Excitotoxic motoneuron degeneration induced by glutamate receptor agonists and mitochondrial toxins in organotypic cultures of chick embryo spinal cord

Glutamate receptor‐mediated excitotoxicity and mitochondrial dysfunction appear to play an important role in motoneuron (MN) degeneration in amyotrophic lateral sclerosis (ALS). In the present study we used an organotypic slice culture of chick embryo spinal cord to explore the responsiveness of mature MNs to different excitotoxic stimuli and mitrochondrial inhibition. We found that, in this system, MNs are highly vulnerable to excitotoxins such as glutamate, N‐methyl‐D‐aspartate (NMDA), and kainate (KA), and that the neuroprotective drug riluzole rescues MNs from KA‐mediated excitotoxic death. MNs are also sensitive to chronic mitochondrial inhibition induced by malonate and 3‐nitropropionic acid (3‐NP) in a dose‐dependent manner. MN degeneration induced by treatment with mitochondrial toxins displays structural changes similar to those seen following excitotoxicity and can be prevented by applying either the antiexcitotoxic drug 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione disodium (CNQX) or riluzole. Excitotoxicity results in an increased frequency of normal spontaneous Ca²⁺ oscillations in MNs, which is followed by a sustained deregulation of intracellular Ca²⁺. Tolerance to excitotoxic MN death resulting from chronic exposure to excitotoxins correlates with a reduced excitotoxin‐induced increase in intracellular Ca²⁺ and increased thapsigargin‐sensitive Ca²⁺ stores. J. Comp. Neurol. 516:277–290, 2009. © 2009 Wiley‐Liss, Inc.     

Ampakine CX546 increases proliferation and neuronal differentiation in subventricular zone stem/progenitor cell cultures

Ampakines are chemical compounds known to modulate the properties of ionotropic α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)-subtype glutamate receptors. The functional effects attributed to ampakines involve plasticity and the increase in synaptic efficiency of neuronal circuits, a process that may be intimately associated with differentiation of newborn neurons. The subventricular zone (SVZ) is the main neurogenic niche of the brain, containing neural stem cells with brain repair potential. Accordingly, the identification of new pharmaceutical compounds with neurogenesis-enhancing properties is important as a tool to promote neuronal replacement based on the use of SVZ cells. The purpose of the present paper is to examine the possible proneurogenic effects of ampakine CX546 in cell cultures derived from the SVZ of early postnatal mice. We observed that CX546 (50 μm) treatment triggered an increase in proliferation, evaluated by BrdU incorporation assay, in the neuroblast lineage. Moreover, by using a cell viability assay (TUNEL) we found that, in contrast to AMPA, CX546 did not cause cell death. Also, both AMPA and CX546 stimulated neuronal differentiation as evaluated morphologically through neuronal nuclear protein (NeuN) immunocytochemistry and functionally by single-cell calcium imaging. Accordingly, short exposure to CX546 increased axonogenesis, as determined by the number and length of tau-positive axons co-labelled for the phosphorylated form of SAPK/JNK (P-JNK), and dendritogenesis (MAP2-positive neurites). Altogether, this study shows that ampakine CX546 promotes neurogenesis in SVZ cell cultures and thereby may have potential for future stem cell-based therapies.     

Developmental expression of N-methyl-D-aspartate glutamate receptor 1 splice variants in the chick retina

Glutamate is the major excitatory neurotransmitter in the vertebrate retina. The N-methyl-D-aspartate glutamate receptor (NMDAR) is assembled as a tetramer containing NR1 and NR2, and possibly NR3 subunits, NR1 being essential for the formation of the ion channel. The NMDAR1 (NR1) gene encodes for mRNAs that generate at least eight functional variants by alternative splicing of exon 5 (cassette N1), 21 (cassette C1), or 22 (cassettes C2 or C2'). NR1 splice variants were identified in the mature chick retina, and their variation during embryonic development (ED) was analyzed. NR1 was shown to lack N1 in early ED, shifting to N1-containing variants in the mature retina, which could contribute to explaining the distinct biochemical properties of retinal NMDARs compared with the CNS. Sequence analysis of C-terminal variants containing C1 and C2 cassettes suggests a membrane-targeting mechanism for avian NMDARs distinct from that in mammals. An NR1 variant containing a novel alternative C-terminal splice exon named C3 was found, which encodes six amino acids containing a predicted casein kinase II phosphorylation site. This new variant is expressed in the retina during a restricted period of ED, coincident with the generation of spontaneous calcium activity waves, which precedes synapse formation in the retina, suggesting its participation in this process.     

