Behavioural and neuronal activation after microinjections of AMPA and NMDA into the perifornical lateral hypothalamus in rats

The perifornical lateral hypothalamic area (PeFLH), which houses orexin/hypocretin (OX) neurons, is thought to play an important role in arousal, feeding, and locomotor activity. The present study examined behavioural effects of activating PeFLH neurons with microinjections of ionotropic glutamate receptor agonists. Three separate unilateral microinjections of either (1) AMPA (1 and 2 mM in 0.1 μL artificial cerebrospinal fluid, ACSF) and ACSF, or (2) NMDA (1 and 10 mM in 0.1 μL ACSF), and ACSF were made into the PeFLH of adult male rats. Following each injection, the rats were placed into an open field for behavioural scoring for 45 min. Rats were perfused after the third injection for immunohistochemistry for c-Fos and OX to assess the level of activation of OX neurons. Behavioural analyses showed that, as compared to ACSF conditions, AMPA injections produced a dose-dependent increase in locomotion and rearing that persisted throughout the 45 min recording period, and an increase in drinking. Injection of NMDA at 10 mM, but not 1 mM, induced a transient increase in locomotion and an increase in feeding. Histological analyses showed that while both agonists increased the number of neurons immunoreactive for c-Fos in the PeFLH, only AMPA increased the number of neurons immunoreactive for both c-Fos and OX. There were positive correlations between the number of c-Fos/OX-immunoreactive neurons and the amounts of locomotion, rearing, and drinking. These results support the role of ionotropic glutamate receptors on OX and other neurons in the PeFLH in the regulation of locomotor and ingestive behaviours.     

Cell-specific expression of type II calcium/calmodulin-dependent protein kinase isoforms and glutamate receptors in normal and visually deprived lateral geniculate nucleus of monkeys

In situ hybridization histochemistry and immunocytochemistry were used to map distributions of cells expressing mRNAs encoding α, β, γ, and δ isoforms of type II calcium/calmodulin-dependent protein kinase (CaMKII), α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA)/kainate receptor subunits, (GluR1–7), and N-methyl-D-aspartate (NMDA) receptor subunits, NR1 and NR2A-D, or stained by subunit-specific immunocytochemistry in the dorsal lateral geniculate nuclei of macaque monkeys. Relationships of specific isoforms with particular glutamate receptor types may be important elements in neural plasticity. CaMKII-α is expressed only by neurons in the S laminae and interlaminar plexuses of the dorsal lateral geniculate nucleus, but may form part of a more widely distributed matrix of similar cells extending from the geniculate into adjacent nuclei. CaMKII-β, -γ, and -δ isoforms are expressed by all neurons in principal and S laminae and interlaminar plexuses. In principal laminae, they are down-regulated by monocular deprivation lasting 8–21 days. All glutamate receptor subunits are expressed by neurons in principal and S laminae and interlaminar plexuses. The AMPA/kainate subunits, GluR1, 2, 5, and 7, are expressed at low levels, although GluR1 immunostaining appears selectively to stain interneurons. GluR3 is expressed at weak, GluR 6 at moderate and GluR 4 at high levels. NMDA subunits, NR1 and NR2A, B, and D, are expressed at moderate to low levels. GluR4, GluR6 and NMDA subunits are down-regulated by visual deprivation. CaMKII-α expression is unique in comparison with other CaMKII isoforms which may, therefore, have more generalized roles in cell function. The results demonstrate that all of the isoforms are associated with NMDA receptors and with AMPA receptors enriched with GluR4 subunits, which implies high calcium permeability and rapid gating. J. Comp. Neurol. 390:278–296, 1998. © 1998 Wiley-Liss, Inc.     

Kaitocephalin Antagonism of Glutamate Receptors Expressed in Xenopus Oocytes

d-aspartate (NMDA) or α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainic acid (kainate, KA) receptors. Nevertheless, the effects of kaitocephalin on the function of these receptors were unknown. In this work, we report some pharmacological properties of synthetic (−)-kaitocephalin on rat brain glutamate receptors expressed in Xenopus laevis oocytes and on the homomeric AMPA-type GluR3 and KA-type GluR6 receptors. Kaitocephalin was found to be a more potent antagonist of NMDA receptors (IC₅₀ = 75 ± 9 nM) than of AMPA receptors from cerebral cortex (IC₅₀ = 242 ± 37 nM) and from homomeric GluR3 subunits (IC₅₀ = 502 ± 55 nM). Moreover, kaitocephalin is a weak antagonist of the KA-type receptor GluR6 (IC₅₀ ∼ 100 μM) and of metabotropic (IC₅₀ > 100 μM) glutamate receptors expressed by rat brain mRNA.     

