Ampakine CX546 bolsters energetic response of astrocytes: a novel target for cognitive-enhancing drugs acting as alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor modulators.

Glutamate was previously shown to enhance aerobic glycolysis i.e. increase glucose utilization and lactate production with no change in oxygen levels, in mouse cortical astrocytes by a mechanism involving glutamate uptake. It is reported here that a similar response is produced in both hippocampal and cerebellar astrocytes. Application of the cognitive-enhancing drug CX546 promoted further enhancement of glucose utilization by astrocytes from each brain area following glutamate exposure. alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors represent the purported molecular target of cognitive-enhancing drugs such as CX546, and the presence of AMPA receptor subunits GluR1-4 was evidenced in astrocytes from all three regions by immunocytochemistry. AMPA itself did not stimulate aerobic glycolysis, but in the presence of CX546, a strong enhancement of glucose utilization and lactate production was obtained in cortical, hippocampal and cerebellar astrocytes. The effect of CX546 was concentration-dependent, with an EC(50) of 93.2 microm in cortical astrocytes. AMPA-induced glucose utilization in the presence of CX546 was prevented by the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the negative modulator GYKI 52466. In addition, the metabolic effect of CX546 in the presence of AMPA was mimicked by the AMPA receptor modulator cyclothiazide. Our data suggest that astrocyte energetics represents a novel target for cognitive-enhancing drugs acting as AMPA receptor modulators.

Intracellular polyamines mediate inward rectification of Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors.

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that lack the glutamate receptor GluR2 subunit are Ca(2+)-permeable and exhibit inwardly rectifying current responses to kainate and AMPA. A proportion of cultured rat hippocampal neurons show similar Ca(2+)-permeable inwardly rectifying AMPA receptor currents. Inward rectification in these neurons was lost with intracellular dialysis and was not present in excised outside-out patches but was maintained in perforated-patch whole-cell recordings, suggesting that a diffusible cytoplasmic factor may be responsible for rectification. Inclusion of the naturally occurring polyamines spermine and spermidine in the recording pipette prevented loss of rectification in both whole-cell and excised-patch recordings; Mg2+ and putrescine were without effect. Inward rectification of Ca(2+)-permeable AMPA receptors may reflect voltage-dependent channel block by intracellular polyamines.

Growth factor-mediated Fyn signaling regulates α-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor expression in rodent neocortical neurons

Src-family protein tyrosine kinases (PTKs) transduce signals to regulate neuronal development and synaptic plasticity. However, the nature of their activators and molecular mechanisms underlying these neural processes are unknown. Here, we show that brain-derived neurotrophic factor (BDNF) and platelet-derived growth factor enhance expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor 1 and 2/3 proteins in rodent neocortical neurons via the Src-family PTK(s). The increase in AMPA receptor levels was blocked in cultured neocortical neurons by addition of a Src-family-selective PTK inhibitor. Accordingly, neocortical cultures from Fyn-knockout mice failed to respond to BDNF whereas those from wild-type mice responded. Moreover, the neocortex of young Fyn mutants exhibited a significant in vivo reduction in these AMPA receptor proteins but not in their mRNA levels. In vitro kinase assay revealed that BDNF can indeed activate the Fyn kinase: It enhanced tyrosine phosphorylation of Fyn as well as that of enolase supplemented exogenously. All of these results suggest that the Src-family kinase Fyn, activated by the growth factors, plays a crucial role in modulating AMPA receptor expression during brain development.

Glycine-induced long-term potentiation is associated with structural and functional modifications of α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid receptors

Global long-term potentiation (LTP) was induced in organotypic hippocampal slice cultures by a brief application of 10 mM glycine. Glycine-induced LTP was occluded by previous theta burst stimulation-induced potentiation, indicating that both phenomena share similar cellular processes. Glycine-induced LTP was associated with increased [3H]α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) binding in membrane fractions as well as increased amount of a selective spectrin breakdown product generated by calpain-mediated spectrin proteolysis. Antibodies against the C-terminal (C-Ab) and N-terminal (N-Ab) domains of GluR1 subunits were used to evaluate structural changes in AMPA receptor properties resulting from glycine-induced LTP. No quantitative or qualitative changes were observed in Western blots from membrane fractions prepared from glycine-treated slices with C-Ab. In contrast, Western blots stained with N-Ab revealed the formation of a 98-kDa species of GluR1 subunits as well as an increased amount of immunoreactivity after glycine-induced LTP. The amount of spectrin breakdown product was positively correlated with the amount of the 98-kDa species of GluR1 after glycine treatment. Functional modifications of AMPA receptors were evaluated by determining changes in the effect of pressure-applied AMPA on synaptic responses before and after glycine-induced LTP. Glycine treatment produced a significant increase in AMPA receptor function after potentiation that correlated with the degree of potentiation. The results indicate that LTP induction produces calpain activation, truncation of the C-Ab domain of GluR1 subunits of AMPA receptors, and increased AMPA receptor function. They also suggest that insertion of new receptors takes place after LTP induction.

