Organization of the guinea-pig uterine innervation. Distribution of immunoreactivities for different neuronal markers. Effects of chemical- and pregnancy-induced sympathectomy

Summary The structural organization of the guinea-pig uterine innervation was investigated by an immunofluorescence method using neurofibrillary protein (NF) and neuron-specific enolase (NSE) as general neuronal markers. NF- and NSE-immunoreactive nerve trunks and non-varicose nerves formed continuous networks similar to nerves with analogue morphology and with immunoreactivities for tyrosine hydroxylase (TH; adrenergic nerves) and neuropeptide Y (NPY). NF- and NSE-immunoreactive non-varicose nerves occurred in the myometrium and along vessels, where TH- and NPY-immunoreactive varicose nerves were also comparatively frequent. After chemical sympathectomy all TH- and NPY-immunoreactive varicose nerves and most NF- and NSE-immunoreactive non-varicose nerves disappeared, suggesting colocalization of TH, NPY, NF and NSE immunoreactivities. During pregnancy all NF-, NSE-, TH- and NPY-immunoreactive nerve structures disappeared in the foetus-bearing uterine horns whereas in the cervix and non-foetus-bearing uterine horns only the myometrial TH- and NPY-immunoreactive varicose nerves disappeared. After parturition there was a complete structural restoration of all types of immunoreactive nerves in previously non-foetus-related tissue. The reinnervation of this tissue followed a similar time-course to that after chemical sympathectomy. In contrast, the reinnervation of previously foetus-related tissue was much slower and incomplete.In conclusion, the whole autonomic uterine innervation undergoes overt structural changes during pregnancy and these changes are related to the foetus-bearing regions.     

Effects of infantile/prepubertal chronic estrogen treatment and chemical sympathectomy with guanethidine on developing cholinergic nerves of the rat uterus.

The innervation of the uterus is remarkable in that it exhibits physiological changes in response to altered levels in the circulating levels of sex hormones. Previous studies by our group showed that chronic administration of estrogen to rats during the infantile/prepubertal period provoked, at 28 days of age, an almost complete loss of norepinephrine-labeled sympathetic nerves, similar to that observed in late pregnancy. It is not known, however, whether early exposure to estrogen affects uterine cholinergic nerves. Similarly, it is not known to what extent development and estrogen-induced responses in the uterine cholinergic innervation are affected by the absence of sympathetic nerves. To address this question, in this study we analyzed the effects of infantile/prepubertal chronic estrogen treatment, chronic chemical sympathectomy with guanethidine, and combined sympathectomy and chronic estrogen treatment on developing cholinergic nerves of the rat uterus. Cholinergic nerves were visualized using a combination of acetylcholinesterase histochemistry and the immunohistochemical demonstration of the vesicular acetylcholine transporter (VAChT). After chronic estrogen treatment, a well-developed plexus of cholinergic nerves was observed in the uterus. Quantitative studies showed that chronic exposure to estrogen induced contrasting responses in uterine cholinergic nerves, increasing the density of large and medium-sized nerve bundles and reducing the intercept density of fine fibers providing myometrial and perivascular innervation. Estrogen-induced changes in the uterine cholinergic innervation did not appear to result from the absence/impairment of sympathetic nerves, because sympathectomy did not mimic the effects produced by estrogen. Estrogen-induced responses in parasympathetic nerves are discussed, considering the direct effects of estrogen on neurons and on changes in neuron-target interactions.     

Voice changes after thyroidectomy: role of the external laryngeal nerve.

Hitherto voice changes have been regarded as an infrequent complication of thyroidectomy and damage to the recurrent laryngeal nerve has been given as their major cause. Voice function was assessed in 325 patients after thyroidectomy. Permanent changes occurred in 35 (25%) after subtotal thyroidectomy and in 19 (11%) after lobectomy. The commonest cause of voice change appeared to be injury to the external laryngeal nerves on one or both sides. Damage to the recurrent laryngeal nerve, which was routinely identified and protected, was rarely a cause. When the external laryngeal nerves were identified and preserved, permanent voice changes occurred in only 5% of cases; this was similar to the incidence of 3% in controls after endotracheal intubation alone. The course of the external laryngeal nerve is variable, and consequently mass ligation of the vessels at the top of the upper pole will damage it in a high proportion of cases. To minimise this serious complication these nerves should be identified and protected as well as the recurrent nerves and voice function should be assessed early in the postoperative period by laryngoscopy and by a speech therapist.     

