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Catecholamine innervation of the caudal spinal cord in the rat.

By means of the aluminum-formaldehyde (ALFA) fluorescence technique for monoamine visualization the distribution of catecholamines was studied in the caudal spinal cord, particularly in relation to motoneurons innervating pelvic structures. In the lumbosacral cord all parts of the spinal gray matter were found to contain catecholamines. In the dorsal horn the most intense fluorescence was seen in the superficial layers. The motoneuron neuropil exhibited the most prominent catecholamine-fluorescence of the ventral horn layers. In the sixth lumbar segment, which contains the motor nuclei that innervate the pelvic striated muscles as well as one innervating muscles in the lower limb, a differential distribution of the density of catecholamine fluorescence was presented by the individual nuclei. The catecholamine fibers in the motoneuron neuropil were seen closely surrounding the motoneuron somata, suggesting the existence of axosomatic contacts, and by utilizing the fluorescent retrograde tracer True Blue in combination with the ALFA method tentative axosomatic noradrenergic synapses on identified neurons innervating small striated pelvic muscles could be visualized in the light microscope. In the intermediate gray the intermediolateral nucleus in thoracic and upper lumbar segments was the most heavily innervated area, followed by the medial lumbar sympathetic group, which contains the majority of the sympathetic preganglionic neurons innervating the pelvic organs. The parasympathetic intermediolateral nucleus in the upper sacral segments received a catecholamine innervation of moderate density. The catecholamine innervation pattern is discussed in relation to the patterns of other putative transmitters. The distribution of catecholamine fluorescence in relation to nuclei that control the pelvic organs differs from the arrangement of other transmitters in this region. The complexity of the innervation of the pelvic organs and their related striated muscles is thus further stressed.

Animals↗

Morphological identification of the motor neurons innervating the dorsal longitudinal flight muscle of Drosophila melanogaster.

The dorsal longitudinal flight muscle (DLM) of Drosophila is composed of six muscle fibers (DLM1-6 from ventral to dorsal) and innervated by five motor neurons (MNs), DLM1-4 being innervated singly by MN1-4, and DLM5 and 6 being jointly innervated by MN5. This study identifies and describes the five motor neurons that innervate these six muscle fibers. Horseradish peroxidase (HRP) applied intracellularly to single identified DLM fibers resulted in the labeling of the single motor neuron innervating that muscle fiber by retrograde transsynaptic transport. This method allowed positive identification of the motor neuron innervating a particular muscle fiber, since only the innervating neuron was labeled. The axonal pathway, soma, and dendritic distribution of each labeled motor neuron was traced in the thoracic ganglion, and their relative positions were determined. The somata of MN1-4 lie in a cluster located near the lateral surface of the thoracic ganglion at the border of the pro- and mesothoracic regions, ipsilateral to the muscle fibers innervated. The somata of MN1 and 2 lie side by side in a horizontal plane with MN1 in a more anterior position. Those of MN3 and 4 lie ventrally to those of MN1 and 2 in a horizontal plane with MN3 in the more anterior position. The soma of MN5 is located contralaterally to the muscle fiber it innervates, lying in the dorsal outermost layer of the thoracic ganglion next to the midline at the border of the pro- and mesothoracic regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Innervation of footpads of normal and mutant mice lacking sweat glands.

Footpads of normal adult mice are innervated by sympathetic and sensory fibers. The sympathetic fibers associated with sweat glands contain acetylcholinesterase and immunoreactivity for vasoactive intestinal peptide. Although catecholamine histofluorescence is absent, the gland innervation exhibits immunoreactivity for tyrosine hydroxylase. A distinct population of sympathetic fibers, which possess catecholamines and neuropeptide Y as well as tyrosinehydroxylase immunoreactivity, innervates blood vessels. Sensory fibers containing immunoreactivity for substance P and calcitonin gene-related peptide course beneath the epidermis and some form endings in it. Treatment of neonatal mice with the adrenergic neurotoxin, 6-hydroxydopamine, results in loss of sympathetic innervation of sweat glands and blood vessels, permits growth of sensory axons into sweat glands, but does not alter the peptidergic sensory innervation of the dermis and epidermis. Three mouse mutations, Tabby (Ta), crinkled (cr), and downless (dl), disrupt the interactions between the mesenchyme and epidermis that are required for normal development of specific epidermal derivatives, including sweat glands. The sympathetic innervation of blood vessels and sensory innervation of footpad skin of the three mutant mice that lack sweat glands is indistinguishable from normal. The sympathetic fibers that normally innervate sweat glands, however, are not present. These results indicate that in the absence of their normal target, the sympathetic fibers that innervate sweat glands are lacking. Furthermore, they suggest that, although sensory fibers may sprout into sympathetic targets in the footpad, the domains occupied by sensory fibers are not normally accessible to sympathetic axons.

