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Sympathetic innervation improves the contractile performance of neonatal cardiac ventricular myocytes in culture.

We propose that sympathetic innervation could contribute to the improvement in cardiac contractility that normally occurs during neonatal life because these processes are developmentally coincident. Effects of sympathetic innervation were studied in primary cultures of isolated, not previously innervated ventricular cardiomyocytes from neonatal rats. Innervation was produced by addition of autologous neurons from the thoracolumbar sympathetic ganglia, and amplitude and frequency of myocyte contraction were measured by on-line video motion analysis. Sympathetic innervation significantly (P less than 0.0001) increased amplitude of contraction (by 34 +/- 8%) and decreased contraction frequency (by 36 +/- 3%). The effect of innervation on myocyte contractility was not attenuated by adrenoceptor blockade (10(-6) M propranolol and 10(-6) M phentolamine), but could be reproduced using medium conditioned by cocultures of neurons and myocytes. Sympathetic innervation improves the contractility of isolated cardiomyocytes, indicating that autonomic innervation contributes to maturation of cardiac function.

Animals↗

Autonomic innervation of rabbit urinary bladder following estrogen administration.

The effects of estrogen administration on the autonomic innervation of the rabbit urinary bladder were studied. Immature female white rabbits were injected twice daily with estrogen (150 microgram./Kg.) for four consecutive days. Control animals received injections of vehicle alone. The adrenergic innervation was identified using the glyoxalic acid method of catecholamine histofluorescence. The cholinergic innervation was visualized utilizing specific acetylcholinesterase staining. Additionally, the effect of estrogen administration on the response of smooth muscle strips of urinary bladder to specific autonomic agonists was determined. Estrogen administration induced a moderate increase in the adrenergic innervation of the rabbit bladder detrusor, whereas no change could be observed in the cholinergic innervation. It should be noted, however, that whereas the adrenergic innervation in the bladder of the control animal was sparse, the cholinergic innervation in the bladder body was quite dense. Estrogen induced a marked increase in the response to alpha-adrenergic (methoxamine) and muscarinic cholinergic (bethanechol) agonists. No alterations were noted in the response to beta-adrenergic agonists (isoproterenol). These findings indicate that the urinary bladder responds as a target organ for estrogen-induced alterations in autonomic innervation.

Animals↗

Central origin of vagal nerve fibres innervating the fundus and corpus of the stomach in rat.

The origin of vagal nerve fibres innervating the anterior and posterior walls of the fundus and corpus of the rat stomach was investigated using the axon tracing dye, Fast blue. The secretomotor nerve supply to the rat stomach was predominantly ipsilateral. A large majority (98-99%) of the vagal perikarya innervating the anterior fundus and corpus were located on the left side of the brainstem. A large majority (96-99%) of the vagal perikarya innervating the posterior fundus and corpus were located on the right side. Vagal perikarya were arranged in longitudinal, dorsal cell columns which extended beyond the normally accepted cytoarchitectural limits of the dorsal motor nucleus of the vagus (DMV). A few vagal cells innervating the fundus were also found in the nucleus ambiguus. Vagal cell columns innervating the anterior and posterior fundus extended rostrocaudally over a distance of up to 4 mm and projected caudally as far as the cervical spinal cord. Vagal cell columns innervating the anterior and posterior corpus were more compact, extended over a distance of 2-3 mm, and projected rostrally as far as the inferior salivatory nucleus of the glossopharyngeal nerves. Vagal cell columns for the fundus and corpus overlapped in the region of the DMV which lay immediately ventral to the area postrema. Between one-third to one-half of the vagal cells innervating the fundus and corpus were concentrated under the area postrema. A simple form of viscerotopic organisation appears to occur within the vagal cell columns innervating the fundus and corpus.

Animals↗

Afferent and sympathetic innervation of the dome and the base of the urinary bladder of the female rat.

