Raymond E. Papka

Origin and distribution of NADPH-diaphorase-positive neurons and fibers innervating the urinary bladder of the rat.

Nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry was utilized to localize nitric oxide synthase (NOS), and thus sites where nitric oxide (NO) can be synthesized, within peripheral nervous system perikarya and fibers. Recent studies suggest that NO relaxes vascular and non-vascular smooth muscle. In this study, the origin and distribution of NADPH-diaphorase perikarya and fibers in the rat urinary bladder were examined. Results suggest that a small number of NADPH-diaphorase-positive perikarya are present within the bladder wall and within adjacent small ganglia. In addition, NADPH-diaphorase-positive nerve fibers were observed in the adventitial and muscular layers, subjacent to the urothelium and as perivascular fibers. After injection of the retrograde tracer fluorogold (FG) into the bladder wall, numerous FG-labeled perikarya in the major pelvic ganglia and the T13-L2, L6 and S1 dorsal root ganglia were NADPH-diaphorase positive. However, none of the FG-labeled perikarya in the inferior mesenteric ganglia were NADPH-diaphorase positive. The prevalence of NADPH-diaphorase-positive perikarya and fibers suggests that NO may serve a role in bladder function.

Estrogen receptor‐immunoreactive neurons are present in the female rat lumbosacral spinal cord

Presence of an estrogen receptor is crucial for cells to respond to estrogen; thus, estrogen‐responsive neurons should be identifiable by immunohistochemically staining for the estrogen receptor (ER). Even though spinal neurons are involved in sexual behaviors and innervation of genital organs, little information is available about ER‐containing neurons in the spinal cord. Consequently, we have undertaken a study of ER‐containing neurons in the female rat lumbosacral cord, an area involved in reproductive functions and predicted to contain estrogen‐responsive neurons. In addition, since parasympathetic preganglionic neurons in the lumbosacral cord produce nitric oxide (NO), we also sought to determine if ER‐immunoreactive (‐IR) neurons contain the enzymes for NO production. Finally, we compared the distribution of ER‐IR neurons to the presence of uterine cervix‐related neurons. Uterine cervix‐related neurons were identified by expression of FOS‐immunoreactivity after vaginocervical mechanostimulation (VCS).

Distribution of NADPH-diaphorase-positive nerves in the uterine cervix and neurons in dorsal root and paracervical ganglia of the female rat

Nitric oxide synthase (NOS) was selectively stained in nerve fibers of the uterine cervix and neurons of the paracervical (PG) and dorsal root ganglia (DRG) by NADPH diaphorase histochemistry. In the cervix, numerous NADPH-diaphorase-positive nerve fibers were observed in the myometrium, endometrium and around arteries. In addition, a subpopulation of neurons within ganglia that innervate the cervix, i.e., the PG and DRG, were NADPH-diaphorase positive; thus the fibers in the cervix could be sensory and/or autonomic. NADPH-diaphorase/NOS localization identifies sites where nitric oxide (NO) can be synthesized. Since NO relaxes vascular and nonvascular smooth muscle, the prevalence and anatomical localization of NADPH-diaphorase-positive fibers suggest that they could influence functions of the uterine cervix.

CGRP immunoreactivity and NADPH-diaphorase in afferent nerves of the rat penis

Calcitonin gene-related peptide (CGRP)-immunoreactive afferent nerve fibers are abundant in the rat penis. In addition, NADPH-diaphorase, which stains for nitric oxide synthase, has been localized within both autonomic and sensory dorsal root ganglia (DRG) and may be part of an important biochemical pathway involved in penile tumescence. The purpose of this study was: 1) to examine the circuitry of afferent nerves that are CGRP immunoreactive from the L6 DRG, 2) to examine the possibility that there are NADPH-diaphorase-positive afferent fibers from the L6 DRG to the rat penis, and 3) to examine the localization and colocalization of CGRP and NADPH-diaphorase within L6 DRG afferent perikarya. Calcitonin gene-related peptide immunostaining in the penis was eliminated following a bilateral transection of the pudendal nerves, but was unchanged following a bilateral transection of the pelvic splanchnic or hypogastric nerves. The NADPH-diaphorase staining was not altered by any of the nerve transections. Injection of the retrograde axonal tracer fluorogold (FG) into the dorsum penis labeled perikarya in the L6 DRG. Although the majority of FG-labeled perikarya contained neither CGRP nor NADPH-diaphorase, small subpopulations of perikarya contained either CGRP immunoreactivity, NADPH-diaphorase, or both. A unilateral pudendal nerve transection virtually eliminated (> 99%) FG labeling in the ipsilateral L6 DRG. These data suggest that NADPH-diaphorase and CGRP are present, either together or separately, within a subpopulation of penile afferent perikarya. In addition, CGRP-immunoreactive afferent nerve fibers reach the penis primarily via the pudendal nerves. Finally, NADPH-diaphorase-positive penile afferents may be another important source of nitric oxide (NO) for penile tumescence.

