Social experience dependent modification of gene regulation and circuit function

Duke University, Durham NC

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Abstract

RESEARCH STRATEGY NIGMS administrative equipment supplement for Parent Award R01GM146010 (NOT-GM-22-017): PI: Pelin C. Volkan - Social experience-dependent modification of gene regulation and circuit function Animals modulate their social behaviors in response to both internal and GH CS♂ SH CS♂ environmental signals to increase fitness. The molecular and neural circuit-based mechanisms underlying behavioral regulation in response to sensory cues remain unclear. The parent award addresses the role of social experience in modulating gene Canton S ☿ w1118☿ expression, circuit function, and courtship behaviors in D. melanogaster. In D. melanogaster, FruitlessM (FruM) and DoublesexM (DsxM) are critical transcription factors that control male-specific innate and learned courtship behaviors, respectively [1-3]. Socially experience alters male courtship in both wild-type and fru mutants, as well as GH SH 1. GH SH Figure Males modifying fru transcription and chromatin state [4-7]. The awarded proposal monosexually grouped or CS w1118 identify social experience and pheromone signaling-dependent changes in 15 10 around fru and 2) dsx genes, and 3) determine their effects on downstream t 5 expression, circuit function, and behavioral outputs. 0 15 Scientific Justification for Requested Supplement Equipment: 10 5 All objectives of the parent award R01GM146010 necessitate quantitati 0 responses from P1 courtship command neurons in the central brain. To 0.00 0.25 0.50 0. 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 Courtship Index Courtship index Canton S ☿ w1118 ☿ 15is 10 a 5 e 0 w fl s i olated for 7 ssayed for co ther Canton-S female Iso a days before urtship with er” female or ed Canton GH “ Non-courter” “Court ie of 1 11 8 . l t - ber 15 C 1 0 u m S males court anton-S and w1 more with females. SH N 1 18 0 5 requesting funds to purchase the Bruker Ultima Investigator Plus Laser Scanning microscope with a Coherent AXON 920-2 TPC laser to support the parent award 5R01GM146010. Aligned with the goals of the parent proposal, we found that social isolation increases the activity of P1 courtship command neurons and courtship behaviors, AA B as well as altering the transcriptional and chromatin state of fru, dsx, and other neuromodulatory genes in the brain. To causally link the effects of socially regulated genes on courtship circuit activity, all aims depend on 2-photon imaging of courtship command neurons in genetic and functional manipulation of distinct genes and neurons within the C D courtship circuits. Bruker Ultima Investigator Plus Laser Scanning microscope with a Coherent AXON 920-2 TPC laser will allow the detection, resolution, and quantitative analysis of calcium dynamics of critical neurons in the courtship circuits, in response to different social contexts as Figure 2. A) P1 Courtship command neuron dendritic field for calcium recording. B) 2-photon microscopy set up for live well as genetic and functional manipulation. The analyses with calcium imaging. C) Live GCaMP imaging of P1 courtship this system will significantly improve the impact of all the aims command neurons show elevated calcium activity in single housed male brains compared to group housed male brains. of the funded proposal and are necessary for the success of Graph in D) shows quantification of GCaMP signal. the project. My team visited Nilay Yapici's laboratory at Cornell University to learn the 2-photon setup, male brain preparation, and live imaging. Initially, we utilized a Leica DIVE SP2 multiphoton system housed in the Duke Light Microscopy Core Facility, which is shared by multiple labs on campus. However, limited access to the microscope and the high associated cost make this unsustainable for essential optimization procedures and experiments of the required scale and duration. This presents a significant delay and financial challenge for successfully conducting and troubleshooting the necessary experiments within the current budget constraints of Count CS fru 440/lexA Or47b 2/3 Or67d GAL4 fru dsx FoxP Dsk CCKLR17D3 GH SH GH SH GH SH GH SH Figure 3. Transcriptional responses of of some courtship regulatory genes are shown for grouped (GH) and isolated (SH) male brains from wild type (CS), Or47b, Or67d and fru mutants. the parent grant. Acquiring the Ultima Investigator Plus Laser Scanning Microscope system with a Coherent AXON 920-2 TPC laser will overcome cost and access limitations. Additionally, it will enable computational synchronization of infrared videos capturing the male's detection of the female, triggering P1 GCaMP responses— a feature currently lacking in the Core Facility's Leica DIVE SP2 system. In summary, the purchase of the new system will significantly enhance our ability to record neural responses, overcoming crucial limitations and ensuring the successful completion of the project. A key goal