Maintaining the presynaptic glutamate supply for excitatory neurotransmission.

Glutamate released from synapses during excitatory neurotransmission must be rapidly recycled to maintain neuronal communication. This review evaluates data from physiological experiments at hippocampal CA3 to CA1 synapses and the calyx of Held synapse in the brainstem to analyze quantitatively the rates of release and resupply of glutamate required to sustain neurotransmission. We calculate that, without efficient recycling, the presynaptic glutamate supply will be exhausted within about a minute of normal synaptic activity. We also discuss replenishment of the presynaptic pool by diffusion from the soma, direct uptake of glutamate back into the presynaptic terminal, and uptake of glutamate precursor molecules. Diffusion of glutamate from the soma is calculated to be fast enough to resupply presynaptic glutamate in the hippocampus but not at the calyx of Held. However, because the somatic cytoplasm will also quickly run out of glutamate and synapses can function continually even if the presynaptic axon is severed, mechanisms other than diffusion must be present to resupply glutamate for release. Direct presynaptic uptake of glutamate is not present at the calyx of Held but may play a role in glutamate recycling in the hippocampus. Alternatively, glutamine or tricarboxylic acid cycle intermediates released from glia can serve as a precursor for glutamate in synaptic terminals, and we calculate that the magnitude of presynaptic glutamine uptake is sufficient to supply enough glutamate to sustain neurotransmission. The nature of these mechanisms, their relative abundance, and the co-ordination between them remain areas of intensive investigation.

Inducible presynaptic glutamine transport supports glutamatergic transmission at the calyx of Held synapse.

The mechanisms by which the excitatory neurotransmitter glutamate is recycled at synapses are currently unknown. By examining the functional expression of plasma membrane transporters at presynaptic terminals, we aim to elucidate some of the mechanisms of glutamate recycling. Using whole-cell voltage-clamp recordings from rat calyx of Held presynaptic terminals, our data show, for the first time, that the glutamate precursor glutamine causes the direct activation of an electrogenic, sodium-dependent presynaptic transporter, which supplies glutamine for generation of presynaptic glutamate and helps sustain synaptic transmission. Interestingly, the functional expression of this transporter at the presynaptic plasma membrane is dynamically controlled by electrical activity of the terminal, indicating that uptake of neurotransmitter precursors is controlled by the demand at an individual terminal. Induction of the transporter current is calcium-dependent and inhibited by botulinum neurotoxin C, demonstrating the involvement of SNARE-dependent exocytosis in inserting transporters into the plasma membrane when the terminal is active. Conversely, inactivity of the presynaptic terminal results in removal of transporters via clathrin-mediated endocytosis. To investigate whether the presynaptic glutamine transporter supplies the precursor for generating the synaptically released glutamate, we measured miniature EPSCs to assess vesicular glutamate content. When the presynaptic glutamate pool was turned over by synaptic activity, inhibiting the presynaptic glutamine transporters with MeAIB reduced the miniature EPSC amplitude significantly. This demonstrates that presynaptic glutamine transport is centrally involved in the production of glutamate and assists in maintaining excitatory neurotransmission.

Modulation of Gq-protein-coupled inositol trisphosphate and Ca2+ signaling by the membrane potential.

Gq-protein-coupled receptors (GqPCRs) are widely distributed in the CNS and play fundamental roles in a variety of neuronal processes. Their activation results in phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and Ca2+ release from intracellular stores via the phospholipase C (PLC)-inositol 1,4,5-trisphosphate (IP3) signaling pathway. Because early GqPCR signaling events occur at the plasma membrane of neurons, they might be influenced by changes in membrane potential. In this study, we use combined patch-clamp and imaging methods to investigate whether membrane potential changes can modulate GqPCR signaling in neurons. Our results demonstrate that GqPCR signaling in the human neuronal cell line SH-SY5Y and in rat cerebellar granule neurons is directly sensitive to changes in membrane potential, even in the absence of extracellular Ca2+. Depolarization has a bidirectional effect on GqPCR signaling, potentiating thapsigargin-sensitive Ca2+ responses to muscarinic receptor activation but attenuating those mediated by bradykinin receptors. The depolarization-evoked potentiation of the muscarinic signaling is graded, bipolar, non-inactivating, and with no apparent upper limit, ruling out traditional voltage-gated ion channels as the primary voltage sensors. Flash photolysis of caged IP3/GPIP2 (glycerophosphoryl-myo-inositol 4,5-bisphosphate) places the voltage sensor before the level of the Ca2+ store, and measurements using the fluorescent bioprobe eGFP-PH(PLCdelta) (enhanced green fluorescent protein-pleckstrin homology domain-PLCdelta) directly demonstrate that voltage affects muscarinic signaling at the level of the IP3 production pathway. The sensitivity of GqPCR IP3 signaling in neurons to voltage itself may represent a fundamental mechanism by which ionotropic signals can shape metabotropic receptor activity in neurons and influence processes such as synaptic plasticity in which the detection of coincident signals is crucial.

