- Email: [email protected]
Clearance of glutamate inside the synapse and beyond
293
Clearance of glutamate inside the synapse and beyond Dwight E Bergles, Jeffrey S Diamond and Craig E Jahr* The
heated
debate
occupancy
over
the
by transmitter
level
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new evidence is fanning two-photon microscopy
of postsynaptic
not
been
in the synaptic
previously and studies
suggested. In contrast, recordings of extrasynaptic receptor activation
efflux
quantities exocytosis.
from
critical synaptic
the
cleft
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in Neurobiology
cells that
it diffuses
is
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L474, Oregon Health Sciences University, Sam Jackson Park Road, Portland, Oregon 97201, USA *e-mail: [email protected]
Opinion
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escape from the cleft the amount of glutamate
Addresses Vellum Institute,
Current
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the flames. Recent experiments suggest that the concentration
glutamate
significant following
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extinguished
1999,
3181
SW
9:293-298
http://biomednet.com/elecref/0959438800900293 0 Elsevier
Science
Ltd ISSN
0959-4388
Abbreviations AMPA a-amino-3-hydroxy-5-methyl-4-isoxazole-propionic EPSC
LTP NMDA
excitatory postsynaptic long-term potentiation N-methyl-o-aspartate
acid
current
Introduction Glutamate is released at excitatory synapses in the CNS when synaptic vesicles containing 4000-5000 molecules of glutamate fuse with the presynaptic membrane. In the very small volume of the synaptic cleft (- 2 attoliters), concentrations of glutamate as high as several millimolar are attained [ 1,2], assuring activation of postsynaptic glutamate receptors and therefore depolarization of the postsynaptic neuron. However, the synaptic cleft is in continuity with extrasynaptic space, and thus the bolus of glutamate rapidly dissipates both by diffusion and binding to transporters. The dynamics of glutamate release, diffusion, binding and uptake determines the spatiotemporal profile of the concentration of glutamate in the synaptic cleft and perisynaptic space. This review focuses on the clearance of glutamate inside the synaptic cleft and from the extracellular space outside the synapse (Figure 1). Several recent reviews cover many related topics not discussed here [ 1,3-61.
Clearance
within
the cleft
Why do we care about the concentration of glutamate attained in the synaptic cleft or how fast this initial spike of glutamate declines? The immediate reason is that the size and shape of this concentration transient determines how many postsynaptic receptors become bound by transmitter. Receptor occupancy, in turn, dictates the size of the
postsynaptic response as well as the number of unbound receptors available for activation by subsequent exocytosis. If rapid receptor activation is required for fast signaling, a brief pulse of ligand will suffice in synchronizing the activation of a sub-population of available receptors; the higher the peak concentration of such a pulse, the more receptors will be bound. A brief, high-concentration pulse of transmitter is thought to occur at glutamatergic synapses in the CNS [1,2]. The rapid rise in transmitter is ensured by exocytotic release: complete emptying of transmitter from a small synaptic vesicle is thought to require less than 100 ~1s [7,8]. The length of time that glutamate remains elevated in the cleft also affects how many receptors are bound; unless very high concentrations are reached, the longer glutamate is present, the more receptors will be bound, albeit with some asynchrony. The difference between concentration decay time constants of 0.5 ms and 1.5 ms could make the difference between low receptor occupancy and nearly saturated receptors. If the receptors at a synapse are normally near saturation, then the event-to-event variation of the response at that synapse will depend mainly on the stochastic nature of channel opening rather than on variations in receptor occupancy. Alternatively, if low occupancy is the norm, the variation may mainly reflect differences in receptor occupancy owing to vesicle-to-vesicle deviations in the amount of glutamate released. Understanding the origin of such variations is essential because they are an intrinsic feature of signaling at individual synapses as well as a means of identifying pre- or postsynaptic loci of plasticity [9-l 11. On the basis of the amount of inhibition of NMDA-receptor-mediated EPSCs with low-affinity antagonists in hippocampal cultures, the average peak concentration of glutamate attained in the cleft has been estimated to be about 1 mM, with a time constant of decay (z) of 1 ms [12]. Because NMDA and AMPA receptors are usually co-localized and because NMDA receptors have a much higher affinity than AMPA receptors, they will reach different levtransmission. els of occupancy during synaptic Simulations using kinetic models of glutamate-gated ion channels suggested that this concentration transient would very nearly saturate the high-affinity NMDA receptors (95% receptor occupancy), but result in only 60% occupancy of the much lower affinity AMPA receptors [l]. Revisions of this estimate of the concentration transient reflect the more realistic assumption of a biphasic decay in concentration and the use of lower affinity antagonists of AILIPA receptors. These studies [ 1,2] suggest a higher peak concentration (-3 mM) that decays very rapidly (-100 ps) to a more slowly decaying component (0.5 mM, t = l-2 ms). Both estimates, however, predict receptor occupancies similar to those originally suggested [ 121. One caveat is that these simulations used parameters derived
294
Figure
Signalling
mechanisms
1
Presynaptic terminal
n
n
Glutamate
. Glutamate
receptor
Current Opinion in Neurobiology
Release of glutamate from presynaptic terminals diffuses beyond the borders of the synaptic cleft. Depending on the location and density of glutamate transporters, glutamate may attain concentrations high enough to activate extrasynaptic receptors on both pre- and postsynaptic membranes. In the extreme, receptors at neighboring
synapses might be activated. Because astrocytes express high densities of glutamate transporters, synapses surrounded by astrocytic processes are more likely to be insulated from their neighbors than those with little astrocytic investiture.
