Internal state configures olfactory behavior and early sensory processing in Drosophila larvae

Abstract

Animals exhibit different behavioral responses to the same sensory cue depending on their internal state at a given moment. How and where in the brain are sensory inputs combined with state information to select an appropriate behavior? Here, we investigate how food deprivation affects olfactory behavior in Drosophila larvae. We find that certain odors repel well-fed animals but attract food-deprived animals and that feeding state flexibly alters neural processing in the first olfactory center, the antennal lobe. Hunger differentially modulates two output pathways required for opposing behavioral responses. Upon food deprivation, attraction-mediating uniglomerular projection neurons show elevated odor-evoked activity, whereas an aversion-mediating multiglomerular projection neuron receives odor-evoked inhibition. The switch between these two pathways is regulated by the lone serotonergic neuron in the antennal lobe, CSD. Our findings demonstrate how flexible behaviors can arise from state-dependent circuit dynamics in an early sensory processing center.

Fig. 1. Feeding state determines odor responses by opposite modulation of lAL outputs.

(A) For preference assay, 15 larvae are tracked in an odor gradient for 15 min. Trajectories demonstrate avoidance/attraction to GA by fed/food-deprived larvae. (B to D) Electron microscopy (EM) reconstructions (left) and behavioral phenotypes (right) for ORNs, uPNs, and cobra mPN [mushroom body calyx (CA), vertical lobe (VL), larval antennal lobe (lAL; circled), lateral horn (LH)]. (B) GA elicits avoidance in fed state (one-sample t test, P < 0.001) and attraction after food deprivation (P < 0.01). Behavior diverges between states (two-sample t test, ***P < 0.001). Anosmic larvae (Orco^−/−) ignore GA [one-sample/two-sample t tests, not significant (n.s.); n = 12 to 18]. (C) Silencing uPNs (GH146-GAL4) specifically abolishes food-deprived attraction [one-way analysis of variance (ANOVA), *P < 0.05; n = 8 to 10]. (D) Silencing cobra mPN (GMR32E03-GAL4) specifically abolishes fed avoidance (one-way ANOVA, ***P < 0.001; n = 6 to 10). (E to G) Calcium responses to GA (10⁻⁶). (E) ORN-Or82a and ORN-Or45a exhibit similar responses in both states (n = 5 to 6). (F and G) After food deprivation, uPNs exhibit increased responses (n = 7 to 9), whereas cobra mPN exhibits more negative responses (n = 8 to 9). (H) Parallel lAL output pathways (uPNs and cobra mPN) support opposite behavioral responses and receive opposite state-dependent modulation.

Fig. 2. Upon food deprivation, the mPN pathway receives glutamatergic inhibition from pLNs.

(A) RNA interference (RNAi) knockdown of GluClα receptor in cobra mPN specifically abolishes attraction in food-deprived state (Kruskal-Wallis test, ***P < 0.001; n = 4 to 8) without affecting avoidance in fed state (P > 0.05). (B and C) Calcium responses of cobra mPN to GA (10⁻⁶) in wild-type (light) and GluClα-RNAi larvae (dark). GluClα knockdown abolishes state-dependent inhibition of cobra mPN seen in wild-type larvae (n = 8). (D) EM reconstruction of picky local interneurons pLN1/4 [mushroom body calyx (CA), vertical lobe (VL), larval antennal lobe (lAL; circled), lateral horn (LH)]. (E) Silencing either pLN1 or pLN4 with UAS-Kir2.1 abolishes attraction in the food-deprived state (Kruskal-Wallis test, *P < 0.05; n = 8 to 10). Silencing pLN1 (but not pLN4) modestly increases avoidance in fed state (*P < 0.05). (F) pLN1 exhibits enhanced calcium responses to GA (10⁻⁶) in food-deprived state (n = 7). (G) pLNs provide glutamatergic inhibition onto cobra mPN via the GluClα receptor.

Fig. 3. Upon food deprivation, CSD activates uPNs via the excitatory 5-HT7 receptor.

(A) EM reconstruction of uPNs [mushroom body calyx (CA), vertical lobe (VL), larval antennal lobe (lAL; circled), lateral horn (LH)]. (B) Silencing 5-HT7–expressing neurons specifically abolishes food-deprived attraction (one-way ANOVA, **P < 0.01; n = 8). (C) CRISPR knockout of 5-HT7 in uPNs specifically abolishes food-deprived attraction (one-way ANOVA, ***P < 0.001; n = 8 to 14). (D and E) uPN calcium responses to GA (10⁻⁶) in wild-type (light) and uPN-specific 5-HT7 knockout larvae (dark). 5-HT7 knockout abolishes state-dependent enhancement of uPN responses seen in wild-type (n = 6 to 7). (F) EM reconstruction of CSD neuron [neuropil annotations as in (A)]. (G) Silencing CSD (R60F02-GAL4) specifically abolishes food-deprived attraction (Kruskal-Wallis test, *P < 0.05; n = 6 to 8). (H) Disrupting 5-HT synthesis in CSD via Trh knockdown specifically abolishes food-deprived attraction (Kruskal-Wallis test, **P < 0.01; n = 8). (I) CSD exhibits increased calcium responses to GA (10⁻⁸) following food deprivation (n = 8 to 10). (J) Optogenetic activation of CSD in fed larvae reproduces the state-dependent behavioral switch (one-way ANOVA, *P < 0.05; n = 12 to 20). (K) Upon food deprivation, elevated 5-HT from CSD excites uPNs via 5-HT7 receptor, promoting behavioral attraction.

Fig. 4. A simple dynamical model recapitulates state-dependent changes in odor valence.

(A) Connectivity and state-dependent dynamics of lAL model. Each node represents a cell type; node size and intensity represent the predicted activity in fed (orange) and food-deprived (blue) states. Edges terminating in arrowheads and bars represent excitatory and inhibitory connections, respectively; edge thickness indicates connection strength. (B) EM-derived synapse counts between all modeled cell types and model predictions for neuronal activity in the fed (orange) and food-deprived (blue) states. Assignment of excitatory (+) or inhibitory (−) sign to each connection is based on experimental data. The positive connection from CSD to uPNs models the effect of putatively nonsynaptic 5-HT release. We also assume excitatory feedback from uPNs to CSD via the lateral horn (LH). Connections with ≥25 synapses are assigned weight 1, and connections with <25 synapses are assigned weight w < 1. The weak connection from CSD to pLN0 is assigned weight β. wt, wild type. (C) Model predictions for activity of each cell type and overall behavioral output in the fed (orange) and food-deprived (blue) states under simulated perturbations. All model predictions are corroborated by experimental results except for the case of pLN0 inactivation (n.d., no data).