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At least 811 records · Page 45Linked to original sources
PubMed2003

Persistence of tracer in the application site--a potential confounding factor in nerve regeneration studies.

Selective reinnervation of peripheral targets after nerve injury might be assessed by injecting a first tracer in a target before nerve injury to label the original neuronal population, and applying a second tracer after the regeneration period to label the regenerated population. However, altered uptake of tracer, fading, and cell death may interfere with the results. Furthermore, if the first tracer injected remains in the target tissue, available for "re-uptake" by misdirected regenerating axons, which originally innervated another region, then the identification of the original population would be confused. With the aim of studying this problem, the sciatic nerve of adult rats was sectioned and sutured. After 3 days, to allow the distal axon to degenerate avoiding immediate retrograde transport, one of the dyes: Fast Blue (FB), Fluoro-Gold (FG) or Diamidino Yellow (DY), was injected into the tibial branch of the sciatic nerve, or in the skin of one of the denervated digits. Rats survived 2-3 months. The results showed labelled dorsal root ganglion (DRG) cells and motoneurones, indicating that late re-uptake of a first tracer occurs. This phenomenon must be considered when the model of sequential labelling is used for studying the accuracy of peripheral reinnervation.

Amidines↗
PubMed1997

Use of peroxidase substrate Vector VIP for multiple staining in light microscopy.

The study of the distribution of a fiber input to a particular brain area and the visualization of the anatomical relationships of that input with both projection- and interneurons, requires a triple-staining that allows the unequivocal distinction of each of the three components in one and the same histological section. In this regard, we investigated the properties of a recently introduced peroxidase chromogen, VIP (V-VIP; Vector Labs) in combination with two traditional substrates, standard diaminobenzidine (DAB, brown precipitate) and nickel-enhanced DAB (DAB-Ni, black). In rats, the anterograde tracer biotinylated dextran amine (BDA) and the retrograde tracer fluorogold (FG) were injected in the perirhinal cortex and hippocampus, respectively. Transported BDA was detected with an avidin-biotin-peroxidase complex, whereas the transported FG was detected via a PAP method. Tracing with BDA and FG was combined with parvalbumin- or calbindin-immunocytochemistry. We compared various combinations and staining sequences. The best results were obtained with a staining sequence comprising first the BDA stain with DAB-Ni as chromogen, second the FG protocol with the chromogen DAB and finally, parvalbumin- or calbinding-immunocytochemistry using the chromogen V-VIP. The order with which the chromogens were applied appeared to be critical. Partial or even total loss of V-VIP reaction product has been observed after standard dehydration in ethanol. As an alternative, a quick dehydration procedure in toluene yields much better staining. Colour separation is excellent and the sensitivity is high. This procedure may also be used for detection of any other combination of three different labels, taking the usual care to avoid cross-reactivity between antibodies.

3,3'-Diaminobenzidine↗
PubMed1997

Persistent neuronal labeling by retrograde fluorescent tracers: a comparison between Fast Blue, Fluoro-Gold and various dextran conjugates.

The permanence of retrograde neuronal labeling by the fluorescent tracers Fast Blue, Fluoro-Gold, Mini-Ruby, Fluoro-Ruby and Fluoro-Emerald was investigated in adult rat spinal motorneurons at 1, 4, 12 and 24 weeks after tracer application to a transected muscle nerve. After 1 week, the largest number of retrogradely labeled motoneurons was found with Mini-Ruby, Fluoro-Gold and Fluoro-Ruby, while Fluoro-Emerald yielded a smaller number of labeled cells. With increasing survival time, all of these tracers exhibited a marked decrease in the number of labeled neurons. Fast Blue also produced very efficient staining after 1 week and, in addition, the number of Fast Blue-labeled cells remained constant over the entire time period studied. Also in embryonic spinal cord tissue exposed to Fast Blue. the label persisted for at least 6 months after transplantation into adult spinal cord. Double-labeling experiments combining Fast Blue with Fluoro-Gold, Mini-Ruby, Fluoro-Ruby or Fluoro-Emerald showed that all these substances were non-toxic and that the time-related decrease in the number of neurons labeled by the latter tracers was due to degradation or leakage of the dyes. Thus, Fast Blue would be the tracer of choice for motoneuronal labeling in long-term experiments, whereas the usage of the other tracers should be restricted to experiments of limited duration.

