Lumbosacral Ganglia Research Articles

MP49-02 MODULATION OF SENSORY GLIA GQ-GPCR SIGNALING IN VIVO AFFECTS SPONTANEOUS VOIDING AND BLADDER FUNCTION IN AN ANIMAL MODEL OF CHRONIC PELVIC PAIN

You have accessJournal of UrologyCME1 May 2022MP49-02 MODULATION OF SENSORY GLIA GQ-GPCR SIGNALING IN VIVO AFFECTS SPONTANEOUS VOIDING AND BLADDER FUNCTION IN AN ANIMAL MODEL OF CHRONIC PELVIC PAIN Alison Xiaoqiao Xie, Randall Meacham, and Anna Malykhina Alison Xiaoqiao XieAlison Xiaoqiao Xie More articles by this author , Randall MeachamRandall Meacham More articles by this author , and Anna MalykhinaAnna Malykhina More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002624.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Chronic pelvic pain (CPP) is a bothersome and highly prevalent symptom present in many chronic disorders of the pelvis. Afferent hypersensitivity plays an important role in the generation and maintenance of CPP symptoms including visceral pain, urgency and urinary frequency. We previously showed that activation of Gq-G protein coupled receptors (Gq-GPCR) in satellite glial cells (SGCs) decreases lumbosacral nociceptive responses and alleviates visceral hypersensitivity in a mouse model of CPP. The objective of this study was to further investigate the modulatory capacity of satellite glial activation in regulation of spontaneous voiding and bladder function. METHODS: Intravesical instillation of vascular endothelial growth factor (VEGFA) induces sensory nerve remodeling in the urinary bladder, and increases nociceptive responses in the lumbosacral sensory ganglia associated with visceral hypersensitivity. Adult C57BL/6 mice were subjected to three intravesical instillations of VEGFA within a 2-week period. Selective activation of lumbosacral sensory glia was achieved by expressing Gq-coupled Designer Receptors Exclusively Activated by Designer Drugs (Gq-DREADD) in glial acidic fibrillary protein (GFAP+) expressing glia by using Gfap-Gq-DREADD transgenic mice or targeted adeno-associated viral vector (AAV) delivery. Gq-DREADD agonist, clozapine N-oxide (CNO), was used to selectively activate Gq-GPCR signaling in GFAP+ glia in vivo. Micturition patterns were recorded by performing a voiding spot assay (spontaneous voiding) and cystometry in unanesthetized animals (bladder function). RESULTS: Activation of lumbosacral SGCs significantly altered spontaneous voiding patterns and bladder function in vivo in VEGFA-treated mice. Higher urinary output, shorter intermicturition intervals, and fewer non-voiding contractions were observed in VEGFA-treated animals following activation of Gq-GPCR signaling. The observed glia-mediated neuromodulatory effects were long-lasting and dose-dependent. CONCLUSIONS: Our data demonstrated that activation of sensory glia in the peripheral nervous system significantly regulate bladder functions. Targeting neuron-glia interactions in the sensory ganglia can be used to effectively alleviate visceral pain and regulate voiding function in vivo. Further studies are warranted to test a therapeutic potential of targeting glial cell signaling in treating hypersensitivity-related symptoms in CPP disorders. Source of Funding: Colorado Pilot Program Mentored Award, CCTSI. "Beyond the neurons: the role of peripheral glia in neurogenic bladder dysfunction". 2019-2021R01 DK129260-01. "Activating Peripheral Glia to Relieve Visceral Pain in Animal Models of Urological Chronic Pelvic Pain Syndrome (UCPPS)". 2021-2026 © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e853 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Alison Xiaoqiao Xie More articles by this author Randall Meacham More articles by this author Anna Malykhina More articles by this author Expand All Advertisement PDF DownloadLoading ...

Unique Molecular Characteristics of Visceral Afferents Arising from Different Levels of the Neuraxis: Location of Afferent Somata Predicts Function and Stimulus Detection Modalities.

