Stimuli which entrain the circadian clock of the neonatal Syrian hamster in vivo regulate the phosphorylation of the transcription factor CREB in the suprachiasmatic nucleus in vitro

Photic resetting of the adult mammalian circadian clock in vivo is associated with phosphorylation of the Ser¹³³ residue of the calcium/cyclic AMP response-element binding-protein (CREB) in the retinorecipient region of the suprachiasmatic nucleus (SCN). Western blotting and immunocytochemistry were used to investigate whether agonists known to reset the clock of neonatal hamsters in vivo are also able to influence the phosphorylation of CREB in the suprachiasmatic hypothalamus in vitro. Antisera raised against synthetic CREB peptide sequences were used to differentiate between total CREB and the Ser¹³³ phosphorylated form of CREB (pCREB). Western blot analysis of proteins isolated from suprachiasmatic tissue of 1-day-old Syrian hamsters revealed bands at ≈ 45 kDa corresponding to total CREB and pCREB. Treatment of the tissue with a mixture of glutamatergic agonists [N-methyl-d -aspartate (NMDA), amino-methyl proprionic acid (AMPA) and kainate, all at 1 μm ], or native glutamate (1 μm ) had no effect on the total CREB signal, but increased the pCREB signal, indicative of agonist-stimulated phosphorylation of CREB on Ser¹³³. A similar effect was seen following treatment of the suprachiasmatic blocks with either dopamine (1 μm ) or forskolin (1 μm ). Simultaneous treatment with melatonin (1 μm ) significantly attenuated stimulation by forskolin. The effect of the agonists on nuclear pCREB-immunoreactivity (-ir) was investigated in primary cultures which contained a mixture of cell types characteristic of the suprachiasmatic nuclei in vivo. Basal expression of nuclear total CREB-ir was high, whereas expression of pCREB-ir was low. Treatment with glutamate (1 μm ) or dopamine (1 μm ) had no effect on total CREB-ir, but increased pCREB-ir in ≈ 50 and 30% of cells, respectively, whereas forskolin (1 μm ) increased pCREB-ir in almost all cells (> 90%). The effects of all three agonists were rapid (< 15 min), and dose and time dependent. Melatonin reversed the effects of forskolin in mixed cultures, but not in pure astrocyte cultures. Dual-immunocytochemistry (ICC) revealed that glutamate (1 μm ) increased nuclear pCREB-ir in cells immunoreactive for microtubule-associated protein II (MAP II-ir), but not other cells, indicating an effect predominantly on neurons. This occurred equally in γ-amino butyric acid (GABA)-ir and non-GABA-ir neurons. Dopamine (1 μm ) was more selective, increasing pCREB-ir only in GABA-ir neurons, whereas forskolin increased pCREB-ir in all cells. The specific stimulation of pCREB-ir in GABA-ir neurons by dopamine was reversed by melatonin, but melatonin had no effect on the increase in pCREB-ir induced in GABA-ir neurons by glutamate. These results demonstrate that agonists known to entrain the circadian clock in vivo modulate phosphorylation of CREB in GABA-ir neurons derived from the neonatal suprachiasmatic nuclei.     

The Role of N-Methyl-D-Aspartate-Type Glutamatergic Neurotransmission in the Photic Induction of Immediate-Early Gene Expression in the Suprachiasmatic Nuclei of the Syrian Hamster

