Motoneuron glutamatergic receptor expression following recovery from cervical spinal hemisection

Cervical spinal hemisection at C2 (SH) removes premotor drive to phrenic motoneurons located in segments C3–C5 in rats. Spontaneous recovery of ipsilateral diaphragm muscle activity is associated with increased phrenic motoneuron expression of glutamatergic N‐methyl‐D‐aspartate (NMDA) receptors and decreased expression of α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐proprionic acid (AMPA) receptors. Glutamatergic receptor expression is regulated by tropomyosin‐related kinase receptor subtype B (TrkB) signaling in various neuronal systems, and increased TrkB receptor expression in phrenic motoneurons enhances recovery post‐SH. Accordingly, we hypothesize that recovery of ipsilateral diaphragm muscle activity post‐SH, whether spontaneous or enhanced by adenoassociated virus (AAV)‐mediated upregulation of TrkB receptor expression, is associated with increased expression of glutamatergic NMDA receptors in phrenic motoneurons. Adult male Sprague‐Dawley rats underwent diaphragm electromyography electrode implantation and SH surgery. Rats were injected intrapleurally with AAV expressing TrkB or GFP 3 weeks before SH. At 14 days post‐SH, the proportion of animals displaying recovery of ipsilateral diaphragm activity increased in AAV–TrkB‐treated (9/9) compared with untreated (3/5) or AAV–GFP‐treated (4/10; P < 0.027) animals. Phrenic motoneuron NMDA NR1 subunit mRNA expression was approximately fourfold greater in AAV–TrkB‐ vs. AAV–GFP‐treated SH animals (P < 0.004) and in animals displaying recovery vs. those not recovering (P < 0.005). Phrenic motoneuron AMPA glutamate receptor 2 (GluR2) subunit mRNA expression decreased after SH, and, albeit increased in animals displaying recovery vs. those not recovering, levels remained lower than control. We conclude that increased phrenic motoneuron expression of glutamatergic NMDA receptors is associated with spontaneous recovery after SH and enhanced recovery after AAV–TrkB treatment. J. Comp. Neurol. 525:1192–1205, 2017. © 2016 Wiley Periodicals, Inc.     

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.     

Nucleus- and cell-specific expression of NMDA and non-NMDA receptor subunits in monkey thalamus

Subcortical and corticothalamic inputs excite thalamic neurons via a diversity of glutamate receptor subtypes. Differential expression of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainate, and N-methyl-D-aspartate (NMDA) receptor subunits (GluR1–4; GluR5–7; NR1, NR2A–D) on a nucleus- and cell type-specific basis was examined by quantitative in situ hybridization histochemistry and by immunocytochemical staining for receptor subunits and colocalized γ-aminobutyric acid (GABA) or calcium binding proteins. Levels of NMDA subunit expression, except NR2C, are higher than for the most highly expressed AMPA (GluR1,3,4) and kainate (GluR6) receptor subunits. Expression of NR2C, GluR2, GluR5, and GluR7 is extremely low. Major differences distinguish the reticular nucleus and the dorsal thalamus and, within the dorsal thalamus, the intralaminar and other nuclei. In the reticular nucleus, GluR4 is by far the most prominent, and NMDA receptors are at comparatively low levels. In the dorsal thalamus, NMDA receptors predominate. Anterior intralaminar nuclei are more enriched in GluR4 and GluR6 subunits than other nuclei, whereas posterior intralaminar nuclei are enriched in GluR1 and differ among themselves in relative NMDA receptor subunit expression. GABAergic intrinsic neurons of the dorsal thalamus express much higher levels of GluR1 and GluR6 receptor subunits than do parvalbumin- or calbindin-immunoreactive relay cells and low or absent NMDA receptors. Relay cells are dominated by NMDA receptors, along with GluR3 and GluR6 subunits not expressed by GABA cells. High levels of NR2B are found in astrocytes. Differences in NMDA and non-NMDA receptor profiles will affect functional properties of the thalamic GABAergic and relay cells. J. Comp. Neurol. 397:371–393, 1998. © 1998 Wiley-Liss, Inc.     