Amidines↗
PubMed1998

Combined methods of retrograde staining, layer-separation and viscoelastic cell stabilization to isolate retinal ganglion cells in adult rats.

The adult retinal ganglion cells (RGCs) are widely used as a model to study mechanisms of de- and regeneration within the central nervous system (CNS). All regions of the CNS including the retina have the common disadvantage of being composed of heterogeneous populations of cells, many of them like RGCs making less than 1% of the total population. This disadvantage can be circumvented by methodologies aimed at purifying specific cell types. Here we describe a method that combines retrograde labelling with fluorescent dyes and the pull-off technique. By using either of four different fluorescent dyes to retrogradely label RGCs, between 55 and 80% of pre-labelled ganglion cells could be identified with fluorescence microscopy. The pull-off procedure was supplemented by enhancing the viscosity of the collecting medium with methylhydroxypropyl-cellulose (MHPC). It appeared that 45% of all RGCs could be collected in the medium containing MHPC as compared to about 19% of RGCs which could be collected in medium devoid of cellulose. Despite the fact that morphometric measurements indicated that 60% of the cells collected fulfilled the criteria of being RGCs, the population obtained was immensely enriched and sufficed to perform a two dimensional SDS-gel electrophoresis and to determine their protein composition. The collected RGCs displayed a similar protein pattern as compared to that of the total retina, indicating that sublayers of the retina were represented in the probe. The results suggest that combined approaches of in vivo staining and ex vivo separation within suitable media enables the collection of an enriched population of cells which can be processed for biochemical analysis, and perhaps for molecular biology.

Age Factors↗
PubMed1999

Differential sensitivities of the two visual pathways of the chick to labelling by fluorescent retrograde tracers.

This study investigates the neurone structure-specific differences of sensitivities of fluorescent tracers. The tracers were used for retrograde labelling of contralateral projections in the two visual pathways of the chick. Rhodamine B Isothiocyanate (RITC), Fluorogold (FG) and True blue (TB) were injected into either the visual Wulst (thalamofugal pathway) or the nucleus rotundus (Rt; tectofugal pathway) and the retrogradely labelled neurones in the nucleus geniculatus lateralis pars dorsalis (GLd) or the optic tectum, respectively, were counted. Differential retrograde labelling in the two pathways was observed. In the thalamofugal pathway, both the contralateral and ipsilateral GLd cells were labelled by all three tracers (RITC, FG and TB). However, in the tectofugal pathway, whereas RITC labelled both the ipsilateral and contralateral tectal neurones, FG or TB labelled effectively only the ipsilateral tectal neurones. It was clear that FG and TB were taken up by the nerve endings and transported part-way along the axon but failed to be transported to the cell bodies of the contralateral tectal neurones. In addition, red beads and green beads were also injected into Rt and the differential labelling was also observed. Red beads labelled both ipsilateral and contralateral tectal neurones but green beads labelled only the ipsilateral tectal neurones. Since the contralateral tectal projections consist of divergent axon collaterals, the present study suggests that various retrograde tracers are not transported in these axon collaterals to label cell bodies. The contralaterally projecting neurones in the thalamofugal pathway are not axon collaterals and they were labelled by all of the tracers used.

Animals↗
PubMed1996

The brainstem network involved in coordination of inspiratory activity and cholinergic outflow to the airways.

