L6-S1 Dorsal Root Ganglia Research Articles

Up-regulation of phosphorylated CREB but not c-Jun in bladder afferent neurons in dorsal root ganglia after cystitis.

We examined the changes of two transcription factors, CREB and c-Jun, in dorsal root ganglia (DRG) after acute (8 or 48 hours) or chronic (10 days) cyclophosphamide (CYP)-induced cystitis. Results showed an increase in the number of p-CREB-immunoreactive (-IR) cells in the L1 and L2 DRG (5-7-fold; P < or = 0.05) as well as L6 and S1 DRG (2-4-fold; P < or = 0.05) after acute and chronic cystitis. The number of p-CREB-IR cells in the L4-L5 DRG was not altered with cystitis. The number of c-Jun-IR cells increased in the L1-L2 DRG (L1: 10-fold; L2: 8-fold; P < or = 0.05) only with chronic cystitis, although it increased in the L6-S1 DRG with CYP-induced cystitis of acute (2-3-fold; P < or = 0.05) and chronic (6-10-fold; P < or = 0.05) duration. After CYP treatment, the percentage of bladder afferent cells expressing p-CREB immunoreactivity (3-7-fold; P < or = 0.05) increased in L1, L2, L6, and S1 DRG. The increase occurred 8 hours post-CYP injection and was maintained with chronic cystitis. There were few c-Jun-IR cells in the bladder afferent population. These results demonstrate that CYP induces p-CREB and c-Jun expression in DRG in a time-dependent manner. However, c-Jun expression is not associated with bladder afferent neurons. Resiniferatoxin reduced CYP-induced up-regulation of p-CREB in DRG, suggesting that cystitis can reveal an altered CREB phosphorylation that may be mediated by capsaicin-sensitive bladder afferents. Colocalization of p-CREB and Trk receptor(s) showed that a subpopulation of p-CREB-IR cells expressed p-Trk with cystitis. These results suggest that up-regulation of p-CREB may be mediated by a neurotrophin/Trk signaling pathway.

The effects of pregnancy and estrogen on the expression of calcitonin gene-related peptide (CGRP) in the uterine cervix, dorsal root ganglia and spinal cord

Before parturition the uterine cervix undergoes a ripening process (“softens” and dilates) to allow passage of the fetus at term. The exact mechanism(s) responsible for cervical ripening are unknown, though a role for peptidergic sensory neurons is emerging. Previous work demonstrated that administration of substance P (SP) to ovariectomized rats caused events associated with cervical ripening, that production of SP in cervix-related dorsal root ganglion (DRG) is estrogen responsive, and that release of SP from neurons terminating in the cervix and spinal cord peaks prior to parturition. The present study was designed to test the hypothesis that calcitonin gene-related peptide (CGRP), a neuropeptide co-stored with SP in many sensory neurons, undergoes changes with pregnancy and hormonal environment. Immunohistochemistry, in situ hybridization, reverse transcriptase–polymerase chain reaction (RT–PCR) and radioimmunoassay (RIA) were used to investigate CGRP in L6-S1 DRG, spinal cord and cervix during pregnancy and the role of estrogen in CGRP synthesis. CGRP-immunoreactive primary sensory neurons expressed estrogen receptors (ER-α and ER-β). In the cervix, CGRP concentrations decreased, but in the L6-S1 DRG and the spinal cord segments, CGRP levels increased, with peak effects observed at day 20 of gestation. CGRP mRNA synthesis increased in DRG over pregnancy. Sensory neurons of ovariectomized rats treated with estrogen showed increased CGRP mRNA synthesis in a dose-related manner, an effect blocked by the ER antagonist ICI 182 780. From these results, we postulate that synthesis of CGRP in L6-S1 DRG and utilization in the cervix increase over pregnancy and this synthesis is the under influence of the estrogen–ER system. Collectively, these data are consistent with the hypothesis that CGRP plays a role in cervical ripening and, consequently in the birth process.

