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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.


Establishing the conditions for larval aversive olfactory learning and memory. (a) Example of preference for AA over EA observed in larvae exposed to different ratios of EA/AA odorant dilutions. Odorant ratios induce a preference that is 60% or above, when the dilution of AA is set to 1 : 10 and the EA dilution is modified to obtain the indicated EA/AA dilution ratios (in black). On the other hand, when modifying the dilutions of both odorants to obtain an EA/AA ratio of 10 (in red), it leads to an equilibrated preference response (50% + 4.7%) that is different from the other dilutions shown. This data argues in favor of the idea that to control for responses to odorant dilutions is necessary for a balanced response of animals exposed to these stimuli. (b) Three training cycles induce a robust olfactory memory that lasts at least 30 min in larvae. Animals were subjected to a reciprocal training: larvae were exposed to one odorant in presence of salt and then to a second odorant that was not associated with salt. This training cycle was repeated two more times. Afterwards, animals were placed for 3 min in the test plate where the two odorants are present. The number of larvae in the conditioned and nonconditioned side of the chamber was recorded at different time points. Data show that control animals form an aversive memory, while two animals expressing a mutation for the cAMP signaling cascade (dunce1 and rut2080) do not. Each data presented (in a and b) was obtained from at least 10 different experiments, each one consisting of 15 or more larvae, so that the minimum amount of animals for any data point was 174 and 169 larvae in (a) and (b), respectively. ∗, ∗∗∗ indicate P < 0.05 and P < 0.001, as compared to data obtained 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 mutants are different from zero.
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fig2: Establishing the conditions for larval aversive olfactory learning and memory. (a) Example of preference for AA over EA observed in larvae exposed to different ratios of EA/AA odorant dilutions. Odorant ratios induce a preference that is 60% or above, when the dilution of AA is set to 1 : 10 and the EA dilution is modified to obtain the indicated EA/AA dilution ratios (in black). On the other hand, when modifying the dilutions of both odorants to obtain an EA/AA ratio of 10 (in red), it leads to an equilibrated preference response (50% + 4.7%) that is different from the other dilutions shown. This data argues in favor of the idea that to control for responses to odorant dilutions is necessary for a balanced response of animals exposed to these stimuli. (b) Three training cycles induce a robust olfactory memory that lasts at least 30 min in larvae. Animals were subjected to a reciprocal training: larvae were exposed to one odorant in presence of salt and then to a second odorant that was not associated with salt. This training cycle was repeated two more times. Afterwards, animals were placed for 3 min in the test plate where the two odorants are present. The number of larvae in the conditioned and nonconditioned side of the chamber was recorded at different time points. Data show that control animals form an aversive memory, while two animals expressing a mutation for the cAMP signaling cascade (dunce1 and rut2080) do not. Each data presented (in a and b) was obtained from at least 10 different experiments, each one consisting of 15 or more larvae, so that the minimum amount of animals for any data point was 174 and 169 larvae in (a) and (b), respectively. ∗, ∗∗∗ indicate P < 0.05 and P < 0.001, as compared to data obtained 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 mutants are different from zero.

Mentions: Drosophila larvae can be trained to avoid odors associated with different aversive stimuli, including electric shocks or chemicals such as quinine or salt. Reciprocal training using two different odorants diminishes the variability associated, among other factors, with the naïve preference expressed by an animal for one of the odorants. Figure 2(a) shows a typical behavioral response observed in control larvae when exposed to the two odorants EA and AA. Data expressed as preference when animals are exposed to different ratios of EA to AA dilutions show a median close to or above 60% for all experimental conditions (boxes in black) and a big variability. These data were obtained modifying only the dilution of EA while AA was used at a 1 : 10 dilution and reflects how important it is to control for the naïve response of larvae to odorants, as to find dilutions that lead to an equal distribution of animals when in presence of the two odorants. The last data shown (Figure 2(a), box in red) present the naïve response of larvae exposed to EA (1 : 10 dilution) and AA (1 : 100 dilution). In this condition, preference expressed by animals for odorants is 50 ± 4.7%. These are the odorant dilutions used in the rest of this work.