AMPA glutamate receptor subunits in the guinea pig hypothalamus: distribution and colocalization with progesterone receptor

Excitatory amino acids (EAAs), particularly glutamate, have been implicated in the control of luteinizing hormone (LH) secretion through facilitation of gonadotropin-releasing hormone release. The effects of EAAs are mediated by means of ionotropic glutamate receptors, which are divided into N-methyl-D-aspartate (NMDA) and non-NMDA (kainate and AMPA) subtypes. Moreover, ovarian steroids are responsible for inducing the preovulatory surge of LH and are involved in the actions of EAAs on LH release. Progesterone is directly involved in the potentiating effect of ovarian steroids on the stimulating effect of AMPA neurotransmission on gonadotropin secretion. To broaden our understanding of the role of hypothalamic AMPA receptors in the steroid-induced LH surge, we determined the cellular localization of AMPA receptors in the hypothalamus of guinea pigs by using antibodies that recognize the GluR1, GluR2, GluR2/3, or GluR4 subunits, and then we examined the neuroanatomic relationships between these receptors and the progesterone receptor (PR). Different patterns of immunostaining within the preoptic area and hypothalamus were evident with the antibodies to the four subunits with marked contrasts between moderate staining for GluR1, intensely stained structures for GluR2 and GluR2/3, and little specific staining for GluR4. Immunoreactive (IR) neurons were visualized in many regions, including the two regions known to contain a dense population of estradiol-induced PR-IR cells: the preoptic periventricular and ventrolateral hypothalamic nuclei. Approximately 60% of GluR1-IR and 39% of GluR2-IR cells in the preoptic region possessed PR, whereas 46% of GluR1-IR and 54% of GluR2-IR cells in the ventrolateral nucleus expressed PR. These neuroanatomic results suggest that the coordinated actions of progesterone and glutamatergic inputs on mammalian reproductive functions are integrated at the cellular level.     

Peripheral administration of NR2 antagonists attenuates orofacial formalin-induced nociceptive behavior in rats

The present study investigated the role of the peripheral NR2 subunits of N-methyl-d-aspartatic acid (NMDA) receptors in inflammatory orofacial pain. Experiments were carried out using adult male Sprague-Dawley rats weighing 220 to 280 g. Formalin (5%, 50 μl) was applied subcutaneously to the vibrissa pad. For each animal, the number of noxious behavioral responses, including rubbing or scratching of the facial region proximal to the injection site, was recorded for 9 sequential 5 min intervals. NR2 subunit antagonists were injected subcutaneously at 20 min prior to formalin injection. The subcutaneous injection of 100 or 200 μg of memantine significantly suppressed the number of scratches in the second phase of the behavioral responses to formalin. The subcutaneous injection of 0.25, 2.5, or 25 μg of 5,7-dichlorokynurenic acid also produced significant antinociceptive effects in the second phase. The subcutaneous injection of AP-5 at high dose produced significant antinociceptive effects in the second phase. The subcutaneous injection of PPPA and Ro 25-6981 both significantly suppressed the number of scratches in the second phase. The antinociceptive doses of memantine (200 μg), 5,7-dichlorokynurenic acid (25 μg), AP-5 (20 μg), PPPA (2.5 μg), or Ro 25-6981 (50 μg) injected into the contralateral hind paw did not affect the number of scratches in both the first and second phases. Moreover, the peripheral administration of NR2 subunit antagonists, including other NMDA receptor blockers, did not produce any motor dysfunction. These results indicate that a targeted blockade of peripheral NR2 receptors is a potentially important new method of treating inflammatory pain in the orofacial area.     

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.     

