Expression of pituitary adenylate cyclase activating peptide in the uterine cervix, lumbosacral dorsal root ganglia and spinal cord of rats during pregnancy

The uterine cervix is highly innervated by the sensory nerves containing neuropeptides which change during pregnancy and are regulated, in part, by estrogen. These neuropeptides act as transmitters both in the spinal cord and cervix. The present study was undertaken to determine the expression pattern of the neuropeptide pituitary adenylate cyclase activating peptide (PACAP) in the cervix and its nerves during pregnancy and the influence of estrogen on this expression using immunohistochemistry, radioimmunoassay and RT-PCR. PACAP immunoreactivity was detected in nerves in the cervix, lumbosacral (L6-S1) dorsal root ganglia (DRG) and spinal cord. PACAP immunoreactivity was highest at day 15 of pregnancy in the cervix and dorsal spinal cord, but then decreased over the last trimester of pregnancy. However, levels of PACAP mRNA increased in the L6-S1 DRG at late pregnancy relative to early pregnancy. DRG of ovariectomized rats treated with estrogen showed increased PACAP mRNA synthesis in a dose-related manner, an effect partially blocked by the estrogen receptor (ER) antagonist ICI 182,780. We postulate that synthesis of PACAP in L6-S1 DRG and utilization in the cervix and spinal cord increase over pregnancy and this synthesis is the under influence of the estrogen-ER system. Since PACAP is expressed by sensory nerves and may have roles in nociception and vascular function, collectively, these data are consistent with the hypothesis that sensory nerve-derived neuronal factors innervate the cervix and play a role in cervical ripening.     

P2X receptors in the rat uterine cervix,lumbosacral dorsal root ganglia,and spinal cord during pregnancy

ATP, an intracellular energy source, is released from cells during tissue stress, damage, or inflammation. The P2X subtype of the ATP receptor is expressed in rat dorsal root ganglion (DRG) cells, spinal cord dorsal horn, and axons in peripheral tissues. ATP binding to P2X receptors on nociceptors generates signals that can be interpreted as pain from damaged tissue. We have hypothesized that tissue stress or damage in the uterine cervix during late pregnancy and parturition can lead to ATP release and sensory signaling via P2X receptors. Consequently, we have examined sensory pathways from the cervix in nonpregnant and pregnant rats for the presence of purinoceptors. Antiserum against the P2X₃-receptor subtype showed P2X₃- receptor immunoreactivity in axon-like structures of the cervix, in small and medium-sized neurons in the L6/S1 DRG, and in lamina II of the L6/S1 spinal cord segments. Retrograde tracing confirmed the projections of axons of P2X₃-receptor-immunoreactive DRG neurons to the cervix. Some P2X₃-receptor-positive DRG neurons also expressed estrogen receptor- immunoreactivity and expressed the phosphorylated form of cyclic AMP response-element-binding protein at parturition. Western blots showed a trend toward increases of P2X₃-receptor protein between pregnancy (day 10) and parturition (day 22–23) in the cervix, but no significant changes in the DRG or spinal cord. Since serum estrogen rises over pregnancy, estrogen may influence purinoceptors in these DRG neurons. We suggest that receptors responsive to ATP are expressed in uterine cervical afferent nerves that transmit sensory information to the spinal cord at parturition.     

The role of sensory neurons in cervical ripening: effects of estrogen and neuropeptides.

Central nervous system nuclei and circuits, such as the medial preoptic, ventromedial and paraventricular nuclei of the hypothalamus, play important roles in reproduction and parturition, and are influenced by estrogen. Peripheral autonomic and sensory neurons also play important roles in pregnancy and parturition. Moreover, the steroid hormone estrogen acts directly, not only on the reproductive tract organs (uterus and cervix), but also on the central and peripheral nerves by regulating expression of various neuronal genes. The peripheral primary afferent neurons innervating the uterine cervix relay mechanical and biochemical sensory information induced by local cervical events and by passage of fetuses, to the spinal cord and supraspinal centers. Consequently, the birth process in mammals is influenced by the combined action of neurons and hormones. Peripheral sensory stimuli, induced physiologically by fetal expulsion or mechanically by vaginocervical stimulation, alter behavior, as well as autonomic and neuroendocrine systems. Recent evidence indicates that primary afferent neurons innervating the cervix, in addition to their sensory effects, likely exert local "efferent" actions on the ripening cervix near term. These efferent effects may involve estrogen-regulated production of such neuropeptides as substance P and calcitonin gene-related peptide in lumbosacral dorsal root ganglia, and their release in the cervix. Collectively, these findings suggest an interrelationship among estrogen, cervix-related sensory neurons, and local cervical events near term.     

