Central adaptation to odorants depends on PI3K levels in local interneurons of the antennal lobe

Abstract

We have previously shown that driving PI3K levels up or down leads to increases or reductions in the number of synapses, respectively. Using these tools to assay their behavioral effects in Drosophila melanogaster, we showed that a loss of synapses in two sets of local interneurons, GH298 and krasavietz, leads to olfaction changes toward attraction or repulsion, while the simultaneous manipulation of both sets of neurons restored normal olfactory indexes. We show here that olfactory central adaptation also requires the equilibrated changes in both sets of local interneurons. The same genetic manipulations directed to projection (GH146) or mushroom body (201Y, MB247) neurons did not affect adaptation. Also, we show that the equilibrium is a requirement for the glomerulus-specific size changes which are a morphological signature of central adaptation. Since the two sets of local neurons are mostly, although not exclusively, inhibitory (GH298) and excitatory (krasavietz), we interpret that the normal phenomena of sensory perception, measured as an olfactory index, and central adaptation rely on an inhibition/excitation ratio.

Figure 1.

Morphological effects of overexpressing PI3K constructs. A–I, Confocal images of whole-mount mushroom bodies from anterior (A–C) and posterior (D–I) views. Neurons were visualized by immunolabeling of the membrane CD8-GFP reporter. In the 201Y-Gal4 domain, UAS-PI3K and UAS-GSK3 caused an increase or decrease, respectively, in the size of the mushroom body lobes (B, C) and calyces (E, F) compared with control (A–D). Equivalent effects were observed in the calyx when PI3K constructs were overexpressed in the MB247-Gal4 domain (G–I). J, K, Quantification of the effects. L–Q, Morphological effects in PNs of GH146-Gal4 domain. These cells (arrowhead in L) have their dendrites in several AL glomeruli (outlined in L) and send their axons to MB and lateral horn (LH) brain centers (outlined in O). Note the increase (PI3K) and reduction (PI3K^DN) in the size and GFP signal intensity in all targeted areas. Scale bars (in F) A–I, 75 μm; (in Q) L–Q, 50 μm.

Figure 2.

Central adaptation after modification of MB neurons and PNs. A–L, Olfactory response index (OI) to 10⁻² EB (A–F) and 10⁻² IAA (G–L) in 6-d-old adults previously exposed to the same odorant (10⁻¹) (gray and black columns to EB and IAA, respectively) or to paraffin oil (white columns). In all genotypes, preexposed flies were significantly less repelled by the odorant than control individuals. Genotypes were tested randomly and each group of flies was subjected to one odorant concentration choice only. Neither modification in mushroom body neurons (201Y-Gal4 and MB247-Gal4) (A, H) nor modification in projection neurons (GH146) (C, I) affected central adaptation to EB or IAA. However, the effect was prevented when synapse number was putatively increased (PI3K, AKT) or reduced (PI3K^DN, GSK3) in the GH298 (D, J) or krasavietz (E, K) domains. By contrast, central adaptation was normal when both neuronal domains were modified simultaneously (F, L). n.s., Not significant.

Figure 3.

Olfactory central adaptation is odor-specific. A, The krasavietz-Gal4 flies were exposed to 10⁻¹ EB (black bars) or to paraffin oil (white bars) and tested with 10⁻¹ IAA or 10⁻³ benzaldehyde (BZD). B, The same test using GH298-Gal4 flies exposed to IAA. Note that in both cases the phenomenon develops for the odorant of exposure only. C, D, The increase (PI3K) or reduction (GSK3) of synapses in the GH-298 neurons (iLNs) prevents the establishment of central adaptation following the exposure to IAA and the effect is general to the three odorants tested. n.s., Not significant.

Figure 4.

Structural effects of olfactory central adaptation. A, Frontal view (confocal section) of the right AL of a 6-d-old ENG3 fly. Glomeruli were detected by the n-synaptobrevin-GFP reporter. The two glomeruli (V and DM2) selected for volumetric measurements are outlined (brown lines). Scale bar, 10 μm. B, 3D reconstruction of the selected glomeruli and their localization in the AL. C–F, Flies of the following genotypes: GH298-Gal4/ENG3 (C), GH298-Gal4/UASPI3K/ENG3 (D), krasavietz-Gal4/UASPI3K^DN/ENG3 (E), and krasavietz-Gal4/GH298-Gal4/UASPI3K^DN/ENG3 (F) were exposed to paraffin oil (control) or 10⁻¹ EB (experimental) during days 2–5, and their brains processed after the behavioral assay (day 6). Dot plots show volume measurements from V (black and orange) and DM2 (black and blue) glomeruli. C, In exposed GH298-Gal4/ENG3 flies, DM2 showed a significant increment in volume, whereas V remained unaffected. No significant changes occurred in V or DM2 glomeruli when GH298 (D) or krasavietz (E) neurons expressed the PI3K and PI3KDN constructs, respectively. Note that both genotypes were also ineffective to produce central adaptation. F, In the double Gal4 driver genotype, however, the relative glomerular volume of DM2, but not that of V, was increased in preexposed flies compared with controls (C). n = 10–12 glomeruli/genotype/experimental group. n.s., Not significant.