Allosteric Interactions between NMDA Receptor Subunits Shape the Developmental Shift in Channel Properties.

During development of the central nervous system, there is a shift in the subunit composition of NMDA receptors (NMDARs) resulting in a dramatic acceleration of NMDAR-mediated synaptic currents. This shift coincides with upregulation of the GluN2A subunit and triheteromeric GluN1/2A/2B receptors with fast deactivation kinetics, whereas expression of diheteromeric GluN1/2B receptors with slower deactivation kinetics is decreased. Here, we show that allosteric interactions occur between the glutamate-binding GluN2 subunits in triheteromeric GluN1/2A/2B NMDARs. This allosterism is dominated by the GluN2A subunit and results in functional properties not predicted by those of diheteromeric GluN1/2A and GluN1/2B NMDARs. These findings suggest that GluN1/2A/2B NMDARs may maintain some signaling properties of the GluN2B subunit while having the kinetic properties of GluN1/2A NMDARs and highlight the complexity in NMDAR signaling created by diversity in subunit composition.

Extracellular Glutamate in the Nucleus Accumbens Is Nanomolar in Both Synaptic and Non-synaptic Compartments.

In the CNS, glutamate is both phasically and tonically released into the extracellular space and must be removed by excitatory amino acid transporters (EAATs) to prevent excitotoxic accumulation. There remains uncertainty, however, regarding the functional steady-state concentration, with estimates ranging from tens of nanomolar to tens of micromolar. Efforts to reconcile these disparate values have led to a hypothesis that the extracellular space comprises distinct compartments in which basal glutamate concentrations are maintained independently. We used electrophysiology and two-photon Ca2+ imaging to test this hypothesis in the nucleus accumbens (NAc), where it has been proposed that micromolar extracellular glutamate is necessary for normal function. We found that the average concentration of synaptic glutamate is nanomolar, in agreement with previous electrophysiological estimates. Furthermore, this held true when glutamate uptake was inhibited, indicating that extracellular glutamate is not compartmentalized by EAATs.

Postsynaptic, not presynaptic NMDA receptors are required for spike-timing-dependent LTD induction.

Long-term depression (LTD) between cortical layer 4 spiny stellate cells and layer 2/3 pyramidal cells requires the activation of NMDA receptors (NMDARs). In young rodents, this form of LTD has been repeatedly reported to require presynaptic NMDARs for its induction. Here we show that at this synapse in the somatosensory cortex of 2- to 3-week-old rats and mice, postsynaptic, not presynaptic NMDARs are required for LTD induction. First, we find no evidence for functional NMDARs in L4 neuron axons using two-photon laser scanning microscopy and two-photon glutamate uncaging. Second, we find that genetic deletion of postsynaptic, but not presynaptic NMDARs prevents LTD induction. Finally, the pharmacology of the NMDAR requirement is consistent with a nonionic signaling mechanism.

Activation of axonal receptors by GABA spillover increases somatic firing.

Axons can be depolarized by ionotropic receptors and transmit subthreshold depolarizations to the soma by passive electrical spread. This raises the possibility that axons and axonal receptors can participate in integration and firing in neurons. Previously, we have shown that exogenous GABA depolarizes cerebellar granule cell axons through local activation of GABA(A) receptors (GABA(A)Rs) and the soma through electrotonic spread of the axonal potential resulting in increased firing. We show here that excitability of granule cells is also increased by release of endogenous GABA from molecular layer interneurons (MLIs) and spillover activation of parallel fiber GABA(A)Rs in mice and rats. Changes in granule cell excitability were assessed by excitability testing after activation of MLIs with channelrhodopsin or electrical stimulation in the molecular layer. In granule cells lacking an axon, excitability was not changed, suggesting that axonal receptors are required. To determine the distance over which subthreshold potentials may spread, we estimated the effective axonal electrical length constant (520 µm) by excitability testing and focal uncaging of RuBi-GABA on the axon at varying distances from the soma. These data suggest that GABA(A)R-mediated axonal potentials can participate in integration and firing of cerebellar granule cells.

Activation of extrasynaptic NMDARs at individual parallel fiber-molecular layer interneuron synapses in cerebellum.