The afferent and sympathetic innervation of different regions of the urinary bladder (bladder dome vs. bladder base) was examined in the female rat using simultaneous injections of two fluorescent tracers. Retrogradely labeled cells were found in the dorsal root ganglia (DRG; L1-L3 and L6-S1), the sympathetic chain (SC; T12-L6), the inferior mesenteric ganglia (IMG) and the major pelvic ganglia (MPG). There were very few double-labeled cells, indicating that the dome and the base of the bladder receive innervation (afferent or sympathetic) from separate and distinct neuronal populations. Most of the sympathetic innervation of the bladder arose from the SC (dome: 77%; base: 89%) and it was carried equally by the hypogastric and pelvic nerves. The distributions of SC postganglionic neurons innervating the dome and the base of the bladder were very similar. In contrast, the contribution of IMG neurons was almost entirely restricted to the dome of the bladder (22%), with less than 1% innervating the base. Tyrosine hydroxylase-immunoreactive (TH) neurons in the MPG displayed a strong sexual dimorphism. Many TH neurons were found in the male MPG, but very few in the female MPG. In the female, these TH neurons projected almost exclusively to the bladder base of the female rat and were responsible for 10% of the sympathetic innervation of the base. Less than 1% innervated the dome. Therefore, prevertebral ganglia (IMG and MPG) show a strong regional selectivity in the innervation of the bladder of the female rat. The possible functional implications of this organization are discussed.

Animals↗

Origin of noradrenergic innervation of the spleen in rats.

Noradrenergic (NA) sympathetic innervation of the spleen was examined in young adult Sprague-Dawley rats (200-250 g) following surgical removal of the superior mesenteric-celiac ganglia (SM-CG) and/or bilateral transection of the subdiaphragmatic vagus nerve. Sham-operated and unoperated rats served as controls. NA sympathetic innervation of spleens from sham-operated and unoperated controls, and from vagotomized rats, was qualitatively similar, with fibers distributing to the capsule, trabeculae, vasculature, and parenchyma of the white pulp. Complete ganglionic extirpation resulted in almost total denervation of NA fibers in all compartments of the spleen. High-performance liquid chromatography with electrochemical detection (LCEC) for catecholamines (CA) and quantitative morphometry of the density of NA varicosities confirmed these observations. LCEC revealed a greater than 85% depletion of norepinephrine (NE) in the spleen following superior mesenteric-celiac ganglionectomy. Stereological evaluation of NA varicosities with a point counting method revealed a decline of 99% in the volume density of NA terminals that occurred uniformly in all compartments of spleens from ganglionectomized rats. In addition, stereological analysis revealed a loss of total NA varicosities (approximately 31% decrease) in spleens from sham-operated rats. This loss in volume density occurred largely due to a loss in parenchymal fibers (approximately 45% decrease). Bilateral subdiaphragmatic vagotomy blocked the effect on NA innervation produced by the surgical stress of sham operation. Retrograde tracing following injection of either fluorogold or true blue into the spleen, coupled with immunocytochemical localization of tyrosine hydroxylase (TH), demonstrated abundant fluorogold (true blue)-labeled neurons in the SM-CG; many, but not all, of these neurons also were TH-positive. These findings indicate that the SM-CG neurons supply NA innervation to the spleen, providing sympathetic innervation as the second neuron in the classical two-neuron sympathetic chain, and suggest additional non-NA innervation of the spleen as well. This study also suggests that surgical stress of sham operation may alter directly the NA innervation of the spleen, possibly by inducing temporary retraction of NA fibers of the parenchymal compartment, which is likely to reduce the availability of NE for interaction with cells of the immune system that possess adrenoceptors and are present adjacent to NA varicosities in this region.4+ Bilateral vagotomy ameliorated the effects of sham operation on NA innervation; since the vagal nerve does not distribute fibers to the spleen, this effect is likely to occur through altered feedback circuits effecting sympathetic outflow, or through altered neuroendocrine outflow.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenergic Fibers↗

Immunohistochemical study of the innervation of pulmonary vessels and smooth muscles in the respiratory tract of two frog species.

The innervation of the respiratory tract of amphibians is still poorly understood. Therefore, the respiratory tracts of the frogs Rana esculenta and Discoglossus pictus have been investigated in order to describe non-adrenergic non-cholinergic (NANC) and adrenergic innervation, and the localization of neuromediators that are possibly involved. Immunohistochemical staining of many bioactive substances was found in neuroepithelial cells of the buccopharynx, larynx, lung septa, nerves and neurons throughout the airway system. The findings indicate the occurrence of vasoactive intestinal peptide (VIP)-immunopositive nerve fibers in fibromuscular septa and the vasculature, nitrergic innervation of the large pulmonary veins showing a plexus of nNOS-immunopositive nerve fibers that also innervate the lung wall and the localization of neuronal nitric oxide synthase (nNOS) in neurons in the lung wall. In addition, laryngeal blood vessels and small arteries in the wall of septa that form capillary networks are supplied by enkephalin-immunopositive nerve terminals. We conclude that the airway system of the two frog species studied is innervated by a parasympathetic NANC system. Adrenergic innervation was also found that was immunostained for tyrosine hydroxylase. Adrenergic fibers were mainly present in muscles in septal edges, arteries present in septa and the wall of the lung. It is suggested that nNOS-positive and leu-enkephalin-positive neurons mediate vasodilation via the release of NO, but the nature of the NANC innervation remains obscure. Despite the many pharmacological studies of the lungs of amphibians, the physiological role of pulmonary autonomic innervation remains poorly understood.

