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At least 703 records · Page 39Linked to original sources
PubMed2004

Premotor nitric oxide synthase immunoreactive pathway connecting lumbar segments with the ventral motor nucleus of the cervical enlargement in the dog.

In this study we investigate the occurrence and origin of punctate nitric oxide synthase immunoreactivity in the neuropil of the ventral motor nucleus in C7-Th1 segments of the dog spine, which are supposed to be the terminal field of an ascending premotor propriospinal nitric oxide synthase-immunoreactive pathway. As the first step, nitric oxide synthase immunohistochemistry was used to distinguish nitric oxide synthase-immunoreactive staining of the ventral motor nucleus. Dense, punctate nitric oxide synthase immunoreactivity was found on control sections in the neuropil of the ventral motor nucleus. After hemisection at Th10-11, axotomy-induced retrograde changes consisting in a strong upregulation of nitric oxide synthase-containing neurons were found mostly unilaterally in lamina VIII, the medial part of lamina VII and in the pericentral region in all segments of the lumbosacral enlargement. Concurrently, a strong depletion of the punctate nitric oxide synthase immunopositivity in the neuropil of the ventral motor nucleus ipsilaterally with the hemisection was detected, thus revealing that an uncrossed ascending premotor propriospinal pathway containing a fairly high number of nitric oxide synthase-immunoreactive fibers terminates in the ventral motor nucleus. Application of the retrograde fluorescent tracer Fluorogold injected into the ventral motor nucleus and analysis of alternate sections processed for nitric oxide synthase immunocytochemistry revealed the presence of Fluorogold-labeled and nitric oxide synthase-immunoreactive axons in the ventrolateral funiculus and in the lateral and medial portions of the ventral column throughout the thoracic and upper lumbar segments. A noticeable number of Fluorogold-labeled and nitric oxide synthase-immunoreactive somata detected on consecutive sections were found in the lumbosacral enlargement, mainly in laminae VIII-IX, the medial part of lamina VII and in the pericentral region (lamina X), ipsilaterally with the injection of Fluorogold into the ventral motor nucleus. In summary, the present study provides evidence for a hitherto unknown ascending premotor propriospinal nitric oxide synthase-immunoreactive pathway connecting the lumbosacral enlargement with the motoneurons of the ventral motor nucleus in the dog.

Afferent Pathways↗
PubMed2004

Substance P (NK1) and somatostatin (sst2A) receptor immunoreactivity in NTS-projecting rat dorsal horn neurones activated by nociceptive afferent input.

Spinal neurones that receive inputs from primary afferent fibres and have axons projecting supraspinally to the medulla oblongata may represent a pathway through which nociceptive and non-nociceptive peripheral stimuli are able to modulate cardiorespiratory reflexes. Expression of the neurokinin-1 (NK1) receptor is believed to be an indicator of lamina I cells that receive nociceptive inputs from substance P releasing afferents, and similarly, sst2A receptor expression may be a marker for neurones receiving somatostatinergic inputs. In this study, immunoreactivity for these two receptors was investigated in rat spinal neurones retrogradely labelled by injections of cholera toxin B or Fluorogold into the nucleus of the solitary tract (NTS). In addition, nociceptive activation of these labelled cells was studied by immunodetection of Fos protein in response to cutaneous and visceral noxious chemical stimuli. NK1 and sst2A receptors in lamina I were localised to mainly separate populations of retrogradely labelled cells with fusiform, flattened and pyramidal morphologies. Examples of projection neurones expressing both receptors were, however observed. With visceral stimulation, many retrogradely labelled cells expressing c-fos were immunoreactive for the NK1 receptor, and a smaller population was sst2A positive. In contrast, with cutaneous stimulation, only NK1 positive retrogradely labelled cells showed c-fos expression. These data provide evidence that lamina I neurones receiving noxious cutaneous and visceral stimuli via NK1 receptor activation project to NTS and so may be involved in coordinating nociceptive and cardiorespiratory responses. Moreover, a subpopulation of projection neurones that respond to visceral stimuli may receive somatostatinergic inputs of peripheral, local or supraspinal origins.

Afferent Pathways↗
PubMed2005

The turtle thalamic anterior entopeduncular nucleus shares connectional and neurochemical characteristics with the mammalian thalamic reticular nucleus.