Distribution of the mineralocorticoid and the glucocorticoid receptor mRNAs in the rat hippocampus

The cellular localization of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) gene expression in the rat hippocampus was studied by in situ hybridization using 35S-labeled RNA-probes, complementary to either 513 bases of the rat brain mineralocorticoid receptor (MR)-mRNA or 500 bases of the rat liver glucocorticoid receptor (GR)-mRNA. Neurons in CA1, CA2, and the dentate gyrus expressed both receptor genes at high levels. The MR-mRNA was demonstrated in all pyramidal cell fields (CA1-4) of the hippocampal formation and the granular neurons of the dentate gyrus. In contrast, GR-mRNA was mainly restricted to CA1 and CA2 pyramidal cell fields and the dentate gyrus. This pattern of hybridization was found to agree with the cellular distribution of the two types of corticosteroid receptors detected previously in the hippocampus by autoradiography of the radio-labeled receptors and by immunocytochemistry of the receptor protein. These observations suggest that the corticosteroid receptors described previously as type 1 and type 2 are encoded by MR- and GR-mRNA, respectively. Although both the MR and GR genes are co-expressed in some hippocampal neurons, the unique patterns of distribution of the two receptor mRNAs in the hippocampal formation suggest that the genes for these receptors are differentially regulated. Moreover, the microanatomy of MR and GR expression provides insight into molecular mechanisms underlying the characteristic action of various steroids on behaviors involved in stress and circadian regulation.     

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.     

Stoichiometries of AMPA receptor subunit mRNAs in rat brain fall into discrete categories

In situ hybridization was used to estimate the relative concentrations of mRNAs encoding different subunits (GluR1-4) of α-amino 3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors in rat brain and to test the hypothesis that within-region expression profiles reflect a limited number of recurring patterns. Fractional subunit mRNA concentrations were calculated for 33 brain regions, and cluster analysis methods were applied to test for statistically meaningful groupings in the data. Four relatively homogeneous classes were identified and designated as AMPA receptor (AR) categories, numbered according to dominant subunit mRNAs. The AR-1 class (47% GluR1 mRNA) was expressed by structures near the mesodiencephalic border, including basal ganglia-related areas. The AR-2 class (57% GluR2 mRNA) was expressed in cortex and tectum. The AR-1,2 class (31% GluR1, 45% GluR2) was found in the largest number of regions, including such dissimilar cell fields as hippocampus and substantia nigra pars compacta. The AR-2,3 grouping (33% GluR2, 31% GluR3) was associated with the sensory relay and reticular thalamic nuclei. It is suggested that AR-1,2 and AR-2, the most closely related categories in clustering space, are largely telencephalic receptors with the former predominant in the subcortex and the latter in the cortex. The AR-2,3 class is associated with ascending sensory stations, whereas AR-1 appears to include several smaller categories expressed by specialized systems. If the balance of subunit mRNAs is reflected at the protein level, then the present data suggest that forebrain AMPA-type glutamate receptors can be classified into a limited number of recurring types. J. Comp. Neurol. 385:491–502, 1997. © 1997 Wiley-Liss, Inc.     

Differential distributions of the NMDA receptor channel subunit mRNAs in the mouse retina

In the retina, the ε2 and ζ1 subunit mRNAs of the NMDA receptor channel were expressed from embryonic stages and found in ganglion cell layer and whole layer of inner nuclear layer at postnatal day 21 (P21). The ε1 subunit mRNA appeared postnatally and was distributed in ganglion cell layer and an inner third of inner nuclear layer at P21. These findings suggest that molecular organization of the NMDA receptor channel may alter during the retinal development.     

