Out of the cleft: the source and target of extra-synaptic glutamate in the CA1 region of the hippocampus.

Glutamate is the central nervous system's principal excitatory transmitter, and its levels in the brain's extracellular space are under tight spatial and temporal regulation. During evoked vesicular release, glutamate levels can peak in the millimolar range within the synaptic cleft. Active transport, enzymatic degradation and diffusion all work to reduce this level so as to avert the continual activation or desensitization of receptors, and give postsynaptic neurons a high signal-to-noise input. However, it has been shown that a much lower, nanomolar to micromolar ambient concentration of glutamate remains within the interstitium. It is therefore possible that such a tonic level may be affecting cellular excitation. Indeed, even such low quantities of the amino acid are sufficient to activate NMDA receptors (NMDARs). The potential tonic activation of NMDARs is of particular interest as these channels have unique electrical properties and are involved in intracellular cascades implicated in a wide range of cellular processes, from learning and memory to excitotoxicity. Sah et al. (1989) were the first to show that NMDARs could be active without synaptic stimulation. Studying rat hippocampal slices, they recorded from CA1 pyramidal neurons in whole-cell configuration under voltage clamp at −35 mV. After the addition of APV (dAP5), a competitive NMDAR antagonist, the group noted a substantial (hundreds of picoamps) net outward shift in the holding current. This effect was magnesium and voltage dependent, hallmarks of an NMDAR-mediated conductance. Notably, they also observed that APV reversibly reduced action potential output in response to current injection, suggesting a role for the tonic conductance in enhancing neuronal excitability. This Journal Club article focuses on The Journal of Physiology publication by Le Meur et al. (2007), who recently added to this area of interest. Voltage clamping CA1 pyramidal neurons at +40 mV, they observed a net inward shift in holding current after the addition of APV, and an I–V curve of the APV-sensitive component typical of NMDARs. Importantly, the change in current after APV was only tens of picoamps, an order of magnitude less than that seen by Sah et al. (1989). In different cells, they superfused NBQX and LY341495 and saw no change in the tonic current, suggesting that the observed change is dependent on NMDARs and not other ionotropic or metabotropic glutamate receptors. If ambient glutamate was indeed activating this NMDAR-dependent current, then increasing its concentration should have the opposite effect of adding APV. Indeed, after blocking glutamate uptake with TBOA, there was an immediate net outward shift in the holding current. Interestingly, in experiments performed in the presence of TBOA, blockers of AMPARs did cause a small effect, and this was amplified by cyclothiazide, a drug that inhibits AMPAR desensitization. This suggested that in their slice recording conditions, the regulation of ambient glutamate levels and receptor desensitization both act to prevent the tonic activation of non-NMDA glutamate receptors. A tonic current mediated by glutamate receptors contrasts sharply with the standard dogma of glutamate signalling, which involves evoked, vesicular release from one neuron to another within a highly structured synapse. While this ambient glutamate could theoretically originate via spillover from high-frequency synaptic activity, work subsequent to Sah et al. (1989) has shown that it is instead mediated by neighbouring glia. Le Meur et al. (2007) therefore focused on confirming this glial origin in their experimental preparation. They first treated slices with bafilomycin A1 to prevent vesicular release. In these slices, there was no difference from control slices in the change in holding current induced by TBOA or MK801, a non-competitive NMDAR antagonist. This confirmed that the glutamate source was indeed not synaptic. In the next experiment, they used L-methionine sulfoximine (MSO) to inhibit glutamine synthetase, a glial enzyme responsible for converting glutamate to glutamine. This treatment has been shown to drastically increase glutamate concentrations in glial cells. While the amplitude of the basal tonic current was unchanged, currents induced by TBOA were doubled in slices first treated with MSO. These data support the notion that the ambient glutamate is of glial origin, and underscore the idea that glia play a far greater role than a simple ‘support network’ for neurons. A number of the above experiments have been done previously and similar results were obtained (Angulo et al. 2004). If the tonic conductance is not activated by vesicular release, one could hypothesize that the