Altered inducible nitric oxide synthase expression and nitric oxide production in the bladder of cats with feline interstitial cystitis.

PURPOSE: Alterations in nitric oxide (NO) levels have been demonstrated in some humans with interstitial cystitis (IC) as well as in chemically induced animal models of cystitis. Thus, in the current study we investigated whether inducible NO synthase (iNOS) mediated NO production is altered in the bladder of cats with a naturally occurring model of IC termed feline IC (FIC). MATERIALS AND METHODS: We examined iNOS expression using Western immunoblotting and baseline NO production using an NO microsensor from smooth muscle and mucosal bladder strips in 9 healthy cats and 6 diagnosed with FIC. RESULTS: There was a significant increase in baseline NO production in cats with FIC compared with that in healthy cats in smooth muscle and mucosal strips. This production was not ablated in the absence of extracellular Ca (100 microM egtazic acid) or following incubation with the calmodulin antagonist trifluoroperazine (20 microM), indicating iNOS mediated Ca independent NO production. Release was significantly decreased following incubation with the NOS antagonist L-NAME (N-nitro-L-arginine methyl ester) (100 microM). Furthermore, immunoblotting revealed a trend toward increased iNOS expression in smooth muscle and mucosal strips from FIC cats but not from healthy cats. CONCLUSIONS: In light of previous findings that the barrier property of the urothelial surface is disrupted in FIC and iNOS mediated increase in NO alters barrier function in other types of epithelium our findings suggest that iNOS dependent NO production may have a role in epithelial barrier dysfunction in FIC.

Alterations in P2X and P2Y purinergic receptor expression in urinary bladder from normal cats and cats with interstitial cystitis.

Purinergic mechanisms appear to be involved in motor as well as sensory functions in the urinary bladder. ATP released from efferent nerves excites bladder smooth muscle, whereas ATP released from urothelial cells can activate afferent nerves and urothelial cells. In the present study, we used immunohistochemical techniques to examine the distribution of purinoceptors in the urothelium, smooth muscle, and nerves of the normal cat urinary bladder as well as possible changes in the expression of these receptors in cats with a chronic painful bladder condition termed feline interstitial cystitis (FIC) in which ATP release from the urothelium is increased. In normal cats, a range of P2X (P2X(1), P2X(2), P2X(3), P2X(4), P2X(5), P2X(6), and P2X(7)) and P2Y (P2Y(1), P2Y(2), and P2Y(4)) receptor subtypes was expressed throughout the bladder urothelium. In FIC cats, there is a marked reduction in P2X(1) and loss of P2Y(2) receptor staining. Both P2X(3) and P2Y(4) are present in nerves in normal cat bladder, and no obvious differences in staining were detected in FIC. Smooth muscle in the normal bladder did not exhibit P2Y receptor staining but did exhibit P2X (P2X(2), P2X(1)) staining. In the FIC bladder smooth muscle, there was a significant reduction in P2X(1) expression. These findings raise the possibility that purinergic mechanisms in the urothelium and bladder smooth muscle are altered in FIC cats. Because the urothelial cells appear to have a sensory function in the bladder, it is possible that the plasticity in urothelial purinergic receptors is linked with the painful bladder symptoms in IC.

Feline interstitial cystitis results in mechanical hypersensitivity and altered ATP release from bladder urothelium.

ATP can be released from a variety of cell types by mechanical stimulation; however, the mechanism for this release and the influence of pathology are not well understood. The present study examined intracellular signaling mechanisms involved in swelling-evoked (exposure to a hypotonic solution) release of ATP in urothelial cells from normal cats and cats diagnosed with interstitial cystitis (feline interstitial cystitis; FIC). Using the luciferin-luciferase bioluminescent assay, we demonstrate that swelling-evoked ATP release is significantly elevated in FIC cells. In both normal and FIC cells, ATP release was significantly decreased (mean 70% decrease) by application of blockers of stretch-activated channels (amiloride or gadolinium), as well as brefeldin A and monensin (mean 90% decrease), suggesting that ATP release occurs when ATP-containing vesicles fuse with the plasma membrane. Swelling-evoked release was reduced after removal of external calcium (65%), and release was blocked by incubation with BAPTA-AM or agents that interfere with internal calcium stores (caffeine, ryanodine, heparin, or 2-aminoethoxydiphenyl borate). In addition, agents known to act through inositol 1,4,5-triphosphate (IP3) receptors (thapsigargin, acetylcholine) release significantly more ATP in FIC compared with normal urothelium. Taken together, these results suggest that FIC results in a novel hypersensitivity to mechanical stimuli that may involve alterations in IP3-sensitive pathways.