The respiratory rhythm modulates cholinergic outflow to the tracheal smooth muscle through the parasympathetic nerves. To determine the basis of this modulation, we combined the retrograde tracer technique to identify bulbospinal cells projecting to phrenic motoneurons, and the transneuronal labeling method to visualize medullary neurons that innervate airway-related vagal preganglionic cells. Following injections of fluorogold into the ventral horns of the cervical spinal cord and injections of pseudorabies virus (PRV) into the wall of the extrathoracic trachea of superior cervical ganglioctomized Sprague-Dawley rats. A large number of the double-labeled cells were identified along the ventral aspect of the medulla oblongata. Most frequently, double-labeled neurons were seen in the medial tegmental field, particularly in the parapyramidal region, within the gigantocellular nuclei, and the caudal raphe nuclei. Less frequently, double-labeled neurons were found in the ventrolateral medulla. No double-labeled cell was observed in the dorsal aspect of the medulla oblongata. This study indicates that a subset of medullary neurons that project to phrenic motoneurons also innervate the airway-related vagal preganglionic cells, allowing the coupling of inspiratory activity and parasympathetic outflow to the airways.

Animals↗
PubMed1998

A target specific pathway from nitric oxide synthase immunoreactive preganglionic sympathetic to superior cervical ganglion neurons innervating the submandibular salivary gland.

The correlations between nitric oxide synthase (NOS)-immunoreactive (NOS-ir) preganglionic sympathetic neurons (PSNs) in the thoracic spinal cord and the neurons in the superior cervical ganglia (SCG) were studied with special reference to target specificity. NOS-ir neurons were distributed in the intermediate gray of the spinal cord and most numerous in the intermediolateral subnucleus of the PSNs. NOS-ir PSNs received direct synaptic contacts from tyrosine hydroxylase-ir, 5-hydroxytryptamine-ir, gamma-amino butyric acid-ir, and phosphate-activated glutaminase-ir axons. A majority of PSNs projecting to SCG were NOS-ir but some were NOS-negative. Large SCG neurons surrounded by a dense network of NOS-ir axons from PSNs projected to the submandibular salivary gland. NPY-ir small SCG neurons devoid of NOS-ir PSN innervation projected to blood vessels. SIF cells in the SCG were NOS-negative and provided with a meshwork of NOS-ir axons. The present results suggest a subpopulation of SCG neurons may be concerned with a distinct functional category in the rat under the influence of NOS-ir preganglionic sympathetic neurons.

Animals↗
PubMed1998

Central histaminergic neurons regulate rabbit tracheal tension through the cervical sympathetic nerve.

We previously showed that stimulation of the posterior hypothalamus decreases tracheal tension and involves central histaminergic neurons. In the present study, we reveal that central histaminergic neurons project to the rostral ventrolateral medulla and affect cervical sympathetic nervous activity in rabbits. Administration of histamine into the fourth ventricle increased cervical sympathetic nervous activity and decreased tracheal tension. These effects were inhibited by administration of a histamine H receptor antagonist, pyrilamine, into the fourth ventricle. Unilateral injection of DL-homocysteic acid into the tuberomammillary nucleus increased cervical sympathetic nervous activity, an effect was antagonized by bilateral injection of pyrilamine into the rostral ventrolateral medulla. The pulse correlogram between the stimulation pulse applied to the tuberomammillary nucleus and the cervical sympathetic nerve activity showed a mode at 150 to 200 ms, which was reduced by pyrilamine administration into the fourth ventricle. Fibers anterogradely labeled by Phaseolus vulgaris leucoagglutinin (PHA-L) injected into the tuberomammillary nucleus were distributed in the A1, A2, C1, and C2 areas which are determined by tyrosine hydroxylase-immunohistochemistry. PHA-L positive neurons were in close contact with tyrosine hydroxylase-immunoreactive neurons in these four areas. Cell bodies in the tuberomammillary nucleus retrogradely labeled with fluorogold from the rostral ventrolateral medulla were immunoreactive with histamine. These results suggest that an excitatory efferent pathway projects from the tuberomammillary nucleus to the cervical sympathetic nerve and that the histaminergic neurons of this pathway influence tracheal tension through the rostral ventrolateral medulla.

Animals↗
PubMed1999

The origin of catecholaminergic nerve fibers in the subdiaphragmatic vagus nerve of rat.

