Ionotropic glutamate receptor subunit distribution on hypoglossal motoneuronal pools in the rat

The expression of ionotropic glutamate receptor subunits in the motoneuronal pools of the hypoglossal nucleus was studied using specific antibodies against subunits of the -amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate and N-methyl-D-aspartate (NMDA) subtypes. The highest numbers of intensely immunolabelled motoneurons were found in the dorsal tier and caudoventromedial part of the hypoglossal nucleus with all antibodies except that against the GluR1 AMPA subunit. Labelling for the GluR1 subunit was weak except for caudally located groups of motoneurons which innervate tongue muscles related to respiratory activity. By contrast, most motoneurons were intensely immunostained with antibodies against GluR2/3 and GluR4 subunits of the AMPA subtype. The low staining observed using an antibody specific for the GluR2 subunit (which prevents Ca²⁺-entry through AMPA channels) strongly suggests that AMPA receptors in hypoglossal motoneurons are Ca²⁺-permeable. Immunolabelling for the GluR5/6/7 kainate receptor subunits was found in many motoneuronal somata as well as in thin axon-like profiles and puncta that resembled synaptic boutons. Most motoneurons were intensely immunostained for the NMDA receptor subunit NR1. These results show that the hypoglossal nucleus contains five heterogeneous pools of motoneurons which innervate functionally defined groups of tongue muscles. The uneven expression of the different receptor subunits analysed here could reflect diverse phenotypic properties of hypoglossal motoneurons which might be expected to generate different patterns of motor responses under different physiological or pathological conditions.     

Expression and localization of ionotropic glutamate receptor subunits in the goldfish retina--an in situ hybridization and immunocytochemical study

The expression and distribution of AMPA, kainate and NMDA glutamate receptor subunits was studied in the goldfish retina. For the immunocytochemical localization of the AMPA receptor antisera against GluR2, GluR2/3 and GluR4 were used, and for in situ hybridization rat specific probes for GluR1 and GluR2 and goldfish specific probes for GluR3 and GluR4 were used. The localization of the low affinity kainate receptor and NMDA receptor was studied using antisera against GluR5-7 and NR1. All AMPA receptor subtypes were demonstrated to be present in the goldfish retina both by immunocytochemistry and in situ hybridization. In situ hybridization revealed expression of all AMPA receptors subunit at the inner border of the INL. Only GluR3 was also strongly expressed in the outer border of the INL. Some of the ganglion cells displayed a strong signal for GluR1, GluR3 and GluR4. GluR1-immunoreactivity was present in subsets of bipolar, amacrine, and ganglion cells. GluR2 and GluR2/3-immunoreactivity was mainly localized in the outer plexiform layer. GluR2 and GluR2/3-immunoreactivity are associated with the photoreceptor synaptic terminals. GluR4-immunoreactivity is present on Müller cells in the inner retina and on dendrites of bipolar cells in the OPL, whereas GluR5-7-immunoreactivity was prominently present on horizontal cell axon terminals. Finally, NR1-immunoreactivity was confined to amacrine cells, the inner plexiform layer and ganglion cells. This study shows that there is a strong heterogeneity of glutamate receptor subunit expression in the various layers of the retina. Of the AMPA receptor subunits GluR3 seems to be expressed the most widely in all layers with strong glutamatergic synaptic interactions whereas all the other subunits seem to have a more restricted expressed pattern.     

Modification of ionotropic glutamate receptor–mediated processes in the rat hippocampus following repeated,brief seizures