Substance P in the uterine cervix, dorsal root ganglia and spinal cord during pregnancy and the effect of estrogen on SP synthesis

Prior to parturition the non-pliable uterine cervix undergoes a ripening process (“softens” and dilates) to allow a timely passage of the fetus at term. The exact mechanism(s) triggering and involved in cervical ripening are unknown, though evidence for a role for sensory neurons and their contained neuropeptides is emerging. Moreover, an apparent increase in neuropeptide immunoreactive nerves occurs in the cervix during pregnancy, maternal serum estrogen levels rise at term and uterine cervix-related L6-S1 dorsal root ganglia (DRG) sensory neurons express estrogen receptor (ER) and neuropeptides. Thus, we sought to test the hypothesis that the neuropeptide substance P (SP) changes biosynthesis and release over pregnancy, that estrogen, acting via the ER pathway, increases synthesis of SP in DRG, and that SP is utilized in cervical ripening at late pregnancy. Using immunohistochemistry, in situ hybridization, reverse transcriptase–polymerase chain reaction (RT–PCR) and radioimmunoassay (RIA), we investigated coexpression of ER-α/β and SP; differential expression of ER-α and -β mRNA in DRG neurons; SP synthesis in DRG; and changes in SP concentration in the cervix, DRG and spinal cord over pregnancy. In addition, the effect of exogenous estrogen on SP synthesis in L6-S1 DRG of ovariectomized rats was examined. SP-immunoreactive neurons expressed ER-α and ER-β. SP synthesis (expressed as β-PPT mRNA label) was prominent in small DRG neurons. SP concentration increased in the L6-S1 DRG and spinal cord segments, with a peak at Day 20 of gestation, but decreased in the cervix during the first two trimesters, with a rise over the last trimester to Day 10 levels. SP and ER-α mRNA synthesis increased in DRG over pregnancy but ER-β mRNA levels were largely unchanged. When ovariectomized rats were treated with exogenous estrogen, SP mRNA synthesis in the DRG increased in a dose-related manner, an effect blocked by ER blocker ICI 182 780. From these results, we postulate that synthesis of SP in L6-S1 DRG and utilization in the cervix increase over pregnancy and this synthesis is under influence of the estrogen–ER system, most likely ER-α. We postulate that SP may play a role in cervical ripening and, consequently in the birth process.

Anatomical location of Macrophage Migration Inhibitory Factor in urogenital tissues, peripheral ganglia and lumbosacral spinal cord of the rat

BackgroundPrevious work suggested that macrophage migration inhibitory factor (MIF) may be involved in bladder inflammation. Therefore, the location of MIF was determined immunohistochemically in the bladder, prostate, major pelvic ganglia, sympathetic chain, the L6-S1 dorsal root ganglia (DRG) and the lumbosacral spinal cord of the rat.ResultsIn the pelvic organs, MIF immunostaining was prominent in the epithelia. MIF was widely present in neurons in the MPG and the sympathetic chain. Some of those neurons also co-localized tyrosine hydroxylase (TH). In the DRGs, some of the neurons that stained for MIF also stained for Substance P. In the lumbosacral spinal cord, MIF immunostaining was observed in the white mater, the dorsal horn, the intermediolateral region and in the area around the central canal. Many cells were intensely stained for MIF and glial fibrillary acidic protein (GFAP) suggesting they were glial cells. However, some cells in the lumbosacral dorsal horn were MIF positive, GFAP negative cells suggestive of neurons.ConclusionsTherefore, MIF, a pro-inflammatory cytokine, is localized to pelvic organs and also in neurons of the peripheral and central nervous tissues that innervate those organs. Changes in MIF's expression at the end organ and at peripheral and central nervous system sites suggest that MIF is involved in pelvic viscera inflammation and may act at several levels to promote inflammatory changes.

Up-regulation of tyrosine kinase (Trka, Trkb) receptor expression and phosphorylation in lumbosacral dorsal root ganglia after chronic spinal cord (T8-T10) injury.