from studies of receptor kinetics in outside-out patches. If receptor properties are significantly altered by patch excision, occupancy predictions could be in error.
diffusion alone is not sufficient for clearance because ultimately the ambient level of extracellular glutamate would slowly rise to toxic levels. This dilemma is solved by a high-capacity glutamate-uptake system. A family of sodium-dependent, electrogenic glutamate transporters are expressed by both neurons and glia, and they could theoretically lower extracellular glutamate to the low nanomolar range [15,16]. These very low levels of glutamate are unlikely to be attained in the CNS because release, both spontaneous and action potential-driven, is ongoing. However, this housekeeping function of glutamate transporters is very important in maintaining concentrations at levels low enough to avoid significant receptor desensitization and excitotoxicity.
Recently, a very different method has been used to estimate occupancy of synaptic NMDA receptors. Twophoton laser scanning microscopy has been used to image intra-spine calcium transients caused by calcium flux through synaptically activated NMDA receptors at individual spines on CA1 hippocampal pyramidal neurons in acute brain slices [13’]. NMDA receptors unbind glutamate very slowly. If they are saturated by the contents of a single vesicle, then a second release event occurring within a few milliseconds would be unable to increase spine calcium further because practically all of the NMDA receptors will still be occupied by glutamate released during the first event. Surprisingly, when release events occurred 10 ms apart, significant increases in intra-spine calcium were observed, leading Mainen et al. [ 13’1 to conclude that NMDA receptors are at most 56% occupied by single release events. These results suggest that the glutamate transient in the cleft is smaller than that estimated with low-affinity antagonists or that the transient is smaller in slice than in culture. At glutamatergic synapses, the decay of the glutamate transient in the cleft is dependent on diffusion and uptake by membrane-bound transporters. Early models of transmitter clearance indicated that very rapid emptying of the cleft could be achieved by diffusion alone [14]. However,
Glutamate transporters also influence the glutamate transient on the time scale of synaptic events, at least at some synapses. EPSCs in Purkinje cells [17], unipolar brush cells [18] and cells in the chick cochlear nucleus [19] are prolonged by transporter blockers. However, in hippocampal slices, blockers of glutamate transporters seem to have no effect on the kinetics of either the rapid AMPA-receptor-mediated EPSC or the slower, higher affinity NMDA-receptor-mediated EPSC [20-Z]. If glutamate clearance is faster than deactivation kinetics of AMPA receptors or otherwise not rate-limiting (as a result of release asynchrony or cable filtering), subtle changes in clearance might be obscured. Transporter inhibition at synapses in hippocampal cultures, for example, results in augmented glutamate transients [2,23,24] without outright
Clearance
of glutamate
alterations in unitary EPSCs. At calyceal synapses in the chick cochlear nucleus and at cultured hippocampal synapses, the degree of prolongation of the EPSC by transporter antagonists depends on the probability of release, suggesting that glutamate released from nearby release sites can pool [19,24]. Owing to the slow cycling rate of glutamate transporters (14 s-1) [ZS], this transporter-dependent augmentation of glutamate clearance requires expression of transporters at very high densities as well as a rapid glutamate-binding rate. Both high expression levels [26’,27] and rapid binding kinetics [26’,28,29”,30] are characteristics of native glutamate transporters. The location of glutamate transporters important in altering the cleft concentration of glutamate is still unresolved and may vary across tissues. In the hippocampus, little evidence for involvement of neuronal transporters has been found [31] despite immunohistochemical and in S&U hybridization data indicating that neurons, particularly their somatic and dendritic membranes, contain transporters [32-341. Transporters expressed by hippocampal astrocytes, both in culture [24] and slices [26’], appear to be the main pathway for clearance of glutamate, although the existence of presynaptic transporters [35] has not been tested physiologically. The prominence of astrocytic transporters in brain regions is suggested as well by knockout and antisense experiments in which decreases in expression levels of glial transporters result in dramatic pathologies, whereas the lack of the most widely expressed neuronal transporter has fewer consequences [36,37’,38]. Exceptions to the lack of neuronal transport are found in cerebellum and retina. In cerebellar cortex, Purkinje cells express high levels of a unique transporter, EAAT4 [39], in their dendritic membranes that is thought to sequester about 20% of glutamate released by climbing fiber stimulation [29”,30,40]. In addition, both photoreceptor and retinal bipolar cell transporters are activated by glutamate release [41,42]. The function served by Purkinje cell uptake is unclear because both parallel and climbing fiber inputs are encased in Bergmann glia membranes that contain a high density of glutamate transporters. Furthermore, transporter currents activated by these inputs are undetectable in whole-cell recordings using physiological ions and are presumably too small to affect Purkinje cell excitability [29”]. At the tonically active synapse between photoreceptors and ON bipolar cells in the retina, however, uptake is robust enough to polarize the membranes of both cell types. Photoreceptor presynaptic terminals are hyperpolarized by the chloride conductance that is associated with all glutamate transporters [41] but is particularly high in those transporters expressed by photoreceptors [43]. This hyperpolarization would tend to decrease voltage-dependent calcium currents and thus serve as negative feedback on release. In ON bipolar cells, the depolarizing light response that results from decreased glutamate release from photoreceptors may be augmented by the decreased hyperpolarizing effect of the transporter-associated anion current [42] in addition to the metabotropic glutamate receptor activity [44]. Although
inside
the synapse
and beyond
Bergles,
Diamond
and Jahr
295
our knowledge is incomplete and there are clear differences across different tissues, it seems that much of the glutamate released during synaptic transmission is taken up by transporters expressed by glial cells.