Viscera receive innervation from sensory ganglia located adjacent to multiple levels of the brainstem and spinal cord. Here we examined whether molecular profiling could be used to identify functional clusters of colon afferents from thoracolumbar (TL), lumbosacral (LS), and nodose ganglia (NG) in male and female mice. Profiling of TL and LS bladder afferents was also performed. Visceral afferents were back-labeled using retrograde tracers injected into proximal and distal regions of colon or bladder, followed by single-cell qRT-PCR and analysis via an automated hierarchical clustering method. Genes were chosen for assay (32 for bladder; 48 for colon) based on their established role in stimulus detection, regulation of sensitivity/function, or neuroimmune interaction. A total of 132 colon afferents (from NG, TL, and LS ganglia) and 128 bladder afferents (from TL and LS ganglia) were analyzed. Retrograde labeling from the colon showed that NG and TL afferents innervate proximal and distal regions of the colon, whereas 98% of LS afferents only project to distal regions. There were clusters of colon and bladder afferents, defined by mRNA profiling, that localized to either TL or LS ganglia. Mixed TL/LS clustering also was found. In addition, transcriptionally, NG colon afferents were almost completely segregated from colon TL and LS neurons. Furthermore, colon and bladder afferents expressed genes at similar levels, although different gene combinations defined the clusters. These results indicate that genes implicated in both homeostatic regulation and conscious sensations are found at all anatomic levels, suggesting that afferents from different portions of the neuraxis have overlapping functions.SIGNIFICANCE STATEMENT Visceral organs are innervated by sensory neurons whose cell bodies are located in multiple ganglia associated with the brainstem and spinal cord. For the colon, this overlapping innervation is proposed to facilitate visceral sensation and homeostasis, where sensation and pain are mediated by spinal afferents and fear and anxiety (the affective aspects of visceral pain) are the domain of nodose afferents. The transcriptomic analysis performed here reveals that genes implicated in both homeostatic regulation and pain are found in afferents across all ganglia types, suggesting that conscious sensation and homeostatic regulation are the result of convergence, and not segregation, of sensory input.

Maternal Infections Associated with Bad Obstetric Outcome: Cytomegalovirus and Herpes Simplex Virus.

Cytomegalovirus (CMV) infection is with global distribution and range of 30-100%. CMV infection is the most common congenital viral infection worldwide . The sign of congenital CMV infection encountered most frequently is hearing loss, which wills 12.6% of congenitally infected newborns and congenital CMV infection form 10-20% as a cause of total hearing loss in children. About10% of the live-born infants with congenital CMV infection are symptomatic at birth. Where as an additional 10% of the infected newborns will develop permanent sequelae in the following years. Herpes simplex virus (HSV) is an abundant, enveloped, and double stranded DNA virus, belonging to the family of Herpesviridae transmitted across mucosal membranes and non intact skin, with ability to induce chronic latent infection. Susceptibility to HSV infection is associated with HLA antigens. The virus type incidence is about the same for both HSV-1 and HSV-2 strains, while HSV-2 is the most predominant in recurrent infections. HVS -1 is the common strain in labial herpetic infection, while HSV-2 is the predominant in genital infection. The site of latency is the trigeminal ganglia for labial herpetic infection and lumbosacral ganglia for genital herpetic infection. Changes in sexual behaviors of young adults may partly explain its higher incidence.

Virus-specific immune memory at peripheral sites of herpes simplex virus type 2 (HSV-2) infection in guinea pigs.

Despite its importance in modulating HSV-2 pathogenesis, the nature of tissue-resident immune memory to HSV-2 is not completely understood. We used genital HSV-2 infection of guinea pigs to assess the type and location of HSV-specific memory cells at peripheral sites of HSV-2 infection. HSV-specific antibody-secreting cells were readily detected in the spleen, bone marrow, vagina/cervix, lumbosacral sensory ganglia, and spinal cord of previously-infected animals. Memory B cells were detected primarily in the spleen and to a lesser extent in bone marrow but not in the genital tract or neural tissues suggesting that the HSV-specific antibody-secreting cells present at peripheral sites of HSV-2 infection represented persisting populations of plasma cells. The antibody produced by these cells isolated from neural tissues of infected animals was functionally relevant and included antibodies specific for HSV-2 glycoproteins and HSV-2 neutralizing antibodies. A vigorous IFN-γ-secreting T cell response developed in the spleen as well as the sites of HSV-2 infection in the genital tract, lumbosacral ganglia and spinal cord following acute HSV-2 infection. Additionally, populations of HSV-specific tissue-resident memory T cells were maintained at these sites and were readily detected up to 150 days post HSV-2 infection. Unlike the persisting plasma cells, HSV-specific memory T cells were also detected in uterine tissue and cervicothoracic region of the spinal cord and at low levels in the cervicothoracic ganglia. Both HSV-specific CD4+ and CD8+ resident memory cell subsets were maintained long-term in the genital tract and sensory ganglia/spinal cord following HSV-2 infection. Together these data demonstrate the long-term maintenance of both humoral and cellular arms of the adaptive immune response at the sites of HSV-2 latency and virus shedding and highlight the utility of the guinea pig infection model to investigate tissue-resident memory in the setting of HSV-2 latency and spontaneous reactivation.

