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. 2010 Feb 8:4:1.
doi: 10.3389/neuro.05.001.2010. eCollection 2010.

SAT1, A Glutamine Transporter, is Preferentially Expressed in GABAergic Neurons

Tom Tallak Solbu  1 , Mona Bjørkmo, Paul Berghuis, Tibor Harkany, Farrukh A Chaudhry
Affiliations

SAT1, A Glutamine Transporter, is Preferentially Expressed in GABAergic Neurons

Tom Tallak Solbu et al. Front Neuroanat. .

Abstract

Subsets of GABAergic neurons are able to maintain high frequency discharge patterns, which requires efficient replenishment of the releasable pool of GABA. Although glutamine is considered a preferred precursor of GABA, the identity of transporters involved in glutamine uptake by GABAergic neurons remains elusive. Molecular analyses revealed that SAT1 (Slc38a1) features system A characteristics with a preferential affinity for glutamine, and that SAT1 mRNA expression is associated with GABAergic neurons. By generating specific antibodies against SAT1 we show that this glutamine carrier is particularly enriched in GABAergic neurons. Cellular SAT1 distribution resembles that of GAD67, an essential GABA synthesis enzyme, suggesting that SAT1 can be involved in translocating glutamine into GABAergic neurons to facilitate inhibitory neurotransmitter generation.

Keywords: GAD67; SNAT1; Slc38a1; glutamate/GABA-glutamine cycle; interneuron; neurotransmitter; parvalbumin; system A.