This study investigated the role of N-methyl-D-aspartate (NMDA)-type glutamatergic neurotransmission in mediating the photic induction of immediate-early gene expression in the Suprachiasmatic nucleus (SCN) of the Syrian hamster. Activation of c-fos, c-jun and egr-1 was assessed by immunocytochemical detection of their protein products. To characterize the circadian basis to the inductive effects of light, hamsters were allowed to free-run in constant dim red light and received a 1 h light pulse at different circadian phases relative to activity onset (defined as CT 12). In control animals which did not receive light pulses, c-fos and egr-1 expression was absent or restricted to a small area of the dorsolateral region of the SCN, and expression of c-jun could not be detected in the SCN. In hamsters killed after presentation of a light pulse at either CT 14 or CT 20, there was a marked increase in c-fos and egr-1 immunoreactivities throughout the ventrolateral division of the SCN. In contrast, light pulses given at CT4 or CT 8 failed to activate immediate-early gene expression. Light pulses did not induce c-jun immunoreactivity at any circadian phase tested. Staining for c-fos was maximal 1 h after the start of the light pulse and had started to decline by 2 h. At this later time, c-jun expression was still undetectable. To compare the distribution of retinal afferents with that of c-fos induction, hamsters held on a light schedule of 16 h light: 8 h dark received an intraocular injection of cholera toxin-horseradish peroxidase conjugate 3 days before exposure to a 1 h light pulse given 2 h after lights off. Comparison of adjacent sections processed for c-fos immunoreactivity or for cholera toxin-horseradish peroxidase revealed that light-induced c-fos expression was precisely restricted to retinal terminal fields in the SCN. Light pulses also induced c-fos expression in the retinoreceptive ventral lateral geniculate nucleus and intergeniculate leaflet but not in the retinal fields of the dorsal lateral geniculate nucleus, indicating that the expression of cfos in response to light is spatially specific. The aim of the subsequent experiments was to investigate the role of NMDA-type glutamatergic neurotransmission in mediating the effects of light on c-fos expression in the SCN. To determine whether NMDA had the potential to activate c-fos expression in the SCN, hamsters were infused with 2.5 nmol NMDA or vehicle via an intracerebroventricular (icv) cannula positioned adjacent to the nuclei. In contrast to the effects of light, icv NMDA activated c-fos expression at both CT8 and CT 14. The distribution of immunoreactivity was more widespread than that observed after light, extending throughout the SCN and adjacent hypothalamus. To test whether NMDA receptors had a physiological role in the photic response, hamsters were treated systemically with the non-competitive NMDA antagonist MK801 (dose range 0.6 to 6.0 mg/kg body wt, ip) or vehicle prior to exposure to a 1 h light pulse given at CT 14 or CT 20. Expression of c-fos was still detectable in the dorsolateral SCN but MK801 blocked expression in the ventral portion of the retinoreceptive zone of the SCN. MK801 (10 or 100 nmol) delivered centrally (icv) also prevented light-induced c-fos expression in the ventral region of the SCN bordering the optic chiasm, though staining again persisted in the dorsolateral region. The induction of c-fos by icv NMDA, and the partial blockade of light-induced c-fos expression by the antagonist MK801, are consistent with the hypothesis that glutamate mediates the effects of light on SCN activity. However, the persistent photic induction of c-fos expression in a subfield of retinal afferents following treatment with MK801 suggests that other, non-NMDA-type mechanisms may contribute to photic entrainment.     

A presynaptic N‐methyl‐d‐aspartate autoreceptor in rat hippocampus modulating amino acid release from a cytoplasmic pool

A possible role of the N-methyl-d -aspartate receptor (NMDA-R) as a presynaptic autoreceptor was investigated using Percoll-purified hippocampus nerve terminals (synaptosomes). This preparation contained only a neglectable amount of postsynaptic structures. Two main effects of NMDA were observed. First, NMDA dose-dependently (10–100 μm ) and in the absence of Mg²⁺, stimulated basal release of aspartate and glutamate, but not of GABA. MK801 (10 μm ), an open NMDA-R-channel blocker, reduced this effect even below control levels, indicating endogenous NMDA-R activation. By superfusing synaptosomes, which prevents a tonic receptor occupation, also basal GABA release was stimulated by NMDA. The NMDA-induced potentiation of amino acid superfusate levels was blocked both by MK801 and Mg²⁺ (1 m m ), was slow in onset and returned to baseline after NMDA-removal. The NMDA-effect was also found in the absence of extracellular Ca²⁺, suggesting that amino acids were released from a non-vesicular (cytoplasmic) pool. Secondly, in KCl-depolarized synaptosomes exposed to 1 m m Mg²⁺, NMDA did not affect the release of the amino acids. MK801, however, reduced the KCl-evoked Ca²⁺-independent release of aspartate and glutamate, but not of GABA. l -trans-PDC, the selective inhibitor of the glutamate/aspartate transporter, prevented this MK801-effect, suggesting a coupling between NMDA-Rs and these transporters. These data provide evidence for a presynaptic NMDA autoreceptor in rat hippocampus. We speculate on the role of this NMDA-R to depolarize the presynaptic membrane by Na⁺-entry, which may induce reversal of amino acid transporters and thereby releasing amino acids from a cytoplasmic pool.     