Calcitonin gene-related peptide-receptor component protein expression in the uterine cervix, lumbosacral spinal cord, and dorsal root ganglia

The neuropeptide calcitonin gene-related peptide (CGRP) may play a role in neurogenic inflammation, tissue remodeling of the uterine cervix, promoting vasodilation, parturition, and processing of sensory information in the spinal cord. CGRP-immunoreactive nerves of the cervix and spinal cord have been studied but cellular identification of the CGRP receptor has received little attention. CGRP-receptor component protein (CGRP-RCP) is a small protein associated with the CGRP receptor; thus, immunostaining for the CGRP-RCP can be used to identify sites of the CGRP receptor. We determined sites of CGRP-RCP immunoreactivity relative to the presence of CGRP-ir nerve fibers in the female rat uterine cervix, spinal cord, and dorsal root ganglia. CGRP-RCP immunoreactivity was expressed in the dorsal horn of the spinal cord, venules of the uterine cervix, and perikarya of sensory neurons in dorsal root ganglia. CGRP-immunoreactive fibers were adjacent to CGRP-RCP-immunoreactive vessels in the cervix and among CGRP-RCP-immunoreactive structures in the dorsal horn of the spinal cord. This suggests CGRP-RCP is associated with structures innervated by CGRP nerves and these interactions may be changed in tissues in response to an appropriate stimulus.     

Formation of Neuromuscular Junctions and Synthesis of Sensory Neuropeptides in the Co-cultures of Dorsal Root Ganglion and Cardiac Myocytes

Aim The interactions between primary sensory neurons and cardiac myocytes are still unclear. In the present study, the co-culture model of dorsal root ganglion (DRG) explant and cardiac myocytes was used to characterize the morphological relationship between primary sensory nerve endings and cardiac myocytes and to investigate whether cardiac myocytes could induce substance P (SP) and calcitonin gene-related peptide (CGRP) synthesis in DRG neurons and release from DRG neurons in the neuromuscular co-cultures. Methods The formation of neuromuscular junctions was observed with scanning electron microscopy (SEM). SP and CGRP expression were detected by immunocytochemistry. Basal SP and CGRP release and capsaicin-evoked SP and CGRP release were analyzed by radioimmunoassay (RIA). Results In this study, neuromuscular junctions were observed in the co-cultures of DRG explant and cardiac myocytes. SP-immunoreactive (IR) and CGRP-IR neurons were detected in both neuromuscular co-cultures and DRG explant cultures, but the number of SP-IR and CGRP-IR neurons migrating from DRG explant was significantly increased in neuromuscular co-cultures. Capsaicin-evoked SP and CGRP release but not basal SP and CGRP release in neuromuscular co-cultures increased significantly as compared with that in the cultures of DRG explant alone. Conclusions The results implicated that the morphological relationship between sensory nerve terminal and cardiac myocyte is much more close in vitro than it is in vivo. Cardiac myocytes may induce sensory neuropeptide synthesis and capsaicin-evoked neuropeptide release in neuromuscular co-cultures. Further experiment needs to be performed about the significance of neuropeptide synthesis and capsaicin-evoked neuropeptide release induced by target cardiac myocytes. Zhen Liu and Huaxiang Liu contributed equally to this article.     

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.     

Changes of the neuropeptides content and gene expression in spinal cord and dorsal root ganglion after noxious colorectal distension