Cellular localization of three vesicular glutamate transporter mRNAs and proteins in rat spinal cord and dorsal root ganglia

Glutamate is transported into synaptic vesicles by vesicular glutamate transporter (VGLUT) proteins. Three different VGLUTs, VGLUT1, VGLUT2, and VGLUT3, have recently been characterized, and they are considered to represent the most specific marker so far for neurons using glutamate as transmitter. We analyzed the cellular localization of VGLUT1-3 in the rat spinal cord and dorsal root ganglia (DRGs) in control rats and after dorsal rhizotomy. Using in situ hybridization, VGLUT1 mRNA containing neurons were shown in the dorsomedial part of the intermediate zone, whereas VGLUT2 mRNA-expressing neurons were present in the entire intermediate zone, both populations most likely representing interneurons. VGLUT3 mRNA could not be detected in the spinal cord. In the ventral horn, a dense plexus of VGLUT1-immunoreactive (ir) nerve terminals was present, with large varicosities abutting on presumed motoneurons. In the dorsal horn a similarly dense plexus was seen, except in laminae I and II. A very dense plexus of VGLUT2-ir fibers was distributed in the entire gray matter of the spinal cord, with many fibers lying close to presumed motoneurons. Few VGLUT3-ir fibers were distributed in the white and gray matter, including lamina IX. However, a dense VGLUT3-ir plexus was seen in the sympathetic intermedio-lateral column (IML). Multiple-labeling immunohistochemistry revealed that the VGLUT1-, VGLUT2-, and VAChT-containing varicosities in lamina IX all represent separate entities. There was no colocalization of VGLUT3 with VAChT or 5-HT in varicose fibers of the ventral horn, but some VGLUT3-ir fibers in the IML were 5-HT-positive. Lesioning of the dorsal roots resulted in an almost complete disappearance of VGLUT1-ir fibers around motoneurons and a less pronounced decrease in the remaining gray matter, whereas the density of VGLUT2- and VAChT-ir fibers appeared unaltered after lesion. Many VGLUT1-ir neurons were observed in DRGs; they were almost all large and did not colocalize calcitonin gene-related peptide (CGRP), and there was no overlap between these markers in fibers in the superficial dorsal horn. VGLUT2 was, at most, seen in a few DRG neurons. Taken together, these results suggest that the VGLUTs mRNAs are present in distinct subsets of neuronal populations at the spinal level. VGLUT1 is mainly present in primary afferents from large, CGRP-negative DRG neurons, VGLUT2 has mainly a local origin, and VGLUT3 fibers probably have a supraspinal origin.     

Expression of glutamate receptors in the human and rat basal ganglia: Effect of the dopaminergic denervation on AMPA receptor gene expression in the striatopallidal complex in parkinson's disease and rat with 6-OHDA lesion

The overactivity of subthalamopallidal and corticostriatal glutamatergic neurons observed in Parkinson's disease (PD) suggests that antagonists of glutamate receptor could be used to alleviate the motor symptoms of the disease. In this study, we analysed two features of the striatopallidal complex: (1) the distribution of α-amino-3 hydroxy-5-methyl-4-isoxasol-propionate (AMPA) and kainate receptors and their corresponding mRNA by immunohistochemistry and in situ hybridisation and (2) the effect of dopaminergic denervation on AMPA receptor gene expression in PD patients and rats with 6-hydroxydopamine (6-OHDA)-induced degeneration of the nigrostriatal dopaminergic system. All AMPA receptor mRNAs and proteins (GluR1–4) were detected in the internal segment of the globus pallidus (GPi). Among kainate receptors, only KA1 and KA2 were detectable and only at a low level. Only GluR4 protein was detected in the neuropil of the GPi. In the striatum, GluR1, GluR2, and GluR3 were detected in about 70% of medium-sized and large neurons. By contrast, GluR4 mRNA was detected in only a small number of large and medium-sized neurons. Among kainate receptors, GluR6, GluR7, and KA2 were detected in about 50–60% of medium-sized neurons, whereas GluR5 and KA1 were restricted to 1–2% and 20–30% of these neurons, respectively. These results suggest that antagonists of AMPA and kainate receptors could be effective in alleviating motor symptoms in Parkinson's disease by blocking the overstimulation of pallidal and striatal neurons by glutamate. A significant decrease in GLuR1 gene expression (−33%) was observed in the neurons of the GPi in PD patients and in rat entopeduncular nucleus ipsilateral to the 6-OHDA lesion (−20%). GluR2, GluR3, and GluR4 mRNA levels in the GPi and GluR1–4 mRNA levels in the striatum were unchanged in PD patients and 6-OHDA-lesioned rats compared with their respective controls. These data suggest that dopamine positively regulates only GluR1 gene expression in the GPi. © 1996 Wiley-Liss, Inc.     