Comparison of excitotoxic profiles of ATPA, AMPA, KA and NMDA in organotypic hippocampal slice cultures

The excitotoxic profiles of (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propionic acid (ATPA), (RS)-2-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainic acid (KA) and N-methyl-D-aspartate (NMDA) were evaluated using cellular uptake of propidium iodide (PI) as a measure for induced, concentration-dependent neuronal damage in hippocampal slice cultures. ATPA is in low concentrations a new selective agonist of the glutamate receptor subunit GluR5 confined to KA receptors and also in high concentrations an AMPA receptor agonist. The following rank order of estimated EC(50) values was found after 2 days of exposure: AMPA (3.7 mM)>NMDA (11 mM)=KA (13 mM)>ATPA (33 mM). Exposed to 30 microM ATPA, 3 microM AMPA and 10 microM NMDA, CA1 was the most susceptible subfield followed by fascia dentata and CA3. Using 8 microM KA, CA3 was the most susceptible subfield, followed by fascia dentata and CA1. In 100 microM concentrations, all four agonists induced the same, maximal PI uptake in all hippocampal subfields, corresponding to total neuronal degeneration. Using glutamate receptor antagonists, like GYKI 52466, NBQX and MK-801, inhibition data revealed that AMPA excitotoxicity was mediated primarily via AMPA receptors. Similar results were found for a high concentration of ATPA (30 microM). In low GluR5 selective concentrations (0.3-3 microM), ATPA did not induce an increase in PI uptake or a reduction in glutamic acid decarboxylase (GAD) activity of hippocampal interneurons. For KA, the excitotoxicity appeared to be mediated via both KA and AMPA receptors. NMDA receptors were not involved in AMPA-, ATPA- and KA-induced excitotoxicity, nor did NMDA-induced excitotoxicity require activation of AMPA and KA receptors. We conclude that hippocampal slice cultures constitute a feasible test system for evaluation of excitotoxic effects and mechanisms of new (ATPA) and classic (AMPA, KA and NMDA) glutamate receptor agonists. Comparison of concentration-response curves with calculation of EC(50) values for glutamate receptor agonists are possible, as well as comparison of inhibition data for glutamate receptor antagonists. The observation that the slice cultures respond with more in vivo-like patterns of excitotoxicity than primary neuronal cultures, suggests that slice cultures are the best model of choice for a number of glutamate agonist and antagonist studies.     

Stimulation of the N-methyl-d-aspartate receptor promotes the biochemical differentiation of cerebellar granule neurons and not astrocytes

Cerebellar granule cells are believed to be glutamatergic, but, as they receive excitatory amino acidergic input from mossy fibers, they also possess N-methyl-d-aspartate (NMDA) receptors. The possible involvement of these NMDA receptors in the biochemical differentiation of cultured granule neurons was studied in terms of the specific activity of phosphate-activated glutaminase, an enzyme important in the synthesis of the putative neurotransmitter pool of glutamate. When the partially depolarized cells were treated with NMDA for the last 3 days (i.e. between 2 and 5 days in vitro), it elevated specific activity of glutaminase in the dose- and time-dependent manners. The half-maximal effect was obtained at about 10 μM NMDA, whereas the maximum concentration, which produced about a 2.7-fold increase in 5-day-old cultures, was about 50 μM NMDA. This increase in glutaminase was completely blocked by the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid, and by the NMDA receptor-linked Ca²⁺ ion channel blockers, MK 801 and Mg²⁺. The effect of NMDA was not related to the survival of the granule cells, as the experiments were carried out before the dependence on high K⁺ for the survival of granule cells develops in culture, and during the period of investigation none of the compounds used compromised the survival of these cells. The enhancement of glutaminase activity was due to an induction in enzyme protein, since it was completely blocked by cycloheximide and actinomycin D. In contrast to granule neurons, the treatment with NMDA had no significant effect on the activity of glutaminase and glutamine synthetase in cultured cerebellar astroglial cells. Our present results on glutaminase enzyme would indicate that an increase in the cellular concentration of free Ca²⁺ mediated through the NMDA induced increase in Ca²⁺ conductance, leads to long term changes in differentiating cerebellar granule neurons, and it is possible that this kind of physiological stimulation of granule cells is normally provided in vivo by the presynaptic glutamatergic mossy fibers.     

