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PubMed2003

Preautonomic neurons in the paraventricular nucleus of the hypothalamus contain estrogen receptor beta.

Central actions of estrogen (E2) include, among others, modulation of autonomic and cardiovascular function. Despite the well-known influence of sex steroid hormones on the incidence of cardiovascular disorders, little is known about the neural substrates and receptors mediating central E2 actions on autonomic function. The paraventricular nucleus of the hypothalamus (PVN) is an important site for the integration of neuroendocrine and autonomic function. Interestingly, while this region was originally found to lack the classical ERalpha receptor, recent studies demonstrated a high degree of expression of the ERbeta subtype. To determine specifically whether autonomic-related neurons in the PVN express ERbeta, thus constituting a neuronal substrate for central E2 actions on autonomic function, we carried out an immunohistochemical study of ERbeta expression in a subpopulation of PVN neurons that innervate the rostroventrolateral medulla (RVLM). ERbeta immunostained neurons were found in medial and caudal aspects of the PVN, overlapping with the distribution of RVLM-projecting neurons. Overall, approximately 50% of RVLM-projecting PVN neurons expressed ERbeta immunoreactivity. Interestingly, the degree of colocalization was found to be sex-dependent (higher expression in males), and varied according to the topographical distribution of neurons within the PVN. ERbeta immunoreactivity was also observed in magnocellular compartments of the PVN, although this appeared to be consistently weaker than that observed in autonomic-related subnuclei. These studies demonstrate for the first time ERbeta expression in identified autonomic-related neurons in the PVN, and suggest that these neurons constitute an important neuronal substrate mediating E2 actions on autonomic and cardiovascular control.

Animals↗
PubMed2003

Bilaterally-projecting efferent neurones to the basilar papilla in the barn owl and the chicken.

The efferent innervation of the auditory basilar papilla of birds and mammals is provided by a dedicated population of brainstem neurones that are separate from those supplying the vestibular organs. This study addresses the question whether a population of bilaterally-projecting efferents, contacting hair cells in both basilar papillae, is consistently present in birds. The chicken and the barn owl were chosen, two species where the total number of efferents was already known and which represent two extremes of an auditory generalist and an auditory specialist, respectively. Fluorogold and Choleratoxin, two potent retrograde tracers, were injected into one cochlear duct each of all individuals. Labelled neurones were subsequently identified in the brainstem using standard fluorescence techniques. A small proportion (up to 2% of the total population) of double-labelled cells was found in both species. The great majority of those double-labelled neurones could be assigned to the ventrolateral group of efferents, which has previously been shown to project exclusively to the auditory basilar papilla. Thus, in birds, like in mammals, a small subgroup of auditory efferents innervates both basilar papillae.

Animals↗
PubMed2003

Cavernous nerve injury elicits GAP-43 mRNA expression but not regeneration of injured pelvic ganglion neurons.

Recovery of erectile dysfunction after cavernous nerve injury takes a long period. To elucidate this mechanism, unilateral cavernous nerve of male rat was cut, and the expression level of a nerve regeneration marker, the growth associated protein-43 (GAP-43) mRNA was evaluated by in situ hybridization and RT-PCR. While GAP-43 mRNA expression was transiently increased in the injured neurons of the major pelvic ganglion (MPG) at 7 days after nerve injury, continuous increase of GAP-43 mRNA was observed in the contralateral MPG from 7 days to 6 months after the nerve injury. Histochemical double-labeling studies for either neuronal NOS (nNOS) or tyrosine hydroxylase (TH) and the GAP-43 mRNA expression demonstrated that in injured MPG the transient up-regulation of GAP-43 mRNA was mainly seen in nNOS negative and/or TH positive neurons, suggesting non-parasympathetic post-ganglionic neurons, and also demonstrated that in contralateral MPG GAP-43 mRNA positive neurons were gradually increased in nNOS positive but TH negative neurons, suggesting parasympathetic post-ganglionic neurons. When a retrograde tracer Fluorogold (FG) was injected into the penile crus 7 days before histological experiments, FG-positive neurons were, if any, hardly seen in nNOS-positive neurons of the injured MPG for at least 6 months, whereas numerous FG-positive cells were seen in nNOS-positive neurons of the contralateral MPG. These results suggest that post-ganglionic projecting neurons of the intact side, which express increased GAP-43 mRNA, would be most likely to contribute to the recovery of the erectile function after unilateral cavernous nerve injury possibly by a plastic change such as nerve sprouting.