Animals↗

Innervation of nonmystacial vibrissae in the adult rat.

Vibrissal follicle-sinus complexes (F-SCs) in the mystacial pad of rodents are heavily innervated by different types of sensory nerve endings. One site in mystacial F-SCs, the inner conical body (ICB), is uniquely well innervated only in those species, such as the rat, that rhythmically whisk their mystacial vibrissae. In this study, we examined the innervation of rat nonmystacial F-SCs, which are not whisked. Supraorbital, posteroorbital, lateral cervical, median cervical, submental, and carpal forelimb F-SCs were cut on a cryostat and were either prepared for anti-human protein gene product (PGP 9.5) immunofluorescence or stained using the Winkelmann silver technique. Much of the innervation of the nonmystacial F-SCs is similar to that of mystacial F-SCs. All are innervated by a large deep vibrissal nerve (DVN) and several smaller superficial vibrissal nerves (SVNs). As in the mystacial pad, the SVNs show a distribution of Merkel and free nerve endings qualitatively similar to the rete ridge collar of all the nonmystacial F-SCs as well as provide circumferentially oriented endings to the ICBs to all but median-cervical and carpal F-SCs. Not only was the ICB innervation relatively sparse in median-cervical and carpal F-SCs, but a large portion of the carpal ICB innervation also ascended from the DVNs, which make only a small ICB contribution in other locations. Similar to mystacial pad F-SCs, the DVNs provided Merkel and lanceolate endings to the level of the ring sinus as well as reticular and irregular lanceolate-like endings to the level of the cavernous sinus. However, all but the posteroorbital F-SCs have relatively few lanceolate endings. Carpal F-SCs also have relatively few ring-sinus Merkel endings, which are diffusely distributed, are limited to the superficial portion of the outer root sheath. They also lack reticular and irregular lanceolate-like endings in the cavernous sinus. However, carpal F-SCs have a unique set of corpuscular endings in the ICB, ring sinus, and cavernous sinus that are rarely seen in other F-SCs. PGP 9.5 immunofluorescence also revealed two sets of fine-caliber profiles at the level of the ICB and ring sinus that were not previously seen in mystacial F-SCs. Although there was no correlation between ICB innervation and whisking, the regional variations in F-SC innervation suggest that functional differences may exist between vibrissae at different locations in the body.

Animals↗

Innervation-dependent and fiber type-specific transcriptional regulation of the slow myosin heavy chain 2 promoter in avian skeletal muscle fibers.

Skeletal muscle fiber type is regulated, in part, by innervation leading to transcriptional regulation of fiber type-specific genes. Here, we report the initial characterization of the transcriptional regulation of the slow myosin heavy chain 2 (MyHC2) promoter in innervated and noninnervated slow medial adductor (MA) and fast pectoralis major (PM) muscle fibers in cell culture. The proximal 1358 bp of slow MyHC2 upstream DNA contains a functional E-box and binding sites for myocyte enhancer factor 2 (MEF2) and nuclear factor of activated T cells (NFAT). Mutagenesis studies indicated that both MEF2 and NFAT binding sites are required for innervation-induced slow MyHC2 promoter activity in MA muscle fibers. However, MEF2 transcription factor activity was unaffected by innervation and did not demonstrate fiber type-specific interactions with the slow MyHC2 MEF2 binding site. NFAT transcription factor activity did increase in innervated MA muscle fibers and not in PM muscle fibers, indicating innervation and muscle fiber type-specific regulation. However, transfection of constitutively active NFAT indicated that NFAT is insufficient to induce slow MyHC2 gene expression in either fast PM or slow MA muscle fibers without innervation. These results indicate the requirement for MEF2 and NFAT in innervation-induced slow MyHC2 gene expression and suggest that additional innervation-dependent and fiber type-specific control of slow MyHC2 gene expression resides in MA and PM muscle fibers, respectively.

Animals↗

Effects of age and GDNF on noradrenergic innervation of the hippocampal formation: studies from intraocular grafts.

