Activation of NR1/NR2B NMDA receptors

N-methyl-D-aspartate (NMDA) receptors are highly expressed in the central nervous system and are involved in excitatory synaptic transmission as well as synaptic plasticity. Despite considerable structural and biophysical research, the mechanism behind activation of the NMDA receptor is still poorly understood. By analyzing patch clamp recordings of one channel activated by glutamate, we determined the burst structure and open probability for recombinant rat NR1/NR2B receptors. We used partial agonists at the glutamate and glycine binding sites to show that at least two kinetically distinct subunit-associated conformational changes link co-agonist binding to the opening of the NMDA receptor pore. These data suggest that NR1 and NR2B subunits, respectively, undergo a fast and slow agonist-dependent conformational change that precedes opening of the pore. We propose a new working model of receptor activation that can account for macroscopic as well as microscopic NMDA receptor properties.     

NMDA receptors and their interacting proteins in the postsynaptic density

Glutamate receptors and their cytoplasmic interacting proteins are thought to play an important role in the process of excitatory synaptic transmission in the mammalian central nervous system. NMDA receptors are a subtype of ionotropic glutamate receptors and specifically interact with PDZ-domain containing proteins such as PSD-95. This interaction is important in the clustering of NMDA receptors on the postsynaptic membrane and the molecular complex containing NMDA receptors and PSD-95 is likely to be the core structure of the postsynaptic density. In the first part of this review, biological significance of the diversity of NMDA receptor subunits is discussed. In the second part, implications obtained from our recent analyses on the structural remodeling of the postsynaptic density are presented.     

Inhibition of glutamate transporters couples to Kv4.2 dephosphorylation through activation of extrasynaptic NMDA receptors

Activation of glutamate receptors is known to modulate K⁺ channel surface trafficking, phosphorylation, and function, and increasing evidence has implicated K⁺ channels in plastic changes in glutamatergic synapses. Kv4.2 channels control the amplitude of back-propagating action potentials and shape postsynaptic responses in hippocampus, and synaptic glutamate receptor activation leads to increased phosphorylation of Kv4.2 channels that is associated with enhanced synaptic plasticity. Thus, we investigated the possibility that activation of extrasynaptic NMDA-type glutamate receptors couples to Kv4.2 channel dephosphorylation. In hippocampal neurons, we found that selective activation of extrasynaptic NMDA receptors dephosphorylates Kv4.2 channels, and driving synaptic activity increases phosphorylation of Kv4.2. We also observed that Ca²⁺ entry through NMDA receptors is necessary for dephosphorylation of Kv4.2 channels. Consistent with a synaptic and extrasynaptic localization at hippocampal synapses, a fraction of Kv4.2 channel clusters was found to localize outside of pre- and postsynaptic markers. Excitatory amino acid transporters (EAATs) regulate ambient extracellular glutamate levels that active extrasynaptic NMDA receptors, and inhibition of glutamate uptake by blocking EAATs with the non-selective transporter inhibitor dl-threo-β-benzyloxyaspartic acid (TBOA) or the EAAT1/3 selective inhibitor l-serine O-sulfate (SOS) dephosphorylates Kv4.2 channels. These findings in conjunction with previous reports support the interesting possibility that synaptic and extrasynaptic NMDA receptors bi-directionally regulate phosphorylation levels of Kv4.2 channels in hippocampus. Moreover, we observed that EAAT activity controls extrasynaptic NMDA receptor modulation of Kv4.2 channel dephosphorylation.     

