Melatonin attenuates neuronal NADPH-d/NOS expression in the hypoglossal nucleus of adult rats following peripheral nerve injury

Oxidative stress and massive production of nitric oxide (NO) have been implicated in the neuropathogenesis following peripheral nerve injury. This study was aimed to ascertain whether melatonin would exert its neuroprotective effect on the lesioned hypoglossal neurons after peripheral axotomy, since it is known to reduce the oxidative damage in a variety of experimental neuropathologies in which NO is involved. Right-sided hypoglossal nerve transection was performed in adult rats following which the animals were given two different doses of melatonin administered intraperitoneally for 3, 7, 14, 21 and 30 successive days. Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry were carried out to detect the neuronal NADPH-d/NOS expression in the hypoglossal nucleus (HN). At various time intervals following axotomy, the neurons in the affected HN were induced to express NADPH-d/NOS reactivity on the lesioned side peaking at 14 days. However, the enzyme expression was markedly depressed by melatonin treatment in a dose-dependent manner in terms of frequency of labelled neurons and staining intensity. It is suggested that the suppressive effect of melatonin on NADPH-d/NOS expression may be attributed to its antioxidant properties. Hence, in consideration of therapeutic strategies for reducing the oxidative stress following peripheral nerve injury, melatonin may prove to be beneficial.     

Distinct roles of oxidative stress and antioxidants in the nucleus dorsalis and red nucleus following spinal cord hemisection

Oxidative stress plays an important role in the pathogenesis of neurodegeneration after the acute central nervous system injury. We reported previously that increased nitric oxide (NO) production following spinal cord hemisection tends to lead to neurodegeneration in neurons of the nucleus dorsalis (ND) that normally lacks expression of neuronal NO synthase (nNOS) in opposition to those in the red nucleus (RN) that constitutively expresses nNOS. We wondered whether oxidative stress could be a mechanism underlying this NO involved neurodegeneration. In the present study, we examined oxidative damage evaluated by the presence of 4-hydroxynonenal (HNE) and iron accumulation and expression of putative antioxidant enzymes heme oxygenase-1 (HO-1) and superoxide dismutase (SOD) in neurons of the ND and RN after spinal cord hemisection. We found that HNE expression was induced in neurons of the ipsilateral ND from 1 to 14 days following spinal cord hemisection. Concomitantly, iron staining was seen from 7 to 14 days after lesion. HO-1, however, was only transiently induced in ipsilateral ND neurons between 3 and 7 days after lesion. In contrast to the ND neurons, HNE was undetectable and iron level was unaltered in the RN neurons after spinal cord hemisection. HO-1, SOD-Cu/Zn and SOD-Mn were constitutively expressed in RN neurons, and lesion to the spinal cord did not change their expression. These results suggest that oxidative stress is involved in the degeneration of the lesioned ND neurons; whereas constitutive antioxidant enzymes may protect the RN neurons from oxidative damage.     

Colocalization of ionotropic glutamate receptor subunits with NADPH-diaphorase-containing neurons in the rat mesopontine tegmentum

Tegmental cholinergic neurons vary their discharge patterns across the sleep-wake cycle, and glutamate is suggested to play an important role in determining these firing patterns. Cholinergic and noncholinergic neurons in the mesopontine tegmentum have different susceptibilities to various excitotoxins, presumably because of heterogeneity in the expression of glutamate receptor subtypes in this area. By using a double-labeling procedure that combines nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) histochemistry and avidin-biotin-peroxidase immunocytochemistry with diaminobenzidine as the chromogen, we compared the colocalization of AMPA receptor subunits GluR1, GluR2/3, and GluR4, kainate receptor subunits GluR5/6/7, and an NMDA receptor subunit NMDAR1 on NADPH-diaphorase-positive (cholinergic) neurons in the mesopontine tegmentum. Throughout the brainstem, neurons immunoreactive for GluR2/3 and NMDAR1 were most numerous, whereas neurons labeled for GluR1, GluR4, and GluR5/6/7 were less common. Specifically within the mesopontine tegmentum, the proportion of double-labeled neurons in the diaphorase-containing cell population was highest with GluR1 (43%) and lowest with GluR5/6/7 (12%). Regardless of the receptor subunit type, the greatest numbers of double-labeled neurons were observed in the pedunculopontine tegmental nucleus pars compacta and the fewest in the dorsal aspect of the laterodorsal tegmental nucleus. In addition, there were regional differences in the relative expression of receptor subunits and diaphorase-positive neurons across the subdivisions of the tegmental cholinergic column. Because each ionotropic subunit confers distinctive properties to a receptor channel, the present results suggest that mesopontine cholinergic neurons have nonuniform responses to glutamate and are also discriminable from basal forebrain cholinergic neurons in terms of glutamate receptor configuration. © 1996 Wiley-Liss, Inc.     