Animals↗
PubMed2003

Neuronal nitric oxide synthase is expressed in the axotomized ganglion cells of the rat retina.

This study investigated the expression and cellular localization of neuronal nitric oxide synthase in the rat retina following optic nerve transection (ONT). In the normal rat retina, nNOS immunoreactivity was localized to amacrine cells and displaced amacrine cells. A few bipolar cells were also labeled. In the axotomized retina, ganglion cells showed nNOS immunoreactivity from 3 days after ONT, and these cells increased in number, peaking 5 days after ONT. Quantitative evaluation using immunoblotting confirmed that nNOS expression showed a peak value (255% of control levels) 5 days after ONT and decreased to 137% of controls by 28 days. These findings suggest that axotomized ganglion cells degenerate via NO-mediated excitotoxicity.

Amacrine Cells↗
PubMed2003

P2X3-immunoreactive primary sensory neurons innervating lumbar intervertebral disc in rats.

The P2X(3) receptor is normally localized in a sub-population of small-diameter dorsal root ganglion (DRG) neurons, and is thought to be related to pain perception. The aim of this study in rats was to examine P2X(3)-immunoreactivity in DRG neurons innervating the lumbar disc and in DRG neurons innervating cutaneous tissues. Fluoro-Gold was applied to the L5-L6 disc, the plantar skin of the hind paw (L4-L5 dermatomes), and the back skin (L1-L2 dermatomes). It has been reported that the L5-L6 disc is innervated by T13-L5 DRG neurons. We performed immunostaining using antibodies against the P2X(3) receptor of T13-L5 DRGs to examine the L5-L6 disc, L4 and L5 DRGs to examine plantar skin and L1 and L2 DRGs to examine back skin. The P2X(3)-immunoreactivity was detected in 22.0 and 22.8% of neurons, labeled by Fluoro-Gold applied to plantar and back skin, respectively. However, P2X(3)-immunoreactivity was detected in only 4.0% of the neurons projecting to the L5-L6 disc. The proportion of P2X(3)-immunoreactive neurons was significantly larger in the DRG neurons innervating the plantar or the back skin, than in the DRG neurons innervating the lumbar disc. These results suggest that the P2X(3) receptors are abundant in DRG neurons innervating cutaneous tissues, but not in neurons innervating the lumbar disc. It is likely therefore that the P2X(3) receptor is less related to the mechanism of discogenic pain, than to cutaneous tissue pain.

Animals↗
PubMed1996

Distribution of origin of nitric oxide synthase-immunoreactive nerve fibers in the rat epididymis.

Distribution of neuronal nitric oxide synthase-immunoreactive (nNOS-IR) nerve fibers and somata in the rat epididymis and major pelvic ganglia was studied by immunohistochemical methods. In the epididymis, the supply of nNOS-IR fibers was highest in the cauda and became progressively fewer toward the caput. In the cauda and corpus, nNOS-IR fibers were distributed throughout the subepithelial tissues and around the epithelial. The pattern of distribution of vasoactive intestinal polypeptide (VIP)- and tyrosine hydroxylase (TH)-immunoreactive fibers in the epididymis was similar but the latter was generally more numerous in a given region as compared to that of nNOS-IR fibers. A population of neurons in the major pelvic ganglia were nNOS-IR-, TH- or VIP-IR. Double-labeling studies revealed that few neurons in the major pelvic ganglia contained both nNOS-IR and TH-IR. Whereas nNOS-IR and VIP-IR appeared to co-localize in the same population of the pelvic ganglion cells. Similarly, nNOS-IR fibers in the epididymis were mostly VIP-positive and TH-negative. Unilateral injection of the fluorescent tracer Fluorogold into the junction between the vas deferens and the cauda labeled a population of neurons in the right and left major pelvic ganglia, some of which were also nNOS-IR. A small number of dorsal root ganglion cells contained Fluorogold and very few expressed NOS-IR. It may be concluded that nNOS-IR nerve fibers in the rat epididymis arise mainly from neurons in the major pelvic ganglia the major of which express VIP-IR but not TH-IR. The extensive supply of nNOS-immunoreactive fibers around the epithelium and throughout the subepithelial tissues suggests that NO may be closely associated with smooth muscle contraction.