Allosteric interaction between zinc and glutamate binding domains on NR2A causes desensitization of NMDA receptors

Fast desensitization is an important regulatory mechanism of neuronal NMDA receptor function. Previous work suggests that fast desensitization of NR1/NR2A receptors is caused by ambient zinc, and that a positive allosteric interaction occurs between the extracellular zinc-binding amino terminal domain and the glutamate-binding domain of NR2A. The relaxation of macroscopic currents in the presence of zinc reflects a shift to a new equilibrium due to increased zinc affinity following the binding of glutamate. Here we demonstrate that this allosteric coupling reflects interactions within the NR2A subunit, and that the affinity of zinc for its binding site is regulated by glutamate binding and not by glycine binding nor by channel pore opening. We fit an explicit model to experimental data over a wide range of parameters, demonstrating that allosteric theory can quantitatively account for the fast zinc-dependent component of desensitization for NR1/NR2A NMDA receptors. We subsequently use this model to evaluate the effects of extracellular zinc on NR1/NR2A excitatory postsynaptic currents (EPSCs) by simulating the response to a brief synaptic-like pulse of glutamate. Modelling results show that zinc at a steady-state concentration of at least 100 n m has a significant effect on the amplitude of NMDA EPSCs but that concurrent release of 10 μ m zinc with synaptic glutamate release has little effect on the amplitude of a single NR1/NR2A NMDA EPSC. These data suggest that while steady-state zinc can regulate the amplitude of synaptic NMDA currents, zinc co-released with glutamate will only have significant impact under conditions of high frequency activity or at concentrations high enough to cause voltage-dependent channel block.     

Corticostriatal paired-pulse potentiation produced by voltage-dependent activation of NMDA receptors and L-type Ca(2+) channels.

AMPA and N-methyl-D-aspartate (NMDA) receptor-mediated synaptic responses expressed differential paired-pulse plasticity when examined in the same cell using intracellular or whole cell voltage-clamp recordings. Electrical stimulation of corticostriatal afferents in brain slices bathed in artificial cerebrospinal fluid containing bicuculline produces excitatory postsynaptic potentials and excitatory postsynaptic currents (EPSCs) mediated primarily by AMPA receptors. Cell-to-cell variation existed in AMPA receptor paired-pulse plasticity, but within-cell plasticity was stable over a range of stimulation intensities. Addition of 6-cyano-7-nitroquinoxalene-2,3-dione blocked most of the synaptic response leaving behind a small AP-5-sensitive component. Increasing the stimulation intensity produced large, long-lasting NMDA receptor-mediated responses. In contrast to AMPA receptor-mediated responses, NMDA receptor responses consistently showed an increase in paired-pulse potentiation with increasing stimulation intensity. This relationship was restricted to interstimulus intervals shorter than 100 ms. Paired-pulse potentiation of NMDA receptor responses was voltage-dependent and reduced by removal of extracellular Mg(2+). Block of postsynaptic L-type Ca(2+) channels with nifedipine produced a voltage-dependent reduction of NMDA receptor excitatory postsynaptic currents (EPSCs) and a voltage-dependent reduction of NMDA receptor paired-pulse potentiation. These data indicate depolarization during the first NMDA receptor response causes facilitation of the second by removing voltage-dependent block of NMDA receptors by Mg(2+) and by activating voltage-dependent Ca(2+) channels.     

Rapid redistribution of glutamate receptors contributes to long-term depression in hippocampal cultures.

Synaptic strength can be altered by a variety of pre- or postsynaptic modifications. Here we test the hypothesis that long-term depression (LTD) involves a decrease in the number of glutamate receptors that are clustered at individual synapses in primary cultures of hippocampal neurons. Similar to a prominent form of LTD observed in hippocampal slices, LTD in hippocampal cultures required NMDA receptor activation and was accompanied by a decrease in the amplitude and frequency of miniature excitatory postsynaptic currents. Immunocytochemical analysis revealed that induction of LTD caused a concurrent decrease in the number of AMPA receptors clustered at synapses but had no effect on synaptic NMDA receptor clusters. These results suggest that a subtype-specific redistribution of synaptic glutamate receptors contributes to NMDA receptor-dependent LTD.     

