Roles for Drosophila mushroom body neurons in olfactory learning and memory

Abstract

Olfactory learning assays in Drosophila have revealed that distinct brain structures known as mushroom bodies (MBs) are critical for the associative learning and memory of olfactory stimuli. However, the precise roles of the different neurons comprising the MBs are still under debate. The confusion surrounding the roles of the different neurons may be due, in part, to the use of different odors as conditioned stimuli in previous studies. We investigated the requirements for the different MB neurons, specifically the alpha/beta versus the gamma neurons, and whether olfactory learning is supported by different subsets of MB neurons irrespective of the odors used as conditioned stimuli. We expressed the rutabaga (rut)-encoded adenylyl cyclase in either the gamma or alpha/beta neurons and examined the effects on restoring olfactory associative learning and memory of rut mutant flies. We also expressed a temperature-sensitive shibire (shi) transgene in these neuron sets and examined the effects of disrupting synaptic vesicle recycling on Drosophila olfactory learning. Our results indicate that although we did not detect odor-pair-specific learning using GAL4 drivers that primarily express in gamma neurons, expression of the transgenes in a subset of alpha/beta neurons resulted in both odor-pair-specific rescue of the rut defect as well as odor-pair-specific disruption of learning using shi(ts1).

Figure 1.

Spatial expression patterns in the brains of flies carrying different GAL4 enhancer detector elements and a UAS-lacZ transposon. Frontal sections through the adult brain were stained for 18 h in a humidified chamber at 37°C for β-galactosidase activity and examined. Sections at the level of the mushroom body α/β and γ lobes are shown. (A,C) GAL4 lines 17d and c739 showed expression in the mushroom body α/β lobes, although the expression level of 17d-GAL4 was less because this line marks only a subset of α/β neurons when compared to c739-GAL4, which stained the α/β lobes more completely. (B,D) GAL4 lines NP1131 and H24 showed common expression in the γ lobes of the mushroom bodies with H24-GAL4 exhibiting robust expression in the antennal lobes (AL) as well. (E,F) Double GAL4 line c739; H24 showed expression in both the α/β lobes and the γ lobes.

Figure 2.

Restoring rut in γ MB neurons produces a partial rescue of the learning-deficiency of rut mutants for all odors tested. Three-minute memory for flies carrying GAL4 drivers that express in γ neurons. (A,B,C) Flies bearing H24-GAL4 in combination with the UAS-rut transgene demonstrated significant rescue of rut memory defect over the mutant control group rut²⁰⁸⁰; H24-GAL4 (P ≤ 0.0006), but were significantly impaired compared to control flies that were CS; H24-GAL4 for all odor combinations tested (P ≤ 0.0006). n = 18 per group except n = 12 for the MCH-BEN experiment. (D,E,F) Flies of the genotype rut²⁰⁸⁰; NP1131-GAL4; UAS-rut performed significantly better than control flies that were rut²⁰⁸⁰; NP1131-GAL4 (P ≤ 0.0049), but were significantly impaired compared to control flies that were CS; NP1131-GAL4 for all odor combinations tested (P ≤ 0.0001). n = 18 per group except n = 17 for MCH-BEN experiment. This suggests that driving rut in γ neurons results in partial rescue of the rut learning defect irrespective of odor combination used. In all experiments, the P.I.’s were subjected to a one-way ANOVA with genotype as the main effect, followed by post hoc analysis with the Bonferroni/Dunn test. Statistical significance is indicated by asterisks (**).

Figure 3.

Restoring rut in a subset of MB α/β neurons results in partial rescue of the learning-deficiency of rut mutants for two odor pairs. Three-minute memory rescue in lines carrying GAL4 drivers that express in α/β neurons. (A,B,C) Rut rescue with c739-GAL4 using different odor combinations. Flies bearing c739-GAL4 in combination with the UAS-rut transgene demonstrated no significant rescue of rut memory defect over the mutant control group rut²⁰⁸⁰; c739-GAL4 for odor combinations MCH-BEN (P = 0.0886, n = 12), MCH-OCT (P = 0.7554, n = 10), and OCT-BEN (P = 0.6218, n = 12). For all odor combinations tested, rut²⁰⁸⁰; c739-GAL4; UAS-rut flies performed significantly differently from control flies that were CS; c739-GAL4 (P < 0.0001), but were indistinguishable from control flies that were rut²⁰⁸⁰; c739-GAL4. (D–F) Rut rescue with 17d-GAL4, a line that expresses in a subset of α/β neurons, using different odor combinations. Flies bearing the GAL4 driver 17d in combination with the UAS-rut transgene demonstrated significant rescue of the rut memory defect over the mutant control group rut²⁰⁸⁰; 17d-GAL4 for odor combinations MCH-BEN (P < 0.0001, n = 24) and OCT-BEN (P = 0.0047, n = 12), but not MCH-OCT (P = 0.2577, n = 12). For all odor combinations tested, rut²⁰⁸⁰; 17d-GAL4; UAS-rut flies performed significantly more poorly than control flies that were CS; 17d-GAL4 (P < 0.0001). In all experiments, the P.I.’s were subjected to a one-way ANOVA with genotype as the main effect, followed by post hoc analysis with the Bonferroni/Dunn test. Statistical significance (**) or nonsignificance (N.S.) is indicated.