Animals↗
PubMed1997

Anterograde tracing of retinohypothalamic afferents with Fluoro-Gold.

The anterograde neuronal tracing properties of Fluoro-Gold (FG) were characterized in this study by its ability to label the retinohypothalamic tract (RHT) upon pressure injection of the substance into the vitrous body of the eye in the Djungarian hamster, Phodopus sungorus. Tracing was compared to the anterograde neuronal transport of cholera toxin B subunit (CTB), Fast blue (FB), Phaseolous vulgaris leucoagglutinin (PHA-L) and biocytin. After survival times that ranged from 24 h to 4 weeks, a major projection was found to the bilateral hypothalamic suprachiasmatic nuclei (SCN). Labeling was also found in the anterior medial preoptic nucleus and, in relatively sparse amounts, in the lateral geniculate nucleus, superior colliculus and lateral habenular nucleus. Similar results were obtained upon injection of CTB or FB, respectively, into the eye, whereas the application of PHA-L or biocytin did not label retinal afferents. The combined injection of FG and CTB or FB into the same eye labeled retino-afferent fibers only when FG was applied three days before the injection of the other tracers. Retrogradely labelled neurons were sen occasionally in the hypothalamus which may provide a sparse retinopetal projection. Additional experiments combining FG tracing and the immunofluorescent detection of the neuropeptides substance P (SP) or vasoactive intestinal polypeptide (VIP) in the SCN showed that FG-containing punctae were accumulated in the vicinity of immunoreactive cell bodies. Our data demonstrate that FG may be used as an anterograde axonal tracer of the retinohypothalamic pathway.

Amidines↗
PubMed1997

Evidence for a dopaminergic innervation of the subthalamic nucleus in the rat.

The dopaminergic connection from the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA) to the subthalamic nucleus in the rat was investigated using anterograde and retrograde tracers. Iontophoretic injection of the retrograde tracer fluoro-gold (FG) into the subthalamic nucleus resulted in a substantial number of labeled neurons in the SNc. Immunohistochemistry of tyrosine hydroxylase (TH) confirmed the dopaminergic nature of these labeled neurons. Retrogradely labeled neurons were also found in the VTA. Injection of the anterograde tracer biocytin into the SNc produced biocytin-labeled terminals in the subthalamic nucleus hence providing clear evidence for a dopaminergic innervation of this nucleus. Quantitative analysis of labeled axons revealed that there were 15-38 terminal branches per axon, each branch being 50-150 microm long. The overall dimensions of one terminal arborization were 400 x 250 x 150 microm. There was no clear-cut topographical organization of the projection, but a slight mediolateral difference in the density of terminals. This direct dopaminergic projection is thought to interact with cortical and pallidal inputs in the subthalamic nucleus, which implies that the functions of the subthalamic nucleus are more complex than previously assumed.

Animals↗
PubMed1997

Parabrachial nucleus neurons providing axons to both the thalamus and the spinal cord in the rat.

After injecting Fluoro-gold (FG) and tetramethylrhodamine-dextran amine (TMR-DA), respectively, into the medial part of the ventrobasal thalamus and the upper segments of the cervical cord of the rat, a small number of neuronal cell bodies were double-labeled retrogradely with both FG and TMR-DA in the parabrachial nuclear complex (BPN) ipsilateral to the injection into the thalamus. The cell bodies double-labeled with FG/TMR-DA were seen mainly in the Kölliker-Fuse subnucleus and additionally in the external medial subnucleus.

Animals↗
PubMed1997

Cholinergic innervation of the song control nuclei by the ventral paleostriatum in the zebra finch: a double-labeling study with retrograde fluorescent tracers and choline acetyltransferase immunohistochemistry.

