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Muscarinic ACh Receptors Contribute to Aversive Olfactory Learning in Drosophila.

Silva B, Molina-Fernández C, Ugalde MB, Tognarelli EI, Angel C, Campusano JM - Neural Plast. (2015)

Bottom Line: Our results show that pharmacological and genetic blockade of mAChRs in MB disrupts olfactory aversive memory in larvae.This effect is not explained by an alteration in the ability of animals to respond to odorants or to execute motor programs.These results show that mAChRs in MB contribute to generating olfactory memories in Drosophila.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio Neurogenética de la Conducta, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda 340, 8331150 Santiago, Chile.

ABSTRACT
The most studied form of associative learning in Drosophila consists in pairing an odorant, the conditioned stimulus (CS), with an unconditioned stimulus (US). The timely arrival of the CS and US information to a specific Drosophila brain association region, the mushroom bodies (MB), can induce new olfactory memories. Thus, the MB is considered a coincidence detector. It has been shown that olfactory information is conveyed to the MB through cholinergic inputs that activate acetylcholine (ACh) receptors, while the US is encoded by biogenic amine (BA) systems. In recent years, we have advanced our understanding on the specific neural BA pathways and receptors involved in olfactory learning and memory. However, little information exists on the contribution of cholinergic receptors to this process. Here we evaluate for the first time the proposition that, as in mammals, muscarinic ACh receptors (mAChRs) contribute to memory formation in Drosophila. Our results show that pharmacological and genetic blockade of mAChRs in MB disrupts olfactory aversive memory in larvae. This effect is not explained by an alteration in the ability of animals to respond to odorants or to execute motor programs. These results show that mAChRs in MB contribute to generating olfactory memories in Drosophila.

No MeSH data available.


Genetic and pharmacological blockade of mAChRs disrupt aversive olfactory memory. (a) Flies exposed to the mAChR antagonist atropine (100 μM, green bars) are not able to form the aversive olfactory memory as compared to control animals (blue bars). Each data presented was obtained from at least 16 different experiments, each one consisting of 15 or more larvae, so that the minimum amount of animals included in any data point was 317 larvae. ∗, ∗∗ indicate P < 0.05 and P < 0.01, as compared to data in control animals at the same time point (two-way ANOVA followed by Bonferroni multiple comparison post hoc test). ϕ indicates data different from zero in control animals (P < 0.05, Wilcoxon signed rank test). None of the values obtained in RNAi expressing animals was different from zero. (b) Atropine effect is dose-dependent: while no effect on memory is observed in flies exposed to an antagonist concentration of 10 nM, a strong reduction in aversive memory is observed at 100 μM. Partial reduction although with big variability is observed in flies exposed to 1 μM atropine. Values shown for memory performance correspond to data obtained 5 min after training, when training and memory test were carried out in presence of indicated concentrations of the drug. Each type of data was obtained from at least 10 experiments, each one including 15 or more larvae. The minimum amount of larvae in any data point was 151 animals. ∗ indicates P < 0.05 as compared to control (one-way ANOVA followed by Dunn's multiple comparison test). (c) Expression of an RNAi for mAChR (RNAimAChR) in MB disrupts olfactory memory formation. Each data presented was obtained from at least 15 different experiments, each one consisting of 15 or more larvae, so that the minimum amount of animals included in any data point shown was 299 larvae. ∗ indicates P < 0.05 as compared to data in genetic control animals at the same time point (two-way ANOVA followed by Bonferroni multiple comparison post hoc test). Genetic controls show values for performance index different from zero at 0, 5, and 15 min (P < 0.05, Wilcoxon signed rank test). None of the values obtained in RNAi expressing animals was different from zero.
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fig4: Genetic and pharmacological blockade of mAChRs disrupt aversive olfactory memory. (a) Flies exposed to the mAChR antagonist atropine (100 μM, green bars) are not able to form the aversive olfactory memory as compared to control animals (blue bars). Each data presented was obtained from at least 16 different experiments, each one consisting of 15 or more larvae, so that the minimum amount of animals included in any data point was 317 larvae. ∗, ∗∗ indicate P < 0.05 and P < 0.01, as compared to data in control animals at the same time point (two-way ANOVA followed by Bonferroni multiple comparison post hoc test). ϕ indicates data different from zero in control animals (P < 0.05, Wilcoxon signed rank test). None of the values obtained in RNAi expressing animals was different from zero. (b) Atropine effect is dose-dependent: while no effect on memory is observed in flies exposed to an antagonist concentration of 10 nM, a strong reduction in aversive memory is observed at 100 μM. Partial reduction although with big variability is observed in flies exposed to 1 μM atropine. Values shown for memory performance correspond to data obtained 5 min after training, when training and memory test were carried out in presence of indicated concentrations of the drug. Each type of data was obtained from at least 10 experiments, each one including 15 or more larvae. The minimum amount of larvae in any data point was 151 animals. ∗ indicates P < 0.05 as compared to control (one-way ANOVA followed by Dunn's multiple comparison test). (c) Expression of an RNAi for mAChR (RNAimAChR) in MB disrupts olfactory memory formation. Each data presented was obtained from at least 15 different experiments, each one consisting of 15 or more larvae, so that the minimum amount of animals included in any data point shown was 299 larvae. ∗ indicates P < 0.05 as compared to data in genetic control animals at the same time point (two-way ANOVA followed by Bonferroni multiple comparison post hoc test). Genetic controls show values for performance index different from zero at 0, 5, and 15 min (P < 0.05, Wilcoxon signed rank test). None of the values obtained in RNAi expressing animals was different from zero.

