Effects of N-methyl-D-aspartate (dizocilpine) and alpha-amino-3-hydroxy-4-isoxazolepropionate (LY215490) receptor antagonists on the voiding reflex induced by perineal stimulation in the neonatal rat.

The present study was undertaken to examine the role of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and N-methyl-D-aspartate glutamate receptors in the regulation of voiding reflexes induced by perineal stimulation in the neonatal rat. Four-, six- and 10-day-old awake rats were used in the experiments and perineal stimulation was applied using the tip of a 1-ml syringe to evoke voiding. Voided volume and residual volume were measured. In 10-day-old rats, LY215490 (3-10 mg/kg, i.p.), a competitive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor antagonist, significantly inhibited reflex voiding, increasing the residual volume 34-53-fold. A 3 mg/kg dose decreased the urine release by 55%, whereas 10 mg/kg totally suppressed the voiding reflex induced by the perineal stimulation. LY215490 (10 mg/kg, i.p.) produced similar effects in four- and six-day-old rats. Dizocilpine (1-3 mg/kg, i.p.), a non-competitive N-methyl-D-aspartate receptor antagonist, also significantly decreased the urine release (62-82%) and increased residual volume (180-230-fold). Combined administration of LY215490 (1 mg/kg, i.p.) and dizocilpine (0.3 mg/kg, i.p.) to 10-day-old rats, in doses that individually had no effect on perineal stimulation-evoked voiding, depressed voided volume by 65%. These results indicate that, in neonatal rats, glutamatergic transmission in the spinal cord has an essential role in reflex micturition induced by perineal stimulation, and that facilitatory interactions between alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and N-methyl-D-aspartate glutamatergic mechanisms are important for voiding, as noted previously in adult rats.

Effects of spinal cord injury on neurofilament immunoreactivity and capsaicin sensitivity in rat dorsal root ganglion neurons innervating the urinary bladder.

The effect of chronic spinal cord transection on neurofilament immunoreactivity and capsaicin sensitivity of L6 and S1 dorsal root ganglion neurons innervating the urinary bladder was examined using an antibody (RT97) against 200,000 mol. wt subunit of neurofilament protein and a cobalt uptake assay, respectively. Bladder afferent neurons labelled by axonal transport of a fluorescent dye (Fast Blue) injected into the bladder wall were identified in sections of intact dorsal root ganglia and among dissociated neurons in short-term culture. Approximately two thirds of bladder afferent neurons from spinal intact rats were neurofilament-poor (i.e. C-fibre neurons). These neurons were on average 37% smaller in cross-sectional area than neurofilament-rich neurons (A delta-fibre neurons). In spinal intact rats, 78% of neurofilament-poor dissociated bladder afferent neurons were sensitive to capsaicin, while only 6.2% of neurofilament-rich neurons were capsaicin-sensitive. Dissociated bladder afferent neurons from spinal transected animals had larger diameters (34.2 +/- 1.1 microns) than those from spinal intact animals (29.2 +/- 1.2 microns). In tissue sections from dorsal root ganglia, the mean cross-sectional area of bladder afferent neuron profiles in spinal transected animals was also larger by approximately 35% than in spinal intact animals. Immunoreactivity to neurofilament protein which occurred in 32% of bladder afferent neurons in spinal intact animals was detected in a larger percentage (56% to 62%) of neurons from spinal transected animals. Conversely, the population of capsaicin-sensitive dissociated neurons was reduced from 55% in spinal intact rats to 38% in spinal transected rats. These results indicate that spinal cord injury induces functional and morphological plasticity in C-fibre visceral afferent neurons innervating the urinary bladder.

Increased expression of neuronal nitric oxide synthase in bladder afferent pathways following chronic bladder irritation.