Neurochemical and key connectional characteristics of the anterior entopeduncular nucleus (Enta) of the turtle (Testudo horsfieldi) were studied by axonal tracing techniques and immunohistochemistry of parvalbumin, gamma-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD). We showed that the Enta, which is located within the dorsal peduncle of the lateral forebrain bundle (Pedd), has roughly topographically organized reciprocal connections with the dorsal thalamic visual nuclei, the nucleus rotundus (Rot) and dorsal lateral geniculate nucleus (GLd). The Enta receives projections from visual telencephalic areas, the anterior dorsal ventricular ridge and dorsolateral cortex/pallial thickening. Most Enta neurons contained GABA and parvalbumin, and some of them were retrogradely labeled when the tracer was injected into the visual dorsal thalamic nuclei. Further experiments using double immunofluorescence revealed colocalization of GAD and parvalbumin in the vast majority of Enta neurons, and many of these cells showed retrograde labeling with Fluoro-gold injected into the Rot and/or GLd. According to these data, the Enta may be considered as a structural substrate for recurrent inhibition of the visual thalamic nuclei. Based on morphological and neurochemical similarity of the turtle Enta, caiman Pedd nucleus, the superior reticular nucleus in birds, and the thalamic reticular nucleus in mammals, we suggest that these structures represent a characteristic component which is common to the thalamic organization in amniotes.

Alligators and Crocodiles↗
PubMed2004

Quantification of neurons in the myenteric plexus: an evaluation of putative pan-neuronal markers.

Accurate estimates of the total number of neurons located in the wall of the gut are essential for studies of the enteric nervous system (ENS). Though several stains and antibodies are used routinely as pan-neuronal markers, controversies of relative sensitivity and completeness have been difficult to resolve, at least in part because comparisons often must be made across experiments and laboratories. Therefore, we evaluated the efficacy of four putative pan-neuronal markers for the ENS, under comparable conditions. Neurons in the myenteric plexus of wholemounts taken from the small intestines of Fischer 344 rats were stained using Cuprolinic Blue, anti-HuC/D, anti-protein gene product 9.5, or FluoroGold injections followed by permanent labeling with an antibody to the FluoroGold molecule. All four markers had useful features, but both protein gene product 9.5 and FluoroGold were found to be problematic for obtaining reliable counts. As a result, only neurons labeled with either Cuprolinic Blue or anti-HuC/D were compared quantitatively. Based on counts from permanently labeled tissue, Cuprolinic Blue and HuC/D were similarly effective in labeling all neurons. Because the two protocols have different strengths and weaknesses, Cuprolinic Blue and HuC/D provide a complementary set of labels to study the total neuronal population of the ENS.

Animals↗
PubMed2004

Microarray analysis of fluoro-gold labeled rat dopamine neurons harvested by laser capture microdissection.

The cellular heterogeneity of brain tissue presents a challenge to gene expression profiling of specific neuronal cell types. The present study employed a fluorescent neural tracer to specifically label midbrain dopamine neurons and non-dopamine cortical neurons. The labeled cells were then used to visually guide harvesting of the cells by laser capture microdissection (LCM). RNA extracted from the two populations of harvested cells was then amplified, labeled and co-hybridized to high density cDNA microarrays for two-color differential expression profiling. Many of the genes most highly enriched in the dopamine neurons were found to be genes previously known to define the dopamine neuronal phenotype. However, results from the microarray were only partially validated by quantitative RT-PCR analysis. The results indicate that LCM harvesting of specific neuronal phenotypes can be effectively guided in a complex cellular environment by specific pre-labeling of the target cell populations and underlie the importance of independent validation of microarray results.

Animals↗
PubMed2005

Improved detection of fluorogold-labeled neurons in long-term studies.

