of behavioral and neuroendocrine responses to stress. The family of CRF-related peptides (CRF and paralogs: urocortin (Ucn)-I, -II, and -III) and associated receptors (CRFR1 and CRFR2) are also expressed in peripheral tissues such as the skin and gastrointestinal tract. Local signaling may exert multiple effects of stress-induced exacerbation of many complex syndromes, including psoriasis and visceral hypersensitivity. Interstitial cystitis/painful bladder syndrome (IC/PBS), a chronic visceral pain syndrome characterized by urinary frequency, urgency, and pelvic pain, is reported to be exacerbated by stress. Functional changes in the epithelial lining of the bladder, a vital blood-urine barrier called the urothelium, may play a role in IC/PBS. This study investigated the expression and functional activity of CRF-related peptides in the urothelium of normal cats and cats with feline interstitial cystitis (FIC), a chronic idiopathic cystitis exhibiting similarities to humans diagnosed with IC/PBS. Western blots analysis showed urothelial (UT) expression of CRFR1 and CRFR2. Enzyme immunoassay revealed release of endogenous ligands (CRF and Ucn) by UT cells in culture. Evidence of functional activation of CRFR1 and CRFR2 by receptor-selective agonists (CRF and UCN3 respectively) was shown by i) the measurement of ATP release using the luciferin-luciferase assay and ii) the use of membrane-impermeant fluorescent dyes (FM dyes) for fluorescence microscopy to assess membrane exocytotic responses in real time. Our findings show evidence of CRF-related peptide signaling in the urothelium. Differences in functional responses between FIC and normal UT indicate that this system is altered in IC/PBS.

Modulation of Axonal Excitability by High-Frequency Biphasic Electrical Current

The modulation of axonal excitability by high-frequency biphasic (HFB) electrical current was analyzed using a lumped-circuit model of the myelinated axon based on Schwarz-Reid-Bostock (SRB) equations. The results show that axonal excitability could be either increased or decreased by HFB current depending on the current intensity. The increase of axonal excitability is due to the high level of sodium channel activation, whereas the activation of both fast and slow potassium channels plays an important role in decreasing axonal excitability. As the HFB current intensity increases, the location determining the axonal excitability changes from the nodes under the electrode within the main lobe region of the activating function to the nodes away from the electrode in the side lobe region of the activating function. This simulation study also shows that the modulation of axonal excitability by HFB electrical current could be potentially useful to selectively activate the small nerve fibers in a compound nerve trunk without activating the large fibers. Understanding how HFB electrical current modulates the axonal excitability will further elucidate the possible mechanisms underlying the nerve conduction block induced by HFB electrical current.