Cellular expression of ionotropic glutamate receptor subunits in subpopulations of neurons in the rat substantia nigra pars reticulata

In order to characterize the expression of ionotropic glutamate receptor immunoreactivity in subpopulations of neurons in the rat substantia nigra pars reticulata (SNr), double labeling experiments were performed. Neurons in the reticulata were found to display GluR1, GluR2, GluR2/3, GluR4, N-methyl-D-aspartate receptor 1 (NMDAR1) and NMDAR2B immunoreactivity. Some of the reticulata neurons were shown to display GluR1 and GluR2 immunoreactivity or GluR2 and GluR4 immunoreactivity at the single cell level. In addition, subpopulations of reticulata neurons were characterized on the basis of the strong expression of parvalbumin (PV) and GABA transaminase immunoreactivity. All of the reticulata neurons that displayed strong immunoreactivity for PV or GABA transaminase also displayed immunoreactivity for GluR1, GluR2/3, GluR4, NMDAR1 and NMDAR2B. A tiny portion (around 15%) of reticulata neurons that display NMDAR1 immunoreactivity was found to be PV- or GABA-transaminase-negative. The present results indicate that native alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)-type receptors and NMDA-type receptors in the rat substantia nigra are composed of heteromeric receptor subunits. The present findings further demonstrate that most of the AMPA-type and NMDA-type glutamate receptor subunits are primarily expressed by subpopulations of neurons in the rat SNr.     

Distinct distribution and time-course changes in neuronal nitric oxide synthase and inducible NOS in the paraventricular nucleus following lipopolysaccharide injection

Nitric oxide (NO) is known to be involved in the modulation of neuroendocrine function. To clarify the role of different isoforms of NO synthase (NOS) in the neuroendocrine response to immune challenge, the expressions of neuronal NOS (nNOS) and inducible NOS (iNOS) genes in the hypothalamus following lipopolysaccharide (LPS) injection were examined using in situ hybridization. NOS activity was also determined by NADPH-diaphorase (NADPH-d) histochemistry. LPS (25 mg/kg) or sterile saline was injected intraperitoneally to male Wistar rats and the rats sacrificed 30 min, or 1, 2, 3, 5, 12 or 24 h after injection. nNOS mRNA expression in the paraventricular nucleus (PVN) was significantly increased 2 h after LPS injection. iNOS mRNA, which was not detected until 2 h after LPS injection, was significantly increased in the PVN 3 h after LPS injection. Both RNA expressions had returned to basal levels by 12 h after LPS injection. The number of NADPH-d positive cells was significantly increased 5 h after LPS injection. iNOS expression was more robust in parvocellular PVN, while nNOS was distributed mainly in the magnocellular PVN. Double in situ hybridization histochemistry revealed that some of the iNOS- (48.4%) or nNOS-positive cells (34. 3%) in the parvocellular PVN expressed CRF mRNA. The results demonstrate that LPS-induced sepsis causes significant increases in nNOS and iNOS gene expression with different time-courses and distributions, and that iNOS mRNA was more frequently co-localized with CRF-producing parvocellular neurons in the PVN. Thus, NO produced by iNOS and nNOS may play an important role in the neuroendocrine response to an immune challenge. Distinct differences in the distribution and time-course changes of iNOS and nNOS suggest different roles for the hypothalamic-pituitary-adrenal axis and/or neurohypophyseal system.     