Clearance
outside
the cleft
In both hippocampus and cerebellar cortex, it appears that the majority of glutamate released from each vesicle diffuses out of the synaptic cleft. This escape of glutamate from the cleft provides a mechanism for activation of glutamate receptors that are located extrasynaptically, the most prominent of these being metabotropic glutamate receptors [4548], but also AMPA receptors in Bergmann glia [49,50]. In addition, this ‘spillout’ of glutamate may lead to ‘spillover’, the activation of glutamate receptors in neighboring synaptic clefts. The extent of these phenomena depends on the amount and rate of escape, the distance between adjacent synapses, and the location and abundance of glutamate transporters. Significant spillover could underlie, in part, the observation of synaptic events that are mediated only by NMDA receptors, termed ‘silent synapses’ [4,10,51-53,54’], while pooling of transmitter from adjacent release sites can lead to increased postsynaptic receptor occupancy [SS] and slower clearance times [18,19,56]. What is the evidence that the majority of glutamate does diffuse out of the cleft? D-aspartate uptake studies show that the majority of label ends up in astrocytic processes [35]. As D-aspartate is a good substrate for glutamate transporters but not for degradative pathways for glutamate, these results suggest that the capacity of glutamate uptake is greater in astrocytes than neurons. Physiological studies also suggest glutamate escapes from the cleft. Stimulation of Schaffer collateral afferents in CA1 hippocampus [26’] and cerebellum climbing fiber [49] and parallel fiber [SO] terminals elicits glutamate transporter currents in astrocytes and Bergmann glia. As glial processes are by definition outside the cleft, activation of these currents requires spillout. In the CA3 region of hippocampus, enhanced release from mossy fibers [48] or slowed diffusion [45] activates metabotropic glutamate receptors, despite their pre-terminal location [46,47]. In addition, elevating mossy fiber release with forskolin [57] increases glutamate transporter currents in nearby astrocytes [58]. The argument that the majority of glutamate diffuses out of the cleft is based primarily on the lack of evidence for uptake within the cleft. Although neurons clearly express glutamate transporters [32-341, electrophysiological evidence for uptake has been obtained only from Purkinje cells [29”], photoreceptors [41] and retinal bipolar cells [42]. In hippocampus, no evidence has been found for transporter currents in CA1 pyramidal neurons, either following synaptic release or by exogenous substrate applications ([31]; but see [59]). In the cerebellar cortex, it is estimated that about 20% of glutamate released by climbing fibers is taken up by Purkinje cell transporters [29”]; the rest is presumably taken up by Bergmann glia [49,50]. In contrast to the apparent low expression of functional transporters in neurons in the
296
Signalling
mechanisms
brain, astrocytes express very high densities of transporters. Estimates of the density of transporters ranges from a minimum of 2500 per pm2 from electrophysiological experiments [26’] to 10,000 per pm2 using biochemical techniques [27]. This density of membrane protein rivals that of the nicotinic receptor at the neuromuscular junction. The high level of transporter expression by astrocytes and the perisynaptic location of their processes have been exploited to monitor changes in the amount of transmitter released from nearby presynaptic terminals. The transporter currents recorded from astrocytes in stratum radiatum of the CA1 region of hippocampus in response to Schaffer collateral stimulation follow changes in the probability of release caused by alterations in extracellular divalent cation concentrations as well as by repetitive stimulation. However, these synaptically activated transporter currents were unaffected by inducing LTP (long-term potentiation) [S&60]. The simplest interpretation of these results is that the expression of LTP at these synapses is not the result of enhanced release of glutamate. Why should uptake be predominantly the province of glia instead of neurons? One possibility is that glia can use their higher density of transporters more efficiently than neurons because of the more favorable glutamate concentration gradient. Packaging glutamate into synaptic vesicles in nerve terminals relies on a very low affinity uptake system that, in turn, requires very high cytoplasmic concentrations of glutamate. Such cytoplasmic concentrations would impede plasmalemmal uptake by lowering its electrochemical gradient. Astrocytes, however, rapidly convert glutamate into glutamine via glutamine synthetase, thereby maintaining a low cytoplasmic glutamate concentration and a favorable gradient for uptake. Another possibility is that, at least in some conditions, spillout of glutamate is desirable. A dearth of neuronal uptake ensures spillout and favors perisynaptic receptor activation. The lack of neuronal uptake in hippocampus is particularly puzzling in light of the anatomical relationship between excitatory synapses and astrocytic processes in hippocampus and cerebral cortex. In hippocampus and cerebral cortex, very few synapses are entirely surrounded by astrocytic processes ([27,61]; KM Harris, R Ventura, SocNewosci Ah- 1998, 24:827), unlike the cerebellar cortex, where Bergmann glia practically encase both climbing and parallel fiber synapses on Purkinje cells, thereby providing a diffusional barrier between neighboring synapses. Rather, the nearest neighboring membranes at the synaptic cleft perimeter are usually neuronal. Although detailed ultrastructural analyses of the occurrence of astrocytic processes between synapses in hippocampus have not yet been published (although see KM Harris, R Ventura, Sot Nezlrosci Abstr 1998, 24:827), it appears that many synapses in hippocampus and in cerebral cortex are not well insulated by astrocytic uptake from glutamate released at neighboring synapses [61]. Given the anatomy of the neuropil, one