Differential expression of NMDA and AMPA receptor subunits in DARPP-32-containing neurons of the cerebral cortex, hippocampus and neostriatum of rats

Dopamine and cyclic adenosine 3',5'-monophosphate-regulated phosphoprotein, 32 kDa (DARPP-32) is a key element of dopamine/D1/DARPP-32/protein phosphatase-1 (PP-1) signaling cascades of mammalian brain. We are interested in the expression patterns of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in DARPP-32-containing neurons, which may constitute morphological basis for interaction between dopamine and ionotropic glutamate receptors in dopaminoceptive cells. Double immunofluorescence was performed to visualize neurons showing coexpression of DARPP-32 with NMDA or AMPA receptor subunits (i.e., NR1, NR2a/b, glutamate receptor subunit 1 [GluR1], GluR2/3, and GluR4) in the forebrains of rats. Distribution of DARPP-32-positive neurons completely or partially overlapped with that of NMDA receptor- or AMPA receptor-immunoreactive ones in the frontal and parietal cortex, hippocampus and neostriatum, and neurons double-labeled with DARPP-32/NR1, DARPP-32/NR2a/b, DARPP-32/GluR1, DARPP-32/GluR2/3, or DARPP-32/GluR4 immunoreactivity were numerously observed. Semiquantification analysis indicated that most of DARPP-32-containing neurons (86-98%) expressed NR1, NR2a/b and GluR2/3, while less of them (14-90%) expressed GluR1 and GluR4. Although high rates (90-98%) of DARPP-32-positive cells expressed NMDA receptors in all regions above, variant percentages of them expressing AMPA receptor subunits were observed among the cortex (54-90%), hippocampus (59-97%) and neostriatum (14-97%). The study presents differential expression patterns of NMDA and AMPA receptors in DARPP-32-postive neurons in these forebrain regions. Taken together with previous reports, the present data suggest that interaction between dopamine and glutamate receptors may occur in the dopaminoceptive neurons with distinct receptor compositions and may be involved in modulating neuronal properties and excitotoxicity in mammalian forebrain.     

Differential expression of NMDA and AMPA/KA receptor subunits in the inferior olive of postnatal rats

We have employed immunohistochemistry to determine the expression patterns of receptor subunits of N-methyl-d-aspartate (NMDA-NR1 and NR2A/B) and alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid/kainic acid (AMPA/KA-GluR1, GluR2, GluR2/3, GluR4, and GluR5/6/7) in the inferior olive of postnatal rats up to adulthood. Immunoreactivity for distinct receptor subunits was predominantly localized in the soma and dendrites of neurons. Semi-quantification showed that the overall immunoreactivity in the inferior olive of adults was intense for GluR1, moderate for NR1 and NR2A/B, and low for GluR2, GluR2/3, GluR4, and GluR5/6/7. At P7, GluR1 was restricted to the dorsomedial cell column, subnucleus beta, principal nucleus and ventrolateral protrusion while the other subunits were found in all subnuclei of the inferior olive. The immunoreactivities for all glutamate receptor subunits ranged from low to moderate. As the rats matured, the immunoreactivity of GluR4 decreased after the second postnatal week, while those of the other subunits showed a general trend of increase, reaching adult level during the third postnatal week. Double immunofluorescence revealed that all NR1-containing neurons exhibited NR2A/B immunoreactivity, indicating that native NMDA receptors comprise of hetero-oligomeric combinations of NR1 and NR2A/B. Furthermore, co-localization of NMDA and AMPA/KA receptor subunits was demonstrated in individual neurons of the inferior olive. All NR1-containing neurons exhibited GluR1 immunoreactivity, and all NR2A/B-containing neurons showed GluR5/6/7 immunoreactivity. Our data suggest that NMDA and AMPA/KA receptors are involved in glutamate-mediated neurotransmission, contributing to synaptic plasticity and reorganization of circuitry in the inferior olive during postnatal development.     

Stimulation of N-methyl-D-aspartate receptors, AMPA receptors or metabotropic glutamate receptors leads to rapid internalization of AMPA receptors in cultured nucleus accumbens neurons