Previous studies have demonstrated changes in urinary bladder neurotrophic factors after bladder dysfunction. We have hypothesized that retrograde transport of neurotrophin(s) from the bladder to lumbosacral dorsal root ganglia (DRG) may play a role in bladder reflex reorganization after spinal cord injury (SCI). In this study, we determined whether the expression of tyrosine kinase receptors (TrkA, TrkB) is altered in lumbosacral DRG after SCI through immunofluorescence techniques. Complete transection of the spinal cord (T8-T10) was performed in female Wistar rats (120-150 g), and animals were studied 5-6 weeks after SCI. One week before killing, Fast Blue (FB) was injected into the bladder to label bladder afferent cells in the L1, L2, L6, and S1 DRG. After SCI, a significant increase in the number of TrkA-immunoreactive (IR) positive cells was detected in the L6-S1 DRG (L6: 1.9-fold, P < or = 0.01; S1: 1.7-fold, P < or = 0.05) and in the L1 DRG (3.0-fold; P < or = 0.01) but not in the L4-L5 DRG compared with spinal-intact (control) rats. After SCI, a significant increase in the number of TrkB-IR cells was also detected in the L6-S1 DRG (L6: 2.2-fold, P < or = 0.01; S1: 1.5-fold, P < or = 0.05) and in the L1-L2 DRG (L1: 1.5-fold, P < or = 0.01; L2: 1.3-fold, P < or = 0.05) but not in the L4-L5 DRG compared with control rats. After SCI, the percentage of FB-labeled cells expressing TrkA immunoreactivity (approximately 68%) or TrkB immunoreactivity (approximately 65%) in L1 and L6 DRG significantly (P < or = 0.01) increased compared with control (20-30%) DRG. After SCI, the percentage of TrkA-IR cells expressing phosphorylated (p)-Trk immunoreactivity significantly increased (1.5- to 2.3-fold increase) in the L1, L6, and S1 DRG. The percentage of TrkB-IR cells expressing p-Trk immunoreactivity after SCI also increased (1.3-fold increase) in the L1 and L6 DRG. These results demonstrate that (1) TrkA and TrkB immunoreactivity is increased in bladder afferent cells after SCI and (2) TrkA and TrkB receptors are phosphorylated in DRG after SCI. Neuroplasticity of lower urinary tract reflexes after SCI may be mediated by both nerve growth factor and brain-derived neurotrophic factor.

Estrogen receptor-α and -β coexist in a subpopulation of sensory neurons of female rat dorsal root ganglia

Immunoreactivities for estrogen receptor-α (ER-α) and ER-β are expressed in sensory neurons of the dorsal root ganglia (DRG). It has not been established, however, if the two receptor subtypes coexist in the same neuron. Double-staining immunohistochemical techniques were used to determine if subpopulations of neurons in the lumbosacral DRG exist based on their content of ERs. Results indicate that some neurons (approximately 17%) of the L6–S1 DRG contain ER-α –, some (approximately 23%) contain ER-β – immunoreactivity and some (approximately 5%) express immunoreactivity for both subtypes of the ER. These results suggest that many sensory neurons can respond to estrogens, but estrogens may produce different morphofunctional effects in different neurons based on their expression of ER subtypes.

Nitric oxide modulates Ca(2+) channels in dorsal root ganglion neurons innervating rat urinary bladder.