Monte Carlo simulation of spontaneous miniature excitatory postsynaptic currents in rat hippocampal synapse in the presence and absence of desensitization

 Using the Monte Carlo method, spontaneous fast excitatory postsynaptic currents (mEPSCs) at a hippocampal synapse were simulated by releasing 150–20,000 glutamate molecules from a point source centred 15 nm above a rectangular grid of 14 × 14 α-amino-3-hydroxy-methyl-isoxazole (AMPA) receptors and assuming the channel kinetics to be as reported by Jonas et al. [J Physiol (Lond) 472:615; 1993]. The relationship between the amplitudes of mEPSCs and their time constants of decay is positive, but not pronounced in physiological conditions (except when the number of molecules released is very high). It increases as desensitization is reduced and becomes highly pronounced when it is eliminated. mEPSCs are prolonged with repeated opening of AMPA channels due to enhancement of two concentration-dependent processes: (1) binding of glutamate molecules by AMPA receptors, and (2) occupancy of both activatable bound states. In contrast, the time constant of decay of the patch currents evoked by a short glutamate pulse is independent of glutamate concentration and current amplitude in control conditions, and only moderately concentration dependent in the absence of desensitization. The fast application protocol thus fails to reproduce synaptic currents reliably when there is repeated binding of glutamate molecules to AMPA receptors. During an mEPSC, the occupancy of desensitized states increases rapidly and it strongly depends on the number of glutamate molecules released. Desensitization reaches its maximum after an mEPSC decays to very low levels, and recovers very slowly (from tens to hundreds of milliseconds), and in a concentration-dependent manner. In conclusion, under physiological conditions the desensitization of AMPA receptors plays a major role in shaping the time course of mEPSCs by minimizing the repeated opening of AMPA channels. Received: 17 April 1997 / Received: after revision: 11 August 1997 / Accepted: 1 September 1997     

Temperature dependence of NR1/NR2B NMDA receptor channels

N-methyl-D-aspartate (NMDA) receptors are highly expressed in the CNS, mediate the slow component of excitatory transmission and play key roles in synaptic plasticity and excitotoxicity. These ligand-gated ion channels are heteromultimers composed of NR1 and NR2 subunits activated by glycine and glutamate. In this study, patch-clamp recordings were used to study the temperature sensitivity of recombinant NR1/NR2B receptors expressed in human embryonic kidney (HEK) 293 cells. Rate constants were assessed by fitting a six-state kinetic scheme to time courses of transient macroscopic currents induced by glutamate at 21.9-46.5 degrees C. Arrhenius transformation of the rate constants characterizing NMDA receptor channel activity indicates that the most sensitive were the rate constants of desensitization (temperature coefficient Q(10)=10.3), resensitization (Q(10)=4.6) and unbinding (Q(10)=3.6). Other rate constants and the amplitude of single-channel currents were less temperature sensitive. Deactivation of responses mediated by NR1/NR2B receptors after a brief application of glutamate was best fit by a double exponential function (tau(fast): Q(10)=3.7; tau(slow): Q(10)=2.7). From these data, we conclude that desensitization/resensitization of the NMDA receptor and glutamate unbinding are especially temperature sensitive and imply that at physiological temperatures the channel kinetics play an important role in determining amplitude and time course of NMDA receptor-mediated postsynaptic currents and these receptors mediated synaptic plasticity.     

Presynaptic and postsynaptic NMDA receptors mediate distinct effects of brain-derived neurotrophic factor on synaptic transmission