might expect that spillover plays an important role in hippocampal and cortical physiology. Indeed, it has been suggested that spillover can explain, in part, ‘silent synapses’ [4,53,54’,62’]. Although these events, in many cases, probably take place at sites where few AMPA receptors are expressed [63,64], this does not rule out effects of spillover at some synapses. Kullmann and colleagues [53,54’] have reported evidence that spillover causes significant activation of NMDA receptors at neighboring, otherwise inactive synapses when recordings are performed at room temperature. However, when the temperature was raised to near-physiological levels, the effect of spillover was diminished [54’], possibly because of the high temperature-dependence of transport [28,31]. Consistent with this, the effect of temperature was partially reversed by the addition of dihydrokainate [54’], a glutamate transporter blocker selective for astrocytic transporters. These are curious results given the apparent lack of uptake into neurons and sparse astrocytic insulation of synaptic contacts. One explanation is that there could be dihydrokainate-sensitive transporters expressed in presynaptic terminals. Although immunocytochemical studies also suggest a scarcity of transporters expressed presynaptically [32-351, it remains possible that neuronal transporters expressed in presynaptic terminals are not labeled by currently available antibodies (see W Chen, R Hadley, C Gruber, N Irwin, PA Rosenberg, Sot NezlroscZ Abstr 1998, 242066). Another puzzle is the relatively slow time course of the synaptically activated transporter currents in hippocampal astrocytes and Bergmann glia. The ZO-80% rise time of these currents is 3-4 ms and they decay with a 7 of about 20 ms at room temperature [‘26’,49]. These values are much slower than the kinetics of transporter currents evoked by applications of high concentrations of glutamate to outside-out patches (ZO-80% rise times of -0.1 ms, half decay times of 2-3 ms). The slow rise time of the synaptic currents requires asynchronous binding caused by low concentrations of glutamate and possibly slow diffusion times to transporters some distance from release sites. The slow decay phases indicate that extrasynaptic glutamate is elevated for many milliseconds. However, the concentrations of glutamate after, say, ten milliseconds need not be high. Ail that is required is that the number of free glutamate molecules remains elevated. Because the number of synapses monitored by a single astrocyte is probably in the tens of thousands, a significant contribution from any one synapse is not required. If transport efficiency (ratio of binding events to actual transport) is low, repeated binding of transmitter by transporters could slow diffusional spread and prolong the synaptically activated transporter current [30,31,62’].
Conclusions Recent findings indicate that glutamate is not imprisoned in rhe synaptic cleft following release but can rapidly diffuse out and therefore may act at a distance. Electrophysiological recordings of EPSCs from neurons
Clearance
of glutamate
directly monitor the transient presence of glutamate in the cleft. Synaptic responses of glia sense extrasynaptic glutamate concentrations and enable monitoring beyond the cleft. To clearly interpret the recent data, however, a better understanding of diffusion in the extracellular space and the kinetics and efficiency of transport are required. Recent advances in microscopy promise to compliment conventional electrophysiological approaches. This combination should permit, for example, the simultaneous recording of activity in adjacent synapses and the determination of the efficacy of glutamate at a distance.
Acknowledgements This work was NS21419 (CEJ).
supported
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Asztely F, Erdemli G, Kullmann DM: Extrasynaptic glutamate spillover in the hippocampus: dependence on temperature and the role of active glutamate uptake. Neuron 1997, 18:281-293. Coefficient of variation and failures analyses of EPSCs in CA1 pyramidal cells are used to suggest that NMDA EPSCs have twice the quanta1 content of AMPA EPSCs. This difference is reduced at elevated temperatures by a mechanism apparently involving glutamate transporters, leadmg the authors to argue that the discrepancy in quanta1 content is attributable to glutamate spillover activating NMDA receptors on neighboring synapses. In addition, the discrepancy in quanta1 content is preserved over a wide range of release probability, suggesting that spillover of transmitter may not result in pooling. 55.
Silver RA, Cull-Candy receptor occupancy synapse with single 1996,494:231-250.
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Trussell LO, Zhang S, Raman receptors upon multiquantal 1993, IO:1 185-l 196.
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Weisskopf MG, Castillo PE, Zalutsky hippocampal mossy fiber long-term Soence 1994, 265:1878-i 882.
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Mennerick S, Dhond RP, Benz A, Xu W, Rothstem lsenberg KE, Zorumski CF: Neuronal expression transporter GLT-1 in hippocampal microcultures. 18:4490-4499.
