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Immunohistochemical properties and spinal connections of pelvic autonomic neurons that innervate the rat prostate gland.

Autonomic innervation of the prostate gland supplies the acini, and non-vascular and vascular smooth muscle. The activity of each of these tissues is enhanced by sympathetic outflow, whereas the role of the parasympathetic nervous system in this organ is unclear. In the present study, a range of methods was applied in rats to determine the location of autonomic neurons supplying this gland, the immunohistochemical properties of these neurons, the spinal connections made with the postganglionic pathways and the distribution of various axon types within the gland. Injection of the retrograde tracer, FluoroGold, into the ventral gland visualised neurons within the major pelvic ganglion and sympathetic chain. Fluorescence immunohistochemical studies on the labelled pelvic neurons showed that most were nonadrenergic (also containing neuropeptide Y, NPY), the others being non-noradrenergic and containing either vasoactive intestinal peptide (VIP) or NPY. Sympathetic dye-labelled neurons were identified by the presence of varicose nerve terminals stained for synaptophysin on their somata following lesion of sacral inputs. Parasympathetic innervation of dye-labelled neurons was identified by continued innervation after hypogastric nerve lesion. Most noradrenergic prostate-projecting neurons were sympathetic, as were many of the non-noradrenergic VIP neurons. Parasympathetic prostate-projecting neurons were largely non-noradrenergic and contained either VIP or NPY. All substances found in retrogradely labelled somata were located in axons within the prostate gland but had slightly different patterns of distribution. The studies have shown that there are a significant number of non-noradrenergic sympathetic prostate-projecting neurons, which contain VIP.

Animals↗

Immunohistochemical characterisation of sympathetic and parasympathetic pelvic neurons projecting to the distal colon in the male rat.

The pelvic ganglia are mixed ganglia containing both sympathetic and parasympathetic neurons that receive spinal input via the hypogastric (lumbar cord) and pelvic nerves (sacral cord), respectively. A recent study has utilised immunohistochemistry against synaptophysin (a protein associated with small vesicles) to visualise the preganglionic terminals in these ganglia. By selectively cutting the hypogastric or pelvic nerves and allowing subsequent terminal degeneration, the populations of parasympathetic and sympathetic preganglionic terminals, respectively, can be visualised. The present study has used this method in conjunction with retrograde labelling of pelvic neurons from the distal colon and double label immunofluorescence against tyrosine hydroxylase and vasoactive intestinal polypeptide (VIP) to identify and characterise the sympathetic and parasympathetic neurons projecting to the distal colon from the major pelvic ganglia of the male rat. Approximately equal numbers of distal colonic-projecting pelvic neurons are sympathetic and parasympathetic. Almost all noradrenergic neurons are sympathetic. Of the VIP neurons that project to the distal colon approximately one third are sympathetic, one third parasympathetic and the remaining third are possibly innervated by both the lumbar and sacral cord. Extrapolation from our results also suggests that the majority of non-noradrenergic neuropeptide Y neurons (which are known to comprise the remainder of the neurons) are parasympathetic. These studies have demonstrated that the pelvic ganglia are a major source of sympathetic innervation to the distal bowel and have further shown that the distal colon is another target for the non-noradrenergic sympathetic neurons of the pelvic ganglia.

Animals↗

2-hydroxystilbamidine isethionate: a new fluorochrome for use in general pathology. I. The selective staining of DNA, mucosubstances and elastic fibres.

2 mg of 2-Hydroxystilbamidine isethionate when dissolved in 50 ml 0.1 M citric acid produced nuclear fluorescence in paraffin sections. Pre-hydrolysis in 5N HCl at room temperature increased selectivity of nuclear fluorescence. The addition of 100-200 mg sodium metabisulphite to the fluorochrome solution and preoxidation in periodic acid produced selective fluorescence of mucosubstances. Pre-oxidation with potassium permanganate induced selective fluorescence of elastic fibres. Yellow nuclear fluorescence contrasted clearly with blue/white fluorescence of mucosubstances and elastic fibres when excited with UV light. Unwanted nuclear fluorescence was quenched with 5% iron alum solution. Mast cells selectively fluoresced in acid alcoholic solutions of the fluorochrome. The procedures described were simple and rapid and produced permanent fluorescent preparations. The metachromatic fluorescence of nuclei in contrast to that of mucosubstances and elastic fibres eliminated the need for counterstaining.

Animals↗

Neurons containing retrogradely transported Fluoro-Gold exhibit a variety of lysosomal profiles: a combined brightfield, fluorescence, and electron microscopic study.

