Parathyroid hormone-related protein inhibits stimulated uterine contraction in vitro.

The effect of parathyroid hormone-related protein (PTHrP) fragments 1-34, 38-64, and 67-86 on acetylcholine-stimulated rat uterine contraction was examined in vitro. In addition, the possibility that PTHrP-(38-64) or (67-86) influenced relaxation caused by PTHrP-(1-34) was also investigated. Contraction of uterine horns was stimulated with 10(-6) or 10(-5) M acetylcholine. PTHrP-(1-34) reduced the magnitude of acetylcholine-stimulated uterine contraction. This effect was dose related over a concentration range of 10(-9)-10(-6) M. Neither PTHrP-(38-64) or PTHrP-(67-86) at concentrations of 10(-8)-10(-6) M affected uterine contraction stimulated by 10(-6) M acetylcholine. These fragments did not affect the relaxation caused by 10(-7) M PTHrP-(1-34). These results demonstrate that (1) PTHrP-(1-34) at 10(-6) M influences contraction of the rat myometrium and (2) the muscle relaxant activity of PTHrP is associated with the first 34 N-terminal amino acids.

Organization of calcitonin gene-related peptide-immunoreactive terminals in the primate dorsal horn.

The present paper is concerned with the arrangement of axons and synaptic terminals immunostained for calcitonin gene-related peptide (CGRP), a primary afferent marker, in the primate (Macaca fascicularis) dorsal horn. The CGRP axons and terminals are uniformly distributed in laminae I and II outer (o) but they are concentrated laterally and distributed intermittently in the reticulated region of lamina V. A prominent bundle of labeled axons is seen in the sacral cord dorsal to the central canal. Emphasis is given to the relation of CGRP-immunoreactive terminals to other terminals, both labeled and unlabeled, in laminae I and IIo. In this regard, adjacent CGRP-immunoreactive terminals are often united by puncta adhaerentia. Of particular interest is the observation that CGRP-immunoreactive terminals can be found presynaptic to other terminals which sometimes resemble central primary afferent endings. In addition CGRP-immunoreactive terminals end on other CGRP terminals. Both findings suggest that primary afferent terminals interact synaptically with other primary afferent terminals.

Calcitonin gene-related peptide immunostained axons provide evidence for fine primary afferent fibers in the dorsal and dorsolateral funiculi of the rat spinal cord.

The hypothesis being tested in the present paper is that there are large numbers of fine primary afferent axons in the dorsal and dorsolateral funiculi of the lumbar spinal cord of the rat. The data show numerous calcitonin gene-related peptide labeled fine myelinated and unmyelinated axons in these funiculi. Approximately 95% of the labeled axons disappear after dorsal rhizotomy. Accordingly, the hypothesis is confirmed. Thus it is becoming apparent that fine primary afferent fibers are more widely distributed in spinal white matter than had been previously recognized. Implications are that it is not possible to find areas in the spinal white matter that contain only large myelinated sensory axons and that significant numbers of fine primary afferent fibers will be lost even if lesions are restricted to the dorsal funiculus. The sizable population of fine myelinated primary afferent axons in the dorsal funiculus is emphasized. An obvious question, suggested by significant differences in average diameters of the axons in the different pathways, is whether there are differences in the types of information carried by the fine afferent fibers in their different locations in the white matter of the lumbar cord.

Changes in dorsal horn synaptic disc numbers following unilateral dorsal rhizotomy.

The present study estimates the numbers of synaptic discs and numbers of degenerating synaptic terminals in laminae I-IV of the rat S2 dorsal horn ipsi- and contralateral to unilateral dorsal rhizotomy. These data allow us to estimate the loss of synapses of primary afferents and to correlate this loss with the rate of axon disappearance in the proximal stump of a transected S2 dorsal root. Our first findings are that 47% of the ipsilateral synapses and 27% of the contralateral synapses disappear within a day following unilateral rhizotomy. Conclusions are that the predominant synaptic population in this part of the rat spinal cord is of primary afferent origin and that there is an extensive bilateral projection of the dorsal root fibers. The contralateral projection is confirmed by the appearance of numerous degenerating terminals on the contralateral side. We also find that synaptic loss and appearance of degenerating terminals occur relatively synchronously in laminae I-IV. Finally we find that the time course of the synaptic loss correlates primarily with the disappearance of unmyelinated fibers in the proximal stump of the transected dorsal root.