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Luscher C, Malenka RC, Nicoll during LTP with glial transporter
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SG, Takahashi T: Non-NMDA glutamate and open probability at a rat cerebellar and multiple release sites. J Physiol (Land)
J: Three-dimensional Anat Embryo/ (Bed)
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RA, Nicoll RA: Mediation of potentiation by cyclic AMP. release monitored LTP. Neuron 1998, JD, Danbolt NC, of the glutamate J Neurosci 1998,
RA: Monitoring glutamate release currents. Neuron 1998, 21:435-441. analysis of dendritic 1985, 171:245-252.
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Ill. Glial
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Rusakov DA, Kullmann DM: Extrasynaptic glutamate diffusion in the hippocampus: ultrastructural constraints, uptake, and receptor activation. J Neurosci 1998, 18:3158-3170. A clever, quantltatlve treatment ot synaptic morphology based on electron micrographs of CA1 neuropil. The authors quantify the tortuosity and extracellular volume fraction of the extrasynaptic space and argue that it can be modeled as an isotropic medium. This information, together with measurements of the average distance between neighboring synapses (465 nm), is used to argue that activation of NMDA receptors in neighboring synapses is likely. A possible role for transporters in regulating this process is discussed. 63.
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Nusser Z, Lujan R, Laube G, Roberts JD, Molnar E, Somogyi P: Cell type and pathway dependence of synaptic AMPA receptor number and variability in the hippocampus. Neuron 1998, 21:545-559.
Clearance of glutamate inside the synapse and beyond Dwight E Bergles, Jeffrey S Diamond and Craig E Jahr* The
heated
debate
occupancy
over
the
by transmitter
level
has
new evidence is fanning two-photon microscopy
of postsynaptic
not
been
in the synaptic
previously and studies
suggested. In contrast, recordings of extrasynaptic receptor activation
efflux
quantities exocytosis.
from
critical synaptic
the
cleft
does
of glutamate Determining
synaptic
to issues plasticity.
cleft
of synaptic
not attain
levels from glial indicate
and
the
distance
specificity
and
the
in Neurobiology
cells that
it diffuses
is
induction
of
L474, Oregon Health Sciences University, Sam Jackson Park Road, Portland, Oregon 97201, USA *e-mail: [email protected]
Opinion
using of
escape from the cleft the amount of glutamate
Addresses Vellum Institute,
Current
- indeed,
the flames. Recent experiments suggest that the concentration
glutamate
significant following
receptor
extinguished
1999,
3181
SW
9:293-298
http://biomednet.com/elecref/0959438800900293 0 Elsevier
Science
Ltd ISSN
0959-4388
Abbreviations AMPA a-amino-3-hydroxy-5-methyl-4-isoxazole-propionic EPSC
LTP NMDA
excitatory postsynaptic long-term potentiation N-methyl-o-aspartate
acid
current
Introduction Glutamate is released at excitatory synapses in the CNS when synaptic vesicles containing 4000-5000 molecules of glutamate fuse with the presynaptic membrane. In the very small volume of the synaptic cleft (- 2 attoliters), concentrations of glutamate as high as several millimolar are attained [ 1,2], assuring activation of postsynaptic glutamate receptors and therefore depolarization of the postsynaptic neuron. However, the synaptic cleft is in continuity with extrasynaptic space, and thus the bolus of glutamate rapidly dissipates both by diffusion and binding to transporters. The dynamics of glutamate release, diffusion, binding and uptake determines the spatiotemporal profile of the concentration of glutamate in the synaptic cleft and perisynaptic space. This review focuses on the clearance of glutamate inside the synaptic cleft and from the extracellular space outside the synapse (Figure 1). Several recent reviews cover many related topics not discussed here [ 1,3-61.
Clearance
within
the cleft
Why do we care about the concentration of glutamate attained in the synaptic cleft or how fast this initial spike of glutamate declines? The immediate reason is that the size and shape of this concentration transient determines how many postsynaptic receptors become bound by transmitter. Receptor occupancy, in turn, dictates the size of the
postsynaptic response as well as the number of unbound receptors available for activation by subsequent exocytosis. If rapid receptor activation is required for fast signaling, a brief pulse of ligand will suffice in synchronizing the activation of a sub-population of available receptors; the higher the peak concentration of such a pulse, the more receptors will be bound. A brief, high-concentration pulse of transmitter is thought to occur at glutamatergic synapses in the CNS [1,2]. The rapid rise in transmitter is ensured by exocytotic release: complete emptying of transmitter from a small synaptic vesicle is thought to require less than 100 ~1s [7,8]. The length of time that glutamate remains elevated in the cleft also affects how many receptors are bound; unless very high concentrations are reached, the longer glutamate is present, the more receptors will be bound, albeit with some asynchrony. The difference between concentration decay time constants of 0.5 ms and 1.5 ms could make the difference between low receptor occupancy and nearly saturated receptors. If the receptors at a synapse are normally near saturation, then the event-to-event variation of the response at that synapse will depend mainly on the stochastic nature of channel opening rather than on variations in receptor occupancy. Alternatively, if low occupancy is the norm, the variation may mainly reflect differences in receptor occupancy owing to vesicle-to-vesicle deviations in the amount of glutamate released. Understanding the origin of such variations is essential because they are an intrinsic feature of signaling at individual synapses as well as a means of identifying pre- or postsynaptic loci of plasticity [9-l 11. On the basis of the amount of inhibition of NMDA-receptor-mediated EPSCs with low-affinity antagonists in hippocampal cultures, the average peak