Visceral pain/hypersensitivity is a cardinal symptom of functional gastrointestinal disorders. With their peripheral and central (spinal) projections, sensory neurons in the dorsal root ganglia (DRG) are the "gateway" for painful signals emanating from both somatic and visceral structures. In contrast to somatic pain, the neurochemical pathways involved in visceral pain/hypersensitivity have not been well studied. We hypothesized the neuropeptide changes in spinal cord and DRG during visceral pain would mirror similar changes in somatic nociception. Noxious (painful) colorectal distension (CRD) was done by distending a rectal balloon up to 60 mm Hg phasically for 1 h in Sprague-Dawley rats. The spinal content of calcitonin gene-related peptide (CGRP), substance P (SP), galanin and vasoactive intestinal peptide (VIP) as well as their mRNAs in DRG were measured at 0, 4 and 24 h after the CRD. Visceromotor reflex (VMR) was measured by recording the electromyogram at the abdominal muscle in response to CRD. Distal colorectum was removed for evaluating the presence of inflammation. No significant evidence of histological inflammation was seen in the colonic mucosa/submucosa after repeated CRD, which is confirmed by myeloperoxidase assay. The spinal content of CGRP and SP decreased significantly 4 h after CRD, while galanin and VIP levels increased gradually and reached highest level at 24 h (p<0.05). The mRNAs in DRG of the neuropeptides were significantly upregulated after CRD (p<0.05). VMR recording showed the rat's colon became hypersensitive 4 h after CRD, a sequence parallel to the spinal changes of CGRP and SP in timeframe. Noxious mechanical distension of the colorectum causes an acute change in the spinal levels of excitatory neurotransmitters (CGRP and SP), probably reflecting central release of these peptides from sensory neurons and contributing to the hypersensitivity following the noxious CRD. This is followed by a slower change in the levels of the inhibitory neurotransmitter galanin and VIP. Such stimulation results in significant alternation of the gene expression in DRG, reflecting the plasticity of the neuronal response. In the absence of visceral inflammation, the aforementioned neuropeptides are important mediators in the processing of visceral pain/hypersensitivity.     

Calcitonin gene-related peptide and substance P contribute to reduced blood pressure in sympathectomized rats

CGRP and substance P (SP) are produced in dorsal root ganglia (DRG) sensory neurons and modulate vascular tone. Sympathetic and sensory nerves compete for NGF, a potent stimulator of CGRP and SP, and it has been suggested that sympathetic hyperinnervation in spontaneously hypertensive rats may reduce the availability of NGF to sensory nerves, thus reducing CGRP and SP. The purpose of this study was to determine whether destruction of peripheral sympathetic nerves in normal rats would increase the availability of NGF for sensory neurons and enhance expression of CGRP and SP. Sympathectomy was produced in rats by guanethidine sulfate administration. Control rats received saline. Sympathectomized rats displayed reductions in blood pressure (BP) and atria norepinephrine levels, whereas NGF levels in the DRG, spleen, and ventricles were increased. Sympathectomy also enhanced CGRP and SP mRNA and peptide content in DRG. Administration of CGRP and SP receptor antagonists increased the BP in sympathectomized rats but not in the controls. Thus sympathectomy enhances sensory neuron CGRP and SP expression that contributes to the BP reduction.     

Sensory nerves and neuropeptides in uterine cervical ripening

At the time of parturition (fetal delivery) the uterine cervix must "ripen," becoming soft, pliable, and dilated to accommodate the fetus' delivery. The fundamental processes underlying cervical ripening remain poorly understood. Knowledge that abundant autonomic and sensory nerves supply the uterine cervix, that transection of afferent nerves supplying the cervix blocks parturition, and that some of the changes in the cervix resemble those seen in inflammatory reactions suggests nerves may have a role in the cervical ripening changes. The present study utilized immunohistochemistry, plasma extravasation, and solution hybridization-nuclease protection assay to elucidate the complement of primary afferent nerves and some receptors in the rat cervix during pregnancy, and to determine if they may have roles in the ripening process at term. This study revealed an abundance of nerves associated with the cervical vasculature and myometrial smooth muscle containing immunoreactivity for substance P, calcitonin gene-related peptide, secretoneurin, and nitric oxide synthase throughout pregnancy. Many of these are small unmyelinated capsaicin-sensitive C-fibers. Substance P- (NK1-) and calcitonin gene-related peptide receptors were apparent on uterine cervix vasculature from pregnant, parturient, and postpartum rats. NK1 receptor mRNA was maximal at 20 days of pregnancy. Plasma extravasation of i.v. administered Evans Blue or Monastral Blue was most pronounced at parturition (shortly after NK1 mRNA is maximal); this was similar to plasma extravasation evoked by i.v. administration of substance P or capsaicin-treatment. This study revealed new data about the nervous system of the rat uterine cervix and that these nerves and their transmitters could very well be part of a neurogenic inflammatory process involved in cervical ripening.     

