Calcium-permeable AMPA Receptors Research Articles

Ca2+ imaging of mouse neocortical interneurone dendrites: contribution of Ca2+-permeable AMPA and NMDA receptors to subthreshold Ca2+dynamics.

In this second study, we have combined two-photon calcium imaging with whole-cell recording and anatomic reconstructions to directly characterize synaptically evoked calcium signals in three types of mouse V1 supragranular interneurones: parvalbumin-positive fast spikers (FS), calretinin-positive irregular spikers (IS), and adapting cells (AD). We observed that subthreshold synaptic activation evoked calcium signals locally restricted to individual dendritic compartments. These signals were mediated by NMDA receptors (NMDARs) in AD and IS cells, whereas in FS cells, calcium-permeable AMPA receptors (CP-AMPARs) provided an additional and kinetically distinct influx. Furthermore, even a single, subthreshold synaptic activation evoked a larger dendritic calcium influx than backpropagating action potentials. Our results demonstrate that NMDARs dominate subthreshold calcium dynamics in interneurones and reveal the functional contribution of CP-AMPARs to a specific subclass of cortical interneurone. These data highlight different strategies in dendritic signal processing by distinct classes of interneurones.

Ionotropic glutamate receptors: still a target for neuroprotection in brain ischemia? insights from in vitro studies

Although experimental studies have widely shown that the pharmacological blockade of ionotropic glutamate receptors reduces ischemic damage, clinical trials with classical AMPA and NMDA glutamate receptor antagonists have provided negative results. To address the involvement of ionotropic glutamate receptors in ischemic damage, corticostriatal brain slices were prepared from adult rats. Extracellular recordings were performed in the striatum after stimulation of the glutamatergic corticostriatal fibres. In vitro ischemia was induced for a 10-min period by omitting oxygen and glucose from the external medium. Under control conditions, ischemia produced an irreversible loss of the corticostriatal field potential amplitude, AP5, a competitive NMDA receptor antagonist, induced a slight rescue of the potential, while ifenprodil, a positive modulator of the proton sensor of the NMDA receptors, allowed a complete recovery from the ischemic insult. Similar neuroprotection was achieved by utilizing either CNQX, a broad spectrum AMPA receptors antagonist, or Joro spider toxin, a selective blocker of calcium permeable AMPA receptors. Interestingly, while CNZX also fully suppressed physiological excitatory transmission, Joro spider toxin was ineffective on this parameter. Finally, lamotrigine and remacemide, two antiepileptic drugs that differentially affect excitatory transmission, exerted neuroprotective effects against ischemia. Noticeably, a combination of low concentrations of these two drugs exerted a stronger neuroprotection than a single drug given in isolation. Thus, it might be possible to reach a neuroprotective action by utilizing doses of these compounds low enough to avoid side effects. Our experimental data still support the idea that a negative modulation of excitatory transmission can be neuroprotective against ischemia. In addition, our findings support the concept that it is possible to produce a significant neuroprotective action in the absence of a relevant interference with normal synaptic transmission.

Coexistence of NMDA and AMPA receptor subunits with nNOS in the nucleus tractus solitarii of rat

We previously showed that most neuronal nitric oxide synthase (nNOS)-containing neurons in the nucleus tractus solitarii (NTS) contain NMDAR1, the fundamental subunit for functional N-methyl- d-aspartate (NMDA) receptors. Likewise, we found that almost all nNOS-containing neurons in the NTS contain GluR1, the calcium permeable AMPA receptor subunit. These data suggest that AMPA and NMDA receptors may colocalize in NTS neurons that contain nNOS. However, other investigators have suggested that non-NMDA receptors are located primarily on second-order neurons and NMDA receptors are located predominantly on higher-order neurons in NTS. We now seek to test the hypothesis that NMDA receptors, AMPA receptors and nNOS are colocalized in NTS cells. We performed triple fluorescent immunohistochemical staining of nNOS, NMDAR1 and GluR1, and performed confocal laser scanning microscopic analysis of the NTS. The distributions of nNOS immunoreactivity (IR), NMDAR1-IR and GluR1-IR in the NTS were similar to those we reported earlier. Superimposed images revealed that almost all NMDAR1-IR cells contained GluR1-IR and almost all GluR1-IR cells contained NMDAR1-IR. Some double-labeled cells were additionally labeled for nNOS-IR. All nNOS-IR neurons contained both GluR1-IR and NMDAR1-IR. These studies support our hypothesis that NMDA and AMPA receptors are colocalized in NTS neurons and are consistent with a role of both types of ionotropic receptors in transmission of afferent signals in NTS. In addition, these data provide support for an anatomical link between ionotropic glutamate receptors and nitric oxide in the NTS.

