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PubMed1996

Transplanted sweat glands from mature and aged donors determine cholinergic phenotype and altered density of host sympathetic nerves.

Contact with sweat gland acini causes sympathetic neurons to switch from a catecholaminergic to a cholinergic phenotype during development and following experimental manipulations. Substantial reductions of cholinergic innervation have been shown in the sweat glands of ageing rats and humans. Using in oculo transplantation, we have now studied whether sweat gland target tissues retain the capacity to regulate changes in the phenotype of sympathetic neurons observed in maturity and old age, including a switch from catecholaminergic to cholinergic characters. Markers have been used which indicate changes in nerve fibre morphology (the pan-neuronal marker, PGP9.5) as well as neurotransmitter expression (acetylcholinesterase (AChE), vasocative intestinal polypeptide (VIP) and tyrosine hydroxylase (TH)). Sweat glands from young and old donor rats became reinnervated by an organotypic pattern of cholinergic host nerves. Surgical sympathectomy demonstrated that these cholinergic nerve fibres originate from sympathetic neurons of the host superior cervical ganglion (SCG). Retrograde tracing combined with staining for VIP (a marker associated with cholinergic phenotype in neurons supplying sweat glands) showed that SCG neurons projecting to irises with sweat gland implants may be induced to express VIP. We hypothesise that these neurons have been switched from their normal catecholaminergic phenotype to a cholinergic one by contact with the sweat gland implants. Transplants from old donors attracted a density of reinnervation by young host nerves which was appropriate to the age of the donor, thus old sweat glands received a significantly reduced density of innervation compared to young glands. Despite the reduced density of innervation, there was no obvious difference in the ability of young and old implants to induce the switch to a cholinergic phenotype, suggesting that different mechanisms regulate nerve growth and neurotransmitter phenotype.

Adrenergic Fibers↗
PubMed1996

Identification of uterine-related sympathetic neurons in the rat inferior mesenteric ganglion: neurotransmitter content and afferent input.

The rat uterus is innervated by sensory and autonomic nerves. Sensory and sympathetic fibers travel in the hypogastric nerves and are associated with the thoracolumbar spinal cord levels T13-L3. The inferior mesenteric ganglion (IMG) contains the somata of sympathetic postganglionic neurons and some of these may project axons to the uterus. Sensory and parasympathetic fibers travel in the pelvic nerve and are associated with the lumbosacral cord levels L6-S1 and pelvic ganglion (PG). We previously reported data concerning the neurochemical anatomy of the PG with regard to the uterine innervation; the present study was undertaken to characterize the neurochemical anatomy of the IMG with regard to it involvement in uterine innervation. A retrograde axonal tracer was used to verify projections of axons of IMG neurons to the uterus. Immunostaining of cryostat sections of the IMG revealed neurons immunoreactive for neuropeptide Y (NPY) and for tyrosine hydroxylase (TH). Immunostaining for the synaptic terminal protein synapsin I (SYN) revealed numerous fine terminals immediately surrounding the principal neurons and in the interneuronal spaces. Varicosities immunoreactive for calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), enkephalin (ENK), substance P (SP) and galanin (GAL) appear to be associated with principal neurons. Additional varicosities stained for nicotinamide adenine dinucleotide phosphate (reduced)-diaphorase (NADPH-d) and nitric oxide synthase (NOS), thus indicating sites of neuronal nitric oxide synthesis. This study revealed that the IMG contains uterine-related neurons and that some of the retrogradely labeled uterine-related neurons contain NPY, TH or both NPY/TH. In addition, uterine-related neurons received abundant afferent inputs indicated by SYN-immunoreactive (-ir) terminals and some of these varicosities labeled for GAL, CGRP, VIP, ENK, or NADPH-d/NOS.

Animals↗
PubMed1996

Association of substance P and its receptor with efferent neurons projecting to the greater curvature of the rat stomach.