Mentions: Two different approaches were used to evaluate the contribution of mAChRs to aversive olfactory memory: one pharmacological and one genetic. For the first one, Drosophila larvae were exposed to atropine (100 μM), a well-known mAChR antagonist, while being trained up to the memory assays. Data obtained show that this pharmacological treatment abolishes the ability of larvae to generate olfactory aversive memory (Figure 4(a)). On the other hand, our data show that the effect of atropine is dose-dependent (Figure 4(b)). Since treatment with 100 μM atropine does not affect the olfactory acuity of larvae (Figure S1), these results suggest that mAChRs contribute to the generation of olfactory aversive memory. It has been shown that, as in adult flies, larval MB contribute to the expression of motor programs [30, 33]. Thus, it is important to control for locomotion in animals treated with atropine. Experiments carried out in animals exposed to atropine 100 μM demonstrate that this manipulation does not affect motor output in these animals (larvae exposed to atropine covered 113.1 ± 14.4 mm versus 95.0 ± 8.5 mm in control animals, n = 12 and 10 larvae resp., t-test, P > 0.05). Thus, all these data support the proposition that mAChRs are contributing to the generation of an aversive olfactory memory in Drosophila larvae, but it does not address where mAChRs are acting to modulate this memory.


Muscarinic ACh Receptors Contribute to Aversive Olfactory Learning in Drosophila.

Silva B, Molina-Fernández C, Ugalde MB, Tognarelli EI, Angel C, Campusano JM - Neural Plast. (2015)