Immunity to Ocular and Genital Herpes Simplex Viruses Infections

Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012, Article ID 732546, 2 pages doi:10.1155/2012/732546 Editorial Immunity to Ocular and Genital Herpes Simplex Viruses Infections Aziz Alami Chentoufi, 1 Lbachir BenMohamed, 2 Philippe Van De Perre, 3 and Ali A. Ashkar 4 1 Pathology and Clinical Laboratory Medicine, Department of Immunology, King Fahad Medical City, Riyadh 11525, Saudi Arabia of Cellular and Molecular Immunology, Gavin Herbert Eye Institute, College of Medicine, University of California, Irvine, CA 92697-4375, USA 3 INSERM U 1058, Universit´ e Montpellier 1, 34090 Montpellier, France 4 Department of Pathology and Molecular Medicine, McMaster Immunology Research Centre, McMaster University, Room MDCL4015, 1280 Main Street West, Hamilton, ON, Canada L8S 4K1 2 Laboratory Correspondence should be addressed to Aziz Alami Chentoufi, aachentoufi@kfmc.med.sa Received 20 November 2012; Accepted 20 November 2012 Copyright © 2012 Aziz Alami Chentoufi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Herpes simplex virus type 1 and type 2 are amongst the most common human viral infection. Despite decades of research, currently, there is no vaccine or sterilizing therapy against human herpes. Both innate and adaptive immunities are required for protection against herpes infec- tions. However, the immunity and immunopathology of herpes infections are not yet fully characterized. To better understand the immunopathology of herpes infections and ultimately design an efficient prophylactic or therapeutic herpes vaccine or treatment, it is fundamental to define the cellular and molecular immune mechanisms and the immune correlates required for an efficient control of these ubiquitous pathogens. Over 400,000 Americans suffer from ocular herpes caused by HSV-1. Each year, nearly 50,000 new and recurring ocular herpes cases are diagnosed in the United States, with the more serious stromal keratitis accounting for about 25 percent. About 1 billion people— one-sixth of the world’s population—are infected with HSV- 2, the most common cause of genital herpes. In the United States, an estimated 50 million people carry the virus, and up to 3 million of those people suffer recurrent outbreaks of painful genital herpes. While the majority of HSV-1 and HSV-2 seropositive individuals is asymptomatic, a nonneg- ligible number of symptomatic individuals have as often as four recurrent herpes per year and required treatments. In addition, genital herpes has played an important role in driving the prevalence of other sexually transmitted infection such as HIV. Current drug therapies, often used to suppress genital herpes, can also treat ocular herpes but do not prevent future outbreaks. There is currently no prophylactic or therapeutic vaccine against herpes in the market. The impairment of T-cell function in acute and latent infection has been reported at many levels including abnormal antigen presentation in the periphery and in the sensory ganglia (trigeminal and lumbosacral ganglia), latently infected neuronal cells resistance to apoptosis by CD8+ T-cells, unbalanced immunoregulatory T-cell func- tion, and deregulation of Th1/Th2/Th17 axes. Despite the identification of many HSV-1 and HSV-2-antigens and their derived CD4+ and CD8+ T-cell epitopes, numerous antigen- specific therapies have failed when evaluated for their utility in the prevention and treatment of HSV infection. In this special issue, we invited authors to submit original research and review articles highlighting the recent advances that have broadened our understanding of immunity and immunopathology of herpes infection and HSV vaccine strategies. Also, we welcomed papers that seek to define immunoregulatory and effector properties of T-cells and dendritic cells to provide new insights as to their potential for clinical use. We were interested in articles that explore salient aspects of protective immunity throughout HSV latency, reactivation, ocular and genital herpetic disease, negative immunosynergy between HSV and HIV or other sexually transmitted diseases, and immunotherapeutical approaches. In a paper of this special issue, P. Stuart and T. Keadle documented the numerous recurrent models of herpetic stromal keratitis (HSK) that have been developed and how data generated from these models differs in some important