Muscarinic acetylcholine receptor activation enhances hippocampal neuron excitability and potentiates synaptically evoked Ca(2+) signals via phosphatidylinositol 4,5-bisphosphate depletion.

Using single cell Ca(2+) imaging and whole cell current clamp recordings, this study aimed to identify the signal transduction mechanisms involved in mACh receptor-mediated, enhanced synaptic signaling in primary cultures of hippocampal neurons. Activation of M(1) mACh receptors produced a 2.48 +/- 0.26-fold enhancement of Ca(2+) transients arising from spontaneous synaptic activity in hippocampal neurons. Combined imaging of spontaneous Ca(2+) signals with inositol 1,4,5-trisphosphate (IP(3)) production in single neurons demonstrated that the methacholine (MCh)-mediated enhancement required activated G(q/11)alpha subunits and phospholipase C activity but did not require measurable increases in IP(3). Electrophysiological studies demonstrated that MCh treatment depolarized neurons from -64 +/- 3 to -45 +/- 3 mV and increased action potential generation. Depletion of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) enhanced neuronal excitability and prolonged the action of MCh. These studies suggest that, in addition to producing the second messengers IP(3) and diacylglycerol, mACh receptor activation may directly utilize PIP(2) hydrolysis to regulate neuronal excitability.

Active release of glycine or D-serine saturates the glycine site of NMDA receptors at the cerebellar mossy fibre to granule cell synapse.

The current and calcium influx generated by NMDA receptors depend on the concentration of the coagonist glycine, or its analogue d-serine, in the synaptic cleft. If there is no release of glycine, the ionic stoichiometry of the glial GlyT1 glycine transporters expressed near NMDA receptors in the brain should be able to lower the extracellular glycine concentration to below the EC50 for coactivation of NMDA receptors. We examined whether changing the glycine or d-serine concentration in the superfusion solution altered the NMDA receptor mediated component of the synaptic current at the rat cerebellar mossy fibre to granule cell synapse. Adding up to 100 microM glycine or d-serine had no effect, implying that the glycine site is saturated. Using the competitive glycine site antagonist 7-chlorokynurenate, and plausible values for the kinetic parameters of NMDA receptors, we estimate that during activation of the mossy fibres the concentration of glycine or d-serine in the synaptic cleft is at least 4.6 microM or 1.5 microM, respectively, requiring active release of glycine or d-serine.

Control of intracellular chloride concentration and GABA response polarity in rat retinal ON bipolar cells.

GABAergic modulation of retinal bipolar cells plays a crucial role in early visual processing. It helps to form centre-surround receptive fields which filter the visual signal spatially at the bipolar cell dendrites in the outer retina, and it produces temporal filtering at the bipolar cell synaptic terminals in the inner retina. The observed chloride transporter distribution in ON bipolar cells has been predicted to produce an intracellular chloride concentration, [Cl(-)](i), that is significantly higher in the dendrites than in the synaptic terminals. This would allow dendritic GABA-gated Cl(-) channels to generate the depolarization needed for forming the lateral inhibitory surround of the cell's receptive field, while synaptic terminal GABA-gated Cl(-) channels generate the hyperpolarization needed for temporal shaping of the light response. In contrast to this idea, we show here that in ON bipolar cells [Cl(-)](i) is only slightly higher in the dendrites than in the synaptic terminals, and that GABA-gated channels in the dendrites may generate a hyperpolarization rather than a depolarization. We also show that [Cl(-)](i) is controlled by movement of Cl(-) through ion channels in addition to transporters, that changes of [K(+)](o) alter [Cl(-)](i) and that voltage-dependent equilibration of [Cl(-)](i) in bipolar cells will produce a time-dependent adaptation of GABAergic modulation with a time constant of 8 s after illumination-evoked changes of membrane potential. Time-dependent adaptation of [Cl(-)](i) to voltage changes in retinal bipolar cells may add a previously unsuspected layer of temporal processing to signals as they pass through the retina.