Expression of N-Methyl-D-Aspartate receptor subunit mRNAs in the human brain: Striatum and globus pallidus

N-methyl-D-aspartate receptors (NRs) play an important role in basal ganglia function. By using in situ hybridization with ribonucleotide probes, we investigated the regional and cellular distribution of NR subunit mRNA expression in the human basal ganglia: caudate nucleus, putamen, lateral globus pallidus (LGP), and medial globus pallidus (MGP). Analysis of both film autoradiograms and emulsion-dipped slides revealed distinct distribution patterns for each subunit. On film autoradiograms, the signal for NR1, NR2B, and NR2C in the striatum (STR) was higher than in globus pallidus (GP). The NR2D probe gave a stronger signal in GP than in STR. For NR2A we found a signal in all regions. Analysis of emulsion-dipped sections demonstrated that in striatal neurons, the NR2B signal was higher than in GP neurons. In GP neurons, NR2D was more abundant than in striatal neurons. Despite the relatively low signal on film for NR2C in GP, we found a slightly higher signal in GP per neuron than in STR since in the pallidal areas neurons were sparse but intensely labeled. NR1 and NR2A were more evenly distributed over neurons of STR and GP. Between the different parts of STR and GP, we observed only minor differences in the expression of NRs. In MGP a subpopulation of neurons exhibiting low NR2D signals could be separated from the majority of neurons showing an intense NR2D signal. Since the physiological properties of NRs are dependent on subunit composition, these data suggest a high degree of regional specialization of NR properties in the human basal ganglia. J. Comp. Neurol. 390:63–74, 1998. © 1998 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.     

Identification of neurones in the female rat hypothalamus that express oestrogen receptor-alpha and vesicular glutamate transporter-2

Oestrogen exerts its effects in the brain by binding to and activating two members of the nuclear receptor family, oestrogen receptor (ER)-alpha and ER-beta. Evidence suggests that oestrogen-receptive neurones participate in the generation of reproductive behaviours and that they convey the oestrogen message to gonadotropin-releasing hormone (GnRH) neurones. The aim of the present study was to identify the neurochemical phenotype of a subset of oestrogen receptor-expressing neurones. To this aim, we focused on the glutamate neuronal system, which is one of the most important stimulators of GnRH synthesis and release. We used the presence of vesicular glutamate transporter-2 (VGLUT2) mRNA as a specific marker to identify glutamate neurones and employed dual in situ hybridization to localize ERalpha mRNA-(35S-labelling) and VGLUT2 mRNA-(digoxigenin-labelling) expressing neurones within the hypothalamus. The results show that the overall distribution of VGLUT2 mRNA and ERalpha mRNA are consistent with previous data in the literature. Dual-labelled neurones were localized in the ventrolateral part of the ventromedial nucleus where 81.3 +/- 3.4% of the ERalpha mRNA containing neurones expressed VGLUT2 mRNA, in the anteroventral periventricular nucleus (30% colocalization) and in the medial preoptic nucleus (19% colocalization). Only 4.4% of the ERalpha expressing neurones in the arcuate nucleus contained VGLUT2 mRNA. These findings reveal that certain subpopulations of oestrogen-receptive neurones are glutamatergic in select hypothalamic areas that are known to regulate reproductive behaviour and GnRH neurones in the female rat. Thus, the oestrogen signal could be propagated through glutamate neurones to distant sites and influence the activity of the postsynaptic neurones.     