Role of NMDA and AMPA glutamate receptors in the induction and the expression of dopamine-mediated sensitization in 6-hydroxydopamine-lesioned rats

Rats with unilateral 6-hydroxydopamine (6-OHDA) lesions exhibit behavioral sensitization following repeated treatment with dopamine agonists, a phenomenon called "priming." Priming has two distinct phases: induction and expression. Priming induction using three injections with D1/D2 agonist apomorphine (0.5 mg/kg) or D1 agonist SKF38393 (10 mg/kg) allows priming expression, robust contralateral rotational behavior and striatal Fos expression, following a challenge with the D2 agonist quinpirole (0.25 mg/kg). We examined the roles of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) glutamate receptors on dopamine agonist priming. Administration of the NMDA antagonist (+)5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine maleate (MK801) (0.5 mg/kg) blocked apomorphine-priming of quinpirole-mediated responses, while MK801 dose-dependently attenuated SKF38393-priming of quinpirole-mediated striatal Fos expression and had no effect on SKF38393-priming of quinpirole-mediated rotational behavior. In contrast, administration of the AMPA antagonist 2,3-dihydroxy-6-nitro-7sulfamoyl-benzo[f]quinoxaline (NBQX) (5 or 10 mg/kg) potentiated apomorphine- and SKF38393-priming of quinpirole-mediated striatal Fos expression, but had no effect on their priming of quinpirole-mediated rotational behavior. In SKF38393-primed 6-OHDA rats, administration of MK801 (0.5 mg/kg) blocked the expression of quinpirole-mediated responses, while administration of NBQX (10 mg/kg) or the noncompetitive AMPA antagonist 4-(8-methyl-9H-1,3-dioxolo[4,5-h][2,3]benzodiazepin-5-yl)-benzenamine dihydrochloride (GYKI52466) (5 or 15 mg/kg) had no effect. These results suggest that NMDA and AMPA glutamate receptors have differing roles in dopamine agonist priming-with NMDA receptors required for D1/D2 priming induction and D2-mediated priming expression, and AMPA receptors inhibiting priming induction of D2-mediated immediate early gene expression in the striatum, but not affecting priming induction of D2-mediated rotational behavior or the expression of D2-mediated responses.     

The effects of excitatory amino acids on intracellular calcium in single mouse striatal neurons in vitro

Using microspectrofluorimetry and the calcium-sensitive dye fura-2, we examined the effect of excitatory amino acids on [Ca2+]i in single striatal neurons in vitro. N-methyl-D-aspartic acid (NMDA) produced rapid increases in [Ca2+]i. These were blocked by DL-2-amino-5-phosphonovaleric acid (AP5), by Mg2+, by phencyclidine, and by MK801. The block produced by Mg2+ and MK801 could be relieved by depolarizing cells with veratridine. When external Ca2+ was removed, NMDA no longer increased [Ca2+]i. Furthermore, the effects of NMDA were not blocked by concentrations of La3+ that blocked depolarization induced rises in [Ca2+]i. Substitution of Na+o by Li+ did not block the effects of NMDA. Concentrations of L-glutamate greater than or equal to 10(-6) M also increased [Ca2+]i. The effects of moderate concentrations of glutamate were blocked by AP5 but not by La3+ or by substitution of Na+ by Li+. The effects of glutamate were blocked by removal of external Ca2+ but were not blocked by concentrations of Mg2+ or MK801 that completely blocked the effects of NMDA. The glutamate analogs kainic acid (KA) and quisqualic acid also increased [Ca2+]i. The effects of KA were blocked by removal of external Ca2+ but not by La3+, Mg2+, MK801, or replacement of Na+ by Li+. Although AP5 was able to block the effects of KA partially, very high concentrations were required. These results may be explained by considering the properties of glutamate-receptor-linked ionophores. Excitatory amino acid induced increases in [Ca2+]i are consistent with the possibility that Ca2+ mediates excitatory amino acid induced neuronal degeneration.     

Subunit selective decrease of AMPA and metabotropic glutamate receptor mRNA expression in rat brain by systemic administration of the NMDA receptor blocker MK-801

Glutamate mediates its effects in mammals through both ionotropic and metabotropic receptors. Antagonists of ionotropic N-methyl-d-aspartate (NMDA) glutamate receptors elicit neuroprotective and neurotropic effects that have been attributed to Ca²⁺ block through the membrane ion channel. Nonetheless, molecular and biochemical effects of NMDA receptor antagonism on other glutamate receptor subunits remain poorly understood. We investigated the effects of acute administration of the noncompetitive NMDA receptor antagonist MK-801 on the mRNA expression of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and metabotropic glutamate receptor (mGluR) subunits to determine the contribution of different glutamate receptors in response to blockade of NMDA receptor channels. In situ hybridization to rat brain sections revealed that AMPA receptor subunits GluR3 and GluR4, and mGluR3 were modestly but significantly decreased ∼10–20%, 8 h following 5 mg/kg MK-801 administration. A time course and dose response study revealed that the effect on mGluR3 was reversed by 24 h and occurred significantly at a dose range from 1 to 5 mg/kg. These results indicate that selected AMPA and mGluR subunit mRNAs respond at the RNA level to the blockade of NMDA receptors.     