Recent studies have suggested that factors in the target tissue influence the degree of plasticity and regeneration following aging and/or specific insults. We have investigated whether young or aged targets differ in their noradrenergic innervation from fetal locus coeruleus (LC) neurons, and also if a specific growth factor, glial cell line-derived neurotrophic factor (GDNF) can affect this innervation pattern. Tissue pieces of fetal brainstem and young (3 months) or old (18 months) iris tissue were transplanted simultaneously into the anterior chamber of the eye of adult hosts. We found that aged iris transplants became innervated to a significantly lesser degree by the cografted LC neurons than young iris transplants. Fetal hippocampal tissue was then grafted to adult hosts, and a fetal brainstem graft containing LC neurons was placed adjacent to the first graft, either at 3 or 21 months post-grafting. Thus, old/young chimeras of the noradrenergic coeruleo-hippocampal pathway were created. Aged hippocampal grafts received a much less dense innervation from co-grafted LC neurons than young hippocampal grafts. Tyrosine hydroxylase-positive-immunoreactive innervation was only found in the outskirts of aged grafts, while the young hippocampal grafts contained an even innervation pattern. The innervation density of hippocampal grafts was significantly enhanced by GDNF treatment. These findings demonstrate that target-derived factors may regulate neuronal plasticity, and that the age of the target is more important for innervation properties than the age of the neuron innervating a particular target.

Aging↗

Refinement of innervation accuracy following initial targeting of peripheral gustatory fibers.

During development, axons of the chorda tympani nerve navigate to fungiform papillae where they penetrate the lingual epithelium, forming a neural bud. It is not known whether or not all chorda tympani axons initially innervate fungiform papillae correctly or if mistakes are made. Using a novel approach, we quantified the accuracy with which gustatory fibers successfully innervate fungiform papillae. Immediately following initial targeting (E14.5), innervation was found to be incredibly accurate: specifically, 94% of the fungiform papillae on the tongue are innervated. A mean of five papillae per tongue were uninnervated at E14.5, and the lingual tongue surface was innervated in 17 places that lack fungiform papillae. To determine if these initial errors in papillae innervation were later refined, innervation accuracy was quantified at E16.5 and E18.5. By E16.5 only two papillae per tongue remained uninnervated. Innervation to inappropriate regions was also removed, but not until later, between E16.5 and E18.5 of development. Therefore, even though gustatory fibers initially innervate fungiform papillae accurately, some errors in targeting do occur that are then refined during later embryonic periods. It is likely that trophic interactions between gustatory neurons and developing taste epithelium allow appropriate connections to be maintained and inappropriate ones to be eliminated.

Animals↗

The influence of innervation on the differentiation of contractile speeds of developing chick muscles.

1. The role of innervation of the differentiation of contractile speeds was studied in the slow anterior latissimus dorsi (ALD) and fast posterior latissimus dorsi (PLD) muscle of the chick. 2. These muscles become innervated during the 12th and 15th day of embryonic development. At this time both muscles contract and relax extremely slowly and their contractile speeds are very similar. By the 18th day their contraction and relaxation becomes more rapid. It is at this time that the contractile characteristics of both muscles also become very different from each other, ALD being about 3 times slower than PLD. Thus innervation percedes differentiation of contractile speeds by several days. 3. The influence of innervation on the contractile characteristics of developing slow and fast muscles was studied during muscle regeneration in adults. When a slow ALD muscle was minced and implanted in place of a fast PLD the newly regenerated ALD became innervated by a PLD nerve and resembled a fast PLD. Conversely, when PLD muscles were minced and replaced ALD the regenerated PLD was innervated by ALD nerve and became slow. 4. Histological examination revealed that the regenerated ALD became focally innervated, and the regenerated PLD multiply innervated. 5. Thus, the contractile speeds are not predetermined properties of the muscle fibre. Both contractile characteristic and the pattern of innervation of developing muscles are determined by the motor nerve.

Animals↗

Neuronal pathways for spinal reflexes activated by group I and group II muscle afferents in the spinal segment (Co1) innervating the tail in the low spinalized cat.

