Cholinergic neurons of the pelvic autonomic ganglia and uterus of the female rat: distribution of axons and presence of muscarinic receptors.

Acetylcholine (ACh) stimulates contraction of the uterus and dilates the uterine arterial supply. Uterine cholinergic nerves arise from the paracervical ganglia and were, in the past, characterized based on acetylcholinesterase (AChE) histochemistry. However, the histochemical reaction for acetylcholinesterase provides only indirect evidence of acetylcholine location and is a nonspecific marker for cholinergic nerves. The present study: (1) reevaluated cholinergic neurons of the paracervical ganglia, (2) examined the cholinergic innervation of the uterus by using retrograde axonal tracing and antibodies against molecules specific to cholinergic neurons, choline acetyltransferase and the vesicular acetylcholine transporter, and (3) examined muscarinic receptors in the paracervical ganglia using autoradiography and a radiolabeled agonist. Most ganglionic neurons were choline acetyltransferase- and vesicular acetylcholine transporter-immunoreactive and were apposed by choline acetyltransferase/vesicular acetylcholine transporter-immunoreactive terminals. Retrograde tracing showed that some cholinergic neurons projected axons to the uterus. These nerves formed moderately dense plexuses in the myometrium, cervical smooth muscle and microarterial system of the uterine horns and cervix. Finally, the paracervical ganglia contain muscarinic receptors. These results clearly reveal the cholinergic innervation of the uterus and cervix, a source of these nerves, and demonstrate the muscarinic receptor content of the paracervical ganglia. Cholinergic nerves could play significant roles in the control of uterine myometrium and vasculature.

Opioid-related changes in nociceptive threshold and in tissue levels of enkephalins after target disruption of the gene for neutral endopeptidase (EC 3.4.24.11) in mice.

Neutral endopeptidase EC 3.4.24.11 (NEP) is localized in peptidergic neurons and various colocalized peptides or other humoral mediators may serve as substrates. Target disruption of the NEP gene was reported to enhance the lethal response to endotoxin shock in mice. We examined thermonociceptive thresholds and enkephalin (ENK) tissue levels in transgenic NEP (-/-) and control wild type NEP (+/+) mice. Hot plate (52 degrees C) latency was 13.1 +/- 1.4 s in NEP (+/+) mice (n = 16) while latency increased significantly (P = 0.031) to 17.7 +/- 1.6 s in NEP (-/-) mice. Naloxone (10 mg/kg) had no effect on hot plate latency in NEP (+/+) mice (12.5 s, n = 8), but significantly decreased the latency in NEP (-/-) mice compared to untreated NEP (-/-) deficient mice (10.5 s, n = 8). Morphine (3 or 10 mg/kg) analgesic response was similar in knockout mice and wild type mice. Methionine-ENK (MET-ENK) and leucine-ENK (LEU-ENK) levels were determined in extracts from cortex, brain stem, hypothalamus, striatum, spinal cord, trigeminal ganglion and heart in treated and untreated mice. ENK-levels varied in a regionally-dependent manner and were significantly decreased in hypothalamus and spinal cord. We conclude that deletion of the NEP gene results in an opioid-related increase in thermonociceptive threshold. Regional differences in opioid metabolism indicate that NEP evokes tissue-specific patterns of ENK-regulation. NEP selectively controls opioid biosynthesis in hypothalamus and spinal cord presumably by feedback regulation.

A preliminary study of reconstruction of the monkey temporomandibular joint with autogenous tissues.

PURPOSE: This study investigated the ability of autogenous pericranium-muscle and cranial bone to restore form and function of the temporomandibular joint (TMJ). MATERIALS AND METHODS: A right TMJ discectomy, condylectomy, vertical ramus osteotomy with superior repositioning of the proximal fragment, and a cranial bone graft to the mandibular angle were performed in 12 cynomolgus monkeys. Clinical, histologic, and autoradiographic evaluation was done at intervals beginning at 3 days and continuing up to 78 weeks. RESULTS: No significant postoperative sequela were identified in any of the monkeys. Jaw mobility and function were restored. Gross morphologic condylar changes were observed. The pericranium-muscle grafts did not differentiate into normal disc-like structures and a cartilage cap did not cover the condyle stump head. CONCLUSIONS: The results of this study suggest that the previous TMJ function was restored. However, the histologic changes observed were consistent with advanced degenerative joint disease.

Ontogeny of neurotransmitter systems in the paracervical ganglion and uterine cervix of the rat.