In situ measurement of neuronal nitric oxide synthase activity in the spinal cord by NADPH-diaphorase histochemistry

NADPH-diaphorase (NADPH-d) histochemistry has provided a simple method to stain neuronal nitric oxide synthase (nNOS)-containing neurons in the central nervous system. In the spinal cord, NO formation following activation of N-methyl-D-asparate (NMDA) receptors plays a crucial role in nociceptive processing. To investigate the molecular mechanisms, we attempted to evaluate nNOS activity in situ using isolated intact spinal cord preparation and NADPH-d histochemistry. NADPH-d activity in the superficial layer of the spinal cord increased gradually with ages from P10 to P30 and NMDA enhanced the NADPH-d staining in a time- and concentration-dependent manner. The NMDA-stimulated NADPH-d staining was inhibited by NMDA receptor antagonists, but not by non-NMDA and metabotropic glutamate receptor antagonists. The NADPH-d staining showed a pronounced stereospecificity for beta-NADPH and completely suppressed by dichlorophenolindophenol, an artificial electron acceptor. NMDA-evoked NO formation in the spinal cord was confirmed by the fluorescent NO indicator diaminofluorescein-FM (DAF-FM). These results demonstrate that NADPH-d activity in the superficial spinal cord is ascribed to nNOS activity and is dependent on NMDA. A combination of isolated intact spinal cord preparations and NADPH-d histochemistry may provide a unique system to elucidate biochemical and molecular mechanisms for nNOS activation in the spinal cord.     

Local injection of kainic acid causes widespread degeneration of NADPH-d neurons and induction of NADPH-d in neurons, endothelial cells and reactive astrocytes

Nitric oxide (NO), a diffusable gas, is a messenger molecule that mediates vascular dilatation and neural transmission. The enzyme nitric oxide synthase (NOS) present in neurons is activated by Ca²⁺ influx associated with activation of glutamate receptors. Cultured cortical neurons containing NOS are selectively vulnerable to injury by kainic acid (KA). However, the relationship between NOS neurons and excitotoxicity under in vivo condition is not entirely clear. In the present study, we examined the time course and spatial distribution of changes in NOS neurons caused by an intracortical microinjection of KA in adult rats. NADPH-diaphorase (NADPH-d) histochemistry was used as a marker for NOS and the neuronal changes were correlated with changes in glial cells and endothelial cells. We demonstrated a rapid loss of NADPH-d neurons in the lesion center and degeneration of NADPH-d neurons and nerve terminals throughout ipsilateral cortex and hippocampus; the striatal neurons appeared to be unaffected. Subsequent to cortical neuronal degeneration, new NADPH-d activity appeared in proliferative reactive astrocytes and in endothelial cells at lesion periphery, and in neuronal groups at lesion periphery, in ipsilateral entorhinal cortex and bilateral hippocampus. These findings indicate that neurons expressing NADPH-d in cerebral cortex and hippocampus are selectively vulnerable to KA toxicity in vivo. The subsequent induction of NOS in neural and non-neural cells may be regarded as an adaptive response to the kainate-induced brain lesion.     

Distribution of N-methyl-D-aspartate and non-N-methyl-D-aspartate glutamate receptor subunits on respiratory motor and premotor neurons in the rat

Glutamate is required for the transmission of inspiratory drive in respiratory premotor and motor neurons. The glutamate receptors (GluRs) responsible for this essential function have yet to be anatomically characterized. We mapped the GluR subtypes expressed by respiratory premotor and motor neurons by using combined immunohistochemistry and retrograde labeling in adult rats. Phrenic motoneurons and bulbospinal ventral respiratory group (VRG) neurons were retrogradely labeled and immunolabeled with subunit-specific antibodies against the N-methyl-D-aspartate (NMDA) receptor subtype (NMDAR1) and the non-NMDA receptor subtypes, α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA; GluR1, GluR2/3, GluR4) and kainate (GluR5–7). Phrenic motoneurons and bulbospinal VRG neurons showed positive immunolabeling for all five GluR subunits. These results support the hypothesis that NMDA and non-NMDA receptor subtypes underlie the excitation of bulbospinal VRG neurons and phrenic motoneurons. Furthermore, immunolabeling for each receptor subtype demonstrated a unique distribution along the neuronal membrane. Immunoreactivity for AMPA receptor subunits was distributed throughout somata and proximal dendrites, NMDAR1 subunit immunolabeling was localized to somata, and GluR5–7 subunit immunolabeling was confined largely to dendrites. The differential distribution of AMPA, kainate, and NMDA receptors on the somal and dendritic surface of respiratory neurons suggests that the location of glutamatergic synapses along the neuronal surface is an important determinant of glutamate-mediated postsynaptic currents. Consequently, different patterns of glutamatergic excitation of respiratory neurons could be achieved by selective activation of different profiles of GluR subtypes on different portions of the neuronal membrane. J. Comp. Neurol. 389:94–116, 1997. © 1997 Wiley-Liss, Inc.     