Fluorogold (FG) is a widely used neuroanatomical tracer. However, because FG-labeled neurons become undetectable over time, its use has been limited in long-term studies. We investigated whether the detection of FG in retrogradely labeled neurons in long-term studies can be improved by immunohistochemistry (IHC) using an antibody to FG. We performed intraperitoneal injections of a FG solution to retrogradely label all parasympathetic preganglionic neurons (PPNs) and motoneurons (MNs) in the S1 spinal cord segment in adult rats. At 1, 6, and 12 weeks after the tracer injection, sections were immunohistochemically processed for FG and choline acetyltransferase (ChAT), an endogenous marker for all PPNs and MNs. Stereological counts demonstrated no cell loss of FG-labeled PPNs and MNs at 6 and 12 weeks. Cell size measurements showed that FG-immunolabeled neurons were smaller at 12 weeks, but not at 6 weeks. However, it is likely that there was no neuronal atrophy, but loss/degradation of the dye at a timepoint between 6 and 12 weeks, as ChAT-immunolabeled neurons showed no cell size reduction at 12 weeks. Our results suggest that the use of an antibody against FG improves the detection of FG for reliable neuronal counts and that the dye is not toxic to the retrogradely labeled neurons. We conclude that FG-labeling is a useful tool to determine neuronal counts in long-term studies, but should be used cautiously for neuronal size measurements.

Animals↗
PubMed2006

Spatial clustering analysis in neuroanatomy: applications of different approaches to motor nerve fiber distribution.

Spatial organization of the nerve fibers in the peripheral nerves may be important for the studies of axonal regeneration, the degenerative nerve diseases and the construction of interfaces with peripheral nerves, such as nerve prostheses. Functional topography of motor axons related to the gastrocnemius muscle was revealed in the ventral spinal roots by retrograde tracing. Gastrocnemius muscles of adult rats were injected with the tracer Fluoro-Gold. After 3 days of survival the animals were sacrificed and their ventral roots were harvested, sectioned, and imaged on a fluorescence microscope. Maps of the traced fibers were automatically analyzed using a novel approach, local spatial clustering statistics, that tested for occurrences of clusters of motor fibers and visualized them. Thresholds indicating the presence of clustering at various scales of observation were computed based on series of Monte Carlo simulations of random spatial point patterns. Clusters were visualized by kernel interpolation. The approach was tested on simulated data and subsequently applied to the motor fiber maps in the ventral roots. Results revealed clustering of the motor fibers innervating the gastrocnemius muscle at the level of the L6 ventral spinal root. The analysis was validated using Voronoi tessellation and nearest neighbor analysis.

Animals↗
PubMed2004

Characterization of cellular and neurological damage following unilateral hypoxia/ischemia.

Rodent models of stroke are often used to investigate the mechanisms that lead to ischemic neuronal damage. In this study, we used a model of cerebral hypoxia with ischemia to produce unilateral damage in C57Bl/6 mice. Lesion volume, ascertained by TTC staining, increased with longer durations of hypoxia. Additionally, cresyl violet, TUNEL, and FluoroJade staining showed a statistically significant increase in cellular damage in the ipsilateral cortex, CA1 pyramidal layer, and dentate gyrus of the hippocampus of ipsilateral hypoxic/ischemic tissue versus sham tissue. Astrocyte reactivity, determined by GFAP staining, was significantly higher in the ipsilateral H/I cortex and contralateral hippocampus compared to sham cortex and hippocampus, respectively. Increased microglia activation was evident in the H/I-treated cortex and hippocampus versus sham cortex and hippocampus, particularly within areas undergoing degeneration. To examine whether this model produces motor deficits, a battery of tests were administered before and after hypoxia. Following 45 min H/I, locomotor activity, rotarod performance and performance on an inverted wire hang test were all significantly decreased. These data indicate that the histological evidence of neuronal damage is consistent with functional deficits and suggest that this model may be useful for investigating strategies designed to protect neurons from hypoxia/ischemia-induced damage.

Animals↗
PubMed2006

Correlative microXRF and optical immunofluorescence microscopy of adherent cells labeled with ultrasmall gold particles.