Animals↗

Anatomical study of accessory nerve innervation relating to functional neck dissection.

PURPOSE: The present study sought to clarify correlations of accessory nerve innervation in the neck region and innervation of the sternocleidomastoid and trapezius muscles with postoperative dysfunction after functional neck dissection by macroscopic observation. MATERIALS AND METHODS: The materials used in this study were 35 cadavers provided for anatomical practice to the Department of Anatomy, Tokyo Dental College. The accessory nerve was identified at the anterior margin of the trapezius muscle, and its innervation in the posterior triangle of the neck was examined in detail. RESULTS: The superficial cervical vein vascularizes the anterior margin of the trapezius muscle near an area where the main trunk of the accessory nerve innervates the trapezius muscle. The results showed 3 types of accessory nerve innervation of the sternocleidomastoid muscle: Type A, the not penetrating type; Type B, the partially penetrating type; and Type C, the completely penetrating type. In addition, 5 types of innervation of the trapezius muscle by the main trunk and branches of the accessory nerve were apparent, with the number of branches innervating the muscle ranging from 0 to 4. CONCLUSIONS: Dysfunction after functional neck dissection can thus be avoided by paying attention to not only the main trunk of the accessory nerve, but also the branches. Moreover, when identifying accessory nerve innervation of the trapezius muscle, the superficial cervical vein may offer a useful surgical landmark.

Accessory Nerve↗

Survival and mitogenesis of neuroepithelial cells are influenced by noradrenergic but not cholinergic innervation in cultured embryonic rat neopallium.

alpha-Adrenoreceptors are present in the ventricular (VZ) and subventricular (SVZ) zones of the mammalian embryonic forebrain, and contribute towards cell cycle controls in the germinal neuroepithelium [Pabbathi et al., Brain Res. 760 (1997) 22-33.]. Since noradrenaline-containing fibres from the locus coeruleus (LC) and other brainstem nuclei innervate many parts of the CNS from an early stage of embryogenesis, we have used heterochronic cocultures to investigate whether noradrenergic innervation may be the source of trophic support. Dorsal neopallium from E13 rat embryo was cultured in proximity to E15 rostral pons (RP), enabling rapid innervation of the neuroepithelium by differentiated noradrenergic neurons of the LC. The projection of interconnecting fibres into germinal areas of the cortex was established in vitro by retrograde tracing and dopamine beta-hydroxylase (DBH) immunocytochemistry. When functional innervation was prevented by transection of axons connecting the explants, the number of apoptotic (TUNEL-positive) cells in the neopallium was increased. Bromodeoxyuridine (BrdU) double-labelling confirmed that germinal cells were amongst those which underwent apoptosis. When cortical explants were innervated by a source of non-monoaminergic (cholinergic) axons from the E18 basal forebrain diagonal band/septum complex (DS), numbers of apoptotic cells were comparable to those in non-innervated cultures. alpha1 and alpha2 adrenoreceptor antagonists included in the medium of cortical cocultures innervated by noradrenergic axons reversed the survival-promoting effect of innervation. Blockade of alpha1 but not of alpha2 receptors also reduced the numbers of S-phase nuclei in the cortex. The results provide evidence that local delivery of neurotransmitter from embryonic noradrenergic axons terminating in the neocortex can regulate survival and proliferation of neuroepithelial cells.

Acetylcholine↗

Alterations in sympathetic innervation of thymus and spleen in aged mice.