Identifications, classification, and evolution of the vertebrate alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunit genes.

The AMPA receptor (AMPAR), a pharmacologically defined ionotropic glutamate receptor, mediates fast excitatory synaptic transmission in the vertebrate central nervous system. Mammalian and avian AMPARs are assembled from the products of four genes (GRIA1-GRIA4) conserved in their translated sequences and gene organizations. Teleost fish also express AMPAR subunits; however, the AMPAR genes have not been extensively investigated in lower vertebrates. To elucidate the evolution of vertebrate AMPAR genes, reverse-transcriptase PCR-based surveys of subunits expressed in the brains of eight nonmammalian vertebrates were performed. The newly cloned vertebrate AMPAR subunits were classified by their sequence identities to the mammalian AMPAR subunits. The results of molecular and phylogenetic analyses indicated that the members of the AMPAR gene family increased from two in the jawless hagfish to four in the tetrapods and the shark and to more than four in the teleost fish. The sizes of AMPAR gene families correlate well with those of many multigene families observed in various vertebrates. Moreover, all vertebrates expressed at least one AMPAR subunit bearing an arginine (R) at the Q/R site, at which no invertebrate glutamate receptor subunit has been found to have an R residue, suggesting that the low calcium-permeable AMPARs appeared at early evolutionary stages of vertebrate central nervous systems. Uniquely, the loop 1 (L1) regions between hydrophobic domain 1 and hydrophobic domain 2 of the hagfish putative GRIA2 and all the teleost GRIA1 subunits were much longer than those of the remaining known ionotropic glutamate receptor subunits. The length and sequence of the L1 of teleost GRIA1 subunits were heterogeneous, suggesting that the amino acid residues in L1 were not highly selected.

Differential Mechanisms of Transmission at Three Types of Mossy Fiber Synapse

The axons of the dentate gyrus granule cells, the so-called mossy fibers, innervate their inhibitory interneuron and pyramidal neuron targets via both anatomically and functionally specialized synapses. Mossy fiber synapses onto inhibitory interneurons were comprised of either calcium-permeable (CP) or calcium-impermeable (CI) AMPA receptors, whereas only calcium-impermeable AMPA receptors existed at CA3 principal neuron synapses. In response to brief trains of high-frequency stimuli (20 Hz), pyramidal neuron synapses invariably demonstrated short-term facilitation, whereas interneuron EPSCs demonstrated either short-term facilitation or depression. Facilitation at all CI AMPA synapses was voltage independent, whereas EPSCs at CP AMPA synapses showed greater facilitation at -20 than at -80 mV, consistent with a role for the postsynaptic unblock of polyamines. At pyramidal cell synapses, mossy fiber EPSCs possessed marked frequency-dependent facilitation (commencing at stimulation frequencies >0.1 Hz), whereas EPSCs at either type of interneuron synapse showed only moderate frequency-dependent facilitation or underwent depression. Presynaptic metabotropic glutamate receptors (mGluRs) decreased transmission at all three synapse types in a frequency-dependent manner. However, after block of presynaptic mGluRs, transmission at interneuron synapses still did not match the dynamic range of EPSCs at pyramidal neuron synapses. High-frequency stimulation of mossy fibers induced long-term potentiation (LTP), long-term depression (LTD), or no change at pyramidal neuron synapses, interneuron CP AMPA synapses, and CI AMPA synapses, respectively. Induction of LTP or LTD altered the short-term plasticity of transmission onto both pyramidal cells and interneuron CP AMPA synapses by a mechanism consistent with changes in release probability. These data reveal differential mechanisms of transmission at three classes of mossy fiber synapse made onto distinct targets.

