Identification and analysis of a glutamatergic local interneuron lineage in the adult Drosophila olfactory system

Abstract

Background: The antennal lobe of Drosophila is perhaps one of the best understood neural circuits, because of its well-described anatomical and functional organization and ease of genetic manipulation. Olfactory lobe interneurons - key elements of information processing in this network - are thought to be generated by three identified central brain neuroblasts, all of which generate projection neurons. One of these neuroblasts, located lateral to the antennal lobe, also gives rise to a population of local interneurons, which can either be inhibitory (GABAergic) or excitatory (cholinergic). Recent studies of local interneuron number and diversity suggest that additional populations of this class of neurons exist in the antennal lobe. This implies that other, as yet unidentified, neuroblast lineages may contribute a substantial number of local interneurons to the olfactory circuitry of the antennal lobe.

Results: We identified and characterized a novel glutamatergic local interneuron lineage in the Drosophila antennal lobe. We used MARCM (mosaic analysis with a repressible cell marker) and dual-MARCM clonal analysis techniques to identify this novel lineage unambiguously, and to characterize interneurons contained in the lineage in terms of structure, neurotransmitter identity, and development. We demonstrated the glutamatergic nature of these interneurons by immunohistochemistry and use of an enhancer-trap strain, which reports the expression of the Drosophila vesicular glutamate transporter (DVGLUT). We also analyzed the neuroanatomical features of these local interneurons at single-cell resolution, and documented the marked diversity in their antennal lobe glomerular innervation patterns. Finally, we tracked the development of these dLim-1 and Cut positive interneurons during larval and pupal stages.

Conclusions: We have identified a novel neuroblast lineage that generates neurons in the antennal lobe of Drosophila. This lineage is remarkably homogeneous in three respects. All of the progeny are local interneurons, which are uniform in their glutamatergic neurotransmitter identity, and form oligoglomerular or multiglomerular innervations within the antennal lobe. The identification of this novel lineage and the elucidation of the innervation patterns of its local interneurons (at single cell resolution) provides a comprehensive cellular framework for emerging studies on the formation and function of potentially excitatory local interactions in the circuitry of the Drosophila antennal lobe.

Figure 1

Gal4-OK371 labels a ventrolateral glutamatergic local interneuron cluster. (A) Gal4-OK371-driven GFP expression in the adult brain labels clusters of cells that innervate (A1) the antennal lobe (blue arrowheads) and the sub-oesophageal ganglia (magenta arrowheads). (A3) Different neuropil regions are innervated (fan-shaped body, cyan arrowhead) or absent in few neuropil regions; one such example is (A2) the γ lobe of the mushroom body (yellow arrowhead). (B1, D1) Gal4-OK371-driven GFP expression labels a cluster of cells located ventrolateral to the adult antennal lobe (demarcated by dotted lines). (B2, B3) These cells are positive for DVGLUT immunoreactivity (magenta). (C) Magnification of the boxed region in (B). (C2, C3) These cells are positive for DVGLUT expression (blue arrowheads). (D1-D3) This cluster of cells is GABA (magenta) negative (yellow arrowhead). Images are oriented as shown in (B3): D = dorsal; L = lateral. Scale bar = 10 μm.

Figure 2

The ventrolateral glutamatergic cell cluster is born from a single lineage. (A) MARCM-derived neuroblast clone labelled with Gal4-OK371 generated by heat shock at 12 ALH. (A1, A2) A cluster of cells (yellow arrowheads) located ventrolateral to the antennal lobe (demarcated by white dots) is labelled. (A2) The antennal glomeruli were labelled with staining with mAbnc82 (Brp) (magenta). (A1 inset) A few confocal sections stacked to enable clearer visualization of somata. (A1, A2) Commissural contralateral projections (indicated by white arrow). (B) Confocal sections were used to reconstruct the interneuron cluster using Amira http://www.amira.com. Scale bar = 10 μm. Images are oriented as shown in (A2): D = dorsal; L = lateral.