Cultured motor neurons possess calcium-permeable AMPA/kainate receptors

We examined the biology of AMPA/kainate-induced motor neuron degeneration using dissociated spinal cord cultures and motor neuron-specific antibodies which enable characterization of individual motor neurons in culture. Cobalt, which is thought to pass through Ca2+-permeable AMPA/kainate receptors following kainate exposure, labeled motor neurons in spinal cord cultures. The analysis of AMPA subunit distribution in dissociated motor neurons revealed a unique pattern of glutamate receptor (GluR) subunits in those cells; the GluR1 subunit was found in all spinal cord neurons, but the GluR2 subunit was not found in identified dissociated motor neurons. These data suggest that selective sensitivity of motor neurons to non-NMDA receptor activation is due, at least in part, to the presence of Ca2+-permeable AMPA/kainate receptors.     

Immunocytochemical localization of glutamate receptor subunits in the brain stem and cerebellum of the turtle Chrysemys picta

The regional distribution of ionotropic (AMPA and NMDA) and metabotropic (mGluR1alpha) glutamate receptor subunits was examined in the brain stem and cerebellum of the pond turtle, Chrysemys picta, by using immunocytochemistry and light microscopy. Subunit-specific antibodies that recognize NMDAR1, GluR1, GluR4, and mGluR1alpha were used to identify immunoreactive nuclei in the brain stem and cerebellum. Considerable immunoreactivity in the turtle brain stem and cerebellum was observed with regional differences occurring primarily in the intensity of staining with the antibodies. The red nucleus, lateral reticular nucleus and cerebellum labeled intensely for NMDAR1 and moderately for GluR1. The cerebellum also labeled strongly for mGluR1alpha. All of the cranial nerve nuclei labeled intensely for NMDAR1 and to varying degrees for GluR1, GluR4, and mGluR1alpha. Counterstaining revealed the presence of neuronal somata where there were no immunoreactive neurons in individual nuclei. This finding suggests that there are subpopulations of immunoreactive neurons within a given nucleus that bear different glutamate receptor subunit compositions. The results suggest that the glutamate receptor subunit distribution in the brain stem and cerebellum of turtles is similar to that reported for rats. Additionally, there is considerable colocalization of NMDA and AMPA receptors as revealed by light microscopy. These results have implications for the organization of neural circuits that control motor behavior in turtles, and, generally, for the function of brain stem and cerebellar neural circuits in vertebrates.     

Phrenic motoneuron expression of serotonergic and glutamatergic receptors following upper cervical spinal cord injury

Following cervical spinal cord injury at C(2) (SH hemisection model) there is progressive recovery of phrenic activity. Neuroplasticity in the postsynaptic expression of neurotransmitter receptors may contribute to functional recovery. Phrenic motoneurons express multiple serotonergic (5-HTR) and glutamatergic (GluR) receptors, but the timing and possible role of these different neurotransmitter receptor subtypes in the neuroplasticity following SH are not clear. The current study was designed to test the hypothesis that there is an increased expression of serotonergic and glutamatergic neurotransmitter receptors within phrenic motoneurons after SH. In adult male rats, phrenic motoneurons were labeled retrogradely by intrapleural injection of Alexa 488-conjugated cholera toxin B. In thin (10μm) frozen sections of the spinal cord, fluorescently-labeled phrenic motoneurons were visualized for laser capture microdissection (LCM). Using quantitative real-time RT-PCR in LCM samples, the time course of changes in 5-HTR and GluR mRNA expression was determined in phrenic motoneurons up to 21 days post-SH. Expression of 5-HTR subtypes 1b, 2a and 2c and GluR subtypes AMPA, NMDA, mGluR1 and mGluR5 was evident in phrenic motoneurons from control and SH rats. Phrenic motoneuron expression of 5-HTR2a increased ~8-fold (relative to control) at 14 days post-SH, whereas NMDA expression increased ~16-fold by 21-days post-SH. There were no other significant changes in receptor expression at any time post-SH. This is the first study to systematically document changes in motoneuron expression of multiple neurotransmitter receptors involved in regulation of motoneuron excitability. By providing information on the neuroplasticity of receptors expressed in a motoneuron pool that is inactivated by a higher-level spinal cord injury, appropriate pharmacological targets can be identified to alter motoneuron excitability.     