Synchrotron-based X-ray fluorescence microscopy (microXRF) is a powerful tool to study the two-dimensional distribution of a wide range of biologically relevant elements in tissues and cells. By growing mouse fibroblast cells directly on formvar-carbon coated electron microscopy grids, microXRF elemental maps with well-defined subcellular resolution were obtained. In order to colocalize the elemental distribution with the location of specific cellular structures and organelles, we explored the application of a commercially available secondary antibody conjugated to FluoroNanogold, a dual-label that combines a regular organic fluorophore with a 1.4 nm Au-cluster as xenobiotic label for microXRF imaging. Adherent mouse fibroblast cells were grown on silicon nitride windows serving as biocompatible XRF support substrate, and labeled with FluoroNanogold in combination with primary antibodies specific for mitochondria or the Golgi apparatus, respectively. Raster scanning of the in-air dried cells with an incident X-ray energy of 11.95 keV, sufficient to ensure excitation of the Au Lalpha line, provided two-dimensional maps with submicron resolution for Au as well as for most biologically relevant elements. MicroXRF proved to be sufficiently sensitive to image the location and structural details of the Au-labeled organelles, which correlated well with the subcellular distribution visualized by means of optical fluorescence microscopy.

Animals↗
PubMed2004

Invulnerability of retinal ganglion cells to NMDA excitotoxicity.

NMDA excitotoxicity has been proposed to mediate the death of retinal ganglion cells (RGCs) in glaucoma and ischemia. Here, we reexamine the effects of glutamate and NMDA on rat RGCs in vitro and in situ. We show that highly purified RGCs express NR1 and NR2 receptor subunits by Western blotting and immunostaining, and functional NMDA receptor channels by whole-cell patch-clamp recording. Nevertheless, high concentrations of glutamate or NMDA failed to induce the death of purified RGCs, even after prolonged exposure for 24 h. RGCs co-cultured together with ephrins, astrocytes, or mixed retinal cells were similarly invulnerable to glutamate and NMDA, though their NMDA currents were 4-fold larger. In contrast, even a short exposure to glutamate or NMDA induced the rapid and profound excitotoxic death of most hippocampal neurons in culture. To determine whether RGCs in an intact retina are vulnerable to excitotoxicity, we retrogradely labeled RGCs in vivo using fluorogold and exposed acutely isolated intact retinas to high concentrations of glutamate or NMDA. This produced a substantial and rapid loss of amacrine cells; however, RGCs were not affected. Nonetheless, RGCs expressed NMDA currents in situ that were larger than those reported for amacrine cells. Interestingly, the NMDA receptors expressed by RGCs were extrasynaptically localized both in vitro and in situ. These results indicate that RGCs in vitro and in situ are relatively invulnerable to glutamate and NMDA excitotoxicity compared to amacrine cells, and indicate that important, as yet unidentified, determinants downstream of NMDA receptors control vulnerability to excitotoxicity.

Amacrine Cells↗
PubMed2006

Enhanced survival and regeneration of axotomized retinal neurons by repeated delivery of cell-permeable C3-like Rho antagonists.

Inactivation of Rho GTPase with a single intraocular injection of Rho antagonists stimulates survival and regeneration of retinal ganglion cells (RGCs) after optic nerve injury. However, this effect is short-lived. Here we tested the impact of multiple injections of C3-like Rho antagonists on RGC viability and axon regeneration after optic nerve lesion. Our data show that both neuronal survival and axon regeneration were enhanced with repeated delivery of cell-permeable C3. We found an approximately 1.5-fold increase in RCG survival when additional Rho antagonist injections were performed after the first week from the time of lesion. In contrast, increased regeneration required early inactivation of Rho and injections performed in the second week did not further enhance regenerative outcome. These results reveal differences in the length of the therapeutic windows through which Rho inactivation acts on RGC survival or regeneration after axotomy.

ADP Ribose Transferases↗
PubMedDate not supplied

Upregulation of the phosphorylated form of CREB in spinothalamic tract cells following spinal cord injury: relation to central neuropathic pain.

Spinal cord injury (SCI) often leads to the generation of chronic intractable neuropathic pain. The mechanisms that lead to chronic central neuropathic pain (CNP) following SCI are not well understood, resulting in ineffective treatments for pain relief. Studies have demonstrated persistent hyperexcitability of dorsal horn neurons which may provide a substrate for CNP. We propose a number of similarities between CNP mechanisms and mechanisms that occur in long-term potentiation, in which hippocampal neurons are hyperexcitable. One biochemical similarity may be activation of the transcription factor, cyclic AMP response element-binding protein (CREB), via phosphorylation (pCREB). The current study was designed to examine whether tactile allodynia that develops in segments rostral to SCI (at-level pain) correlates with an increase in CREB phosphorylation in specific neurons known to be involved in allodynia, the spinothalamic tract (STT) cells. This study determined that, in animals experiencing at-level allodynia 35 days after SCI, pCREB was upregulated in the spinal cord segment rostral to the injury. In addition, pCREB was found to be upregulated specifically in STT cells in the rostral segment 35 days after SCI. These findings suggest one mechanism of maintained central neuropathic pain following SCI involves persistent upregulation of pCREB expression within STT cells.