Regional distribution of the cells expressing glycine receptor β subunit mRNA in the rat brain

The expression of mRNA of the β subunit of the glycine receptor was investigated in the rat by in situ hybridization histochemistry using an oligonucleotide probe specific to the sequence of the β subunit. Neurons expressing β subunit mRNA were widely and abundantly distributed in the rat brain from the olfactory bulb to the spinal cord. The pattern of distribution of cells containing β subunit mRNA in the lower brainstem was very similar to that of cells containinga₁ subunit mRNA. In addition, β subunit mRNA was strongly expressed by the neurons of the cerebral cortex, hippocampal formation and diencephalon as well as by the Purkinje cells wherea₁ subunit mRNA expression is rare. These findings indicated that the glycine receptor is heterogeneous. The sites where strong labeling was noted were as follows. In the forebrain and diencephalon, strongly labeled neurons were abundant in the olfactory region, hippocampal formation, cerebral cortex, and thalamus. In the hippocampal formation, neurons in the subiculum, pyramidal cells in Ammon's horn, and neurons in the polymorphic layer of the dentate gyrus were strongly labeled. In the thalamus, the anterodorsal, reticular, parafascicular, and the subthalamic nuclei were strongly labeled. In the brainstem, the red nucleus, almost all of the motor neurons in the cranial motor nuclei innervating striated muscles, the trigeminal mesencephalic nucleus, the ventral tegmental nucleus of Gudden, and the pontine nucleus were strongly labeled. In the cerebellum, Purkinje cells in the Purkinje cell layer and all of the cerebellar nuclei were strongly labeled.     

Lipopolysaccharide administration in vivo induces differential expression of cAMP-specific phosphodiesterase 4B mRNA splice variants in the mouse brain

Many inflammatory processes involve cAMP. Pharmacological manipulation of cAMP levels using specific phosphodiesterase (PDE) inhibitors provokes an antiinflammatory response. The aim of this study was to investigate changes in the pattern and levels of expression of mRNAs coding for the cAMP-specific PDE4 family and subfamilies in mouse brain during the immediate acute immune response provoked by an intraperitoneal injection of lipopolysaccharide (LPS). PDE4B, and furthermore the splice variants PDE4B2 and PDE4B3, were the only mRNAs that showed altered expression. Whereas PDE4B2 presented increased expression at both 3 and 8 hr postinjection, PDE4B3 mRNA showed decreased expression that reached a minimum 8 hr postinjection. PDE4B2 mRNA upregulation was observed mainly in endothelial and macrophage/neutrophil cell populations in the leptomeninges, and the downregulation of PDE4B3 was observed mainly in oligodendrocytes throughout the brain. Our results clearly illustrate the distinctive anatomical distribution and cellular localization of the PDE4Bs during neuroinflammation and emphasize the importance of PDE4B splice-variant-specific inhibitors as therapeutic tools.     

Distinct spatiotemporal distributions of the N-Methyl-D-aspartate receptor channel subunit mRNAs in the mouse cervical cord

The distribution of five N-methyl-D -aspartate (NMDA) receptor channel subunit mRNAs in the mouse spinal cord from embryonic day 13 (E13) through postnatal day 56 (P56) was semiquantitatively examined at the cervical level via in situ hybridization with subunit-specific oligonucleotide probes. Signals for the ξ1 subunit mRNA were restricted to the most ventral portion of the spinal cord during embryonic stages. They extended to all laminae of the spinal cord except for the lamina 2 (substantia gelatinosa) during postnatal development. A wide expression of the ?2 subunit mRNA was found in the spinal gray matter from E13 through neonatal stages, but the signals became restricted to the lamina 2 by P21. No significant signals for the ?3 subunit mRNA were detected in the spinal cord at any developmental stages. The ?4 subunit mRNA was distributed widely in the spinal cord during embryonic and early postnatal periods but decreased nearly to background levels by P21. In contrast to the differential distribution of the ? subunit mRNAs, the ξ1 subunit mRNA was found ubiquitously at each developmental stage examined. These findings suggest that the molecular organization of the ? subunits may be difference between the dorsal horn and the remaining regions in the mature spinal cord, which provides a molecular basis for functional heterogeneity of the NMDA receptor channel. Moreover, this spatial heterogeneity might be generated through drastic alterations in the subunit composition of the channel complex during spinal cord development. © 1994 Wiley-Liss, Inc.