It is known that the vagus nerve contains catecholaminergic fibers. However, the origin of these fibers has not been systematically examined. In this study, we addressed this issue using retrograde tracing from the subdiaphragmatic vagus nerve combined with immunocytochemistry. The cervical and thoracic sympathetic trunk ganglia, the nodose ganglia and the dorsal motor nucleus of the vagus nerve were examined following injection of Fluoro-Gold or cholera toxin horseradish peroxidase conjugate into the trunks of the subdiaphragmatic vagus nerve of rats. Numerous retrogradely labeled neurons were seen in the nodose ganglion and the dorsal motor nucleus of the vagus nerve. Very few labeled neurons were found in the sympathetic ganglia (less than 0.06% of the neurons in either superior cervical ganglion or cervicothoracic ganglion were retrogradely labeled). Double labeling with immunofluoresence for catecholamine synthesizing enzymes revealed that: (1) 92% of all Fluoro-Gold retrogradely labeled tyrosine hydroxylase immunoreactive neurons were found in parasympathetic sources (75% in the dorsal motor nucleus of the vagus nerve and 17% in the nodose ganglia), and only 8% in the cervicothoracic sympathetic ganglia; (2) 12% of the retrogradely labeled catecholaminergic neurons in the dorsal motor nucleus of the vagus nerve were also dopamine-beta-hydroxylase immunopositive neurons; (3) 70% of the retrogradely labeled neurons in the sympathetic ganglia were tyrosine hydroxylase immunopositive and 54% of these catecholaminergic neurons contained dopamine-beta-hydroxylase, while 30% of the retrogradely labeled neurons were non-catecholaminergic neurons. These results indicate that catecholaminergic fibers in the abdominal vagus nerve are primarily dopaminergic and of parasympathetic origin, and that only an extremely small number of these fibers, mostly noradrenergic in nature, arise from postganglionic sympathetic neurons.

Animals↗
PubMed2001

Cocaine- and amphetamine-regulated transcript peptide-immunoreactivity in dorsal motor nucleus of the vagus neurons of immature rats.

Cocaine- and amphetamine-regulated transcript (CART) peptide, a family of neuropeptides, is shown to inhibit food intake upon intracerebroventricular injection to the rat. CART peptide-immunoreactivity (irCART) was detected in neurons of the dorsal motor nucleus of the vagus (DMNV) of postnatal day one (P1) rats, the earliest day examined. The number of labeled DMNV neurons reached the peak between P5 and P8 rats and gradually declined thereafter. Few irCART neurons were noted in the DMNV between P22 and P90 rats. Double-labeling the medullary sections from P5 and P8 rats with CART-antiserum and choline acetyltransferase (ChAT)-antiserum revealed that irCART neurons in the DMNV were ChAT-immunoreactive (irChAT), but not all irChAT neurons were irCART. Intraperitoneal injection of the retrograde tracer Fluorogold to P3 and P5 rats labeled DMNV neurons, the majority of which were also irCART. The number of irCART neurons in other regions of the brain and spinal cord generally showed an increase in adult rats as compared to that of the same regions in immature rats. Our result suggests that expression of irCART in DMNV neurons undergoes developmental changes such that few neurons appear to contain irCART in mature rats. As a corollary, CART may be a signaling molecule to the gastrointestinal tract during the critical period of early development.

Age Factors↗
PubMed2001

Excitotoxic lesions of the rostral thalamic reticular nucleus do not affect the performance of spatial learning and memory tasks in the rat.

Rats with cytotoxic lesions of the rostral pole of the thalamic reticular nucleus were compared with surgical control animals on a series of spatial learning and memory tests. While evidence was found for an initial, transient impairment on forced-choice alternation in a T-maze, this rapidly disappeared, and overall performance was unaffected. Subsequent experiments found no evidence that lesions of the rostral reticular nucleus affected the acquisition or performance of tests in the radial arm maze and the Morris water maze. Thus, it appears that the rostral pole of the thalamic reticular nucleus often does not play a necessary role in the performance of tests of spatial learning and memory, in spite of its interconnections with other regions that are required for normal spatial memory.

Animals↗
PubMed1999

Sexual experience sensitizes mating-related nucleus accumbens dopamine responses of female Syrian hamsters.