Co-localization of nitric oxide synthase and vasoactive intestinal peptide immunoreactivity in neurons of the major pelvic ganglion projecting to the rat rectum and penis

We demonstrate the existence of nerve fibers possessing substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactivity in the mouse cervical ventral roots. The distribution of the SP and CGRP fibers was similar, but CGRP fibers were generally more numerous. Both types entered the ventral pia mater or formed hairpin loops, but they did not enter the spinal cord directly through these roots. SP and CGRP fibers in the ventral roots were thin and had many varicosities. We suggest that these SP and CGRP fibers are involved not only in a sensory mechanism, but also in other functions, via the release of SP and CGRP from varicosities in the ventral roots.     

CGRPα-expressing sensory neurons respond to stimuli that evoke sensations of pain and itch

Calcitonin gene-related peptide (CGRPα, encoded by Calca) is a classic marker of nociceptive dorsal root ganglia (DRG) neurons. Despite years of research, it is unclear what stimuli these neurons detect in vitro or in vivo. To facilitate functional studies of these neurons, we genetically targeted an axonal tracer (farnesylated enhanced green fluorescent protein; GFP) and a LoxP-stopped cell ablation construct (human diphtheria toxin receptor; DTR) to the Calca locus. In culture, 10-50% (depending on ligand) of all CGRPα-GFP-positive (+) neurons responded to capsaicin, mustard oil, menthol, acidic pH, ATP, and pruritogens (histamine and chloroquine), suggesting a role for peptidergic neurons in detecting noxious stimuli and itch. In contrast, few (2.2±1.3%) CGRPα-GFP(+) neurons responded to the TRPM8-selective cooling agent icilin. In adult mice, CGRPα-GFP(+) cell bodies were located in the DRG, spinal cord (motor neurons and dorsal horn neurons), brain and thyroid-reproducibly marking all cell types known to express Calca. Half of all CGRPα-GFP(+) DRG neurons expressed TRPV1, ～25% expressed neurofilament-200, <10% contained nonpeptidergic markers (IB4 and Prostatic acid phosphatase) and almost none (<1%) expressed TRPM8. CGRPα-GFP(+) neurons innervated the dorsal spinal cord and innervated cutaneous and visceral tissues. This included nerve endings in the epidermis and on guard hairs. Our study provides direct evidence that CGRPα(+) DRG neurons respond to agonists that evoke pain and itch and constitute a sensory circuit that is largely distinct from nonpeptidergic circuits and TRPM8(+)/cool temperature circuits. In future studies, it should be possible to conditionally ablate CGRPα-expressing neurons to evaluate sensory and non-sensory functions for these neurons.     

Gestational changes in calcitonin gene-related peptide, nerve growth factor, and its receptors in rat dorsal root ganglia.

In dorsal root ganglia (DRG) cell cultures, levels of calcitonin gene-related peptide (CGRP) are increased in the presence of ovarian hormones and nerve growth factor (NGF). In addition, injection of ovariectomized rats with ovarian hormones led to an increase in levels of two NGF receptors, TrkA and p75(NTR), in DRG. Thus, we hypothesized that increased levels of ovarian hormones during pregnancy may elevate the synthesis of CGRP and NGF receptors in the DRG. DRG harvested from rats on specific days of pregnancy, on Day 2 postpartum, and after ovariectomy were subjected to radioimmunoassay, Western blot analysis, and NGF immunoassay to determine levels of CGRP, TrkA and p75(NTR), and NGF, respectively. CGRP levels in rat DRG were significantly higher during pregnancy than at Day 2 postpartum or in ovariectomized rats. Levels of both TrkA and p75(NTR) in DRG increased during pregnancy and remained elevated at Day 2 postpartum, but CGRP levels declined. Levels of NGF reached a statistically significant peak at Day 18 of gestation, and were not significantly reduced at Day 2 postpartum. Increased levels of ovarian steroid hormones during pregnancy may be involved in the synthesis of CGRP, however, the postpartum decreases in CGRP synthesis appear to be unrelated to NGF and its receptors.     