concentration of glutamate attained in the cleft has been estimated to be about 1 mM, with a time constant of decay (z) of 1 ms [12]. Because NMDA and AMPA receptors are usually co-localized and because NMDA receptors have a much higher affinity than AMPA receptors, they will reach different levtransmission. els of occupancy during synaptic Simulations using kinetic models of glutamate-gated ion channels suggested that this concentration transient would very nearly saturate the high-affinity NMDA receptors (95% receptor occupancy), but result in only 60% occupancy of the much lower affinity AMPA receptors [l]. Revisions of this estimate of the concentration transient reflect the more realistic assumption of a biphasic decay in concentration and the use of lower affinity antagonists of AILIPA receptors. These studies [ 1,2] suggest a higher peak concentration (-3 mM) that decays very rapidly (-100 ps) to a more slowly decaying component (0.5 mM, t = l-2 ms). Both estimates, however, predict receptor occupancies similar to those originally suggested [ 121. One caveat is that these simulations used parameters derived
294
Figure
Signalling
mechanisms
1
Presynaptic terminal
n
n
Glutamate
. Glutamate
receptor
Current Opinion in Neurobiology
Release of glutamate from presynaptic terminals diffuses beyond the borders of the synaptic cleft. Depending on the location and density of glutamate transporters, glutamate may attain concentrations high enough to activate extrasynaptic receptors on both pre- and postsynaptic membranes. In the extreme, receptors at neighboring
synapses might be activated. Because astrocytes express high densities of glutamate transporters, synapses surrounded by astrocytic processes are more likely to be insulated from their neighbors than those with little astrocytic investiture.
from studies of receptor kinetics in outside-out patches. If receptor properties are significantly altered by patch excision, occupancy predictions could be in error.
diffusion alone is not sufficient for clearance because ultimately the ambient level of extracellular glutamate would slowly rise to toxic levels. This dilemma is solved by a high-capacity glutamate-uptake system. A family of sodium-dependent, electrogenic glutamate transporters are expressed by both neurons and glia, and they could theoretically lower extracellular glutamate to the low nanomolar range [15,16]. These very low levels of glutamate are unlikely to be attained in the CNS because release, both spontaneous and action potential-driven, is ongoing. However, this housekeeping function of glutamate transporters is very important in maintaining concentrations at levels low enough to avoid significant receptor desensitization and excitotoxicity.
Recently, a very different method has been used to estimate occupancy of synaptic NMDA receptors. Twophoton laser scanning microscopy has been used to image intra-spine calcium transients caused by calcium flux through synaptically activated NMDA receptors at individual spines on CA1 hippocampal pyramidal neurons in acute brain slices [13’]. NMDA receptors unbind glutamate very slowly. If they are saturated by the contents of a single vesicle, then a second release event occurring within a few milliseconds would be unable to increase spine calcium further because practically all of the NMDA receptors will still be occupied by glutamate released during the first event. Surprisingly, when release events occurred 10 ms apart, significant increases in intra-spine calcium were observed, leading Mainen et al. [ 13’1 to conclude that NMDA receptors are at most 56% occupied by single release events. These results suggest that the glutamate transient in the cleft is smaller than that estimated with low-affinity antagonists or that the transient is smaller in slice than in culture. At glutamatergic synapses, the decay of the glutamate transient in the cleft is dependent on diffusion and uptake by membrane-bound transporters. Early models of transmitter clearance indicated that very rapid emptying of the cleft could be achieved by diffusion alone [14]. However,
Glutamate transporters also influence the glutamate transient on the time scale of synaptic events, at least at some synapses. EPSCs in Purkinje cells [17], unipolar brush cells [18] and cells in the chick cochlear nucleus [19] are prolonged by transporter blockers. However, in hippocampal slices, blockers of glutamate transporters seem to have no effect on the kinetics of either the rapid AMPA-receptor-mediated EPSC or the slower, higher affinity NMDA-receptor-mediated EPSC [20-Z]. If glutamate clearance is faster than deactivation kinetics of AMPA receptors or otherwise not rate-limiting (as a result of release asynchrony or cable filtering), subtle changes in clearance might be obscured. Transporter inhibition at synapses in hippocampal cultures, for example, results in augmented glutamate transients [2,23,24] without outright
Clearance
of glutamate
alterations in unitary EPSCs. At calyceal synapses in the chick cochlear nucleus and at cultured hippocampal synapses, the degree of prolongation of the EPSC by transporter antagonists depends on the probability of release, suggesting that glutamate released from nearby release sites can pool [19,24]. Owing to the slow cycling rate of glutamate transporters (14 s-1) [ZS], this transporter-dependent augmentation of glutamate clearance requires expression of transporters at very high densities as well as a rapid glutamate-binding rate. Both high expression levels [26’,27] and rapid binding kinetics [26’,28,29”,30] are characteristics of native glutamate transporters. The location of glutamate transporters important in altering the cleft concentration of glutamate is still unresolved and may vary across tissues. In the hippocampus, little evidence for involvement of neuronal transporters has been found [31] despite immunohistochemical and in S&U hybridization data indicating that neurons, particularly their somatic and dendritic membranes, contain transporters [32-341. Transporters expressed by hippocampal astrocytes, both in culture [24] and slices [26’], appear to be