The seizure-induced molecular and functional alterations of glutamatergic transmission in the hippocampus have been investigated. Daily repeated epileptic seizures were induced for 12 days by intraperitoneal administration of 4-aminopyridine (4-AP; 4.5 mg/kg) in adult Wistar rats. The seizure symptoms were evaluated on the Racine's scale. One day after the last injection, the brains were removed for in vitro electrophysiological experiments and immunohistochemical analysis. The glutamate receptor subunits NR1, NR2A, NR2B, GluR1, GluR1_flop, GluR2, and KA-2 were studied using the histoblotting method. The semi-quantitative analysis of subunit immunoreactivities in hippocampal layers was performed with densitometry. In the hippocampus, increase of GluR1, GluR1_flop and NR2B immunostaining was observed in most of the areas and layers. The significant decrease of GluR2 staining intensity was observed in the CA1 and dentate gyrus. Calcium permeability of hippocampal neurons was tested by a cobalt uptake assay in hippocampal slices. The uptake of cobalt increased in the CA1 area and dentate gyrus, but not in the CA3 region following 4-AP treatment. Effects of AMPA and NMDA (N-methyl-d-aspartate) glutamate receptor antagonists (1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466) and D-APV respectively) were measured in hippocampal slices using extracellular recording. Analysis of the population spikes revealed the reduced effectiveness of the AMPA receptor antagonist GYKI 52466, while the effect of the NMDA receptor antagonist d-(2R)-amino-5-phosphonovaleric acid was similar to controls. The results demonstrated that repeated convulsions induced structural and functional changes in AMPA receptor–mediated transmission, while NMDA and kainate receptor systems displayed only alterations in receptor subunit composition.     

Modulation of the glutamatergic receptors (AMPA and NMDA) and of glutamate vesicular transporter 2 in the rat facial nucleus after axotomy

Facial nerve axotomy is a good model for studying neuronal plasticity and regeneration in the peripheral nervous system. We investigated in the rat the effect of axotomy on the different subunits of excitatory glutamatergic AMPA (GLuR1-4), NMDA (NR1, NR2A-D) receptors, post-synaptic density 95, vesicular glutamate transporter 2, beta catenin and cadherin. mRNA levels and/or protein production were analyzed 1, 3, 8, 30 and 60 days after facial nerve axotomy by in situ hybridization and immunohistofluorescence. mRNAs coding for the GLuR2-4, NR1, NR2A, B, D subunits of glutamatergic receptors and for post-synaptic density 95, were less abundant after axotomy. The decrease began as early as 1 or 3 days after axotomy; the mRNAs levels were lowest 8 days post-lesion, and returned to normal or near normal 60 days after the lesion. The NR2C subunit mRNAs were not detected in either lesioned or intact facial nuclei. Immunohistochemistry using specific antibodies against GLuR2-3 subunits and against NR1 confirmed this down-regulation. There was also a large decrease in vesicular glutamate transporter 2 immunostaining in the axotomized facial nuclei at early stages following facial nerve section. In contrast, no decrease of NR2A subunit and of post-synaptic density 95 could be detected at any time following the lesion. beta Catenin and cadherin immunoreactivity pattern changed around the cell body of facial motoneuron by day 3 after axotomy, and then, tends to recover at day post-lesion 60 days. Therefore, our results suggest a high correlation between restoration of nerve/muscle synaptic contact, synaptic structure and function in facial nuclei. To investigate the mechanisms involved in the change of expression of these proteins following axotomy, the facial nerve was perfused with tetrodotoxin for 8 days. The blockade of action potential significantly decreased GLuR2-3, NR1and NR2A mRNAs in the ipsilateral facial nuclei. Thus, axotomy-induced changes in mRNA abundance seemed to depend partly on disruption of activity.     

Expression of glutamate receptor subunits 2/3 and 4 in the hypoglossal nucleus of the rat after neurectomy

The immunoreactivity of glutamate receptor subunits 2/3 (GluR2/3) and 4 (GluR4) was studied following neurectomy of the hypoglossal nucleus (NH). After a short period of survival (at 1, 2, and 7 days postoperation, dpo), GluR2/3 immunoreactivity was barely dectectable in the operated side of HN. During these periods, GluR4 immunoreactivity was present, but was greatly reduced when compared with the GluR4 immunoreactivity in the unoperated side. The data suggest that of the 4 subunits of the AMPA receptor, GluR2/3 is the most susceptible receptor to the early stage of hypoglossal neurectomy, and GluR4 tolerated the lesion more than the others. It is also suggested that both GluR2/3 and 4 may play a very important neuroprotective role in the early stage of neuronal degeneration after axotomy, especially the former. Following a midterm survival period (14, 21, and 35 dpo), GluR2/3 immunoreactivity gradually reappeared in some neurons on the operated side of HN, which may indicate functional recovery. However, the number of GluR4-immunopositive neurons on the operated side of HN was greatly reduced. The reason for such a reduction is not known, but, from the speculative point of view, it is possible that the disappearance of GluR4-positive neurons may be related to their excitotoxic property, especially at 35 dpo, when neuronal cell death had already occurred. Following a long-term period of survival (i.e., 56, 90, and 120 dpo), the numbers of surviving neurons remained fairly constant, suggesting the possible cessation of neuronal death. Received: 15 April 1997 / Accepted: 13 June 1997     