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, 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.

Colon and anal sphincter contractions evoked by microstimulation of the sacral spinal cord in cats.

The colon and anal sphincter contractions induced by microstimulation of the S2 spinal cord were investigated by measuring the intraluminal pressure change via saline filled balloons in alpha-chlorolose anesthetized cats. Stimulation of sacral ventral roots (S1-S3) revealed that the S2 efferent outflow usually produces the largest colon response. Stimulation of the S2 ventral root or the spinal cord both indicated that 15 Hz stimulation was the optimal frequency for evoking colon contractions. Colon and anal sphincter contractions were also influenced by stimulation intensity and pulsewidth. Locations in S2 spinal cord, where microstimulation evoked a distal/proximal colon pressure response that was greater than the anal sphincter response, included the area of sacral parasympathetic nucleus (SPN), the area medial to the SPN extending to the dorsal commissure, and areas deep in the ventral horn. Anal sphincter relaxation was evoked by microstimulation in more restricted locations in S2 spinal cord, which appeared to overlap with those evoking anal sphincter contractions. These results suggest a possible method to evoke colon contraction and defecation by microstimulation of the S2 spinal cord with multiple microelectrodes.

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.

Increased c-fos expression in spinal lumbosacral projection neurons and preganglionic neurons after irritation of the lower urinary tract in the rat.

Chemical irritation of the lower urinary tract (LUT) induces c-fos expression in neurons in the lumbosacral (L(6) and S(1)) spinal cord. This study used axonal tracing with fluorescent dyes to identify the types of spinal neurons expressing Fos immunoreactivity (IR) after LUT irritation in the rat. Fos-IR was detected in lateral and medial superficial dorsal horn, the sacral parasympathetic nucleus (SPN) and lamina X around the central canal. Fos-IR was detected in spinal neurons projecting to supraspinal sites (brainstem and hypothalamus), in preganglionic neurons (PGN) and in unlabeled segmental interneurons. A substantial percentage (20%) of dye labeled PGN exhibited Fos-IR after LUT irritation; and a larger percentage (36%) exhibited Fos-IR after electrical stimulation of the pelvic nerve which contains afferent pathways from all of the pelvic organs. The majority (average 55%) of Fos-positive neurons projecting to supraspinal sites were also located in the region of the SPN. A selective distribution of different types of neurons was detected in this region: PGN were located ventral to the spinal projection neurons which in turn were located ventral to the majority of unidentified Fos-positive neurons. The distribution of Fos-positive PGN and projection neurons was similar in spinal intact and spinal transected animals indicating that c-fos expression was mediated by monosynaptic afferent input or input from segmental interneurons and was not due to activation of supraspinal micturition reflex pathways.

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.

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.

Developmental and injury induced plasticity in the micturition reflex pathway.

The storage and periodic elimination of urine are dependent upon neural circuits in the brain and spinal cord that co-ordinate the activity of the urinary bladder, the urethra and the striated urethral sphincter. This study utilized anatomical, electrophysiological and pharmacological techniques to examine: (1) the organization of the parasympathetic excitatory reflex mechanisms that control the urinary bladder of the rat and the cat; and (2) the changes in these reflexes during postnatal development and after spinal cord injury. In normal adult cats and rats, the parasympathetic excitatory input to the bladder is dependent upon a spinobulbospinal reflex pathway that is activated by myelinated (Adelta) bladder afferents and that passes through an integrative center (the pontine micturition center, PMC) in the rostral brain stem. Transneuronal tracing studies using pseudorabies virus as well as physiological methods have revealed that the PMC is located in close proximity to the locus coeruleus. Single unit recordings indicate that neurons in the PMC respond to afferent input from the bladder and are excited prior to or during reflex bladder contractions. Glutamic acid is the major excitatory transmitter in the micturition reflex pathway. Glutamatergic transmission which is mediated by AMPA/kainate and NMDA receptors can be modulated by a variety of other transmitters. In neonatal animals, a spinal micturition reflex is activated by somatic afferent fibers from the perigenital region. This reflex is suppressed during postnatal development, but can be unmasked in adult animals following spinal cord injury. Spinal injury also causes the emergence of a spinal bladder-to-bladder reflex which in the cat is activated by capsaicin-sensitive C-fiber bladder afferents. Patch clamp studies in spinal cord slice preparations indicate that developmental and spinal cord injury induced plasticity in sacral parasympathetic reflex pathways is due in part to alterations in glutamatergic excitatory transmission between interneurons and preganglionic neurons. Changes in the electrical properties of bladder afferent pathways may also contribute to the reorganization of bladder reflexes in paraplegic animals.