Age is associated with reduced immune reactivity, contributing to increased rates of infectious disease and cancer in old age. We have begun to assess the potential for sympathetic nervous system involvement in age-related immune dysfunction by characterizing sympathetic noradrenergic (NA) innervation in lymphoid organs in old animals. In the present study noradrenergic innervation of spleen and thymus was examined histologically and neurochemically in 2-, 12- and 24-month old BALB/c mice. In the thymus of 2-month old animals, NA nerve fibers were found in the subcapsular, cortical, and cortico-medullary regions associated with blood vessels and septa; occasional branches from these nerve fibers entered the parenchyma. With increasing age and thymic involution, NA nerve fibers increased in density; by 24 months of age, dense plexuses were compacted among septa and blood vessels, and numerous linear, varicose nerve fibers were observed branching into the parenchyma. Thymic norepinephrine (NE) concentration (per mg wet weight) increased approximately 4-fold in 12-month old animals and 15-fold in 24-month old animals. Taking the reduced thymus weight into account, total thymic NE at 12- and 24-month of age was equivalent to total thymic NE at 2-month of age, suggesting that NA innervation is maintained as the thymus involutes. In the spleen from 2-month old animals, NA innervation entered the white pulp with the central artery to innervate the periarteriolar lymphatic sheath and the marginal zone. At 12-month of age, histologically and neurochemically there was no change in splenic NA innervation. By 24-month of age, NE was increased significantly, independent of changes in spleen weight. Histologically, increased catecholamine-containing fibers were apparent at 24-month of age, particularly in the parenchyma surrounding the central artery. The alterations in sympathetic NA innervation of lymphoid organs with age suggest that the sympathetic nervous system and NE may play a role in age-associated immune dysregulation. Alternatively, the changes in NA innervation may be secondary to functional changes within the immune system.

Age Factors↗

Comparative formaldehyde-induced and glyoxylic acid-induced fluorescence histochemical studies on the intrinsic adrenergic innervation of the intestine and the liver of normal and vagotomized cats.

The inbuilt intrinsic adrenergic nervous apparatus of the intestine and liver of the cat was studied using 1. the formaldehyde-induced fluorescence histochemical method and 2. the glyoxylic acid-induced fluorescence histochemical method for serial microtome sections and whole mount tissue layers or smear preparations. In addition, the effect of I) total abdominal infra-diaphragmatic vagotomy with or without associated Finney-type gastro-duodenostomy and II) unilateral (left or right) and bilateral cervical vagotomy with or without tracheostomy on the intrinsic adrenergic innervation was tested. Fluorescing varicose axons, both "free" (i.e. unrelated to the blood vessels) and gathered to typical perivascular nerve plexuses were observed in all segments and all layers of the wall of the intestine. The density of the adrenergic innervation varied remarkably from an area to another, even in the same segment and tissue layer, which makes comparative estimations of the density of the innervation very difficult. However, the intrinsic adrenergic innervation of the circular muscle layer of the colon and the rectum seems to be consistently quite rich, and in the rectum, also the longitudinal muscle layer is relatively heavily innervated. It thus seems obvious that (in the cat) also the direct adrenergic innervation of the external smooth muscle layers is of considerable importance, specially in the rectum. In contrast, the results of the present study clearly indicate that the liver parenchyma (of the cat) is devoid of functional intrinsic adrenergic innervation. Vagotomies did not cause any changes in the intrinsic adrenergic innervation of the intestine and liver: even after complete vagotomy no reduction was observed in the number of fluorescing axons or in the intensity of the fluorescence. Consequently, the vagal contribution of adrenergic axons to the liver and the intestine must be negligible, at least in the cat. The use of the glyoxylic acid-induced fluorescence histochemical method and whole-mount tissue layers was found most suitable for mapping and comparative estimation of the density of the intrinsic adrenergic nerve net, and is therefore recommended for other similar or related studies.

Adrenergic Fibers↗

Triad proteins and intracellular Ca2+ transients during development of human skeletal muscle cells in aneural and innervated cultures.