The effect of a nitric oxide (NO) donor on high-voltage-activated Ca(2+) channel currents (I(Ca)) was examined using the whole cell patch-clamp technique in L(6)-S(1) dorsal root ganglion (DRG) neurons innervating the urinary bladder. The neurons were labeled by axonal transport of a fluorescent dye, Fast Blue, injected into the bladder wall. Approximately 70% of bladder afferent neurons exhibited tetrodotoxin (TTX)-resistant action potentials (APs), and 93% of these neurons were sensitive to capsaicin, while the remaining neurons had TTX-sensitive spikes and were insensitive to capsaicin. The peak current density of nimodipine-sensitive L-type Ca(2+) channels activated by depolarizing pulses (0 mV) from a holding potential of -60 mV was greater in bladder afferent neurons with TTX-resistant APs (39.2 pA/pF) than in bladder afferent neurons with TTX-sensitive APs (28.9 pA/pF), while the current density of omega-conotoxin GVIA-sensitive N-type Ca(2+) channels was similar (43-45 pA/pF) in both types of neurons. In both types of neurons, the NO donor, S-nitroso-N-acetylpenicillamine (SNAP) (500 microM), reversibly reduced (23.4-26.6%) the amplitude of I(Ca) elicited by depolarizing pulses to 0 mV from a holding potential of -60 mV. SNAP-induced inhibition of I(Ca) was reduced by 90% in the presence of omega-conotoxin GVIA but was unaffected in the presence of nimodipine, indicating that NO-induced inhibition of I(Ca) is mainly confined to N-type Ca(2+) channels. Exposure of the neurons for 30 min to 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM), an inhibitor of NO-stimulated guanylyl cyclase, prevented the SNAP-induced reduction in I(Ca). Extracellular application of 8-bromo-cGMP (1 mM) mimicked the effects of NO donors by reducing the peak amplitude of I(Ca) (28.6% of reduction). Action potential configuration and firing frequency during depolarizing current pulses were not altered by the application of SNAP (500 microM) in bladder afferent neurons with TTX-resistant and -sensitive APs. These results indicate that NO acting via a cGMP signaling pathway can modulate N-type Ca(2+) channels in DRG neurons innervating the urinary bladder.

Differential expression of estrogen receptor alpha and beta in rat dorsal root ganglion neurons

Estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) mRNAs are both expressed in rat dorsal root ganglion (DRG) neurons, but the distribution of these two mRNAs differs markedly. Radiolabeled probes highly specific to ERalpha or ERbeta mRNAs were used for in situ hybridization studies; two antibodies specific to ERalpha protein were used for immunocytochemistry and specific primers were used for reverse transcription polymerase chain reaction (RT-PCR) studies. These revealed that ERbeta mRNA is widely expressed in the DRG of both male and female rats, with virtually all neurons showing positive signals. In contrast, ERalpha mRNA, as well as nuclear localized ERalpha protein, is more restricted in its localization and is present in many, but not all, of the small-sized (<600 microm(2)) DRG neurons, but is only rarely present in larger neurons. The L6-S1 DRG levels, which contain sensory neurons that innervate reproductive tissues, are relatively enriched in ERalpha compared to L3-L5 DRG levels, which contain sensory neurons that innervate hind limb regions. Long-term estrogen treatment of ovariectomized rats (21-28 days) dramatically reduces immunocytochemically detectable ERalpha protein in the DRG relative to that in ovariectomized controls. RT-PCR studies also showed that long-term estrogen treatment of ovariectomized rats downregulates the levels of ERalpha mRNA, but upregulates the levels of ERbeta mRNA in the DRG. Interestingly, in intact cycling female rats, ERalpha and ERbeta mRNA levels in the DRG were both higher during proestrus compared to metestrus. These findings suggest that the changes in expression of estrogen receptors which occur dynamically during the estrus cycle differ from those induced by long-term estrogen treatment of ovariectomized animals.

The peptide content of colonic afferents decreases following colonic inflammation