the main pathway for clearance of glutamate, although the existence of presynaptic transporters [35] has not been tested physiologically. The prominence of astrocytic transporters in brain regions is suggested as well by knockout and antisense experiments in which decreases in expression levels of glial transporters result in dramatic pathologies, whereas the lack of the most widely expressed neuronal transporter has fewer consequences [36,37’,38]. Exceptions to the lack of neuronal transport are found in cerebellum and retina. In cerebellar cortex, Purkinje cells express high levels of a unique transporter, EAAT4 [39], in their dendritic membranes that is thought to sequester about 20% of glutamate released by climbing fiber stimulation [29”,30,40]. In addition, both photoreceptor and retinal bipolar cell transporters are activated by glutamate release [41,42]. The function served by Purkinje cell uptake is unclear because both parallel and climbing fiber inputs are encased in Bergmann glia membranes that contain a high density of glutamate transporters. Furthermore, transporter currents activated by these inputs are undetectable in whole-cell recordings using physiological ions and are presumably too small to affect Purkinje cell excitability [29”]. At the tonically active synapse between photoreceptors and ON bipolar cells in the retina, however, uptake is robust enough to polarize the membranes of both cell types. Photoreceptor presynaptic terminals are hyperpolarized by the chloride conductance that is associated with all glutamate transporters [41] but is particularly high in those transporters expressed by photoreceptors [43]. This hyperpolarization would tend to decrease voltage-dependent calcium currents and thus serve as negative feedback on release. In ON bipolar cells, the depolarizing light response that results from decreased glutamate release from photoreceptors may be augmented by the decreased hyperpolarizing effect of the transporter-associated anion current [42] in addition to the metabotropic glutamate receptor activity [44]. Although
inside
the synapse
and beyond
Bergles,
Diamond
and Jahr
295
our knowledge is incomplete and there are clear differences across different tissues, it seems that much of the glutamate released during synaptic transmission is taken up by transporters expressed by glial cells.
Clearance
outside
the cleft
In both hippocampus and cerebellar cortex, it appears that the majority of glutamate released from each vesicle diffuses out of the synaptic cleft. This escape of glutamate from the cleft provides a mechanism for activation of glutamate receptors that are located extrasynaptically, the most prominent of these being metabotropic glutamate receptors [4548], but also AMPA receptors in Bergmann glia [49,50]. In addition, this ‘spillout’ of glutamate may lead to ‘spillover’, the activation of glutamate receptors in neighboring synaptic clefts. The extent of these phenomena depends on the amount and rate of escape, the distance between adjacent synapses, and the location and abundance of glutamate transporters. Significant spillover could underlie, in part, the observation of synaptic events that are mediated only by NMDA receptors, termed ‘silent synapses’ [4,10,51-53,54’], while pooling of transmitter from adjacent release sites can lead to increased postsynaptic receptor occupancy [SS] and slower clearance times [18,19,56]. What is the evidence that the majority of glutamate does diffuse out of the cleft? D-aspartate uptake studies show that the majority of label ends up in astrocytic processes [35]. As D-aspartate is a good substrate for glutamate transporters but not for degradative pathways for glutamate, these results suggest that the capacity of glutamate uptake is greater in astrocytes than neurons. Physiological studies also suggest glutamate escapes from the cleft. Stimulation of Schaffer collateral afferents in CA1 hippocampus [26’] and cerebellum climbing fiber [49] and parallel fiber [SO] terminals elicits glutamate transporter currents in astrocytes and Bergmann glia. As glial processes are by definition outside the cleft, activation of these currents requires spillout. In the CA3 region of hippocampus, enhanced release from mossy fibers [48] or slowed diffusion [45] activates metabotropic glutamate receptors, despite their pre-terminal location [46,47]. In addition, elevating mossy fiber release with forskolin [57] increases glutamate transporter currents in nearby astrocytes [58]. The argument that the majority of glutamate diffuses out of the cleft is based primarily on the lack of evidence for uptake within the cleft. Although neurons clearly express glutamate transporters [32-341, electrophysiological evidence for uptake has been obtained only from Purkinje cells [29”], photoreceptors [41] and retinal bipolar cells [42]. In hippocampus, no evidence has been found for transporter currents in CA1 pyramidal neurons, either following synaptic release or by exogenous substrate applications ([31]; but see [59]). In the cerebellar cortex, it is estimated that about 20% of glutamate released by climbing fibers is taken up by Purkinje cell transporters [29”]; the rest is presumably taken up by Bergmann glia [49,50]. In contrast to the apparent low expression of functional transporters in neurons in the
296
Signalling
mechanisms