In addition to its effects on neuronal survival and differentiation, brain-derived neurotrophic factor (BDNF) plays an important role in modulating synaptic transmission and plasticity in many brain areas, most notably the neocortex and hippocampus. These effects may underlie a role for BDNF in learning and memory as well as developmental plasticity. Consistent with localization of the tropomyosin-related kinase B receptor to both sides of the synapse, BDNF appears to have pre- and postsynaptic effects, but the underlying cellular mechanisms are unclear and it is not known whether pre- and postsynaptic modulations by BDNF occur simultaneously. To address these issues, we recorded dual-component (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [AMPA] and N-methyl-D-aspartate [NMDA]) miniature excitatory postsynaptic currents (mEPSCs) from cortical and hippocampal pyramidal neurons and dentate gyrus granule cells from acute brain slices. BDNF had no effect on the fast component of mEPSC decay or on the peak amplitude, suggesting that BDNF did not modulate postsynaptic AMPA receptors, although BDNF rapidly modulated NMDA receptors, as seen by an enhancement of the slow component of mEPSC decay that was prevented by blocking postsynaptic NMDA receptors. At the same time, BDNF acted presynaptically to enhance mEPSC frequency. Surprisingly, the effect on frequency was also NMDA receptor dependent, but required activation of presynaptic, not postsynaptic, NMDA receptors. BDNF also enhanced action potential-dependent glutamate release via presynaptic NMDA receptors, an effect that was unmasked when voltage-gated calcium channels were partially inhibited. Our results indicate that BDNF acutely modulates presynaptic release and postsynaptic responsiveness through simultaneous effects on pre- and postsynaptic NMDA receptors.     

Excitation of cerebellar interneurons by group I metabotropic glutamate receptors

Cerebellar basket and stellate neurons (BSNs) provide feed-forward inhibition to Purkinje neurons (PNs) and thereby play a principal role in determining the output of the cerebellar cortex. During low-frequency transmission, glutamate released at parallel fiber synapses excites BSNs by binding to AMPA receptors; high-frequency transmission also recruits N-methyl-d-aspartate (NMDA) receptors. We find that, in addition to these ligand-gated receptors, a G-protein-coupled glutamate receptor subtype participates in exciting BSNs. Stimulation of metabotropic glutamate receptor 1alpha (mGluR1alpha) with the mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) leads to an increase in spontaneous firing of BSNs and indirectly to an increase in the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded in PNs. Under conditions in which ligand-gated glutamate receptors are blocked, parallel fiber stimulation generates a slow excitatory postsynaptic current (EPSC) in BSNs that is inhibited by mGluR1alpha-selective antagonists. This slow EPSC is capable of increasing BSN spiking and indirectly increasing sIPSCs frequency in PNs. Our findings reinforce the idea that distinct subtypes of glutamate receptors are activated in response to different patterns of activity at excitatory synapses. The results also raise the possibility that mGluR1alpha-dependent forms of synaptic plasticity may occur at excitatory inputs to BSNs.     

Presynaptic NMDA receptor-mediated modulation of excitatory neurotransmission in the mouse dorsal motor nucleus of the vagus

Activity of neurons in the dorsal motor nucleus of the vagus nerve (DMV) is closely regulated by synaptic input, and regulation of that input by glutamate receptors on presynaptic terminals has been proposed. Presynaptic N-methyl-d-aspartic acid (NMDA) receptors have been identified in a number of brain regions and act to modulate neurotransmitter release, but functional presynaptic NMDA receptors have not been adequately studied in the DMV. This study identified the presence and physiological function of presynaptic NMDA receptors on synaptic input to DMV neurons. Whole-cell patch-clamp recordings from DMV neurons in acute slices from mice revealed prevalent miniature excitatory postsynaptic currents, which were significantly increased in frequency, but not amplitude, by application of NMDA. Antagonism of NMDA receptors with dl-2-amino-5-phosphonopentanoic acid (100 μM) resulted in a decrease in miniature excitatory postsynaptic current frequency and an increase in the paired pulse ratio of responses following afferent stimulation. No consistent effects of presynaptic NMDA receptor modulation were observed on GABAergic inputs. These results suggest that presynaptic NMDA receptors are present in the dorsal vagal complex and function to facilitate the release of glutamate, preferentially onto DMV neurons tonically, with little effect on GABA release. This type of presynaptic modulation represents a potentially novel form of glutamate regulation in the DMV, which may function to regulate glutamate-induced activity of central parasympathetic circuits.     