Sciatic nerve injury induces functional pro-nociceptive chemokine receptors in bladder-associated primary afferent neurons in the rat

Visceral sensory afferents during disease or following injury often produce vague, diffuse body sensations, and pain referred to somatic targets. Alternatively, injury due to trauma or disease of somatic nerve targets can also lead to referred pain in visceral targets via a somatovisceral reflex. Both phenomenons are thought to be due to convergence of visceral and somatic afferents within the spinal cord. To investigate a potential peripheral influence for referred pain in visceral targets following somatic nerve injury, we examined whether a sciatic nerve injury known to produce hindpaw tactile hyperalgesia alters the frequency of micturition and the sensitivity of bladder-associated sensory neurons to pro-nociceptive chemokines. Adult female Sprague-Dawley rats received injections of cholera toxin B subunit conjugated to 555 into urinary bladder wall to retrogradely label visceral primary afferent neurons. After 7 days, the right sciatic nerve of these animals was subjected to a lysophosphatidylcholine (LPC)-induced focal demyelination injury. Pre- and post-injury tactile sensitivity in the hind paw and micturition frequency were assayed. Animals were allowed to survive for 14–28 days. Lumbosacral and lumbar dorsal root ganglia (DRG) ipsilateral to the nerve injury were acutely dissociated from sham and nerve injured animals. Bladder wall-associated sensory neurons identified via the retrograde marker were assayed for fluxes in intracellular calcium following administration of pro-nociceptive chemokines. The assayed chemokines included monocyte chemoattractant protein-1 (MCP1/CCL2) and stromal cell derived factor-1 alpha (SDF1/CXCL12). LPC nerve injured animals exhibited tactile hyperalgesia and increased micturition frequency for at least 28 days. Focal demyelination of the sciatic nerve also increased the number of injured L4L5 and non-injured L6–S2 bladder-associated sensory neurons that responded to MCP1 and SDF1 when compared with sensory neurons derived from uninjured naïve and sham-injured control animals. Taken together, these data suggest that some visceral hypersensitivity states may have a somatic origin. More importantly, nociceptive somatovisceral sensation may be mediated by upregulation of chemokine signaling in visceral sensory neurons.

Nerve-Sparing Laparoscopic Radical Excision of Deep Endometriosis with Rectal and Parametrial Resection

A pelvic laparoscopic nerve-sparing approach for treatment of deep endometriosis starts retroperitoneally from the promontory. Blunt dissection of loose fatty tissue of the rectosacral and pararectal spaces to the level of the rectosacral- fascia and rectal wings enables identification of the sympathetic superior hypogastric plexus, hypogastric nerves, and lumbosacral trunks and ganglia. Moreover, identification of the parasympathetic pelvic splanchnic nerves at their origin from the sacral roots enables safe dissection of the rectal wings and of the posterior parametrial and lower mesorectal planes. Identification of the origin of pelvic plexuses, is performed. This technique seems to be feasible, with an adequate surgical radicality (and a lower rate of l postoperative dysfunctions, improving the patient's quality of life. A pelvic laparoscopic nerve-sparing approach for treatment of deep endometriosis starts retroperitoneally from the promontory. Blunt dissection of loose fatty tissue of the rectosacral and pararectal spaces to the level of the rectosacral- fascia and rectal wings enables identification of the sympathetic superior hypogastric plexus, hypogastric nerves, and lumbosacral trunks and ganglia. Moreover, identification of the parasympathetic pelvic splanchnic nerves at their origin from the sacral roots enables safe dissection of the rectal wings and of the posterior parametrial and lower mesorectal planes. Identification of the origin of pelvic plexuses, is performed. This technique seems to be feasible, with an adequate surgical radicality (and a lower rate of l postoperative dysfunctions, improving the patient's quality of life.