Peripheral injury produces long term changes in peptide content in dorsal root ganglion (DRG) cells that contribute to the inflammatory process in the periphery and neuronal plasticity in the spinal cord. We report here the proportion of colonic afferents labeled for calcitonin gene-related peptide (CGRP), substance P (SP) or somatostatin (Som) in the T13-L2 and L6-S2 DRG and changes in the percentage of SP or CGRP labeled afferents 6, 24, and 72 h following induction of experimental colitis. Following injection of fluorogold (FG) into the descending colon, significantly more FG labeled DRG cells were observed in the T13-L2 than L6-S2 DRG. In noninflamed rats, in both spinal regions, 60–70% of the colonic afferents that were labeled with FG were double labeled for SP. Similar results were obtained when double labeling for CGRP. Only 20–30% of the FG labeled afferents were double labeled for Som. Following experimental colitis induced by intracolonic zymosan, there was a significant decrease in the percentage of cells double labeled for SP in the T13-L2 and L6-S2 DRG at 6, 24, and 72 h. The percentage of CGRP double labeled cells was decreased in the T13-L2 DRG at all time points, but only at 24 h in the L6-S2 DRG. The cell bodies of CGRP labeled colonic afferents were significantly larger than SP or Som in control rats. Inflammation did not affect the mean size of the double labeled cells. These results suggest that colonic inflammation increases SP and CGRP release in the spinal cord and the colon that is manifest as a decrease in peptide content in the cell bodies of the colonic afferents during the first 72 h following injury.

Retrograde and transganglionic transport of horseradish peroxidase-conjugated cholera toxin B subunit, wheatgerm agglutinin and isolectin B4 from Griffonia simplicifolia I in primary afferent neurons innervating the rat urinary bladder

In the present study, we investigated and compared the ability of the cholera toxin B subunit, wheat germ agglutinin and isolectin B4 from Griffonia simplicifolia I conjugated to horseradish peroxidase, to retrogradely and transganglionically label visceral primary afferents after unilateral injections into the rat urinary bladder wall. Horseradish peroxidase histochemical or lectin-immunofluorescence histochemical labelling of bladder afferents was seen in the L6–S1 spinal cord segments and in the T13–L2 and L6–S1 dorsal root ganglia. In the lumbosacral spinal cord, the most intense and extensive labelling of bladder afferents was seen when cholera toxin B subunit–horseradish peroxidase was injected. Cholera toxin B subunit–horseradish peroxidase-labelled fibres were found in Lissauer's tract, its lateral and medial collateral projections, and laminae I and IV–VI of the spinal gray matter. Labelled fibres were numerous in the lateral collateral projection and extended into the spinal parasympathetic nucleus. Labelling from both the lateral and medial projections extended into the dorsal grey commissural region. Wheat germ agglutinin–horseradish peroxidase labelling produced a similar pattern but was not as dense and extensive as that of cholera toxin B subunit–horseradish peroxidase. The isolectin B4 from Griffonia simplicifolia I–horseradish peroxidase-labelled fibres, on the other hand, were fewer and only observed in the lateral collateral projection and occasionally in lamina I. Cell profile counts showed that a larger number of dorsal root ganglion cells were labelled with cholera toxin B subunit–horseradish peroxidase than with wheat germ agglutinin– or isolectin B4–horseradish peroxidase. In the L6–S1 dorsal root ganglia, the majority (81%) of the cholera toxin B subunit-, and almost all of the wheat germ agglutinin- and isolectin B4-immunoreactive cells were RT97-negative (an anti-neurofilament antibody that labels dorsal root ganglion neurons with myelinated fibres). Double labelling with other neuronal markers showed that 71%, 43% and 36% of the cholera toxin B subunit-immunoreactive cells were calcitonin gene-related peptide-, isolectin B4-binding- and substance P-positive, respectively. A few cholera toxin B subunit cells showed galanin-immunoreactivity, but none were somatostatin-, vasoactive intestinal polypeptide-, or neuropeptide Y-immunoreactive or contained fluoride-resistant acid phosphatase. The results show that cholera toxin B subunit–horseradish peroxidase is a more effective retrograde and transganglionic tracer for pelvic primary afferents from the urinary bladder than wheat germ agglutinin–horseradish peroxidase and isolectin B4–horseradish peroxidase, but in contrast to somatic nerves, it is transported mainly by unmyelinated fibres in the visceral afferents.

Increased expression of neuronal nitric oxide synthase in bladder afferent and spinal neurons following spinal cord injury.