Glutamate exocytosis from astrocytes controls synaptic strength

The release of transmitters from glia influences synaptic functions. The modalities and physiological functions of glial release are poorly understood. Here we show that glutamate exocytosis from astrocytes of the rat hippocampal dentate molecular layer enhances synaptic strength at excitatory synapses between perforant path afferents and granule cells. The effect is mediated by ifenprodil-sensitive NMDA ionotropic glutamate receptors and involves an increase of transmitter release at the synapse. Correspondingly, we identify NMDA receptor 2B subunits on the extrasynaptic portion of excitatory nerve terminals. The receptor distribution is spatially related to glutamate-containing synaptic-like microvesicles in the apposed astrocytic processes. This glial regulatory pathway is endogenously activated by neuronal activity-dependent stimulation of purinergic P2Y1 receptors on the astrocytes. Thus, we provide the first combined functional and ultrastructural evidence for a physiological control of synaptic activity via exocytosis of glutamate from astrocytes.     

Slow synaptic inhibition mediated by metabotropic glutamate receptor activation of GIRK channels

Glutamate is the predominant excitatory neurotransmitter in the vertebrate CNS. Ionotropic glutamate receptors mediate fast excitatory actions whereas metabotropic glutamate receptors (mGluRs) mediate a variety of slower effects. For example, mGluRs can mediate presynaptic inhibition, postsynaptic excitation, or, more rarely, postsynaptic inhibition. We previously described an unusually slow form of postsynaptic inhibition in one class of projection neuron in the song-control nucleus HVc of the songbird forebrain. These neurons, which participate in a circuit that is essential for vocal learning, exhibit an inhibitory postsynaptic potential (IPSP) that lasts several seconds. Only a portion of this slow IPSP is mediated by GABA(B) receptors. Since these cells are strongly hyperpolarized by agonists of mGluRs, we used intracellular recording from brain slices to investigate the mechanism of this hyperpolarization and to determine whether mGluRs contribute to the slow synaptic inhibition. We report that mGluRs hyperpolarize these HVc neurons by activating G protein-coupled, inwardly-rectifying potassium (GIRK) channels. MGluR antagonists blocked this response and the slow synaptic inhibition. Thus, glutamate can combine with GABA to mediate slow synaptic inhibition by activating GIRK channels in the CNS.     

Creation of AMPA-silent synapses in the neonatal hippocampus

In the developing brain, many glutamate synapses have been found to transmit only NMDA receptor-mediated signaling, that is, they are AMPA-silent. This result has been taken to suggest that glutamate synapses are initially AMPA-silent when they are formed, and that AMPA signaling is acquired through activity-dependent synaptic plasticity. The present study on CA3-CA1 synapses in the hippocampus of the neonatal rat suggests that AMPA-silent synapses are created through a form of activity-dependent silencing of AMPA signaling. We found that AMPA signaling, but not NMDA signaling, could be very rapidly silenced by presynaptic electrical stimulation at frequencies commonly used to probe synaptic function (0.05-1 Hz). Although this AMPA silencing required a rise in postsynaptic Ca(2+), it did not require activation of NMDA receptors, metabotropic glutamate receptors or voltage-gated calcium channels. The AMPA silencing, possibly explained by a removal of postsynaptic AMPA receptors, could subsequently be reversed by paired presynaptic and postsynaptic activity.     

Realistic modelling of receptor activation in hippocampal excitatory synapses: analysis of multivesicular release, release location, temperature and synaptic cross-talk