Correlation between kinetics and RNA splicing of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors in neocortical neurons.

In the cortex fast excitatory synaptic currents onto excitatory pyramidal neurons and inhibitory nonpyramidal neurons are mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors exhibiting cell-type-specific differences in their kinetic properties. AMPA receptors consist of four subunits (GluR1-4), each existing as two splice variants, flip and flop, which critically affect the desensitization properties of receptors expressed in heterologous systems. Using single cell reverse transcription PCR to analyze the mRNA of AMPA receptor subunits expressed in layers I-III neocortical neurons, we find that 90% of the GluR1-4 in nonpyramidal neurons are flop variants, whereas 92% of the GluR1-4 in pyramidal neurons are flip variants. We also find that nonpyramidal neurons predominantly express GluR1 mRNA (GluR1/GluR1-4 = 59%), whereas pyramidal neurons contain mainly GluR2 mRNA (GluR2/GluR1-4 = 59%). However, the neuron-type-specific splicing is exhibited by all four AMPA receptor subunits. We suggest that the predominance of the flop variants contributes to the faster and more extensive desensitization in nonpyramidal neurons, compared to pyramidal cells where flip variants are dominant. Alternative splicing of AMPA receptors may play an important role in regulating synaptic function in a cell-type-specific manner, without changing permeation properties.

Linking supply to demand: the neuronal monocarboxylate transporter MCT2 and the alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid receptor GluR2/3 subunit are associated in a common trafficking process.

MCT2 is the major neuronal monocarboxylate transporter (MCT) that allows the supply of alternative energy substrates such as lactate to neurons. Recent evidence obtained by electron microscopy has demonstrated that MCT2, like alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA) receptors, is localized in dendritic spines of glutamatergic synapses. Using immunofluorescence, we show in this study that MCT2 colocalizes extensively with GluR2/3 subunits of AMPA receptors in neurons from various mouse brain regions as well as in cultured neurons. It also colocalizes with GluR2/3-interacting proteins, such as C-kinase-interacting protein 1, glutamate receptor-interacting protein 1 and clathrin adaptor protein. Coimmunoprecipitation of MCT2 with GluR2/3 and C-kinase-interacting protein 1 suggests their close interaction within spines. Parallel changes in the localization of both MCT2 and GluR2/3 subunits at and beneath the plasma membrane upon various stimulation paradigms were unraveled using an original immunocytochemical and transfection approach combined with three-dimensional image reconstruction. Cell culture incubation with AMPA or insulin triggered a marked intracellular accumulation of both MCT2 and GluR2/3, whereas both tumor necrosis factor alpha and glycine (with glutamate) increased their cell surface immunolabeling. Similar results were obtained using Western blots performed on membrane or cytoplasm-enriched cell fractions. Finally, an enhanced lactate flux into neurons was demonstrated after MCT2 translocation on the cell surface. These observations provide unequivocal evidence that MCT2 is linked to AMPA receptor GluR2/3 subunits and undergoes a similar translocation process in neurons upon activation. MCT2 emerges as a novel component of the synaptic machinery putatively linking neuroenergetics to synaptic transmission.