Changes in the distribution of neuronal nitric oxide synthase immunoreactivity (NOS-IR) after chronic (5-6 weeks) spinal cord injury (SCI) were examined in bladder afferent and spinal neurons in the region of the sacral parasympathetic nucleus (SPN) in the L6-S1 spinal segments. Bladder afferent neurons in the L1, L2, L6 and S1 dorsal root ganglia (DRG) were identified by retrograde axonal transport following injection of fluorogold (FG) into the urinary bladder. A differential distribution of NOS-IR was detected in DRG cells at different segmental levels of spinal cord intact animals with significantly greater numbers of NOS-IR cells present in thoracic (T8, T12; 30-40 NOS-IR cell profiles/section) and rostral lumbar (L1) DRGs (18 NOS-IR cell profiles/ section) compared to caudal lumbosacral (L5-S1) DRGs (0.2-0.4 NOS-IR cell profiles/section). A significant increase in the number of NOS-IR cells was detected in the L6-S1 DRG (p < or = 0.001; 12-20 NOS-IR cell profiles/section) and in the L1-L2 DRG (15-40 NOS-IR cell profiles/section) but not in the L5 DRG following SCI. In these ganglia, an average of 41.2 +/- 7.8% (L6) and 36.3 +/- 0.9% (S1) of FG-labeled bladder afferent neurons were NOS-IR. In contrast, in spinal cord intact animals, no FG-labeled bladder afferent neurons were NOS-IR. Following SCI, NOS-IR fibers were detected along the lateral edge of the dorsal horn extending from Lissauer's tract to the region of the SPN (lateral collateral pathway of Lissauer) of the L6 and S1 spinal segments. These NOS-IR fibers were not detected in adjacent spinal segments (L5, S2). SCI also significantly (p < or = 0.001) increased the number of spinal neurons in the region of the SPN (presumptive preganglionic neurons) in the L6-S1 spinal segments exhibiting NOS-IR. These results indicate that NOS-IR in bladder afferent and spinal neurons is plastic and can be up-regulated by chronic SCI. Changes in the neurochemical properties of these neurons after SCI may be mediated by pathological changes in the target organ (i.e., urinary bladder) and/or spinal cord.

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 ≤ .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 μl) 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. © 1996 Wiley-Liss, Inc.

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.

Central nervous system neurons infected by pseudorabies virus injected into the rat urinary bladder following unilateral transection of the pelvic nerve.

Following the transection of one pelvic nerve and both hypogastric nerves, the urinary bladder of male Sprague-Dawley rats was injected with pseudorabies virus (PRV; Bartha strain). The central stump of the transected pelvic nerve was labelled with fast blue (FB), and rats were maintained for 2, 2.5, and 3 days following viral infection. Tissue was processed with antisera against PRV and choline acetyltransferase (CAT). In the L6-S1 spinal cord, neurons in the ipsilateral intermediolateral area (IML) were labelled after 2 days. After 2.5 days, labelled neurons were also found in the dorsal gray commissure (DGC), the ipsilateral superficial dorsal horn, and the contralateral IML area. After 3 days, many labelled neurons appeared in the superficial dorsal horns and, bilaterally, in the L6-S1 dorsal root ganglia. In both IMLs, two groups of PRV-labelled neurons were found: 1) CAT-positive preganglionic cells and 2) smaller, CAT-negative cells located slightly dorsal to the preganglionic neurons. No other doubly stained neurons were found in the spinal cord. Contralateral DRG neurons stained for either PRV or FB or both. Ipsilateral DRG neurons stained only for PRV. PRV-immunoreactive (IR) neurons appeared in the brainstem only after 3 days. These were located primarily in the pontine micturition centers (equal numbers), the ventral locus coeruleus, and the raphe and lateral reticular areas.

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.