Synaptic activity at calcium-permeable AMPA receptors induces a switch in receptor subtype.

Activity-dependent change in the efficacy of transmission is a basic feature of many excitatory synapses in the central nervous system. The best understood postsynaptic modification involves a change in responsiveness of AMPAR (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor)-mediated currents following activation of NMDA (N-methyl-D-aspartate) receptors or Ca2+-permeable AMPARs. This process is thought to involve alteration in the number and phosphorylation state of postsynaptic AMPARs. Here we describe a new form of synaptic plasticity--a rapid and lasting change in the subunit composition and Ca2+ permeability of AMPARs at cerebellar stellate cell synapses following synaptic activity. AMPARs lacking the edited GluR2 subunit not only exhibit high Ca2+ permeability but also are blocked by intracellular polyamines. These properties have allowed us to follow directly the involvement of GluR2 subunits in synaptic transmission. Repetitive synaptic activation of Ca2+-permeable AMPARs causes a rapid reduction in Ca2+ permeability and a change in the amplitude of excitatory postsynaptic currents, owing to the incorporation of GluR2-containing AMPARs. Our experiments show that activity-induced Ca2+ influx through GluR2-lacking AMPARs controls the targeting of GluR2-containing AMPARs, implying the presence of a self-regulating mechanism.

Inhibition of delayed rectifier K+ conductance in cultured rat cerebellar granule neurons by activation of calcium-permeable AMPA receptors.

Activation of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors in cerebellar granule cells during perforated-patch whole-cell recordings activated an inward current at negative voltages which was followed, after a delay, by the inhibition of an outward potassium current at voltages positive to -20 mV. The activated inward current was inwardly rectifying suggesting that the AMPA receptors were Ca2+-permeable. This was confirmed by direct measurements of intracellular calcium where Ca2+ rises were seen following AMPA receptor activation in Na+-free external solution. Ca2+ rises were equally large in the presence of 100 microM Cd2+ to block voltage-gated Ca2+ channels. Specific voltage-protocols, allowing selective activation of the delayed rectifier potassium current (KV) and the transient A current (KA), showed that kainate inhibited KV, but not to any great extent KA. The inhibition of KV was blocked by the AMPA receptor antagonist CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) and was no longer observed when the KV current was abolished with high concentrations of Ba2+. The responses to kainate were not altered by pre-treating the cells with pertussis toxin, suggesting that the AMPA receptor stimulation of the G-protein Gi cannot account for the effects observed. Replacing extracellular Na+ with choline did not alter the inhibition of KV by kainate, however, removing extracellular Ca2+ reduced the kainate response. The inhibition of KV by kainate was unaffected by the presence of 100 microM Cd2+. The guanylyl cyclase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), did not alter kainate inhibition of KV. It is concluded that ion influx (particularly Ca2+ ions) through AMPA receptor channels following receptor activation leads to an inhibition of KV currents in cerebellar granule neurons.

Intermediate Zone Cells Express Calcium-Permeable AMPA Receptors and Establish Close Contact with Growing Axons

Recent studies have shown that cells in the intermediate zone (IZ) of the embryonic neocortex originate in the basal telencephalon and migrate tangentially in the cortical wall (;; ). We had previously observed growing cortical axons closely apposed to calbindin-positive, tangentially oriented cells in the IZ (), and it has been shown that neurites in the IZ express a glutamate transporter (). To test if glutamate released by corticofugal growth cones could influence the tangential IZ cells, we characterized the glutamate receptors expressed by IZ cells using patch-clamp techniques, histochemical labeling, and immunostaining on slices of embryonic mice forebrain. We show that tangential IZ cells express inwardly rectifying kainate responses, but not NMDA responses, and accumulate cobalt after AMPA receptor activation. We conclude that IZ cells express calcium-permeable AMPA receptors. This property correlates with our observation that the GluR2 subunit is not expressed in the IZ. AMPA receptors are activated by a millimolar concentration of glutamate. To know whether this high level of glutamate could occur at the surface of IZ cells, we examined contacts made by corticofugal growth cones and calbindin-positive IZ cells using electron microscopy. We show vesicle-containing neurites tightly apposed to calbindin-positive IZ cells over remarkably long length. This suggests that glutamate released by growing corticofugal axons could reach high concentrations close to AMPA receptors of tangential IZ cells and efficiently activate them to control the intracellular calcium in embryonic IZ cells.

