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

Abstract

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.

Figure 1.

Membrane depolarization rapidly and reversibly induces a calcium and exocytosis-dependent glutamine-mediated current in presynaptic terminals. A, A differential interference contrast image and the corresponding fluorescence image of a Lucifer yellow-filled calyx of Held presynaptic terminal (arrow) surrounding an MNTB neuron (#) in a brainstem slice. Glutamine (10 mm) or glutamate (200 μm) was applied by pressure ejection from one of two puffer pipettes (*) placed equidistant from the terminal. B, The 5 s puff application of glutamate did not elicit a membrane current before or after activation of Ca²⁺ channels by depolarization trains (inset; Calibration: 1 nA, 10 ms). Glutamine induced a membrane current after Ca²⁺-channel activation. C, The magnitude of I_Gln over time in control (black trace) and with Pitstop-1 in the patch pipette (gray trace). Stimulation of the presynaptic calcium channels was performed before each puff application, as indicated by the arrows. D, Averaged data showing I_Gln induced by stimulation of presynaptic Ca²⁺ currents or release of caged calcium in the presynaptic terminal. Induction was reversed by cessation of stimulation, by the removal of external Ca²⁺, or by the inclusion of BoNT/C in the patch-pipette. Reversal of induction was prevented by Pitstop-1. **p < 0.01. ***p < 0.001.

Figure 2.

The induced presynaptic glutamine response is mediated by amino acid transporters. A, Single-cell representative traces of 5 s puff application of glutamine (10 mm) inducing a membrane current that was substantially inhibited by the amino acid transporter substrate MeAIB (20 mm). B, Averaged data show that MeAIB or removal of external Na⁺ eliminates I_Gln, whereas 200 μm TBOA or 1 mm d-aspartate has no effect. **p < 0.01. ***p < 0.001. C, The magnitude of I_Gln after activation of Ca²⁺ currents in a single cell as MeAIB is applied in the bath solution and included in the puffer pipette, showing rapid inhibition. D, Current–voltage relationship of I_Gln indicates transporter rather than ion channel activation (n ≥ 3 for each voltage).

Figure 3.

Presynaptic glutamine transport sustains glutamatergic transmission. A, mEPSC amplitudes recorded from voltage-clamped postsynaptic MNTB principal neurons after stimulation at 200 Hz to promote turnover of the presynaptic glutamate pool, before and during bath application of 20 mm MeAIB (application bar, black trace; n = 5) and in control (200 Hz stimulation but no MeAIB; gray trace; n = 5). B, mEPSC amplitude is significantly inhibited by MeAIB during 200 Hz stimulation but is not reduced during 0.1 Hz stimulation. *p < 0.05. C, Representative graph showing cumulative frequency of mEPSC amplitudes from a single cell stimulated at 200 Hz before and 10–15 min after MeAIB application.