Glutamine Transport via Neurotransmitter Transporter 4 (NTT4, SLC6A17) Maintains Presynaptic Glutamate Supply at Excitatory Synapses in the Central Nervous System

The glutamate-glutamine cycle is thought to be the principle metabolic pathway that recycles glutamate at excitatory synapses. In this cycle, synaptically released glutamate is sequestered by astrocytes and converted to glutamine before being returned to the presynaptic terminal for conversion back into glutamate to replenish the neurotransmitter pool. While many aspects of this cycle have been extensively studied, a key component remains unknown: the nature of the transporter responsible for the presynaptic uptake of glutamine. We hypothesise that neurotransmitter transporter 4 (NTT4/ SLC6A17 ) plays this role. Accordingly, we generated NTT4 knockout mice to assess its contribution to presynaptic glutamine transport and synaptic glutamate supply. Using biochemical tracing of [1- ¹³ C] glucose and [1,2- ¹³ C] acetate in awake mice, we observe a reduction of neuronal glutamate supply when NTT4 is absent. In addition, direct electrical recording of hippocampal mossy fibre boutons reveals a presynaptic glutamine transport current that is entirely inhibited by genetic or pharmacological elimination of NTT4. The role of NTT4 in neurotransmission was demonstrated by electrophysiological recordings in acute hippocampal slices, which reveal that NTT4 is required to maintain vesicular glutamate content and to sustain adequate levels of glutamate supply during periods of high-frequency neuronal activity. Finally, behavioural studies in mice demonstrate a deficit in trace fear conditioning; a hippocampus-dependent memory paradigm, and abnormalities in nest building, anxiety behaviour, and social preference. These results demonstrate that NTT4 is a presynaptic glutamine transporter which is a central component of the glutamate-glutamine cycle. NTT4 and hence the glutamate-glutamine cycle maintain neuronal glutamate supply for excitatory neurotransmission during high-frequency synaptic activity, and are key regulators of memory retention and normal behaviour.