We studied neuronal pathways for spinal reflexes activated by group-I and group-II muscle afferents in the spinal segments innervating the tail in unanesthetized and spinalized (L1) cats. Experiments were performed on 25 adult cats of both sexes. The effects of stimulating nerves innervating six tail muscles on both sides were recorded from tail motoneurons in the first coccygeal spinal segment (Co1) using glass microelectrodes. Stable recordings were obtained from 150 tail motoneurons. Stimulation of group-1 muscle afferents (stimulus intensity <1.8 T) often produced EPSPs (82/150) after stimulating nerves innervating neighboring tail muscles. Motoneurons innervating the long-tendoned muscles, M. extensor caudae lateralis and M. flexor caudae longus (ECL and FCL), received heteronymous monosynaptic connections from group-I muscle afferents innervating the ipsilateral tail muscles. The motoneurons innervating segmental muscles, M. extensor caudae medialis and M. flexor caudae brevis (ECM and FCB), received heteronymous monosynaptic connections from group-I muscle afferents innervating tail muscles on both sides. The motoneurons innervating tail muscles originated from the Ossa coxae, M. abductor caudae externus and M. abductor caudae internus (ACE and ACI), received monosynaptic connection from group-I muscle afferents innervating most of the tail muscles on both sides. Crossed disynaptic inhibitory pathways activated by primary muscle afferent inputs were observed in ECM, ACE, FCL, and FCB motoneurons. The effects of group-II afferent inputs were not dependent on the kind of motoneuron, and alternative excitatory and inhibitory pathways were not clearly observed in the tail motoneuron pool. It is suggested that variability of the neuronal pathways from group-I and -II muscle afferents to tail motoneurons corresponds to functional relationships among tail muscles, depending on the tail movements.

Animals↗

Preganglionic innervation of the pancreas islet cells in the rat.

The position and number of preganglionic somata innervating the insulin-secreting beta-cells of the endocrine pancreas were investigated in Wistar rats. This question was approached by comparing the innervation of the pancreas of normal rats with the innervation of the pancreas in alloxan-induced diabetic animals. The presumption was made that alloxan treatment destroys the beta-cells of the islet of Langerhans and results in a selective degeneration of the beta-cells innervation. Cell bodies of preganglionic fibers innervating the pancreas were identified by retrograde transport of horseradish peroxidase following pancreas injections. It was found that 25% of the cells innervating the pancreas in the left dorsal vagal motor nucleus, 50% of the cells in the ambiguous nucleus and 50% of the cells innervating the pancreas, that originate in segments C3-C4 of the spinal cord, fail to become labeled after alloxan treatment. The position and distribution of these cell groups are described in detail and are assumed to be involved in preganglionic beta-cell innervation. A second cell population in the ventral horn and intermediolateral column of the segments T3-L2 of the cord also was labeled in normal rats and was not affected by the alloxan treatment. These thoracic cell groups are thus considered as sympathetic preganglionic somata that maintain direct connections to the pancreas. Additional preliminary information is presented dealing with the general aspects of sympathetic and parasympathetic organization of the pancreas innervation.

Acetylcholinesterase↗

The adrenergic innervation of pulmonary vasculature in the normal and pulmonary hypertensive rat.

It would appear that susceptibility to chronic proliferative pulmonary hypertension in response to chronic alveolar hypoxia is most severe in species in which adrenergic innervation of pulmonary arteries is reduced or lacking. Intrapulmonary arteries of the rat have been reported to lack adrenergic innervation by some workers but not others. Since the rat develops severe proliferative pulmonary hypertension in response to prolonged alveolar hypoxia, the different divisions of the lung vasculature of Sprague-Dawley rats were thoroughly examined to determine the presence or absence of an adrenergic innervation. The degree of innervation in normal rats was compared with that of rats developing pulmonary hypertension. Both in normal and experimental pulmonary hypertensive rats the pulmonary arteries, all their branches and the small pulmonary veins with a smooth muscle media were found to be devoid of adrenergic innervation. In contrast, the cardiac-like muscle in the media of large pulmonary veins, the bronchial arteries and the vasa vasorum of larger vessels were richly innervated by adrenergic nerves. Thus the increase in medial smooth muscle which occurs in pulmonary arteries during chronic alveolar hypoxia is independent of a pre-existing adrenergic innervation or of such an innervation newly derived from that of adjacent vessels or structures. This is in contrast to systemic vessels where it has been suggested that increased adrenergic activity and density of innervation may augment hypertrophy of the media in hypertensive animals. Adrenergic nerves are suggested to have a protective action on pulmonary vessels.

Adrenergic Fibers↗

The pattern and timing of cutaneous hair follicle innervation in the rat pup and human fetus.