Postnatal development and distribution of peptide-containing nerves in the genital system of the male rat. An immunohistochemical study.

The distribution and relative density of peptide-containing nerves was studied in the rat in order to assess the progression of neuronal changes during the postnatal development of the male genital system from the prepubertal age to adulthood. Testis, caput and cauda epididymis, ductus deferens, seminal vesicles, prostate and penis from 8-, 20-, 38-, and 70-day-old rats were sectioned and were immunostained with antisera to the neuropeptides calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY), and to a general neuronal marker, protein gene product 9.5 (PGP 9.5). The testicular parenchyma and caput epididymis did not show any immunoreactivity. Very scattered CGRP-containing nerves were present in 8-day-old rats; numerous VIP-, CGRP-, and NPY-peptide-containing nerves were observed in the cauda epididymis, ductus deferens, accessory glands and penis of 20-day-old rats. The number of nerves increased in 35-day-old rats while no changes were observed in more adult rats. A parallel increase was seen for the immunostain for PGP 9.5. These data suggest that peptide-containing nerves appear in the genital system after birth and reach a full development before the completion of puberty. Peptide-containing nerves were visible first in the interstitial area and then spread in the muscular coat of the ducts, glands and of the blood vessels. While CGRP- and NPY-containing nerves were distributed in the vicinity of the muscle cells, VIP-containing nerves were also observed in the subepithelial regions, suggesting a possible role of this neuropeptide in the control of epithelial functions.     

Effects of pregnancy on the extrinsic innervation of the guinea pig uterus. A histochemical, immunohistochemical and ultrastructural study

Summary The extrinsic innervation of the guinea pig uterus was studied by immunohistochemical, ultrastructural and enzyme histochemical methods.The extrinsic innervation was organized in two major ways. One consisted of nerve trunks and non-varicose nerve fibres running in the suspensory ligament, and the other of a plexus of varicose nerve fibres surrounding vessels, and non-vessel-related non-varicose nerve fibres in the mesouterus. The use of different neuronal and Schwann cell markers showed that the extrinsic innervation was predominantly adrenergic and contained only few peptidergic nerves. Acetylcholinesterase-positive (cholinergic) nerves were only found around the uterine artery.In late pregnancy, the extrinsic nerves of the mesouterus adjacent to foetus-containing uterine horns underwent pronounced degenerative changes comprising both Schwann cell and axonal structures. In comparison, no changes were found in extrinsic nerves of mesouteri adjacent to non-foetus-bearing uterine horns or in extrinsic nerves in the suspensory ligaments. Further, chemical sympathectomy produced axonal degeneration but no changes in the Schwann cells.In conclusion, the pregnancy-induced nerve degeneration is of a very special type different from that following chemical sympathectomy and represents a local phenomenon related to the conceptus. Hypothetically, this could be of importance for counteracting disturbances in placental blood flow.     

The effects of ciguatoxin on the nerves of the teleost fish, Sillago ciliata.

The absolute refractory period, relative refractory period, and the duration and magnitude of the supernormal period were measured after incubation of fish nerves with ciguatoxin and other channel modifying compounds, tetrodotoxin, veratridine, verapamil, and lignocaine. In vitro electrophysiological studies were carried out on the lateral line nerve of the whiting, Sillago ciliata Cuvier. Electrophysiological changes in fish nerves after exposure to ciguatoxin (0.3 MU.ml-1) and veratridine (1 x 10(-5) M) are similar to changes that occur in mammalian nerves and include an increase in the absolute refractory period, the relative refractory period, and the magnitude and duration of supernormality. The effects of ciguatoxin (0.3 MU.ml-1) in fish nerves were antagonised by tetrodotoxin (5 x 10(-10) M), verapamil (5 x 10(-7) M), and lignocaine (1 x 10(-5) g/ml-1). The nerves of Sillago ciliata used in this study responded to ciguatoxin and its antagonists in a similar manner to mammalian nerves, suggesting that these teleost nerves have no specific electrophysiological mechanism to cope with this toxin.     