Retrograde tracing and immunocytochemistry were used to identify and map the distribution of substance P (SP) and its receptor (NK-1r) associated with gastric motor neurons in the dorsal motor nucleus of the vagus (DMV) in the rat brain stem. The presence of peptide and receptor in surrounding regions within the dorsal vagal complex were also observed. Injection of the retrograde tracer Fluorogold (FG) into the greater curvature of the stomach produced bilateral labelling of neurons within the DMV. The majority of the NK-1r immunoreactivity appeared as an intricate lattice of fibres with a small number of immunoreactive cell bodies. The NK-1r-labelled fibres were detected within the DMV in close association with FG-labelled neurons and in the region between the DMV and nucleus of the solitary tract (NTS). A proportion of FG-labelled neuronal cell bodies were also labelled with NK-1r (7% of total). The greatest density of NK-1r-labelled fibres was observed at the rostral end of the FG-labelled neuron columns in the DMV (close to the IV ventricle) in the region where gastric vagal afferents terminate. Little NK-1r labelling was observed at the caudal end of the FG-labelled neuron tracts adjacent to the central canal. In the coronal plane, the NK-1r-labelled fibres were seen at the edges of the DMV extending into overlying NTS. Substance P was visualized as a dense network of fibres spanning the entire length of the DMV and in close association with FG-labelled neurons. Substance P staining was also detected in the NTS and in the ventral AP. Most of the association between SP/NK-1r immunoreactive fibres was observed within the DMV and at the border between the DMV and NTS. These findings suggest that SP directly regulates a subpopulation of efferent neurons in the DMV which project to the greater curvature of stomach.

Animals↗
PubMed1996

Transplanted sweat glands from mature and aged donors determine cholinergic phenotype and altered density of host sympathetic nerves.

Contact with sweat gland acini causes sympathetic neurons to switch from a catecholaminergic to a cholinergic phenotype during development and following experimental manipulations. Substantial reductions of cholinergic innervation have been shown in the sweat glands of ageing rats and humans. Using in oculo transplantation, we have now studied whether sweat gland target tissues retain the capacity to regulate changes in the phenotype of sympathetic neurons observed in maturity and old age, including a switch from catecholaminergic to cholinergic characters. Markers have been used which indicate changes in nerve fibre morphology (the pan-neuronal marker, PGP9.5) as well as neurotransmitter expression (acetylcholinesterase (AChE), vasocative intestinal polypeptide (VIP) and tyrosine hydroxylase (TH). Sweat glands from young and old donor rats became reinnervated by an organotypic pattern of cholinergic host nerves. Surgical sympathectomy demonstrated that these cholinergic nerve fibres originate from sympathetic neurons of the host superior cervical ganglion (SCG). Retrograde tracing combined with staining for VIP (a marker associated with cholinergic phenotype in neurons supplying sweat glands) showed that SCG neurons projecting to irises with sweat gland implants may be induced to express VIP. We hypothesise that these neurons have been switched from their normal catecholaminergic phenotype to a cholinergic one by contact with the sweat gland implants. Transplants from old donors attracted a density of reinnervation by young host nerves which was appropriate to the age of the donor, thus old sweat glands received a significantly reduced density of innervation compared to young glands. Despite the reduced density of innervation, there was no obvious difference in the ability of young and old implants to induce the switch to a cholinergic phenotype, suggesting that different mechanisms regulate nerve growth and neurotransmitter phenotype.

Adrenergic Fibers↗
PubMed1990

Sympathetic sprouting into neonatally denervated contralateral target: superior cervical ganglion neuronal numbers and sizes.