Repeated 4-aminopyridine induced seizures diminish the efficacy of glutamatergic transmission in the neocortex

Systemic administration of the potassium channel blocker 4-aminopyridine (4-AP) elicits acute convulsions. Synchronized tonic–clonic activity develops during the first hour after the treatment. However, subsequent chronic spontaneous seizures do not appear which suggests changes in neuronal excitability. The aim of our present work was to evaluate alterations in the glutamatergic transmission in the somatosensory cortex of rats following daily, brief convulsions elicited by 4-AP treatment. Changes in general neuronal excitability and pharmacological sensitivity of glutamate receptors were tested in ex vivo electrophysiological experiments on brain slices. In parallel studies quantitative changes in subunit composition of glutamate receptors were determined with immunohistoblot technique, together with the analysis of kainate induced Co²⁺ uptake. The results of our coordinated electrophysiological, receptor-pharmacological and histoblot studies demonstrated that repeated, daily, short convulsions resulted in a significant decrease of the general excitability of the somatosensory cortex together with changes in ionotropic glutamate receptor subunits. The relative inhibitory effect of the AMPA receptor antagonist, however, did not change. The NMDA receptor antagonist exerted somewhat stronger effect in the slices from convulsing animals. 4-AP pretreatment resulted in the attenuation of kainate induced Co²⁺ uptake, which suggests either reduction in non-NMDA receptors numbers or reduction in their Ca²⁺ permeability. Repeated seizures decreased GluR1–4 AMPA receptor subunit levels in all cortical layers with a relaitve increase in GluR1 subunits. While the principle NR1 NMDA receptor subunit showed no significant change, the staining density of NR2A subunit increased. These changes in ionotropic glutamate receptors are consistent with reduced excitability at glutamatergic synapses following repeated 4-AP induced seizures.     

NMDA and non-NMDA receptor-mediated excitotoxicity are potentiated in cultured striatal neurons by prior chronic depolarization.

The excitatory input from cortex and/or thalamus to striatum appears to promote the maturation of glutamate receptors on striatal neurons, but the mechanisms by which it does so have been uncertain. To explore the possibility that the excitatory input to striatum might influence glutamate receptor maturation on striatal neurons, at least in part, by its depolarizing effect on striatal neurons, we examined the influence of chronic KCl depolarization on the development of glutamate receptor-mediated excitotoxic vulnerability and glutamate receptors in cultured striatal neurons. Dissociated striatal neurons from E17 rat embryos were cultured for 2 weeks in Barrett's medium containing either low (3 mM) or high (25 mM) KCl. The vulnerability of these neurons to NMDA receptor agonists (NMDA and quinolinic acid), non-NMDA receptor agonists (AMPA and KA), and a metabotropic glutamate receptor agonist (trans-ACPD) was examined by monitoring cell loss 24 h after a 1-h agonist exposure. We found that high-KCl rearing potentiated the cell loss observed with 500 microM NMDA or 250 microM KA and yielded cell loss with 250 microM AMPA that was not evident under low KCl rearing. In contrast, neither QA up to 5 mM nor trans-ACPD had a significant toxic effect in either KCl group. ELISA revealed that chronic high KCl doubled the abundance of NMDA NR2A/B, AMPA GluR2/3, and KA GluR5-7 receptor subunits on cultured striatal neurons and more than doubled AMPA GluR1 and GluR4 subunits, but had no effect on NMDA NR1 subunit levels. These receptor changes may contribute to the potentiation of NMDA and non-NMDA receptor-mediated excitotoxicity shown by these neurons following chronic high-KCl rearing. Our studies suggest that membrane depolarization produced by corticostriatal and/or thalamostriatal innervation may be required for maturation of glutamate receptors on striatal neurons, and such maturation may be important for expression of NMDA and non-NMDA receptor-mediated excitotoxicity by striatal neurons. Striatal cultures raised under chronically depolarized conditions may, thus, provide a more appropriate culture model to study the role of NMDA or non-NMDA receptor subtypes in excitotoxicity in striatum.     

Ionotropic and metabotropic glutamate receptor protein expression in glioneuronal tumours from patients with intractable epilepsy

Glioneuronal tumours are an increasingly recognized cause of chronic pharmaco-resistant epilepsy. In the present study the immunocytochemical expression of various glutamate receptor (GluR) subtypes was investigated in 41 gangliogliomas (GG) and 16 dysembryoplastic neuroepithelial tumours (DNT) from patients with intractable epilepsy. Immunocytochemistry with antibodies specific for ionotropic NR1, NR2A/B (NMDA) GluR1, GluR2 (AMPA), GluR5-7 (kainate), and metabotropic mGluR1, mGluR2-3, mGluR5, mGluR7a subtypes demonstrated in both GG and DNT the presence of an highly differentiated neuronal population, containing subunits from each receptor class. More than 50% of tumours contained a high percentage of neuronal cells immunolabelled for NMDA, AMPA and kainate receptor subunits. A high percentage of neurones showed strong expression of NR2A-B, which co-localized with NR1. Group I mGluRs (mGluR1 and mGluR5) were highly represented in the neuronal component of the tumours. Immunolabelling for several GluRs was also present in the glial component. Increased expression of mGluR2-3, mGluR5 and GluR5-7 was observed in reactive astrocytes in the perilesional zone compared to normal cortex. The neurochemical profile of glioneuronal tumours, with high expression of specific GluR subtypes, supports the central role of glutamatergic transmission in the mechanisms underlying the intrinsic and high epileptogenicity of these lesions.     