We examined the effects of prior sexual experience on extracellular concentrations of dopamine in the nucleus accumbens of female hamsters. Nucleus accumbens dopamine was measured by in vivo microdialysis during mating in female Syrian hamsters that had previously been given six prior sexual encounters with a male, three prior encounters, or were sexually naive. High levels of sexual behavior were observed in all three groups, which were accompanied by increases in dialysate dopamine during periods when the male was present. However, females that received six prior sexual encounters had significantly elevated and prolonged increases in dialysate dopamine compared with those of the sexually naive females or females with only three prior sexual encounters with a male. The data indicate that the mesolimbic system can be sensitized by repeated experiences associated with a motivated behavior.

Animals↗
PubMed2000

Collateral projection from the amygdalo--hippocampal transition area and CA1 to the hypothalamus and medial prefrontal cortex in the rat.

Amygdaloid and hippocampal neurons projecting to both the medial prefrontal cortex and hypothalamus by way of axon collaterals were examined in the rat by double labeling method using fluorescence retrograde tracers. Fluoro-gold was injected in the medial prefrontal cortex, while Fluoro-red was injected into the ventromedial and ventral premammillary nuclei of the hypothalamus. The results indicated that neurons which sent axon collaterals to both the medial prefrontal cortex and hypothalamus constituted 50 or 30% of populations of medial prefrontal cortex-projecting neurons in the amygdalo-hippocampal transition area or in CA1, respectively. Possible roles of the neurons with axon collaterals in sexually related aggressive and/or defensive behavior were discussed.

Amygdala↗
PubMed2001

Somatic noxious mechanical stimulation induces Fos expression in the postsynaptic dorsal column neurons in laminae III and IV of the rat spinal dorsal horn.

This study was conducted to ascertain the possible expression of Fos-like immunoreactivity (Fos-LI) in the postsynaptic dorsal column (PSDC) neurons in response to noxious mechanical stimulation of the forepaw glabrous area of normal rats. For this purpose, Fos immunohistochemistry along with Fluoro-Gold (FG) retrograde tracing was utilized. After repeated noxious pinching of the forepaw glabrous area, there was a marked increase in number of Fos-LI neurons in the dorsal horn, including Rexed's laminae III and IV, at C5-T1 spinal cord segments ipsilateral to the stimulation. Between segments C5 and T1, about 40% of the Fos-LI neurons in laminae III and IV were distributed at segment C7. In the rats subjected to the noxious pinch coupled with FG injection into the right cuneate nucleus, PSDC neurons double labeled with Fos and FG were localized in the ipsilateral laminae III and IV extending from segment C5 to T1, with about 70% of them distributed at segments C6 and C7. At segment C6 or C7, double-labeled neurons made up about 10% of the PSDC neurons that projected their axons to the cuneate nucleus. Most of the double-labeled neurons appeared fusiform with their primary dendrites projected dorso-ventrally. The present results suggest that the morphologically distinct, subclasses of PSDC neurons in spinal laminae III and IV may contribute to the central transmission of mechanical nociceptive information through the dorsal column into the cuneate nucleus.

Animals↗
PubMed2001

Small primary sensory neurons innervating epidermis and viscera display differential phenotype in the adult rat.

Target tissues contribute to the phenotype and function of sensory neurons. Due to lack of appropriate markers for trkA expressing sensory axons and terminals, the detailed peripheral projection of these neurons is unclear. In this study, the peripheral projections of trkA immunoreactive neurons are characterized using the combined techniques of immunohistochemistry and retrograde tracing. We found approximately 65% of all neurons projecting to the adrenal gland and kidney are trkA immunoreactive, whereas 6, 14 and 37% of neurons innervating whisker follicle, epidermis and footpad, respectively, are immunoreactive for trkA. A low proportion of trkA immunoreactive neurons innervating epidermis indicates that the majority of sensory neurons innervating epidermis are independent of trkA signalling for their normal function. We further investigated whether these epidermal projecting neurons can bind isolectin IB4. We found approximately 70% of all neurons innervating epidermis are IB4 binding neurons, but they did not express trkA. Thus, NGF sensitive neurons primarily project to viscera but not epidermis or other skin structures, whereas IB-4 positive neurons primarily project to epidermis in the adult rat.