Cutting-edge technology. III. Imaging and the gastrointestinal tract: mapping the human enteric nervous system

Monitoring membrane potentials by multisite optical recording techniques using voltage-sensitive dyes is ideal for direct analysis of network signaling. We applied this technology to monitor fast and slow excitability changes in the enteric nervous system and in hundreds of neurons simultaneously at cellular and subcellular resolution. This imaging technique presents a powerful tool to study activity patterns in enteric pathways and to assess differential activation of nerves in the gut to a number of stimuli that modulate neuronal activity directly or through synaptic mechanisms. The optical mapping made it possible to record from tissues such as human enteric nerves, which were, until now, inaccessible by other techniques.     

Reductions of neural activities to upper airway muscles after elevations in static lung volume.

We evaluated the hypothesis that the tonic discharge of pulmonary stretch receptors significantly influences the respiratory-modulated activities of cranial nerves. Decerebrate and paralyzed cats were ventilated with a servo-respirator, which produced changes in lung volume in parallel with integrated phrenic activity. Activities of the facial, hypoglossal, and recurrent laryngeal nerves and nerves to the thyroarytenoid muscle and triangularis sterni were recorded. After a stereotyped pattern of lung inflation, tracheal pressure was held at 1, 2, 4, or 6 cmH2O during the subsequent ventilatory cycle. Increases in tracheal pressure caused progressive reductions in both inspiratory and expiratory cranial nerve activities and progressive elevations in triangularis sterni discharge; peak levels of phrenic activity declined modestly. Similar changes were observed in normocapnia and hypercapnia. We conclude that the tonic discharge of pulmonary stretch receptors is an important determinant of the presence and magnitude of respiratory-modulated cranial nerve activity. This reflex mechanism may maintain upper airway patency and also regulate expiratory airflow.     

Locomotion in the pulmonate snail Melampus--I. The motor pattern

1. The patterned neural activity that drives muscular locomotor movements in Melampus is generated within the central nervous system. 2. In the transition from quiescent state to crawling, the pattern recorded in nerves and connectives changes from short-duration bursts in many units to the 60-100 sec cycle of events recorded during tethered crawling in the semi-intact snail. 3. Extracellularly recorded bursts and individually recognizable spikes in pedal nerves are correlated with movements that occur at each stage of the cyclically repeated crawl-steps. 4. Intracellularly recorded pedal neurons involved in locomotion receive excitatory drive, inhibitory drive, or alternating excitatory and inhibitory drive during the step cycle.     

Reversal of axonal transport in regenerating nerves.

Abstract— Orthograde and retrograde axonal transport were studied in rat sciatic nerves which had been crushed and either allowed to regenerate, or prevented from doing so by tightly ligaturing the nerve. At various intervals after crushing the nerve. L-[³H]leucine was injected into the lumbosacral spinal cord. and the subsequent transport of labeled protein in motoneuron axons was quantitated by measuring the accumulation of labeled protein at collection crushes made proximal to the original nerve crush. Accumulations proximal to the collection crushes (orthograde transport) 9-11 h after injection (p.i.). decreased within I day of nerve injury, but returned to normal values as regeneration proceeded. In non-regenerating nerves accumulations remained depressed for at least 30 days. Accumulations distal to the collection crushes (retrograde transport) 9-11 h pi. increased over the first 5 days following injury but returned to normal values as regeneration proceeded. In non-regenerating nerves accumulations remained elevated. The time-course of retrograde transport of newly-synthesized protein also returned to normal during nerve regeneration. It is suggested that changes in retrograde transport during regeneration may inform the neuron cell body of the progress of regeneration and elicit appropriate metabolic responses. among which may be the changes in orthograde transport that follow axotomy.     

Influence of renal denervation on chronopharmacology of furosemide in rats.