The advantages of axonally transported Fluoro-Gold as a retrograde fluorescent marker are numerous. The objective of the present study was to determine whether transported Fluoro-Gold is visible in either semi-thin sections for light microscopy or thin sections for electron microscopy. Rats received injections of Fluoro-Gold into either the striatum or thoracic spinal cord. After appropriate survival times, labelled neurons were observed with the fluorescence microscope in brain regions that are known to project to the injected areas. Sections that contained labelled cells were embedded in plastic and examined with a fluorescence microscope. Semi-thin sections of unosmicated tissue displayed high-resolution fluorescent labelling of somata and dendrites. In contrast, osmicated tissue did not fluoresce, but numerous dark granules were observed in the dendritic and perikaryal cytoplasm of labelled neurons in toluidine blue stained sections that were examined with brightfield optics. The unosmicated tissue did not display these granules, and this finding suggested that the granules are composed of membranes. Neurons in other brain regions that are known not to project to the injection sites did not contain these dark granules. Adjacent thin sections examined with the electron microscope displayed numerous electron-dense, lysosome-like organelles in the cytoplasm of labelled neurons. The electron density of these organelles was greater than that of lysosomes in unlabelled neurons. Three types of distinctive organelles were observed in these preparations: (1) relatively dense concentric lamellar bodies of various sizes; (2) heterogeneous or lipofuscin-like lysosomes; and (3) coarse grained lysosomes. Control sections and unlabelled neurons did not display these organelles. Therefore, these organelles appear to correlate with Fluoro-Gold localized within the somata and dendrites of retrogradely labelled neurons. It is not known if they are the Fluoro-Gold itself, or represent a physiological effect on membranes. The results of this study indicate that Fluoro-Gold may be useful for tract tracing at the electron microscopic level.

Biological Transport↗

Is there a dopaminergic projection from the A11 catecholamine cell group to the amygdala?

The dopaminergic nature of the pathway from the subparafascicular thalamic nucleus and its adjacent region to the amygdala was reexamined by means of retrograde fluorescent tracers coupled with tyrosine hydroxylase (TH) immunofluorescence. After injecting a small amount of tracer into the amygdala, retrogradely labeled cells were found in the subparafascicular thalamic nucleus and its adjacent periventricular region. TH immunofluorescence showed that these labeled cells completely lacked TH immunoreactivity. Similar results were obtained when a larger amount of tracer was applied to the amygdala. The present study, in contrast to the previous report describing the dopaminergic innervation of the amygdala by the cells in and around the subparafascicular area (A11 catecholamine cell group), indicates that the A11 cell group does not contribute to a dopaminergic input to the amygdala.

Amygdala↗

Chloroquine, hydroxystilbamidine, and dapsone inhibit resorption of fetal rat bone in organ culture.

Three potential inhibitors of lysosomal enzyme release, chloroquine, hydroxystilbamidine, and dapsone were tested for their effects on the release of previously incorporated 45Ca and beta (beta)-glucuronidase from fetal rat long bones cultured in a chemically defined medium. At concentrations of 10(-5) to 10(-8)M, all three agents were able to inhibit the stimulation of bone resorption by parathyroid hormone (PTH) or prostaglandin E2 (PGE2). Inhibition was seen at concentrations which did not alter the uptake of (3H)-2-deoxy-glucose or the incorporation of (3H)-thymidine in bone. While the inhibitors blocked the stimulation of beta-glucuronidase release by PTH and PGE2, they could also cause a direct increase in total beta-glucuronidase content and release. Hence the usual strong correlation between the release of beta-glucuronidase and 45Ca was no longer seen in the presence of inhibitors. These data indicate that chloroquine, hydroxystilbamidine, and dapsone are potent inhibitors of bone resorption which may act by blocking the release of lysosomal enzymes in cells stimulated by PTH or PGE2, but may have a different effect on other cell populations.

Animals↗

Axotomized, adult basal forebrain neurons can innervate fetal frontal cortex grafts: a double fluorescent tracer study in the rat.