Targeting of GLUR4-containing AMPA receptors to synaptic sites during in vitro classical conditioning

The synaptic delivery of GluR4-containing AMPA receptors during in vitro classical conditioning of a neural correlate of an eyeblink response was examined by fluorescence imaging of punctate staining for glutamate receptor subunits and the presynaptic marker synaptophysin. There was a significant increase in GluR4-containing AMPA receptors to synaptic sites after conditioning as determined by colocalization of GluR4 subunit puncta with synaptophysin. Moreover, the trafficking of these receptor subunits requires NMDA receptor activation as it was blocked by D,L-2-amino-5-phosphonovaleric acid (AP-5). In contrast, colocalization of NR1 subunits with synaptophysin was stable regardless of whether the preparations had undergone conditioning or had been treated by AP-5. The enhanced colocalization of GluR4 and synaptophysin was accompanied by an increase in both the total number and size of puncta for both proteins, suggesting greater synthesis and aggregation during conditioning. Western blot analysis confirmed upregulation of synaptophysin and GluR4 following conditioning. These data support the hypothesis that GluR4-containing AMPA receptors are delivered to synaptic sites during conditioning. Further, they suggest coordinate presynaptic and postsynaptic modifications during in vitro classical conditioning.     

AMPA receptor subunit expression in the cuneate nucleus of adult squirrel monkeys after peripheral nerve injury

The primate somatosensory system provides an excellent model system with which to investigate adult neural plasticity. Here, we report immunohistochemical staining data for the GluR1 and GluR2/3 AMPA receptor subunits in the cuneate nucleus of adult squirrel monkeys one week after median nerve compression. These data are compared to subunit changes in the area 3b cortex of the same animals. We report differences between control and deprived brainstem implying that deprivation induced changes in subunit expression mirror those reported in the cortex. There are significant increases in GluR1 receptor subunit staining intensity and significant decreases in GluR2/3 receptor subunit staining intensity. This pattern of expression resembles receptor configurations reported in developing sensory systems. Taken together, these results suggest that the brainstem and the cortex initially progress through a phase of developmental recapitulation prior to the onset of NMDA mediated adult somatosensory reorganization.     

Reduced NR2A expression and prolonged decay of NMDA receptor-mediated synaptic current in rat vagal motoneurons following axotomy

To elucidate characteristic changes in the N -methyl- d -aspartate (NMDA) receptor on neurons following axotomy, subunit expressions and functional features of the NMDA receptor were examined in the dorsal motor nucleus of vagus (DMV) of rats receiving vagal axotomy at the neck. Western blotting analysis demonstrated that the expression of NR2A decreased 2–3 days after in vivo axotomy, while expression of NR1 and NR2B, NR2C and NR2D subunits did not change significantly. To examine the functional changes, patch clamp recordings in whole-cell mode were employed on the axotomized DMV neurons identified by retrograde labelling with fluorescent dye. The amplitude ratios of ifenprodil-sensitive components of NMDA response and d , l -2-amino-5-phosphovaleric acid (APV)-sensitive evoked postsynaptic current increased after axotomy. In addition, APV-sensitive postsynaptic currents exhibited a longer decay time in identified axotomized vagal motoneurons than in control neurons. No significant differences in the current density of the NMDA response and the peak amplitude of APV-sensitive synaptic currents were observed between axotomized and intact DMV neurons. In conclusion, a decrease in NR2A expression results in the appearance of functional characteristics of the NMDA receptor predominantly containing the NR2B subunit. This might lead to a long-term increase of the susceptibility of neurons to excitotoxicity.     