Our previous studies have suggested that the adrenergic nervous system is involved in the mechanism responsible for the time-dependent change in the urinary excretion of furosemide in rats. To examine a potential role of renal nerves in this phenomenon, renal denervation or sham operation was performed using unilaterally nephrectomized rats. Furosemide (30 mg/kg) was given orally at 12 am or 12 pm. Urine was collected for 8 hours after furosemide dosing, and urinary excretions of furosemide and sodium were determined. Urinary furosemide excretion and diuretic effects of the agent (urine volume and urinary sodium) were significantly greater at 12 am than at 12 pm in the sham-operated group of rats. However these administration time-dependent changes in urinary furosemide and its diuretic effects disappeared in the renal-denervated group of animals. These results suggest that the renal nerves contribute to the time-dependent changes in the urinary excretion of furosemide and its subsequent diuretic effects.     

Na,K-atpase alterations in diabetic rats: relationship with lipid metabolism and nerve physiological parameters.

Type 1 diabetes induces several metabolic and biochemical disturbances which result in the alteration ofNa,K-ATPase, an enzyme implicated in the physiopathology of neuropathy Several fatty acid supplementations lessen this alteration. The aims of this study were to determine the possible relationships between Na,K-ATPase activity in nerves and red blood cells (RBCs) and, on one hand, the fatty acid alterations induced by diabetes in these tissues and plasma and on the other, on nerve physiological parameters. Two groups of rats, control and diabetic (n = 15), were sacrified 8 weeks after induction of diabetes with streptozotocin. Nerve conduction velocity (NCV), nerve blood flow (NBF), Na,K-ATPase activity and membrane fatty acid composition of sciatic nerves, red blood cells (RBCs) and plasma were measured. NCV, NBF and Na,K-ATPase activity in RBCs and in sciatic nerves were significantly decreased in diabetic rats. We revealed a positive correlation between Na,K-ATPase activity in sciatic nerves and both NBF and NCV and between Na,K-ATPase activity in RBCs and NBF and the same activity in sciatic nerve. Diabetes induced major changes in plasma fatty acids and RBC membranes and less important changes in sciatic nerve membranes. Na,K-ATPase activity correlated negatively with C20: 4 (n-6) and C22: 4 (n-6) levels in nerves and with C18: 2 (n-6) levels in RBCs. During diabetes, changes in the membrane fatty acid composition suggest the existence of a tissue-specific regulation, and the decrease in Na,K-ATPase activity correlates with the alteration in the level of specific fatty acids in RBCs and sciatic nerves. Modifications in the lipidic environment of Na,K-ATPase would be involved in the alteration of its activity. Na,K-ATPase activity seems to be implicated in the decrease of both NCV and NBF during diabetes.     

Supersensitivity to ACh in muscles after prolonged nerve block.

Sciatic nerves of rats and tibial nerves of rabbits were kept anaesthetized in situ for periods of 3-11 days by applying silastic cuffs containing lidocaine base or marcaine hydrochloride. To insure a more uniform release of the rapidly diffusing lidocaine base, the drug was contained in compartments at some distance from the nerve and the cuffs were covered with polystyrene. The completeness of anaesthesia and the functional state of the nerve were tested by stimulating the exposed nerves proximal and distal to the cuff and by observing the behaviour of the muscle prior to killing the animals. The ACh sensitivity was tested by electrophoretic application of ACh from micropipettes and by recording the results changes of the resting membrane potential in individual muscle fibres. The ACh sensitivity was found to be present in the extrajunctional area of all muscle fibres including those displaying miniature end-plate potentials. These was no difference between the behaviour of muscles from rats and rabbits and between the action of lidocaine base and marcaine hydrochloride. Previous reports on the absence of extrajunctional ACh sensitivity in muscles of rabbits whose nerves had been treated by lidocaine base were explained by a relatively rapid loss of the drug from the usual type of nerve cuffs (more than 70% of the drug lost in one day), permitting a premature recovery of the nerves from anaesthesia.     

Early increase in CGRP- and VIP-immunoreactive nerves in the skin of streptozotocin-induced diabetic rats.