Denervation-induced intraspinal synaptogenesis of calcitonin gene-related peptide containing primary afferent terminals.

The purpose of the present study is to provide evidence that chronic spinal denervation leads to an increase in numbers of synaptic terminals from a specific population of primary afferent fibers. Rats were unilaterally deafferented for 35 days (chronic denervation) by dorsal rhizotomies performed from T2 to T8 and T10 to L5, which isolates or spares the T9 root. The contralateral T9 root was spared by similar surgery 5 days (acute denervation) prior to sacrifice. The survival time on the chronic side presumably allows sprouting of T9 primary afferents to occur, whereas the time on the acute side does not. The terminals were labeled with calcitonin gene-related peptide (CGRP), which is a compound that labels a specific population of primary afferent fibers and terminals, and stereological methods were used to determine the numbers of immunolabeled terminals in laminae I and IIo on the chronic and acute sides of the T9 spinal cord. The findings are that the chronic side had approximately twice as many terminals as the acute side. This difference is statistically significant. These findings are compatible with the hypothesis that chronic denervation leads to synaptogenesis from surviving primary afferent fibers.

Numbers of synapses in laminae I-IV of the rat dorsal horn.

The present study determines numerical densities (NVsyn) and total numbers of synaptic discs in laminae I-IV of the rat S2 dorsal horn. Previous methods for NVsyn have the advantage of being relatively simple, but these assume that the discs are round, flat, and of uniform size. In our material, serial reconstructions indicate that these assumptions are not met. Accordingly we use a stereological method that is not as dependent on these assumptions. This method is to divide the surface density of the discs by the mean surface area of a disc (NVsyn = SVsyn/Ssyn). We refer to this as a reconstruction method because synaptic discs are reconstructed from serial sections. We also calculate numerical densities by several previously used standard methods, and the findings are similar but not identical. We find that numerical density and total synaptic numbers are smallest in lamina I, and densities and total numbers are not significantly different when lamina II is compared to laminae III and IV. Thus the intense labeling of terminals with certain compounds that characterize lamina I and II does not imply an increase in total synaptic numbers or in synaptic density. In addition there is a general increase in synaptic densities and numbers as one proceeds from lamina I to lamina IV. Another point is that the numerical density of synapses in the dorsal horn is approximately that of the cerebral cortex. These data will serve as a basis from which to judge the effects of denervations and other manipulations that purportedly change synaptic numbers.

Ascending unmyelinated primary afferent fibers in the dorsal funiculus.

The primary purpose of the present study is to obtain evidence as to the destination of the recently discovered unmyelinated primary afferent fibers in the mammalian dorsal funiculus. To do this rat dorsal roots were transected unilaterally from segments T8 or T9 caudally, and the numbers of axons were determined in the C3 fasciculus gracilis in normal animals and from both sides of the rhizotomied animals. In addition, C3 fasciculus gracilis counts were done in animals that had complete T6 or T10 spinal transections. The data indicate that there is an 80% loss of unmyelinated axons ipsilaterally and a 60% loss contralaterally in the fasciculus gracilis of the rhizotomied animals. These findings are interpreted as indicating that a significant fraction of the unmyelinated fibers in the fasciculus gracilis ascend, presumably to the nucleus gracilis in the brain stem, and also that a significant number of these fibers branch. We also provide evidence for contralateral myelinated primary afferent fiber projection in the fasciculus gracilis and show that the myelinated primary afferent fibers seem to be a more diverse population than the unmyelinated primary afferent fibers in the C3 fasciculus gracilis.

Spinal projection neurons to the laterodorsal pontine tegmental nucleus: relationship to preganglionic neurons and nitric oxide synthase.