An atypical clinical presentation for the first isolation of Canid herpesvirus 1 in Argentina

, with a host range restricted to domestic and wild canids (Remond et al., 1996). CaHV-1 was first recognized as the agent responsible for causing a highly fatal hemorrhagic viral disease in newborn puppies in 1965. Apart from being an important disease in newborn puppies, CaHV-1 also affects reproduction of dogs in other ways: the virus may cause vesicular lesions in the vestibulum and vagina of the bitch, as well as on the penis and the preputial mucosa of dogs and may cause embryonic resorption, abortion, and fetal death (Carmichael, 1970). In addition, the virus is associated with respiratory (kennel cough syndrome) and ocular disease in dogs (Erles and Brownlie, 2005; Ledbetter et al., 2009). Oronasal and venereal transmission are the common routes of infection but transplacental infection has also been described. Lesions in the vestibulum and vagina of bitches may recur when bitches come into pro-estrus and regress when they go into anestrus (Carmichael, 1970; Hashimoto et al., 1982). Affected animals may remain latent carriers and the virus was identified in the lumbosacral ganglia, tonsils, parotid salivary glands, and liver of dogs that showed no sign of herpesvirus infection (Burr et al., 1996). Several studies suggest that CaHV-1 is enzootic in the

Naturally Occurring Multiple Perineuriomas in a Chicken (Gallus domesticus)

A 2-year-old, male Japanese native fowl (Gallus gallus domesticus) was presented with an inability to feed and torticollis. At a necropsy, there were cylindrical enlargements and yellow discoloration of multiple peripheral nerves, including nerves of the lumbosacral plexus, brachial plexus, and spinal ganglia. On histologic examination, these lesions consisted of diffuse proliferations of spindle cells with characteristic onion bulb-like structures around residual axons. The spindle cells were immunohistochemically positive for glucose transporter 1 (GLUT1) and negative for S-100 alpha/beta proteins. On the basis of microscopic, histologic, and immunohistochemical findings, the tumors were diagnosed as multiple perineuriomas.

Convergence of bladder and colon sensory innervation occurs at the primary afferent level

Dichotomizing afferents are individual dorsal root ganglion (DRG) neurons that innervate two distinct structures thereby providing a form of afferent convergence that may be involved in pelvic organ cross-sensitization. To determine the distribution of dichotomizing afferents supplying the distal colon and bladder of the Sprague–Dawley rat and the C57Bl/6 mouse, we performed concurrent retrograde labeling of urinary bladder and distal colon afferents using cholera toxin subunit B (CTB) fluorescent conjugates. Animals were perfused 4–5 days after sub-serosal organ injections, and the T10-S2 DRG were removed, sectioned, and analyzed using confocal microscopy. In the rat, CTB-positive afferents retrogradely labeled from the bladder were nearly three times more numerous than those labeled from the distal colon, while in the mouse, each organ was equally represented. In both species, the majority of colon and bladder afferents projected from lumbosacral (LS) ganglia and secondarily from thoracolumbar (TL) ganglia. In the rat, 17% of the total CTB-positive neurons were retrogradely labeled from both organs with 11% localized in TL, 6% in LS, and 0.8% in thoracic (TH) ganglia. In the mouse, 21% of the total CTB-positive neurons were dually-labeled with 12% localized in LS, 4% in TH, and 4% in TL ganglia. These findings support the existence of dichotomizing pelvic afferents, which provide a pre-existing neuronal substrate for possible immediate and maintained pelvic organ cross-sensitization and ultimately may play a role in the overlap of pelvic pain disorders.