of Aim 1 is to identify changes in fru gene regulation by social experience and pheromone signaling and its effects on courtship circuit function and behavioral outputs. Social isolation increases activity in P1 courtship command neurons in the central brain, resulting in heightened courtship behaviors toward females (Figs. 1 and 2). We found that both fru and dsx show a general upregulation in gene expression in socially isolated male brains (Fig. 3). However, single-cell RNAseq analysis from fru- positive neurons in the central brain reveals only a few clusters where fru expression is significantly influenced by social isolation (Fig. 4). To establish a causal link between fru regulation and neural activity, we plan to conduct knockdown or overexpression of fruM in neural clusters regulated by social experience and assess their effects on P1 neuron activity and courtship in grouped and isolated males. Our investigation into courtship behaviors in pheromone receptor mutants revealed the involvement of neural circuits activated by Or47b neurons in suppressing courtship after group housing (Fig. 5). To establish a causal link between Or47b neural circuits and the modulation of courtship with social experience, we aim to record GCaMP responses from P1 neurons in grouped and isolated wild-type and Or47b mutant male brains. We will also examine males with Figure 4ed. Dot plot showing GH/SH log2fold change of some either activated or silenced Or47b neurons. Given the crucial role of P1 neurons differentially expressed genes in in initiating courtship, live imaging of GCaMP responses is critical and requires different fru-positive neural clusters. Many of these genes have known unlimited access to a two-photon microscope system. The Ultima Investigator functions in modulating courtship. Plus Laser Scanning Microscope system with Coherent AXON 920-2 TPC laser, GO:0008049 (male courtship behaviors) provides the necessary equipment for this investigation. A key goal of Aim 2 is to identify changes in dsx gene regulation by social experience in wild type, fru and pheromone receptor mutants and its effects on courtship circuit function and behavioral outputs. Isolated fru mutants display no courtship, yet they learn to court when grouped with other Male-Female courtship flies [5]. Interestingly, fru-dsx double mutants do not exhibit social experience- dependent courtship learning [5]. We propose that social experience- dependent transcriptional changes in fru mutants influence P1 neural activity and courtship responses. Compared to wild-type males, fru mutants exhibit increased dsx expression in grouped males and decreased expression in isolation (Fig. 3). Additionally, we've identified socially regulated neuromodulatory genes in wild-type and fru mutant brains (Fig. 3 and 4). We GH SH GH SH GH SH are currently screening courtship in double mutants of fru-dsx, Or47b- fru, and CS Or47b 2/3 Or67d GAL4 Or67d-fru to pinpoint pheromone circuits driving differential transcriptional Figure 5. GH index in wild Courtship (CI) type (CS), Or47b and Or67d mutants responses of dsx to social context. To establish causal links between socially that are grouped (GH) and isolated (SH). regulated genes (e.g., dsx) and circuit responses, we will utilize the Ultima Investigator Plus Laser Scanning Microscope system to record P1 neuron GCaMP responses in grouped and isolated wild-type and fru mutant males, as well as fru mutants where candidate genes are genetically manipulated. A key goal of Aim 3 is to identify changes in transcriptional profiles in the central brain in response to social experience, pheromone signaling and FruM function, and their effect on courtship and courtship circuit activity. Our recent RNAseq experiments revealed differential expression in genes related to courtship regulation, synaptic plasticity, learning/memory, and neuromodulation in response to social experience and pheromone circuit activity (Figs. 3 and 4) [8]. To establish causal links between gene expression modulation and neural circuit activity regulated by social experience and pheromone circuit function, we plan to record GCaMP responses in P1 courtship command neurons through various genetic experiments. This will enable the establishment of causal links between genes transcriptionally regulated by social experience and pheromone circuits, shedding light on the modulation of circuit and behavioral function. These experiments necessitate the use of the Ultima Investigator Plus Laser Scanning Microscope system with Coherent AXON 920-2 TPC laser. Anticipated future costs and training with the requested equipment: The initial training cost is incorporated into the Bruker Ultima Investigator Plus Laser Scanning microscope system. Bruker will provide ongoing service for the scope at no cost, eliminating upkeep expenses. Any additional maintenance issues will be addressed using available discretionary funds and funds from the parent grant. Pka-C1 S S h hab D d o n p c 1R1 mGluR Rdl rut sb Dop1R2 Kl AGO1 FoxP fru cac