Postsynaptic plasticity of cholinergic synapses underlies the induction and expression of appetitive memories in Drosophila
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Evaluation Summary:
Synaptic plasticity can take place on the presynaptic and/or postsynaptic sites, and these two sites of plasticity are known to involve distinct mechanisms. Using a combined approach of physiology, Drosophila genetics, and behaviour, this study provides evidence that postsynaptic mechanisms underlie plasticity for olfactory learning. This complements the field knowledge that olfactory associative learning largely relies on the presynaptic mechanism in mushroom body neurons. The paper also emphasizes the similarities in learning and memory mechanisms between vertebrates and invertebrates.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
In vertebrates, several forms of memory-relevant synaptic plasticity involve postsynaptic rearrangements of glutamate receptors. In contrast, previous work indicates that Drosophila and other invertebrates store memories using presynaptic plasticity of cholinergic synapses. Here, we provide evidence for postsynaptic plasticity at cholinergic output synapses from the Drosophila mushroom bodies (MBs). We find that the nicotinic acetylcholine receptor (nAChR) subunit α5 is required within specific MB output neurons (MBONs) for appetitive memory induction, but is dispensable for aversive memories. In addition, nAChR α2 subunits mediate memory expression and likely functions downstream of α5 and the postsynaptic scaffold protein Dlg. We show that postsynaptic plasticity traces can be induced independently of the presynapse, and that in vivo dynamics of α2 nAChR subunits are changed both in the context of associative and non-associative memory formation, underlying different plasticity rules. Therefore, regardless of neurotransmitter identity, key principles of postsynaptic plasticity support memory storage across phyla.
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Author Response
Reviewer #3 (Public Review):
In invertebrates, learning-dependent plasticity was reported to take place predominantly in presynaptic neurons. In Drosophila appetitive olfactory learning, cholinergic synapses between presynaptic Kenyon cells and postsynaptic MBONs undergo behaviourally relevant associative plasticity, and it was shown to reside largely in Kenyon cell output sites. This study provided several lines of evidence for postsynaptic plasticity in MBONs. The authors nicely showed the requirement of Kenyon cell output during training, strongly suggesting that behaviourally relevant associative plasticity also resides downstream of Kenyon cell output. This is further supported by impaired appetitive memory by downregulating nAChR subunits (a2, a5) and scaffold protein Dlg in specific MBONs. Live imaging …
Author Response
Reviewer #3 (Public Review):
In invertebrates, learning-dependent plasticity was reported to take place predominantly in presynaptic neurons. In Drosophila appetitive olfactory learning, cholinergic synapses between presynaptic Kenyon cells and postsynaptic MBONs undergo behaviourally relevant associative plasticity, and it was shown to reside largely in Kenyon cell output sites. This study provided several lines of evidence for postsynaptic plasticity in MBONs. The authors nicely showed the requirement of Kenyon cell output during training, strongly suggesting that behaviourally relevant associative plasticity also resides downstream of Kenyon cell output. This is further supported by impaired appetitive memory by downregulating nAChR subunits (a2, a5) and scaffold protein Dlg in specific MBONs. Live imaging experiments demonstrated that the learning-dependent depression in M4-MBON was reduced upon knocking down the a2 nAChR subunit. Using in-vivo FRAP experiments, the authors showed recovery rates of nAChR-a2::GFP were altered by the co-application of olfactory stimulation and DA. All these lines of evidence point to the significance of nAChR subunits in MBONs for postsynaptic plasticity.
On the technical side, this study achieved a very high standard, such as the measurement of lowexpressed receptor mobility by in-vivo FRAP. The authors conducted a wide array of experiments for collecting data supporting postsynaptic mechanisms. The downside of this multitude is somewhat compromised coherence. To give an example, the authors duplicated many behaviour and imaging experiments in different MBONs for non-associative learning (Fig. 7 and 8), which is primarily out of the scope of this paper (cf. title).
