# Sensory Processing to Sequential Action Control Durafshan Sakeena Syed ## Module 3 Instructor Guide — Sensory Processing to Sequential Action Control ### Conceptual Framework - Sensory-driven activation – mechanosensory input triggers grooming behavior - Command-like neuron activation – specific neural populations bias or initiate motor programs - Hierarchical suppression – competing actions are organized into structured sequences Students investigate how these levels interact to produce ordered, flexible grooming behavior in *Drosophila melanogaster*. ### Pedagogical Approach - Observation – record natural grooming behavior - Quantification – annotate and measure sequences - Manipulation – test causal roles using optogenetics - Interpretation – relate behavior to neural circuits ### Learning Objectives - Describe hierarchical organization of motor behavior - Quantify behavioral sequences using time sampling - Compare sensory-driven vs circuit-driven activation - Interpret behavioral data in neural circuit context - Evaluate selection and suppression of competing motor programs ### Suggested Teaching Timeline (2–3 hours) - 20 min: Introduction & Background - 40 min: Data Collection (Dusting & Optogenetics) - ~60 min: Data Analysis (Scoring & Graphing) - 30 min: Discussion & Interpretation ### Experimental Overview #### Conditions - Dust stimulation – natural mechanosensory activation - Optogenetic activation – artificial activation of neurons #### Key Experimental Question - How do sensory input and specific neurons influence grooming sequence, timing, and selection? #### Expected Outcomes - Grooming follows an anterior → posterior progression - Head grooming often occurs first - Behavioral sequences are structured, not random #### Optogenetic activation can: - Induce specific actions in clean flies - Override ongoing grooming sequences - Alter timing and transition probabilities Variability across flies is expected and can be used to discuss biological variability and experimental noise. ## Data Analysis Options - **Level 1 (Introductory):** manual sampling, % time, bar graphs - **Level 2 (Intermediate):** frame scoring, ethograms, transition analysis - **Level 3 (Advanced):** MATLAB/Python, transition matrices, automated classification Optional statistics: - Mann–Whitney U test (between groups) - Paired t-test (within-fly comparisons) ## Common Pitfalls & Troubleshooting - Weak optogenetic activation → check retinal feeding and light conditions - Scoring inconsistencies → enforce “dominant behavior per second” rule - High variability → expected; use as discussion point ## Key Conceptual Takeaways - Grooming is hierarchical, not a simple reflex - Sensory input biases but does not fully determine behavior - Command neurons can initiate and dominate actions - Sequences emerge from competition and suppression - Motor control is dynamic and flexible ## Preparation Genetic crosses and fly husbandry (~15 days before the experiment) ### Fly stocks: - Obtain fly stocks from [Bloomington Drosophila Stock Center](https://flystocks.bio.indiana.edu/index.html). - Keep different fly lines in separate vials/bottles. For optogenetic (or thermogenic) experiments: Use flies carrying UAS-CsChrimson (for optogenetics) or UAS-TrpA1 (for thermogenetics). These need to be crossed with GAL4 or Split-GAL4 driver lines. ### Virgin Collection (for setting up crosses): 1. Identify virgins directly: Collect newly eclosed females (wings still folded, no sex combs on legs). 2. Alternative method (for beginners): - Collect pupae and place a single pupa per vial. - After eclosion, identify males and females. - All females eclosed in individual vials will be virgins (since no males were present). - These vials may also contain unfertilized eggs laid by virgin females. > [!TIP] > > ### Tip > > The largest number of virgins will eclose in the morning [Drosophila Workers Unite!](https://marksteinlab.org/dwu/) is an excellent resource by Michele Markstein on how to do fly pushing/identifying gender differences/virgins and setting up the crosses. ### Setting Up Crosses: 1. Collect males from the desired GAL4 or Split-GAL4 driver lines. 2. Place 6 virgin females (from UAS-CsChrimson or UAS-TrpA1) and 3–6 males (from a given driver line) together in one vial with fly food. 3. Maintain at room temperature (or recommended growth temperature for your experiment). ## Transferring Parents Transfer parent flies into new food vials every 5 days. This prevents the parental generation from mixing with the first generation of progeny. > [!TIP] > > ### Tip > > To separate flies safely, you can immobilize/anesthetize them by > placing the vial on ice or in the refrigerator for ~1 minute. ### Collecting Experimental Flies Collect males from the first-generation progeny (correct genotype) for experiments. These flies will be ready for dusting or optogenetic/thermogenetic activation experiments. ### Fly stocks: | Fly Genotype | Source | Cat# | |:-------------------|:--------------------------------|:-----| | Iav GAL4 | Bloomington Stock Center (BDSC) | | | R38B08 GAL4 | BDSC | | | DNg12 | BDSC | | | DNg11 | BDSC | | | MagoNote | BDSC/Simpson lab | | | wPN | BDSC/Simpson lab | | | UASCsChrimson | BDSC | | | UAS TrpA1 | BDSC | | | AD-DBD-empty SPLIT | BDSC | | | Canton S | BDSC | | ### Preparing flies for experiment (~3 days before the experiment) Perform behavioral experiments on flies that are 3–6 days old after eclosion. Control Flies (Wild-Type or Non-Optogenetic): These can be used directly for dusting experiments without any special preparation. Experimental Flies (Optogenetic with CsChrimson): Flies carrying UAS-CsChrimson must be fed on retinal-containing food for 3 days prior to the experiment. Retinal is required to make the Chrimson channel sensitive to light activation.