Mushroom body input connections form independently of sensory activity in Drosophila melanogaster

Abstract

Associative brain centers, such as the insect mushroom body, need to represent sensory information in an efficient manner. In Drosophila melanogaster, the Kenyon cells of the mushroom body integrate inputs from a random set of olfactory projection neurons, but some projection neurons-namely those activated by a few ethologically meaningful odors-connect to Kenyon cells more frequently than others. This biased and random connectivity pattern is conceivably advantageous, as it enables the mushroom body to represent a large number of odors as unique activity patterns while prioritizing the representation of a few specific odors. How this connectivity pattern is established remains largely unknown. Here, we test whether the mechanisms patterning the connections between Kenyon cells and projection neurons depend on sensory activity or whether they are hardwired. We mapped a large number of mushroom body input connections in partially anosmic flies-flies lacking the obligate odorant co-receptor Orco-and in wild-type flies. Statistical analyses of these datasets reveal that the random and biased connectivity pattern observed between Kenyon cells and projection neurons forms normally in the absence of most olfactory sensory activity. This finding supports the idea that even comparatively subtle, population-level patterns of neuronal connectivity can be encoded by fixed genetic programs and are likely to be the result of evolved prioritization of ecologically and ethologically salient stimuli.

Keywords: Kenyon cells; antennal lobe; mushroom body; olfaction; projection neurons; sensory activity.

Figure 1.. Odor-evoked activity is decreased in the mushroom body calyx of Orco^−/− flies.

(A-C) Calcium imaging in Kenyon cells shows odor-evoked activity in Orco^−/− flies in response to isopentyl acetate, an odor that activates multiple Odorant Receptors, but not in response to acetic acid, an odor that activates Ionotropic Receptors. (A) The calcium indicator GCaMP6f was expressed in all Kenyon cells using the R13F02 transgene. Guided by baseline fluorescence, the region where Kenyon cells extend their dendrites — the calyx of the mushroom body — was identified in Orco^+/+ and Orco^−/− female flies that are two- or three-day old (left column, dashed white line). Example heatmaps show ΔF/F₀ in response to isopentyl acetate (middle column) and acetic acid (right column). The color bars denote the range of ΔF/F₀ in each sample. Scale bar is 20 μm. (B) The ΔF/F₀ values recorded in the main calyx in response to isopentyl acetate (pink) and acetic acid (gray) were averaged across eight trials collected in eight different animals and are shown as traces; the shaded area of each trace represents the standard error of mean. (C) The median ΔF/F₀ values during odor presentation were averaged across trials in Orco^+/+ (green) and Orco^−/− (blue) flies (n = 8); the long black bars represent the median whereas the short black bars represent the 25th and 75th percentile of the data. The statistical significance, or ‘p-value’, was measured using the Mann-Whitney U test; the asterisk indicates values that were statistically different (p < 0.05). See also Figure S1.

Figure 2.. Antennal lobes are morphologically similar in Orco^+/+ and Orco^−/− flies.

(A) The brains of two- or three-day old Orco^+/+ and Orco^−/− female flies were fixed, immuno-stained (using the nc82 monoclonal antibody against Bruchpilot) and imaged; each of the 51 glomeruli forming the antennal lobe were reconstructed and identified based on theirs shapes and locations; each glomerulus receives input from either Odorant Receptors-expressing neurons (pink), Ionotropic Receptors/Gustatory Receptors-expressing neurons (dark gray) or unidentified receptor neurons (light gray). Scale bar is 25 μm. (B-C) The reconstructed volumes of the entire antennal lobe (B) or individual glomeruli (C) were compared across genotypes (green: Orco^+/+ (n = 5); blue: Orco^−/− (n = 5)); the long black bars represent the median whereas the short black bars represent the 25th and 75th percentile of the data. (C) The volumes of a given glomerulus in both genotypes are linked with a black line. The statistical significance, or ‘p-value’, was measured using the Mann-Whitney U test; the statistical significance of the differences in glomerular volumes are provided in Table S1.

Figure 3.. Projection neurons are morphologically similar in Orco^+/+ and Orco^−/− flies.

(A) The projection neurons innervating the DL4 (left), VA2 (middle) and DP1m (right) glomeruli were photo-labeled in Orco^+/+ and Orco^−/− female flies that were two- or three-day old, and the presynaptic terminals — called ‘boutons’ — formed by these neurons in the calyx of the mushroom body were imaged. Scale bar is 15 μm. (B-G) The number of neurons photo-labeled (B), the number of primary branches (C) and forks (D) formed by the photo-labeled neurons, as well as the total and average branch length (E, F) was quantified in Orco^+/+ (green; DL4: n = 10; VA2: n = 10; DP1m: n = 10) and Orco^−/− flies (blue; DL4: n = 10; VA2: n = 10; DP1m: n = 9); the total bouton volume was quantified (G); the long black bars represent the median whereas the short black bars represent the 25th and 75th percentile of the data. The statistical significance, or ‘p-value’, was measured using the Mann-Whitney U test; the asterisks indicate values that were statistically different (*: p < 0.05 and **: p < 0.01). See also Figure S2.

