SNAT3-mediated glutamine transport in perisynaptic astrocytes in situ is regulated by intracellular sodium.

The release of glutamine from astrocytes adjacent to synapses in the central nervous system is thought to play a vital role in the mechanism of glutamate recycling and is therefore important for maintaining excitatory neurotransmission. Here we investigate the nature of astrocytic membrane transport of glutamine in rat brainstem slices, using electrophysiological recording and fluorescent imaging of pHi and Nai+. Glutamine application to perisynaptic astrocytes induced a membrane current, caused by activation of system A (SA) family transporters. A significant electroneutral component was also observed, which was mediated by the system N (SN) family transporters. This response was stimulated by glutamine (KM of 1.57 mM), histidine, and asparagine, but not by leucine or serine, indicating activation of the SNAT3 isoform of SN. We hypothesized that increasing the [Na+ ]i would alter the SNAT3 transporter equilibrium, thereby stimulating glutamine release. In support of this hypothesis, we show that SNAT3 transport can be driven by changing cation concentration and that manipulations to raise [Na+ ]i (activation of excitatory amino acid transporters (EAATs), SA transporters or AMPA receptors) all directly influence SNAT3 transport rate. A kinetic model of glutamine fluxes is presented, which shows that EAAT activation causes the release of glutamine, driven mainly by the increased [Na+ ]i . These data demonstrate that SNAT3 is functionally active in perisynaptic astrocytes in situ. As a result, astrocytic Nai+ signaling, as would be stimulated by neighboring synaptic activity, has the capacity to stimulate astrocytic glutamine release to support glutamate recycling.

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.

Activation of glutamate transport evokes rapid glutamine release from perisynaptic astrocytes.

Stimulation of astrocytes by neuronal activity and the subsequent release of neuromodulators is thought to be an important regulator of synaptic communication. In this study we show that astrocytes juxtaposed to the glutamatergic calyx of Held synapse in the rat medial nucleus of the trapezoid body (MNTB) are stimulated by the activation of glutamate transporters and consequently release glutamine on a very rapid timescale. MNTB principal neurones express electrogenic system A glutamine transporters, and were exploited as glutamine sensors in this study. By simultaneous whole-cell voltage clamping astrocytes and neighbouring MNTB neurones in brainstem slices, we show that application of the excitatory amino acid transporter (EAAT) substrate d-aspartate stimulates astrocytes to rapidly release glutamine, which is detected by nearby MNTB neurones. This release is significantly reduced by the toxins L-methionine sulfoximine and fluoroacetate, which reduce glutamine concentrations specifically in glial cells. Similarly, glutamine release was also inhibited by localised inactivation of EAATs in individual astrocytes, using internal DL-threo-ß-benzyloxyaspartic acid (TBOA) or dissipating the driving force by modifying the patch-pipette solution. These results demonstrate that astrocytes adjacent to glutamatergic synapses can release glutamine in a temporally precise, controlled manner in response to glial glutamate transporter activation. Since glutamine can be used by neurones as a precursor for glutamate and GABA synthesis, this represents a potential feedback mechanism by which astrocytes can respond to synaptic activation and react in a way that sustains or enhances further communication. This would therefore represent an additional manifestation of the tripartite relationship between synapses and astrocytes.