Either nitric oxide or nerve growth factor is required for dorsal root ganglion neurons to survive during embryonic and neonatal development

During early embryonic (E12) development almost all dorsal root ganglion (DRG) neurons express the neuronal isoform of nitric oxide synthase (nNOS). At this stage, the axons of these neurons are rudimentary and have not made contact with peripheral tissue targets. As their axons establish contact with peripheral targets such as the skin, the number of neurons expressing nNOS decrease that correspond to increased immunoreactivity for nerve growth factor (NGF) in the skin, and its high affinity receptor, tyrosine kinase A (trkA) in both skin and DRG neurons. During late postnatal development, very few DRG neurons express nNOS; however, axotomy or NGF deprivation of cultured DRG neurons induce nNOS and NOS blockade causes neuronal death. In contrast, NGF-deprived embryonic and neonatal DRG neurons die by apoptosis, while NOS blockade has no effect. Overall, these observations suggest that NGF and nitric oxide (NO) interact during embryonic and postnatal development to facilitate neuronal selection and survival. The roles of NO, NGF and its receptor trkA in DRG neurons during different stages of development are discussed.     

Responses of nitric oxide synthase expression in the gracile nucleus to sciatic nerve injury in young and aged rats

Neuronal nitric oxide synthase (nNOS) is induced in dorsal root ganglion neurons following axotomy in young rats, and is also increased in the gracile nucleus neurons of intact aged rats. The present study examined the influence of sciatic nerve axotomy on nNOS expression in the gracile nucleus in young compared to aged rats. The unilateral transection of the sciatic nerve was performed in young (4 months) and old (24 months) Fischer rats. Sections of rat medulla obtained 14 days after axotomy were immunolabelled using a polyclonal antibody directed against nNOS and stained by nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry, a marker of nNOS activity. In young rats, unilateral axotomy produced increased NADPHd containing neurons in the rostral region and the caudal region of the ipsilateral gracile nucleus compared to the side with intact sciatic nerve. In old rats, the NADPHd containing neurons in the ipsilateral gracile nucleus were moderately increased by axotomy over the age changes seen in the contralateral side. Similar results were obtained with nNOS immunoreactivity in young rats, but more cells were seen with NADPHd staining compared to nNOS immunostaining in old rats. The results suggest that unilateral sciatic axotomy causes an increase in nNOS expression in the ipsilateral gracile nucleus of young rats, which is still seen in old rats as an increase over normal aging changes.     

Role of nitric oxide in pentylenetetrazol-induced seizures: age-dependent effects in the immature rat

PURPOSE: Seizure susceptibility and consequences are highly age dependent. To understand the pathophysiologic mechanisms involved in seizures and their consequences during development, we investigated the role of nitric oxide (NO) in severe pentylenetetrazol (PTZ)-induced seizures in immature rats. METHODS: Four cortical electrodes were implanted in 10-day-old (P10) and 21-day-old (P21) rats, and seizures were induced on the following day by repetitive injections of subconvulsive doses of PTZ. The effects of NG-nitro-l-arginine methyl ester (l-NAME; 10 mg/kg) and 7-nitroindazole (7NI; 40 mg/kg), two NO synthase (NOS) inhibitors, and l-arginine (l-arg; 300 mg/kg), the NOS substrate, were evaluated regarding the mean PTZ dose, seizure type and duration, and mortality rate. RESULTS: At P10, the postseizure mortality rate increased from 18-29% for the rats receiving PTZ only to 100% and 89% for the rats receiving l-NAME and 7NI, respectively; whereas l-arg had no effect. Conversely, at P21, NOS inhibitors did not affect the 82-89% mortality rate induced by PTZ alone, whereas l-arg decreased the mortality rate to 29%. In addition, all NO-related drugs increased the duration of ictal activity at P10, whereas at P21, l-arg and l-NAME affected the first seizure type, producing clonic seizures with l-arg and tonic seizures with l-NAME. CONCLUSIONS: The relative natural protection of very immature rats (P10) against PTZ-induced deaths could be linked to a high availability of l-arg and, hence, endogenous NO. At P21, the modulation of seizure type by NO-related compounds may be related to the maturation of the brain circuitry, in particular the forebrain, which is involved in the expression of clonic seizures.     