In hippocampus and other regions, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors are inserted into synapses during long-term potentiation and removed during long-term depression. However, little is known about regulation of AMPA receptor trafficking in the nucleus accumbens (NAc), despite growing evidence that glutamate-dependent forms of plasticity in the NAc contribute to drug addiction. Using postnatal rat NAc cultures and an immunocytochemical method that selectively detects newly internalized GluR1, we studied the regulation of AMPA receptor internalization in NAc neurons by glutamate agonists. Newly internalized GluR1 was detected during 15 or 30 min of incubation at room temperature, indicating a basal rate of GluR1 turnover. The rate of GluR1 internalization was increased by glutamate (50 microM) within 5 min of its addition. Glutamate-induced GluR1 internalization was partially blocked by either an AMPA receptor antagonist (CNQX; 20 microM) or an N-methyl-D-aspartate (NMDA) receptor antagonist (APV; 50 microM). Both NMDA (50 microM) and AMPA (50 microM) increased GluR1 internalization in a Ca(2+)-dependent manner. The NMDA effect was blocked by APV while the AMPA effect was blocked by APV or CNQX. We interpret these findings to suggest that NMDA and AMPA ultimately trigger GluR1 internalization through the same NMDA receptor-dependent pathway. The effect of glutamate was also partially blocked by the group 1 metabotropic glutamate receptor antagonist N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC; 50 microM), while the group 1 agonist 3,5-dihydroxyphenylglycine (DHPG; 50 microM) stimulated GluR1 internalization. These data suggest that AMPA receptors on NAc neurons may be subject to rapid regulation of their surface expression in response to changes in the activity of glutamate inputs from cortical and limbic regions.     

Co-localization of NMDA receptors and AMPA receptors in neurons of the vestibular nuclei of rats

We are interested in studying the co-localization of NMDA glutamate receptor subunits (NR1, NR2A/B) and AMPA glutamate receptor subunits (GluR1, GluR2, GluR2/3 and GluR4) in individual neurons of the rat vestibular nuclei. Immunoreactivity for NR1, NR2A/B, GluR1, GluR2, GluR2/3 and GluR4 was found in the somata and dendrites of neurons in the four major subdivisions (superior, medial, lateral, and spinal vestibular nuclei) and in two minor groups (groups x and y) of the vestibular nuclei. Double immunofluorescence showed that all the NR1-containing neurons exhibited NR2A/B immunoreactivity, indicating that native NMDA receptors are composed of NR1 and NR2A/B in a hetero-oligomeric configuration. Co-expression of NMDA receptor subunits and AMPA receptor subunits was demonstrated by double labeling of NR1/GluR1, NR1/GluR2/3, NR1/GluR4 and NR2A/B/GluR2 in individual vestibular nuclear neurons. All NR1-containing neurons expressed GluR2/3 immunoreactivity, and all NR2A/B-containing neurons expressed GluR2 immunoreactivity. However, only about 52% of NR1-immunoreactive neurons exhibited GluR1 immunoreactivity and 46% of NR1-containing neurons showed GluR4 immunoreactivity. The present data reveal that NMDA receptors are co-localized with variants of AMPA receptors in a large proportion of vestibular nuclear neurons. These results suggest that cross-modulation between NMDA receptors and AMPA receptors may occur in individual neurons of the vestibular nuclei during glutamate-mediated excitatory neurotransmission and may in turn contribute to synaptic plasticity within the vestibular nuclei.     

Subcellular and subsynaptic distribution of the NR1 subunit of the NMDA receptor in the neostriatum and globus pallidus of the rat: co-localization at synapses with the GluR2/3 subunit of the AMPA receptor

Glutamatergic neurotransmission in the neostriatum and the globus pallidus is mediated through NMDA-type as well as other glutamate receptors and is critical in the expression of basal ganglia function. In order to characterize the cellular, subcellular and subsynaptic localization of NMDA receptors in the neostriatum and globus pallidus, multiple immunocytochemical techniques were applied using antibodies that recognize the NR1 subunit of the NMDA receptor. In order to determine the spatial relationship between NMDA receptors and AMPA receptors, double labelling was performed with the NR1 antibodies and an antibody that recognizes the GluR2 and 3 subunits of the AMPA receptor. In the neostriatum all neurons with characteristics of spiny projection neurons, some interneurons and many dendrites and spines were immunoreactive for NR1. In the globus pallidus most perikarya and many dendritic processes were immunopositive. Immunogold methods revealed that most NR1 labelling is associated with asymmetrical synapses and, like the labelling for GluR2/3, is evenly spread across the synapse. Double immunolabelling revealed that in neostriatum, over 80% of NR1-positive axospinous synapses are also positive for GluR2/3. In the globus pallidus most NR1-positive synapses are positive for GluR2/3. In both regions many synapses labelled only for GluR2/3 were also detected. These results, together with previous data, suggest that NMDA and AMPA receptor subunits are expressed by the same neurons in the neostriatum and globus pallidus and that NMDA and AMPA receptors are, at least in part, colocalized at individual asymmetrical synapses. The synaptic responses to glutamate in these regions are thus likely be mediated by both AMPA and NMDA receptors at the level of individual synapses.     