NMDA receptors (NMDARs) expressed by cerebellar molecular layer interneurons (MLIs) are not activated by single exocytotic events but can respond to glutamate spillover following coactivation of adjacent parallel fibers (PFs), indicating that NMDARs are perisynaptic. Several types of synaptic plasticity rely on these receptors but whether they are activated at isolated synapses is not known. Using a combination of electrophysiological and optical recording techniques in acute slices of rat cerebellum, along with modeling, we find that repetitive activation of single PF-MLI synapses can activate NMDARs in MLIs. High-frequency stimulation, multivesicular release (MVR), or asynchronous release can each activate NMDARs. Frequency facilitation was found at all PF-MLI synapses but, while some showed robust MVR with increased release probability, most were limited to univesicular release. Together, these results reveal a functional diversity of PF synapses, which use different mechanisms to activate NMDARs.

Distribution of extracellular glutamate in the neuropil of hippocampus.

Reported values of extracellular glutamate concentrations in the resting state depend on the method of measurement and vary ∼1000-fold. As glutamate levels in the micromolar range can cause receptor desensitization and excitotoxicity, and thus affect neuronal excitability, an accurate determination of ambient glutamate is important. Part of the variability of previous measurements may have resulted from the sampling of glutamate in different extracellular compartments, e.g., synaptic versus extrasynaptic volumes. A steep concentration gradient of glutamate between these two compartments could be maintained, for example, by high densities of glutamate transporters arrayed at the edges of synapses. We have used two photon laser scanning microscopy and electrophysiology to investigate whether extracellular glutamate is compartmentalized in acute hippocampal slices. Pharmacological blockade of NMDARs had no effect on Ca(2+) transients generated in dendritic shafts or spines of CA1 pyramidal neurons by depolarization, suggesting that ambient glutamate is too low to activate a significant number of NMDARs. Furthermore, blockade of transporters did not flood the synapse with glutamate, indicating that synaptic NMDARs are not protected from high concentrations of extrasynaptic glutamate. We suggest that, in the CA1 region of hippocampus, glutamate transporters do not create a privileged space within the synapse but rather keep ambient glutamate at very low levels throughout the neuropil.

NMDA receptor agonists fail to alter release from cerebellar basket cells.

Previous studies of NMDA receptor (NMDAR) expression on axons of cerebellar molecular layer interneurons have produced conflicting results. We made use of the calcium sensitivity of vesicular release machinery to test for NMDAR activity in basket cell axons. Iontophoresis of l-aspartate, an NMDAR agonist, onto basket cell axon collaterals had no effect on evoked IPSCs measured in synaptically coupled Purkinje cells. Furthermore, calcium indicators in basket cell varicosities did not report any change in intracellular calcium following iontophoresis of l-aspartate or two-photon uncaging of glutamate. In contrast, activation of presynaptic purinergic receptors by iontophoresis of ATP decreased evoked IPSC amplitudes and action potential-evoked calcium transients in axonal varicosities, demonstrating the effectiveness of activating presynaptic receptors by iontophoresis. We find no evidence for functional NMDARs in basket cell varicosities.

Axonal GABAA receptors increase cerebellar granule cell excitability and synaptic activity.

We report that activation of GABA(A) receptors on cerebellar granule cell axons modulates both transmitter release and the excitability of the axon and soma. Axonal GABA(A) receptors depolarize the axon, increasing its excitability and causing calcium influx at axonal varicosities. GABA-mediated subthreshold depolarizations in the axon spread electrotonically to the soma, promoting orthodromic action potential initiation. When chloride concentrations are unperturbed, GABA iontophoresis elicits spikes and increases excitability of parallel fibers, indicating that GABA(A) receptor-mediated responses are normally depolarizing. GABA release from molecular layer interneurons activates parallel fiber GABA(A) receptors, and this, in turn, increases release probability at synapses between parallel fibers and molecular layer interneurons. These results describe a positive feedback mechanism whereby transmission from granule cells to Purkinje cells and molecular layer interneurons will be strengthened during granule cell spike bursts evoked by sensory stimulation.

Ca(2+)-dependent enhancement of release by subthreshold somatic depolarization.