Figure 4.

Odor-pair-specific disruption of 3-min memory when MB signaling from α/β neurons is blocked. Three-minute memory performance of flies carrying UAS-shi^ts1 in combination with the GAL4 drivers c739 or 17d with training and testing performed under permissive (25°C) and restrictive (32°C) conditions. The different odor combinations used for the experiments are indicated above the graphs: MCH-BEN (A,D), MCH-OCT (B,E), and OCT-BEN (C,F). (A,B,C) Performance of flies of the genotype c739-GAL4; UAS-shi^ts1 were indistinguishable from controls w; c739-GAL4 and w; UAS-shi^ts1 at the permissive temperature for MCH-BEN (P ≥ 0.2820, n = 6), MCH-OCT (P ≥ 0.4691, n = 10), and OCT-BEN (P ≥ 0.2569, n = 6), but showed a significant disruption of memory at the restrictive temperature for MCH-BEN (P ≤ 0.0127, n = 6), MCH-OCT (P ≤ 0.0014, n = 6), and OCT-BEN (P ≤ 0.0002, n = 9). (D–F) Performance of flies of the genotype 17d-GAL4; UAS-shi^ts1 were indistinguishable from controls w; 17d-GAL4 and w; UAS-shi^ts1 at the permissive temperature for MCH-BEN (P ≥ 0.0981, n = 6), MCH-OCT (P ≥ 0.7679, n = 10), and OCT-BEN (P ≥ 0.0404, n = 13). Significant disruptions of memory were observed for 17d-GAL4; UAS-shi^ts1 flies at the restrictive temperature when compared to controls for MCH-BEN (P ≤ 0.0008) and OCT-BEN (P ≤ 0.0111) but not for MCH-OCT (P ≥ 0.2310, n = 10 for all). In all experiments, the P.I.’s were subjected to a one-way ANOVA with genotype as the main effect, followed by post hoc analysis with the Bonferroni/Dunn test. Comparisons among the three different genotypes were performed, producing three pairwise planned comparisons per experiment at each of the temperatures. Comparisons were not significant unless indicated by asterisks (**).

Figure 5.

Complete rescue of the rut learning defect for all odor combinations is achieved when rut is expressed in both α/β and γ neurons. (A,B,C) Flies of the genotype rut²⁰⁸⁰; c739-GAL4; H24-GAL4/UAS-rut exhibited significantly elevated performance scores compared to the mutant control group rut²⁰⁸⁰; c739-GAL4; H24-GAL4 for all odor combinations (P < 0.0001, n = 6 for all groups). In each case, the performance levels were indistinguishable from control flies of the genotype CS; c739-GAL4; H24-GAL4, indicating complete rescue of the rut learning defect (P ≥ 0.2853, n = 6 for all groups). In all experiments, the P.I.’s were subjected to a one-way ANOVA with genotype as the main effect, followed by post hoc analysis with the Bonferroni/Dunn test. Statistical significance (**) or nonsignificance (N.S.) is indicated.

Figure 6.

Neuron counts and GAL4 expression patterns of the different drivers at the level of the MB cell body region. (A) Expression patterns of 17d-GAL4, c739-GAL4, H24-GAL4, and NP1131-GAL4 in the MB cell body region visualized by crossing the drivers with UAS-GFP∷lacZ.nls 30.1 flies and doubly stained with anti-ELAV and anti-GFP. Whole-mount brains were examined under a confocal microscope, and serial sections were taken at 0.5-μm intervals. Shown are representative projection images of confocal Z-series that were generated using ImageJ (Abramoff et al. 2004). Brains from five flies per GAL4 line were examined with a representative MB cell body region from individual flies shown for each driver. (B) The number of neurons expressing the GFP reporter was counted after image segmentation and identification of neuronal nuclei (Ponomarev and Davis 2003). Neuron counts for 17d-GAL4 were less than half the number of GFP-positive neurons for c739-GAL4 flies (n = 10 Kenyon cell soma regions from five flies). Neuron counts for H24-GAL4 and NP1131-GAL4 were not significantly different from one another (P = 0.5407, n = 10 Kenyon cell soma regions from five flies). Brains for each group showed overall stereotypy and bilateral symmetry with no significant differences in neuron counts between the left and right hemispheres (P ≥ 0.4052, n = 5 for each line).