Analysis of Variance↗
PubMed2006

Participation of TRPV1 and TRPV2 in the rat laryngeal sensory innervation.

Laryngeal sensory innervation is essential to the laryngeal defense system. We investigated the participation of TRPV1 and its homologue TRPV2 in the rat laryngeal sensory innervation using immunohistochemistry and the neuronal tracer, fluoro-gold (FG). After injection of FG into the internal branch of the superior laryngeal nerve, FG-labeled neurons were seen in the rostral part of the nodose ganglion (NG). Neurons immunoreactive for TRPV1 or TRPV2 were distributed throughout the NG. TRPV1 immunoreactivity was seen in 49.0+/-4.5% of the FG-labeled neurons, while TRPV2 immunoreactivity was seen in 12.5+/-4.1% of the FG-labeled neurons. These findings suggest that both TRPV1 and TRPV2 participate in laryngeal nociception, but that TRPV1 may have a particularly important role.

Animals↗
PubMed2006

Fluorogold labeling of descending brain neurons in larval lamprey does not cause cell death.

In our previous double-labeling studies, the fluorescent anatomical tracers Fluorogold (FG) and Texas red dextran amine (TRDA) were used to demonstrate that descending brain neurons, approximately 80% of which are reticulospinal (RS) neurons, in spinal cord-transected larval lamprey regenerate their axons. However, the numbers of FG-labeled descending brain neurons decreased significantly with increasing recovery times, from 2 to 16 weeks. For some FG-labeled mammalian neurons, FG appears to degrade and/or be lost over time, while in other neurons this tracer can kill neurons. In the present study, these possibilities were examined in larval lamprey for FG-labeled descending brain neurons. As in our previous studies, FG was applied to the spinal cord at 40% body length (BL, relative distance from the head) to retrogradely labeled descending brain neurons, and after recovery times of 2, 8, or 16 weeks, HRP, a non-toxic retrograde tracer, was applied to the spinal cord at 20% BL to determine if the numbers of HRP-labeled neurons were reduced. At these three recovery times, the numbers of HRP-labeled descending brain neurons were not significantly different than the numbers of HRP-labeled neurons in control animals that were not labeled with FG. Furthermore, the size and morphology of cell bodies and dendritic trees were not noticeably different in descending brain neurons with and without FG. Thus, in larval lamprey, FG does not appear to kill these neurons, but some FG probably is degraded and/or lost from neurons with increasing recovery times.

Animals↗
PubMed2006

Mesopontine cholinergic projections to the hypoglossal motor nucleus.

Mesopontine cholinergic (ACh) neurons have increased discharge during wakefulness, rapid eye movement (REM) sleep, or both. Hypoglossal (12) motoneurons, which play an important role in the control of upper airway patency, are postsynaptically excited by stimulation of nicotinic receptors, whereas muscarinic receptors presynaptically inhibit inputs to 12 motoneurons. These data suggest that ACh contributes to sleep/wake-related changes in the activity of 12 motoneurons by acting within the hypoglossal motor nucleus (Mo12), but the origins of ACh projections to Mo12 are not well established. We used retrograde tracers to assess the projections of ACh neurons of the mesopontine pedinculopontine tegmental (PPT) and laterodorsal tegmental (LDT) nuclei to the Mo12. In six Sprague-Dawley rats, Fluorogold or B subunit of cholera toxin, were pressure injected (5-20nl) into the Mo12. Retrogradely labeled neurons, identified as ACh using nitric oxide synthase (NOS) immunohistochemistry, were found bilaterally in discrete subregions of both PPT and LDT nuclei. Most retrogradely labeled PPT cells (96%) were located in the PPT pars compacta region adjacent to the ventrolateral tip of the superior cerebellar peduncle. In the LDT, retrogradely labeled neurons were located exclusively in its pars alpha region. Over twice as many ACh neurons projecting to the Mo12 were located in the PPT than LDT. The results demonstrate direct mesopontine ACh projections to the Mo12. These projections may contribute to the characteristic of wakefulness and REM sleep increases, as well as REM sleep-related decrements, of 12 motoneuronal activity.