We have previously shown depletion of nerves and neuropeptides in skin biopsies of diabetic patients, even in the absence of clinical signs and symptoms of sensory and autonomic neuropathy, but were unable to examine the changes occurring at an early stage of the disease. Therefore, the distribution and relative density of peptide-containing nerves was studied in streptozotocin-treated rats in order to assess the progression of neural changes in the initial stages of diabetes. Skin samples dissected from the lip and footpad of diabetic rats, 2, 4, 8 and 12 weeks after streptozotocin injection and age matched controls were sectioned and were immunostained with antisera to the neuropeptides substance P, calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY), and to a general neural marker, protein gene product 9.5 (PGP 9.5). No change was apparent in the distribution or relative density of immunoreactive cutaneous nerve fibres 2, 4 and 8 weeks after streptozotocin treatment. By 12 weeks there was a marked increase in the number of CGRP-immunoreactive fibres present in epidermis and dermis, and of VIP-immunoreactive fibres around sweat glands and blood vessels. A parallel increase was seen in nerves displaying PGP 9.5 immunoreactivity. No differences were detected in nerves immunoreactive for either substance P in the epidermis and dermis, and NPY around blood vessels. The alterations in the peptide immunoreactivities may be similar in the initial stages of human diabetes.     

Objectivity and Illusion in Evolutionary Ethics: Comments on Waller

In this paper I argue that any adequate evolutionary ethical theory needs to account for moral belief as well as for dispositions to behave altruistically. It also needs to be clear whether it is offering us an account of the motivating reasons behind human behaviour or whether it is giving justifying reasons for a particular set of behaviours or, if both, to distinguish them clearly. I also argue that, unless there are some objective moral truths, the evolutionary ethicist cannot offer justifying reasons for a set of behaviours. I use these points to refute Waller's claims that the illusion of objectivity plays a dispensable role in Ruse's theory, that my critique of Ruse's Darwinian metaethics is built on a false dilemma, that there is nothing to be distressed about if morality is not objective, and that ethical beliefs are subject to a kind of causal explanation that undermines their objectivity in a way that scientific beliefs are not.     

Myelin labeled with mercuric chloride. Asymmetric localization of phosphatidylethanolamine plasmalogen

We have recorded modified X-ray diffraction patterns to 15 Å spacing from sciatic nerves treated with mercuric chloride (HgCl₂) at concentrations of 0.5 to 32 mm in water or in saline. The observed changes in repeat period and in the intensities of the low-order reflections indicate closer packing of membranes at their cytoplasmic surfaces after treatments with HgCl₂. In addition, HgCl₂ at 0.25 mm or more prevents swelling in water at the extracellular boundaries. By comparing the distinctive diffraction patterns from nerves treated under different conditions with HgCl₂, we have interpreted the changes in intensities of the higher order X-ray reflections and have calculated electron density profiles of the modified membranes. The most striking difference between membrane profiles before and after treatment with HgCl₂ is the large increase in electron density in the region of the lipid headgroup peak in the cytoplasmic half of the bilayer. The magnitude and location of this increase suggests labeling of myelin lipid. To examine this possibility, we analyzed the lipids from mercury-treated sciatic nerves.Thin-layer chromatography of lipids extracted from nerves treated with HgCl₂ shows a marked decrease of phosphatidylethanolamine, which exists in myelin primarily as plasmalogen. At the same time, a new spot identified as lysophosphatidylethanolamine appears. An identical result was obtained by treating extracted lipids with HgCl₂, suggesting that the same sites of interaction are present in the intact membrane as in the dispersed lipids. Previous studies on plasmalogens indicate that mercury adds to the β-carbon of the α,β-unsaturated ether group to produce a lyso-lipid and an aldehyde with bound mercury (Norton, 1959). From a correlation of our X-ray structural analysis and the chemical studies, we conclude that phosphatidylethanolamine plasmalogen is preferentially localized in the cytoplasmic half of the myelin membrane bilayer.     