In many neurons, subthreshold somatic depolarization can spread electrotonically into the axon and modulate subsequent spike-evoked transmission. Although release probability is regulated by intracellular Ca(2+), the Ca(2+) dependence of this modulatory mechanism has been debated. Using paired recordings from synaptically connected molecular layer interneurons (MLIs) of the rat cerebellum, we observed Ca(2+)-mediated strengthening of release following brief subthreshold depolarization of the soma. Two-photon microscopy revealed that, at the axon, somatic depolarization evoked Ca(2+) influx through voltage-sensitive Ca(2+) channels and facilitated spike-evoked Ca(2+) entry. Exogenous Ca(2+) buffering diminished these Ca(2+) transients and eliminated the strengthening of release. Axonal Ca(2+) entry elicited by subthreshold somatic depolarization also triggered asynchronous transmission that may deplete vesicle availability and thereby temper release strengthening. In this cerebellar circuit, activity-dependent presynaptic plasticity depends on Ca(2+) elevations resulting from both sub- and suprathreshold electrical activity initiated at the soma.

Presynaptically expressed long-term potentiation increases multivesicular release at parallel fiber synapses.

At a number of synapses, long-term potentiation (LTP) can be expressed by an increase in presynaptic strength, but it is unknown whether presynaptic LTP is expressed solely through an increase in the probability that a single vesicle is released or whether it can increase multivesicular release (MVR). Here, we show that presynaptic LTP decreases inhibition of AMPA receptor EPSCs by a low-affinity antagonist at parallel fiber-molecular layer interneuron (PF-MLI) synapses. This indicates that LTP induction results in larger glutamate concentration transients in the synaptic cleft, a result indicative of MVR, and suggests that MVR can be modified by long-term plasticity. A similar decrease in inhibition was observed when release probability (PR) was increased by forskolin, elevated extracellular Ca2+, and paired-pulse facilitation. Furthermore, we show that MVR may occur under baseline physiological conditions, as inhibition increased when P(R) was lowered by reducing extracellular Ca2+ or by activating presynaptic adenosine receptors. These results suggest that at PF-MLI synapses, MVR occurs under control conditions and is increased when PR is elevated by both short- and long-term plasticity mechanisms.

Selective expression of ligand-gated ion channels in L5 pyramidal cell axons.

NMDA receptor (NMDAR)-dependent strengthening of neurotransmitter release has been widely observed, including in layer 5 (L5) pyramidal cells of the visual cortex, and is attributed to the axonal expression of NMDARs. However, we failed to detect NMDAR-mediated depolarizations or Ca(2+) entry in L5 pyramidal cell axons when focally stimulated with NMDAR agonists. This suggests that NMDARs are excluded from the axon. In contrast, local GABA(A) receptor activation alters axonal excitability, indicating that exclusion of ligand-gated ion channels from the axon is not absolute. Because NMDARs are restricted to the dendrite, NMDARs must signal to the axon by an indirect mechanism to alter release. Although subthreshold somatic depolarizations were found to spread electrotonically hundreds of micrometers through the axon, the resulting axonal potential was insufficient to open voltage-sensitive Ca(2+) channels. Therefore, if NMDAR-mediated facilitation of release is cell autonomous, it may depend on voltage signaling but apparently is independent of changes in basal Ca(2+). Alternatively, this facilitation may be even less direct, requiring a cascade of events that are merely triggered by NMDAR activation.

Dendritic NMDA receptors activate axonal calcium channels.

NMDA receptor (NMDAR) activation can alter synaptic strength by regulating transmitter release from a variety of neurons in the CNS. As NMDARs are permeable to Ca(2+) and monovalent cations, they could alter release directly by increasing presynaptic Ca(2+) or indirectly by axonal depolarization sufficient to activate voltage-sensitive Ca(2+) channels (VSCCs). Using two-photon microscopy to measure Ca(2+) excursions, we found that somatic depolarization or focal activation of dendritic NMDARs elicited small Ca(2+) transients in axon varicosities of cerebellar stellate cell interneurons. These axonal transients resulted from Ca(2+) entry through VSCCs that were opened by the electrotonic spread of the NMDAR-mediated depolarization elicited in the dendrites. In contrast, we were unable to detect direct activation of NMDARs on axons, indicating an exclusive somatodendritic expression of functional NMDARs. In cerebellar stellate cells, dendritic NMDAR activation masquerades as a presynaptic phenomenon and may influence Ca(2+) -dependent forms of presynaptic plasticity and release.