Differential expression of NMDA and AMPA receptor subunits in rat dorsal and ventral hippocampus

Several studies have demonstrated anatomical and functional segregation along the dorsoventral axis of the hippocampus. This study examined the possible differences in the AMPA and NMDA receptor subunit composition and receptor binding parameters between dorsal and ventral hippocampus, since several evidence suggest diversification of NMDA receptor-dependent processes between the two hippocampal poles. Three sets of rat dorsal and ventral hippocampus slices were prepared: 1) transverse slices for examining a) the expression of the AMPA (GluRA, GluRB, GluRC) and NMDA (NR1, NR2A, NR2B) subunits mRNA using in situ hybridization, b) the protein expression of NR2A and NR2B subunits using Western blotting, and c) by using quantitative autoradiography, c(1)) the specific binding of the AMPA receptor agonist [(3)H]AMPA and c(2)) the specific binding of the NMDA receptor antagonist [(3)H]MK-801, 2) longitudinal slices containing only the cornus ammonis 1 (CA1) region for performing [(3)H]MK-801 saturation experiments and 3) transverse slices for electrophysiological measures of NMDA receptor-mediated excitatory postsynaptic potentials. Ventral compared with dorsal hippocampus showed for NMDA receptors: 1) lower levels of mRNA and protein expression for NR2A and NR2B subunits in CA1 with the ratio of NR2A /NR2B differing between the two poles and 2) lower levels of [(3)H]MK-801 binding in the ventral hippocampus, with the lowest value observed in CA1, apparently resulting from a decreased receptor density since the B(max) value was lower in ventral hippocampus. For the AMPA receptors CA1 our results showed in ventral hippocampus compared with dorsal hippocampus: 1) lower levels of mRNA expression for GluRA, GluRB and GluRC subunits, which were more pronounced in CA1 and in dentate gyrus region and 2) lower levels of [(3)H]AMPA binding. Intracellular recordings obtained from pyramidal neurons in CA1 showed longer NMDA receptor-mediated excitatory postsynaptic potentials in ventral hippocampus compared with dorsal hippocampus. In conclusion, the differences in the subunit mRNA and protein expression of NMDA and AMPA receptors as well as the lower density of their binding sites observed in ventral hippocampus compared with dorsal hippocampus suggest that the glutamatergic function differs between the two hippocampal poles. Consistently, the lower value of the ratio NR2A/NR2B seen in the ventral part would imply that the ventral hippocampus NMDA receptor subtype is functionally different than the dorsal hippocampus subtype, as supported by our intracellular recordings. This could be related to the lower ability of ventral hippocampus for long-term synaptic plasticity and to the higher involvement of the NMDA receptors in the epileptiform discharges, observed in ventral hippocampus compared with dorsal hippocampus.     

Nociceptive stimulation induces glutamate receptor down-regulation in the trigeminal nucleus

The dorsal horn of the subnucleus caudalis of the spinal trigeminal nucleus is a relay of oro-facial pain transmission; increase in subnucleus caudalis neuronal activity in response to tissue injury affects the level of chemical mediators participating in nociceptive processing. We investigated, by means of immunocytochemistry, the expression of N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) glutamate receptor subunits in this nucleus in a model of inflammation. Rats injected with formalin in the whisker pad were compared with saline-injected control rats. One and two days after formalin injection, the immunostaining of cell bodies and neuropil of the AMPA receptor subunits GluR1 and GluR2/3 was markedly decreased in the ipsilateral superficial laminae of the subnucleus caudalis compared to the contralateral side. Side differences were not evident in the saline-treated animals. The down-regulation of AMPA GluR1 and GluR2/3 was no longer detectable in the subnucleus caudalis three days after formalin injection. No side difference was detected in the N-methyl-D-aspartate receptor subunit NR2A/B immunoreactivity of the subnucleus caudalis at any time-point in the formalin-injected animals. The modulation of AMPA receptor may be related to the decrease of hyperalgesia evident 1 h after formalin injection, in spite of the increasing perioral inflammation evident later on and characteristic of the formalin model. The present findings point out a selective down-regulation of AMPA receptor subunits in the transduction of trigeminal pain. These data also support the involvement of glutamate receptor subunits in the processing of trigeminal inflammation induced by noxious chemical stimulation.     