Role of spinal glutamatergic transmission in the ascending limb of the micturition reflex pathway in the rat.

This study was undertaken to evaluate the role of glutamate receptors at spinal synapses on the ascending limb of the micturition reflex. In urethane-anesthetized female rats, a tungsten electrode was inserted stereotaxically into the dorsal part of the rostral pons to record field potentials which were evoked by electrical stimulation of the pelvic nerve (PLN) (1-15 V, 0.05 ms pulse duration at 100-300 Hz, 5-30 ms train duration). The effects of glutamate receptor antagonists administered intrathecally (i.t.) on the PLN-evoked field potentials in the dorsal part of the rostral brainstem were examined. PLN stimulation evoked short latency (10-22 ms) negative field potentials (85 +/- 4 microV) in a limited area of the dorsal part of the rostral pons (bregma -9.0 to -8.4, L 0.5 to 1. 5, H 4.2 to 5.4). The i.t. administration of LY215490 (0.1-30 microg), a competitive alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist, reduced the amplitude of the evoked potentials in a dose-dependent manner; 84 +/- 6%, 59 +/- 11% (P < .001), 31 +/- 10% (P < .001), 17 +/- 9% (P < .001) of control after 0.1, 1, 10, 30 microg of LY215490, respectively. The i.t. administration of MK-801 (1-100 microg), a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, also reduced the amplitude of the evoked potentials in a dose-dependent manner; 93 +/- 21%, 76 +/- 14%, 52 +/- 9% (P < .001), 39 +/- 9% (P < .001) of control after 1, 10, 30, 100 microg of MK-801, respectively. Combined administration of LY215490 (0.1 microg) and MK-801 (1 microg), in doses which individually did not elicit a significant effect, markedly reduced the amplitude of the evoked potentials (27 +/- 9% of control, P = . 0002). These results suggest that AMPA and NMDA glutamatergic synaptic mechanisms play a key role in the spinal processing of afferent input from the bladder and that these mechanisms function synergistically in the ascending limb of the spinobulbospinal micturition reflex pathway.

Effects of LY215490, a competitive alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist, on the micturition reflex in the rat.

The effects of glutamate receptor antagonists on urinary bladder and external urethral sphincter- (EUS) electromyogram (EMG) activity were evaluated in unanesthetized decerebrate rats. In normal rats, LY215490, an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist, in small i.v. doses (1-3 mg/kg) decreased bladder contraction amplitude (BC-Amp) by 29% and EUS-EMG by 41%; whereas a large dose (10 mg/kg) completely abolished bladder and EUS-EMG activity. LY215490 injected intrathecally in small doses (0.01-0.1 microg) decreased BC-Amp by 20% and EUS-EMG by 62%; whereas large doses (1-10 microg) completely abolished bladder and EUS-EMG activity. LY215490 (0.1 microg i.t.) increased bladder capacity by 28% and decreased voiding efficiency by 44%. Combined i.t. administration of small doses of LY215490 (0.1 microg) and MK-801 (1 microg), an N-methyl-D-aspartate (NMDA) receptor antagonist, which individually had little effect on BC-Amp, markedly suppressed bladder activity. In chronic spinal rats, LY215490 (10 mg/kg i.v.) abolished EUS-EMG activity and decreased BC-Amp by 41%. Intrathecal injections of LY215490 were also less effective in chronic spinal rats; a 10-microg dose producing only a partial block (53%) of BC-Amp, but complete block of EUS-EMG. In chronic spinal rats, MK-801 (1 mg/kg i.v.) abolished EUS-EMG activity and decreased BC-Amp by 36%. Pretreatment with MK-801 (1 mg/kg i.v.) did not enhance the effect of LY215490 on bladder activity in chronic spinal rats. These data suggest that AMPA glutamate receptors have a major role in the excitatory pathways controlling bladder and EUS activity in spinal cord intact rats. However, in chronic spinal rats, AMPA and NMDA receptors are essential for EUS reflexes, but are responsible for only a part of reflex bladder activity.