Dihydropyridine receptors (DHPRs), ryanodine receptors (RyRs), and triadin are major components of triads of mature skeletal muscle and play crucial roles in Ca2+ release in excitation-contraction (E-C) coupling. We investigated the expression and localization of these proteins as well as intracellular Ca2+ transients during development of human muscle cells cultured aneurally and innervated with rat spinal cord. mRNAs encoding skeletal muscle isoforms of the DHPR alpha1 subunit (alpha1S-DHPR), the RyR, and triadin were scarce in myoblasts and increased remarkably after myotube formation. Immunocytochemically, alpha1S-DHPR was expressed after myoblast fusion and localized mainly within the cytoplasmic area of aneural myotubes whereas the cardiac isoform (alpha1C-DHPR) was abundant along the plasma membrane. RyRs and triadin were both detected after myotube formation and colocalized in the cytoplasm of aneural myotubes and innervated muscle fibers. Along the plasma membrane of aneural myotubes, colocalization of alpha1C-DHPR with the RyR was more frequently observed than that of alpha1S-DHPR. In innervated muscle fibers, alpha1S-DHPR and RyR were colocalized first along the plasma membrane and later in the cytoplasmic area and formed regular double rows of cross-striation. The alpha1C-DHPR diminished after innervation. In Ca2+ imaging, spontaneous irregular slow Ca2+ oscillations were observed in aneurally cultured myotubes whereas nerve-driven regular fast oscillations were observed in innervated muscle fibers. Both caffeine and depolarization induced Ca2+ transients in aneurally cultured myotubes and innervated muscle fibers. In aneurally cultured myotubes, depolarization-induced Ca2+ transients were highly dependent on extracellular Ca2+, suggesting immaturity of the Ca2+ release system. This dependence remarkably decreased after innervation. Our present results show that these proteins are expressed differently in aneurally cultured myotubes than in adult skeletal muscle, that Ca2+ release in aneurally cultured myotubes is different from in adult skeletal muscle, and that innervation induces formation of a mature skeletal muscle-like excitation-contraction coupling system in cultured human muscle cells.

Animals↗

Cholinergic phenotype developed by noradrenergic sympathetic neurons after innervation of a novel cholinergic target in vivo.

Mammalian sympathetic neurons in vivo may express either a noradrenergic or cholinergic phenotype. In view of the opposing effect of noradrenaline and acetylcholine on most autonomic target organs, the target-appropriate expression of neurotransmitter is critical. We have examined the maturation of the sympathetic innervation of rat sweat glands to define the developmental mechanisms regulating neurotransmitter choice in vivo. Eccrine sweat glands and their sympathetic innervation develop together postnatally in the rat. Early postnatal innervation expresses only noradrenergic properties, but as the glands and their innervation mature, noradrenergic properties decrease dramatically and cholinergic features appear in the same population of neurons. To investigate the role of the sweat gland in this change we have used a transplantation paradigm which allows sweat glands to be innervated by sympathetic neurons that would normally innervate noradrenergic target organs and remain noradrenergic throughout life. We observe that the sympathetic neurons that innervate the novel cholinergic target alter their neurotransmitter properties and develop a cholinergic phenotype. These results indicate that target organs are able to induce appropriate neurotransmitter traits in the neurons that innervate them.

Acetylcholinesterase↗

Innervation of the female levator ani muscles.

OBJECTIVE: The objective of this study was to characterize the innervation of the human female levator ani muscles. STUDY DESIGN: Detailed dissections of the peripheral innervation of the iliococcygeal, pubococcygeal, puborectal, and coccygeal muscles were performed in 12 fresh-frozen female cadavers (aged, 32-100 years) with the use of transabdominal, gluteal, and perineal approaches. Both the pudendal nerve and the sacral nerve roots that enter the pelvis from the cephalic side were followed from their origin at the sacral foramina to their termination. Pelvic floor innervation was described with reference to fixed bony landmarks, particularly the coccyx, the ischial spine and the inferior pubis. Photographs were taken, and nerve biopsies were performed to confirm the gross findings histologically. Biopsy specimens were stained with Masson's trichrome. RESULTS: In each dissection, a nerve originated from the S3 to S5 foramina (S4 alone, 30%; from S3 and S4, 40%; from S4 and S5, 30%), crossed the superior surface of the coccygeal muscle (3.0 +/- 1.4 cm medial to the ischial spine [range, 1.0-4.2 cm]), traveled on the superior surface of the iliococcygeal muscle innervating it at its approximate midpoint, and continued on to innervate both the pubococcygeal and puborectal muscles at their approximate midpoint. The pudendal nerve originated from the S2 to S4 foramina, exited the pelvis through the greater sciatic foramen, traversed Alcock's canal, and branched to innervate the external anal sphincter, the external urethral sphincter, the perineal musculature, the clitoris, and the skin. Despite specific attempts to locate pudendal branches to the levator ani, none could be demonstrated. Nerve biopsy specimens that were obtained at gross dissection were confirmed histologically. CONCLUSION: Gross dissections suggest that the female levator ani muscle is not innervated by the pudendal nerve but rather by innervation that originates the sacral nerve roots (S3-S5) that travels on the superior surface of the pelvic floor (levator ani nerve). Because definitive studies (eg, nerve transection or neurotracer studies) cannot be performed in humans, further studies that will use appropriate animal models are necessary to confirm and extend our findings.