Ultrastructural analysis of the glutamatergic system in the outer plexiform layer of zebrafish retina

l-Glutamate, the photoreceptor neurotransmitter, depolarizes horizontal cells and OFF-bipolar cells by ionotropic receptors and hyperpolarizes ON-bipolar cells by metabotropic receptors. Despite extensive light microscopy on the distribution of glutamate receptors in zebrafish retina, there are little ultrastructural data. Given the importance of zebrafish in studies on the genetic manipulation of retinal development and function, precise data on the synaptic neurochemical organization of the zebrafish retina is needed. Immunohistochemical techniques were used to determine the ultrastructural localization of glutamate receptor subunits GluR2, GluR4, NMDA2B (NR2B) and mGluR1α in zebrafish outer plexiform layer (OPL). These antibodies were chosen because of an apparent conservation of localization of GluR2, GluR4 and mGluR1α in the vertebrate OPL, while there is some support for NMDA receptors in the OPL. GluR2-immunoreactivity (IR) was in all horizontal cell dendrites that invaginated cone pedicles and rod spherules. Three arrangements of dendrites contained GluR-IR in rod spherules: classical-type with GluR2-IR on lateral horizontal cell dendrites, a butterfly-shaped horizontal cell dendrite, and a goblet-shaped dendrite, likely of bipolar cell origin. GluR4-IR was restricted to dendrites of OFF-bipolar cells that innervated rod and cone terminals. NR2B-IR was restricted to a subtype of cone ON-bipolar cell. mGluR1α-IR was restricted to ON mixed rod/cone (Mb) bipolar cells whose dendrites innervated rod and cone synaptic terminals. The presence of mGluR1α on Mb bipolar cell dendrites is consistent with a role in retrograde endocannabinoid suppression. The subunit composition of glutamate receptors should affect the kinetics and pharmacology of these cells to glutamate receptor activation.     

Expression profile of AMPA receptor subunit mRNA in single adult rat brain and spinal cord neurons in situ

The differential expression of AMPA receptor subunit mRNA was analyzed in the adult rat brain and spinal cord using quantitative RT-PCR with laser capture microdissection. The expression of all four AMPA receptor subunits was demonstrable, with the mRNA expression level for GluR2 being the highest in all the brain areas and neuronal subsets examined. Both the absolute expression level of GluR2 mRNA and its expression relative to the other subunits were the lowest in motoneurons. The unique AMPA receptor expression profile of motoneurons may render them selectively vulnerable to AMPA receptor-mediated excitotoxicity.     

Early onset of NMDA receptor GluR epsilon 1 (NR2A) expression and its abundant postsynaptic localization in developing motoneurons of the mouse hypoglossal nucleus

Oro-facial sensorimotor function conducted by the brainstem is vital to newborn mammals, and N-methyl-D-aspartate (NMDA) receptors play an important role in the regulation. Here we examined the expression of NMDA receptor subunits in the mouse hypoglossal nucleus from embryonic day 13 (E13) through postnatal day 21 (P21). Compared with other brainstem regions, early onset of GluRepsilon1 (NR2A) mRNA expression was conspicuous to the embryonic hypoglossal nucleus. The expression peaked at P1-P7, when other brainstem regions just started to express it. At P1, GluRepsilon1 subunit was localized to asymmetrical synapses on motoneuron dendrites, particularly, on the postsynaptic junction membrane. In developing motoneurons, expressions of GluRepsilon2 (NR2B), GluRepsilon4 (NR2D), and GluRzeta1 (NR1) mRNAs were accompanied. Until P21, however, all of these subunits were down-regulated with particular reduction for GluRepsilon2 and GluRepsilon4 mRNAs. Similar patterns of temporal expressions were observed in motoneurons of other brainstem motor nuclei. Taking that high levels of GluRepsilon1, GluRepsilon2, and GluRzeta1 subunits are also found in the adult hippocampus and cerebral cortex, it can be assumed that NMDA receptors in developing motoneurons are highly potent and potentially involved in structural and functional development of the brainstem motor system.     