ambient glutamate was activating extrasynaptic NMDARs, specifically. Le Meur et al. (2007) first tested this idea with pharmacological tools to block particular NMDAR subunits, as such an approach has been used previously to discriminate synaptic from extrasynaptic NMDA (Fellin et al. 2004) as well as GABAA receptors. EPSC amplitude and holding current were compared in the presence of these blockers. However, these studies did not clearly differentiate between the NMDAR subunits mediating synaptic versus tonic currents. PPDA, an NMDAR2C- and D-preferring blocker, inhibited the tonic slightly more than the synaptic current, but infenprodil (an NMDAR2B antagonist) inhibited neither, and NVP-AAM077 (an NMDAR2A-preferring antagonist) inhibited them equally. In a key experiment, they then turned to the use-dependent general NMDAR antagonist, MK801. After recording control EPSCs at +40 mV in the presence of NBQX, they stopped synaptic stimulation and washed in MK801. Stimulation was resumed, and the first EPSC after MK801 superfusion was compared with the last one before it. The holding current was monitored during the same time frame. If the NMDARs mediating the tonic current were extrasynaptic, one would expect to see a change in the holding current during MK801 wash-in (as these receptors would be open), and less of a change in the synaptic current (these receptors would have been closed, owing to a lack of glutamate in the cleft). This is indeed what Le Meur et al. (2007) observed. While the tonic current was approximately halved, the peak amplitudes of the EPSCs before and after wash-in did not differ. There was, however, a decrease in the decay time constant of the EPSC after MK801 addition, which is consistent with the use-dependent blocker shortening the mean channel open-time, demonstrating the efficacy of MK801 at these synapses. While the idea that extrasynaptic NMDARs are mediating the tonic current has been suggested previously (Fellin et al. 2004), this open channel block experiment elegantly revealed the existence of separate tonic and phasic populations of glutamate receptors. With mounting evidence for ambient glutamate present both in vitro and in vivo, many questions remain regarding the signalling roles of extrasynaptic receptor activation. This publication focused on a tonic glutamate-mediated current. However, Angulo et al. (2004) and others have described transient currents of the order of hundreds of milliseconds which were also suggested to be due to glial glutamate release. This issue of the dynamics of extra-synaptic glutamate is critical in determining its potential roles in neuronal signalling. Such observations argue for the importance of understanding glial physiology with respect to the spatial and temporal variations in extracellular glutamate concentration, including mechanisms and modulation of release and reuptake. Equally intriguing questions remain regarding the postsynaptic effects of ambient glutamate. NMDAR activation may affect neuronal activity on multiple time scales. The small tonic NMDAR component did not affect the overall input–output relationship of CA1 neurons in this study, which contrasts with measurements of Sah et al. (1989). However, tonic NMDAR activation may be particularly important in shaping synaptic inputs. As NMDARs are expressed in dendritic membranes, they are poised to interact with local synaptic inputs prior to somatic integration. Moreover, the magnesium block of NMDARs results in negative slope conductance profiles at voltages near and below action potential generation, which means that depolarization from a nearby EPSP will boost the amount of inward currents flowing through open NMDARs. The effects of such boosting may be EPSP amplification or possibly regenerative dendritic spiking. Furthermore, there have been reports that glial-dependent NMDAR activation may be highly temporally correlated in nearby neurons, suggesting a possible role of synchronous modulation of excitability across multiple neuronal elements (Fellin et al. 2004; Angulo et al. 2004). At longer time scales, NMDAR activation by ambient glutamate may result in important intracellular signalling due to the influx of Ca2+. NMDAR-mediated Ca2+ influx may act locally to modulate ion channels or biochemical processes, as well as distally by affecting signalling cascades and gene transcription. Hardingham et al. (2002) further suggest that activation of extrasynaptic NMDARs, unlike synaptic receptors, cause cAMP-response element binding protein (CREB) dephosphorylation and mitochondrial dysfunction and cell death. These dramatic findings highlight the importance of current studies like the one discussed in this Journal Club article, as glutamate activity outside the synapse may be a critical parameter in the function of neuronal circuits.