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The model genetic organism Drosophila melanogaster, commonly known as the fruit fly, uses many of the same neurotransmitters as mammals and very similar mechanisms of neurotransmitter storage, release and recycling. This system offers a variety of powerful molecular-genetic methods for the study of transporters, many of which would be difficult in mammalian models. We review here progress made using Drosophila to understand the function and regulation of neurotransmitter transporters and discuss future directions for its use.

Abstract: The sublaterodorsal nucleus (SLD) in the pons of the rat is a locus supporting short-latency induction of a REM sleep-like state following local application of a GABAA receptor antagonist or kainate, glutamate receptor agonist. One putatively relevant source of these neurotransmitters is from the region of the deep mesencephalic nucleus (DpMe) just ventrolateral to the periaquiductal gray, termed the dorsal DpMe (dDpMe). Here, the amino acid neurotransmitter innervation of SLD from dDpMe was studied utilizing anterograde tract-tracing with biotinylated dextranamine (BDA) and fluorescence immunohistochemistry visualized with laser scanning confocal microscopy. Both markers for inhibitory and excitatory amino acid neurotransmitters were found in varicose axon fibers in SLD originating from dDpMe. Vesicular glutamate transporter2 (VGLUT2) represented the largest number of anterogradely labeled varicosities followed by vesicular GABA transporter (VGAT). Numerous VGAT and VGLUT2 labeled varicosities were observed apposed to dDpMe-labeled axon fibers indicating both excitatory and inhibitory presynaptic, local modulation within the SLD. Some double-labeled BDA/VGAT varicosities were seen apposed to small somata labeled for glutamate consistent with being presynaptic to the phenotype of REM sleep-active SLD neurons. Results found support the current theoretical framework of the interaction of dDpMe and SLD in control of REM sleep, while also indicating operation of mechanisms with a greater level of complexity. [Copyright &y& Elsevier]

Abstract: There is accumulating evidence that glutamate and GABA release are key mechanisms of ischaemic events in the CNS. However, data on the expression of involved transporters for these mediators are inconsistent, potentially impeding further neuroprotective approaches. Here, we applied immunofluorescence labelling to characterise the expression pattern of vesicular glutamate (VGLUT) and GABA transporters (VGAT) after acute focal cerebral ischaemia and in two models of retinal ischaemia. Mice were subjected to filament-based focal cerebral ischaemia predominantly involving the middle cerebral artery territory, also leading to retinal ischaemia due to central retinal artery occlusion (CRAO). Alternatively, retinal ischaemia was induced by a transient increase of the intraocular pressure (HIOP). One day after ischaemia onset, diminished immunolabelling of neuronal nuclei and microtubule-associated protein 2-positive structures were found in the ipsilateral neocortex, subcortex and the retina, indicating neuronal degeneration. VGLUT1 expression did not change significantly in ischaemic tissues whereas VGLUT2 was down-regulated in specific areas of the brain. VGLUT3 expression was only slightly down-regulated in the ischaemia-affected neocortex, and was found to form clusters on fibrils of unknown origin in the ischaemic lateral hypothalamus. In contrast, retinae subjected to CRAO or HIOP displayed a rapid loss of VGLUT3-immunoreactivity. The expression of VGAT appears resistant to ischaemia as there was no significant alteration in all the regions analysed. In summary, these data indicate a region- and subtype-specific change of VGLUT expression in the ischaemia-affected CNS, whose consideration might help to generate specific neuroprotective strategies. [Copyright &y& Elsevier]

