Daniel L. McNeill

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.

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.

Vagal afferent fibers excite upper cervical neurons and inhibit activity of lumbar spinal cord neurons in the rat

&NA; Effects of electrical stimulation of the cervical vagus nerve were determined in cervical or lumbar spinal neurons in 27 rats anesthetized with pentobarbital. Ipsilateral cervical vagus stimulation (ICVS) increased activity of 44 neurons in the C1 segment. At the same stimulation parameters, contralateral cervical vagus stimulation (CCVS) either increased, decreased or did not affect activity of C1 neurons that were excited by ICVS. For C1 cells excited by both ICVS and CCVS, the mean latency for activation was significantly longer for CCVS than for ICVS, and ICVS produced a greater degree of excitation than CCVS. In segments C2‐C6, 16 of 18 neurons were excited by ICVS and 2 were inhibited. However, CCVS did not excite the C2‐C6 neurons but either inhibited or did not affect activity. In 6 cervical cells, a CCVS conditioning stimulus reduced the level of excitation by ICVS (test stimulus). Transection of the C2 or C3 dorsal roots did not significantly affect the excitatory vagal input to C1 cells. Excitatory somatic receptive fields were classified for 60 cervical spinal cells that responded to vagal stimulation. Most (87%) cells were excited by noxious pinch; 29 were wide dynamic range (WDR) cells and 21 were high threshold cells. In contrast to upper cervical neurons, spinothalamic tract (STT) and spinal cells in lumbar segments were not excited by ICVS or CCVS at the stimulation parameters used in this study, but were primarily inhibited by vagal stimulation. Results of this study showed that a group of cells in upper cervical segments were excited by vagal afferents. This excitatory vagal input reaches the C1 segment primarily via an ipsilateral, supraspinal route.

Projection of nodose ganglion cells to the upper cervical spinal cord in the rat

Afferent fibers mediating pain from myocardial ischemia classically are believed to travel in sympathetic nerves to enter the thoracic spinal cord. After sympathectomies, angina pectoris still may radiate to the neck and inferior jaw. Sensory fibers from those regions are thought to enter the central nervous system through upper spinal cord segments. We postulated that axons from nodose ganglion cells might project to cervical cord segments. The purpose of this study was to determine the density and pathway of vagal afferent innervation to the upper cervical spinal cord. Following an injection of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the upper cervical spinal cord, approximately 5.8% of cells in the nodose ganglion contained reaction product. Cervical vagotomy did not diminish the density of WGA-HRP labeled cells in the nodose ganglion. However, a spinal cord hemisection cranial to the injection site eliminated labeling of nodose cells. These data indicate that a portion of vagal afferent neurons project from the nodose ganglion to the upper cervical spinal cord. In addition, vagal afferent fibers reach the spinal cord via a central route rather than through dorsal root ganglia.

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.

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.

Acute and chronic ethanol alter somatosensory-evoked potentials in conscious rats

This study examined the effects of acute and chronic ethanol on cortical somatosensory-evoked potentials (SEPs) of laboratory rats. Evoked potentials were recorded following stimulation of the left hindpaw before and after injection of either saline or ethanol. Animals were then chronically exposed to ethanol in vapor inhalation chambers for five weeks. Recordings of SEPs before and after acute ethanol injection were then obtained 24 h and again seven days after withdrawal from ethanol exposure. The results indicate that acute ethanol produced a dose-dependent reduction in SEP amplitude, but did not alter peak latencies. Chronic ethanol exposure and withdrawal resulted in a significant increase in preinjection baseline response amplitudes when measured at 24 h after withdrawal, but not at seven days, and this treatment did not alter response latency or the effects of acute ethanol administration.

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.