During the sensitive period of song learning, the content of acetylcholine and the enzyme activity of choline acetyltransferase (ChAT) increase remarkably in the song control nuclei of a young male zebra finch. Cholinergic fibers innervate the two main song control nuclei of the forebrain: the higher vocal center (HVC) and the robust nucleus of the archistriatum (RA). The present study combines the retrograde tracer, Fluoro-Red (FRe), with ChAT immunohistochemistry. The results indicate that the cholinergic fibers which innervate the RA and HVC originate from the ventral paleostriatum (VP) in the basal forebrain, and that there is an anterior-posterior topography in the location of the cholinergic neurons in the VP that project to the HVC and RA, although there are a few neurons which project to both nuclei. These findings suggest that the VP is homologous to the nucleus basalis of Meynert of the basal forebrain cholinergic system of mammals which is associated with learning and memory processes, and that the cholinergic neurons in the VP play an important role in avian song learning.

Animals↗
PubMed1997

Identification of retinal ganglion cells projecting to the lateral hypothalamic area of the rat.

The objective of the present study was to identify the retinal ganglion cells projecting to the lateral hypothalamic area of the rat. The retinohypothalamic tract has been divided into a medial and a lateral component on anatomical and developmental grounds. The medial component projects to the suprachiasmatic nucleus and adjacent structures such as the anterior hypothalamic and retrochiasmatic areas. The lateral component terminates in the lateral hypothalamic are dorsal to the supraoptic nucleus. Injections of the retrograde tracer FluoroGold were made into the retinorecipient region of the lateral hypothalamic area and retinal whole mounts were immunohistochemically processed for retrogradely labeled retinal ganglion cells. With FluoroGold injections confined to the lateral hypothalamic area, retrogradely labeled retinal ganglion cells are located almost exclusively in the superior temporal quadrant of the retina. Their size and morphology indicates that they are a homogeneous subset of type III cells, but a definitive classification would require a more complete fill of dendritic arbors than is available in our retrograde material. In contrast, injections involving fibers of passage in the optic tract, or centered in the medial terminal nucleus of the accessory optic system, label cells distributed across the entire retinal surface. Unlike the retinal ganglion cells projecting to the suprachiasmatic nucleus [Moore et al., J. Comp. Neurol., 352 (1995) 351-366], the cells labeled after restricted lateral hypothalamic injections are not distributed evenly across the retinal surface. The difference in location of the retinal ganglion cells projecting to the lateral hypothalamic area supports the view that this retinohypothalamic projection is anatomically and functionally distinct from the projection to the suprachiasmatic nucleus and adjacent medial hypothalamus.

Animals↗
PubMed1997

Reconstruction of the striato-nigro-striatal circuitry by simultaneous double dopaminergic grafts: a tracer study using fluorogold and horseradish peroxidase.

The main strategy in neural transplantation for Parkinson's disease (PD) has been the ectopic placement of dopaminergic grafts in the striatum in order to restore dopaminergic innervation to the host striatum. Although intrastriatal transplants usually improve asymmetric rotational behavior in the 6-hydroxydopamine lesioned rodent model of PD, they are less likely to completely restore the more complex sensorimotor behavioral deficits induced by dopamine loss. Re-establishment of the nigrostriatal circuitry and dopaminergic reinnervation of the substantia nigra may be necessary to promote a more complete restoration of function in the dopamine depleted brain and improve the clinical efficacy of dopaminergic transplants. Recently, we demonstrated the reconstruction of the nigrostriatal pathway by simultaneous intrastriatal and intranigral dopaminergic transplants [Mendez et al., J. Neurosci. 16 (1996) 7216-7227.]. Using this strategy, it was found that placing a graft of embryonic ventral mesencephalic tissue in the striatum promoted the growth and guidance of axons from a similar graft placed homotopically in the ventral mesencephalon. Since it is apparent that developing tissue has the ability to promote axonal growth and guidance along the nigrostriatal pathway, the double grafting strategy may contribute to re-establishing host-graft connectivity. The current study provides evidence of reconstruction of the striato-nigro-striatal loop circuitry by simultaneous intrastriatal and intranigral dopaminergic transplants. Injection of the retrograde tracer fluorogold (FG) into the striatum resulted in fluorescent labeled cells within the intranigral grafts. Similarly injection of FG into the nigra resulted in fluorescent labeled cells within the intrastriatal graft and surrounding striatum. Injection of the anterograde tracer horseradish peroxidase (HRP) resulted in the presence of HRP reaction product throughout the target striatum. These results strongly support the re-establishment of nigrostriatal and striatonigral connections between simultaneous intrastriatal and intranigral dopaminergic transplants and suggest reconstruction of the striato-nigro-striatal loop circuitry.