Figure 4.. Kenyon cells are morphologically similar in Orco^+/+ and Orco^−/− flies.

(A) Individual α/β (left), α’/β’ (middle), and γ Kenyon cells (right) were photo-labeled in Orco^+/+ (top) and Orco^−/− (bottom) female flies that were two- or three-day old, and the post-synaptic terminals formed by these neurons in the mushroom body calyx — called ‘claws’ — were imaged. Scale bar is 15 μm. (B-E) The total and average branch length formed by a Kenyon cell was measured (B,C), the number of claws formed by a Kenyon cells was counted (D), and the average length of a claw was measured (E) in Orco^+/+ (green) and Orco^−/− flies (blue); the long black bars represent the median whereas the short black bars represent the 25th and 75th percentile of the data. The statistical significance, or ‘p-value’, was measured using the Mann-Whitney U test; the asterisk indicates values that were statistically different (p < 0.05). See also Figure S3.

Figure 5.. Connection frequencies are similar in Orco^+/+ and Orco^−/− flies.

(A) A total of 887 and 899 connections between projection neurons and Kenyon cells were mapped in Orco^+/+ and Orco^−/− female flies that were two- or three-day old; all connections are reported in two connectivity matrices (Orco^+/+: left panel and green; Orco^−/−: right panel and blue). In each matrix, a row corresponds to a Kenyon cell (250 Kenyon cells per matrix) and each column corresponds to one of the 51 types of projection neuron; each colored bar indicates the input connections of a given Kenyon cell, and the color indicates the number of connections found between a particular Kenyon cell and a given type of projection neuron (blue/green: one connection; red: two connections; black: three connections). The bar graphs above the matrices represent the frequencies at which a particular type of projection neuron connects to Kenyon cells. (B) The frequencies at which different types of projection neuron connect to Kenyon cells in both data sets is shown (green: Orco^+/+; blue: Orco^−/−). Projection neurons are identified based on the glomeruli they innervate: ‘OR glomeruli’ refers to the projection neurons innervating glomeruli that receive input from Odorant Receptors-expressing neurons; ‘IR/GR glomeruli’ refers to projection neurons innervating glomeruli that receive input from Ionotropic Receptors/Gustatory Receptors-expressing neurons. The frequencies of connections measured for a given type of projection neuron in both genotypes are linked with a black line. (C) The p-value measured for each glomerulus was plotted against the ratio of frequencies (ratio = frequency of connections in Orco^−/− / frequency of connections in Orco^+/+) measured for each glomerulus (pink: projection neuron(s) receiving input from an OR glomerulus, dark gray: projection neurons receiving input from an IR or GR glomerulus; light gray: unknown). The statistical significance, or ‘p-value’, measured for each glomerulus was measured using the Fischer’s exact test; to control for false positives, p-values were adjusted with a false discovery rate using a Benjamini-Hochberg procedure. A ratio of 1 indicates that there is no shift in frequencies between the Orco^+/+ and Orco^−/− flies whereas a ratio smaller than 1 indicates that a given type of projection neuron connects more frequently in Orco^−/− and a ratio greater than 1 indicates that a given type of projection neuron connects more frequently in Orco^+/+.

Figure 6.. Distributions of connectivity frequencies.

The distributions of connectivity frequencies obtained in the experimental datasets — Orco^+/+ (green) and Orco^−/− (blue) — as well as the connectivity frequencies reported in the hemibrain connectome (gray) were plotted and compared across all Kenyon cells (top), α/β Kenyon cells (middle top), α’/β’ Kenyon cells (middle bottom) and γ Kenyon cells (bottom). The statistical significance, or ‘p-value’ measured for each glomerulus was measured using the Fischer’s exact test; none of the values were statistically different across the Orco^+/+ and Orco^−/− data sets (p < 0.01). See also Table S2-S5.

Figure 7.. Mushroom body input connectivity is globally similar in Orco^+/+ and Orco^−/− flies.

(A) The Jensen-Shannon distances were measured between the experimental matrices, their shuffle version as well as their fixed-shuffle version. The color bar denotes the range in the distances measured. (B) Principal components were extracted using either the Orco^+/+ (left panel, green) or the Orco^−/− (right panel, blue) connectivity matrices — using the experimental and fixed-shuffle versions — and the fraction of the variance explained by each component was measured (dark circles: experimental matrices, light circles: fixed-shuffle versions); error bars represent 95% confidence interval. See also Figure S4.