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Figures

Figure 1
Figure 1
Characterization of antibodies raised against SAT1. (A) Two rabbits were immunized with a GST fusion protein containing the N-terminus of SAT1. The sera obtained were tested for specificity by immunoblotting on extracts from COS cells transiently transfected with SAT1 (1), SAT2 (2) or a mock vector (3). Both antibodies revealed an immunoreaction restricted to extracts from SAT1 transfected cells (results shown only for anti-SAT1-N1). (B) The SAT1 selective sera were affinity purified and tested on rat brain extracts separated by SDS-PAGE. A broad band close to 50 kDa was obtained by both antibodies (1 and 2). Two additional slender bands of higher molecular weight were detected by anti-SAT1-N2 antibody (2). (C) Differentiated PC12 cells transiently transfected with FLAG-tagged SAT1 were double stained with SAT1-N1 antibody (C′) and a commercial antibody against FLAG-epitope (C″). Superimposition of the images shows that both antibodies stain the same subset of PC12 cells. Note that the staining occurs in the cell bodies as well as in their processes. Note also that a cell not immunostained by anti-FLAG is not immunostained for SAT1 either. (D) SAT1 (green; D′) is targeted to the processes also labeled for synaptophysin (red; D″), a vesicular protein, demarcating axonal endings of differentiated SAT1 transfected PC12 cells. Note that an adjacent putative untransfeced cell stains strongly for synaptophysin, but eludes immunostaining by SAT1 antibody. (E) Double labeling of differentiated SAT1 transfected PC12 for SAT1 and syntaxin-6 (stx6), a marker of the trans-Golgi network, shows pronounced colocalization in the perinuclear region. All sections have been counterstained by DAPI (blue). Scale bar: 20 μm.
Figure 2
Figure 2
Prominent immunoperoxidase staining for SAT1 in a subset of neurons in several brain regions. (A) Immuneperoxidase staining of free-floating parasagittal brain sections reveals SAT1 localization in many subregions. The highest staining intensity is found in thalamus, mesencephalic region and cerebellum, while cortex, hippocampus and striatum over all have comparatively lower levels of staining. (B) The strong SAT1-like immunoreactivity is abolished when the antibodies are preincubated with the GST fusion protein used to immunize rabbits (result shown for anti-SAT1-N1). (C,D) Prominent cellular SAT1 immunoreactivity is detected in the reticular thalamic nucleus (Rt) by both antibodies (anti-SAT1-N1 and anti-SAT1-N2, in C and D, respectively). The ventral posterolateral thalamic nucleus (VPL) also contains scattered SAT1 immunoreactive cells although less intensely stained. Very few SAT1 immunolabeled cells are detected in the internal capsule (ic). (E) In the brain stem, SAT1 immunoreactivity is enriched in the pontine nucleus (Pn). SAT1 immunostained cells are also detected in the lateral superior olive (LSO) and facial nucleus (7n). Strongly immunostained neuron-like cells appear in the oral part of the pontine reticular nucleus (PnO) in which SAT1 immunoreactivity is distributed in the cell body as well as their processes (inset). (F) The medulla oblongata harbors scattered neurons in the reticular formation strongly immunostained for SAT1. The inset shows some neuron-like cells enriched with strong SAT1 immunostaining distributed throughout their entire cellular extension including their processes. (G) Prominent SAT1 immunolabeling is detected in neuron-like cells in the lateral olfactory tract nucleus. (H) In the spinal cord, moderate to strong SAT1 immunostaining is detected in neuron-like cells in the grey matter (Gm). No immunostained cell bodies are detected in the spinal white matter (W). (I) In the cerebellar granule cell layer (G), SAT1 expression is particularly prominent in some scattered neurons with large cell bodies, resembling Golgi interneurons (I), while the granule cells (g) are faintly immunolabeled (inset). Purkinje cells are also immunostained by the affinity-purified antibodies against SAT1 (arrows; lower inset). The cerebellar nuclei (Cbn) contain scattered cells strongly immunostained for SAT1. Hardly any stained cells are detected in the white matter (W). Designations: *, canalis centralis; 7n, facial nucleus; Cb, cerebellum; Cbn, cerebellar nucleus (nucleus interpositus); cc, corpus callosum; CPu, caudate putamen; Cx, cortex; DCn, dorsal cochlear nucleus; Fr, frontal cortex; G, granule cell layer; g, granule cell; Gm, grey matter; GP, globus pallidus; Hc, hippocampus; I, interneuron; IC, inferior colliculus; lo, lateral olfactory tract; LL, lateral lemniscus nucleus; LSO, lateral superior olive; LV, lateral ventricle; M, molecular layer; P, Purkinje cell layer; Pir, piriform cortex; Pn, pontine nuclei; PnO, pontine reticular nucleus, oral part; Pu, Purkinje cell; Rt, reticular thalamic nucleus; SC, superior colluculus; SN, substantia nigra; Th, thalamus; Tu, olfactory tubercule; VPL, ventral posterolateral thalamic nucleus; W, white matter; ZI, zona incerta. Scale bars: 20 μm (insets I), 40 μm (G, insets E and F), 100 μm (C–F,H,I), 350 μm (A).
Figure 3
Figure 3
Basal ganglionic neurons exhibit strong SAT1 immunoreactivity in their cell bodies as well as their processes. (A) Globus pallidus (GP) is highly enriched with SAT1 immunoreactive neurons. Smaller, scattered neuron-like cells strongly immunostained for SAT1 are also detected in the caudatoputamen (CPu) (arrows). (B) High power light microscopy of neurons in the globus pallidus demonstrates SAT1 immunostaining in the cell bodies as well as in their processes. Note that the immunostaining is restricted to the plasma membrane (arrow). (C) In the reticular part of substantia nigra (SNR), neuronal cell bodies and their processes are strongly immunolabeled for SAT1. The compact part of the substantia nigra (SNC) shows SAT1 immunostaining diffusely in the neuropil and in some smaller cell bodies. (D) A neuron-like cell is stained for SAT1 in the caudatoputamen. Note that the staining appears in the cell body and its processes (arrows). CPu, caudatoputamen; GP, globus pallidus; SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata. Scale bars: 20 μm (B,D), 80 μm (A,C).
Figure 4
Figure 4
Differential SAT1 immunoreactivity among cortical neuronal subpopulations. (A) SAT1 immunoreactive cells are detected in all layers of the neocortex. Layers III and V stand out as broad bands diffusely stained in the neuropil. (B) In the layer V, strong immunostaining for SAT1 is detected in scattered small neuron-like cells (arrows) while larger pyramidal-like cells (arrow heads) are moderately or weakly immunolabeled. I–VI: layers 1–VI. Scale bars: 100 μm (B), 200 μm (A).
Figure 5
Figure 5
SAT1 immunostaining is enriched in hippocampal interneurons. (A) The hippocampal formation contains dispersed cells strongly immunostained for SAT1 while the principal cells are weakly immunostained. The neuropil is moderately stained in the vicinity of the principal cells (P and G) and in stratum lacunosum-moleculare (LM). (B) SAT1 immunostaining is abolished on preincubation of SAT1 antibody with the fusion protein used for immunization. (C) High power light microscopy confirms strong SAT1 immunostaining of non-pyramidal cells and their processes between faintly labeled pyramidal cells of CA1. Intracellular stained structures are visible. (D) Two SAT1 immunostained cells in the stratum oriens of CA1. Note that SAT1 immunoreactivity extends to the distal parts of their processes. (E) The dentate area also possesses scattered cells strongly stained for SAT1. Note their prominently SAT1 stained processes. (F) A SAT1 immunostained neuron with its cell body located in the basal part of the granule cell layer. Note that it extends a immunostained process vertically between unstained granule cells (g) and a horizontal process along the inner border of the granule cell layer (arrow). G, granule cell layer; g, granule cell; H, hilus; LM, stratum lacunosum-moleculare; M, stratum molecular of the area dentata; O, stratum oriens; P, stratum pyramidale; p, pyramidal cell; R, stratum radiatum. Scale bars: 20 μm (C, D, F), 75 μm (E), 150 μm (A,B).
Figure 6
Figure 6
SAT1 immunostaining is pronounced in GAD67/PARV+ neurons and is targeted to VGAT containing axonal segments. Young adult mice heterozygously expressing green fluorescence protein (GFP) knocked into the GAD67 locus were perfusion fixed and immunofluorescently labeled for SAT1 and cellular markers. (A) In the neocortex (L2/3) and hippocampus, SAT1 immunoreactivity accumulates in the somatodendritic domains of a subset of GABAergic interneurons also containing PARV (arrows). Arrowheads indicate SAT1/GAD67gfp/+-positive GABAergic interneurons that fall into other subclasses of interneurons. (B) Co-localization of SAT1 and PARV immunoreactivities in the basal ganglia. Tripled labelled neurons and their processes appear in white/light pink color. Note that virtually all PARV-containing neurons in the globus pallidus (GP) and reticular thalamic nucleus (RTN) express SAT1. Open rectangle indicates the location of images in the majority of GABAergic neurons in RTN (C). (C) Channel separation reveals co-localization of SAT1 and PARV immunoreactivities in RTN. Arrows point to select triple-labelled cells. (D) SAT1 immunoreactive GABAergic neurons lacking PARV immunoreactivity in the substantia nigra pars reticulata. (E,F) Hippocampal PARV+ interneurons were isolated and cultured for 12 days in vitro. Considerable SAT1 immunoreactivity was only found in cells co-expressing perineuronal net components (as revealed by Wisteria floribunda lectin binding), and the vesicular GABA transporter (VGAT). Arrows point to triple-labelled varicose axonal structures. Abbreviations: CPu, caudate putamen; GP, globus pallidus; or, stratum oriens; pyr, stratum pyramidale; rad, stratum radiatum; RTN, reticular thalamic nucleus. Scale bars = 4 μm (F), 14 μm (E), 30 μm (A,C,D), 100 μm (B).
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References