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)

Establishing the conditions for larval aversive olfactory learning and memory. (a) Example of preference for AA over EA observed in larvae exposed to different ratios of EA/AA odorant dilutions. Odorant ratios induce a preference that is 60% or above, when the dilution of AA is set to 1 : 10 and the EA dilution is modified to obtain the indicated EA/AA dilution ratios (in black). On the other hand, when modifying the dilutions of both odorants to obtain an EA/AA ratio of 10 (in red), it leads to an equilibrated preference response (50% + 4.7%) that is different from the other dilutions shown. This data argues in favor of the idea that to control for responses to odorant dilutions is necessary for a balanced response of animals exposed to these stimuli. (b) Three training cycles induce a robust olfactory memory that lasts at least 30 min in larvae. Animals were subjected to a reciprocal training: larvae were exposed to one odorant in presence of salt and then to a second odorant that was not associated with salt. This training cycle was repeated two more times. Afterwards, animals were placed for 3 min in the test plate where the two odorants are present. The number of larvae in the conditioned and nonconditioned side of the chamber was recorded at different time points. Data show that control animals form an aversive memory, while two animals expressing a mutation for the cAMP signaling cascade (dunce1 and rut2080) do not. Each data presented (in a and b) was obtained from at least 10 different experiments, each one consisting of 15 or more larvae, so that the minimum amount of animals for any data point was 174 and 169 larvae in (a) and (b), respectively. ∗, ∗∗∗ indicate P < 0.05 and P < 0.001, as compared to data obtained 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 mutants are different from zero.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: Establishing the conditions for larval aversive olfactory learning and memory. (a) Example of preference for AA over EA observed in larvae exposed to different ratios of EA/AA odorant dilutions. Odorant ratios induce a preference that is 60% or above, when the dilution of AA is set to 1 : 10 and the EA dilution is modified to obtain the indicated EA/AA dilution ratios (in black). On the other hand, when modifying the dilutions of both odorants to obtain an EA/AA ratio of 10 (in red), it leads to an equilibrated preference response (50% + 4.7%) that is different from the other dilutions shown. This data argues in favor of the idea that to control for responses to odorant dilutions is necessary for a balanced response of animals exposed to these stimuli. (b) Three training cycles induce a robust olfactory memory that lasts at least 30 min in larvae. Animals were subjected to a reciprocal training: larvae were exposed to one odorant in presence of salt and then to a second odorant that was not associated with salt. This training cycle was repeated two more times. Afterwards, animals were placed for 3 min in the test plate where the two odorants are present. The number of larvae in the conditioned and nonconditioned side of the chamber was recorded at different time points. Data show that control animals form an aversive memory, while two animals expressing a mutation for the cAMP signaling cascade (dunce1 and rut2080) do not. Each data presented (in a and b) was obtained from at least 10 different experiments, each one consisting of 15 or more larvae, so that the minimum amount of animals for any data point was 174 and 169 larvae in (a) and (b), respectively. ∗, ∗∗∗ indicate P < 0.05 and P < 0.001, as compared to data obtained 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 mutants are different from zero.
Mentions: Drosophila larvae can be trained to avoid odors associated with different aversive stimuli, including electric shocks or chemicals such as quinine or salt. Reciprocal training using two different odorants diminishes the variability associated, among other factors, with the naïve preference expressed by an animal for one of the odorants. Figure 2(a) shows a typical behavioral response observed in control larvae when exposed to the two odorants EA and AA. Data expressed as preference when animals are exposed to different ratios of EA to AA dilutions show a median close to or above 60% for all experimental conditions (boxes in black) and a big variability. These data were obtained modifying only the dilution of EA while AA was used at a 1 : 10 dilution and reflects how important it is to control for the naïve response of larvae to odorants, as to find dilutions that lead to an equal distribution of animals when in presence of the two odorants. The last data shown (Figure 2(a), box in red) present the naïve response of larvae exposed to EA (1 : 10 dilution) and AA (1 : 100 dilution). In this condition, preference expressed by animals for odorants is 50 ± 4.7%. These are the odorant dilutions used in the rest of this work.

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.