Long-Term Presence of Virus-Specific Plasma Cells in Sensory Ganglia and Spinal Cord following Intravaginal Inoculation of Herpes Simplex Virus Type 2

The tissue sites of long-term herpes simplex virus type 2 (HSV-2)-specific antibody production in mice and guinea pigs were identified. In addition to secondary lymphoid tissue and bone marrow, HSV-specific plasma cells were detected in spinal cords of mice up to 10 months after intravaginal inoculation with a thymidine kinase-deficient HSV-2 strain and in lumbosacral ganglia and spinal cords of guinea pigs inoculated with HSV-2 strain MS. The long-term retention of virus-specific plasma cells in the peripheral and central nervous systems following HSV infection may be important for resistance to reinfection of neuronal tissues or may play a role in modulation of reactivation from latency.

Double Labelling Immunohistochemistry on the Peripheral Autonomic Neurons Projecting to the Bulbospongiosus Muscle in Male Impuberal Pigs

The functional involvement of striated perineal muscles (bulbospongiosus, external anal sphincter, ischiocavernosus and external urethral sphincter) in the activity of urogenital and digestive organs, has required definition of the central and peripheral sites of their neurons. Preliminary observations (Botti et al., 2001), carried out using the retrograde neuronal tracer Fast Blue (FB), have documented the central and peripheral site of the motor and sensitive neurons projecting to the bulbospongiosus muscle (BSM) in the swine species. It has been documented that the somatic motor nucleus is located ipsilaterally (S1–S2) in a dorsolateral area with respect to the central canal. The peripheral autonomic neurons projecting to the blood vessels of the BSM are located bilaterally in the lumbosacral ganglia (L2–S4) of the sympathetic chain and in the caudal mesenteric ganglion, while the primary sensitive neurons are located bilaterally in the sacral spinal ganglia (S1–S3). The purpose of this research is to define, using double immunofluorescence labelling, the neurochemical characteristics of some peripheral autonomic neurons projecting to the BSM.

Distribution and changes with age of calcitonin gene-related peptide- and substance P-immunoreactive nerves of the rat urinary bladder and lumbosacral sensory neurons.

In the distal parts of the urinary tract, nerves containing calcitonin gene-related peptide (CGRP) or substance P (SP) are sensory with their cell bodies located in lumbosacral dorsal root ganglia. These two neuropeptides are recognised as being present in pelvic sensory nerves, and may be involved in the mediation of pain, stretch and/or vasodilatation. We have used indirect immunohistochemical techniques to examine the distribution and regional variation of nerves immunoreactive (-ir) for CGRP and SP in the urinary bladder and in neurons in lumbosacral dorsal root ganglia (L1-L2 & L6-S1) of young adult (3 months) and aged (24 months) male rats. Semi-quantitative estimations of nerve densities were made for CGRP-ir and SP-ir fibres innervating the dome, body and base of the urinary bladder. Quantitative studies were also used to examine the effects of age on the percentage of dorsal root ganglion neurons immunoreactive for CGRP and SP. There were very few immunoreactive axons in the dome and the overall density of innervation increased progressively towards the base of the bladder. The density of innervation in the aged rats revealed a slight reduction in CGRP and SP innervation of the detrusor muscle but was otherwise comparable to the young group. However, immunostaining of the lumbosacral dorsal root ganglia revealed that the percentage of CGRP- and SP-ir neuronal profiles showed a significant (P < 0.05) reduction from (mean +/- S.D) 44.5 +/- 2; 23.3 +/- 2 in young adult to 25.0 +/- 2.9; 14.8 +/- 1.6 in aged rats, respectively. These findings suggest that the involvement of CGRP and SP in urinary bladder innervation is relatively unchanged in old age, but their expression in dorsal root ganglion neurons is affected by age. The afferent micturition pathway from the pelvic region via these lumbosacral ganglia may be perturbed as a result.

Role of polymorphonuclear leukocytes in resolution of HSV-2 infection of the mouse vagina