The central neural pathways involved in micturition in the neonatal rat as revealed by the injection of pseudorabies virus into the urinary bladder.

Pseudorabies virus was injected into the wall of the urinary bladder in 2- and 12-day-old rats in order to examine developmental changes in the central neural pathways controlling micturition. Forty-eight hours after virus injection, virus-labeled neurons were identified in the lumbosacral spinal cord but not in the brain. Sixty to seventy-two hours after virus injection in both 2- and 12-day-old rats, infected neurons were detected in Barrington's nucleus, nucleus paragigantocellularis, nucleus reticularis gigantocellularis, A5 area, nucleus raphe obscurus, locus subcoeruleus, periaquaductal gray, red nucleus, paraventricular nucleus and cerebral cortex. These results in neonatal rats are similar to those reported in adult rats. Thus, it is likely that the supraspinal neural circuitry which underlies micturition in the adult animal is already organized in the neonatal rat during the early postnatal period even though the supraspinal micturition reflex pathway does not become functional until the third postnatal week.

Interactions between NMDA and AMPA/kainate receptors in the control of micturition in the rat.

In unanesthetized decerebrate rats, GYKI 52466 (1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride), an AMPA/kainate receptor antagonist, and MK-801 (dizocilpine), an NMDA receptor antagonist, acted synergistically to depress the micturition reflex. MK-801 (1 mg/kg i.v.) and GYKI 52466 (4 mg/kg i.v.) administered separately had no or only a small depressant effect on reflex bladder contractions but markedly depressed external urethral sphincter activity. However, in MK-801-treated rats, GYKI 52466 decreased the amplitude, frequency and duration of reflex bladder contractions. These results suggest that both AMPA/kainate and NMDA glutamate receptors are important in the micturition reflex pathway and that these receptors may be activated in parallel at some site in the pathway so that excitatory transmission via only one receptor type is sufficient to mediate reflex activation of the bladder.

Effects of GYKI 52466 and CNQX, AMPA/kainate receptor antagonists, on the micturition reflex in the rat.

GYKI 52466, a non-competitive AMPA/kainate glutamate receptor antagonist, administered in graded doses (0.5-8 mg/kg, i.v.) at 10 min intervals, decreased the amplitude and duration of reflex bladder contractions (maximal inhibition = 63%), the intercontraction interval (maximal inhibition = 83%) and external urethral sphincter activity (maximal inhibition = 91%) in urethane-anesthetized (1.2 g/kg, s.c.) intact rats during continuous transurethral cystometrograms. On the other hand, in unanesthetized decerebrate rats, the drug did not alter reflex bladder activity but did produce a significant depression of sphincter activity (maximal inhibition = 59%). The depressant effects of single doses of GYKI 52466 (4 mg/kg, i.v.) on external urethral sphincter EMG activity occurred with similar time courses in both urethane-anesthetized (1.2 g/kg, s.c.) intact and unanesthetized decerebrate rats during continuous transurethral cystometrograms. In urethane-treated (0.6 g/kg, i.p.) decerebrate rats, GYKI 52466 (0.5-4 mg/kg, i.v.) depressed bladder contraction amplitude and sphincter EMG activity, similar to the effects in urethane-anesthetized (1.2 g/kg, s.c.) intact rats. CNQX (0.01-1 mg/kg, i.v.), a competitive AMPA/kainate receptor antagonist, administered to urethane-anesthetized (1.2 g/kg, s.c.) intact or unanesthetized decerebrate rats did not alter the bladder or the external urethral sphincter activity during continuous transurethral cystometrograms, possibly due to the inability of the drug to readily cross the blood-brain barrier. The present results indicate that glutamatergic excitatory transmission mediated by AMPA/kainate receptors is essential for the activation of external urethral sphincter activity during micturition in anesthetized and unanesthetized preparation. However, the depressant effect of GYKI 52466 on reflex bladder activity is only unmasked by urethane anesthesia, raising the possibility that urethane interacts with AMPA/kainate glutamate receptors in the spinobulbospinal micturition reflex pathway.