Functional significance of calcium-permeable AMPA receptor channels in cerebellar Bergmann glial cells

Functional significance of calcium-permeable AMPA receptor channels in cerebellar Bergmann glial cells

Time Course and Permeation of Synaptic AMPA Receptors in Cochlear Nuclear Neurons Correlate with Input

AMPA receptors mediate rapid glutamatergic synaptic transmission. In the mammalian cochlear nuclei, neurons receive excitatory input from either auditory nerve fibers, parallel fibers, or both fiber systems. The functional correlates of differences in the source of input were examined by recording AMPA receptor-mediated, miniature EPSCs (mEPSCs) in whole-cell voltage-clamp mode from identified neurons. Bushy, octopus, and T-stellate cells of the ventral cochlear nucleus (VCN) and tuberculoventral cells of the dorsal cochlear nucleus (DCN) receive most of their excitatory input from the auditory nerve; fusiform cells receive excitatory inputs from both the auditory nerve and parallel fibers; cartwheel cells receive excitatory input from parallel fibers alone. mEPSCs from bushy, octopus, T-stellate, and tuberculoventral cells had significantly faster decay time constants (0.35-0.40 msec) than did those from fusiform and cartwheel cells (1.32-1.79 msec). Some fusiform cells had two populations of mEPSCs with distinct time courses. mEPSCs in cells with auditory nerve input alone were inhibited by philanthotoxin, a blocker of calcium-permeable AMPA receptors, whereas mEPSCs in cells with parallel fiber input were not. Thus AMPA receptors postsynaptic to the auditory nerve differ from those postsynaptic to parallel fibers both in channel-gating kinetics and in their permeability to calcium. These results confirm the conclusion that synaptic AMPA receptors are specialized according to the source of input (Hunter et al., 1993; Rubio and Wenthold, 1997; Wang et al., 1998).

Postsynaptic Glutamate Receptors and Integrative Properties of Fast-Spiking Interneurons in the Rat Neocortex

The glutamate-mediated synaptic responses of neocortical pyramidal cell to fast-spiking interneuron (pyramidal-FS) connections were studied by performing paired recordings at 30-33 degrees C in acute slices of 14- to 35-day-old rats (n = 39). Postsynaptic fast-spiking (FS) cells were recorded in whole cell configuration with a patch pipette, and presynaptic pyramidal cells were impaled with sharp intracellular electrodes. At a holding potential of -72 mV (near the resting membrane potential), unitary excitatory postsynaptic potentials (EPSPs) had a mean amplitude of 2.1 +/- 1.3 mV and a mean width at half-amplitude of 10.5 +/- 3.7 ms (n = 18). Bath application of the N-methyl-D-aspartate (NMDA) receptor antagonist D(-)2-amino-5-phosphonovaleric acid (D-AP5) had minor effects on both the amplitude and the duration of unitary EPSPs, whereas the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) almost completely blocked the synaptic responses. In voltage-clamp mode, the selective antagonist of AMPA receptors 1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-7,8-methylenedioxy-3, 4-dihydro-5H-2,3-benzodiazepine (GYKI 53655; 40-66 microM) blocked 96 +/- 1.9% of D-AP5-insensitive unitary excitatory postsynaptic currents (EPSCs), confirming the predominance of AMPA receptors, as opposed to kainate receptors, at pyramidal-FS connections (n = 3). Unitary EPSCs mediated by AMPA receptors had fast rise times (0.29 +/- 0.04 ms) and amplitude-weighted decay time constants (2 +/- 0.8 ms; n = 16). In the presence of intracellular spermine, these currents showed the characteristic rectifying current-voltage (I-V) curve of calcium-permeable AMPA receptors. A slower component mediated by NMDA receptors was observed when unitary synaptic currents were recorded at a membrane potential more positive than -50 mV. In response to short trains of moderately high-frequency (67 Hz) presynaptic action potentials, we observed only a limited temporal summation of unitary EPSPs, probably because of the rapid kinetics of AMPA receptors and the absence of NMDA component in these subthreshold synaptic responses. By combining paired recordings with extracellular stimulations (n = 11), we demonstrated that EPSPs elicited by two different inputs were summed linearly by FS interneurons at membrane potentials below the action potential threshold. We estimated that, in our in vitro recording conditions, 8 +/- 5 pyramidal cells (n = 18) should be activated simultaneously to make FS interneurons fire an action potential from -72 mV. The low level of temporal summation and the linear summation of excitatory inputs in FS cells favor the role of coincidence detectors of these interneurons in neocortical circuits.