Differential co-localization of neurokinin-3 receptor and NMDA/AMPA receptor subunits in neurons of the substantia nigra of C57/BL mice

By using a double immunofluorescence method we examined co-localization of neurokinin-3 receptor (NK-3R) and N-methyl-D-aspartate (NMDA)/alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunits in neurons of the substantia nigra of adult mice. Overlapping distribution of NMDA receptor subunit 1 (NR1)/AMPA receptor subunits 1-4 (GluR1-4) and NK-3R-immunoreactive neurons were found in the substantia nigra pars compacta. It revealed that all (100%) of NK-3R-positive neurons displayed NR1, GluR2 or GluR3 immunoreactivity, 80% of them showed GluR1 immunoreactivity. In contrast, these neurons exhibiting both NK-3R and GluR4 immunoreactivity were hardly detected although GluR4-positive neurons were still distributed in the substantia nigra. The co-expression of NK-3R and NMDA/AMPA receptor subunits in the nigral neurons has provided a structural basis for functional modulation of neuronal glutamate receptors by neurokinin-3, suggesting that neurokinin peptides may be involved in modulation of neuronal properties and excitotoxicity in the substantia nigra of basal ganglia.     

Co-localization of AMPA-type glutamate receptor immunoreactivity in neurons of the rat subthalamic nucleus

In order to determine the precise cellular localization of the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)-type glutamate receptor subunit immunoreactivity in the rat subthalamic nucleus, single and double immunofluorescence was performed. Intense level of GluR1, GluR2, GluR2/3 and GluR4 immunoreactivity was found in almost all neurons of the subthalamic nucleus. By double immunofluorescence, the subthalamic neurons in the same sections that displayed a strong immunoreactivity for GluR1 were found to display a robust GluR2 immunoreactivity and the subthalamic neurons that displayed GluR2 immunoreactivity were also found to express GluR4 immunoreactivity. The present results thus demonstrate that individual neurons of the subthalamic nucleus are likely to co-express GluR1 and GluR2, and GluR2 and GluR4 immunoreactivity. The native AMPA channels in the subthalamic neurons may, therefore, be composed of heteromeric subunits. The present results provide information of the neuroanatomical localization of AMPA receptor subunits in neurons of the subthalamic nucleus. The localization of AMPA receptor subunits may be related to functional characteristics of AMPA channels in the subthalamic neurons.     

Development of calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in cultured neocortical neurons visualized by cobalt staining

The developmental expression of calcium (Ca²⁺)-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors in cultured neocortical neurons was evaluated by using cobalt uptake, a histochemical method that identifies cells expressing Ca²⁺-permeable, non-N-methyl-D-aspartate (non-NMDA) receptors. At a concentration of 500 μM, AMPA was found to stimulate cobalt uptake only late in development, resulting in staining of 2.7% ± 0.3% of the neurons maintained in culture for 12 days in vitro (DIV). When AMPA receptor desensitization was blocked with 50 μM cyclothiazide, the developmental profile of cobalt uptake mediated by 25 μM AMPA changed dramatically. The cobalt staining now appeared in young cultures (5 DIV), and the percentage of stained cells increased from 3.4% ± 0.2% at 5 DIV to 21.7% ± 1.6% at 12 DIV. The effect of 200 μM kainate was similar to that seen with 25 μM AMPA plus 50 μM cyclothiazide, resulting in 17.7% ± 0.3% stained neurons at 12 DIV. The cobalt uptake was specific to AMPA and kainate receptors because NMDA receptors and voltage-gated calcium channels were found not to mediate any cobalt staining. In addition, 10 μM 6-nitro-7-sulphamoylbenzo- [f]-quinoxaline-2,3-dione (NBQX) was able to prevent all staining at 5 and 8 DIV and most of the staining at 12 DIV, indicating that the non-NMDA ionotropic glutamate receptors are involved in cobalt uptake into the neurons. The AMPA receptor-selective antagonist GYKI 53655 was used to differentiate between cobalt influx through AMPA- or kainate-preferring receptors. After pretreatment with concanavalin A (con A), an inhibitor of kainate receptor desensitization, cobalt uptake was assessed after stimulation by 200 μM kainate in the presence of 25 μM GYKI 53655. No cobalt staining was observed under these conditions, indicating that most if not all of the cobalt influx induced by kainate was mediated through AMPA receptor channels. J. Neurosci. Res. 54:273–281, 1998. © 1998 Wiley-Liss, Inc.     