Immunocytochemical techniques were used to examine alterations in the expression of neuronal nitric oxide synthase (NOS) in bladder pathways following acute and chronic irritation of the urinary tract of the rat. Chemical cystitis was induced by cyclophosphamide (CYP) which is metabolized to acrolein, an irritant eliminated in the urine. Injection of CYP (n = 10, 75 mg/kg, i.p.) 2 hours prior to perfusion (acute treatment) of the animals increased Fos-immunoreactivity (IR) in neurons in the dorsal commissure, dorsal horn, and autonomic regions of spinal segments (L1-L2 and L6-S1) which receive afferent inputs from the bladder, urethra, and ureter. Fos-IR in the spinal cord was not changed in rats receiving chronic CYP treatment (n = 15, 75 mg/kg, i.p., every 3rd day for 2 weeks). In control animals and in animals treated acutely with CYP, only small numbers of NOS-IR cells (0.5-0.7 cell profiles/sections) were detected in the L6-S1 dorsal root ganglia (DRG). Chronic CYP administration significantly (P < or = .002) increased bladder weight by 60% and increased (7- to 11-fold) the numbers of NOS-immunoreactive (IR) afferent neurons in the L6-S1 DRG. A small increase (1.5-fold) also occurred in the L1 DRG, but no change was detected in the L2 and L5 DRG. Bladder afferent cells in the L6-S1 DRG labeled by Fluorogold (40 microliters) injected into the bladder wall did not exhibit NOS-IR in control animals; however, following chronic CYP administration, a significant percentage of bladder afferent neurons were NOS-IR: L6 (19.8 +/- 4.6%) and S1 (25.3 +/- 2.9%). These results indicate that neuronal gene expression in visceral sensory pathways can be upregulated by chemical irritation of afferent receptors in the urinary tract and/or that pathological changes in the urinary tract can initiate chemical signals that alter the chemical properties of visceral afferent neurons.

Increased expression of neuronal nitric oxide synthase in dorsal root ganglion neurons after systemic capsaicin administration.

Nitric oxide synthase which is constitutively expressed in some neurons can be induced in other neurons by pathological conditions. For example, sciatic nerve or pelvic nerve transection induced nitric oxide synthase expression in lumbosacral dorsal root ganglion cells. This occurred in small to medium sized dorsal root ganglion neurons suggesting that this change might be limited to, or most prominent in, C-fiber afferents. These afferents are also very sensitive to the neurotoxin, capsaicin, which can deplete neuropeptide stores in C-fibers and in high doses cause degeneration of C-fiber afferent pathways. The present study was undertaken to determine if a chemically induced injury elicited in small diameter afferent neurons by systemic administration of capsaicin can induce a change in nitric oxide synthase expression similar to that elicited by peripheral nerve injury. Following capsaicin pretreatment (three days) a significant increase in the number of nitric oxide synthase-immunoreactive cells was detected in dorsal root ganglia. The increase was most prominent (34-63 fold) in the L5-S1 dorsal root ganglia but also substantial (2-17 fold) in cervical, caudal thoracic and rostral lumbar dorsal root ganglia. These results indicate that the expression of nitric oxide synthase-immunoreactivity in afferent neurons in the dorsal root ganglia is plastic and can be upregulated in response to chemical stimulation and/or injury. It is possible that nitric oxide formed by enhanced expression of nitric oxide synthase may play a role in capsaicin-induced neurotoxicity.

Increased expression of neuronal nitric oxide synthase (NOS) in visceral neurons after nerve injury.

The distribution of nitric oxide synthase immunoreactivity (NOS-IR) and the changes in this distribution after peripheral axotomy were examined in lumbosacral afferent and preganglionic neurons (PGNs) innervating the pelvic viscera of the male rat. The visceral neurons in L6-S1 and L1-L2 dorsal root ganglia (DRG) and in the spinal cord were identified by retrograde axonal transport following injection of Fluorogold (FG) into the major pelvic ganglion (MPG). Axotomy was performed by removing the MPG on one side 2-4 weeks prior to sacrificing the animals. A differential distribution of NOS-IR was detected in DRG cells at different segmental levels of control animals. Significantly greater numbers of NOS-IR cells were present in thoracic (T8, T10, T12; 30-44 cell profiles/section) and rostral lumbar DRGs (L1-L2; 3-15 NOS-IR cell profiles/section) compared to caudal lumbosacral (L5-S1) DRGs (0.2-0.7 cell profiles/section). A significant increase in the number of NOS-IR cells was detected in the L6-S1 DRG (p < or = 0.001; 11 NOS-IR cell profiles/section) but not in the L2 or L5 DRG ipsilateral to axotomy. In these ganglia, an average of 37.0 +/- 4.0% (L6) and 20.6 +/- 2.2% (S1), respectively, of FG-labeled pelvic afferent neurons were NOS-IR compared to 1.1 +/- 0.5% (L6) and 2.5 +/- 1.4% (S1) contralateral to the axotomy. Following axotomy, a significantly greater percentage of dye-labeled pelvic visceral afferents in the L1 and L2 DRG also exhibited NOS-IR in comparison to the contralateral side. Following axotomy, NOS-IR fibers were detected along the lateral edge of the dorsal horn extending from Lissauer's tract to the region of the sacral parasympathetic nucleus (SPN) on the ipsilateral side of the L6 and S1 spinal segments. These NOS-IR fibers were not detected in adjacent spinal segments (L4, L5, or S2). Axotomy also changed the numbers of NADPH-d-positive and NOS-IR cells in the region of the SPN in the L6 spinal segment. Contralateral to the axotomy 38.3 +/- 4.0% of PGNs in the L6 spinal segment were colabeled with NOS-IR; however, ipsilateral to axotomy, a significantly greater percentage (61.0 +/- 3.0%; p < or = 0.01) of PGNs exhibited NOS-IR. Axotomy did not alter the distribution of PGNs in the S1 segment exhibiting NOS-IR. These results indicate that NOS-IR in visceral afferent and PGNs is plastic and can be upregulated by peripheral nerve injury.