Pathway-specific patterns of the co-existence of substance P,calcitonin gene-related peptide,cholecystokinin and dynorphin in neurons of the dorsal root ganglia of the guinea-pig

Summary The co-existence of immunoreactivities to substance P (SP), calcitonin gene-related peptide (CGRP), cholecystokinin (CCK) and dynorphin (DYN) in neurons of the dorsal root ganglion (DRG) of guinea-pigs has been investigated with a double-labelling immunofluorescence procedure. Four main populations of neurons could be identified that contained different combinations of these peptides and had distinctive peripheral projections: (1) Neurons that contained immunoreactivity to SP, CGRP, CCK and DYN were distributed mainly to the skin. (2) Neurons with immunoreactivity to SP, CGPR and CCK, but not DYN, were distributed mainly to the small blood vessels of skeletal muscles. (3) Neurons with immunoreactivity to SP, CGRP and DYN, but not CCK, were distributed mainly to pelvic viscera and airways. (4) Neurons containing immunoreactivity to SP and CGRP, but not CCK and DYN, were distributed mainly to the heart, systemic blood vessels, blood vessels of the abdominal viscera, airways and sympathetic ganglia. Other small populations of DRG neurons containing SP, CGRP or CCK alone also were detected. Perikarya containing these combinations of neuropeptides were not found in autonomic ganglia. The peripheral axons of neurons containing immunoreactivity to at least SP and CGRP were damaged by chronic treatment with capsaicin. However, some sensory neurons containing CCK alone were not affected morphologically by capsaicin.These results clearly show that individual DRG neurons can contain many different neuropeptides. Furthermore, the combination of neuropeptides found in any particular neuron is related to its peripheral projection.     

Bilateral activation of STAT3 by phosphorylation at the tyrosine-705 (Y705) and serine-727 (S727) positions and its nuclear translocation in primary sensory neurons following unilateral sciatic nerve injury

Unilateral sciatic nerve compression (SNC) or complete sciatic nerve transection (CSNT), both varying degrees of nerve injury, induced activation of STAT3 bilaterally in the dorsal root ganglia (DRG) neurons of lumbar (L4-L5) as well as cervical (C6–C8) spinal cord segments. STAT3 activation was by phosphorylation at the tyrosine-705 (Y705) and serine-727 (S727) positions and was followed by their nuclear translocation. This is the first evidence of STAT3(S727) activation together with the well-known activation of STAT3(Y705) in primary sensory neurons upon peripheral nerve injury. Bilateral activation of STAT3 in DRG neurons of spinal segments anatomically both associated as well as non-associated with the injured nerve indicates diffusion of STAT3 activation inducers along the spinal cord. Increased levels of IL-6 protein in the CSF following nerve injury as well as activation and nuclear translocation of STAT3 in DRG after intrathecal injection of IL-6 shows that this cytokine, released into the subarachnoid space can penetrate the DRG to activate STAT3. Previous results on increased bilateral IL-6 synthesis and the present manifestation of STAT3 activation in remote DRG following unilateral sciatic nerve injury may reflect a systemic reaction of the DRG neurons to nerve injury.     

Characterization of VR1 within the BMBF-Leitproject: 'Molecular Pain Research'

Agents that activate cannabinoid CB1 receptors for marijuana's active principal, THC, or vanilloid VR1 receptors for red chilli peppers' pungent ingredient, capsaicin, modulate pain perception. Stimulation of presynaptic CB1 leads to inhibition of glutamate release in the spinal cord, whereas VR1 stimulation causes release of substance P and CGRP from DRG neurons. VR1 undergoes rapid desensitization by its agonists, which makes VR1-expressing neurons insensitive to subsequent stimulation and results in analgesia. Thus, both CB1 and VR1, which are coexpressed in several spinal and DRG neurons, are targets for analgesic drug development. CB1 and VR1 also share endogenous agonists, namely anandamide, NADA and some of their analogs, and may be regarded as metabotropic and ionotropic receptors for the same family of mediators, with opposing roles in pain perception. The development of 'hybrid' CB1/VR1 agonists as potent analgesics and the functional relationships between CB1 and VR1 in sensory neurons will be discussed.     

Sustained neurochemical plasticity in central terminals of mouse DRG neurons following colitis