Modulation of dopamine D1-mediated turning behavior and striatal c-fos expression by the substantia nigra

In order to study the possible contribution of the substantia nigra (SN) in the positive interaction between dopamine D₁ receptor agonists and glutamate antagonists in unilaterally 6-hydroxydopamine (6-OHDA) lesioned rats, the effect of the D₁ agonist, SKF 38393, was studied in combination with intranigral infusions of glutamate antagonists of the NMDA (MK 801, CPP) or AMPA (NBQX) type of receptor. Local infusion into the SN of the 6-OHDA lesioned side of MK 801, CPP or NBQX at doses inducing no or minimal behavioral effects significantly increased the turning behavior and the expression of c-fos induced, in the lesioned caudate-putamen (CPu), by a parenteral administration of SKF 38393. The same result was obtained after intra-SN infusion of the GABA agonist, muscimol. High doses of MK 801, CPP or muscimol infused into the SN produced intense contralateral turning per se and induced a sparse c-fos expression in the lesioned CPu which was antagonized by parenteral administration of MK 801. The results indicate that a depression of SN pars reticulata efferent neurons potentiates D₁-mediated responses and suggest that this area may play a role in the positive interaction between glutamate antagonists and D₁ receptor agonists. © 1995 Wiley-Liss, Inc.     

Stimulation of N-methyl-d-aspartate receptors induces apoptosis in rat brain

To evaluate the contribution of apoptotic mechanisms to excitotoxin-induced neurodegeneration as well as to characterize the glutamate receptor subtypes involved, biochemical and morphological effects of intrastriatally administered NMDA receptor agonist N-methyl-d-aspartate (NMDA) or quinolinic acid (QA) were studied. Receptor autoradiography showed that NMDA (75–300 nmol) caused a loss of 18–68% of striatal D₁ dopamine (DA) and 10–43% of NMDA receptors 7 days after drug administration. Treatment with QA (60–240 nmol) also led to a loss of 60–73% of D₁ DA and 37–44% of NMDA receptors in the ipsilateral striatum. Agarose gel electrophoresis revealed that both NMDA and QA induced internucleosomal DNA fragmentation in the striatum 12 to 48 h after drug administration. NMDA- and QA-induced internucleosomal DNA fragmentation was attenuated by the protein synthesis inhibitor cycloheximide in a dose-dependent manner. Terminal transferase-mediated deoxyuridine triphosphate (d-UTP)-digoxigenin nick end labeling (TUNEL)-positive nuclei were found in the ipsilateral striatum in response to NMDA or QA treatment. In addition, many fragmented nuclei were observed in the NMDA or QA-treated striatum and propidium iodide staining showed profound nuclear condensation in the NMDA or QA-treated striatum. NMDA- and QA-induced internucleosomal DNA fragmentation and TUNEL-positive nuclei as well as nuclear condensation were abolished by the NMDA receptor antagonist MK-801, but not by the AMPA/KA receptor antagonist NBQX. MK-801, but not NBQX, also prevented NMDA or QA-induced striatal cell death. These results suggest that apoptotic mechanisms are involved in excitotoxin-induced striatal cell death. The initiation of an apoptotic cascade by NMDA or QA appears to be mediated by stimulation of NMDA but not AMPA/KA receptors.     