Animals↗
PubMed1998

Localization of mGluR1a-like immunoreactivity and mGluR5-like immunoreactivity in identified populations of striatal neurons.

Metabotropic glutamate receptors are important mediators of excitatory amino acid neurotransmission in the striatum. Two-color immunofluorescence histochemistry and immunohistochemistry in combination with retrograde tract-tracing techniques were used to examine the distribution of metabotropic glutamate receptor subtypes 1a and 5 (mGluR1a and mGluR5) among identified subpopulations of striatal projection neurons and interneurons. The majority of striatopallidal and striatonigral neurons were double-labeled for both mGluR1a or mGluR5. Approximately 60% to 70% of either striatonigral or striatopallidal neurons expressed mGluR1a- or mGluR5-like immunoreactivity. The percentage of double-labeled striatopallidal or striatonigral projection neurons did not differ among striatal quadrants. Striatal interneurons expressing parvalbumin or somatostatin or choline acetyltransferase exhibited varying degrees of expression of mGluR1a or mGluR5. Virtually all (94%) parvalbumin-immunoreactive striatal neurons expressed mGluR1a-like immunoreactivity with a majority (79%) of these neurons expressing mGluR5-like immunoreactivity. A high percentage (89%) of striatal choline acetyltransferase-immunoreactive neurons were double-labeled for mGluR1a-like immunoreactivity. Approximately 65% of striatal choline acetyltransferase-immunoreactive neurons expressed mGluR5-like immunoreactivity. A majority (65%) of somatostatin-immunoreactive striatal interneurons expressed mGluR1a-like immunoreactivity with a slightly lower percentage (55%) expressing mGluR5-like immunoreactivity. These findings indicate considerable heterogeneity among striatal projection and interneurons with respect to mGluR1a and mGluR5 expression. There may be subpopulations of striatonigral and striatopallidal projection neurons. These results are consistent as well with prior data indicating subpopulations of the different classes of striatal interneurons.

Animals↗
PubMed1998

Neurochemical organization of paratrigeminal nucleus projections to the dorsal vagal complex in the rat.

The paratrigeminal nucleus, located in the spinal trigeminal tract rostral to the obex, is important in the integration of visceral and somatosensory afferent information and may modulate autonomic function through its projections to the dorsal vagal complex. Anterograde and retrograde neuroanatomical tracers were used in conjunction with immunohistochemistry to determine the neurochemical organization of the efferent pathway from the paratrigeminal nucleus to the dorsal vagal complex in the rat. Double-labelling studies demonstrated that leu-enkephalin, 28-kDa calbindin, and neuronal nitric oxide synthase were present in neurons in the paratrigeminal nucleus that project to the dorsal vagal complex. The results of this study are consistent with the hypothesis that neurochemically distinct pathways from the paratrigeminal nucleus are involved in the sensory modulation of autonomic function.

Animals↗
PubMed1998

Thalamic paraventricular nucleus neurons collateralize to innervate the prefrontal cortex and nucleus accumbens.

The prefrontal cortex and nucleus accumbens are primary recipients of medial thalamic inputs, prominently including projections from the thalamic paraventricular nucleus. It is not known if paraventricular neurons collateralize to innervate both the prefrontal cortex and nucleus accumbens. We used dual retrograde tract tracing methods to examine this question. A small population of paraventricular neurons was found to innervate the prefrontal cortex and medial nucleus accumbens. These data suggest that the thalamic paraventricular nucleus may coordinately influence activity in the prefrontal cortex and ventral striatum.

Amidines↗
PubMed1998

The retinal ganglion cells that drive the pupilloconstrictor response in rats.