We thank the reviewer for their positive assessment and constructive criticism. We have thought a lot about removing data on non-associative learning (Fig. 7 and 8.), however feel that they do add important experiments that are not feasible to address for the other MBONs due to technical constraints (complexity of training protocols and localization of imaging area). We also decided, as reviewer 1 was happy with these experiments, that it is important to show that the receptor plasticity is not confined to associative appetitive memory but also is important for other postsynaptic memory storage mechanisms. As a response to this reviewer, we have adjusted the title to:
Postsynaptic plasticity of cholinergic synapses underlies the induction and expression of appetitive and familiarity memories in Drosophila
We also now include familiarity learning in the abstract. Moreover, we now expanded our explanation on to why we conduct these additional experiments and now state:
line 436ff: ‘Our data so far suggest that regulation of α2 subunits downstream of α5 are involved in postsynaptic plasticity mechanisms underlying appetitive, but not aversive memory storage. Besides associative memories, non-associative memories, such as familiarity learning, a form of habituation, are also stored at the level of Drosophila MBs. We next asked whether postsynaptic plasticity expressed through α5 and α2 subunit interplay, was exclusive to appetitive memory storage, or would represent a more generalizable mechanism that could underlie other forms of learning represented in the MBs. We turned to the α’3 compartment at the tip of the vertical MB lobe that has previously been shown to mediate odor familiarity learning. This form of learning allows the animal to adapt its behavioral responses to new odors and permits for assaying direct odor-related plasticity at the level of a higher order integration center. Importantly, this compartment follows different plasticity rules, because the odor serves as both the conditioned (activating KCs) and unconditioned stimulus (activating corresponding dopaminergic neurons)15. While allowing us to test whether the so far uncovered principles could also be relevant in a different context, it also provides a less complex test bed to further investigate whether α5 functions upstream of α2 dynamics.’
We also would like to emphasize that - if the reviewer feels that keeping these data / this information as part of our manuscript would prevent publication - we are prepared to remove these data from the manuscript, and submit these data in their own right (potentially as a research advance subsequently).
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Evaluation Summary:
Synaptic plasticity can take place on the presynaptic and/or postsynaptic sites, and these two sites of plasticity are known to involve distinct mechanisms. Using a combined approach of physiology, Drosophila genetics, and behaviour, this study provides evidence that postsynaptic mechanisms underlie plasticity for olfactory learning. This complements the field knowledge that olfactory associative learning largely relies on the presynaptic mechanism in mushroom body neurons. The paper also emphasizes the similarities in learning and memory mechanisms between vertebrates and invertebrates.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the …
Evaluation Summary:
Synaptic plasticity can take place on the presynaptic and/or postsynaptic sites, and these two sites of plasticity are known to involve distinct mechanisms. Using a combined approach of physiology, Drosophila genetics, and behaviour, this study provides evidence that postsynaptic mechanisms underlie plasticity for olfactory learning. This complements the field knowledge that olfactory associative learning largely relies on the presynaptic mechanism in mushroom body neurons. The paper also emphasizes the similarities in learning and memory mechanisms between vertebrates and invertebrates.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Neural circuits of the fruit fly mushroom body provide an interesting system to study molecular processes underlying learning and formation of memories since the input-output relationship of the circuit is quite well characterized and - importantly- genetic tools to manipulate specific circuit components are available. The current manuscript focuses on the role of different subunits of the nicotinergic Acetylcholine receptors.
The authors use a series of state-of-the-art techniques and several elegant - and partly innovative, explorative - approaches to address a specific set of neurons as models (the MBONs M4/M6), that are relevant for appetitive memories. The logic of the manuscript is overall well developed, and the experiments provided are widely in support of the model the authors propose. They first …
Reviewer #1 (Public Review):
Neural circuits of the fruit fly mushroom body provide an interesting system to study molecular processes underlying learning and formation of memories since the input-output relationship of the circuit is quite well characterized and - importantly- genetic tools to manipulate specific circuit components are available. The current manuscript focuses on the role of different subunits of the nicotinergic Acetylcholine receptors.
The authors use a series of state-of-the-art techniques and several elegant - and partly innovative, explorative - approaches to address a specific set of neurons as models (the MBONs M4/M6), that are relevant for appetitive memories. The logic of the manuscript is overall well developed, and the experiments provided are widely in support of the model the authors propose. They first corroborate that synaptic transmission of M4/M6 is critical for appetitive memories and subsequently test different subunits of the nicotinergic Acetylcholine receptors using RNAi. Interestingly, only the alpha-5 subunit shows learning defects (acquisition or induction), while alpha-1,2, and 5 (and Dlg) show defects after 3 hours (what the authors call "memory expression"). This result indicated the differential requirement for acquisition versus that expression.
The subsequent and particularly elegant and intriguing set of experiments aims to recapitulate the processes while circumventing a direct synaptic transmission from the KCs. The authors therefore optogenetically activate DANs, while blocking KCs neurotransmitter release and measure neuronal activity in the MBONs. Only when ACh is locally applied an effect of synaptic facilitation can be observed. While this experiment is not particularly critical in the context of the current manuscript it provides a very different, complementary support for the proposed overall model.