The postnatal development of hair follicle innervation was studied in the rat hindlimb using a silver stain which detects large and medium calibre cutaneous nerve fibres. The pattern and timing of innervation in relation to postnatal changes in follicle growth were studied providing new data on nerve-target interactions in the developing peripheral nervous system. Sensory axons begin to leave the dermal plexus and grow towards follicles at P (postnatal day) 3 but do not start to innervate them until P7 or achieve an adult appearance until P19. The first terminals are circumferential, followed some days later by the appearance of palisade endings. The number of axons innervating a hair follicle increases steadily with age until P19 and there is no evidence of exuberant innervation of follicles during development. Hair follicle density in the rat is maintained during development due to waves of small, vellus follicle growth later in postnatal life as the skin grows. The percentage of follicles innervated however, decreases from the second postnatal week onwards presumably because late developing vellus hairs do not become innervated. Comparative analysis in human fetal abdominal skin using the same silver stain reveals a similar sequence and pattern of innervation to the rat over the period of 22 to 35 weeks EGA (estimated gestational age). Human skin does not, however, undergo the late waves of follicle growth seen in the rat. Follicular density decreases and the percentage of innervated follicles increases in the third trimester of fetal life.

Animals↗

Growth-associated protein-43 and protein gene-product 9.5 innervation in human pancreas: changes in chronic pancreatitis.

Growth-associated protein-43, an established marker of neuronal plasticity during development and in injury, was used to characterize innervation in the normal human pancreas and changes in chronic alcohol-induced pancreatitis by using light microscopic immunocytochemistry and computer-assisted image analysis. Immunostaining for the pan-neuronal marker protein gene-product 9.5 served as a reference for the characterization of total innervation in both groups. In normal human pancreas, strong protein gene-product 9.5 immunostaining revealed all nerve fibres in nerve trunks, all neuronal cell bodies and the entire parenchymal innervation. In contrast, growth-associated protein-43 immunoreactivity was restricted to a few nerve fibres in interlobular nerve trunks and to fine varicose nerve fibres supplying the parenchyma, blood vessels, pancreatic ducts and intrinsic ganglia. In cell bodies of intrinsic neurons, growth-associated protein-43 immunoreactivity was absent or extremely faint. In chronic pancreatitis, the protein gene-product 9.5 innervation exhibited region-specific changes. In areas with reduced parenchyma, the protein gene-product 9.5 innervation was sparse. In fibrotic regions, which are characteristic for advanced stages of chronic pancreatitis, enlarged nerve trunks showing neuroma-like formations were heavily stained for protein gene-product 9.5. In fibrotic tissue, protein gene-product 9.5-containing nerve fibres were extremely rare. The growth-associated protein-43 innervation in chronic pancreatitis was characterized by a dramatic increase, which was most pronounced in the enlarged nerve trunks. Such nerve trunks were frequently surrounded by infiltrates of immune cells, which in some cases formed follicle-like structures. Digital image analysis of adjacent sections and double fluorescence immunocytochemistry revealed that growth-associated protein-43 immunoreactivity was present in the vast majority of protein gene-product 9.5-immunoreactive nerve fibres. In contrast to the normal pancreas, a major subpopulation of intrinsic neurons immunostained for growth-associated protein-43. The expression of growth-associated protein-43 in the terminal fields of pancreatic nerve suggests that the innervation of the normal human pancreas undergoes continual and toposelective remodelling. The increase in the density of growth-associated protein-43 immunoreactive nerve fibres in enlarged nerve trunks paralleled by augmented expression of growth-associated protein-43 in intrinsic neurons and reduced parenchymal growth-associated protein-43-immunoreactive innervation underline the dramatic plasticity of pancreatic innervation in chronic pancreatitis.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗

The onset of dopaminergic innervation during ontogeny decreases melanotrope proliferation in the intermediate lobe of the rat pituitary.