Neuroprotective effects of the beta-catenin stabilization in an oxygen- and glucose-deprived human neural progenitor cell culture system

β-Catenin stabilization achieved either via GSK-3β specific inhibition or involving canonical Wnt signalling pathway, contributes to neuroprotection in an oxygen-glucose deprivation (4 h OGD) in vitro hypoxia model performed on human cortical neural progenitor cells previously differentiated into neurons and glia. Neuroprotection mechanisms include both acquiring tolerance to injury throughout preconditioning (72 h prior to OGD) or being pro-survival during 24 h reoxygenation after the insult. Four hours of OGD induced apoptotic cell death elevation to 73 ± 1% vs. 12% measured in control and the LDH level, indicative of necrotic cell injury, elevation by 67 ± 7% (set to 100%). A significant reduction in apoptosis occurred at 24 h reoxygenation with indirubin supplement which was 49 ± 6% at 2.5 μM BIO while LDH level was only 47 ± 5% of OGD. Kenpaullone was efficient in reducing both cell deaths at 5 μM (apoptosis 38 ± 1% and necrosis 33 ± 3% less than in OGD). Wnt agonist reduced apoptosis to 45 ± 3% at 0.01 μM, while LDH value was decreased to a level of 53 ± 5% of control. Our findings suggest that GSK-3beta inhibitors/β-catenin stabilizers may ultimately be useful drugs in neuroprotection and neuroregeneration therapies in vivo.     

Development of excitatory amino acid induced cytotoxicity in cultured neurons

The neurotoxicity of the excitatory amino acids (EAAs) L-glutamate (L-glu), L-aspartate (L-asp), N-methyl-D-aspartate (NMDA), kainate (KA), quisqualate (QA) and RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolopropionate (AMPA) was followed as a function of development in primary cultures of cerebral cortex neurons and cerebellar granule cells. These two types of neurons express, respectively, glutamate receptor subtypes with sensitivity to all of these excitatory amino acids or only to glutamate and aspartate. None of the EAAs were toxic in cerebral cortex neurons at 2 days in culture, whereas at culture day 4 the neurons became sensitive to glutamate, at day 5 to KA followed by sensitivity to QA at day 6, and finally to NMDA, L-asp and AMPA at day 7. The rank order of potency of the EAAs was in cerebral cortex neurons cultured for 12 days: L-asp (ED50 = 0.5 microM) = L-glu (ED50 = 1 microM) greater than AMPA (ED50 = 10 microM) greater than NMDA (ED50 = 65 microM) greater than QA = KA (ED50 = 100 microM). Cerebellar granule cells were insensitive to all of the EAAs at 3 and 5 days in culture but at day 8 the cells became sensitive to toxicity induced by L-glu (ED50 = 70 microM) and L-asp (ED50 = 30 microM). In order to determine ED50 values for L-asp and L-glu accurately, media in these experiments also contained 500 microM of the glutamate uptake inhibitor L-aspartate-beta-hydroxamate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for caspase-mediated cleavage of AMPA receptor subunits in neuronal apoptosis and Alzheimer's disease.

In Alzheimer's disease (AD) synapses degenerate and neurons die in brain regions involved in learning and memory processes. Although the cellular and molecular mechanisms underlying the neurodegenerative process in AD are unclear, increasing evidence suggests roles for amyloid beta-peptide (Abeta) and biochemical cascades associated with a form of programmed cell death called apoptosis. Cysteine proteases of the caspase family are activated in neurons undergoing apoptosis and apparently play a major role in the cell death process by cleaving yet-to-be-identified substrates. We now report that caspase activity is increased in brain tissue and neurons from AD patients, and in cultured hippocampal neurons undergoing apoptosis after exposure to amyloid beta-peptide (Abeta). Western blot analyses using antibodies against different subunits of 2-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) types of ionotropic glutamate receptors indicate that AMPA receptor subunits (GluR1, GluR2/3, and GluR4), but not NMDA receptor subunits (NR1 and NR2A), are proteolytically cleaved after exposure of hippocampal neurons to apoptotic insults, including Abeta, and that the caspase inhibitor zVAD-fmk suppresses such cleavage. Western blot analysis of brain tissue from AD patients and age-matched controls revealed evidence for increased proteolysis of AMPA receptor subunits in AD. Our data suggest roles for caspase-mediated cleavage of AMPA receptor subunits in modifying neuronal responsivity to glutamate and in the neurodegenerative process in AD.     