Sensitization of dorsal root ganglia (DRG) neurons is an important mechanism underlying the expression of chronic abdominal pain caused by intestinal inflammation. Most studies have focused on changes in the peripheral terminals of DRG neurons in the inflamed intestine but recent evidence suggests that the sprouting of central nerve terminals in the dorsal horn is also important. Therefore, we examine the time course and reversibility of changes in the distribution of immunoreactivity for substance P (SP), a marker of the central terminals of DRG neurons, in the spinal cord during and following dextran sulphate sodium (DSS)-induced colitis in mice. Acute and chronic treatment with DSS significantly increased SP immunoreactivity in thoracic and lumbosacral spinal cord segments. This increase developed over several weeks and was evident in both the superficial laminae of the dorsal horn and in lamina X. These increases persisted for 5 weeks following cessation of both the acute and chronic models. The increase in SP immunoreactivity was not observed in segments of the cervical spinal cord, which were not innervated by the axons of colonic afferent neurons. DRG neurons dissociated following acute DSS-colitis exhibited increased neurite sprouting compared with neurons dissociated from control mice. These data suggest significant colitis-induced enhancements in neuropeptide expression in DRG neuron central terminals. Such neurotransmitter plasticity persists beyond the period of active inflammation and might contribute to a sustained increase in nociceptive signaling following the resolution of inflammation.     

Parturition and recruitment of macrophages in cervix of mice lacking the prostaglandin F receptor

Parturition does not occur in transgenic mice lacking the prostaglandin F receptor (Ptgfr(-/-)) because luteolysis is forestalled and progesterone production persists. Ovariectomy of pregnant Ptgfr(-/-) mice leads to a decline in circulating progesterone and delivery of live pups. The objective of the present study was to test the hypothesis that immigration of macrophages and increased innervation of the cervix of Ptgfr(-/-) mice was associated with ripening and parturition. The cervix of pregnant Ptgfr(-/-) mice was studied on Days 15-21 after breeding; additional groups were ovariectomized on Day 19 of pregnancy, and the cervix obtained on Day 20 of pregnancy before birth or the next day at about 24 h after birth. On Days 18-19 of pregnancy, macrophage numbers and nerve fiber density increased more than 3-fold compared with findings in nonpregnant or Day 15 or 21 pregnant Ptgfr(-/-) mice. The magnitude and time course of these changes were comparable to those found in wild-type controls that delivered on Day 19 after breeding. Thus, the mechanism regulating macrophage immigration, innervation, and cervical remodeling in Ptgfr(-/-) mice with delayed parturition is similar to wild-type controls that deliver at term. By contrast, ovariectomy forestalled the decrease in cervical macrophages in Ptgfr(-/-) mice. By Day 21 after breeding, macrophage numbers more than double those after ovariectomy, relative to those found in pregnant Ptgfr(-/-) mice, whereas nerve fiber density was the same regardless of birth. Density of collagen structure in these mice directly matched macrophage traffic in the cervix. The findings indicate that the prostaglandin F(2alpha) receptor and progesterone withdrawal are a necessary part of the final common pathway for ripening of the cervix and the process of parturition.     

Distribution,immunohistochemical characteristics and nerve pathways of primary sensory neurons supplying the porcine vas deferens

The present study investigated: (1) the distribution and chemical coding of primary sensory neurons supplying the vas deferens in juvenile pigs by the use of retrograde tracing combined with double-labelling immunofluorescence, (2) nerve pathways from dorsal root ganglia (DRG) to the vas deferens by means of denervation procedures involving transection of the hypogastric or pelvic nerve combined with a retrograde tracing method, and (3) possible interactions of the substance P (SP)/calcitonin gene-related peptide (CGRP)-immunoreactive varicose nerve fibres on vas deferens projecting neurons (VDPN) in the anterior pelvic ganglion (APG). The vast majority of VDPN were found mainly in the lumbar L2, L3 and sacral S2, S3 pairs of DRG and showed a clear ipsilaterally organized projection pattern. Immunohistochemistry revealed that most of these neurons contained SP and/or CGRP, occasionally coexpressed with galanin. Interestingly, pronounced differences in the expression of SP and/or CGRP were observed between the lumbar and sacral VDPN in that most of the lumbar but less than half of the sacral neurons stained for these peptides. Denervation experiments showed that the neurons located within the lumbar DRG project through the ipsilateral hypogastric nerve, whereas those found within the sacral DRG send their processes through the ipsilateral and contralateral pelvic nerve. In the nerve-lesioned animals, especially in those with the hypogastric nerve cut, a dramatic reduction in the number of SP and/or CGRP-containing nerve terminals surrounding the efferent VDPN within the APG was observed. This study has disclosed the distribution and, for the first time, chemical coding and nerve pathways of vas deferens-projecting primary sensory neurons in a mammalian species, the pig. The results obtained also provide some novel information about the possible morphological and functional relationship between vas deferens-projecting primary sensory and pelvic efferent nerve cells.