NADPH-diaphorase-positive nerves and the role of nitric oxide in CGRP relaxation of uterine contraction

We previously demonstrated calcitonin gene-related peptide (CGRP) immunoreactivity in sensory nerves in the rat uterus and that CGRP inhibits stimulated uterine contraction in vitro. The present study was undertaken to: 1) examine possible roles nitric oxide (NO) may have in the inhibitory action of CGRP on uterine contraction and 2) identify sites where NO may be synthesized. The relaxing effect of CGRP on SP-stimulated uterine contraction was established in vitro on uterine horns from diethylstilbestrol-treated rats. These experiments were repeated with or without an arginine analog [NG-monomethyl-L-arginine (L-NMMA)] that inhibits NO formation. The localization of the synthetic enzyme for NO production, NO synthase, was accomplished by histochemically staining for NADPH-diaphorase. Calcitonin gene-related peptide (10(-7) M) significantly reduced SP (10(-5) or 10(-6) M)-stimulated uterine contraction. The L-NMMA (10(-3) M) blocked the relaxing action of CGRP on SP-stimulated uterine contraction. The L-NMMA alone had no effect on SP-stimulated uterine contraction. NADPH-diaphorase-positive nerve fibers were located in the myometrium, endometrium, and adjacent to the vasculature. These data demonstrate that: 1) L-NMMA suppresses the relaxant effect of CGRP on myometrial activity and 2) NADPH-diaphorase (indicative of NO synthase) is localized in uterine nerve fibers. These data suggest that the inhibitory action of CGRP may be dependent on NO formation and that the enzyme necessary for NO production is present in nerves in areas optimal to affect myometrial activity.

Calcitonin gene-related peptide in the rat uterus: Presence in nerves and effects on uterine contraction

The influence of calcitonin gene-related peptide (CGRP) on rat uterine activity was examined in concert with the anatomical distribution of CGRP-like immunoreactivity in the uterus. CGRP-like immunoreactivity was localized in nerve fibers; these peptide-containing nerves were abundant throughout the mesometrium of the uterine horn and appeared to innervate mesometrial smooth muscle and vascular smooth muscle. In the uterine wall, CGRP-like immunoreactive fibers were prevalent in the myometrium, endometrium and the endocervix. Fibers in the endometrium and endocervix appeared to form a plexus subjacent to the epithelium and some fibers penetrated the epithelium as an intraepithelial plexus. The action of CGRP (10(-9) to 10(-6) M) on acetylcholine (10(-6) or 10(-5) M)-stimulated uterine activity was examined in vitro. Exogenously applied CGRP induced a dose-dependent relaxation of acetylcholine-stimulated uterine contractions. CGRP had no effect on basal uterine tension. The localization of CGRP-like immunoreactivity in nerves and the relaxing effect of CGRP suggests a role for CGRP-containing nerve fibers in the regulation of uterine activity.

Identification of uterine-related sympathetic neurons in the rat inferior mesenteric ganglion: neurotransmitter content and afferent input

The rat uterus is innervated by sensory and autonomic nerves. Sensory and sympathetic fibers travel in the hypogastric nerves and are associated with the thoracolumbar spinal cord levels T13-L3. The inferior mesenteric ganglion (IMG) contains the somata of sympathetic postganglionic neurons and some of these may project axons to the uterus. Sensory and parasympathetic fibers travel in the pelvic nerve and are associated with the lumbosacral cord levels L6-S1 and pelvic ganglion (PG). We previously reported data concerning the neurochemical anatomy of the PG with regard to the uterine innervation; the present study was undertaken to characterize the neurochemical anatomy of the IMG with regard to it involvement in uterine innervation. A retrograde axonal tracer was used to verify projections of axons of IMG neurons to the uterus. Immunostaining of cryostat sections of the IMG revealed neurons immunoreactive for neuropeptide Y (NPY) and for tyrosine hydroxylase (TH). Immunostaining for the synaptic terminal protein synapsin I (SYN) revealed numerous fine terminals immediately surrounding the principal neurons and in the interneuronal spaces. Varicosities immunoreactive for calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), enkephalin (ENK), substance P (SP) and galanin (GAL) appear to be associated with principal neurons. Additional varicosities stained for nicotinamide adenine dinucleotide phosphate (reduced)-diaphorase (NADPH-d) and nitric oxide synthase (NOS), thus indicating sites of neuronal nitric oxide synthesis. This study revealed that the IMG contains uterine-related neurons and that some of the retrogradely labeled uterine-related neurons contain NPY, TH or both NPY/TH. In addition, uterine-related neurons received abundant afferent inputs indicated by SYN-immunoreactive (-ir) terminals and some of these varicosities labeled for GAL, CGRP, VIP, ENK, or NADPH-d/NOS.