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.

Ontogeny of nitric oxide synthase in the lumbosacral spinal cord of the neonatal rat.

The present experiments were performed to determine the temporal pattern of expression of nitric oxide synthase (NOS) immunoreactivity in cells and fibers in the lumbosacral spinal cord during early postnatal development and to examine the relationship between NADPH-diaphorase (NADPH-d) activity and NOS-immunoreactivity (IR). At postnatal days 0-1 and 4-5, NADPH-d and NOS-IR were detected in L6-S1 segments of the spinal cord in cells and fibers in the region of the sacral parasympathetic nucleus (SPN), dorsal commissure and around the central canal but were absent in the superficial layers of the dorsal horn. Fiber staining on the lateral edge of the dorsal horn (the lateral collateral pathway, LCP) in a region containing primary afferent projections from the pelvic viscera and in a fiber tract in the dorsolateral funiculus was also not detectable. At days 4-5 some stained cells were detected in the deeper laminae of the dorsal horn. At postnatal days 10-12 and 20-22, cells in the region of the SPN, around the central canal and in the superficial laminae of the dorsal horn exhibited NADPH-d and NOS-IR. NADPH-d and NOS-IR fiber staining in the superficial laminae of the dorsal horn and the dorsolateral funiculus was observed at postnatal days 10-12 and increased in staining intensity by postnatal days 20-22. NADPH-d fiber staining in the LCP was not prominent at postnatal days 10-12; however, prominent fiber staining at this site did occur by postnatal days 20-22 and in adult animals. In postnatal days 20-22 and in adult animals NADPH-d activity and NOS-IR had a similar distribution except in the LCP where NADPH-d stained fibers did not exhibit NOS-IR. These data indicate that NADPH-d and NOS-IR in the spinal cord exhibit marked changes during the early postnatal development. The changes in afferent projections in the LCP may be related to maturation of visceral reflex pathways including micturition.

Differential distribution of nitric oxide synthase in neural pathways to the urogenital organs (urethra, penis, urinary bladder) of the rat.

Axonal tracing techniques were used in combination with histochemical methods (NADPH-diaphorase activity and nitric oxide synthase immunoreactivity) to examine the distribution of nitric oxide synthase (NOS) in the neural pathways to the urogenital organs of the male rat. The major goal of this study was to compare the histochemical properties of the efferent and afferent neurons innervating the urethra with the properties of neurons innervating the penis and bladder. In the major pelvic ganglion (MPG) large percentages of postganglionic neurons innervating the urethra (44%) and the penis (97%) exhibited NADPH-diaphorase (NADPH-d) staining whereas only a small percentage (3.5%) of neurons innervating the bladder were N-d positive. Urethral neurons stained for N-d were on average smaller (33.3 microns diameter) than unstained neurons (54.5 microns diameter). The histochemical difference between the three types of neurons was also reflected in NOS-immunoreactivity (IR); however, the absolute percentage of neurons exhibiting NOS-IR was low: penis (21%), urethra (11%) and bladder (0%). Axonal varicosities staining for N-d or NOS-IR were noted in the MPG in close proximity to unidentified neurons and neurons innervating the urogenital organs. A considerable number of afferent neurons in the lumbosacral dorsal root ganglia (DRG) stained for N-d (64 cells/L6, 35 cells/S1 section); however, only small numbers of neurons (average 1 cell/section) exhibited NOS-IR. N-d activity was detected in a large percentage of urethral (55%) and bladder (80%) afferent neurons in the L6-S1 dorsal root ganglia (DRG) but in relatively few (12%) penile afferent neurons in the L6 ganglia. These results suggest that the contribution of nitric oxide (NO) to neurotransmission varies considerably in different urogenital organs. NO could have a significant role in postganglionic efferent pathways to the urethra and penis but very likely has no role in the efferent pathways to the bladder. Similarly, the prominence of N-d staining in some DRG neurons (e.g. urethra and bladder) but not others (penile) also raises the possibility of a varying role of NO in afferent pathways. However, in these neurons N-d staining was not paralleled by NOS-IR, which was present in only a small percentage of neurons. Thus, N-d staining may not reflect the presence of NO in afferent pathways to the pelvic viscera.