Adult↗

The adrenergic innervation and adrenergic receptor activity of the feline urinary bladder and urethra in the normal state and after hypogastric and/or parasympathetic denervation.

After long-term parasymphatetic denervation, detrusor muscle hypertrophy was found regardless of whether the hypogastric nerves had been sectioned simultaneously or not. After hypogastric denervation only, there was no difference in gross appearance from normal bladders. Adrenergic innervation was studied by means of a histochemical fluorescence technique. After hypogastric denervation, there was usually a slight increase in the detrusor innervation, and a clear decrease in the trigone and urethral adrenergic innervation. After parasympathetic denervation, the detrusor showed an increase in adrenergic innervation, while the innervation of the trigone and the urethra was unchanged. After simultaneous parasympathetic and hypogastric denervation, the adrenergic innervation of the detrusor was similar to that of the bladders subjected to parasympathetic denervation only. The adrenergic innervation of the trigone and urethra was similar to that found after hypogastric denervation only. Adrenergic receptor functions were studied by a method in which changes in the isometric tension of muscle strips were recorded following different pharmacological treatments. In the normal state, noradrenalin and phenylephrine caused contraction and isoprenaline relaxation of the trigone-urethra. In the detrusor, noradrenalin caused relaxation of strips contracted by carbacholine. Relaxed detrusor muscle strips were usually not contracted by noradenalin. In 2 out of 8 normal cats, however, a contraction was observed after very large doses of noradrenalin. After hypogastric denervation, the adrenoreceptor functions were consistent with those of the normal state. After parasympathetic denervation, the trigone-urethral receptor functions were unchanged compared with the normal receptor functions. In the detrusor, isoprenaline caused relaxation and noradrenalin and phenylephrine contraction. After simultaneous parasympathetic and hypogastric denervation, the results were consistent with those obtained after parasympathetic denervation only. Thus, the present study showed an increased adrenergic detrusor innervation and alpha-receptor activity after parasympathetic denervation regardless of whether the hypogastric nerves had been sectioned simultaneously or not. Although alpha-receptors might exist in the normal detrusor, beta-receptors dominate strongly. After parasympathetic denervation, there seems to be a change in the relation between alpha- and beta-receptors in favour of alpha-receptors.

Animals↗

Evidence for recurrent laryngeal nerve contribution in motor innervation of the human cricopharyngeal muscle.

OBJECTIVE: To study the functional motor nerve supply of the upper esophageal sphincter in humans. STUDY DESIGN: Intraoperative electromyographic study. METHODS: The contribution of the recurrent laryngeal nerve and the pharyngeal plexus in the motor nerve innervation of the cricopharyngeal muscle and the inferior pharyngeal constrictor muscle was examined intraoperatively. RESULTS: Electromyography showed that there is a considerable overlap in the innervation of the cricopharyngeal muscle and the inferior pharyngeal constrictor muscle. The recurrent laryngeal nerve functionally contributes to the motor innervation of the cricopharyngeal muscle in all patients and contributes to the motor innervation of the inferior pharyngeal constrictor muscle in most patients. The pharyngeal plexus functionally contributes to the motor innervation of the inferior pharyngeal constrictor muscle but does not always contribute to the motor innervation of the cricopharyngeal muscle. CONCLUSIONS: This is the first report which provides evidence that the recurrent laryngeal nerve functionally contributes to the motor innervation of the cricopharyngeal and inferior pharyngeal constrictor muscle. Furthermore, this study shows that intraoperative electromyography in humans is a feasible method to analyze the physiology of the motor innervation of the upper esophageal sphincter.

Adult↗

Autonomic innervation of receptors and muscle fibres in cat skeletal muscle.