Genetic and pharmacological blockade of mAChRs disrupt aversive olfactory memory. (a) Flies exposed to the mAChR antagonist atropine (100 μM, green bars) are not able to form the aversive olfactory memory as compared to control animals (blue bars). Each data presented was obtained from at least 16 different experiments, each one consisting of 15 or more larvae, so that the minimum amount of animals included in any data point was 317 larvae. ∗, ∗∗ indicate P < 0.05 and P < 0.01, as compared to data in control animals at the same time point (two-way ANOVA followed by Bonferroni multiple comparison post hoc test). ϕ indicates data different from zero in control animals (P < 0.05, Wilcoxon signed rank test). None of the values obtained in RNAi expressing animals was different from zero. (b) Atropine effect is dose-dependent: while no effect on memory is observed in flies exposed to an antagonist concentration of 10 nM, a strong reduction in aversive memory is observed at 100 μM. Partial reduction although with big variability is observed in flies exposed to 1 μM atropine. Values shown for memory performance correspond to data obtained 5 min after training, when training and memory test were carried out in presence of indicated concentrations of the drug. Each type of data was obtained from at least 10 experiments, each one including 15 or more larvae. The minimum amount of larvae in any data point was 151 animals. ∗ indicates P < 0.05 as compared to control (one-way ANOVA followed by Dunn's multiple comparison test). (c) Expression of an RNAi for mAChR (RNAimAChR) in MB disrupts olfactory memory formation. Each data presented was obtained from at least 15 different experiments, each one consisting of 15 or more larvae, so that the minimum amount of animals included in any data point shown was 299 larvae. ∗ indicates P < 0.05 as compared to data in genetic control animals at the same time point (two-way ANOVA followed by Bonferroni multiple comparison post hoc test). Genetic controls show values for performance index different from zero at 0, 5, and 15 min (P < 0.05, Wilcoxon signed rank test). None of the values obtained in RNAi expressing animals was different from zero.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: Genetic and pharmacological blockade of mAChRs disrupt aversive olfactory memory. (a) Flies exposed to the mAChR antagonist atropine (100 μM, green bars) are not able to form the aversive olfactory memory as compared to control animals (blue bars). Each data presented was obtained from at least 16 different experiments, each one consisting of 15 or more larvae, so that the minimum amount of animals included in any data point was 317 larvae. ∗, ∗∗ indicate P < 0.05 and P < 0.01, as compared to data in control animals at the same time point (two-way ANOVA followed by Bonferroni multiple comparison post hoc test). ϕ indicates data different from zero in control animals (P < 0.05, Wilcoxon signed rank test). None of the values obtained in RNAi expressing animals was different from zero. (b) Atropine effect is dose-dependent: while no effect on memory is observed in flies exposed to an antagonist concentration of 10 nM, a strong reduction in aversive memory is observed at 100 μM. Partial reduction although with big variability is observed in flies exposed to 1 μM atropine. Values shown for memory performance correspond to data obtained 5 min after training, when training and memory test were carried out in presence of indicated concentrations of the drug. Each type of data was obtained from at least 10 experiments, each one including 15 or more larvae. The minimum amount of larvae in any data point was 151 animals. ∗ indicates P < 0.05 as compared to control (one-way ANOVA followed by Dunn's multiple comparison test). (c) Expression of an RNAi for mAChR (RNAimAChR) in MB disrupts olfactory memory formation. Each data presented was obtained from at least 15 different experiments, each one consisting of 15 or more larvae, so that the minimum amount of animals included in any data point shown was 299 larvae. ∗ indicates P < 0.05 as compared to data in genetic control animals at the same time point (two-way ANOVA followed by Bonferroni multiple comparison post hoc test). Genetic controls show values for performance index different from zero at 0, 5, and 15 min (P < 0.05, Wilcoxon signed rank test). None of the values obtained in RNAi expressing animals was different from zero.
Mentions: Two different approaches were used to evaluate the contribution of mAChRs to aversive olfactory memory: one pharmacological and one genetic. For the first one, Drosophila larvae were exposed to atropine (100 μM), a well-known mAChR antagonist, while being trained up to the memory assays. Data obtained show that this pharmacological treatment abolishes the ability of larvae to generate olfactory aversive memory (Figure 4(a)). On the other hand, our data show that the effect of atropine is dose-dependent (Figure 4(b)). Since treatment with 100 μM atropine does not affect the olfactory acuity of larvae (Figure S1), these results suggest that mAChRs contribute to the generation of olfactory aversive memory. It has been shown that, as in adult flies, larval MB contribute to the expression of motor programs [30, 33]. Thus, it is important to control for locomotion in animals treated with atropine. Experiments carried out in animals exposed to atropine 100 μM demonstrate that this manipulation does not affect motor output in these animals (larvae exposed to atropine covered 113.1 ± 14.4 mm versus 95.0 ± 8.5 mm in control animals, n = 12 and 10 larvae resp., t-test, P > 0.05). Thus, all these data support the proposition that mAChRs are contributing to the generation of an aversive olfactory memory in Drosophila larvae, but it does not address where mAChRs are acting to modulate this memory.

Bottom Line: Our results show that pharmacological and genetic blockade of mAChRs in MB disrupts olfactory aversive memory in larvae.This effect is not explained by an alteration in the ability of animals to respond to odorants or to execute motor programs.These results show that mAChRs in MB contribute to generating olfactory memories in Drosophila.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio Neurogenética de la Conducta, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda 340, 8331150 Santiago, Chile.

ABSTRACT
The most studied form of associative learning in Drosophila consists in pairing an odorant, the conditioned stimulus (CS), with an unconditioned stimulus (US). The timely arrival of the CS and US information to a specific Drosophila brain association region, the mushroom bodies (MB), can induce new olfactory memories. Thus, the MB is considered a coincidence detector. It has been shown that olfactory information is conveyed to the MB through cholinergic inputs that activate acetylcholine (ACh) receptors, while the US is encoded by biogenic amine (BA) systems. In recent years, we have advanced our understanding on the specific neural BA pathways and receptors involved in olfactory learning and memory. However, little information exists on the contribution of cholinergic receptors to this process. Here we evaluate for the first time the proposition that, as in mammals, muscarinic ACh receptors (mAChRs) contribute to memory formation in Drosophila. Our results show that pharmacological and genetic blockade of mAChRs in MB disrupts olfactory aversive memory in larvae. This effect is not explained by an alteration in the ability of animals to respond to odorants or to execute motor programs. These results show that mAChRs in MB contribute to generating olfactory memories in Drosophila.

No MeSH data available.