Complement and glutamate neurotoxicity

Using mice genetically deficient in the complement (C)-system component C5, this study explored a potential novel role of the C-system in Ca²⁺-mediated control of glutamate AMPA receptor functions. We found that Ca²⁺ preincubation of frozen brain tissue sections enhances AMPA binding capacity more dynamically in C5 deficient (C5^?) than congenic C5 sufficient (C5⁺) mice. The Ca²⁺-mediated response was mostly localized to the CA3 and CA1 subdivisions of the pyramidal layers of the hippocampal formation. In C5^? mice, kainic acid (KA) excitotoxicity that models hippocampal neurodegeneration abolished the Ca²⁺-mediated induction of hippocampal AMPA binding. The changes in AMPA binding preceded temporally and overlapped anatomically the appearance of apoptotic features in the same hippocampal neuron layers. C5^? mice showed greater hippocampal neurodegeneration then C5^? mice. NMDA binding controlled for specificity of glutamate-mediated changes and found no C5 genotypic influences. The study gives further credence to the role of the C-system in modifying the intensity and outcome during response to conditions leading to hippocampal neurodegeneration.     

Development of NMDA receptor-channel complex and L-type calcium channels in mouse hippocampus

In vitro binding autoradiography was used to examine the pattern and intensity of binding of [³H]glutamate to NMDA receptors, [³H]MK 801 to NMDA receptor associated channels in and [³H]PN-200 110 to L-type calcium channels in the hippocampus of mice aged 3–70 days. The distribution of NMDA receptors and NMDA receptor associated channels was similar but not identical at the tested ages. Beginning with postnatal day 8, high binding levels were confined mostly to the hippocampal strata: the oriens and radiatum (CA1 and CA3 with [³H]MK 801 labeling but only CA1 with NMDA displaced [³H]glutamate labeling), the moleculare (higher labeling with [³H]MK 801 than with NMDA displaced [³H]glutamate binding), and the lucidum. The binding values for NMDA receptor-channel complex rose in the examined period (especially within the second and third week), reaching a plateau at the end of the third postnatal week. Sharp growth of binding within the second and third week of life was about 50% greater with [³H]MK 801 than with NMDA displaced [³H]glutamate labeling. L-type calcium channels were found to be most abundant in the strata: the oriens of the CA3 field, the moleculare, and the lucidum. The time course of binding value changes for the calcium channel was similar to the time course found for the NMDA receptor-channel complex. © 1993 Wiley-Liss, Inc.     

Autoradiographic comparison of the distribution of [H]MK801 and [H]CNQX in the human cerebellum during development and aging

The autoradiographic distribution of (NMDA) and -a-amino-3-hydroxyl-5-methyl-4-isoxazoleproprionic acid/quisqualate (AMPA/QUIS) receptors was determined in cerebellum obtained at autopsy from 37 human individuals, aged from 24 weeks gestation to 95 years. [³H]MK801 was used to label the NMDA receptor and [³H]CNQX to label the AMPA/QUIS receptor. AMPA/QUIS receptors were concentrated in the cerebellar molecular layer, and NMDA receptors in the granular layer. Significant (3- to 4-fold) increases in binding were seen for both ligands from the fetal to neonatal periods in the molecular layer (CNQX) and in both molecular and granular layers (MK801). MK801 binding in the molecular layer continued to increase with age up to the tenth decade and together with binding in the granular layer, increased 2-fold between 10–40 years. The Purkinje cell layer was negative for MK801 binding until the 6–7th decade when it became positive. [³H]CNQX binding in the molecular layer increased significantly with age between the fetal period and the tenth decade, whereas in the granular layer binding increased from neonate to 40 years, but then decreased significantly from 60 years to the tenth decade. Lamination of the molecular and granular layers was absent during the fetal period and appeared with both ligands during the neonatal period. These marked differences in age-related expression of ligand binding sites in the granular layer during development and aging are of potential significance in relation both to selective vulnerability to ischaemia, and synaptic plasticity and remodelling related to neuronal loss in senescence.     

The NMDA and AMPA/KA receptors are involved in glutamate-induced alterations of occludin expression and phosphorylation in brain endothelial cells.

Glutamate levels increase dramatically in cerebral ischemia and stroke. This may lead to opening of the blood-brain barrier (BBB) and induce further brain damage. Because endothelial tight junctions are critical elements of the BBB integrity, the aim of this study was to investigate the mechanisms of glutamate-induced alterations of the tight-junction protein occludin in cultured brain microvascular endothelial cells (BMECs). Transient exposure to glutamate resulted in cellular redistribution of occludin, followed by a decrease in the total level of this protein and diminished barrier function of BMECs. Inhibition of the N-methyl-D-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate (AMPA/KA) receptors attenuated glutamate-induced changes in occludin redistribution but not in the total protein levels. Treatment with glutamate also increased tyrosine phosphorylation and decreased threonine phosphorylation of occludin. Inhibition of the NMDA receptors by MK-801 partially protected against glutamate-induced elevation of occludin tyrosine phosphorylation. In addition, pretreatment with MK-801-attenuated glutamate-mediated disruption of endothelial barrier function. Blocking of the AMPA/KA receptors by 6,7-dinitroquinoxaline-2.3-dione (DNQX) protected against hypophosphorylation of threonine residues of occludin; however, it did not affect disruption of endothelial integrity. These findings indicate the opposite effects of the NMDA and AMPA/KA receptors on occludin phosphorylation and disruption of the BBB functions.     