Remodeling of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit composition in hippocampal neurons after global ischemia

Transient global ischemia induces selective delayed cell death, primarily of principal neurons in the hippocampal CA1. However, the molecular mechanisms underlying ischemia-induced cell death are as yet unclear. The present study shows that global ischemia triggers a pronounced and cell-specific reduction in GluR2 [the subunit that limits Ca2+ permeability of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors] in vulnerable CA1 neurons, as evidenced by immunofluorescence of brain sections and Western blot analysis of microdissected hippocampal subfields. At 72 h after ischemia (a time before cell death), virtually all CA1 pyramidal neurons exhibited greatly reduced GluR2 immunolabeling throughout their somata and dendritic processes. GluR2 immunolabeling was unchanged in pyramidal cells of the CA3 and granule cells of the dentate gyrus, regions resistant to ischemia-induced damage. Immunolabeling of the AMPA receptor subunit GluR1 was unchanged in CA1, CA3, and dentate gyrus. Western analysis indicated that GluR2 subunit abundance was markedly reduced in CA1 at 60 and 72 h after the ischemic insult; GluR1 abundance was unchanged in all subfields at all times examined. These findings, together with the previous observation of enhanced AMPA-elicited Ca2+ influx in postischemic CA1 neurons, show that functional GluR2-lacking, Ca2+-permeable AMPA receptors are expressed in vulnerable neurons before cell death. Thus, the present study provides an important link in the postulated causal chain between global ischemia and delayed death of CA1 pyramidal neurons.

7-Chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine S,S-dioxide (IDRA 21), a congener of aniracetam, potently abates pharmacologically induced cognitive impairments in patas monkeys.

We report here on the ability of IDRA 21 and aniracetam, two negative allosteric modulators of glutamate-induced DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor desensitization, to attenuate alprazolam-induced learning deficit in patas monkeys working in a complex behavioral task. In one component of a multiple schedule (repeated acquisition or "learning"), patas monkeys acquired a different four-response chain each session by responding sequentially on three keys in the presence of four discriminative stimuli (geometric forms or numerals). In the other component (performance) the four-response chain was the same each session. The response chain in each component was maintained by food presentation under a fixed-ratio schedule. When alprazolam (0.1 or 0.32 mg/kg p.o.) was administered alone, this full allosteric modulator of gamma-aminobutyric acid type A (GABAA) receptors produced large decreases in the response rate and accuracy in the learning component of the task. IDRA 21 (3 or 5.6 mg/kg p.o.) and aniracetam (30 mg/kg p.o.) administered 60 min before alprazolam, having no effect when given alone, antagonized the large disruptive effects of alprazolam on learning. From dose-response studies, it can be estimated that IDRA 21 is approximately 10-fold more potent than aniracetam in antagonizing alprazolam-induced learning deficit. We conclude that IDRA 21, a chemically unrelated pharmacological congener of aniracetam, improves learning deficit induced in patas monkeys by the increase of GABAergic tone elicited by alprazolam. Very likely IDRA 21 exerts its behavioral effects by antagonizing AMPA receptor desensitization.

Calcium-permeable AMPA receptors mediate long-term potentiation in interneurons in the amygdala

Fear conditioning is a paradigm that has been used as a model for emotional learning in animals'. The cellular correlate of fear conditioning is thought to be associative N-methyl-D-aspartate (NMDA) receptor-dependent synaptic plasticity within the amygdala(1-3). Here we show that glutamatergic synaptic transmission to inhibitory interneurons in the basolateral amygdala is mediated solely by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. In contrast to AMPA receptors at inputs to pyramidal neurons, these receptors have an inwardly rectifying current-voltage relationship, indicative of a high permeability to calcium(4 5), Tetanic stimulation of inputs to interneurons caused an immediate and sustained increase in the efficacy of these synapses. This potentiation required a rise in postsynaptic calcium, but was independent of NMDA receptor activation. The potentiation of excitatory inputs to interneurons was reflected as an increase in the amplitude of the GABAA-mediated inhibitory synaptic current in pyramidal neurons. These results demonstrate that excitatory synapses onto interneurons within a fear conditioning circuit show NMDA-receptor independent long-term potentiation. This plasticity might underlie the increased synchronization of activity between neurons in the basolateral amygdala after fear conditioning(6).