Mechanisms of Calcium Influx into Hippocampal Spines: Heterogeneity among Spines, Coincidence Detection by NMDA Receptors, and Optical Quantal Analysis

Dendritic spines receive most excitatory inputs in the vertebrate brain, but their function is still poorly understood. Using two-photon calcium imaging of CA1 pyramidal neurons in rat hippocampal slices, we investigated the mechanisms by which calcium enters into individual spines in the stratum radiatum. We find three different pathways for calcium influx: high-threshold voltage-sensitive calcium channels, NMDA receptors, and an APV-resistant influx consistent with calcium-permeable AMPA or kainate receptors. These pathways vary among different populations of spines and are engaged under different stimulation conditions, with peak calcium concentrations reaching >10 microM. Furthermore, as a result of the biophysical properties of the NMDA receptor, the calcium dynamics of spines are exquisitely sensitive to the temporal coincidence of the input and output of the neuron. Our results confirm that individual spines are chemical compartments that can perform coincidence detection. Finally, we demonstrate that functional studies and optical quantal analysis of single, identified synapses is feasible in mammalian CNS neurons in brain slices.

The Distribution of Neurons Expressing Calcium-Permeable AMPA Receptors in the Superficial Laminae of the Spinal Cord Dorsal Horn

The superficial dorsal horn is a major site of termination of nociceptive primary afferents. Fast excitatory synaptic transmission in this region is mediated mainly by release of glutamate onto postsynaptic AMPA and NMDA receptors. NMDA receptors are known to be Ca2+-permeable and to provide synaptically localized Ca2+ signals that mediate short-term and long-term changes in synaptic strength. Less well known is a subpopulation of AMPA receptors that is Ca2+-permeable and has been shown to be synaptically localized on dorsal horn neurons in culture (Gu et al., 1996) and expressed by dorsal horn neurons in situ (Nagy et al., 1994; Engelman et al., 1997). We used kainate-induced cobalt uptake as a functional marker of neurons expressing Ca2+-permeable AMPA receptors and combined this with markers of nociceptive primary afferents in the postnatal rat dorsal horn. We have shown that cobalt-positive neurons are located in lamina I and outer lamina II, a region strongly innervated by nociceptors. These cobalt-positive neurons colocalize with afferents labeled by LD2, and with the most dorsal region of capsaicin-sensitive and IB4- and LA4-positive afferents. In contrast, inner lamina II has a sparser distribution of cobalt-positive neurons. Some lamina I neurons expressing the NK1 receptor, the receptor for substance P, are also cobalt positive. These neurons are likely to be projection neurons in the nociceptive pathway. On the basis of all of these observations, we propose that Ca2+-permeable AMPA receptors are localized to mediate transmission of nociceptive information.

Low expression of GluR2 AMPA receptor subunit protein by human motor neurons.