Age-related changes in GluR2 and NMDAR1 glutamate receptor subunit protein immunoreactivity in corticocortically projecting neurons in macaque and patas monkeys

A distinct subpopulation of neurons forming long corticocortical projections in the association neocortex is highly vulnerable to the degenerative process in Alzheimer's disease. However, the degree to which age-related molecular and morphologic alterations of identifiable neuronal populations reflects early cellular degeneration leading to functional deficits has not yet been fully investigated in the aging brain. We performed an immunohistochemical analysis of neurons forming short and long corticocortical projections in young and old monkeys using antibodies to the GluR2 and NMDAR1 glutamate receptor subunit proteins. Projection neurons differed in their expression of these receptor subunits, as GluR2 was less prevalent than NMDAR1 among retrogradely labeled neurons. Long and short corticocortical pathways in old animals demonstrated a considerable decrease in the proportions of projection neurons containing GluR2 and NMDAR1, an observation that was particularly consistent in the case of GluR2. No age-related differences were observed in distribution of neurofilament protein in either type of projection neurons. These data suggest that cortical neurons furnishing long and short corticocortical projections display consistent neurochemical changes during aging and that a differential decrease in cellular expression of glutamate receptor subunit proteins occurs. The fact that in aging these neurons have lower levels of GluR2 than in young individuals, but comparatively higher levels of NMDAR1 than GluR2, may render them prone to calcium-mediated excitotoxicity, which in humans may be related to the selective vulnerability of such neurons during the course of Alzheimer's disease. Also, it is apparent that age-related neuronal changes are quite subtle and involve subcellular components of the cortical circuits rather than major morphologic alterations.     

Distribution of ionotropic glutamate receptor subunit mRNAs in the rat hypothalamus

The excitatory amino acid neurotransmitter glutamate participates in the control of most (and possibly all) neuroendocrine systems in the hypothalamus. This control is exerted by binding to two classes of membrane receptors, the ionotropic and metabotropic receptor families, which differ in their structure and mechanisms of signal transduction. To gain a better understanding about the precise sites of action of glutamate and the subunit compositions of the receptors involved in the glutamatergic neurotransmission in the hypothalamus and septum, in situ hybridization was used with 35S-labeled cRNA probes for the different ionotropic receptor subunits, including glutamate receptor subunits 1-4 (GluR1-GluR4), kainate-2, GluR5-GluR7, N-methyl-D-aspartate (NMDA) receptor 1 (NMDAR1), and NMDAR2A-NMDAR2D. The results showed that subunits of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate-preferring, kainate-preferring, and NMDA-preferring receptor subunits are distributed widely but heterogeneously and that the GluR1, GluR2, kainate-2, NMDAR1, NMDAR2A, and NMDAR2B subunits are the most abundant in the hypothalamus. Thus, GluR1 subunit mRNA was prominent in the lateral septum, preoptic area, mediobasal hypothalamus, and tuberomammillary nucleus, whereas kainate-2 subunit mRNA was abundant in the medial septum-diagonal band, median and anteroventral preoptic nuclei, and supraoptic nuclei as well as the magnocellular portion of the posterior paraventricular nucleus. Regions that contained the highest levels of NMDAR1 subunit mRNA included the septum, the median preoptic nucleus, the anteroventral periventricular nucleus, and the supraoptic and suprachiasmatic nuclei as well as the arcuate nucleus. Together, the extensive distribution of the different GluR subunit mRNAs strengthen the view that glutamate is a major excitatory neurotransmitter in the hypothalamus. The overlap in the distribution of the various subunit mRNAs suggests that many neurons can express GluR channels that belong to different families, which would allow a differential regulation of the target neurons by glutamate.     