BACKGROUND: The paracervical ganglia (PG) are components of the pelvic plexus that provides sensory and motor innervation to the reproductive system of the female rat. Several neurotransmitters including norepinephrine (NE), acetylcholine (ACh), neuropeptide Y (NPY), and vasoactive intestinal polypeptide (VIP) are present in neurons of the adult PG and in axons innervating the adult uterus and uterine cervix. The current study was undertaken to describe the onset of immunoreactivity of these neurotransmitters and neuropeptides during development. METHODS: Female rats, ages E18 to P36, were prepared for immunohistochemistry for TH (tyrosine hydroxylase, a marker of noradrenergic neurons), NPY, or VIP as well as the histochemical demonstration of acetylcholinesterase (AChE). RESULTS: All four markers were detected in neurons of the PG at E18. Changes in the appearance of these markers from E18 to P36 reflected previously described growth changes in the PG. Axons containing AChE, TH, NPY, or VIP were first detected within the cervix at E20. Immunopositive axons first appeared as thick, unbranched structures at the outermost portion of the cervical myometrium. Over time, these axon bundles ramified to form discrete varicose axons. The ingrowth was similar for axons containing each of the four markers. CONCLUSIONS: The relative density of each neuronal type in the PG was reflected in the density of axons containing the same marker in the cervix. Changes in neurotransmitter/neuropeptide staining of PG neurons or axons in the cervix were not observed as the animals approached puberty.

Galanin-immunoreactive nerves in the female rat paracervical ganglion and uterine cervix: distribution and reaction to capsaicin.

The presence and distribution of galanin-immunoreactivity was examined in the uterine cervix and paracervical autonomic ganglia of the female rat. Some animals were treated with capsaicin to determine if galanin-immunoreactivity was present in small-diameter primary afferent nerves. Other animals were treated with the noradrenergic neurotoxin 6-hydroxydopamine to ascertain if galanin-immunoreactivity was present in sympathetic noradrenergic nerves. Galanin-immunoreactive nerve fibers were sparse in the cervical myometrium and vasculature, but numerous in the paracervical ganglion where they appeared to innervate principal neurons. Immunoreactivity was also present in dorsal root ganglia, dorsal horn of spinal cord, and inferior mesenteric ganglia. Capsaicin treatment resulted in a marked reduction of galanin-immunoreactivity in the spinal cord dorsal horn, but not in the dorsal root ganglia, paracervical ganglia, or cervix (although there was a substantial reduction of substance P-, neurokinin A-, and calcitonin gene-related peptide-immunoreactivity in the dorsal horn. dorsal root ganglia, and uterine cervix). 6-Hydroxydopamine treatment did not cause any appreciable change in the galanin-immunoreactivity in any tissues. We conclude that galanin-like immunoreactivity is expressed in nerve fibers innervating the paracervical ganglia and uterine cervix of the female rat. This immunoreactivity is probably present in afferent nerves and could play a role in neuroendocrine reflexes and in reproductive function.

Healing following condylar shave in the monkey temporomandibular joint.

Condylar shaves were performed in monkeys, and the reparative process was studied by histologic and autoradiographic techniques. The condylar surface was found to repair mainly with fibrous connective tissue produced by cells derived from the underlying marrow spaces. The residual condylar cartilage did not play a significant role in this process.

Effects of capsaicin on reproductive function in the female rat: role of peptide-containing primary afferent nerves innervating the uterine cervix in the neuroendocrine copulatory response.

Nerves immunoreactive for the peptides substance P, neurokinin A, calcitonin gene-related peptide or cholecystokinin-octapeptide innervate the uterine cervix in the rat. Nerve terminals are associated with the myometrial and vascular smooth muscle and are distributed throughout the endocervix. These nerves degenerate following neonatal capsaicin treatment indicating that they are small-diameter, unmyelinated, C-type primary afferent nerves. Adult female rats, treated with capsaicin as neonates, exhibit decreased fertility following mating and diminished sensitivity to the induction of pseudopregnancy following copulomimetic electrical stimulation of the cervix. The results also demonstrate that hypothalamo-adenohypophyseal-ovarian interactions, corpus luteum progesterone secretion and uterine sensitivity to progesterone are normal in capsaicin-treated rats. Taken together, these data suggest that the reproductive dysfunction observed in capsaicin-treated rats is due to destruction of the afferent limb of the neuroendocrine copulatory response that facilitates the luteal progesterone secretion necessary to support pregnancy or pseudopregnancy. Thus, it is concluded that the afferent limb of this neuroendocrine response in the rat consists primarily of unmyelinated, peptide-containing, C-type primary afferent nerves.

Distribution of subgroups of neuropeptide Y-immunoreactive and noradrenergic nerves in the female rat uterine cervix.