The ability of axonal regeneration of identified adult basal forebrain (BFB) neurons was examined after homotopic grafting of fetal neocortical tissue to a lesion cavity in the frontal neocortex. Using a four step experimental procedure, adult rats first received an injection of the fluorescent dye Fluoro-Gold (FG) into the sensorimotor cortex in order to label those neurons with projections to the area by retrograde axonal transport. After one week the injection area was removed by aspiration, leaving a cavity in the neocortex. One week later a block of fetal (E14) frontal cortical tissue was placed in the cavity. The animals were then allowed to survive for 6 weeks before a second fluorescent tracer, Nuclear Yellow (NY), was injected into the transplant. The animals were sacrificed 24 h later and analyzed by fluorescence microscopy. Both single labeled, FG and NY containing neurons and double labeled neurons containing both tracers were found in the BFB. The results demonstrate that adult BFB neurons can reestablish cortical projections into fetal cortical grafts (double labeled neurons), and they suggest that other BFB neurons, not initially innervating the lesioned cortical area, have sprouted into the transplant (NY labeled neurons).

Animals↗

Kala-azar in Bihar.

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Adult↗

Protective effects of adenoviral cardiotrophin-1 gene transfer on rubrospinal neurons after spinal cord injury in adult rats.

Cardiotrophin-1 (CT-1), a muscle-derived cytokine, supports the survival of motoneurons in vivo and in vitro. The present study investigated whether adenoviral huCT-1 gene transfer protected injured neurons from cell death or atrophy and promoted regeneration of rubrospinal tract (RST) after spinal cord injury in adult rats. Administration of the adenoviral CT-1 vector (Adv-CT1) to C3-4 lateral funiculus hemisection cavity, that completely interrupted RST, led to sustained CT-1 expression. Providing Adv-CT1, which rescued 20% of neurons, could prevent the loss of injured rubrospinal neurons 8 weeks post-injury. Retrograde tracing with FluoroGold showed that 1.2% of RST neurons regenerated at least two segments caudal to the injury site. Anterograde tracing with biotinylated dextran amine revealed that the RST axons terminated in white matter and gray matter. Behavioral testing revealed a significant functional recovery in limb usage. This observation indicated that adenoviral CT-1 gene transfer into the injured cord promoted survival and regeneration of rubrospinal neurons in adult rats.

Adenoviridae↗

[Quantitative determination of specific reinnervation after suture of the facial nerve in the rat].

INTRODUCTION: In recent studies we identified two morphological phenomena in the facial nucleus of the brainstem that play an important role in the recovery of facial movement: hyperinnervation and misdirected reinnervation. While the hyperinnervation could easily be quantified by cell counting, the extent of misdirected reinnervation could not be estimated accurately. In the present study we developed a method for accurate quantification of this misdirected reinnervation. MATERIAL AND METHODS: In 6 rats we injected the fluorescent tracer FluoroGold into the whiskerpad. After 4 days the facial nerve was transected and a facial-facial anastomosis (FFA) performed. Eight weeks later the fluorescent tracer Fast Blue was injected in the same site of the whiskerpad, as done before with the FluoroGold. Following sacrifice, the brainstems of the animals were removed and labeled motoneurons in the facial nucleus counted. RESULTS: Three different types of labeled motoneurons could be identified: (a) white fluorescent motoneurons (labeled preoperatively only with fluoroGold, (b) blue fluorescent motoneurons (labeled only postoperatively with Fast Blue) and (c) green-grey fluorescent motoneurons double-labeled pre- and postoperatively. The double-labeled motoneurons were seen to project to the same sites in the whiskerpad pre- and postoperatively, demonstrating no misdirected reinnervation. In our experiment we counted 478 +/- 45 green-grey labeled neurons from a total number of 1446 +/- 131 postoperatively labeled cells (double-labeled and single FB-labeled). These findings show that 33% of the regenerating motoneurons were correctly redirected after FFA in our animal model and 67% were misdirected.

Animals↗

Neurogenesis of cuneothalamic neurons and NO-containing neurons in the cuneate nucleus of the rat.

The genesis of the cuneothalamic neurons (CTNs) in the rat cuneate nucleus was determined by a double-labeling method using 5'-bromodeoxyuridine (BrdU), the thymidine analogue, and Fluoro-Gold (FG), a retrograde fluorescent tracer. BrdU-positive cells were observed in the cuneate nucleus in all rats receiving BrdU injection at embryonic days (E) E13--E16; none was detected in rats given BrdU injection at E12. At E13 and E14, BrdU-positive cells were randomly distributed. However, at E15, the number of BrdU-positive cells was clearly reduced and the majority of them was located at the dorsolateral or peripheral region of the nucleus. FG/BrdU double-labeling study showed the existence of BrdU-labeled CTNs when the mother rat received BrdU injection at E13 and E14, being more numerous at E13 in which the neurons were scattered throughout the nucleus. At E14, however, the majority of the BrdU-labeled CTNs were located superficially in the nucleus. Double-labeled cells were undetected in rats that had been exposed to BrdU at E15 and E16. Quantitative data showed that the majority (ca 70-80%) of the CTNs were generated at E13, and were markedly decreased at E14 (ca 4-6%). Using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry coupled with BrdU immunohistochemistry, we have shown the NADPH-d/BrdU double-labeled neurons in the nucleus between E13 and E15, with the majority of them occurring at E14, but absent at E16. The present results suggest that the CTNs are generated prior to the NO-containing neurons in the cuneate nucleus.