The region of the rat sacral parasympathetic nucleus (SPN) contains distinct subpopulations of neurons that project supraspinally or are preganglionic neurons. Some preganglionic neurons in the SPN serve as the motor outflow for urinary bladder contraction; other neurons in the SPN project to regions of the rostral pons that subserve micturition reflexes. Previous studies utilizing immunohistochemistry or staining for nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) have demonstrated that numerous neurons in the SPN contain nitric oxide synthase (NOS), the enzyme for nitric oxide synthesis. Thus, the objectives of this study were to determine 1) the distribution of neurons in the region of the SPN that project to the laterodorsal tegmentum (LDT) of the pons, 2) whether spinal neurons projecting to a peripheral autonomic ganglion also project to the LDT, and 3) whether NOS or NADPH-d is present in LDT projection neurons. Preganglionic neurons were identified by injecting the retrograde tracer fluorogold (FG) into the major pelvic ganglion (MPG). Supraspinally projecting neurons were identified by injecting the retrograde tracer fast blue (FB) into the LDT. Numerous FB-labeled neurons were present in the ipsi- and contralateral SPN and were immediately dorsal to FG-labeled preganglionic neurons. Neurons containing both tracers were not observed. Approximately 20% of preganglionic neurons, but no LDT projection neurons, were reactive for NOS and NADPH-d. These data suggest that the region of the SPN is a site for distinct subpopulations of neurons that project to the LDT and to the MPG and that NOS is contained in some preganglionic neurons, but is not a marker for LDT projection neurons.

Peptide immunoreactivity of unmyelinated primary afferent axons in rat lumbar dorsal roots.

The present study demonstrates calcitonin gene-related peptide (CGRP), somatostatin (SOM), bombesin (BOM), and substance P (SP) at the electron microscopic level in lumbar dorsal root axons of normal rats. The highest percentages of labeled axons were for CGRP (14%) and then, in descending order, for SP (8.6%), SOM (6.8%), and BOM (3.1%). The labeled axons were exclusively unmyelinated for SP, SOM, and BOM, and predominantly unmyelinated for CGRP. These data are consistent with the data for labeled sensory cell bodies for these same compounds. We emphasize that these peptides were immunocytochemically visualized in the dorsal roots without experimental manipulation, such as colchicine or dorsal root ligation. Quantitative sampling of this type can be used to assay changes in response to physiological stimuli in numbers of sensory axons that contain identifiable concentrations of these peptides.

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.

Substance P and calcitonin gene-related peptide immunoreactivity in nerves of the rat uterus: localization, colocalization and effects on uterine contractility.

Immunoreactivity to the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) was examined in nerves in the rat uterus as a prelude to studying their effects on uterine contractility. With immunocytochemical techniques, SP immunoreactivity (SP-I) and CGRP-I were localized in myometrial nerves throughout the uterine horns, with nerves immunoreactive for CGRP being the more numerous. Immunocytochemical double labeling studies revealed SP coexisted with CGRP in a subpopulation of CGRP-I nerve fibers, i.e., SP-I was not present in all CGRP-I nerves. Effects of these neuropeptides on uterine contractility were examined on in vitro preparations of uterine horns from diethylstilbestrol-treated rats. SP (10(-4) to 10(-8) M) stimulated uterine contraction in a dose-related manner. CGRP(1-37) and CGRP(8-37) had no effect on basal uterine tension. While CGRP(1-37) (10(-7) M) reduced SP-stimulated (10(-5) M) uterine contraction by 56%, CGRP(8-37) had no effect on SP-stimulated uterine contraction. However, CGRP(8-37) (10(-6) M) significantly reduced the ability of CGRP(1-37) (10(-7) M) to inhibit SP-stimulated uterine contraction. These results demonstrate that SP- and CGRP-I are present in, and coexist in some uterine nerves, presumably afferent nerves. The first 7 amino acids are necessary for the inhibitory effect of CGRP(1-37) on stimulated uterine contraction. In addition, CGRP(8-37) acted as an antagonist to this inhibitory action. SP and CGRP could be coreleased from afferent fibers in an "efferent fashion" and influence uterine contractility. SP having a contractile effect and CGRP having a relaxing effect.

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.

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.

Galanin and calcitonin gene-related peptide immunoreactivity in nerves of the rat uterus: localization, colocalization, and effects on uterine contractility.

