The recently cloned system A transporter SAT1 (formerly known as GlnT) has been shown to mediate high affinity glutamine uptake and to be expressed by many glutamatergic neurones in the rat brain (Varoqui et al. 2000). Glutamine accumulated via SAT1 may act as a precursor for glutamate synthesis in excitatory neurones using glutamate for transmission, but since inhibitory neurones are known to require glutamate for synthesis of GABA, the transporter may also play a role in supplying glutamine to some GABAergic neurones. We have therefore examined the distribution of SAT1 immunoreactivity in the rat brain, using an affinity-purified antibody directed against an N-terminal sequence of SAT1, and performed immunolabelling and in situ hybridisation studies to examine the expression of SAT1 immunoreactivity in populations of glutamatergic and GABA neurones.
Adult rats were humanely killed by perfusion with paraformaldehyde fixative under halothane anaesthesia (5 % in O2) and coronal sections cut using a vibratome. Labelling with the specific antibody detected SAT1 immunoreactivity in widespread and numerous neurones throughout the brain that displayed granular or punctate labelling of their somata. At the ultrastructural level, immunoreaction was associated with presumptive endosomal inclusions and in some cases plasmalemma, in somata, dendrites, pre-terminal axons and synaptic boutons. Glial cell labelling was only rarely observed. The well-labelled somata in areas such as olfactory bulb, neocortex, hippocampus, thalamus, pontine and medullary reticular formation, vestibular, cochlear and deep cerebellar nuclei, dorsal column and spinal trigeminal nuclei suggested SAT1 expression in excitatory and inhibitory neurones. This notion was supported by multiple fluorescence labelling with antisera to various known markers for GABA and glutamate neurones, and RNA probes for glutamic acid decarboxylase (GAD65 and GAD67) and vesicular glutamate transporter (VGLUT2) transcripts. However, some known groups of glutamatergic neurones, such as the cerebellar granule cells, were only modestly labelled for SAT1. The presence of SAT1 immunoreactivity in subpopulations of GABA neurones that were reactive for parvalbumin, but rarely for nitric oxide synthase, calbindin D28k, calretinin, somatostatin or neuropeptide Y, could indicate expression of the transporter in fast spiking inhibitory interneurones.
Our observations suggest that glutamine uptake mediated by SAT1 may be a characteristic of subsets of both glutamatergic and GABAergic neurones in the brain.