    1. Albrecht J., Sonnewald U., Waagepetersen H. S., Schousboe A. (2007). Glutamine in the central nervous system: function and dysfunction. Front. Biosci. 12, 332–34310.2741/2067 - DOI - PubMed
    1. Appell P. P., Behan M. (1990). Sources of subcortical GABAergic projections to the superior colliculus in the cat. J. Comp. Neurol. 302, 143–15810.1002/cne.903020111 - DOI - PubMed
    1. Armano S., Coco S., Bacci A., Pravettoni E., Schenk U., Verderio C., Varoqui H., Erickson J. D., Matteoli M. (2002). Localization and functional relevance of system a neutral amino acid transporters in cultured hippocampal neurons. J. Biol. Chem. 277, 10467–1047310.1074/jbc.M110942200 - DOI - PubMed
    1. Bartho P., Freund T. F., Acsady L. (2002). Selective GABAergic innervation of thalamic nuclei from zona incerta. Eur. J. Neurosci. 16, 999–101410.1046/j.1460-9568.2002.02157.x - DOI - PubMed
    1. Battaglioli G., Martin D. L. (1990). Stimulation of synaptosomal gamma-aminobutyric acid synthesis by glutamate and glutamine. J. Neurochem. 54, 1179–118710.1111/j.1471-4159.1990.tb01946.x - DOI - PubMed