The role of glial glutamate transporters in maintaining the independent operation of juvenile mouse cerebellar parallel fibre synapses.

There is controversy over the extent to which glutamate released at one synapse can escape from the synaptic cleft and affect receptors at other synapses nearby, thereby compromising the synapse-specificity of information transmission. Here we show that the glial glutamate transporters GLAST and GLT-1 limit the activation of Purkinje cell AMPA receptors produced by glutamate diffusion between parallel fibre synapses in the cerebellar cortex of juvenile mice. For a single stimulus to the cerebellar molecular layer of wild-type mice, increasing the number of activated parallel fibres prolonged the parallel fibre EPSC, demonstrating an interaction between different synapses. Knocking out GLAST, or blocking GLT-1 in the absence of GLAST, prolonged the EPSC when many parallel fibres were stimulated but not when few were stimulated. When spatially separated parallel fibres were activated by granular layer stimulation, the EPSC prolongation produced by stimulating more fibres or reducing glutamate transport was greatly reduced. Thus, GLAST and GLT-1 curtail the EPSC produced by a single stimulus only when many nearby fibres are simultaneously activated. However when trains of stimuli were applied, even to a small number of parallel fibres, knocking out GLAST or blocking GLT-1 in the absence of GLAST greatly prolonged and enhanced the AMPA receptor-mediated current. These results show that glial cell glutamate transporters allow neighbouring synapses to operate more independently, and control the postsynaptic response to high frequency bursts of action potentials.

NMDA receptor-mediated currents in rat cerebellar granule and unipolar brush cells.

The properties of N-methyl-D-aspartate (NMDA) receptor-mediated currents at the giant cerebellar mossy-fiber unipolar brush cell (UBC) synapse were compared with those of adjacent granule cells using patch-clamp recording methods in thin slices of rat cerebellar nodulus. In UBCs, NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) decayed as a single exponential whose time constant was independent of membrane potential. The EPSC was reduced in all cells by the NR1/NR2B-selective antagonist ifenprodil, and the Zn(2+) chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) produced a transient potentiation in 50% of cells. In contrast, the NMDA EPSC in granule cells decayed as a double exponential that dramatically switched to a slower rate at positive membrane potentials. The synaptic response in some granule cells also displayed a late second peak at positive potentials, and in others, activation of mossy fibers produced repetitive trains of EPSCs indicating they may be postsynaptic to the UBC network. Single-channel recordings of outside-out somatic patches from UBCs in magnesium-free solution revealed only high-conductance (50 pS) channels whose open time was increased with depolarization, but the opening frequency was decreased to yield a low (p(o) = 0.0298), voltage-independent opening probability. Lowering extracellular calcium (2.5-0.25 mM) had no effects on channel gating, although an increase of single-channel conductance was observed at lower calcium concentrations. Taken together, the data support the notion that the NMDA receptor in UBCs may comprise both NR1/NR2A and NR1/NR2B receptors. Furthermore, the properties of the EPSC in these two classes of feedforward glutamatergic interneurons display fundamental differences that may relate to their roles in synaptic integration.

The amino terminus of the glial glutamate transporter GLT-1 interacts with the LIM protein Ajuba.

We have identified a cytoplasmic LIM protein, Ajuba, which interacts with the amino terminus of GLT-1, the most abundant plasma membrane glutamate transporter in the brain. Ajuba has a cytoplasmic location when expressed alone in COS cells, but translocates to colocalize with GLT-1 at the plasma membrane when GLT-1 is coexpressed. Ajuba is expressed in cerebellum, cortex, hippocampus, and retina and also in organs outside the CNS. Ajuba is found with GLT-1 in astrocytes, cerebellar Bergmann glia and retinal neurons, and antibodies to Ajuba coimmunoprecipitate GLT-1 from brain. For GLT-1 expressed in COS cells, coexpression of Ajuba did not affect the transporter's K(m) or V(max) for glutamate. Since Ajuba is known to activate MAP kinase enzymes, and its homologue Zyxin binds to cytoskeletal proteins, we propose that Ajuba is a scaffolding protein allowing GLT-1 to regulate intracellular signaling or interact with the cytoskeleton.