Animals↗

Projections of diencephalic dopamine neurons into the spinal cord in mice.

The aim of this study is to investigate the pathway of diencephalic dopaminergic (DA) neuronal innervating into the spinal cord in mice, the pathway is postulated relevant to clinical restless legs syndrome (RLS). Tyrosine hydroxylase (TH) immunohistochemistry was used to identify the DA neuron. The fluorescent tracer Fluoro-Gold (FG) was stereotaxically injected into the T10-L5 spinal cord of CBL57 mice (n=20) seven days before the animals were sacrificed. The diencephalic sections were stained with TH antibody and the FG tracer present in the diencephalic DA neurons were examined under fluoresce microscope. The average number of total DA neurons per side in A11, A12, A13 and A14 was 66+/-8, 221+/-12, 350+/-17 and 254+/-21 respectively. After being injected into the spinal cord, FG reached the DA neurons within the A10 and A11 groups, but didn't target to any other DA neuron groups including the A8 and A9 groups in substantia nigra (SN). The diencephalic A11 DA neurons possessed long axons extending over several segments and possibly traversing the entire length of the spinal cord. It is the first time to report A10 and A11 DA neuron projections into the spinal cord in mice.

Animals↗

A single re-implanted ventral root exerts neurotropic effects over multiple spinal cord segments in the adult rat.

Spinal cord injuries, particularly traumatic injuries to the conus medullaris and cauda equina, are typically complex and involve multiple segmental levels. Implantation of avulsed ventral roots into the spinal cord as a repair strategy has been shown to be neuroprotective and promote axonal regeneration by spinal cord neurons into an implanted root. However, it is not well known over what distance in the spinal cord an implanted ventral root can exert its neurotropic effect. Here, we investigated whether an avulsed L6 ventral root acutely implanted into the rat spinal cord after a four level (L5-S2) unilateral ventral root avulsion injury may exert neurotropic effects on autonomic and motor neurons over multiple spinal cord segments at 6 weeks postoperatively. Using retrograde labeling techniques and stereological quantification methods, we demonstrate that autonomic and motor neurons from all four lesioned spinal cord segments, spanning more than an 8 mm rostro-caudal distance, reinnervated the one implanted root. The rostro-caudal distribution suggested a gradient of neurotropism, where the axotomized neurons closest to the implanted site had the highest probability of root reinnervation. These results suggest that implantation of a single ventral root may provide neurotropic effects to injured neurons at the site of lesion as well as in the adjacent spinal cord segments. Our findings may be of translational research interest for the development of surgical repair strategies after multi-level conus medullaris and cauda equina injuries, in which fewer ventral roots than spinal cord segments may be available for implantation.

Amidines↗

Prostaglandin E2 potentiates the excitability of small diameter trigeminal root ganglion neurons projecting onto the superficial layer of the cervical dorsal horn in rats.

The aim of the present study was to investigate how prostaglandin E2 (PGE2) affects the excitability of trigeminal root ganglion (TRG) neurons, projecting onto the superficial layer of the cervical dorsal horn, using fluorescence retrograde tracing and perforated patch-clamp techniques. TRG neurons were retrogradely labeled with fluorogold (FG). The cell diameter of FG-labeled neurons was small (< 30 microm). Under the voltage-clamp mode, application of PGE2 (0.01-10 microM) concentration-dependently increased the magnitude of the peak tetrodotoxin-resistant sodium current (TTX-R I(Na)) and this current was maximal at a concentration of 1 microM. One micromolar PGE2 application caused a hyperpolarizing shift of 8.3 mV in the activation curve for TTX-R I(Na). In the current-clamp mode, the PGE2 (1 microM) application significantly increased the number of action potentials during the depolarizing step pulses as well as the level of overshoot but had no significant effect on the resting membrane potential. These results suggest that the excitability of small diameter TRG neurons seen after 1 microM PGE2 application is involved in an increase in the

Action Potentials↗