Chemically mediated synaptic transmission results from fusion of synaptic vesicles with the presynaptic plasma membrane, subsequent release of the vesicular content into the cleft and binding to postsynaptic receptors. Previous modelling studies of excitatory neurotransmitter glutamate were based on simplified geometries failing to account for the biologically realistic synaptic environment, in particular, the presence of astrocytes, the geometry of extracellular space, and the neurotransmitter uptake mechanism. Using 3-dimensional reconstructions of hippocampal glutamatergic synapses including the surrounding astrocytic processes we have developed a biologically realistic model to analyse receptor activation in different conditions. We used the finite element method to simulate glutamate release, analyse glutamate diffusion following single and multiple vesicle release and binding at the postsynaptic site to AMPA and NMDA receptors. We demonstrate that: (1) the transmitter diffusion is highly temperature-sensitive; (2) release conditions and geometry more specifically affect AMPARs than NMDARs; (3) the sensitivities of AMPARs and NMDARs to simultaneous vesicular release are different; (4) in the case of multivesicle neurotransmitter release with variable delays, the binding of glutamate to AMPARs is additive up to 1 ms after the release, then becomes independent, but to NMDARs the binding is additive up to 33 ms; (5) the number of AMPARs varies more than the number of NMDRs in response to the input firing patterns; (6) the presence of astrocytes effectively blocks synaptic cross-talk; and (7) synaptic cross-talk, mediated by NMDARs but not AMPARs, is only possible after quasi-simultaneous multivesicular release at physiological temperature (35°C) without intervening astrocytes, but not at 25°C. Our simulations demonstrate the importance of temperature and ultrastructural synaptic environment in synaptic transmission and synaptic cross-talk.     

Properties of vertebrate glutamate receptors: calcium mobilization and desensitization.

Glutamate is now recognized as a major excitatory neurotransmitter in the vertebrate CNS, participating in a number of physiological and pathological processes. The importance of glutamate in the mobilization of intracellular Ca2+ as well as the relationship between excitatory and toxic properties has made it important to understand factors that regulate the responsivity of glutamate receptors. In recent years considerable insight has been gained about regulatory sites on NMDA receptors, with the recognition that these receptors are modulated by multiple endogenous and exogenous agents. Less is known about the regulation of responses mediated by AMPA, kainate, ACPD or APB receptors. Desensitization represents a potentially powerful means by which glutamate responses may be regulated. Indeed, two agents closely linked to the physiology of NMDA receptors, glycine and Ca2+, appear to modulate different types of desensitization. In the case of glycine, alteration of a rapid form of desensitization may be important in the role of this amino acid as a necessary cofactor for NMDA receptor activation. Additionally, changes in the affinity of the receptor complex for glycine may underlie the use-dependent decline in NMDA responses under certain conditions. Likewise, Ca2+ is a crucial player in the synaptic and toxic effects mediated by NMDA receptors, and is involved in a slower form of desensitization, in effect helping to regulate its own influx into neurons. The site and mechanism of the Ca2+ regulatory effects remain uncertain with evidence supporting both intracellular and ion channel sites of action. A clear role for Ca(2+)-dependent desensitization in the function of NMDA receptors under physiological conditions has not yet been demonstrated. AMPA receptor desensitization has been an area of intense investigation in recent years. The rapidity and degree of this process, coupled with its apparent rapid recovery, has suggested that desensitization is a key mechanism for the short-term regulation of responses mediated by these receptors. Furthermore, rapid desensitization appears to be one factor determining the time course and efficacy of fast excitatory synaptic transmission mediated by AMPA receptors, highlighting the physiological relevance of the process. The molecular mechanisms underlying desensitization remain uncertain. Traditionally, desensitization, like inactivation of voltage-gated channels, has been thought to represent a conformational change in the ion channel complex (Ochoa et al., 1989). However, it is unknown to what extent desensitization, in particular rapid AMPA receptor desensitization, has mechanistic features in common with inactivation. In voltage-gated channels, conformational changes in the channel protein restrict ion flow through the channel (Stuhmer, 1991).(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of morphological postsynaptic silent synapses in developing hippocampal neurons

Many excitatory synapses are thought to be postsynaptically 'silent', possessing functional NMDA but lacking functional AMPA glutamate receptors. The acquisition of AMPA receptors at silent synapses may be important in synaptic plasticity and neuronal development. Here we characterize a possible morphological correlate of silent synapses in cultured hippocampal neurons. Initially, most excitatory synapses contained NMDA receptors, but only a few contained detectable AMPA receptors. Synapses progressively acquired AMPA receptors as the cultures matured. AMPA receptor blockade increased the number, size and fluorescent intensity of AMPA receptor clusters and rapidly induced the appearance of AMPA receptors at 'silent' synapses. In contrast, NMDA receptor blockade increased the size, intensity and number of NMDA receptor clusters and decreased the number of AMPA receptor clusters, resulting in an increase in the proportion of 'silent' synapses. These results suggest that the number of silent synapses is regulated during development and by changes in synaptic activity.     