Glutamate receptor expression in multiple sclerosis lesions

Blockade of receptors for the excitatory neurotransmitter glutamate ameliorates neurological clinical signs in models of the CNS inflammatory demyelinating disease multiple sclerosis (MS). To investigate whether glutamate excitoxicity may play a role in MS pathogenesis, the cellular localization of glutamate and its receptors, transporters and enzymes was examined. Expression of glutamate receptor (GluR) 1, a Ca(++)-permeable ionotropic AMPA receptor subunit, was up-regulated on oligodendrocytes in active MS lesion borders, but Ca(++)-impermeable AMPA GluR2 subunit levels were not increased. Reactive astrocytes in active plaques expressed AMPA GluR3 and metabotropic mGluR1, 2/3 and 5 receptors and the GLT-1 transporter, and a subpopulation was immunostained with glutamate antibodies. Activated microglia and macrophages were immunopositive for GluR2, GluR4 and NMDA receptor subunit 1. Kainate receptor GluR5-7 immunostaining showed endothelial cells and dystrophic axons. Astrocyte and macrophage populations expressed glutamate metabolizing enzymes and unexpectedly the EAAC1 transporter, which may play a role in glutamate uptake in lesions. Thus, reactive astrocytes in MS white matter lesions are equipped for a protective role in sequestering and metabolizing extracellular glutamate. However, they may be unable to maintain glutamate at levels low enough to protect oligodendrocytes rendered vulnerable to excitotoxic damage because of GluR1 up-regulation.     

Glutamate-induced currents reveal three functionally distinct NMDA receptor populations in rat dorsal horn - effects of peripheral nerve lesion and inflammation

The importance of the N-methyl-D-aspartate (NMDA) receptor in various painful conditions is well established. The effects of peripheral nerve lesion or joint inflammation, as models of different pain states, on NMDA receptor-mediated currents and NMDA receptor subunit mRNA expression were therefore studied in acutely dissociated neurones from the rat spinal cord dorsal horn. In the neuronal population from control rats, all four NR2 subunits and both NR1 splice variants assayed were detected. A majority of neurones expressed mRNA for more than one NR2 subunit, and some neurones expressed all four NR2 subunits as well as both NR1 splice variants. The NR2B subunit was the most commonly expressed, while the NR2C was the rarest. Following nerve lesion, fewer neurones expressed NR2A compared to the control. The dose-response curve for glutamate-evoked NMDA receptor-mediated currents in the neurones was best described by a three-component fit, suggesting that three functionally distinct NMDA receptor populations are present in the dorsal horn. Minor changes in the dose-response curve after nerve lesion could not be ascribed with certainty to the lesion. Changes in other parameters of NMDA receptor-mediated currents were observed neither after nerve lesion nor after joint inflammation.In summary, the present work demonstrates that single dorsal horn neurones express mRNA for several NMDA receptor subunits. The glutamate dose-response curves indicate that there are three major types of NMDA receptors present in dorsal horn neurones. We also report a reduced expression of NR2A following peripheral nerve lesion.     

Cellular and subcellular distribution of the amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunit GluR2 in the rat dorsal vagal complex

Amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) type glutamate receptors are ligand gated ion channels made up of various combinations of four subunits termed GluR1-4. The GluR2 subunit controls several key features of the receptor including calcium permeability and inward rectification. In the present study, we analysed by immunocytochemistry the cellular and subcellular distribution of the GluR2 subunit in neurons of the dorsal vagal complex of the rat. GluR2 immunoreactivity was found both in the neuropile and in neuronal cell bodies. Perikaryal staining was strong in the dorsal motor nucleus of the vagus nerve and moderate in the medial part of the nucleus tractus solitarii as well as in the area postrema. The lateral part of the nucleus tractus solitarii was almost devoid of immunoreactivity except for the interstitial subnucleus which was filled with numerous strongly immunoreactive perikarya and large cell processes. Ultrastructural examination was carried out in the interstitial subnucleus. Peroxidase staining indicative of GluR2 immunoreactivity was observed in neuronal cell bodies and dendrites. No labeled axon terminal or glial cell body was found. Additional experiments performed using pre-embedding immunogold showed that most of the labeling in immunoreactive dendrites was intracytoplasmic.These results indicate that GluR2 immunoreactivity is differentially distributed among neurons in the dorsal vagal complex, thereby suggesting differences in the functional properties of AMPA receptors between neuronal populations. These results also suggest that AMPA receptors, at least those containing the GluR2 subunit, have no major role as presynaptic receptors within this region. Finally, they indicate the existence of large intracellular pools of GluR2 subunits within dendrites of immunoreactive neurons.     

Cholinergic terminals in the ventral horn of adult rat and cat: Evidence that glutamate is a cotransmitter at putative interneuron synapses but not at central synapses of motoneurons

Until recently it was generally accepted that the only neurotransmitter to be released at central synapses of somatic motoneurons was acetylcholine. However, studies on young mice (P0-10) have provided pharmacological evidence indicating that glutamate may act as a cotransmitter with acetylcholine at synapses between motoneurons and Renshaw cells. We performed a series of anatomical experiments on axon collaterals obtained from intracellularly labeled motoneurons from an adult cat and labeled by retrograde transport in adult rats to determine if glutamate is co-localized with acetylcholine by these terminals. We could find no evidence for the presence of vesicular glutamate transporters in motoneuron axon terminals of either species. In addition, we were unable to establish any obvious relationship between motoneuron terminals and the R2 subunit of the AMPA receptor (GluR2). However we did observe a population of cholinergic terminals in lamina VII which did not originate from motoneurons but were immunoreactive for the vesicular glutamate transporter 2 and formed appositions to GluR2 subunits. These were smaller than motoneuron terminals and, unlike them, formed no relationship with Renshaw cells. The evidence suggests that glutamate does not act as a cotransmitter with acetylcholine at central synapses of motoneurons in the adult cat and rat. However, glutamate is present in a population of cholinergic terminals which probably originate from interneurons where its action is via an AMPA receptor.     

Synergistic interactions of dopamine D1 and glutamate NMDA receptors in rat hippocampus and prefrontal cortex: Involvement of ERK1/2 signaling

Interactions between dopamine and glutamate receptors are essential for the prefrontal cortical (PFC) and hippocampal cognitive functions. In order to understand the molecular basis of dopamine/glutamate interactions in rat PFC and hippocampus, we investigated (a) the effect of in vitro dopamine D1 receptor stimulation on glutamate N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits' phosphorylation and (b) the signal transduction pathway underlying these interactions, by examining the involvement of D1–extracellular regulated kinase 1/2 (ERK1/2) and D1/protein kinase A (PKA)/dopamine- and cyclic AMP-regulated phosphoprotein-32 (DARPP-32) signaling pathways. Furthermore, we compared the D1/NMDA/AMPA receptor interactions seen in PFC and hippocampus with those appearing in striatum, in which the D1 receptors' density is the highest within the mammalian brain. Our results showed that stimulation of D1 receptor by the specific agonist SKF38393 (10 μM) in PFC and hippocampal slices significantly increased the phosphorylation state of NR1ser897 and NR2Bser1303 subunits of NMDA receptor and of the GLUR1 (ser831 and ser845) subunit of AMPA receptor, as well as of ERK1/2, but not of DARPP-32. Interestingly, co-stimulation of D1 and NMDA receptors with an ineffective dose of SKF38393 (2 μM) and NMDA (5 μM) respectively, elevated further the phosphorylation level of NMDA and AMPA receptor subunits, as well as of ERK1/2, but not of DARPP-32. The D1- and D1/NMDA-induced phosphorylations were totally inhibited by SL327 (specific ERK1/2 inhibitor). Conversely, in striatal slices our data confirm that the D1-mediated phosphorylation of NMDA and AMPA receptor subunits relies on D1/PKA/DARPP-32 signaling. In conclusion, in PFC and hippocampus: (a) a strong synergistic interaction of D1 and NMDA receptors exists, which results in a significant ERK1/2 pathway activation, (b) the D1 and the D1/NMDA receptor-induced phosphorylation of NMDA and AMPA receptor subunits seems to rely on ERK1/2 signaling and could to some extent underlie the enhancement of NMDA and AMPA receptor currents mediated by D1 receptor activation.     