Neuronal glutamate transporters regulate synaptic transmission in single synapses on CA1 hippocampal neurons

Glutamate is the major excitatory transmitter in CNS although it causes severe brain damage by pathologic excitotoxicity. Efficient neurotransmission is controlled by powerful protection and support afforded by specific high-affinity glutamate transporters in neurons and glia, clearing synaptic glutamate. While the role of glial cells in glutamate uptake is well defined, the role of neuronal transporters remains poorly understood. The evaluation of impact of neuronal transporters on spontaneous and evoked EPSC in hippocampal CA1 neurons within a model ‘single bouton preparation’ by pre- and postsynaptic uptake was addressed.In whole-cell patch clamp experiments the influence of blocking, pre- or both pre- and postsynaptic glutamate transporters (GluT) on spontaneous and evoked postsynaptic currents (sEPSC and eEPSC), was examined by manipulating the content of intracellular solution. Suppressing GluT by non-transportable inhibitor TBOA (10 μM) led to remarkable alteration of glutamate uptake process and was reflected in measurable changes of general properties of synaptic currents.Elimination of intracellular K⁺ concentration required for glutamate transporter operation by using Cs⁺-based internal solution (postsynaptic GluTs are non-functional apriori), causes the deficient of presynaptic glutamate transporters. Applied in such conditions glutamate transporter inhibitor TBOA (10 μM) affected the occurrence of synaptic event and thus unregulated the transmitter release. eEPSCs were generally suppressed both in amplitude (to 48.73 ± 7.03% vs. control) and in success rate (R_suc) by TBOA (from 91.1 ± 7.5% in control to 79.57 ± 13.2%). In contrast, with K⁺-based solution in patch pipette (pre- and postsynaptic GluT are intact), amplitude of eEPSC was substantially potentiated by pre-treatment with TBOA (152.1 ± 11%), whereas (R_suc) was reduced to 79.8 ± 8.3% in average. The identical reduction of event success rate as well as increased pair-pulse ratios (PPF ratio) for eEPSC in both cases indicates the effect of TBOA on presynaptic uptake. sEPSCs simultaneously recorded from neurons, showed the same pattern of regulation but with less potency, indicating the similar processes in most of excitatory synapses.In conclusion, presynaptic transporters are suggested to act mainly as negative feedback signal on presynaptic release and/or referred to vesicle refilling processes.     

Developmental exposure to methylmercury elicits early cell death in the cerebral cortex and long-term memory deficits in the rat

Experiments were performed to assess the neurotoxic effects induced by prenatal acute treatment with methylmercury on cortical neurons. To this purpose, primary neuronal cultures were obtained from cerebral cortex of neonatal rats born to dams treated with methylmercury (4 and 8 mg/kg by gavage) on gestational day 15, the developmental stage critical for cortical neuron proliferation. Prenatal exposure to methylmercury 8 mg/kg significantly reduced cell viability and caused either apoptotic or necrotic neuronal death. Moreover, this exposure level resulted in abnormal neurite outgrowth and retraction or collapse of some neurites, caused by a dissolution of microtubules. The severe and early cortical neuron damage induced by methylmercury 8 mg/kg treatment correlated with long term memory impairment, since adult rats (90 days of age) born to dams treated with this dose level showed a significant deficit in the retention performance when subjected to a passive avoidance task. Prenatal exposure to methylmercury 4 mg/kg significantly increased the neuronal vulnerability to a neurotoxic insult. This was determined by measuring the increment of chromatin condensation induced by glutamate, at a concentration (30 μM) able to induce an excitotoxic damage. This exposure level eliciting apoptotic death did not result in cognitive dysfunctions. In conclusion, the methylmercury-induced disruption of glutamate pathway during critical windows of brain development may interfere with cell fate and proliferation resulting in a more or less severe cortical lesions associated or not with loss of function later in life, depending on the exposure levels. Therefore, the early biochemical effects and long-term behavioral changes elicited by high methylmercury levels suggest that the developing brain is impaired in its ability to recover following toxic insult, and the initial effects on cortical neurons may lead to permanent cognitive dysfunctions.