Extracellular glutamate concentration in hippocampal slice.

Synaptic glutamate transients resulting from vesicular exocytosis are superimposed on a low baseline concentration of glutamate in the extracellular space. Reported values of baseline glutamate concentrations range up to 4 microM. If glutamate were present tonically at low micromolar concentrations, many receptors, especially the high-affinity NMDA receptors (NMDARs), would be activated or desensitized, altering neuronal excitability. Using NMDARs expressed by CA1 pyramidal cells in acute hippocampal slices to monitor extracellular glutamate, we find that its baseline concentration is much lower, near 25 nM. In addition, superfusion of low micromolar concentrations of glutamate had no effect on neurons, indicating that glutamate transport prevents access to receptors within the slice. However, equipotent concentrations of NMDA, a nontransported agonist, depolarized neurons dramatically. We suggest that ambient concentrations of glutamate in vivo are also in the nanomolar range and are too low to cause significant receptor activation.

Glutamatergic and purinergic receptor-mediated calcium transients in Bergmann glial cells.

Astrocytes respond to neuronal activity with [Ca2+]i increases after activation of specific receptors. Bergmann glial cells (BGs), astrocytes of the cerebellar molecular layer (ML), express various receptors that can mobilize internal Ca2+. BGs also express Ca2+ permeable AMPA receptors that may be important for maintaining the extensive coverage of Purkinje cell (PC) excitatory synapses by BG processes. Here, we examined Ca2+ signals in single BGs evoked by synaptic activity in cerebellar slices. Short bursts of high-frequency stimulation of the ML elicited Ca2+ transients composed of a small-amplitude fast rising phase, followed by a larger and slower rising phase. The first phase resulted from Ca2+ influx through AMPA receptors, whereas the second phase required release of Ca2+ from internal stores initiated by P2 purinergic receptor activation. We found that such Ca2+ responses could be evoked by direct activation of neurons releasing ATP onto BGs or after activation of metabotropic glutamate receptor 1 on these neurons. Moreover, examination of BG and PC responses to various synaptic stimulation protocols suggested that ML interneurons are likely the cellular source of ATP.

Exocytosis unbound.

The accepted theory of vesicular release of neurotransmitter posits that only a single vesicle per synapse can fuse with the membrane following action potential invasion, and this exocytotic event is limited to the ultrastructurally defined presynaptic active zone. Neither of these dictums is universally true. At certain synapses, more than a single vesicle can be released per action potential, and there is growing evidence that neuronal exocytosis can occur from sites that are unremarkable in electron micrographs. The first discrepancy extends the dynamic range of synapses, whereas the second enables faster and more robust chemical transmission at sites distant from morphologically defined synapses. Taken together, these attributes expand the capabilities of cellular communication in the nervous system.

Multivesicular release at Schaffer collateral-CA1 hippocampal synapses.

Whether an individual synapse releases single or multiple vesicles of transmitter per action potential is contentious and probably depends on the type of synapse. One possibility is that multivesicular release (MVR) is determined by the instantaneous release probability (Pr) and therefore can be controlled by activity-dependent changes in Pr. We investigated transmitter release across a range of Pr at synapses between Schaffer collaterals (SCs) and CA1 pyramidal cells in acute hippocampal slices using patch-clamp recordings. The size of the synaptic glutamate transient was estimated by the degree of inhibition of AMPA receptor EPSCs with the rapidly equilibrating antagonist gamma-D-glutamylglycine. The glutamate transient sensed by AMPA receptors depended on Pr but not spillover, indicating that multiple vesicles are essentially simultaneously released from the same presynaptic active zone. Consistent with an enhanced glutamate transient, increasing Pr prolonged NMDA receptor EPSCs when glutamate transporters were inhibited. We suggest that MVR occurs at SC-CA1 synapses when Pr is elevated by facilitation and that MVR may be a phenomenon common to many synapses throughout the CNS.

Intrinsic kinetics determine the time course of neuronal synaptic transporter currents.