Localization of ionotropic glutamate receptors in caudate-putamen and nucleus accumbens septi of rat brain: Comparison of NMDA,AMPA, and kainate receptors

Changes in binding of selective radioligands at NMDA ([³H]MK-801), AMPA ([³H]CNQX), and kainate ([³H]kainic acid) glutamate (GLU) ionotropic receptors in rat caudate-putamen (CPu) and nucleus accumbens (NAc) were examined by quantitative autoradiography following: 1) unilateral surgical ablation of frontal cerebral cortex to remove descending corticostriatal GLU projections, 2) unilateral injection of kainic acid (KA) into CPu or NAc to degenerate local intrinsic neurons, or 3) unilateral injections of 6-hydroxydopamine (6-OH-DA) into substantia nigra to degenerate ascending nigrostriatal dopamine (DA) projections. Cortical ablation significantly decreased NMDA receptor binding in ipsilateral medial CPu (20%), and NAc (16%), similar to previously reported losses of DA D₄ receptors. KA lesions produced large losses of NMDA receptor labeling in CPu and NAc (both by 52%), AMPA (41% and 45%, respectively), and kainate receptors (40% and 45%, respectively) that were similar to the loss of D₂ receptors in CPu and NAc after KA injections. Nigral 6-OH-DA lesions yielded smaller but significant losses in NMDA (17%), AMPA (12%), and kainate (11%) receptor binding in CPu. The results indicate that most NMDA, AMPA, and kainate receptors in rat CPu and NAc occur on intrinsic postsynaptic neurons. Also, some NMDA, but not AMPA or kainate, receptors are also found on corticostriatal projections in association with D₄ receptors; these may, respectively, represent excitatory presynaptic NMDA autoreceptors and inhibitory D₄ heteroceptors that regulate GLU release from corticostriatal axons in medial CPu and NAc. Conversely, the loss of all three GLU receptor subtypes after lesioning DA neurons supports their role as excitatory heteroceptors promoting DA release from nigrostriatal neurons. Synapse 30:227–235, 1998. © 1998 Wiley-Liss, Inc.     

Role of desensitization and subunit expression for kainate receptor–mediated neurotoxicity in murine neocortical cultures

The neurotoxic actions of kainate and domoate were studied in cultured murine neocortical neurons at various days in culture and found to be developmentally regulated involving three components of neurotoxicity: (1) toxicity via indirect activation of N‐methyl‐d ‐aspartate (NMDA) receptors, (2) toxicity mediated by α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) receptors, and (3) toxicity that can be mediated by kainate receptors when desensitization of the receptors is blocked. The indirect action at NMDA receptors was discovered because (5R,10S)‐(+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine (MK‐801), an NMDA receptor antagonist, was able to block part of the toxicity. The activation of NMDA receptors is most likely a secondary effect resulting from glutamate release upon kainate or domoate stimulation. 1‐(4‐Aminophenyl)‐3‐methylcarbamyl‐4‐methyl‐3,4‐dihydro‐7,8‐ethylenedioxy‐5H‐2,3benzodiazepine (GYKI 53655), a selective AMPA receptor antagonist, abolished the remaining toxicity. These results indicated that kainate‐ and domoate‐mediated toxicity involves both the NMDA and the AMPA receptors. Pretreatment of the cultures with concanavalin A to prevent desensitization of kainate receptors led to an increased neurotoxicity upon stimulation with kainate or domoate. In neurons cultured for 12 days in vitro a small but significant neurotoxic effect was observed when stimulated with agonist in the presence of MK‐801 and GYKI 53655. This indicates that the toxicity is produced by kainate receptors in mature cultures. Examining the subunit expression of the kainate receptor subunits GluR6/7 and KA2 did, however, not reveal any major change during development of the cultures. J. Neurosci. Res. 55:208–217, 1999. © 1999 Wiley‐Liss, Inc.     