Unilateral superior cervical ganglionectomy results in establishment of an atypical functional projection from the contralateral ganglion to the denervated superior tarsal muscle in neonatal but not in juvenile or adult rats. Previous studies of peripheral nervous system development have attributed enhanced neuroplasticity to either formation of pathways by surplus cells rescued from cell death (hypothanasia-induced hyperplasia), or more vigorous terminal aborization of neurons innervating adjacent target zones (collateral sprouting with associated somatic hypertrophy). In the present study, experiments were performed to determine if neonatal sympathetic sprouting to contralaterally denervated target is associated with superior cervical ganglion neuronal hyperplasia or hypertrophy. Fluoro-Gold injections of right superior tarsal muscle in adult rats following neonatal ganglionectomies labelled cells in the contralateral superior cervical ganglion, whereas cells were labelled strictly ipsilaterally in sham operates. Profile areas of contralaterally projecting neuronal somata were twice as large as those providing ipsilateral innervation. Ganglion volumes, neuronal nuclear diameters and packing densities, and total neuronal numbers in left superior cervical ganglia of animals receiving neonatal right superior cervical ganglionectomy were comparable to those of both right and left ganglia of sham-operated controls. It is concluded that establishment of a contralateral projection does not rescue significant numbers of neurons from naturally occurring cell death. Rather, contralateral sprouting derives from neurons showing pronounced somatic hypertrophy.

Adrenergic Fibers↗
PubMed1995

A study of double-labeled retinal ganglion cells from the superior colliculus in the developing albino rat.

We studied the distribution pattern and percentage of bilaterally projecting, double-labeled retinal ganglion cells in the albino rat by the retrograde fluorescent double labeling. Forty-five albino (Wistar, Japan Clea) rats of either sex and of different stage of development ranging in age from the day of birth (Day 0) to Day 30, were used. With the rats under deep anesthesia, we pressure injected 0.02 microliter of 15% Evans blue (EB) and 0.02 microliter of 4% Fluoro-gold (FG) into the right and left superior colliculi, respectively; for rats older than 5 days, the volume of each tracer was 0.04 microliter. The animals were perfused with formol-saline 48 to 72 h later and the brain and eyeballs were excised and sectioned. Double-labeled cells were found over almost the entire retina, with the concentration in the lower temporal crescent in rats up to day 1; concentration gradually shifted to the ventral half between days 5 and 10. After day 15, double-labeled cells were found only in the ventral-temporal crescent of the retina, which is the pattern in the adult rats. The percentages of retinal ganglion cells that were double-labeled at days 0, 1, 5, 7, 10, 15, 20, 25 and 30 were 60.2, 51.6, 60.5, 57.6, 62.2, 60.7, 55.7, 45.2, and 39.1, respectively. After day 10, the percentage of such cells decreased steadily.

Animals↗
PubMed1994

Neurogenesis of spinothalamic and spinocerebellar tract neurons in the lumbar spinal cord of the rat.

The temporal and spatial neurogenic patterns of spinothalamic and spinocerebellar neurons were determined in spinal cord segment L1 of the rat. Neurogenic patterns were demonstrated with [3H]thymidine administered to fetal rats during the period when neurons with supraspinal projections are known to be generated, i.e. on one of embryonic (E) days E13, E14, or E15. The animals were allowed to survive 50 to 100 days postpartum, then neurons with spinothalamic and spinocerebellar projections were retrogradely filled with fluorescent axonal tracers, Fluoro-Gold or True blue, which were pressure injected into the dorsal thalamus and cerebellum in various combinations in the same and in separate animals. Neurons labeled with each retrograde tracer and [3H]thymidine and neurons labeled with retrograde tracers alone were counted in spinal cord segment L1 in each of the animals. Spinothalamic and spinocerebellar neurons were found to be separate and distinct populations. Statistical analysis of the data showed that spinothalamic and spinocerebellar neurons also have distinctly different patterns of neurogenesis which suggest early determination in each cell line. The temporal neurogenic pattern followed a projection-distance gradient, such that spinothalamic neurons, which have longer axons than spinocerebellar neurons, completed neurogenesis prior to spinocerebellar neurons in each region of the spinal gray.

Animals↗
PubMed1996

Retrograde fluorescent double-labeling study of bilaterally projecting retinal ganglion cells in albino rats at different stages of development.