Abstract: Inactivation of the genes encoding the neuronal, synaptic vesicle-associated proteins synapsin I and II leads to severe reductions in the number of synaptic vesicles in the CNS. We here define the postnatal developmental period during which the synapsin I and/or II proteins modulate synaptic vesicle number and function in excitatory glutamatergic synapses in mouse brain. In wild-type mice, brain levels of both synapsin I and synapsin IIb showed developmental increases during synaptogenesis from postnatal days 5–20, while synapsin IIa showed a protracted increase during postnatal days 20–30. The vesicular glutamate transporters (VGLUT) 1 and VGLUT2 showed synapsin-independent development during postnatal days 5–10, following which significant reductions were seen when synapsin-deficient brains were compared with wild-type brains following postnatal day 20. A similar, synapsin-dependent developmental profile of vesicular glutamate uptake occurred during the same age periods. Physiological analysis of the development of excitatory glutamatergic synapses, performed in the CA1 stratum radiatum of the hippocampus from the two genotypes, showed that both the synapsin-dependent part of the frequency facilitation and the synapsin-dependent delayed response enhancement were restricted to the period after postnatal day 10. Our data demonstrate that while both synaptic vesicle number and presynaptic short-term plasticity are essentially independent of synapsin I and II prior to postnatal day 10, maturation and function of excitatory synapses appear to be strongly dependent on synapsin I and II from postnatal day 20. [Copyright &y& Elsevier]

At inhibitory synapses, glycine and GABA are accumulated into synaptic vesicles by the same vesicular transporter VGAT/VIAAT (vesicular GABA transporter/vesicular inhibitory amino acid transporter), enabling a continuum of glycine, GABA, and mixed phenotypes. Many fundamental aspects of the presynaptic contribution to the inhibitory phenotypes remain unclear. The neuronal transporter GlyT2 is one of the critical presynaptic factors, because glycinergic transmission is impaired in knock-out GlyT2-/- mice and mutations in the human GlyT2 gene slc6a5 are sufficient to cause hyperekplexia. Here, we establish that GlyT2-mediated uptake is directly coupled to the accumulation of glycine into recycling synaptic vesicles using cultured spinal cord neurons derived from GlyT2—enhanced green fluorescent protein transgenic mice. Membrane expression of GlyT2 was confirmed by recording glycine-evoked transporter current. We show that GlyT2 inhibition induces a switch from a predominantly glycine to a predominantly GABA phenotype. This effect was mediated by a reduction of glycinergic quantal size after cytosolic depletion of glycine and was entirely reversed by glycine resupply, illustrating that the filling of empty synaptic vesicles is tightly coupled to GlyT2-mediated uptake. Interestingly, high-frequency trains of stimuli elicit two phases of vesicle release with distinct kinetic requirements for glycine refilling. Thus, our results demonstrate the central role played by GlyT2 in determining inhibitory phenotype and therefore in the physiology and pathology of inhibitory circuits. [ABSTRACT FROM AUTHOR]

Abstract: The functional balance of glutamatergic and GABAergic signaling in neuronal cortical circuits is under homeostatic control. That is, prolonged alterations of global network activity leads to opposite changes in quantal amplitude at glutamatergic and GABAergic synapses. Such scaling of excitatory and inhibitory transmission within cortical circuits serves to restore and maintain a constant spontaneous firing rate of pyramidal neurons. Our recent work shows that this includes alterations in the levels of expression of vesicular glutamate (VGLUT1 and VGLUT2) and GABA (VIAAT) transporters. Other vesicle markers, such as synaptophysin or synapsin, are not regulated in this way. Endogenous regulation at the level of mRNA and synaptic protein controls the number of transporters per vesicle and hence, the level of vesicle filling with transmitter. Bidirectional and opposite activity-dependent regulation of VGLUT1 and VIAAT expression would serve to adjust the balance of glutamate and GABA release and therefore the level of postsynaptic receptor saturation. In some excitatory neurons and synapses, co-expression of VGLUT1 and VGLUT2 occurs. Bidirectional and opposite changes in the levels of two excitatory vesicular transporters would enable individual neocortical neurons to scale up or scale down the level of vesicular glutamate storage, and thus, the amount available for release at individual synapses. Regulated vesicular transmitter storage and release via selective changes in the level of expression of vesicular glutamate and GABA transporters indicates that homeostatic plasticity of synaptic strength at cortical synapses includes presynaptic elements. [Copyright &y& Elsevier]