Localization of NADPH diaphorase in the lumbosacral spinal cord and dorsal root ganglia of the cat.

The distribution of NADPH-d activity in the spinal cord and dorsal root ganglia of the cat was studied to evaluate the role of nitric oxide in lumbosacral afferent and spinal autonomic pathways. At all levels of the spinal cord NADPH-d staining was present in neurons and fibers in the superficial dorsal horn and in neurons around the central canal and in the dorsal commissure. In addition, the sympathetic autonomic nucleus in the rostral lumbar segments exhibited prominent NADPH-d cellular staining whereas the parasympathetic nucleus in the sacral segments was not well stained. The most prominent NADPH-d activity in the sacral segments occurred in fibers extending from Lissauer's tract through laminae I along the lateral edge of the dorsal horn to lamina V and the region of the sacral parasympathetic nucleus. These fibers were very similar to VIP-containing and pelvic nerve afferent projections in the same region. They were prominent in the S1-S3 segments but not in adjacent segments (L6-L7 and Cx1) or in thoracolumbar and cervical segments. NADPH-d activity and VIP immunoreactivity in Lissauer's tract and the lateral dorsal horn were eliminated or greatly reduced after dorsal-ventral rhizotomy (S1-S3), indicating the fibers represent primary afferent projections. A population of small diameter afferent neurons in the L7-S2 dorsal root ganglia were intensely stained for NADPH-d. The functional significance of the NADPH-d histochemical stain remains to be determined; however, if NADPH-d is nitric oxide synthase then this would suggest that nitric oxide may function as a transmitter in thoracolumbar sympathetic preganglionic efferent pathways and in sacral parasympathetic afferent pathways in the cat.

Localization of NADPH diaphorase in bladder afferent and postganglionic efferent neurons of the rat.

NADPH diaphorase histochemistry was used in combination with axonal labelling techniques to determine if NADPH diaphorase is present in afferent and postganglionic efferent pathways to the urinary bladder of the rat. In the L6 and S1 dorsal root ganglia, 80.9 and 78.5%, respectively, of bladder afferent neurons labelled with fluorescent dyes were NADPH diaphorase positive. In the major pelvic ganglion (MPG), many non-labelled neurons and fibers were intensely stained for NADPH diaphorase. Intensely stained cells were clustered near the exit of the penile nerve although stained cells were also scattered throughout the ganglion. Only a small percentage (3.5%) of bladder postganglionic neurons in the MPG were NADPH diaphorase positive. Since NADPH diaphorase activity commonly reflects the presence of nitric oxide synthase, the present findings raise the possibility that nitric oxide may have a role as a neurotransmitter or neuromodulator in afferent pathways from the urinary bladder.

Differences in Fluorogold and wheat germ agglutinin-horseradish peroxidase labelling of bladder afferent neurons.

Rat urinary bladder afferent neurons were significantly smaller (34%) when labelled with Fluorogold (FG) than when labelled with wheat germ agglutinin-horseradish peroxidase (WGA-HRP). This study showed that this difference was due to an artifact of tissue processing (ethanol dehydration) and was not due to uptake and transport of the two tracers by two different subpopulations of bladder afferents.

The effect of rhizotomy on NADPH diaphorase staining in the lumbar spinal cord of the rat.