The onset of dopaminergic innervation and its effects on melanotrope proliferation were investigated in the rat pituitary intermediate lobe. Dopamine, and its synthetic rate-limiting enzyme tyrosine hydroxylase, were first detected immunohistochemically on late post-natal day 3 or early postnatal day 4. Axon density was highest at the neural lobe/intermediate lobe border, and decreased toward the pituitary cleft. By postnatal day 10, the adult pattern of tyrosine hydroxylase immunoreactivity was established and remained through post-natal day 14. Neurointermediate lobe dopamine levels, measured by HPLC, correlated well with the increased axon density observed in the immunohistochemical studies. Dopamine could not be measured by our assay (100 fg limit) until post-natal day 3 (439.32 fg/NIL). Dopamine concentration increased to 2.09 +/- 0.425 ng at PN 4, 86.31 +/- 20.42 ng at PN 7, 168.72 +/- 18.37 ng at PN 10. Melanotrope proliferation was determined by [3H]thymidine incorporation before and after innervation. Concomitant with the onset of innervation, the proliferation index dropped from 13.4 +/- 0.01% to 6.5 +/- 0.002% at PN 4, and continued to decrease until a level of 3 +/- 0.003% was established by PN 10. To confirm the inhibitory action of dopaminergic innervation on melanotrope proliferation, rat neonates were injected intracisternally with 150 mg 6-hydroxydopamine to destroy dopaminergic axons within the intermediate lobe. Measurement of dopamine concentrations in neurointermediate lobes of injected animals showed a decrease in dopamine levels as compared to controls. From PN 4 (0.88 +/- 0.165 ng), DA levels gradually increased during development: at PN 5, [DA] = 0.689 +/- 0.104 ng; PN 6 [DA] = 11.60 +/- 2.24 ng; PN 7 [DA] = 20.93 +/- 3.80 ng; and PN 10 [DA] = 27.95 +/- 3.46 ng. Melanotrope proliferation also increased in 6-hydroxydopamine-treated animals. At PN 4, the onset of innervation reduced the pre-innervation proliferation index to 8.75 +/- 0.002%, only a 30% reduction in contrast to the greater than 50% decrease observed in control animals. A stable proliferation level of approximately 7.5% persisted in all subsequent stages with 6-OHDA administration. Our results demonstrated the time of dopamine innervation onset and a characteristic developmental pattern for axons within the rat intermediate lobe. The onset of innervation and increased dopamine concentration suggests increased dopaminergic control of the melanotropes, illustrated specifically by a decrease in their level of proliferation. This is the first presentation of evidence showing that dopaminergic innervation within the intermediate lobe of the rat pituitary regulates melanotrope proliferation.

Animals↗

Nerve growth factor treatment of adult rats selectively enhances innervation of urinogenital tract rather than vascular smooth muscle.

Following treatment of adult rats with nerve growth factor (0.5 mg/rat, three times a week for 3 weeks), the innervation of cardiovascular and urinogenital tract smooth muscle was investigated using immunoassay and immunohistochemical techniques. Substance P and calcitonin gene-related peptide levels were increased in the vas deferens, but not in the atria or femoral artery. Neuropeptide Y and vasoactive intestinal polypeptide levels were unchanged. In penile tissues, there was a marked increase in the density of substance P-, calcitonin gene-related peptide-, neuropeptide Y-, tyrosine hydroxylase- and vasoactive intestinal polypeptide-containing nerves innervating the urethra and in SP-containing nerves in the tunica with little changes in the innervation of the deep dorsal vein and artery and corpus cavernosum. In the bladder, there was increased innervation of the detrusor by neuropeptide Y- and vasoactive intestinal polypeptide-containing nerves, but a decrease in innervation by substance P-containing nerves in the trigone. There were no changes in the density of innervation of the femoral artery after nerve growth factor treatment. Thus, in the mature rat, sensory and sympathetic nerve innervating urinogenital tract smooth muscle appear to be more responsive to exogenous nerve growth factor than those innervating cardiovascular smooth muscle. This may reflect an ongoing requirement of plasticity of innervation in the urinogenital tract of the sexually mature animal.

Animals↗

Estrogen selectively increases sensory nociceptor innervation of arterioles in the female rat.