Excitatory synaptic inputs to pyramidal neurons of the lateral amygdala

Whole-cell patch clamp recordings were made from pyramidal neurons in the rat lateral amygdala (LA). Synaptic currents were evoked by stimulating in either the external capsule (ec), internal capsule (ic) or basolateral nucleus (BLA). Stimulation of either the ic, ec or BLA evoked a glutamatergic excitatory synaptic current (EPSC) which was mediated by both non-NMDA and NMDA (N-methyl-D-aspartic acid) receptors, The ratio of the amplitude of the NMDA receptor-mediated component measured at +40 mV to the amplitude of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) component measured at -60 mV was similar regardless of whether EPSCs were evoked in the ec, ic or BLA. At resting membrane potentials, excitatory synaptic potentials evoked from either the ec or putative thalamic inputs were unaffected by application of the NMDA receptor antagonist APV. Spontaneous glutamatergic currents had two components to their decay phase. The slow component was selectively blocked by the NMDA receptor antagonist D-APV, indicating that AMPA and NMDA receptors are colocalized in spiny neurons. We conclude that pyramidal cells of the LA receive convergent inputs from the cortex, thalamus and basal nuclei. At all inputs, both AMPA/kainate and NMDA-type receptors are active and colocalized in the postsynaptic density.

Roles of mGluR5 in synaptic function and plasticity of the mouse thalamocortical pathway.

The group I metabotropic glutamate receptor 5 (mGluR5) has been implicated in the development of cortical sensory maps. However, its precise roles in the synaptic function and plasticity of thalamocortical (TC) connections remain unknown. Here we first show that in mGluR5 knockout (KO) mice bred onto a C57BL6 background cytoarchitectonic differentiation into barrels is missing, but the representations for large whiskers are identifiable as clusters of TC afferents. The altered dendritic morphology of cortical layer IV spiny stellate neurons in mGluR5 KO mice implicates a role for mGluR5 in the dendritic morphogenesis of excitatory neurons. Next, in vivo single-unit recordings of whisker-evoked activity in mGluR5 KO adults demonstrated a preserved topographical organization of the whisker representation, but a significantly diminished temporal discrimination of center to surround whiskers in the responses of individual neurons. To evaluate synaptic function at TC synapses in mGluR5 KO mice, whole-cell voltage-clamp recording was conducted in acute TC brain slices prepared from postnatal day 4-11 mice. At mGluR5 KO TC synapses, N-methyl-D-aspartate (NMDA) currents decayed faster and synaptic strength was more easily reduced, but more difficult to strengthen by Hebbian-type pairing protocols, despite a normal developmental increase in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated currents and presynaptic function. We have therefore demonstrated that mGluR5 is required for synaptic function/plasticity at TC synapses as barrels are forming, and we propose that these functional alterations at the TC synapse are the basis of the abnormal anatomical and functional development of the somatosensory cortex in the mGluR5 KO mouse.

Regulation of the intracellular distribution, cell surface expression, and protein levels of AMPA receptor GluR2 subunits by the monocarboxylate transporter MCT2 in neuronal cells.

The neuronal monocarboxylate transporter, MCT2, is not only an energy substrate carrier but it is also purported to be a binding partner for the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR2 subunit. To unravel a putative role of MCT2 in the regulation of GluR2 subcellular distribution, Neuro2A cells and primary cultures of mouse cortical neurons were co-transfected with plasmids containing sequences to express the fluorescent proteins mStrawberry (mStb)-fused MCT2 and Venus-fused GluR2. Subsequently, their subcellular distribution was visualized by fluorescence microscopy. GluR2 was led to form perinuclear and dendritic clusters together with MCT2 when co-transfected in Neuro2A cells or in neurons, following the original distribution of MCT2. MCT2 co-transfection had no effect on the intracellular distribution of several other post-synaptic proteins, although it partially affected the intracellular distribution of GluR1 similarly to GluR2. Both cell surface and total protein expression levels of GluR2 were significantly reduced by co-expression with MCT2. Finally, partial perinuclear and dendritic co-localization between MCT2 and Rab8, a member of the small GTPase family involved in membrane trafficking of AMPA receptors, was also observed in co-transfected neurons. These results suggest that MCT2 could influence AMPA receptor trafficking within neurons by modulating GluR2 sorting between different subcellular compartments.