Disturbance of glutamate neurotransmission may contribute to motor neuron injury in amyotrophic lateral sclerosis. There is evidence that human motor neurons may express a specific profile of glutamate receptors, with low or absent expression of mRNA for the GluR2 AMPA receptor subunit, which has a crucial role in controlling calcium permeability. This study, using an immunocytochemical approach with a GluR2 specific antibody, shows that human upper and lower motor neurons have a very low/absent expression of GluR2 protein, in contrast to many other neuronal groups. Thus, it is likely that human motor neurons express a high proportion of atypical, calcium permeable AMPA receptors which may contribute to selective vulnerability and may allow cell-specific modulation of the actions of glutamate.

Glutamate Potentiates the Toxicity of Mutant Cu/Zn-Superoxide Dismutase in Motor Neurons by Postsynaptic Calcium-Dependent Mechanisms

Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene are responsible for a subset of familial cases of amyotrophic lateral sclerosis. Using a primary culture model, we have demonstrated that normally nontoxic glutamatergic input, particularly via calcium-permeable AMPA/kainate receptors, is a major factor in the vulnerability of motor neurons to the toxicity of SOD-1 mutants. Wild-type and mutant (G41R, G93A, or N139K) human SOD-1 were expressed in motor neurons of dissociated cultures of murine spinal cord by intranuclear microinjection of plasmid expression vector. Both a general antagonist of AMPA/kainate receptors (CNQX) and a specific antagonist of calcium-permeable AMPA receptors (joro spider toxin) reduced formation of SOD-1 proteinaceous aggregates and prevented death of motor neurons expressing SOD-1 mutants. Partial protection was obtained by treatment with nifedipine, implicating Ca2+ entry through voltage-gated calcium channels as well as glutamate receptors in potentiating the toxicity of mutant SOD-1 in motor neurons. Dramatic neuroprotection was obtained by coexpressing the calcium-binding protein calbindin-D28k but not by increasing intracellular glutathione levels or treatment with the free radical spin trap agent, N-tert-butyl-alpha-phenylnitrone. Thus, generalized oxidative stress could have contributed in only a minor way to death of motor neurons expressing the mutant SOD-1. These studies demonstrated that the toxicity of these mutants is calcium-dependent and provide direct evidence that calcium entry during neurotransmission, coupled with deficiency of cytosolic calcium-binding proteins, is a major factor in the preferential vulnerability of motor neurons to disease.

Afferent-specific innervation of two distinct AMPA receptor subtypes on single hippocampal interneurons.

Using the polyamine toxin philanthotoxin, which selectively blocks calcium-permeable AMPA receptors, we show that synaptic transmission onto single hippocampal interneurons occurs by afferent-specific activation of philanthotoxin-sensitive and -insensitive AMPA receptors. Calcium-permeable AMPA receptors are found exclusively at synapses from mossy fibers. In contrast, synaptic responses evoked by stimulation of CA3 pyramidal neurons are mediated by calcium-impermeable AMPA receptors. Both pathways converge onto single interneurons and can be discriminated with Group II mGluR agonists. Thus, single interneurons target AMPA receptors of different subunit composition to specific postsynaptic sites, providing a mechanism to increase the synapse-specific computational properties of hippocampal interneurons.

Activity-Dependent Modulation of Glutamate Receptors by Polyamines

The mechanisms by which polyamines block AMPA and kainate receptors are not well understood, but it has been generally assumed that they act as open-channel blockers. Consistent with this, voltage-jump relaxation analysis of GluR6 equilibrium responses to domoate could be well fit, assuming that spermine, spermidine, and philanthotoxin are weakly permeable open-channel blockers. Analysis of rate constants for binding and dissociation of polyamines indicated that the voltage dependence of block arose primarily from changes in koff rather than kon. Experiments with changes in Na concentration further indicate that the voltage dependence of polyamine block was governed by ion flux via open channels. However, responses to 1 msec applications of L-Glu revealed slow voltage-dependent rise-times, suggesting that polyamines additionally bind to closed states. A kinetic model, which included closed-channel block, reproduced these observations but required that polyamines accelerate channel closure either through an allosteric mechanism or by emptying the pore of permeant ions. Simulations with this model reveal that polyamine block confers novel activity-dependent regulation on calcium-permeable AMPA and kainate receptor responses.