Nerves in the uterine cervix of the rat were examined with regard to co-existence of markers for noradrenaline and neuropeptide Y, and differential tissue innervation by nerves containing different combinations of these markers. Immunohistochemical labeling of single and adjacent serial cryostat sections, and double labeling was employed. Some animals were treated with the noradrenergic neurotoxin, 6-hydroxydopamine. In control animals neuropeptide Y-immunoreactive fibers were numerous in the myometrium and around arteries; noradrenergic fibers were few in the myometrium and moderate in number around arteries. Myometrial neuropeptide Y-immunoreactive fibers were not decreased, but apparently increased, in 6-hydroxy-dopamine-treated rats; in contrast, perivascular neuropeptide Y-immunoreactive fibers were markedly reduced, but not totally absent. Noradrenergic fibers were absent in the myometrium and around arteries following 6-hydroxydopamine treatment. Labeling of adjacent sections and double labeling revealed coincident labeling of markers for neuropeptide Y and noradrenaline in perivascular, but not myometrial, nerves. We concluded that most myometrial neuropeptide Y-immunoreactive nerves did not contain noradrenaline since they were not sensitive to 6-hydroxydopamine and did not stain doubly; however, perivascular neuropeptide Y-immunoreactive fibers which degenerated after 6-hydroxydopamine treatment and did label doubly must co-store noradrenaline. Some neuropeptide Y-immunoreactive perivascular fibers may contain neuropeptide Y but not noradrenaline. Thus, it appears there is a differential innervation of tissues in the cervix by neuropeptide Y/noradrenergic nerves; this could reflect a differential regulation of tissues innervated by these nerves.

Healing following conventional and cryosurgical discoplasty in the monkey temporomandibular joint.

The techniques of conventional discoplasty and the scar producing potential of cryosurgical treatment of the bilaminar region were examined in a study of 17 adult female cynomologous monkeys. Temporomandibular joint (TMJ) tissues were obtained at four and 10 days, four, 20, 52, and 78 weeks after surgery, and compared to TMJ tissues in unoperated control animals. No clinically observable changes in weight, occlusion, or jaw function were identified. All the surgical sites healed normally. At necropsy, lesions of the articular surface of the condyle were apparent in areas that had been surgically "shaved." The anterior, intermediate, and posterior zones of the surgically-treated discs were generally free of change. Two cryosurgically-treated discs showed lesions and perforations. Histologic and autoradiographic observations suggested that the bilaminar area heals quickly with minimal scarring, following both conventional surgical manipulation and cryosurgery. Under the conditions of this experiment the cryosurgical technique was judged not to be superior to the conventional surgical approach.

Paracervical ganglia of the female rat: histochemistry and immunohistochemistry of neurons, SIF cells, and nerve terminals.

The paracervical ganglia of the female rat were studied to elucidate the variety of neural elements in the ganglia. Light and electron microscopy, histochemistry, and immunohistochemistry were employed to reveal subtypes of neurons; small, intensely fluorescent (SIF) cells; and nerve terminals and to examine the relationships between these elements. On the basis of their histochemical markers, four subtypes of principal neurons were identified: acetylcholinesterase (ACHE)-positive, noradrenergic, neuropeptide tyrosine-immunoreactive (NPY-I), and vasoactive intestinal polypeptide-immunoreactive (VIP-I). The NPY-I neurons appeared to be the most numerous and the noradrenergic the least common type of neuron. Four subtypes of chemically coded SIF cells were revealed: catecholamine-containing, NPY-I, and those immunoreactive for calcitonin-gene-related peptide (CGRP-I) and cholecystokinin-octapeptide (CCK-8-I). The SIF cells were present as single cells among and adjacent to principal neurons and as large clusters near the edges of the ganglia or in nearby nerve trunks. Synaptic contacts on SIF cells, or between SIF-cell processes and neurons, were not observed. Seven subtypes of nerve terminals were stained: ACHE-positive, CGRP-I, CCK-8-I, VIP-I, substance P-I, enkephalin-I, and atrial natriuretic factor-I. Nerve terminals enwrapped the neurons as perineuronal plexuses in synaptic-like relationships. These results demonstrate that the paracervical ganglia of the female rat are a complex system of neural elements. For example, several classes of chemically coded neurons, SIF cells, and terminals exist in the ganglia. Each of these components contains a number of substances, some of which are putative neurotransmitters, which could influence activity in the ganglia or in the effector organs innervated by the ganglia.

Localization of peptides in nerve terminals in the paracervical ganglion of the rat by light and electron microscopic immunohistochemistry: enkephalin and atrial natriuretic factor.

Enkephalin- and atrial natriuretic factor-like immunoreactivities were localized in nerve terminals in the female rat paracervical ganglia. Immunoreactive terminals were adjacent to and in synaptic contact with many, but not all, principal neurons. Immunoreactivity was restricted to large dense-core synaptic vesicles. These results suggest that there is a complex synaptic input to the principal neurons of the rat paracervical ganglia and that these substances could act as neurotransmitters or neuromodulators in the ganglion.