A naturally occurring population of polymorphonuclear leukocytes (PMNs) was detected in the vaginal lumen of uninoculated mice. A large population of these cells also infiltrated the vaginal mucosa following intravaginal HSV-2 inoculation. We examined the role of PMNs in preventing infection of the vaginal mucosa, virus clearance, and limiting virus spread to the lumbosacral ganglia. Depletion of PMNs prior to HSV-2 inoculation did not increase the incidence of infection suggesting that the small population of resident PMNs was ineffective in preventing infection by a viral pathogen. Depletion of PMNs impacted virus clearance from the vagina over a range of HSV-2 doses resulting in significantly higher virus titers on days 4 through 6 after inoculation. Virus clearance was delayed in PMN-depleted immune μMT mice suggesting that PMN involvement in HSV-2 clearance did not require specific antibody. PMN-depletion of non-immune mice increased virus spread to the sensory ganglia only in mice inoculated with high virus doses. Immunization of mice with an attenuated strain of HSV-2 protected the sensory ganglia against acute infection with a challenge strain. Although PMN depletion of immune mice significantly increased virus titers in the vagina, the incidence of acute virus replication in the sensory ganglia was not different than in control-treated immune mice suggesting that PMNs were not required for protection of the sensory ganglia in immune animals. Taken together, these results suggest that PMNs were involved in resolving genital HSV-2 infections, but played only a limited role in preventing HSV-2 spread to the sensory ganglia.

Glycine receptors containing the alpha4 subunit in the embryonic sympathetic nervous system, spinal cord and male genital ridge.

Inhibitory glycine receptors (GlyRs) are known to mediate postsynaptic inhibition in spinal cord, brain stem and some higher brain regions. Several developmentally and regionally regulated GlyR isoforms exist, which result from a differential expression of the GlyR alpha (alpha1-alpha4) and beta subunit genes. Currently, very little is known about GlyRs containing the alpha4 subunit, whose existence was predicted from a partial genomic sequence. Here, we describe the isolation of complementary DNA (cDNA) sequences for the mouse and chick GlyR alpha4 subunits. We show that a mouse GlyR alpha4 subunit full-length cDNA directs the formation of functional homo-oligomeric strychnine-sensitive GlyRs in Xenopus laevis oocytes and mammalian cells, and that these resemble GlyRs composed of the alpha1 subunit in pharmacological profile and single-channel properties. In situ hybridization reveals high levels of GlyR alpha4 subunit transcripts in the embryonic (E13) chick spinal cord, lumbosacral sympathetic ganglia and dorsal root ganglia. The avian GlyR alpha4 subunit gene also shows male-specific expression in the developing genital ridge. The pharmacological profile of alpha4 subunit-containing receptors and deduced location of the avian GlyR alpha4 subunit are consistent with it being a component of the embryonic excitatory GlyRs previously identified in sympathetic neurons. Our data also suggest a novel role for GlyRs in the maturation of reproductive organs.

The quantity of latent viral DNA correlates with the relative rates at which herpes simplex virus types 1 and 2 cause recurrent genital herpes outbreaks.

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) have evolved specific anatomic tropisms and site-dependent rates of reactivation. To determine whether reactivation rates depend on distinct abilities of HSV-1 and -2 to establish latency and to express latency-associated transcripts (LATs), virulent strains of each virus were studied in the guinea pig genital model. Following infection with equivalent titers of virus, the quantities of latent HSV-2 genomes and LATs were higher in lumbosacral ganglia, and HSV-2 infections recurred more frequently and lasted longer than HSV-1 infections. In contrast, if the inoculum of HSV-1 was 10 times that of HSV-2, the quantity of HSV-1 DNA and LATs increased correspondingly and HSV-1 infections were as likely to recur as those with HSV-2. The quantity of latent virus DNA correlates with and may be a major determinant of the site-specific patterns and rates of reactivation of HSV-1 and -2.

NGF-Mediated Survival Depends on p21ras in Chick Sympathetic Neurons from the Superior Cervical but Not from Lumbosacral Ganglia

In rat embryonic sympathetic neurons from the superior cervical ganglia (SCG) NGF-mediated survival depends on the activation of the trkA receptor tyrosine kinase and on the activity of the intracellular plasmamembrane-anchored small G-protein p21ras. In contrast, chick sympathetic neurons derived from the more caudally located lumbosacral chain ganglia (LSCG) do not respond to activated p21ras (G12V-Ha-ras mutant). In these neurons endogenous p21ras and its downstream effector MAP kinase are activated but are not essential for NGF-dependent survival. Here we show that also in chick sympathetic neurons of the SCG permanently activated p21ras protein does promote neuron survival. Consistently, their NGF-mediated survival is sensitive to Fab fragments blocking endogenous p21ras activity. These results suggest that sympathetic neurons derived from sympathoenteric (SCG) and sympathoadrenal (LSCG) lineages differ in their requirement for p21ras in the NGF-mediated survival pathways.