Expression of ionotropic glutamate receptor subunit mRNAs in the hypothalamic paraventricular nucleus of the rat

The hypopthalamic paraventricular nucleus (PVN) coordinates multiple aspects of homeostatic regulation, including pituitary-adrenocortical function, cardiovascular tone, metabolic balance, fluid/electrolyte status, parturition and lactation. In all cases, a substantial component of this function is controlled by glutamate neurotransmission. In this study, the authors performed a high-resolution in situ hybridization analysis of ionotropic glutamate receptor subunit expression in the PVN and its immediate surround. N-methyl-D-aspartate (NMDA) receptor 1 (NMDAR1), NMDAR2A, and NMDAR2B mRNAs were expressed highly throughout the PVN and its perinuclear region as well as in the subparaventricular zone. NMDAR2C/2D expression was limited to subsets of neurons in magnocellular and hypophysiotrophic regions. In contrast with NMDA subunit localization, AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate)-preferring and kainate (KA)-preferring receptor subunit mRNAs were expressed heterogeneously in the PVN and surround. Glutamate receptor 1 (GluR1) mRNA labeling was most intense in preautonomic subregions, whereas GluR2, GluR4, GluR5, and KA2 were expressed in hypophysiotrophic cell groups. It is noteworthy that GluR5 mRNA expression was particularly robust in the dorsolateral region of the medial parvocellular PVN, suggesting localization in corticotropin-releasing hormone neurons. All four AMPA subunits and GluR6 and GluR7 mRNAs were expressed highly in the perinuclear PVN region and the subparaventricular zone. These data suggest the capacity for multifaceted regulation of PVN function by glutamate, with magnocellular neurons preferentially expressing NMDA subunits, preautonomic neurons preferentially expressing AMPA subunits, and hypophysiotrophic neurons preferentially expressing KA subunits. Localization of all species in the perinuclear PVN suggests that glutamate input to the immediate region of the PVN may modulate its function, perhaps by communication with local gamma-aminobutyric acid neurons.     

Immunohistochemical localization of NMDA- and AMPA-type glutamate receptor subunits in the basal ganglia of red-eared turtles

Corticostriatal and thalamostriatal projection systems have been shown to utilize glutamate as a neurotransmitter in mammals and birds. Although corticostriatal and thalamostriatal projection systems have been demonstrated in turtles, it is uncertain whether they too use glutamate as their neurotransmitter. Immunohistochemical localization of glutamate and of NMDA- and AMPA-type ionotropic glutamate receptor subunits (NMDAR2A/B, GluR1, GluR2/3, and GluR4) were used to address this issue. Numerous medium-sized neurons that were rich in NMDAR2A/B and GluR2/3 were observed in the striatal part of the basal ganglia of red-eared turtles. Smaller numbers of medium-sized neurons and some large neurons rich in the GluR1 and GluR4 subunits were also observed in the striatum. The striatal neuropil was notably rich in GluR1, GluR2/3 and NMDAR2A/B subunits. The pallidal region was specifically rich in large neurons possessing GluR4 subunits. Consistent with the glutamate receptors on striatal and pallidal neurons, sources of input to the striatum and pallidum in turtle such as the dorsomedial and dorsolateral thalamic nuclei (which appear to correspond to intralaminar thalamic nuclei), telencephalic pallial cell groups, and the apparent subthalamic nucleus homologue were rich in glutamatergic neurons. The results show that the thalamostriatal, corticostriatal and subthalamo-pallidal projection systems of turtles are glutamatergic and that similar basal ganglia cell types in turtles and mammals have largely similar glutamate receptor characteristics. Copyright (R) 2000 S.Karger AG, Basel     