Acetylcholine↗
PubMed2004

Vesicular acetylcholine transporter can be a morphological marker for the reinnervation to muscle of regenerating motor axons.

This study was designed to evaluate whether the vesicular acetylcholine transporter (VAChT), which packages acetylcholine into synaptic vesicles, can be used as a marker for regenerating motor axon terminal. We examined motor axon regeneration in the tongue after hypoglossal nerve axotomy, using an anterograde tracer biotin-dextran (BD), retrograde tracer Fluoro-Gold (FG), electron microscopic (EM) observation, and VAChT immunocytochemistry. BD study demonstrated that outgrowth of thin regenerating axons into the frontal area of the tongue was firstly observed at 14 post-operative days, and presynaptic formation of neuromuscular junction (NMJ) was observed from 21 post-operative days. Under electron microscopic observation, reconstruction of new NMJs was observed within the interval between 21 and 28 days. VAChT-immunoreactive nerve terminals disappeared by 3 days after axotomy, slightly appeared at 14 post-operative days, and thereafter gradually increased in number from 21 to 28 post-operative days. The re-expression of VAChT positive presynaptic terminal was almost the same as those obtained in BD, FG and EM studies. Regenerating axons tip in the crush model of the hypoglossal nerve exhibited prominent VAChT immunoreactivity in growing tip of regenerating axons. These indicate that VAChT is an excellent morphological indicator for regenerating nerve terminals of motor neurons.

Animals↗
PubMed2006

Heterogeneity of nitric oxide synthase-containing neurons in the mouse main olfactory bulb.

The distribution and structural features of nitric oxide [corrected] synthase (NOS) containing intrinsic neurons were studied in the mouse main olfactory bulb (MOB). NOS positive neurons were heterogeneous, including some subpopulations of periglomerular cells, granule cells, interneurons in the external plexiform layer, superficial and deep short-axon cells and stellate cells. NOS positive periglomerular cells were frequently calretinin immunoreactive and, although rarely, calbindin positive. Importantly, some middle and external tufted cells were also confirmed to be NOS positive, some of which were also cholecystokinin (CCK) positive. Retrograde tracer experiments showed that some NOS positive tufted cells, which were also CCK positive, constitute the intrabulbar association system and the projection system to the olfactory tubercle. In addition, another particular subpopulation of NOS positive neurons with no or little CCK immunoreactivity appeared to project to areas covering the dorsal endopiriform nucleus, claustrum and insular cortex. Furthermore, diverse types of neurons other than mitral/tufted cells were also suggested to be projection neurons of the MOB. The present study revealed the diversity of NOS positive neurons in the mouse MOB and further revealed that they were different from those reported previously in the rat MOB in structural and chemical properties.

Animals↗
PubMed2005

Lhx6 delineates a pathway mediating innate reproductive behaviors from the amygdala to the hypothalamus.

In mammals, innate reproductive and defensive behaviors are mediated by anatomically segregated connections between the amygdala and hypothalamus. This anatomic segregation poses the problem of how the brain integrates activity in these circuits when faced with conflicting stimuli eliciting such mutually exclusive behaviors. Using genetically encoded and conventional axonal tracers, we have found that the transcription factor Lhx6 delineates the reproductive branch of this pathway. Other Lhx proteins mark neurons in amygdalar nuclei implicated in defense. We have traced parallel projections from the posterior medial amygdala, activated by reproductive or defensive olfactory stimuli, respectively, to a point of convergence in the ventromedial hypothalamus. The opposite neurotransmitter phenotypes of these convergent projections suggest a "gate control" mechanism for the inhibition of reproductive behaviors by threatening stimuli. Our data therefore identify a potential neural substrate for integrating the influences of conflicting behavioral cues and a transcription factor family that may contribute to the development of this substrate.

Aggression↗