Neural control of urethral outlet activity in vivo: role of nitric oxide.

The present study investigated the role of nitric oxide (NO) in the reflex changes in urethral outlet activity during micturition. Isovolumetric bladder contractions, urethral pressure and external urethral sphincter electromyogram (EUS EMG) activity were recorded independently in urethane-anesthetized rats. During reflex bladder contractions, the urethra exhibited reflex responses characterized by an initial decrease in urethral pressure in conjunction with a rise in bladder pressure. This was followed by a period of high frequency oscillations (HFOs) associated with maximal urethral relaxation and burst type EUS EMG activity. Administration of N-nitro-L-arginine (L-NOARG) 10 mg./kg. intravenously, a nitric oxide synthase inhibitor, reversibly decreased the magnitude (62%, p < 0.05) and duration (40%, p < 0.05) of reflex urethral relaxation (N = 7). In 4 additional experiments, L-NOARG (10 to 15 mg./kg. intravenously) completely eliminated reflex urethral relaxation during micturition, and this effect was reversed in all animals by the administration of L-arginine (100 to 150 mg./kg. intravenously). Administration of N-nitro-D-arginine (D-NOARG) (10 to 30 mg./kg. intravenously) had no effect on reflex urethral relaxation. Neuromuscular blockade (vecuronium bromide 5 mg./kg. intravenously) reversibly decreased resting urethral pressure and eliminated the HFOs. The urethral smooth muscle relaxation that remained after neuromuscular blockade was eliminated following administration of L-NOARG (10 mg./kg. intravenously) in 2 of 3 animals. These results suggest that reflex urethral responses during micturition involve changes in both smooth and striated muscle activity, and that the predominant neurotransmitter mechanisms that mediate reflex urethral smooth muscle relaxation involve NO.

Transneuronal labeling of neurons in the adult rat brainstem and spinal cord after injection of pseudorabies virus into the urethra.

Transneuronal tracing techniques were used to identify sites in the central nervous system involved in the neural control of urethral function. The distribution of virus-infected neurons was examined in the spinal cord and brainstem at various intervals (56-96 hours) following pseudorabies virus (PRV) injection into the urethra. In the lumbosacral (L6-S1) spinal cord at 56 hours, neurons containing PRV immunoreactivity (PRV-IR) were located in the region of the sacral parasympathetic nucleus (SPN), around the central canal, and in the dorsal commissure. Some animals also exhibited PRV-IR in cells in the L6 dorsolateral motor nucleus. At longer survival times (72-96 hours), PRV-IR cells were observed in the superficial and deeper laminae of the dorsal horn, and increased numbers of PRV-IR cells were consistently detected in the region of the SPN, around the central canal, and in the dorsal commissure. PRV-IR fiber-like staining also occurred along the lateral edge of the dorsal horn extending from Lissauer's tract to the region of the SPN. In rostral lumbar segments (L1-L2), PRV-IR cells were located in the region of the dorsal commissure and the intermediolateral cell nucleus (IML), around the central canal, and in the dorsal horn. After 72-84 hours, PRV-IR cells were also noted at more rostral levels of the neuraxis including the medulla, pons, midbrain, and diencephalon. At 72 hours, PRV-IR cells were consistently observed in Barrington's nucleus (pontine micturition center), nucleus raphe magnus (RMg), parapyramidal reticular formation, and the A5 and A7 regions. At 78-84 hours, additional regions exhibited PRV-IR cells, including the periaqueductal gray, locus coeruleus, the dorsal and ventral subcoeruleus alpha, and the red nucleus. A few cells were also located in the lateral hypothalamic area. This distribution of PRV-labeled cells in the spinal cord and brainstem is similar in many respects to the distribution of cells labeled in previous studies by PRV injection into the urinary bladder. This overlap of urethra and bladder neurons is consistent with the results of physiological experiments indicating a close coordination between the central nervous control of bladder and urethral activity.