NADPH-diaphorase and cytosolic urea cycle enzymes in the rat spinal cord

Nitric oxide synthase (NOS), argininosuccinate synthetase (ASS), and argininosuccinate lyase (ASL) compose a cyclic pathway to form nitric oxide (NO). These enzymes, however, are localized differentially in most regions of the brain. To find out whether NOS, ASS, and ASL are colocalized in neurons of the spinal cord, we examined the distribution of these enzymes by using a double-labeling procedure combining fluorescent immunohistochemistry with an assay for reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d). Results indicate that neurons in the dorsal horn, the intermediolateral nucleus, and the central canal region were NADPH-d active (+) and NOS-, ASS-, and ASL-like immunoreactive (-LI). In laminae II and III of the dorsal horn, some NADPH-d (+) neurons were ASL-LI (8–30%) but only a few were ASS-LI (0.5–7%). In the nucleus intermediolateralis, a large portion of NADPH-d (+) neurons were ASL-LI (30–60%), whereas only a small portion of NADPH-d (+) neurons were ASS-LI (10–20%). In the central canal region, some NADPH-d (+) neurons were ASL-LI (15–40%), and a few NADPH-d (+) neurons were ASS-LI (3–16%). Thus, the results suggest that, in the nucleus intermediolateralis and the central canal region, NOS, ASS, and ASL are colocalized and form a cyclic pathway to produce NO, whereas, in the dorsal horn, these enzymes are more characteristically localized in different neurons, which may transport the substrates intercellularly. J. Comp. Neurol. 385:616–626, 1997. © 1997 Wiley-Liss, Inc.     

Developmental expression of NMDA and AMPA receptor subunits in vestibular nuclear neurons that encode gravity-related horizontal orientations

We examined the expression profile of subunits of ionotropic glutamate receptors [N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionate (AMPA)] during postnatal development of connectivity in the rat vestibular nucleus. Vestibular nuclear neurons were functionally activated by constant velocity off-vertical axis rotation, a strategy to stimulate otolith organs in the inner ear. These neurons indicated Fos expression as a result. By immunodetection for Fos, otolith-related neurons that expressed NMDA/AMPA receptor subunits were identified as early as P7, and these neurons were found to increase progressively up to adulthood. Although there was developmental invariance in the percentage of Fos-immunoreactive neurons expressing the NR1, NR2A, GluR1, or GluR2/3 subunits, those expressing the NR2B subunit decreased from P14 onward, and those expressing the GluR4 subunit decreased in adults. These double-immunohistochemical data were corroborated by combined immuno-/hybridization histochemical data obtained from Fos-immunoreactive neurons expressing NR2B mRNA or GluR4 mRNA. The staining of both NR2B and GluR4 in the cytoplasm of these neurons decreased upon maturation. The percentage of Fos-immunoreactive neurons expressing the other ionotropic glutamate receptor subunits (viz. NR1, NR2A, GluR1, and GluR2/3) remained relatively constant throughout postnatal maturation. Triple immunofluorescence further demonstrated coexpression of NR1 and NR2 subunits in Fos-immunoreactive neurons. Coexpression of NR1 subunit with each of the GluR subunits was also observed among the Fos-immunoreactive neurons. Taken together, the different expression profiles of ionotropic glutamate receptor subunits constitute the histological basis for glutamatergic neurotransmission in the maturation of central vestibular connectivity for the coding of gravity-related horizontal head movements.     

Glutamate receptor subunit immunoreactivity in neurons of the rat rostral ventrolateral medulla

Immunohistochemical studies were conducted to assess the subunits of ionotropic and metabotropic glutamate receptor present in the rostral ventrolateral medulla (RVLM) of the rat. Double labeling the medullary sections with polyclonal GluR1, GluR2/3, GluR4, NMDAR1, NMDAR2A/B, mGluR1alpha, and mGluR2/3 antiserum and monoclonal tyrosine hydroxylase (TH) antiserum revealed nearly all TH immunoreactive (irTH) cells and many TH-negative neurons were immunoreactive to GluR2/3 (irGluR2/3), NMDAR1 (irNMDAR1), and NMDAR2A/B (irNMDAR2A/B). A few RVLM neurons were immunoreactive to GluR1 (irGluR1) and GluR4 (irGluR4), but they were generally TH-negative. Immunoreactivity to mGluR1alpha (irmGluR1alpha) appeared to be localized exclusively to fiber-like elements in the RVLM area. Our results show that neurons in the RVLM, including irTH, are endowed mainly with GluR2/3 and NMDAR1 or NMDAR2A/B ionotropic receptor subunits, and that irmGluR1alpha splice variant appears to be located on nerve fibers ramifying within the RVLM. Moreover, TH-negative neurons in the RVLM appear to bear similar subunits of ionotropic glutamate receptors.