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.     

AMPA glutamatergic receptor-immunoreactive subunits are expressed in lumbosacral neurons of the spinal cord and neurons of the dorsal root and pelvic ganglia controlling pelvic functions in the rat

Sacral preganglionic neurons innervate the pelvic organs via a relay in the major pelvic ganglion. Pudendal motoneurons innervate striated muscles and sphincters of the lower urinary, genital and digestive tracts. The activity of these spinal neurons is regulated by sensory afferents of visceral and somatic origins. Glutamate is released by sensory afferents in the spinal cord, and interacts with a variety of receptor subtypes. The aim of the present study was to investigated the presence of AMPA glutamate receptor subunits (GluR1-GluR4) in the neural network controlling the lower urogenital and digestive tracts of male rats. We performed double-immunohistochemistry directed against a neuronal tracer, the cholera toxin beta subunit (Ctbeta) and each of the four receptor subunits. GluR1, GluR2 and GluR3 subunits were present in many sacral preganglionic neurons retrogradely labelled with Ctbeta applied to the pelvic nerve, and in some dorsolateral and dorsomedian motoneurons retrogradely labelled with Ctbeta injected in ischiocavernosus and bulbospongiosus muscles. The four subunits were detected in postganglionic neurons of the major pelvic ganglion retrogradely labelled with Ctbeta injected in the corpus cavernosum, and in some somata of sensory afferents of the L6 dorsal root ganglion labelled with Ctbeta applied to the dorsal penile nerve or injected in corpus cavernosum. The results provide a detailed knowledge of the neural targets expressing the various AMPA receptor subunits and suggest that part of the neural network that controls pelvic organs, including sensory afferents and postganglionic neurons, is sensitive to glutamate through the whole family of AMPA subunits.     

Hormonal regulation of glutamate receptor gene expression in the anteroventral periventricular nucleus of the hypothalamus.

Glutamate plays an important role in mediating the positive feedback effects of ovarian steroids on gonadotropin secretion, and the preoptic region of the hypothalamus is a likely site of action of glutamate. The anteroventral periventricular nucleus (AVPV) of the preoptic region is an essential part of neural pathways mediating hormonal feedback on gonadotropin secretion, and it appears to provide direct inputs to gonadotropin releasing hormone (GnRH)-containing neurons. Immunohistochemistry and in situ hybridization were used in this study to define the distribution and hormonal regulation of glutamate receptor subtypes in the AVPV of juvenile female rats. Neurons that express the NMDAR1 receptor subtype are abundant in the AVPV, as are cells that express AMPA receptor subtypes (GluR1, GluR2, and GluR3 but not GluR4), and the AVPV appears to contain a dense plexus of NMDAR1-immunoreactive presynaptic terminals. However, AVPV neurons do not seem to express detectable levels of kainate receptor (GluR5, GluR6, and GluR7) or metabotropic receptor (mGluR1-6) subtypes. Treatment of ovariectomized juvenile rats with estradiol induced expression of GluR1 mRNA but did not alter levels of GluR2 or GluR3 mRNA. Treatment of estrogen-primed ovariectomized juvenile rats with progesterone caused an initial increase in GluR1 mRNA expression, followed by a small decrease 24 hr after treatment. In contrast, estrogen appears to suppress levels of NMDAR1 mRNA in the AVPV, which remained unchanged after progesterone treatment. Thus, one mechanism whereby ovarian steroids may provide positive feedback to GnRH neurons is by altering the sensitivity of AVPV neurons to glutamatergic activation.     

