A temporally restricted function of the Dopamine receptor Dop1R2 during memory formation
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eLife assessment
The authors design and implement an elegant strategy to delete genomic sequences encoding the dopamine receptor dop1R2 from specific subsets of mushroom body neurons (ab, a'b' and gamma) and show that while none of these manipulations affect short term appetitive or aversive memory, loss of dop1R2 from ab or a'b' block the ability of flies to display measurable forms of longer forms of appetitive memory. These findings are valuable in confirming and/or moderating prior observations, with better genetic perturbation techniques and convincing data to support the authors' main conclusions.
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Abstract
Dopamine is a crucial neuromodulator, which is involved in many brain processes, including learning and the formation of memories. Dopamine acts through multiple receptors and controls an intricate signaling network to regulate different tasks. While the diverse functions of dopamine are intensely studied, the interplay and role of the distinct dopamine receptors to regulate different processes is less well understood. An interesting candidate is the dopamine receptor Dop1R2 (also known as Damb), as it could connect to different downstream pathways. Dop1R2 is reported to be involved in forgetting and memory maintenance, however, the circuits requiring the receptors are unknown. To study Dop1R2 and its role in specific spatial and temporal contexts, we generated a conditional knock-out line using the CRISPR-Cas9 technique. Two FRT sites were inserted, allowing flippase-mediated excision of the dopamine receptor in neurons of interest. To study the function of Dop1R2, we knocked it out conditionally in the Mushroom body of Drosophila melanogaster , a well-studied brain region for memory formation. We show that Dop1R2 is required for later memory forms but not for short-term memories for both aversive and appetitive memories. Moreover, Dop1R2 is specifically required in the the α/β-lobe and the α’/β’-lobe but not in the γ-lobe of the Mushroom body. Our findings show a spatially and temporally restricted role of Dop1R2 in the process of memory formation highlighting the differential requirement of receptors during distinct phases of learning.
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eLife assessment
The authors design and implement an elegant strategy to delete genomic sequences encoding the dopamine receptor dop1R2 from specific subsets of mushroom body neurons (ab, a'b' and gamma) and show that while none of these manipulations affect short term appetitive or aversive memory, loss of dop1R2 from ab or a'b' block the ability of flies to display measurable forms of longer forms of appetitive memory. These findings are valuable in confirming and/or moderating prior observations, with better genetic perturbation techniques and convincing data to support the authors' main conclusions.
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Reviewer #1 (Public Review):
Summary:
In this manuscript, the authors present a novel CRISPR/Cas9-based genetic tool for the dopamine receptor dop1R2. Based on the known function of the receptor in learning and memory, they tested the efficacy of the genetic tool by knocking out the receptor specifically in mushroom body neurons. The data suggest that dop1R2 is necessary for longer-lasting memories through its action on ⍺/ß and ⍺'/ß' neurons but is dispensable for short-term memory and thus in ɣ neurons. The experiments impressively demonstrate the value of such a genetic tool and illustrate the specific function of the receptor in subpopulations of KCs for longer-term memories. The data presented in this manuscript are significant.
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Reviewer #2 (Public Review):
Summary:
This manuscript examines the role of the dopamine receptor, Dop1R2, in memory formation. This receptor has complex roles in supporting different stages of memory, and the neural mechanisms for these functions are poorly understood. The authors are able to localize Dop1R2 function to the vertical lobes of the mushroom body, revealing a role in later (presumably middle-term) aversive and appetitive memory. In general, the experimental design is rigorous, and statistics are appropriately applied. While the manuscript provides a useful tool, it would be strengthened further by additional mechanistic studies that build on the rich literature examining the roles of dopamine signaling in memory formation. The claim that Dop1R2 is involved in memory formation is strongly supported by the data presented, and …
Reviewer #2 (Public Review):
Summary:
This manuscript examines the role of the dopamine receptor, Dop1R2, in memory formation. This receptor has complex roles in supporting different stages of memory, and the neural mechanisms for these functions are poorly understood. The authors are able to localize Dop1R2 function to the vertical lobes of the mushroom body, revealing a role in later (presumably middle-term) aversive and appetitive memory. In general, the experimental design is rigorous, and statistics are appropriately applied. While the manuscript provides a useful tool, it would be strengthened further by additional mechanistic studies that build on the rich literature examining the roles of dopamine signaling in memory formation. The claim that Dop1R2 is involved in memory formation is strongly supported by the data presented, and this manuscript adds to a growing literature revealing that dopamine is a critical regulator of olfactory memory. However, the manuscript does not necessarily extend much beyond our understanding of Dop1R2 in memory formation, and future work will be needed to fully characterize this reagent and define the role of Dop1R2 in memory.
Strengths:
(1) The FRT lines generated provide a novel tool for temporal and spatially precise manipulation of Dop1R2 function. This tool will be valuable to study the role of Dop1R2 in memory and other behaviors potentially regulated by this gene.
(2) Given the highly conserved role of Dop1R2 in memory and other processes, these findings have a high potential to translate to vertebrate species.
Weaknesses:
(1) The authors state Dop1R2 associates with two different G-proteins. It would be useful to know which one is mediating the loss of aversive and appetitive memory in Dop1R2 knockout flies.
(2) It would be interesting to examine 24hr aversive memory, in addition to 24hr appetitive memory.
(3) The manuscript would be strengthened by added functional analysis. What are the DANs that signal through Dop1R. How do these knockouts impact MBONs?