brain, astrocytes express very high densities of transporters. Estimates of the density of transporters ranges from a minimum of 2500 per pm2 from electrophysiological experiments [26’] to 10,000 per pm2 using biochemical techniques [27]. This density of membrane protein rivals that of the nicotinic receptor at the neuromuscular junction. The high level of transporter expression by astrocytes and the perisynaptic location of their processes have been exploited to monitor changes in the amount of transmitter released from nearby presynaptic terminals. The transporter currents recorded from astrocytes in stratum radiatum of the CA1 region of hippocampus in response to Schaffer collateral stimulation follow changes in the probability of release caused by alterations in extracellular divalent cation concentrations as well as by repetitive stimulation. However, these synaptically activated transporter currents were unaffected by inducing LTP (long-term potentiation) [S&60]. The simplest interpretation of these results is that the expression of LTP at these synapses is not the result of enhanced release of glutamate. Why should uptake be predominantly the province of glia instead of neurons? One possibility is that glia can use their higher density of transporters more efficiently than neurons because of the more favorable glutamate concentration gradient. Packaging glutamate into synaptic vesicles in nerve terminals relies on a very low affinity uptake system that, in turn, requires very high cytoplasmic concentrations of glutamate. Such cytoplasmic concentrations would impede plasmalemmal uptake by lowering its electrochemical gradient. Astrocytes, however, rapidly convert glutamate into glutamine via glutamine synthetase, thereby maintaining a low cytoplasmic glutamate concentration and a favorable gradient for uptake. Another possibility is that, at least in some conditions, spillout of glutamate is desirable. A dearth of neuronal uptake ensures spillout and favors perisynaptic receptor activation. The lack of neuronal uptake in hippocampus is particularly puzzling in light of the anatomical relationship between excitatory synapses and astrocytic processes in hippocampus and cerebral cortex. In hippocampus and cerebral cortex, very few synapses are entirely surrounded by astrocytic processes ([27,61]; KM Harris, R Ventura, SocNewosci Ah- 1998, 24:827), unlike the cerebellar cortex, where Bergmann glia practically encase both climbing and parallel fiber synapses on Purkinje cells, thereby providing a diffusional barrier between neighboring synapses. Rather, the nearest neighboring membranes at the synaptic cleft perimeter are usually neuronal. Although detailed ultrastructural analyses of the occurrence of astrocytic processes between synapses in hippocampus have not yet been published (although see KM Harris, R Ventura, Sot Nezlrosci Abstr 1998, 24:827), it appears that many synapses in hippocampus and in cerebral cortex are not well insulated by astrocytic uptake from glutamate released at neighboring synapses [61]. Given the anatomy of the neuropil, one
might expect that spillover plays an important role in hippocampal and cortical physiology. Indeed, it has been suggested that spillover can explain, in part, ‘silent synapses’ [4,53,54’,62’]. Although these events, in many cases, probably take place at sites where few AMPA receptors are expressed [63,64], this does not rule out effects of spillover at some synapses. Kullmann and colleagues [53,54’] have reported evidence that spillover causes significant activation of NMDA receptors at neighboring, otherwise inactive synapses when recordings are performed at room temperature. However, when the temperature was raised to near-physiological levels, the effect of spillover was diminished [54’], possibly because of the high temperature-dependence of transport [28,31]. Consistent with this, the effect of temperature was partially reversed by the addition of dihydrokainate [54’], a glutamate transporter blocker selective for astrocytic transporters. These are curious results given the apparent lack of uptake into neurons and sparse astrocytic insulation of synaptic contacts. One explanation is that there could be dihydrokainate-sensitive transporters expressed in presynaptic terminals. Although immunocytochemical studies also suggest a scarcity of transporters expressed presynaptically [32-351, it remains possible that neuronal transporters expressed in presynaptic terminals are not labeled by currently available antibodies (see W Chen, R Hadley, C Gruber, N Irwin, PA Rosenberg, Sot NezlroscZ Abstr 1998, 242066). Another puzzle is the relatively slow time course of the synaptically activated transporter currents in hippocampal astrocytes and Bergmann glia. The ZO-80% rise time of these currents is 3-4 ms and they decay with a 7 of about 20 ms at room temperature [‘26’,49]. These values are much slower than the kinetics of transporter currents evoked by applications of high concentrations of glutamate to outside-out patches (ZO-80% rise times of -0.1 ms, half decay times of 2-3 ms). The slow rise time of the synaptic currents requires asynchronous binding caused by low concentrations of glutamate and possibly slow diffusion times to transporters some distance from release sites. The slow decay phases indicate that extrasynaptic glutamate is elevated for many milliseconds. However, the concentrations of glutamate after, say, ten milliseconds need not be high. Ail that is required is that the number of free glutamate molecules remains elevated. Because the number of synapses monitored by a single astrocyte is probably in the tens of thousands, a significant contribution from any one synapse is not required. If transport efficiency (ratio of binding events to actual transport) is low, repeated binding of transmitter by transporters could slow diffusional spread and prolong the synaptically activated transporter current [30,31,62’].
Conclusions Recent findings indicate that glutamate is not imprisoned in rhe synaptic cleft following release but can rapidly diffuse out and therefore may act at a distance. Electrophysiological recordings of EPSCs from neurons
Clearance
of glutamate
directly monitor the transient presence of glutamate in the cleft. Synaptic responses of glia sense extrasynaptic glutamate concentrations and enable monitoring beyond the cleft. To clearly interpret the recent data, however, a better understanding of diffusion in the extracellular space and the kinetics and efficiency of transport are required. Recent advances in microscopy promise to compliment conventional electrophysiological approaches. This combination should permit, for example, the simultaneous recording of activity in adjacent synapses and the determination of the efficacy of glutamate at a distance.
Acknowledgements This work was NS21419 (CEJ).
supported
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