Glutamate receptor agonist kainate enhances primary dendrite number and length from immature mouse cortical neurons in vitro

Glutamate is an important regulator of dendrite development that may inhibit, (during ischemic injury), or facilitate (during early development) dendrite growth. Previous studies have reported mainly on the N-methyl-D-aspartate (NMDA) receptor-mediated dendrite growth-promoting effect of glutamate. In this study, we examined how the non-NMDA receptor agonist kainate influenced dendrite growth. E18 mouse cortical neurons were grown for 3 days in vitro and immunolabeled with anti-microtubule-associated protein 2 (MAP2) and anti-neurofilament (NF-H), to identify dendrites and axons, respectively. Exposure of cortical neurons to kainate increased dendrite growth without affecting neuron survival. This effect was dose-dependent, reversible and blocked by the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA)/kainate receptor antagonist NBQX and the low-affinity kainate receptor antagonist NS-102, but not by the AMPA receptor antagonist CFM-2. In addition, the NMDA receptor antagonist MK-801 had no effect on kainate-induced dendrite growth. Immunolabeling and Western blot analysis of kainate receptors using antibodies against the GluR6 and KA2 subunits, demonstrated that the immature cortical neurons used in this study express kainate receptor proteins. These results suggest that kainate-induced non-NMDA receptor activation promotes dendrite growth, and in particular primary dendrite number and length, from immature cortical neurons in vitro, and that kainate receptors may be directly involved in this process. Furthermore, these data support the possibility that like NMDA receptors, kainate receptor activation may also contribute to early neurite growth from cortical neurons in vitro.     

Ultrastructural analysis of NMDA,AMPA, and kainate receptors on unmyelinated and myelinated axons in the periphery

The present study determines the proportions of unmyelinated cutaneous axons at the dermal–epidermal junction in glabrous skin and of myelinated and unmyelinated axons in the sural and medial plantar nerves that immunostain for subunits of the ionotropic glutamate receptors. Approximately 20% of the unmyelinated cutaneous axon profiles at the dermal–epidermal junction immunostain for either N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or kainate receptor subunits. These findings are consistent with previous observations that NMDA and non-NMDA antagonists ameliorate nociceptive behaviors that result from noxious peripheral stimulation. In the sural nerve, where the large majority of myelinated fibers are sensory, approximately half of the myelinated axon profiles immunostain for the NMDA receptor 1 (R1) subunit, 28% immunostain for the glutamate receptor 1 (GluR1) AMPA subunit, and 11% for the GluR5,6,7 kainate subunits. Even higher proportions immunostain for these receptors in the medial plantar nerve, a mixed sensory and motor nerve. In the sural nerve, 20% of the unmyelinated axon profiles immunostain for NMDAR1 and only 7% label for GluR1 or GluR5,6,7. Because the sural nerve innervates hairy skin, these data suggest that glutamate will activate a higher proportion of unmyelinated axons in glabrous skin than in hairy skin. Measurements of fiber diameters indicate that all sizes of myelinated axon profiles, including Aδ and Aβ, are positively labeled for the ionotropic receptors. The presence of glutamate receptors on large-diameter myelinated axons suggests that these mechanosensitive receptors, presumably transducing touch and pressure, may also respond to local glutamate and thus be chemosensitive. J. Comp. Neurol. 391: 78–86, 1998. © 1998 Wiley-Liss, Inc.     

Ionotropic glutamate receptor expression in preganglionic neurons of the rat inferior salivatory nucleus

Glutamate receptor (GluR) subunit composition of inferior salivatory nucleus (ISN) neurons was studied by immunohistochemical staining of retrogradely labeled neurons. Preganglionic ISN neurons innervating the von Ebner or parotid salivary glands were labeled by application of a fluorescent tracer to the lingual-tonsilar branch of the glossopharyngeal nerve or the otic ganglion respectively. We used polyclonal antibodies to glutamate receptor subunits NR1, NR2A, NR2B, (NMDA receptor subunits) GluR1, GluR2, GluR3, GluR4 (AMPA receptor subunits), and GluR5-7, KA2 (kainate receptor subunits) to determine their expression in ISN neurons. The distribution of the NMDA, AMPA and kainate receptor subunits in retrogradely labeled ISN neurons innervating the von Ebner and parotid glands was qualitatively similar. The percentage of retrogradley labeled ISN neurons innervating the parotid gland expressing the GluR subunits was always greater than those innervating the von Ebner gland. For both von Ebner and parotid ISN neurons, NR2A subunit staining had the highest expression and the lowest expression of GluR subunit staining was NR2B for von Ebner ISN neurons and GluR1 for parotid ISN neurons. The percentage of NR2B and GluR4 expressing ISN neurons was significantly different between the two glands. The percentage of ISN neurons that expressed GluR receptor subunits ranged widely indicating that the distribution of GluR subunit expression differs amongst the ISN neurons. While ISN preganglionic neurons express all the GluR subunits, differences in the percentage of ISN neurons expression between neurons innervating the von Ebner and parotid glands may relate to the different functional roles of these glands.     