Alteration by urethane of glutamatergic control of micturition.

The i.v. administration of MK-801 (0.001-3 mg/kg), a non-competitive NMDA receptor antagonist, did not alter reflex bladder activity in unanesthetized decerebrate rat recorded during fast infusion (0.21 ml/min) cystometry or under isovolumetric conditions, but did depress reflex bladder contractions in doses between 0.1 and 3 mg/kg i.v. in the urethane-anesthetized (1.2 g/kg s.c.) intact rat during fast infusion cystometry. The ED50 and the dose to produce maximal inhibition in urethane-anesthetized intact rats were 0.25 mg/kg and 3 mg/kg i.v., respectively. During slow infusion (0.04 ml/min) cystometry, in unanesthetized decerebrate rats, MK-801 (0.1-1 mg/kg i.v. or 6-60 micrograms i.t.) decreased by 12-44% the micturition volume threshold (VT) but did not change the amplitude and duration of the bladder contractions. The administration of a larger i.t. dose (60 micrograms) of MK-801 produced no further decrease in VT but decreased the amplitude of bladder contractions by 24%. External urethral sphincter electromyogram activity was reduced or abolished by MK-801 (0.01-3 mg/kg i.v.) in both unanesthetized decerebrate and urethane-anesthetized intact rats with ED50 of 0.12 mg/kg and 0.05 mg/kg, respectively. These results indicate that NMDA receptors play an important role in both facilitatory and inhibitory central neural control of voiding function and that there is a significant interaction between urethane anesthesia and NMDA glutamatergic transmission. Thus, even though urethane anesthesia has been useful for studying the physiological characteristics of the micturition reflex, it seems inappropriate for analyzing the normal transmitter role of glutamic acid in reflex voiding.

Differential localization of neuronal nitric oxide synthase immunoreactivity and NADPH-diaphorase activity in the cat spinal cord.

The distributions of neuronal nitric oxide synthase immunoreactivity (NOS-IR) and NADPH-diaphorase (NADPH-d) activity were compared in the cat spinal cord. NOS-IR in neurons around the central canal, in superficial laminae (I and II) of the dorsal horn, in the dorsal commissure, and in fibers in the superficial dorsal horn was observed at all levels of the spinal cord. In these regions, NOS-IR paralleled NADPH-d activity. The sympathetic autonomic nucleus in the rostral lumbar and thoracic segments exhibited prominent NOS-IR and NADPH-d activity, whereas the parasympathetic nucleus in the sacral segments did not exhibit NOS-IR or NADPH-d activity. Within the region of the sympathetic autonomic nucleus, fewer NOS-IR cells were identified compared with NADPH-d cells. The most prominent NADPH-d activity in the sacral segments occurred in fibers within and extending from Lissauer's tract in laminae I and V along the lateral edge of the dorsal horn to the region of the sacral parasympathetic nucleus. These afferent projections did not exhibit NOS-IR; however, NOS-IR and NADPH-d activity were demonstrated in dorsal root ganglion cells (L7-S2). The results of this study demonstrate that NADPH-d activity is not always a specific histochemical marker for NO-containing neural structures.

Plasticity in spinal opioid control of lower urinary tract function in paraplegic cats.

Spinal cord injury disrupts micturition reflexes, which produces morbidity. The contribution of endogenous opioid systems to urinary retention were assessed in chronic spinal cats by administering the opioid receptor antagonist, naloxone (5-500 micrograms kg-1, i.p.), to unanesthetized paraplegic cats while monitoring lower urinary tract function and observing hind limb reflexes. While naloxone had no overt effect in acute spinal cats, in chronic spinal cats naloxone induced the release of large volumes of urine and produced marked hind limb hyper-reflexia. Prominent tachyphylaxis and tolerance to the effects of naloxone were evident. Immunohistochemical studies indicated a marked increase in leucine enkephalin and dynorphin in sacral spinal neurons. Together, these data indicate hyperactivity of the endogenous spinal opioid system following recovery from spinal cord injury and, furthermore, suggest that the spinal neural circuitry may become 'dependent' upon elevated levels of endogenous opioid peptides.