Interaction between neuropeptide Y (NPY) and brain-derived neurotrophic factor in NPY-mediated neuroprotection against excitotoxicity : a role for microglia

The neuroprotective effect of neuropeptide Y (NPY) receptor activation was investigated in organotypic mouse hippocampal slice cultures exposed to the glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Exposure of 2-week-old slice cultures, derived from 7-day-old C57BL/6 mice, to 8 microm AMPA, for 24 h, induced degeneration of CA1 and CA3 pyramidal cells, as measured by cellular uptake of propidium iodide (PI). A significant neuroprotection, with a reduction of PI uptake in CA1 and CA3 pyramidal cell layers, was observed after incubation with a Y(2) receptor agonist [NPY(13-36), 300 nm]. This effect was sensitive to the presence of the selective Y(2) receptor antagonist (BIIE0246, 1 microm), but was not affected by addition of TrkB-Fc or by a neutralizing antibody against brain-derived neurotrophic factor (BDNF). Moreover, addition of a Y(1) receptor antagonist (BIBP3226, 1 microm) or a NPY-neutralizing antibody helped to disclose a neuroprotective role of endogenous NPY in CA1 region. Cultures exposed to 8 microm AMPA for 24 h, displayed, as measured by an enzyme-linked immunosorbent assay, a significant increase in BDNF. In such cultures there was an up-regulation of neuronal TrkB immunoreactivity, as well as the presence of BDNF-immunoreactive microglial cells at sites of injury. Thus, an increase of AMPA-receptor mediated neurodegeneration, in the mouse hippocampus, was prevented by neuroprotective pathways activated by NPY receptors (Y(1) and Y(2)), which can be affected by BDNF released by microglia and neurons.

A quantitative analysis of L-glutamate-regulated Na+ dynamics in mouse cortical astrocytes: implications for cellular bioenergetics.

The mode of Na+ entry and the dynamics of intracellular Na+ concentration ([Na+]i) changes consecutive to the application of the neurotransmitter glutamate were investigated in mouse cortical astrocytes in primary culture by video fluorescence microscopy. An elevation of [Na+]i was evoked by glutamate, whose amplitude and initial rate were concentration dependent. The glutamate-evoked Na+ increase was primarily due to Na+-glutamate cotransport, as inhibition of non-NMDA ionotropic receptors by 6-cyano-7-nitroquinoxiline-2,3-dione (CNQX) only weakly diminished the response and D-aspartate, a substrate of the glutamate transporter, produced [Na+]i elevations similar to those evoked by glutamate. Non-NMDA receptor activation could nevertheless be demonstrated by preventing receptor desensitization using cyclothiazide. Thus, in normal conditions non-NMDA receptors do not contribute significantly to the glutamate-evoked Na+ response. The rate of Na+ influx decreased during glutamate application, with kinetics that correlate well with the increase in [Na+]i and which depend on the extracellular concentration of glutamate. A tight coupling between Na+ entry and Na+/K+ ATPase activity was revealed by the massive [Na+]i increase evoked by glutamate when pump activity was inhibited by ouabain. During prolonged glutamate application, [Na+]i remains elevated at a new steady-state where Na+ influx through the transporter matches Na+ extrusion through the Na+/K+ ATPase. A mathematical model of the dynamics of [Na+]i homeostasis is presented which precisely defines the critical role of Na+ influx kinetics in the establishment of the elevated steady state and its consequences on the cellular bioenergetics. Indeed, extracellular glutamate concentrations of 10 microM already markedly increase the energetic demands of the astrocytes.

Toward an Explanation of the Genesis of Ketamine-Induced Perceptual Distortions and Hallucinatory States

The NMDA receptor (NMDAR) channel has been proposed to function as a coincidence-detection mechanism for afferent and reentrant signals, supporting conscious perception, learning, and memory formation. In this paper we discuss the genesis of distorted perceptual states induced by subanesthetic doses of ketamine, a well-known NMDA antagonist. NMDAR blockage has been suggested to perturb perceptual processing in sensory cortex, and also to decrease GABAergic inhibition in limbic areas (leading to an increase in dopamine excitability). We propose that perceptual distortions and hallucinations induced by ketamine blocking of NMDARs are generated by alternative signaling pathways, which include increase of excitability in frontal areas, and glutamate binding to AMPA in sensory cortex prompting Ca++ entry through voltage-dependent calcium channels (VDCCs). This mechanism supports the thesis that glutamate binding to AMPA and NMDARs at sensory cortex mediates most normal perception, while binding to AMPA and activating VDCCs mediates some types of altered perceptual states. We suggest that Ca++ metabolic activity in neurons at associative and sensory cortices is an important factor in the generation of both kinds of perceptual consciousness.