9 Culture of Avian Sympathetic Neurons

This chapter provides a summary of techniques that have proven to be successful over many years in laboratory in the study of sympathetic neuron development, and that can be easily established by newcomers in the field. A particular property of immature sympathetic neurons is the possibility of obtaining exogenous gene expression by transfection. The reason that this method is much less time consuming and difficult than the single-cell injections widely used for primary neuron cultures, details of this procedure are also provided in the chapter. Two major sources of sympathetic neurons are commonly used, the neonatal superior cervical ganglia (SCG) of the rat and mouse and avian lumbosacral chain ganglia. The advantage of the latter ganglia is that ganglia containing mainly proliferating, immature neurons and ganglia containing differentiated, postmitotic neurons are easily accessible, and large numbers of cells can be obtained. Recent data indicate that sympathetic neurons of SCG and trunk sympathetic ganglia belong to different cell lineages and may display different properties.

Detection of Canine Herpesvirus 1 in a wide range of tissues using the polymerase chain reaction

Canine herpesvirus 1 (CHV-1), a member of the alphaherpesvirus sub-family, is known to cause fatal infections in litters of puppies and may also be involved in infertility, abortion, and stillbirths in adult dogs. The purpose of this study was to determine the presence of CHV-1 DNA using the polymerase chain reaction (PCR) in twelve key sites that have been associated with latency for other herpesviruses. A 605 base pair portion of the viral glycoprotein B (gB) gene was amplified using degenerate primers, cloned, and sequenced. Conventional 20mer primers were designed using this sequence information to amplify a 120 bp fragment of gB situated between the original degenerate primers. The specificity of amplification was confirmed by Southern Blot hybridisation using an internal oligonucleotide probe. DNA was extracted from tissue samples taken from twelve dogs at post mortem and from twenty-four blood samples. Nine out of twelve dogs showed evidence of infection with CHV-1; the tissues most commonly affected were lumbo-sacral ganglia ( 5 12 dogs), tonsil ( 5 12 ), parotid salivary gland ( 4 9 ), and liver ( 4 9 ). No positive results were detected within the twenty-four blood samples. These results indicate that exposure to CHV-1 may be much more common than previously suggested.

Prenatal development of rat primary afferent fibers: I. Peripheral projections.

Development of the peripheral innervation patterns of the L1-S1 lumbosacral ganglia and motor segments in embryonic day 12-17 (E12-17) rat embryos was examined using carbocyanine dyes. Individual dorsal root ganglia (DRGs) and/or isolated ventral horn (VH) segments, or individual peripheral nerves, were isolated in rat embryos fixed at different stages and filled with one of three carbocyanine dyes; DiI, DiA, and DiO. Individual experimental preparations included labeling of 1) single DRGs; 2) multiple DRGs with alternating dyes, DiO, DiI, and DiA; 3) single isolated VH segments; 4) multiple VH segments with alternating dyes; 5) single VH segments and the corresponding segmental DRGs with different dyes; and 6) two or more individual peripheral nerves labeled with different dyes. Results from these preparations have shown that the first fibers exited the lumbar ventral horn and DRGs at E12. At E13 major nerve trunks (e.g., femoral and sciatic) were visible as they exited the plexus region. By E14 afferent fibers were present in the epidermis of the proximal hindlimb, and the major nerve trunks extended into the leg. Fibers originating from L3 to L5 (DRG and VH) reached the paw by E14.5-E15, and the epidermis of the most distal toes was innervated by E16-E16.5. While afferent fibers and motor axons of the same segmental origin mixed extensively in the spinal nerve, fibers of different segmental origin combined in the plexus and major nerve trunks with little or no interfascicular mixing. Dermatomes observed at E14 were in general spotty and non-contiguous. However, by E16 the dermatomes resembled mature forms with substantial overlap only between adjacent ones. Thus the adult pattern of spatial relationships between cutaneous afferent fibers in the periphery is established early in development.