Enhanced NR2A subunit expression and decreased NMDA receptor decay time at the onset of ocular dominance plasticity in the ferret.

Enhanced NR2A subunit expression and decreased NMDA receptor decay time at the onset of ocular dominance plasticity in the ferret. The NMDA subtype of glutamate receptor is known to exhibit marked changes in subunit composition and functional properties during neural development. The prevailing idea is that NMDA receptor-mediated synaptic responses decrease in duration after the peak of cortical plasticity in rodents. Accordingly, it is believed that shortening of the NMDA receptor-mediated current underlies the developmental reduction of ocular dominance plasticity. However, some previous evidence actually suggests that the duration of NMDA receptor currents decreases before the peak of plasticity. In the present study, we have examined the time course of NMDA receptor changes and how they correlate with the critical period of ocular dominance plasticity in the visual cortex of a highly binocular animal, the ferret. The expression of NMDA receptor subunits NR1, NR2A, and NR2B was examined in animals ranging in age from postnatal day 16 to adult using Western blotting. Functional properties of NMDA receptors in layer IV cortical neurons were studied using whole cell patch-clamp techniques in an in vitro slice preparation of ferret primary visual cortex. We observed a remarkable increase in NR1 and NR2A, but not NR2B, expression after eye opening. The NMDA receptor-mediated synaptic currents showed an abrupt decrease in decay time concurrent with the increase in NR2A subunit expression. Importantly, these changes occurred in parallel with increased ocular dominance plasticity reported in the ferret. In conclusion, molecular changes leading to decreased duration of the NMDA receptor excitatory postsynaptic current may be a requirement for the onset, rather than the end, of the critical period of ocular dominance plasticity.     

Astrocytic Ca2+ signaling evoked by sensory stimulation in vivo

Although astrocytes are the most abundant cell type in the brain, evidence for their activation during physiological sensory activity is lacking. Here we show that whisker stimulation evokes increases in astrocytic cytosolic calcium (Ca(2+)) within the barrel cortex of adult mice. Increases in astrocytic Ca(2+) were a function of the frequency of stimulation, occurred within several seconds and were inhibited by metabotropic glutamate receptor antagonists. To distinguish between synaptic input and output, local synaptic activity in cortical layer 2 was silenced by iontophoresis of AMPA and NMDA receptor antagonists. The antagonists did not reduce astrocytic Ca(2+) responses despite a marked reduction in excitatory postsynaptic currents in response to whisker stimulation. These findings indicate that astrocytes respond to synaptic input, by means of spillover or ectopic release of glutamate, and that increases in astrocytic Ca(2+) occur independently of postsynaptic excitatory activity.     

Structural Characteristics of Ionotropic Glutamate Receptors as Identified by Channel Blockade

The channels of four types of ionotropic glutamate receptor (NMDA receptors and Ca-permeable AMPA receptors of rat brain neurons, and cation-selective receptors from mollusk neurons and insect postsynaptic muscle membranes) and two subtypes of nicotinic cholinoreceptor (from frog neuromuscular junctions and cat sympathetic ganglia) were studied. The structural characteristics of channels determining their susceptibility to blockade by organic mono- and dications were identified. These studies used homologous series of adamantane and phenylcyclohexyl derivatives. These experiments showed that the receptors studied here could be divided into two groups. The first group included the AMPA receptor and the mollusk and insect receptors. These were characterized by the lack of effect on the part of monocations and a strong relationship between the activity of dications and the distance between nitrogen atoms. The second group included the NMDA receptor and both subtypes of the nicotinic cholinoreceptor (muscular and neuronal). Here, conversely, the activity of monocations and dications, regardless of their lengths, were essentially identical. A model for the binding sites of blockers in channels is proposed, which takes these observations into account.