(4) Also in Figure 2, the lobe-specific knockouts might be moved to supplemental since there is no effect. Instead, consider moving the control sensory tests into the main figure.
(5) Can the single-cell atlas data be used to narrow down the cell types in the vertical lobes that express Dop1R2? Is it all or just a subset?
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Reviewer #3 (Public Review):
Summary:
Kaldun et al. investigated the role of Dopamine Receptor Dop1R2 in different types and stages of olfactory associative memory in Drosophila melanogaster. Dop1R2 is a type 1 Dopamine receptor that can act both through Gs-cAMP and Gq-ERCa2+ pathways. The authors first developed a very useful tool, where tissue-specific knock-out mutants can be generated, using Crispr/Cas9 technology in combination with the powerful Gal4/UAS gene-expression toolkit, very common in fruit flies.
They direct the K.O. mutation to intrinsic neurons of the main associative memory centre fly brain-the mushroom body (MB). There are three main types of MB-neurons, or Kenyon cells, according to their axonal projections: a/b; a'/b', and g neurons.Kaldun et al. found that flies lacking dop1R2 all over the MB displayed impaired …
Reviewer #3 (Public Review):
Summary:
Kaldun et al. investigated the role of Dopamine Receptor Dop1R2 in different types and stages of olfactory associative memory in Drosophila melanogaster. Dop1R2 is a type 1 Dopamine receptor that can act both through Gs-cAMP and Gq-ERCa2+ pathways. The authors first developed a very useful tool, where tissue-specific knock-out mutants can be generated, using Crispr/Cas9 technology in combination with the powerful Gal4/UAS gene-expression toolkit, very common in fruit flies.
They direct the K.O. mutation to intrinsic neurons of the main associative memory centre fly brain-the mushroom body (MB). There are three main types of MB-neurons, or Kenyon cells, according to their axonal projections: a/b; a'/b', and g neurons.Kaldun et al. found that flies lacking dop1R2 all over the MB displayed impaired appetitive middle-term (2h) and long-term (24h) memory, whereas appetitive short-term memory remained intact. Knocking-out dop1R2 in the three MB neuron subtypes also impaired middle-term, but not short-term, aversive memory.
These memory defects were recapitulated when the loss of the dop1R2 gene was restricted to either a/b or a'/b', but not when the loss of the gene was restricted to g neurons, showcasing a compartmentalized role of Dop1R2 in specific neuronal subtypes of the main memory centre of the fly brain for the expression of middle and long-term memories.
Strengths:
(1) The conclusions of this paper are very well supported by the data, and the authors systematically addressed the requirement of a very interesting type of dopamine receptor in both appetitive and aversive memories. These findings are important for the fields of learning and memory and dopaminergic neuromodulation among others. The evidence in the literature so far was generated in different labs, each using different tools (mutants, RNAi knockdowns driven in different developmental stages...), different time points (short, middle, and long-term memory), different types of memories (Anesthesia resistant, which is a type of protein synthesis independent consolidated memory; anesthesia sensitive, which is a type of protein synthesis-dependent consolidated memory; aversive memory; appetitive memory...) and different behavioral paradigms. A study like this one allows for direct comparison of the results, and generalized observations.
(2) Additionally, Kaldun and collaborators addressed the requirement of different types of Kenyon cells, that have been classically involved in different memory stages: g KCs for memory acquisition and a/b or a'/b' for later memory phases. This systematical approach has not been performed before.
(3) Importantly, the authors of this paper produced a tool to generate tissue-specific knock-out mutants of dop1R2. Although this is not the first time that the requirement of this gene in different memory phases has been studied, the tools used here represent the most sophisticated genetic approach to induce a loss of function phenotypes exclusively in MB neurons.
Weaknesses:
(1) Although the paper does have important strengths, the main weakness of this work is that the advancement in the field could be considered incremental: the main findings of the manuscript had been reported before by several groups, using tissue-specific conditional knockdowns through interference RNAi. The requirement of Dop1R2 in MB for middle-term and long-term memories has been shown both for appetitive (Musso et al 2015, Sun et al 2020) and aversive associations (Plaçais et al 2017).
(2) The approach used here to genetically modify memory neurons is not temporally restricted. Considering the role of dopamine in the correct development of the nervous system, one must consider the possible effects that this manipulation can have in the establishment of memory circuits. However, previous studies addressing this question restricted the manipulation of Dop1R2 expression to adulthood, leading to the same findings than the ones reported in this paper for both aversive and appetitive memories, which solidifies the findings of this paper.
(3) The authors state that they aim to resolve disparities of findings in the field regarding the specific role of Dop1R2 in memory, offering a potent tool to generate mutants and addressing systematically their effects on different types of memory. Their results support the role of this receptor in the expression of long-term memories, however in the experiments performed here do not address temporal resolution of the genetic manipulations that could bring light into the mechanisms of action of Dop1R2 in memory. Several hypotheses have been proposed, from stabilization of memory, effects on forgetting, or integration of sequences of events (sensory experiences and dopamine release).
Overall, the authors generated a very useful tool to study dopamine neuromodulation in any given circuit when used in combination with the powerful genetic toolkit available in Drosophila. The reports in this paper confirmed a previously described role of Dop1R2 in the expression of aversive and appetitive LTM and mapped these effects to two specific types of memory neurons in the fly brain, previously implicated in the expression and consolidation of long-term associative memories.
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