Calcium channel antagonists prevent urinary bladder growth and neuroplasticity following mechanical stress

Cytosolic Ca2+ has been postulated to regulate smooth muscle hypertrophy and growth factor production. Consistent with this hypothesis we report that the Ca2+ channel antagonists verapamil and diltiazem prevent bladder and neuronal growth in rats in response to 3 wk of urethral obstruction. Ca2+ channel blockers prevented 30-45% of the increase in bladder weight, protein, and DNA content found in obstructed animals. Similarly, these drugs produced a 15-27% reduction in area profiles for retrogradely labeled (Fluoro-Gold) motoneurons in the major pelvic ganglia and afferents in the L6-S1 dorsal root ganglia after obstruction. The reduced growth in neuronal areas was attributed, in part, to less nerve growth factor (NGF) in bladders of obstructed rats receiving verapamil (8.5 pg/bladder) or diltiazem (14.5 pg/bladder) compared with obstructed animals not given these drugs (58.2 pg/bladder). The alpha 1-adrenergic antagonist, prazosin, while decreasing voiding frequency in obstructed rats, had no significant impact on bladder weight or neuronal size. These reductions in the increase in bladder hypertrophy and NGF content may be due to altered handling of Ca2+.

Characterization of forms of immunoreactive somatostatin in sensory neuron and normal and deafferented spinal cord

In order to determine the contribution made by primary sensory afferents and supraspinal projections to the immunoreactive somatostatin (IRS) content of the spinal cord, measurements were made of the concentration of IRS in the dorsal and ventral halves of the cord in cats subjected to unilateral lumbosacral dorsal rhizotomy (L1-S3) alone or combined with spinal cord transection. The molecular forms of IRS (characterized by gel chromatography) in L7 lumbar spinal cord, L6-S1 dorsal roots, ventral roots and dorsal root ganglia, and sciatic nerve were also determined. S14 was the predominant form in all tissues examined, but two additional molecular forms corresponding to S28 and S11.5 kdalton were present in dorsal root ganglia and spinal cord; S28 but not S11.5 kdalton was detected in both dorsal roots and sciatic nerves. These results indicate that S14 and S28 and S28 are transported along the central and peripheral processes of dorsal root ganglia, but that spinal cord S11.5 kdalton originates in the central nervous system. IRS in the dorsal horn was reduced by ca. 40% following dorsal root section. Neither disruption of descending pathways by spinal transection nor surgical isolation of the lumbar segements lowered cord somatostatin content below that produced by dorsal root section, indicating that most of the somatostatin within the cord arises from the dorsal root and from neurons in local spinal segments. Although the total content of IRS in the dorsal horn was reduced by ca. 40% following dorsal rhizotomy, the pattern of molecular forms was not changed accordingly. Since S14 and S28 but not S11.5 kdalton are transported via the dorsal root, the dorsal root section would be predicted to produce a relatively greater decrease in S14 and S28 than in S11.5 kdalton. Therefore, failure to find a selective loss of S14 and S28 suggests that dorsal rhizotomy affects dorsal horn IRS content not only by removing afferent input but possibly also by modifyinh the processing of IRS by the remaining somatostatinergic neurons.

Segmental localization of sensory cells that innervate the bladder.

The present study labels the neuronal cell bodies that give rise to afferent fibers that innervate the bladder of cat and rat. The method used was the retrograde transport of horseradish peroxidase (HRP) from its injection site in the bladder to cells in various dorsal root ganglia. In the rat, the labelled cells are located in the L1-L2 and L6-S1 dorsal root ganglia. In the cat, the labelled cells are located in the L2-L5 and S1-S4 dorsal root ganglia. This confirms older clinical findings, and for the first time directly demonstrates the afferent cell bodies for the bladder. The bladder afferents are small ganglion cells in both rat and cat, and because there is a correlation between the size of axon and the cell body from which it originates, we conclude that the great majority of bladder afferents are small myelinated or unmyelinated axons. In addition, by restricting the HRP to one side of the bladder, we are able to show that some afferent cell bodies send their distal processes across the midline. These results will be useful in considerations of the neural control of bladder function.