It is well established that the pupillary light reflex (PLR) in rats is mediated by a direct retinal projection to the olivary pretectal nucleus (OPN). Although several authors have commented on the specific subpopulation of retinal ganglion cells (RGC) that project to the rat pretectum, much of this evidence is circumstantial, and depends mostly upon electrophysiological data (e.g., conduction velocity). Here, we have used microinjections of Fluoro-Gold into the OPN (pretectum and superior colliculus as controls) to retrogradely label RGCs projecting to this region. The retinae were whole-mounted, viewed under fluorescence, and the regional distribution pattern, laterality of projection, and cell soma sizes determined. The results show OPN injections label a small subpopulation of RGCs. In the contralateral retinae, labeled RGCs were most numerous and widespread, with 97% projecting to the contralateral pretectum. The highest density of cells in the contralateral retinae was found in the inferior and nasal retinal quadrants. In the ipsilateral retinae, the small number of labeled cells were concentrated in the periphery of the inferior and nasal retinal quadrants. A striking feature of both ipsilateral and contralateral retinae was the paucity of labeled cells found in the dorsal hemiretina (lower visual field). Cell size measurements indicate 90-95% of labeled RGCs had diameters of 9-13 microm, while most of the remaining cells had diameters of 20-25 microm. This would suggest class III cells may be the predominant RGC type mediating pupilloconstriction, although a smaller population of larger cells (e.g., class I and/or II) may also contribute to this pathway. The recent reports utilizing the PLR as an assay for the efficacy of intraretinal grafts has highlighted the significance of the regional distribution results. The extremely low number of labeled cells in the dorsal hemiretina would argue for the placement of such grafts in the ventral hemiretina.

Animals↗
PubMed1998

MK801-a neuroprotectant in rat hypertensive eyes.

Excitatory synaptic transmission in the mammalian CNS and retina is mainly mediated through l-glutamate. The effect of MK-801, a non-competitive antagonist of the NMDA subtype of glutamate receptor was studied on rat retinal ganglion cells in hypertensive eyes. MK-801 was administered intraperitoneally to the first group, 1 day before the increase, and in the second group, 2 days after the intraocular pressure (IOP) elevation. Phosphate-buffered saline was administered to the control group. Animals were sacrificed 2 and 4 weeks post-IOP increase. The retinal ganglion cells were counted and compared between control (right) and experimental (left) eyes. The data presented here suggests that MK-801 has neuroprotective properties.

Animals↗
PubMed1998

Identification of somatostatin sst2(a) receptor expressing neurones in central regions involved in nociception.

Somatostatin is a neuromodulator and neurotransmitter in the central nervous system. Administration of somatostatin to the spinal cord or brain areas involved in nociception has been shown to result in analgesia. Little information is available about the somatostatin receptor types which may be involved in mediating the neuromodulatory and analgesic effects of the peptide. To define the neuronal systems expressing the sst2(a) receptor in brain areas associated with analgesia, immunohistochemical co-localisation studies were carried out in the periaqueductal grey (PAG) and spinal cord using an antibody specific for the sst2(a) receptor. To further define sst2(a) receptor expressing neurones, sst2(a) receptor immunohistochemistry was combined with retrograde tracing using fluorogold. In the PAG, sst2(a) receptor expressing neurones were found to co-express calbindin D28k (36%), the glutamate transporter EAAC-1 (25%), and GABA transporter GAT-1 ( approximately 10%). A total of 65% of sst2(a) positive neurones projected to the thalamus. In the spinal cord, the sst2(a) receptor shows cellular co-localisation with EAAC-1 and GAT-1. Immunohistochemistry and receptor autoradiography using [125I]BIM 23027 after dorsal rhizotomy of the lumbar dorsal roots, L4 and L5, suggests that the somatostatin sst2(a) receptor is not present on primary afferent neurones. Dorsal hemisections of the mid thoracic cord did not alter the immunohistochemical signal for the somatostatin sst2(a) receptor, providing further evidence for an intrinsic localisation of the receptor protein in the dorsal horn of the spinal cord. These data show that the somatostatin sst2(a) receptor exists on morphologically and neurochemically heterogenous neurones and is closely associated with brain areas involved in analgesia and the modulation of nociception.

Animals↗