Cat hindlimb muscles, deprived of their somatic innervation, have been examined with fluorescence and electron microscopy and in teased, silver preparations; normal diaphragm muscles have been examined with electron microscopy only. An autonomic innervation was found to be supplied to both intra- and extrafusal muscle fibres. It is not present in all muscle spindles and is not supplied at all to tendon organs. Fluorescence microscopy revealed a noradrenergic innervation distributed to extrafusal muscle fibres and some spindles. On the basis of the vesicle content of varicosities the extrafusal innervation was identified as noradrenergic (32 axons traced), and the spindle innervation as involving noradrenergic, cholinergic and non-adrenergic axons (14 traced). Some of the noradrenergic axons that innervate spindles and extrafusal muscle fibres are branches of axons that also innervate blood vessels. We cannot say whether there are any noradrenergic axons that are exclusively distributed to intra- or extrafusal muscle fibres. The varicosities themselves may be in neuroeffective association with striated muscle fibres only, or with both striated fibres and the smooth muscle cells in the walls of blood vessels. The functional implications of this direct autonomic innervation of muscle spindles and skeletal muscle fibres are discussed and past work on the subject is evaluated.

Animals↗

In vitro regulation of the innervation pattern of quail muscle fibers by quail and mouse neurons.

Myoblasts from rudiments of slow and fast muscle, anterior latissimus dorsi (ALD) and posterior latissimus dorsi (PLD) respectively, of 9-day-old quail embryos were cultured in vitro for a period of up to 60 days in order to give rise to well-differentiated muscle fibres. These fibres were innervated by neurons from either quail or mouse embryo spinal cord and their innervation pattern was examined by the visualization of acetylcholine receptors (ACh-R) and of acetylcholinesterase (ACh-E) activity at the neuromuscular contacts. In the culture system used, quail neurons always innervated muscle fibres at several sites and only when a fast-type activity was imposed on these neurons did a reduction in the number of the previously established neuromuscular contacts take place. In contrast, in the muscle fibres innervated by mouse neurons, a spontaneous reduction in the number of the previously established neuromuscular contacts occurred but this spontaneous reduction depended upon the level of differentiation reached by the muscle fibres in vitro. In the cultures of muscle fibres previously innervated by mouse neurons, the addition of quail neurons did not provoke any modification in the initial innervation pattern, and no quail ACh-R cluster was observed. In contrast, in the muscle fibres previously innervated by quail neurons, the mouse neurons contacted these fibres, resulting in a decrease in the number of quail ACh-R clusters. These results emphasize the part played by neurons in the establishment of the innervation pattern when muscle fibres have reached a high level of differentiation. In vitro, the slow and fast characteristics of the muscle fibres do not influence this pattern.

Acetylcholinesterase↗

Paralysis of innervated cultured human muscle fibers affects enzymes differentially.

Increased accumulation of muscle-specific isozyme (MSI) of creatine kinase (CK), lactate dehydrogenase (LDH), glycogen phosphorylase (GP), and phosphoglycerate mutase (PGAM) occurs with development and indicates muscle fiber maturation. The expression of MSIs of those four enzymes is greatly enhanced in innervated-contracting as compared to noninnervated and noncontracting cultured human muscle fibers. We have now studied the effect of contractile activity on developmental accumulation of MSIs in innervated-contracting, innervated-paralyzed (2 microM tetrodotoxin for 30 days), and noninnervated-noncontracting cultured human muscle fibers. Muscle acetylcholinesterase (AChE) and total enzyme activities were also studied under the same conditions. We observed a different dependency on contractile activity between total enzymatic activities of CK, LDH, and AChE, which were substantially reduced after paralysis, and GP and PGAM, which were unchanged. The expression of MSIs of CK, GP, PGAM, and LDH was always significantly increased in innervated as compared to noninnervated fibers. While the expression of MSIs of GP and PGAM was the same in contracting-innervated and paralyzed-innervated muscle fibers, the expression of MSIs of CK and LDH in paralyzed-innervated muscle fibers was very slightly decreased as compared to their contracting-innervated controls. Our studies demonstrate that in human muscle: (1) total enzymatic activities and the expression of MSIs of GP and PGAM are regulated by neuronal effect(s); (2) total enzymatic activities of CK, LDH, and AChE depend mainly on muscle contractile activity; and (3) MSIs of CK and LDH are regulated predominantly by neuronal factors and to a much lesser degree by muscle contractile activity.

Acetylcholinesterase↗