Immunoreactivity to the neuropeptides galanin (GAL) and calcitonin gene-related peptide (CGRP) was examined in nerves in the rat uterus as a prelude to studying their effects on uterine contractility. With immunocytochemical techniques, GAL immunoreactivity (GAL-I) and CGRP-I were localized in myometrial nerves throughout the uterine horns and cervix, with nerves immunoreactive for CGRP being more numerous. Immunocytochemical double-labeling studies revealed GAL coexists with CGRP in a subpopulation of CGRP-I nerve fibers, i.e., GAL-I was not present in all CGRP-I nerves. Effects of these neuropeptides on uterine contractility were examined on in vitro preparations of uterine horns from diethylstilbestrol-treated rats. GAL (10(-5) to 10(-8) M) stimulated uterine contraction in a dose-related manner. CGRP had no effect on basal uterine tension, but CGRP (10(-7) M) reduced GAL-stimulated (10(-7) M) uterine contraction by 92.5%. These results demonstrate that GAL- and CGRP-I are present in, and coexist in, some uterine nerves, presumably afferent nerves. GAL and CGRP could be released from afferent fibers in an "efferent fashion" and influence uterine contractility, GAL having a contractile effect and CGRP having a relaxing effect.

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.

Utilization of a polyethylene enclosure for nerve tracing studies in the rat ovary.

A commonly used technique for mapping neuronal connections in the peripheral nervous system involves the injection of a tracer substance into a peripheral organ. The tracer is taken up by nerve terminals at the injection site and transported retrogradely to motor or sensory perikarya where it can be visualized. One of the pitfalls of this technique is that tracer may leak from the injection site, enter nerve terminals adjacent to the injection area and thus cause erroneous interpretations. Similarly, some tracers may enter blood vessels and be taken up by nerve cell bodies indiscriminately. In order to circumvent these potential problems in studies on the innervation of the rat ovary, a polyethylene enclosure for the organ was fabricated from a BEEM capsule. The retrograde tracer, True blue, was then applied topically on the ovarian surface inside the enclosure. In chronic experiments, specifically labeled ovarian nerve cell bodies were identified in dorsal root and preaortic ganglia. Utilization of this simple and effective enclosure eliminates the false-positive and non-specific labeling of perikarya due to diffusion and blood-borne transport of the tracer from the injection area.

Aspartate immunoreactive axons in normal rat L4 dorsal roots.

The present study demonstrates that approximately 15% of the unmyelinated axons and 4% of the myelinated axons in the rat L4 dorsal root are immunostained for the excitatory amino acid aspartate. Thus these primary afferent axons contain enough of the antigen to be labeled. This is the first report that high concentrations of aspartate characterize a subpopulation of dorsal root axons. This allows the suggestion that aspartate is a candidate transmitter for primary afferent neurons. We emphasize that these axons are demonstrated in otherwise normal animals so that changes in percentages of labeled axons in response to various stimuli are not complicated by manipulations usually necessary to demonstrate immunoreactive compounds in the cell body.

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.

Peripheral pathways for neuropeptide Y- and cholecystokinin-8-immunoreactive nerves innervating the rat ovary.

The superior ovarian nerve (SON) or plexus nerve (PN) innervating the rat ovary was transected separately or in combination and the effects of these nerve lesions on intra-ovarian NPY- or CCK-8-immunoreactive nerve fibers was evaluated. Transection of the SON did not affect NPY or CCK-8-immunoreactive nerve fibers. In contrast, section of the PN eliminated nerve fibers immunoreactive for these neuropeptides. This study demonstrates that both NPY- and CCK-8-immunoreactive nerve fibers reach the ovary via the PN.

Intraspinal sprouting of rat primary afferents after deafferentation.

To test intraspinal sprouting of primary afferent fibers, unilateral chronic, followed by contralateral acute, dorsal rhizotomies were done 3 segments above and below a spared segment (T9) in rats. Following perfusion with 4% paraformaldehyde, T9 spinal cord segments were frozen, sectioned and reacted by a modified Gomori procedure for fluoride-resistant acid phosphatase (FRAP) enzyme activity, a marker for primary afferent terminals. As determined by light microscopy, there was greater and extended FRAP reaction product in laminae I and II on the chronically denervated side than on the contralateral acutely denervated side. These data support the hypothesis of axonal sprouting and synaptogenesis of primary afferent fibers after spinal cord deafferentation.