Efficient clearance of synaptically released glutamate from the extracellular space is an absolute requirement for maintaining information processing in the central nervous system. In the cerebellum, clearance of glutamate relies on uptake by Bergmann glial cells and Purkinje cells (PCs). Uptake by PCs can be monitored by recording the synaptic transport current (STC) mediated by the PC-specific transporter excitatory amino acid transporter 4 (EAAT4). The slow time course of the PC STC has been used to argue that glutamate clearance is protracted. We find, however, that the time course of the STC is not affected by altering the amount of glutamate released at individual synapses or by partial transporter blockade, manipulations that would be expected to change the duration of the extracellular glutamate transient. Ion substitution experiments and kinetic modeling of the PC transporter current suggest that physiological levels of intracellular Na(+) and glutamate slow the cycling rate of transporters and thereby lengthen the time course of STCs. The model predicts that PC transporters bind glutamate quickly but that the actual cycling rate of EAAT4 in physiological conditions is slow; therefore, the STC reflects the intrinsic kinetics of the glutamate transporter, not the rate of glutamate clearance.

Patterned expression of Purkinje cell glutamate transporters controls synaptic plasticity.

Glutamate transporters are responsible for clearing synaptically released glutamate from the extracellular space. If expressed at high enough densities, transporters can prevent activation of extrasynaptic receptors by rapidly lowering glutamate concentrations to insignificant levels. We find that synaptic activation of metabotropic glutamate receptors expressed by Purkinje cells is prevented in regions of rat cerebellum where the density of the glutamate transporter EAAT4 is high. The consequences of metabotropic receptor stimulation, including activation of a depolarizing conductance, cannabinoid-mediated presynaptic inhibition and long-term depression, are also limited in Purkinje cells expressing high levels of EAAT4. We conclude that neuronal uptake sites must be overwhelmed by glutamate to activate perisynaptic metabotropic glutamate receptors. Regional differences in glutamate transporter expression affect the degree of metabotropic glutamate receptor activation and therefore regulate synaptic plasticity.

High-concentration rapid transients of glutamate mediate neural-glial communication via ectopic release.

Until recently, communication from neurons to astrocytes was thought to be mediated by low-concentration transients of glutamate caused by spillover from the synaptic cleft. However, quantal events recorded in rat cerebellar Bergmann glial cells (BGs) have fast kinetics, comparable with those recorded in neurons. By combining outside-out patch recordings of BG AMPA receptors and quantitative electron microscopic analysis of glutamate receptor subunit 1 (GluR1) and GluR4 immunogold labeling measurements, at both the soma and membranes surrounding synapses, we estimate the absolute density of functional AMPA receptors. Using a kinetic model of BG AMPA receptors, we find that quantal events recorded in BGs are produced by high-concentration (approximately 1-1.5 mM), fast transients (approximately 0.5 ms decay) of glutamate, similar to transients within the synaptic cleft. Our results indicate that neural signaling to BGs is mediated by ectopic release of transmitter from presynaptic elements directly facing the BG membrane.

Differential control of synaptic and ectopic vesicular release of glutamate.

Exocytosis of synaptic vesicles occurs not only at synaptic active zones but also at ectopic sites. Ectopic exocytosis provides a direct and rapid mechanism for neurons to communicate with glia that does not rely on transmitter spillover from the synaptic cleft. In the cerebellar cortex the processes of Bergmann glia cells encase synapses between presynaptic climbing fiber varicosities and postsynaptic Purkinje cell spines and express both AMPA receptors and electrogenic glutamate transporters. AMPA receptors expressed by Purkinje cells and Bergmann glia cells are activated predominantly by synaptic and ectopic release, respectively, and therefore can be used to compare the properties of the two release mechanisms. We report that vesicular release differs at synaptic and ectopic sites in the magnitude of short-term plasticity and the proportions of Ca2+ channel subtypes that trigger glutamate release. High-affinity glutamate transporter-mediated currents in Bergmann glia cells follow the rules of synaptic release more closely than the rules of ectopic release, indicating that the majority of glutamate is released from conventional synapses. On the other hand, ectopic release produces high-concentration glutamate transients at Bergmann glia cell membranes that are necessary to activate low-affinity AMPA receptors rapidly. Ectopic release may provide a geographical cue to guide Bergmann glia cell membranes to surround active synapses and ensure efficient uptake of glutamate that diffuses out of the synaptic cleft.