In vivo modulation of nitric oxide concentration dynamics upon glutamatergic neuronal activation in the hippocampus

Nitric oxide (^?NO) is a labile endogenous free radical produced upon glutamatergic neuronal activity in hippocampus by neuronal nitric oxide synthase (nNOS), where it acts as a modulator of both synaptic plasticity and cell death associated with neurodegeneration. The low CNS levels and fast time dynamics of this molecule require the use of rapid analytical methods that can more accurately describe its signaling in vivo. This is critical for understanding how the kinetics of ^?NO‐dependent signaling pathways is translated into physiological or pathological functions. In these studies, we used ^?NO selective microelectrodes coupled with rapid electrochemical recording techniques to characterize for the first time the concentration dynamics of ^?NO endogenously produced in hippocampus in vivo following activation of ionotropic glutamate receptors. Both L ‐glutamate (1–100 mM) and N‐methyl‐D ‐aspartate (NMDA; 0.01–5 mM) produced transient, dose‐dependent increases in extracellular ^?NO concentration. The production of ^?NO in the hippocampus by glutamate was decreased by the nNOS inhibitor 7‐NI. Intraperitoneal administration of the NMDA receptor blocker, MK‐801, and the inhibitor of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoazolepropionic acid (AMPA) receptor, NBQX, applied locally greatly attenuated glutamate‐evoked overflow of ^?NO. Thus, ^?NO overflow elicited by activation of glutamate receptors appeared to result from an integrated activation of ionotropic glutamate receptors, both of the NMDA and AMPA receptors subtypes. Additionally, distinct concentration dynamics was observed in the trisynaptic loop with stronger and longer lasting effects of glutamate activation on ^?NO overflow seen in the CA1 region as compared with the dentate gyrus. Overall, the results provide a quantitative and temporal basis for a better understanding of ^?NO activity in the rat hippocampus. © 2010 Wiley‐Liss, Inc.     

Deficient NMDA-mediated glutamate release from synaptosomes of schizophrenics

Previous studies from our laboratory indicated that the veratridine-induced release of glutamate and GABA from synaptosomes derived from brains of schizophrenics was decreased. In the present study, synaptosomes were prepared from frozen brain samples from schizophrenics and from controls. Stimulation by 10 μmol/L 2-amino-3-hydroxy-5-methoxylisoxazole-4-propionic acid (AMPA) produced equal glutamate release from both groups. Release induced by either 10 μmol/L kainic acid (KA) or n-methyl-d-asparate (NMDA) was reduced significantly in the preparations derived from schizophrenics. Similarly, the amount of GABA released by 50 μmol/L glutamate was also reduced in the schizophrenic-derived synaptosomes. However, in membranes derived from the crude synaptosomal pellet, no differences between the controls and schizophrenics were observed in measures of total glutamate binding or its displacement by NMDA. The data demonstrate a deficiency in NMDA (and possibly KA) receptor functioning in schizophrenics and support the “second-generation” theories of schizophrenia as a glutamatergic deficiency disorder.     

Differential effects of zinc on glutamatergic and GABAergic neurotransmitter systems in the hippocampus

Approximately 10% of total zinc in the brain exists in synaptic vesicles of glutamatergic neurons; however, the function of vesicular zinc is poorly understood. The presynaptic action of zinc against excitatory and inhibitory neurotransmission was studied in rat hippocampus using in vivo microdialysis. When the hippocampal CA3 region was perfused with 10-300 microM ZnCl(2), the level of glutamate in the perfusate was decreased, whereas the level of gamma-aminobutyric acid (GABA) was increased. Chelation of endogenous zinc with CaEDTA increased the glutamate level in the perfusate but decreased the GABA level, suggesting that zinc released into the synaptic cleft acts differentially on glutamatergic and GABAergic neurons in the CA3 region. The increase of GABA level by zinc was antagonized by 2,3-dioxo-6-nitro-1,2.3,4-tetrahydrobenzo(f)quinoxaline-7-sulphonamide (NBQX), an antagonist of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors, but not affected by MK801, an antagonist of N-methyl-D-aspartate (NMDA) receptors, and verapamil, a blocker of voltage-dependent calcium channels. The present study suggests that zinc enhances GABA release via potentiation of AMPA/kainate receptors in the CA3 region, followed by a decrease in presynaptic glutamate release in the same region. Zinc seems to be an inhibitory neuromodulator of glutamate release.