Adrenal Glands↗
PubMed2002

Re-examination of the thalamostriatal projections in the rat with retrograde tracers.

Topographical arrangements of thalamostriatal projection neurons was examined in the rat by the retrograde tract-tracing method. After injecting Fluoro-Gold (FG) and/or cholera toxin beta-subunit (CTB) in different regions of the caudate-putamen (CPu), distribution of retrogradely labeled neurons was observed in the thalamus. The main findings were as follows: (1) Retrogradely labeled neurons were seen in the midline-intralaminar thalamic nuclei in all rats examined in the present study. Neurons in the ventral lateral and posterior thalamic nuclear groups were also labeled in the rats which were injected with the tracer into the dorsal part of Cpu, but not in the rats which were injected with the tracer into the nucleus accumbens (Acb) and its adjavent regions in the ventromedial part of the Cpu. (2) Topographical organization was observed in the projections from the midline-intralaminar thalamic nuclei to the CPu. After the tracer injection into the dorsal part of the CPu or the ventral part of the CPu (including the Acb), labeled neurons in the midline-intralaminar thalamic nuclei were distributed predominantly in the lateral part of the intralaminar nuclei or the midline nuclei, respectively. On the other hand, after the tracer injection into the medial or the lateral part of the CPu, labeled neurons in the midline-intralaminar nuclei were distributed mainly in the dorsal or the ventral part of these nuclei, respectively. (3) Topographical organization was also observed in the thalamostriatal projections from the ventral and Pos. After the tracer injection into the rostral part of the CPu, labeled neurons were distributed mainly in the rostral part of the ventral nuclear group. On the other hand, after the tracer injection into the caudal part of the CPu, labeled neurons were distributed mainly in the caudal part of the ventral nuclear group, as well as in the posterior nuclear group.

Animals↗
PubMed2003

Great potentiality of neonatal facial motor neurons for neural plasticity as determined by functionally essential neuronal population.

The present study was undertaken to determine the neuronal population essential for normal and minimal facial function of young adult rats that had received various degrees of crush injuries to the facial nerve in the neonatal period. Using a neuronal tracer, it was found in young adult rats receiving neonatal injuries that the minimum number of tracer-labeled facial motor neurons necessary for normal facial function corresponded to 13-14% of the neurons (2540+/-64) of the age-matched control animals, whereas the minimum number of neurons necessary for minimal facial function corresponded to 5%. On the other hand, the minimum numbers of tracer-labeled facial motor neurons necessary for normal and minimal facial function of young adult rats that received various degrees of crush injuries corresponded to 61 and 27-30%, respectively, of the neurons (2540+/-64) of the uninjured control animals. These results indicate that the facial function of animals with nerves crushed at the neonatal stage can be adequately maintained by a very small population of neurons, implying a great potential of neonatal neurons for neural plasticity.

Animals↗
PubMed2003

Regenerative process of the facial nerve: rate of regeneration of fibers and their bifurcations.

After the main trunk of the mouse facial nerve was injured by crushing, a fiber tracing method was used to quantify the facial motor neurons that extended regenerating nerve fibers to the specific site of the facial nerve branch. The total number of motor neurons retrogradely labeled with a fluorescent tracer, Fluoro-Gold (FG), were 0 on postsurgical days (PSDs) 1 and 2, 75+/-25 on PSD3, 264+/-21 on PSD4, 378+/-19 on PSD6, 428+/-19 on PSD8, 491+/-13 on PSD12 and 532+/-15 on PSD16. Assuming that the FG-positive neurons (535+/-11) of the control mice represent 100%, the FG-labeled neurons accounted for 0, 14, 49, 71, 80, 92 and 99% on the corresponding days. Two different fluorescent tracers were applied to the different facial nerve branches 16 days after facial nerve injuries. Double-labeled neurons were consistently found in the nerve-crushed facial nucleus (3.2%), and their number increased in the nerve-transected facial nucleus (12.2%). The present study indicates that the regenerating facial nerve consists of heterogeneous nerve fibers with varying growth rates and that excessive axonal branching occurs more frequently in the nerve-transected than in the nerve-crushed injuries.

Animals↗