Figure 3

Local interneurons (LNs) of the glutamatergic ventrolateral cluster derive from a novel lineage. (A) Dual binary expression system to label (A1) LNs using Gal4-OK371 > UAS-mCD8::GFP and (A2) projection neurons (PNs) using GH146-LexA::GAD > rCD2::GFP. (A3, A4) A clear separation of the LNs (green) and PNs (magenta) can be seen. (B1-B4) tub-LexA::GAD/+ (or Y); FRTG13, Gal4-GH146, UAS-mCD8/FRT G13, hsFLP, tub-GAL80 ; lexAop-rCD2::GFP/+. Dual MARCM clone induced at 0-4 ALH. Clone labelled with (B1) Tub is (demarcated with yellow dots) and (B2) Gal4-GH146 (yellow dots) is labelled with (B3) anti-GABA; (B4) Merged image of the different channels in B1-B3. (C1-C4) tub-LexA::GAD/+ or Y; FRTG13, Gal4-GH146, UAS-mCD8/FRT G13, hsFLP, tub-GAL80 ; lexAop-rCD2::GFP/+. Dual MARCM clone induced at 0-4 ALH. The clone labelled with (C1) tub (yellow dots) is not labelled with (C2) Gal4-GH146. (C1, C4) Primary neurite bundle from the cell bodies (magenta arrow). (C3) The clonal cells are not labelled with anti-GABA. (C1, C4) Arbours of these cells cross to the contralateral lobe via the commissure (yellow arrow). (D1-D3) tub-LexA::GAD/+ or Y; FRT G13, hsFLP, tub-GAL80/UASmCD8::GFP, Gal4-OK371, FRTG13; lexAop-rCD2::GFP/+. Dual MARCM clone generated by heat shock at 24 ALH. The clone labelled with (D1, D3) tub completely overlaps that labelled with (D2, D3) Gal4-OK371. Glutamatergic ventrolateral cluster (cyan dotted line). (A4) Antennal lobes (demarcated by white dots). D = dorsal; L = lateral. Scale bars = 10 μm.

Figure 4

Diverse neuronal architectures of the ventral local interneurons (vILNs). Single cell vlLN clones showing examples of (A-D) ipsilaterally and (E-F) bilaterally projecting neurons. (A) An ipsilaterally innervating oligoglomerular neuron (arborizing in one to three glomeruli). (B-D) Ipsilaterally innervating multiglomerular (arborizing in a large subset of glomeruli) neurons. (A-D) Arbours terminate in contiguous glomeruli (continuous innervation). (E) Bilaterally innervating LN. The innervation is multiglomerular on the ipsilateral side with only oligoglomerular (one to three) innervation contralaterally. (F) Bilaterally innervating neuron with oligoglomerular arbours in both antennal lobes. (A1, B1, C1, D1, E1 F1) Neuron labelled with the mCD8::GFP reporter. (C1, D1, E1, F1) Tiled images made from cropped portions of appropriate sections to highlight the single cell. (A2, B2, C2, D2, E2, F2) Merged images (of GFP and Brp channels) that are reconstructed (A3, B3, C3, D3, E3 and F3) of confocal sections through the antennal lobe using Amira. White arrowheads indicate the cell bodies. Images are oriented as shown in (B3): D = dorsal, L = lateral. Scale bars = 10 μm.

Figure 5

Developmental profiling of the glutamatergic ventrolateral cluster. (A) Labelling of developing (third instar larval stage) ventral local interneurons (vILNs) with antibodies against neurotactin (Nrt; magenta). (A1-A3) Sections at 7 μm intervals focus on the cell somata of vlLNs labelled with OK371 (demarcated by white dots). (A1) Neuroblast is not labelled with green fluorescent protein (GFP) (blue arrowhead); GFP co-localizes with neurotactin (Nrt) (white arrowhead), and secondary axonal tract is labelled with anti-Nrt (small white arrows). (A4) Merged image of all 35 confocal sections showing the entire ventrolateral lineage. (B) Reconstruction of appropriate confocal sections using Amira. (B1) Larval antennal lobe (LAL) with ventrolaterally located cell bodies labelled with GFP expression in Gal4-OK371. (B2) Reconstruction of the anti-Nrt labelled lineage; the secondary axonal tract (SAT) traverses the LAL (outlined with dots) and terminates near the prospective adult AL (asterisk). (B3) Merge of the images shown in (B1) and (B2); white dotted line demarcates the location of the neuroblast. (C,D) Antennal lobes from (C) 6 and (D) 12 hours APF, showing developing neurons labelled with Gal4-OK371 (yellow arrowheads). (C2. D2). The presumptive adult antennal lobe is labelled with anti-N-Cadherin (yellow dotted lines). (C3, D3)The regressing larval antennal lobe is identified by staining with mAbnc82 (anti-Brp) (white arrowheads). (D1) Tiled image made from cropped portions of appropriate sections to highlight the cells that are part of this cluster. Orientation of the images; D = dorsal; L = lateral; P = posterior. Scale bar = 10 μm.

Figure 6

Transcription factor expression in the ventrolateral lineage interneurons (vILNs). Third instar (A-C) larval and (D-F) adult brains with vlLNs labelled with Gal4-OK371, UAS-mCD8::GFP. (A,D) Antennal lobe (white dots) and neurons (yellow dots) in the lineage. (B, C, E, F) Single confocal sections through the somata from (B,C) larval and (E,F) adult brains labelled with antibodies against (B,E) dLim1 and (C,F) Cut. Labelling is more abundant in developing than in adult brains. (B, C, E and F) Cell bodies co-labelled with the corresponding transcription factor (yellow arrowheads). D = dorsal, L = lateral. Scale bar = 10 μm.