Injection of the fluorescent tracers 10% Evans blue (EB) and 4% fluoro-gold (FG) into the right and the left dorsal lateral geniculate nucleus, respectively, of albino rats at different stages of development demonstrated the presence of double-labeled retinal ganglion cells that projected bilaterally into both the dorsal lateral geniculate nuclei (dLGN). Findings confirmed that the distribution of these double-labeled cells was gradually reduced after birth, being confined to the peripheral temporoventral quarter (temporal-ventral crescent) of the retina after postnatal day 15. We estimated the proportion of double-labeled cells to total labeled cells in the same area at different stages of development (0-90 days); values ranged from 35.3% in the neonate to 5.27% in the adult rat which suggests that the majority of double-labeled cells and/or their axons were lost early in development. That a small number of ganglion cells were observed to project bilaterally in the adult rats suggested that these cells conduct the same visual information to both hemispheres throughout the animal's life.

Animals↗
PubMed1996

Crossed projection neurons are generated prior to uncrossed projection neurons in the lateral superior olive of the rat.

The present study examined in the lateral superior olive (LSO) of the rat whether LSO neurons projecting to the ipsilateral inferior colliculus (IC) might be generated later than those projecting to the contralateral IC. Rat fetuses were exposed in utero to 5-bromodeoxyuridine (BrdU), a thymidine analogue, to label neurons proliferating at different embryonic stages from day E11 through to E20. Upon reaching adulthood, the rats were given unilateral injections of fluoro-gold (FG), a retrograde fluorescent tracer, into the IC. Subsequently, the tissue sections of the brains obtained from the rats were immunostained for BrdU to simultaneously detect neurons that were BrdU-positive and/or FG-positive. BrdU-positive LSO neurons were found in the rats which had been exposed to BrdU during E12-E16. In E12 and E13 BrdU-exposure cases, the vast majority of doubled-labeled (BrdU-positive and FG-positive) neurons were seen on the contralateral side to the FG injection. In E14, E15 and E16 BrdU-exposure cases, in contrast, all double-labeled neurons were found on the ipsilateral side to the FG injection. The distribution of these double-labeled neurons within the nucleus was diffuse in all the BrdU-exposure cases. Thus, the results indicate that LSO neurons are generated during E12-E16, that the crossed projection neurons are generated 1-4 days earlier than the uncrossed projection neurons, and that no topographical relationships exist between the early- and the late-generated populations of the LSO neurons.

Animals↗
PubMed1990

Cortical laminar distribution of rat thalamic ventrolateral fibers demonstrated by the PHA-L anterograde labeling method.

The cortical innervation pattern of rat thalamic ventrolateral (VL) fibers was investigated with the Phaseolus vulgaris-leukoagglutinin (PHA-L) anterograde labeling method. The PHA-L-labeled terminals were distributed in the sensorimotor cortex where the cerebello-cerebral (C-C) evoked responses were recognized. The labeled terminals were detected in all layers except layer VI. In the upper part of layer I, the most densely packed terminals were formed and moderately labeled terminals were seen in layers IV and V. Such a laminar distribution of PHA-L-labeled VL terminals is consistent with the profiles of the C-C responses according to laminar field potential analysis.

Animals↗
PubMed1992

Distribution and origin of extrinsic nerve fibers containing calcitonin gene-related peptide, substance P and galanin in the rat upper rectum.