AMPA Receptors: Players in Calcium-Mediated Neuronal Injury?

AMPA receptors mediate a majority of fast, excitatory neurotransmissions in the CNS. AMPA receptors are multimeric proteins composed of one or more of four subunits, termed GluR1-4. Structure-function studies using recombinant AMPA receptor subunits have demonstrated the importance of an arginine residue at the Q/R site within the second transmembrane domain of the GluR2 subunit in conferring the selectivity of the receptor for monovalent cations. Native AMPA receptors in the adult, mammalian CNS are multimeric proteins that are largely calcium-impermeable because of the presence of the dominant GluR2 subunit. Populations of cells in both the developing and adult brain, however, express AMPA receptors that lack the GluR2 subunit and thus are cal cium-permeable. The function of these calcium-permeable AMPA receptors (CPARs) is unknown. Such cells may, however, be selectively vulnerable to excitotoxic injury. After severe global cerebral ischemia in rats, GluR2 expression is selectively downregulated in the vulnerable CA1 region of the hippocampus before the onset of ischemic cell death. Thus AMPA receptor activation may contribute to the excessive accumulation of intra cellular calcium that is thought to mediate irreversible cell injury. This article reviews the molecular basis of CPARs and discusses their possible role in mediating excitotoxic injury. NEUROSCIENTIST 4:149-153, 1998

Developing rat brainstem motoneurones in organotypic culture express calcium permeable AMPA-gated receptors

Recent studies suggest that calcium permeable excitatory amino acid receptors may play an important role in many developmental processes including dendritic differentiation, synaptogenesis and activity dependent plasticity or excitotoxicity related disorders. In this work, we investigate the existence of calcium permeable AMPA receptors in embryonic rat motoneurones in organotypic slice culture, which display an early sensitivity to AMPA and Kainate. We used excitatory amino acids induced cobalt uptake and show that developing motoneurones express Ca 2+ permeable AMPA receptors. We demonstrate, as already described for other neuronal types in acute slices of adult animals, that the cobalt loading of motoneurones is not suppressed by blockade of synaptic activity by TTX. It is not induced by NMDA stimulation and does not result from the activation of voltage dependent calcium channels. It is specifically suppressed by the non-competitive AMPA antagonist GYKI 53784 (LY303070) and enhanced by the AMPA-receptor desensitization blocker Aniracetam. We conclude that cobalt loading results from the specific activation of AMPA receptors. We further show that, when cobalt loading is induced by threshold doses of Glutamate agonists, cobalt–sulfide deposits are found specifically in primary dendrites, dendritic spines and localized spots on the somatic and peripheral dendritic membrane. We suggest that this particular pattern of staining, different from the Golgi-like staining obtained with excitotoxic doses, may offer new information regarding the membrane density and distribution of calcium permeable AMPA receptors.

Correlation between the calcium permeability of ampa receptors and the expression of calcium-binding proteins

A recently described form of synaptic plasticity results in dynamic changes in the calcium permeability of synaptic AMPA receptors. Since the AMPA receptor GluR2 subunit confers calcium permeability, this plasticity is thought to occur through the dynamic exchange of synaptic GluR2-lacking and GluR2-containing receptors. To investigate the molecular mechanisms underlying this calcium-permeable AMPA receptor plasticity (CARP), we examined whether AMPA receptor exchange was mediated by subunit-specific protein-protein interactions. We found that two GluR2-interacting proteins, the PDZ domain-containing Protein interacting with C kinase (PICK1) and N-ethylmaleimide sensitive fusion protein (NSF), are specifically required for CARP. Furthermore, PICK1, but not NSF, regulates the formation of extrasynaptic plasma membrane pools of GluR2-containing receptors that may be laterally mobilized into synapses during CARP. These results demonstrate that PICK1 and NSF dynamically regulate the synaptic delivery of GluR2-containing receptors during CARP and thus regulate the calcium permeability of AMPA receptors at excitatory synapses.