A conditional GABAergic synaptic vesicle marker for Drosophila

Abstract

Background: Throughout the animal kingdom, GABA is the principal inhibitory neurotransmitter of the nervous system. It is essential for maintaining the homeostatic balance between excitation and inhibition required for the brain to operate normally. Identification of GABAergic neurons and their GABA release sites are thus essential for understanding how the brain regulates the excitability of neurons and the activity of neural circuits responsible for numerous aspects of brain function including information processing, locomotion, learning, memory, and synaptic plasticity, among others.

New method: Since the structure and features of GABA synapses are critical to understanding their function within specific neural circuits of interest, here we developed and characterized a conditional marker of GABAergic synaptic vesicles for Drosophila, 9XV5-vGAT.

Results: 9XV5-vGAT is validated for conditionality of expression, specificity for localization to synaptic vesicles, specificity for expression in GABAergic neurons, and functionality. Its utility for GABAergic neurotransmitter phenotyping and identification of GABA release sites was verified for ellipsoid body neurons of the central complex. In combination with previously reported conditional SV markers for acetylcholine and glutamate, 9XV5-vGAT was used to demonstrate fast neurotransmitter phenotyping of subesophageal ganglion neurons.

Comparison with existing methods: This method is an alternative to single cell transcriptomics for neurotransmitter phenotyping and can be applied to any neurons of interest represented by a binary transcription system driver.

Conclusion: A conditional GABAergic synaptic vesicle marker has been developed and validated for GABA neurotransmitter phenotyping and subcellular localization of GABAergic synaptic vesicles.

Keywords: Drosophila; Epitope tag; GABAergic; Synaptic vesicle; VGAT.

Figure 1.

Strategic design of the conditional GABAergic synaptic vesicle marker RSRT-STOP-RSRT-9XV5-vGAT and conditional neuropil expression in adult brain and larval ventral nerve cord. A) Genomic exon structure of Drosophila vGAT. B) Genome editing at the endogenous vGAT genomic locus included insertion of a FlpStop cassette flanked by R recombinase target sites into the vGAT intron and insertion of a 9XV5 epitope tag near the beginning of exon 2. The FlpStop cassette includes two transcription termination sequences and an upstream splice acceptor sequence followed by translation STOP codons in all three reading frames. Prior to excision of the STOP cassette, 9XV5-vGAT is not expressed. C) After expression of the R recombinase using a binary transcription system driver and a corresponding R recombinase responder transgene, the STOP cassette is excised and 9XV5-vGAT is expressed in GABAergic neurons. D-F) Conditional expression in adult brain. D-D”) 9XV5-vGAT germline excision/9XV5-germline excision. D) 9XV5-vGAT; D’) Syn; D”) overlay. E-E”) RSRT-STOP-RSRT-9XV5-vGAT/+. E) 9XV5-vGAT; E’) Syn; E”) overlay. F-F”) yw. F) vGAT; F’) Syn; F”) overlay. Images in panels D and E were collected and processed identically. G-I) Conditional expression in larval ventral nerve cord). G-G”) 9XV5-vGAT germline excision/9XV5-germline excision. G) 9XV5-vGAT; G’) Syn; G”) overlay. H-H”) RSRT-STOP-RSRT-9XV5-vGAT/+. H) 9XV5-vGAT; H’) Syn; H”) overlay. I-I”) yw. I) vGAT; I’) Syn; I”) overlay. Images in panels G and H were collected and processed identically. Germline excision of the RSRT-STOP-RSRT-9XV5-vGAT STOP cassette results in strong neuropil-specific 9XV5-vGAT immunostaining in the adult brain and larval ventral nerve cord that is not observed prior to STOP cassette excision. The 9XV5-vGAT distribution is nearly indistinguishable from endogenous vGAT. Syn-Synapsin. Scale bar: 100μm.

Figure 2.

Assessment of neurotransmitter and synaptic vesicle specificity of RSRT-STOP-RSRT-9XV5-vGAT in single neuron types. Neuron anatomy is visualized with the plasma membrane marker CD8-mCherry. A-A”) GABAergic neuron R4d. A) CD8-mCherry; A’) 9XV5-vGAT; A”) overlay. 9XV5-vGAT expression exhibits highly restricted localization to the ring region of R4d neurons. B-B”) High resolution 100X images of pre-synaptic terminals of GABAergic neuron R4d. B) Syt-smHA; B’) 9XV5-vGAT; B”) overlay. High resolution images of R4d pre-synaptic terminals reveals near precise overlap of 9XV5-vGAT with the synaptic vesicle marker Syt-smHA. C-C”) GABAergic neuron R4d. C) CD8-mCherry; C’) GFP-Rab3; C”) overlay. GFP-Rab3 is expressed in GABAergic neuron R4d and distributes predominantly to pre-synaptic terminals in a pattern similar to that of 9XV5-vGAT. D-D”) Glutamatergic neuron MBON-6. D) CD8-mCherry; D’) 9XV5-vGAT; D”) overlay. E-E”) Cholinergic neuron LH2094. E) CD8-mCherry; E’) 9XV5-vGAT; E’) overlay. 9XV5-vGAT expression was not detected in the glutamatergic neuron MBON-6 or the cholinergic neuron LH2094. Large arrows-presynaptic terminals; small arrows-dendrites; arrowheads-cell bodies. Scale bars: A, C-50μm; B-10μm; D, E-20μm.

Figure 3.

GABAergic neurotransmitter phenotyping of ellipsoid body neurons. A-A”) SS02704. B-B”) SS02714. C-C”) SS02766. D-D”) SS02705. E-E”) SS02767. F-F”) SS02702. The plasma membrane marker CD8-mCherry (red, left column) allows visualization of the neuroanatomy of each ellipsoid body neuron. The presence of 9XV5-vGAT (arrows, middle column) indicates each ellipsoid body neuron is GABAergic and its subcellular localization reveals the site of presynaptic terminals where SV fusion and GABA release occurs. Scale bars: 20μm.

Figure 4.

Fast neurotransmitter phenotyping of the subesophageal neuron trident/SS46209. A) CD8-mCherry; A’) 9XV5-vGAT); A”) overlay. 9XV5-vGAT expression in trident neurons indicates a GABAergic neurotransmitter phenotype. B) CD8-mCherry; B’) 7XMYC-vAChT; B”) overlay. C) CD8-mCherry; C’) smFLAG-vGlut; C”) overlay. 7XMYC-vAChT and smFLAG-vGlut were not detected in trident neurons. Scale bar: 20μm.

Figure 5.

Fast neurotransmitter phenotyping of the subesophageal neuron G2N-1/SS47082. A) CD8-mCherry; A’) 9XV5-vGAT); A”) overlay. B) CD8-mCherry; B’) 7XMYC-vAChT; B”) overlay. 7XMYC-vAChT expression in G2N-1 neurons indicates a cholinergic neurotransmitter phenotype.C) CD8-mCherry; C’) smFLAG-vGlut; C”) overlay. 9XV5-vGAT and smFLAG-vGlut were not detected G2N-1 neurons. Scale bar: 20μm.

Figure 6.

Fast neurotransmitter phenotyping of the subesophageal neuron genie/SS30377. A) CD8-mCherry; A’) 9XV5-vGAT); A”) overlay. B) CD8-mCherry; B’) 7XMYC-vAChT; B”) overlay. C) CD8-mCherry; C’) smFLAG-vGlut; C”) overlay. smFLAG-vGlut expression in genie neurons indicates a GABAergic neurotransmitter phenotype. 9XV5-vGAT and 7XMYC-vAChT were not detected in genie neurons. Scale bar: 20μm.