Comparative distribution of neuropeptide tyrosine-, vasoactive intestinal polypeptide-, substance P-immunoreactive, acetylcholinesterase-positive and noradrenergic nerves in the reproductive tract of the female rat.

Location, distribution and density of nerve fibers immunoreactive to neuropeptide tyrosine, vasoactive intestinal polypeptide and substance P were studied in the reproductive tract of the female rat and compared with acetylcholinesterase-positive ("cholinergic") and noradrenergic nerves. Plexuses of all types of fibers were present in the vagina, uterine cervix, uterine horn and oviduct. In the tubular reproductive organs all of these types of nerve fibers appeared to innervate vascular and non-vascular smooth muscle and nearly all types of fibers formed plexuses subjacent to the epithelium lining the organs. Individual fibers of all classes appeared to innervate fascicles of smooth muscle in the mesometrium of the uterine horn. A few acetylcholinesterase-positive and substance P-immunoreactive fibers were present in the ovary but no vasoactive intestinal polypeptide-immunoreactive nerves were observed. Noradrenergic and neuropeptide tyrosine-immunoreactive nerves were numerous in the ovary where they were seen in the interstitial gland tissue and associated with follicles and blood vessels. It is suggested that these nerves may influence hemodynamic events and non-vascular smooth muscle in such functions as transport of sperm and ova and parturition. Substance P-immunoreactive nerve fibers are likely to be sensory fibers that could have roles in neurohormonal reflexes.

Autoradiographic localization of 3H-digoxin binding by neural cells in the medulla.

The purpose of this investigation was to localize binding sites for the cardiac glycoside digoxin in the medulla of the rat in vivo. Adult male Sprague-Dawley rats were injected (IV) with 3H-digoxin and killed 30 minutes later. Autoradiographs of medullas showed evidence of 3H-digoxin binding to small- and medium-sized neural cells in the regions of the nucleus solitarius, dorsal motor nucleus of the vagus, area postrema, and in the zone between the area postrema and the underlying neuropil. However, the parasympathetic preganglionic neurons of the dorsal motor nucleus were not labeled. The 3H-digoxin-labeled cells in the medulla were located mainly in the commissural and medial portions of nucleus solitarius at the level of the area postrema. Animals injected with unlabeled digoxin followed by 3H-digoxin showed reduced binding of radioactivity. The small- and medium-sized neurons of the caudal portions of the nucleus solitarius are internuncial in position with respect to cardiovascular afferents of the glossopharyngeal and vagus nerves and sympathetic and parasympathetic cardiovascular efferent neurons of the medulla. The results of this study suggest that these 3H-digoxin-labeled cells, presumably neurons of nucleus solitarius, may possess high affinity binding sites for digoxin. Further, the area postrema, which lacks a blood-brain barrier, may provide a portal of entry for 3H-digoxin into regions of the medulla known to contain neurons that play a role in the regulation of cardiac rhythm.

Radioactivity in pituitaries and cerebrums of neonatal rats following [3H]-estradiol administration.

The uptake of radioactivity by the pituitary, cerebrum and muscle of female and male neonatal and prepuberal rats 1 hour after administration of [3H]-estradiol was measured by liquid scintillation. Tissues were homogenized and aqueous, lipid, and protein-nucleoprotein fractions were separated for quantification of radioactivity. No significant sex differences in the radioactivity associated with the pituitary fractions were noted at any of the ages studied.

Lysosomal acid hydrolase activities in the lungs of fetal, neonatal, adult, and senile mice.

The activities of four lysosomal acid hydrolases (LAH) in the lungs of two strains of mice changed significantly throughout the life cycle. In the CK7B1/6J animals, acid phosphatase (AP) and beta-glucuronidase (beta-G) were maximally active during early neonatal life then gradually declined the adult levels by 4-5 weeks of age. After reaching the adult level, acid phosphatase activities did not change significantly tcreased markedly with advanced age. N-Acetyl-beta-D-glucosaminidase (GAD) activities did not change significantly duringlate fetal, neonatal or young adult stages but increased significantly with advancing age. In the lungs of the CFW animals, the increase in activities of beta-G and GAD between young adult life and advanced age was highly significant, whereas there was no notable change in the activities of acid phosphatase or arylsulfatase (AS). The specific activities of the hydrolases in the lungs of the C57B1/6J strain were quite similar to those in the lungs of the CFW strain. The activities of all four hydrolases were markedly elevated in two spontaneous adenomatous tumors found in the lungs of old mice. The data indicate that LAH play a significant role in lung growth and maturation, and in changes associated with aging.