A new pyrrolyl-quinoxalinedione series of non-NMDA glutamate receptor antagonists: pharmacological characterization and comparison with NBQX and valproate in the kindling model of epilepsy

Antagonists at the ionotropic non-NMDA [AMPA (amino-methyl proprionic acid)/kainate] type of glutamate receptors have been suggested to possess several advantages compared to NMDA (N-methyl-d -aspartate) receptor antagonists, particularly in terms of risk/benefit ratio, but the non-NMDA receptor antagonists available so far have not fulfilled this promise. From a large series of pyrrolyl-quinoxalinedione derivatives, we selected six new competitive non-NMDA receptor antagonists. The basis of selection was high potency and selectivity for AMPA and/or kainate receptors, high in vivo potency after systemic administration, and an acceptable ratio between neuroprotective or anticonvulsant effects and adverse effects. Pharmacological characteristics of these novel compounds are described in this study with special emphasis on their effects in the kindling model of temporal lobe epilepsy, the most common type of epilepsy in humans. In most experiments, NBQX and the major antiepileptic drug valproate were used for comparison with the novel compounds. The novel non-NMDA receptor antagonists markedly differed in their AMPA and kainate receptor affinities from NBQX. Thus, while NBQX essentially did not bind to kainate receptors at relevant concentrations, several of the novel compounds exhibited affinity to rat brain kainate receptors or recombinant kainate receptor subtypes in addition to AMPA receptors. One compound, LU 97175, bound to native high affinity kainate receptors and rat GluR5–GluR7 subunits, i.e. low affinity kainate binding sites, with much higher affinities than to AMPA receptors. All compounds potently blocked AMPA-induced cell death in vitro and, except LU 97175, AMPA-induced convulsions in vivo. In the kindling model, compounds with a high affinity for GluR7 (LU 97175) or compounds (LU 115455, LU 136541) which potently bind to AMPA receptors and low affinity kainate receptor subunits were potent anticonvulsants in the kindling model, whereas the AMPA receptor-selective LU 112313 was the least selective compound in this model, indicating that non-NMDA antagonists acting at both AMPA and kainate receptors are more effective in this model than AMPA receptor-selective drugs. Three of the novel compounds, i.e. LU 97175, LU 115455 and LU 136541, exerted potent anticonvulsant effects without inducing motor impairment in the rotarod test. This combination of actions is thought to be a prerequisite for selective anticonvulsant drug action.     

Dendritic localization of Ca(2+)-permeable AMPA/kainate channels in hippocampal pyramidal neurons.

Although it is well established that cortical and hippocampal gamma-aminobutyric acid (GABA)-ergic neurons generally have large numbers of Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate channels (Ca-A/K channels), their presence on pyramidal neurons is controversial. Ca2+ permeability of AMPA channels is regulated by expression of a particular glutamate receptor subunit (GluR2), which confers Ca2+ impermeability to heteromeric channels. Most electrophysiology studies, as well as in situ hybridization and immunolabeling studies demonstrating expression of GluR2 mRNA or peptide in pyramidal neurons, have provided evidence against the presence of Ca-A/K channels on pyramidal neurons. However, observations that pyramidal neurons often appear to be labeled by kainate-stimulated Co2+ influx (Co2+(+) cells), a histochemical stain that identifies cells possessing Ca-A/K channels, suggests that they may have these channels. The present study futher examines cellular and subcellular distribution of Ca-A/K channels on hippocampal pyramidal neurons in slice as well as in culture. To this end, techniques of kainate-stimulated Co2+ influx labeling, supplemented by AMPA receptor subunit immunocytochemistry and fluorescent imaging of kainate-stimulated intracellular Ca2+ ([Ca2+]i) rises are employed. Co2+ labeling is often seen in pyramidal neuronal dendrites in both slice and in culture. In addition, although GluR1 and 4 staining in these neurons is often seen in the soma and dendrites, GluR2 label, when evident, is generally more restricted to the soma. Finally, measurement of kainate-stimulated [Ca2+]i rises in cultured neurons, assessed by using low affinity Ca2+ indicators in the presence of N-methyl-D-aspartate (NMDA) receptor and voltage-sensitive Ca2+ channel blockade, often shows dendritic rises to precede those in the somata. Thus, these data support the hypothesis that Ca-A/K channels are present in dendritic domains of many pyramidal neurons, and may help to provide resolution of the apparently conflicting data regarding their distribution.