Aging and cholinergic deafferentation alter GluR1 expression in rat frontal cortex

Previously, we demonstrated that plasticity of frontal cortex is altered in aging rats: lesions of the nucleus basalis magnocellularis (NBM) produce larger declines in dendritic morphology in frontal cortex of aged rats compared to young adults. Cholinergic afferents from the NBM modulate glutamatergic transmission in neocortex, and glutamate is known to be involved in dendritic plasticity. To begin to identify possible mechanisms underlying age-related differences in plasticity after NBM lesion, we assessed the effect of cholinergic deafferentation on expression of the AMPA receptor subunit GluR1 in frontal cortex of young adult and aging rats. Young adult, middle-aged, and aged rats received sham or 192 IgG-saporin lesions of the NBM, and an unbiased stereological technique was used to estimate the total number of intensely GluR1-immunopositive neurons in layer II-III of frontal cortex. While the number of GluR1-positive neurons was increased in both middle-aged and aged rats, lesions markedly increased the number of intensely GluR1-immunopositive neurons in frontal cortex of young adult rats only. This age-related difference in lesion-induced expression of AMPA receptor subunit protein could underlie the age-related differences in dendritic plasticity after NBM lesions.     

Distribution of glutamate and preproenkephalin messenger RNAs following transient focal cerebral ischemia

Middle cerebral artery occlusion may result in increased activation of N-methyl-D-aspartate- or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type receptors by glutamate and lead to neuronal cell death. To characterize molecular events that precede cell death following transient focal ischemia, in situ hybridization histochemistry was used to measure levels of glutamate receptor subunit 1 (GluR1), GluR2, GluR3, N-methyl-D-aspartate receptor subunit 1 (NR1) and preproenkephalin messenger RNAs in adult rats at various recirculation times (1.5, 3 and 24 h) following a 90-min period of middle cerebral artery occlusion. At 1.5 and 3 h recirculation, autoradiography showed pronounced but differential decreases in AMPA, NR1 and preproenkephalin messenger RNA expression throughout the infarcted ipsilateral striatum. Non-uniform patterns of in situ hybridization grains emerged such that many striatal neurons were depleted of AMPA and preproenkephalin messenger RNAs, while others retained control levels. In cortical regions destined to undergo infarction, GluR2 and NR1 messenger RNAs were preferentially reduced relative to the contralateral side (to 75+/-8.5% and 66+/-4.5%, respectively); GluR1, GluR3 and preproenkephalin messenger RNAs were unaltered. At 24 h recirculation, depletion of striatal and cortical messenger RNAs became less selective. GluR3 and preproenkephalin messenger RNAs were up-regulated in ipsilateral spared regions of the striatum, and GluR1 and GluR2 messenger RNAs increased bilaterally in the cingulate cortex and in selective nuclei of the amygdala. Histological cell death or neurodegeneration was not detected in areas of reduced glutamate and preproenkephalin messenger RNA expression in either the ipsilateral striatum or cortex before 24 h. These findings suggest that complex and long-lasting decreases in messenger RNA expression occur prior to significant cell loss in regions destined to undergo infarction. Increased formation of Ca2+-permeable AMPA receptor assemblies may occur in "unspared" and "spared" regions via different mechanisms and contribute to alterations in post-ischemic synaptic activity. The possibility arises that there may be altered relationships between glutamatergic and enkephalin synapses, since the dorsolateral striatum, where preproenkephalin messenger RNA expression is acutely reduced, receives innervation by the affected ipsilateral cortical region.