The distributions of nerve fibers containing calcitonin gene-related peptide (CGRP), substance P (SP) and galanin (GAL) were examined in the rat rectum of mutants rats, aganglionic rats (AGRs), which completely lack the intramural nerve cells in the large intestine, and of their normal littermates. The origin of extrinsic peptide-containing nerve fibers was examined using retrograde tracing combined with immunohistochemistry in normal rats. In the rectum of normal rats, CGRP-, SP- and GAL-immunoreactive varicose fibers were observed throughout all layers of the rectal wall, and immunoreactive nerve cells were present in the enteric ganglia of colchicine-treated rats. In the aganglionic rectum of AGR, a rich supply of CGRP-immunoreactive fibers was observed in the mucosa, around the blood vessels, and in the submucous and intermuscular spaces. SP- and GAL-immunoreactive fibers in the aganglionic rectum showed a similar distribution to CGRP-immunoreactive fibers but were less dense. These results suggest that most of CGRP-positive fibers in the rectum are extrinsic whereas a large part of SP- or GAL-positive fibers are intrinsic. Fluoro-gold injected into the upper rectum of normal rat labelled nerve cells (less than 10% of total ganglion cells) in the lumbar (L1 and L2) and lumbosacral (L6 and S1) dorsal root ganglia. More than half of nerve cells in the dorsal root ganglia (L6 and S1) projecting to the rectum were immunoreactive for CGRP, and less than 10% were immunoreactive for SP or GAL. Comparison of serial sections of the dorsal root ganglion revealed that about half of the CGRP-immunoreactive cells were also positive for SP or GAL. These results indicate that SP- or GAL-positive neurons projecting to the rectum are scarce in the dorsal root ganglia. The present investigation suggests that CGRP-containing nerves are visceral afferents forming a major component of the sensory innervation of the rat rectum, and SP- and GAL-containing nerves which share their extrinsic origins appear to form a lesser proportion of the sensory innervation.

Animals↗
PubMed1993

Topographic relationship between anteromedial thalamic nucleus neurons and their cortical terminal fields in the rat.

The present study has examined the topographic relationship between cells in the anteromedial thalamic nucleus (AM) and their cortical terminal fields, with retrograde transport of Fluoro Gold in the rat. Projections to the frontal area 2 originate from the ventrolateral part of the AM and the entire interanteromedial nucleus (IAM). Projections to the anterior cingulate area originate from the peripheral part of the rostral AM and the entire IAM. Fibers to the rostral retrosplenial area arise from the caudodorsal part of the AM, whereas those to the caudal retrosplenial area arise from the rostralmost and the rostrodorsomedial parts. Fibers to the rostral area 29D originate from the rostrocentral part of the AM, whereas those to the caudal area 29D originate from the rostroventrolateral and the ventromedial parts. Projections to the medial half of the entorhinal area originate from the rostrodorsomedial part of the AM. In contrast, projections to the lateral half of the entorhinal area originate from the IAM and the central part of the AM. The results show a complex topographic relationship between cells of origin of the AM and their cortical terminal fields, suggesting complex functional roles played by the AM in learning behavior such as discriminative avoidance behavior.

Animals↗
PubMed1993

The sites of origin of serotoninergic afferent fibers in the trigeminal motor, facial, and hypoglossal nuclei in the rat.

The sites of origin of serotoninergic afferents in the trigeminal motor (Vm), facial (VII), and hypoglossal nuclei (XII) were studied in the rat by fluorescent retrograde labeling with Fluoro-Gold, in combination with immunofluorescence histochemistry for serotonin (5-HT). The results indicated: (1) The nucleus raphe magnus, nucleus raphe pallidus, and nucleus raphe obscurus contained 5-HT neurons projecting to the Vm, VII or XII. (2) The nucleus raphe dorsalis sends 5-HT fibres to the Vm and VII, but not to the XII. (3) The gigantocellular reticular nucleus pars alpha contained 5-HT neurons projecting to the VII.

Afferent Pathways↗
PubMed1988

The distribution of thalamic projection neurons containing cholecystokinin messenger RNA, using in situ hybridization histochemistry and retrograde labeling.

The distribution of cholecystokinin (CCK) synthesizing neurons in the thalamus was studied using in situ hybridization histochemistry. The message coding for CCK is present at different levels of intensity in almost all neurons (95%) of the anterior, ventral, medial and lateral groups of nuclei, with the ventral nuclei having the highest grain density. Many neurons (90%) of the medial and dorsal lateral geniculate nuclei also contained high levels of CCK transcripts. Very few neurons were found to express CCK in the parafascicular and paraventricular nuclei (2% and 10%, respectively), whereas the other intralaminar and midline nuclei had intermediate levels of CCK transcripts (75% of the neurons). The ventral lateral geniculate nucleus and the reticular nucleus were completely devoid of signal. After injection of the fluorescent dye, Fluorogold, into several areas of the cortex and corpus striatum, almost all retrogradely labeled cells in the thalamus (except in the parafascicular nucleus) expressed the CCK gene.