In order to gain insight into the function of the alpha-5 and alpha-2 subunits, the authors next investigated the expression of these genes and report non-uniform patterns between the lobe systems, presumably by using GFP fusion reporters. A weakness in this section is that the technical details are not well described and thus the impact of these results remains a bit elusive. The results indicate that alpha-2 is somehow genetically "downstream" of alpha-5 and Dlg. While many parts of the manuscript are of great impact and clear, this notion - even though extremely interesting - may actually be one of the weakest parts, since no explanation for the phenomenon is provided. One would assume that dopamine signaling and coincidence detection may be involved. It is however true that the authors conceptually take this up to some degree in the discussion, particularly the point that alpha-2 may be a molecular central point to switch.
Using Calcium imaging in vivo the authors show that the physiological formation of a memory trace in M4/6 shows the expected dynamics in an alpha-2-dependent fashion and similarly that alpha-2 itself (by photobleaching) shows the expected expression/localization dynamics.
The final section is indeed an important extension and addresses the generality of the alpha-5 to alpha-2 transition by investigating familiarity rather than associative learning. The results provided are in line that this mechanism appears to be general, a point also taken up in the discussion. What I felt was especially refreshing in the discussion section is the global comparison of NMDA/AMPA as a concept and possibilities of how this task may be resolved in other systems using other transmitters, again maybe not at the molecular depth that may have added an explorative touch.
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Reviewer #2 (Public Review):
This paper investigates memory mechanisms in Drosophila adults using behavioral, optogenetic, molecular, and biophysical methods. The authors identify nAChR subunits important in this process in post-synaptic MB output neurons (MBONs) and show that the alpha 5 subunit is important for appetitive induction but not aversive learning. They also show that the alpha 2 subunit is needed for the subsequent step of memory expression. Using FRAP analysis, the dynamic of these receptors is investigated in post-synaptic neurons in the MB. They speculate on the functional similarities between acetylcholine and glutamatergic signaling during memory formation in vertebrates and invertebrates, with emphasis on pre versus post-synaptic contributions.
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Reviewer #3 (Public Review):
In invertebrates, learning-dependent plasticity was reported to take place predominantly in presynaptic neurons. In Drosophila appetitive olfactory learning, cholinergic synapses between presynaptic Kenyon cells and postsynaptic MBONs undergo behaviourally relevant associative plasticity, and it was shown to reside largely in Kenyon cell output sites. This study provided several lines of evidence for postsynaptic plasticity in MBONs. The authors nicely showed the requirement of Kenyon cell output during training, strongly suggesting that behaviourally relevant associative plasticity also resides downstream of Kenyon cell output. This is further supported by impaired appetitive memory by downregulating nAChR subunits (a2, a5) and scaffold protein Dlg in specific MBONs. Live imaging experiments demonstrated …
Reviewer #3 (Public Review):
In invertebrates, learning-dependent plasticity was reported to take place predominantly in presynaptic neurons. In Drosophila appetitive olfactory learning, cholinergic synapses between presynaptic Kenyon cells and postsynaptic MBONs undergo behaviourally relevant associative plasticity, and it was shown to reside largely in Kenyon cell output sites. This study provided several lines of evidence for postsynaptic plasticity in MBONs. The authors nicely showed the requirement of Kenyon cell output during training, strongly suggesting that behaviourally relevant associative plasticity also resides downstream of Kenyon cell output. This is further supported by impaired appetitive memory by downregulating nAChR subunits (a2, a5) and scaffold protein Dlg in specific MBONs. Live imaging experiments demonstrated that the learning-dependent depression in M4-MBON was reduced upon knocking down the a2 nAChR subunit. Using in-vivo FRAP experiments, the authors showed recovery rates of nAChR-a2::GFP were altered by the co-application of olfactory stimulation and DA. All these lines of evidence point to the significance of nAChR subunits in MBONs for postsynaptic plasticity.
On the technical side, this study achieved a very high standard, such as the measurement of low-expressed receptor mobility by in-vivo FRAP. The authors conducted a wide array of experiments for collecting data supporting postsynaptic mechanisms. The downside of this multitude is somewhat compromised coherence. To give an example, the authors duplicated many behaviour and imaging experiments in different MBONs for non-associative learning (Fig. 7 and 8), which is primarily out of the scope of this paper (cf. title).
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