Stomatogastric nervous system of Galleria mellonella L. (Lepidoptera : Pyralidae): Changes during metamorphosis with special reference to FMRFamide neurons

Scanning electron microscopy and immunohistochemical staining for FMRFamide-like peptides revealed that the stomatogastric nervous system of Galleria mellonella (Lepidoptera : Pyralidae) includes 5 ganglia: the frontal ganglion with 4, the hypocerebral ganglion with 2, the ingluvial ganglion with 2–4, and each of the paired proventricular ganglia with 6–8 immunoreactive perikarya. Immunoreactivity was also found in axons to and within the corpora cardiaca, in the nerves connecting stomatogastric ganglia, as well as in 8 gastric nerves that extend along longitudinal midgut muscles. Adhesion of corpora cardiaca to the hypocerebral ganglion and partial merging and shortening of gastric nerves were the only conspicuous changes of the stomatogastric system that occurred during metamorphosis.     

Perivascular nerves and the regulation of cerebrovascular tone.

Brain perfusion is tightly coupled to neuronal activity, is commonly used to monitor normal or pathological brain function, and is a direct reflection of the interactions that occur between neuronal signals and blood vessels. Cerebral blood vessels at the surface and within the brain are surrounded by nerve fibers that originate, respectively, from peripheral nerve ganglia and intrinsic brain neurons. Although of different origin and targeting distinct vascular beds, these "perivascular nerves" fulfill similar roles related to cerebrovascular functions, a major one being to regulate their tone and, therein, brain perfusion. This utmost function, which underlies the signals used in functional neuroimaging techniques and which can be jeopardized in pathologies such as Alzheimer's disease, stroke, and migraine headache, is thus regulated at several levels. Recently, new insights into our understanding of how neural input regulate cerebrovascular tone resulted in the rediscovery of the functional "neurovascular unit." These remarkable advances suggest that neuron-driven changes in vascular tone result from interactions that involve all components of the neurovascular unit, transducing neuronal signals into vasomotor responses not only through direct interaction between neurons and vessels but also indirectly via the perivascular astrocytes. Neurovascular coupling is thus determined by chemical signals released from activated perivascular nerves and astrocytes that alter vascular tone to locally adjust perfusion to the spatial and temporal changes in brain activity.     

The origin of VIP-containing nerves in the urinary bladder of rat

The innervation of the urinary bladder is known to include a considerable number of nerves containing vasoactive intestinal polypeptide (VIP). The origin of such nerves in the bladder of rat was investigated in this study using the methods of immunocytochemistry and radioimmunoassay combined with surgical sectioning of the hypogastric and/or pelvic nerves to the bladder. Eight days after pelvic nerve sectioning proximal to the main pelvic ganglion, VIP-immunoreactive nerves and VIP content were markedly increased from the level in the sham-operated rat bladder. Sectioning of hypogastric or both nerve pathways led to a less significant increase. It was therefore postulated that the majority of VIP-immunoreactive nerves originate from ganglia located either close to the bladder or within the bladder wall. It is interesting that in these experiments the VIP content of the bladder nerves is inversely related to the changes in motility that would be expected to result from the nerve sections.     

Effect of neuropeptides released from sensory nerves on blood flow in the rat airway microcirculation.

Stimulation of sensory nerves in the airway mucosa causes local release of the neuropeptides substance P and calcitonin gene-related peptide (CGRP). In this study we used a modification of the reference-sample microsphere technique to measure changes in regional blood flow and cardiac output distribution produced in the rat by substance P, CGRP, and capsaicin (a drug that releases endogenous neuropeptides from sensory nerves). Three sets of microspheres labeled with different radionuclides were injected into the left ventricle of anesthetized F344 rats before, immediately after, and 5 min after left ventricular injections of capsaicin, substance P, or CGRP. The reference blood sample was withdrawn from the abdominal aorta and was simultaneously replaced with 0.9% NaCl at 37 degrees C. We found that stimulation of sensory nerves with a low dose of capsaicin causes a large and selective increase in microvascular blood flow in the extrapulmonary airways. The effect of capsaicin is mimicked by systemic injection of substance P but not by CGRP, suggesting that substance P is the main agent of neurogenic vasodilation in rat airways.