Animals↗
PubMed1993

Destruction of the nigrostriatal pathway increases Fos-like immunoreactivity predominantly in striatopallidal neurons.

Unilateral destruction of the nigrostriatal pathway by injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (MFB) produces a long-lasting (> 3 months) increase in the expression of Fos-like immunoreactivity (FLI) in medium-sized neurons (12-18 microns) of the ipsilateral striatum. In order to determine the nature of neurons which contain FLI in the 6-OHDA-denervated striatum, striatonigral and striatopallidal neurons were retrogradely labelled with the fluorescent tracer Fluoro-Gold. Nuclei displaying FLI were frequently found in striatopallidal neurons (72% overlap) but seldom in striatonigral neurons (11% overlap). These results are consistent with studies suggesting that dopamine tonically inhibits striatopallidal neurons which become more active in its absence. Moreover, the preferential localization of FLI in striatopallidal neurons supports the proposal that the AP-1 transcriptional regulating factor may contribute to neuropeptide and/or D2 dopamine receptor increases which occur in these neurons after 6-OHDA lesions.

Animals↗
PubMedDate not supplied

Cell size and cell type analysis of calcitonin gene-related peptide-containing cutaneous and splanchnic sensory neurons in the rat.

Cell size, cell type and calcitonin gene-related peptide (CGRP)-like immunoreactivity were compared between cutaneous and splanchnic sensory neurons by means of a combination of fluorescent tracer and immunohistochemistry. Nineteen percent of cutaneous sensory neurons and 88% of splanchnic sensory neurons were shown to contain CGRP. The former cells were larger than the latter ones, which was also confirmed by the finding that about a half of the former cells contained 200 kDa subunit of neurofilament protein, while only 8% of the latter ones were positively stained. These findings suggest that most of the visceral CGRP-IR sensory neurons are small type B.

Animals↗
PubMedDate not supplied

Median eminence-afferent vasoactive intestinal peptide (VIP) neurons in the hypothalamus: localization by simultaneous tract tracing and immunocytochemistry.

Retrograde tract tracing and immunocytochemistry were used to investigate the CNS source of the VIP that is present in high concentrations in the hypophysial portal blood and has been shown to have a stimulatory effect on pituitary prolactin secretion. Fluoro-gold (FG), which enters the CNS through areas devoid of the blood-brain barrier, such as median eminence, was injected peripherally. Brain sections from FG-treated animals were immunostained for VIP. A small population of VIP-containing cell bodies in the parvocellular and periventricular parts of the paraventricular nucleus (PVN) was also labeled with FG. Vasoactive intestinal peptide-immunoreactive perikarya not labeled with FG were also observed in the PVN, as well as FG-labeled cells that did not contain VIP. The results suggest that some VIP-producing neurons in the PVN project to the median eminence and are, therefore, functionally related to pituitary regulation; the function of other VIP neurons in the PVN is unknown.

Animals↗
PubMed1987

Simultaneous observation of fluorescent retrogradely labeled neurons and the immunofluorescently labeled fibers apposing them using Fluoro-Gold and antisera labeled with the blue fluorochrome 7-amino-4-methylcoumarin-3-acetic acid (AMCA).

In this paper we describe a method allowing the simultaneous observation of fluorescent retrogradely labeled neurons and the immunofluorescently labeled fibers which appose them. This technique employs the fluorescent dye Fluoro-Gold to label neurons retrogradely, and 7-amino-4-methylcoumarin-3-acetic-acid (AMCA) conjugated to a secondary antiserum as a label for fluorescence immunohistochemistry. Both Fluoro-Gold and AMCA are excited by near-ultraviolet (UV) light, but under UV excitation Fluoro-Gold appears yellow and AMCA appears blue. Thus the two fluorochromes are both visible under a single condition of illumination and can be readily distinguished by color. This allows quick and accurate determination of whether or not immunofluorescent fibers appose retrogradely labeled neurons.

Animals↗