CYTOSOLIC ESTROGEN RECEPTOR CONCENTRATIONS IN THE LUMBOSACRAL SPINAL CORD FLUCTUATE DURING THE ESTROUS CYCLE

Estrogen responsive neurons have been anatomically identified with autoradiographic and immunohistochemical techniques and their distribution mapped in the lumbosacral spinal cord of female rats. Such neurons contain estrogen receptors (ERs). The present study was undertaken to: 1) quantify cytosolic estrogen receptor (ER) concentrations in the lumbosacral spinal cord and 2) determine if there is a relationship between cytosolic ER concentrations and fluctuations in serum estradiol (SE2) levels during the estrous cycle. Lumbosacral spinal segments were removed from intact cycling rats during the morning of proestrus, the afternoon of proestrus, and the morning of estrus, metestrus and diestrus. Trunk blood was collected at euthanasia and SE2 levels were determined using radioimmunoassay. Cytosolic ER concentrations were measured using a dextran-charcoal coated tube method. Concentrations of cytosolic ERs were low during estrus and metestrus, increased during diestrus with maximum concentrations during the afternoon of proestrus. These changes in ER concentrations paralleled SE2 levels measured in intact cycling animals; i.e., during estrus SE2 levels were low, but began to rise during metestrus, diestrus, and during the morning of proestrus with a maximum peak increase during the afternoon of proestrus. These data indicate there are fluctuations of cytosolic ER concentrations during the estrous cycle and that these changes coincide with changing SE2 concentrations suggesting that ER content is influenced by SE2.

Coexistence of calcitonin gene-related peptide and galanin immunoreactivity in female rat pelvic and lumbosacral dorsal root ganglia

Coexistence of immunoreactivity for calcitonin gene-related peptide (CGRP) and galanin (GAL) was examined in varicose nerve endings in female rat pelvic paracervical ganglia (PG) and in perikarya of lumbosacral dorsal root ganglia (DRG). Varicose peptide-containing nerves were closely adjacent to somata of neurons in the PG, certain somata being virtually surrounded by immunoreactive varicosities. Some nerve endings were immunoreactive for either CGRP or GAL; in others, immunoreactivity for CGRP and GAL coexisted. Likewise, many perikarya in DRG were CGRP immunoreactive, fewer were GAL immunoreactive, and in some immunoreactivity for CGRP and GAL coexisted. The results suggest there are subpopulations of neuropeptide-containing sensory nerve endings in the PG; some contain CGRP, some contain GAL, and in some CGRP and GAL coexist. These substances contained in sensory nerve endings could have important roles in pelvic ganglionic functions.

CGRP-immunoreactive primary afferent nerve fibers in the rat urinary bladder : effects of dorsal rhizotomy and MK-801

A transection lesion of the suprasacral spinal cord results in a decreased density of calcitonin gene-related peptide (CGRP)-immunoreactive (I) primary afferent nerve fibers in the rat urinary bladder. The fiber density can be restored by postsurgical treatment with the N-methyl-D-aspartate receptor antagonist MK-801. We are attempting to determine the level of the primary afferent neuron at which MK-801 might have a restorative effect on CGRP immunostaining. Thus, the purpose of this study was to determine if MK-801 had a similar restorative effect on immunostaining for CGRP in bladder nerves after a direct lesion of the sacral afferent system, i.e., rhizotomy of the L6 and S1 dorsal roots. To assess the effect of the lesion, the mean length and number of bladder CGRP-I nerve fibers, as well as the number of CGRP-I perikarya in the L6 and S1 dorsal root ganglia (DRG), were measured following bilateral L6 and S1 dorsal rhizotomies. Both the mean length and the numbers of CGRP-I bladder fibers were significantly decreased by the lesion. However, the number of CGRP-I primary afferent perikarya in the L6 and S1 DRG was unchanged from control values. Rats which received rhizotomies and subsequent treatment with MK-801 did not exhibit restoration of the density of CGRP-I bladder fibers nor an alteration in the number of CGRP-I primary afferent perikarya. These data suggest that MK-801-induced restoration of bladder CGRP-I primary afferent nerve fibers may rely on an intact central process.