Differences exist in vascular function and disease susceptibility in males and females, and estrogen is apparently a primary factor. One mechanism by which estrogen may influence vascular function is by affecting vasomotor innervation. We have shown previously that estrogen increases calcitonin gene-related peptide (CGRP)-immunoreactive sensory innervation of the rat mammary gland, but it is not known if this occurs in other tissues. The objective of this study was to determine if estrogen modulates CGRP-immunoreactive innervation of vascular and non-vascular tissues. Ovariectomized adult virgin female rats were implanted with pellets containing 17beta-estradiol or placebo. After 7 days, innervation was examined in the external ear, jejunal mesenteric arterioles, superficial epigastric, femoral, and uterine arteries, and foot skin. Immunofluorescence microscopy of the external ear pinna revealed increased CGRP-immunoreactive sensory innervation in estrogen-treated rats, and this was attributable specifically to increased innervation of arterioles. Tyrosine hydroxylase-immunoreactive innervation was unchanged. Total nerve density, revealed by the pan-neuronal marker PGP 9.5, was also greater after estrogen treatment, implying structural proliferation of nociceptor vasodilator fibers. Mesenteric arteriolar CGRP-immunoreactive nerve density was also selectively increased by estrogen treatment. However, estrogen did not affect CGRP-immunoreactive nerve density of superficial epigastric, femoral, or uterine arteries, or foot skin. Therefore, estrogen increases sensory innervation of arterioles, but not of large arteries or skin. We conclude that sensory nociceptor vasodilatory innervation of arterioles is selectively enriched by estrogen, which may influence cardiovascular function in health and disease.

Animals↗

Nitrergic innervation of the human gut during early fetal development.

Classically, development of the human enteric nervous system has been characterized by the early appearance (between 9 and 12 weeks' gestation) of adrenergic and cholinergic nerves. The development of peptidergic innervation occurs much later. Recent studies have indicated that nitric oxide is involved in the nonadrenergic noncholinergic innervation of the gut, mediating its relaxation. The authors have investigated the ontogeny of nitrergic (nitric oxide synthase-containing) neurons of the developing gut. Bowel segments from the esophagus, pylorus, and ileocecal and rectosigmoid regions of 14 fetuses (gestational age range, 12 to 23 weeks) were studied with nicotinamide adenosine dinucleotide phosphate (NADPH) diaphorase histochemistry. By 12 weeks' gestation, nitrergic neurons had appeared in the myenteric ganglia, at all levels of the gut, and had begun plexus formation. Nitrergic innervation in the submucous plexus becomes evident after 14 weeks. As gestational age increases, nitrergic innervation becomes richer and more organized. Increasing numbers of nitrergic nerve fibers are seen in the circular muscle; some of these fibers project from the myenteric plexus. By 23 weeks' gestation, nitrergic innervation has matured to the pattern observed in the postnatal gut. Thus, the onset and pace of development of nitrergic innervation are similar to adrenergic and cholinergic innervation and occur before peptidergic innervation. This study provides morphological evidence of the ontogenetic significance of nitrergic innervation in the human gut and supports previous suggestions that nitric oxide has a pathophysiological role in developmental gut motility disorders.

Cell Differentiation↗

Absence of cholinergic sympathetic innervation from limb muscle vasculature in rats and mice.

Although the existence of cholinergic sympathetic vasodilatory innervation in limb muscle vasculature is well established for some species, previous pharmacological studies have failed to reveal the presence of such innervation in rats. Recently, Schafer and colleagues [Schafer, M.K., Eiden, L.E., Weihe, E., 1998. Cholinergic neurons and terminal fields revealed by immunohistochemistry for the vesicular acetylcholine transporter. II. The peripheral nervous system. Neuroscience 84(2), 361-376] reported that vesicular acetylcholine transporter immunoreactivity (VAChT-IR), a marker for cholinergic terminals, is present in the innervation of the microvasculature of rat hindlimb skeletal muscle and concluded that rats possess cholinergic sympathetic innervation of limb muscle vasculature. Because of our interest in identifying targets of cholinergic sympathetic neurons, we have analyzed the transmitter properties of the innervation of muscle vessels in rat and mouse limbs. We found that the innervation of vasculature in muscle is noradrenergic, exhibiting robust catecholamine histofluorescence and immunoreactivity for tyrosine hydroxylase (TH) and the peptide transmitters, neuropeptide Y (NPY) and occasionally vasoactive intestinal peptide (VIP). In contrast, cholinergic phenotypic markers,VAChT-IR and acetylcholinesterase (AChE) activity, are absent. Neuron cell bodies in sympathetic ganglia, retrogradely labeled with injections of tracer into limb muscles, also lacked VAChT but contained TH-IR. The innervation of large extramuscular feed arteries in hindlimbs was also devoid of cholinergic markers, as were the cell bodies of sympathetic neurons innervating extramuscular femoral arteries. These results, like those of previous physiological studies, provide no evidence for the presence of cholinergic sympathetic innervation of muscle vasculature in rats or mice.

Acetylcholinesterase↗