In the L6-S1 spinal segments of the rat NADPH diaphorase fiber-like staining extending along the lateral edge of the dorsal horn to the sacral parasympathetic nucleus was eliminated 1-3 weeks following dorsal-ventral L6-S1 rhizotomy. However, other NADPH diaphorase staining in the dorsal horn was not affected by rhizotomy. Rhizotomy also doubled the number (5 versus 10 cells/section) of NADPH diaphorase positive neurons in the region of the sacral parasympathetic nucleus (SPN). These data indicate that NADPH diaphorase is present in primary afferent projections to the SPN and that NADPH diaphorase activity in SPN neurons can be enhanced by neuronal injury. The functional significance of the NADPH diaphorase histochemical stain remains to be determined; however, if NADPH diaphorase is nitric oxide synthase then this would suggest that NO has an important role in the neural pathways to the pelvic viscera.

Localization of NADPH-diaphorase in pelvic afferent and efferent pathways of the rat.

The NADPH diaphorase histochemical reaction was used in combination with retrograde axonal transport of Fluorogold (FG) from the major pelvic ganglion (MPG) to determine if NADPH diaphorase is contained within afferent and preganglionic efferent pathways to pelvic visceral organs. In L6 and S1 dorsal root ganglia, 68.5% and 62.2%, respectively, of FG-labeled afferent neurons were NADPH-diaphorase positive. In the sacral parasympathetic nucleus (SPN) of the L6 and S1 spinal cord segments, 49.4% and 51.7%, respectively of FG labeled preganglionic efferent neurons were NADPH-diaphorase positive. NADPH-diaphorase-positive neurons were also observed in laminae I and II of the dorsal horn, around the central canal and in the dorsal commissure. In addition, fiber-like NADPH diaphorase staining was present in superficial dorsal horn, Lissauer's tract and the lateral edge of the dorsal horn extending into the region of the SPN. If NADPH diaphorase activity in neurons does indicate a physiological function of nitric oxide, then nitric oxide may have a role as a neuromodulator or neurotransmitter within visceral afferent and preganglionic efferent pathways to the pelvic viscera in the rat.

Vasoactive intestinal polypeptide and substance P in primary afferent pathways to the sacral spinal cord of the cat.

An analysis of vasoactive intestinal polypeptide immunoreactivity (VIP-IR) and substance P-IR in the cat spinal cord has revealed marked differences in the distribution of the two peptides. While substance P-IR was located at all levels of the cord, VIP-IR was most prominent in the sacral segments in Lissauer's tract and lamina I on the lateral edge of the dorsal horn. VIP-IR was also present in the sacral cord in (1) laminae V, VII, and X, (2) a thin band on the medial side of the dorsal horn, (3) the dorsal commissure, (4) the lateral band of the sacral parasympathetic nucleus, and (5) in a few animals in Onuf's nucleus. In other segments of the spinal cord VIP-IR was much less prominent but was present in Lissauer's tract and laminae I, II, and X. Substance P-IR was more uniformly distributed at all segmental levels in laminae I-III, V, VII, and X and in the dorsal commissure. In ventrolateral lamina I of the sacral spinal cord both VIP-IR and substance P-IR exhibited a distinctive periodic pattern in the rostrocaudal axis. The peptides were associated with bundles of dorsoventrally oriented axons and varicosities spaced at approximately 210-micron intervals center to center along the length of the spinal cord. The bundles in lamina I continued into lamina V where they further divided into smaller bundles that extended medially through laminae V and VII. The most prominent bundles of VIP axons passed ventrally from lateral laminae V and VII to enter lamina X and the ventral part of the dorsal gray commissure. On the other hand the majority of substance P axons in lamina V turned dorsally to join with axons on the medial side of the dorsal horn and to pass into the dorsal part of the dorsal gray commissure. Rostrocaudal VIP axons were present not only in Lissauer's tract but also in dorsolateral lamina I, in the lateral funiculus and in the ependymal cell layer of the central canal. Following unilateral transection of the sacral dorsal roots (2 weeks-22 months) the density of VIP axons and terminals was markedly reduced in ipsilateral Lissauer's tract and lateral laminae I and V; however, no change was detected in lamina X. Sacral deafferentation reduced substance P-IR in the dorsal gray commissure and in lateral laminae I and V.(ABSTRACT TRUNCATED AT 400 WORDS)

The presence of leucine-enkephalin in the sacral preganglionic pathway to the urinary bladder of the cat.

Leucine-enkephalin (L-ENK) nerve terminals which surround the cholinergic neurons in ganglia of the cat urinary bladder are eliminated after transection of the sacral ventral roots or the pelvic nerve. These findings, coupled with other anatomical and physiological data, suggest that L-ENK may be a cotransmitter with acetylcholine in the sacral preganglionic pathways to the urinary bladder.