Abstract Synapses are specialized contact points between neurons where neuronal signaling take place. The synaptic transmission involves release of chemical signaling molecules (known as neurotransmitters) from one neuron, which then diffuses through a narrow synaptic cleft and act upon a subsequent cell to convey a message. In the central nervous system glutamate is the most ubiquitous excitatory neurotransmitter responsible for more than 80 % of the neuronal signaling in synapses and is pivotal for a variety of brain functions. However, mechanisms involved in the replenishment of the neurotransmitter pools of glutamate have eluded characterization. Glutamine has a pivotal role in many processes in the body. This is reflected in the vast amount of glutamine in the blood and the cerebrospinal fluid. In fact, glutamine contributes to one fifth of the total amino acid content in the blood and two-third of the amino acid concentration in the cerebrospinal fluid. In the CNS, glutamine has been suggested to serve two important functions. First of all, it is necessary for ammonia detoxification, since the brain lacks a complete urea cycle. Secondly, it acts as a precursor to the fast excitatory and inhibitory neurotransmitters glutamate and GABA. In this ultrastructural study, we have investigated whether inhibition of neuronal glutamine has an impact on synapse morphology, alters trafficking of synaptic vesicles and/or mitochondrial function.