Imaging Glomerular Signaling of Unrestrained Olfactory Search in mice Isabelle Cullen, Nelly Nouboussi, Blake Holcomb, Dr. Morgan Brown, Reese Findley, and Dr. Matt Smear NEXT STEPS:: iGluSnFR GENE CONSTRUCT FUTURE DIRECTIONS:: TWO-PHOTON IMAGING AND FREELY ABSTRACT MOVING IMAGING Olfaction is vital for many crucial animal behaviors such as social interaction, GltI cpEGFP avoiding predators, and locating food. Our goal is to understand how an animal D. H. I. navigates toward the source of an odor. However, little is known about how odors are coded to inform olfactory searching behaviors. Air turbulence can cause odor distributions to be highly variable and unpredictable. Although we have previously characterized specific behavioral patterns in turbulent odor plumes, little is known about how odors are translated into movements. Our goal is to capture and understand the sensory input that informs these previously observed behaviors. We do this by injecting iGluSnFR, a fluorescent (Image from AddGene Blog) glutamate reporter, into the mitral cell layer of the olfactory bulb. This reporter tells D. Green Fluorescent Protein Construct attached to Glutamate Binding us how glutamate released from olfactory sensory neuron terminals influences Protein activity of mitral cells. iGluSnFR's fast kinetics allows us to observe and measure Presence of glutamate induces conformational changes in the glutamate glutamate levels as the mouse performs olfactory navigation. By revealing activity binding protein (Gltl), which enhances the fluorescent of the permutated in olfactory sensory neurons during olfactory navigation, this technique can tell us green fluorescent protein (cpEGFP) how odor informs the mouse's brain during active sampling. Following the development of this technique, we will image from iGluSnFR mice performing our E. H. Imaging iGluSnFR I. iGluSnFR in Vivo with olfactory search task to determine the neural computation that connects movement Mice are head-fixed and placed under Odor Presentation and sensation. Understanding how mice translate odor into behavior will inform our the laser. They are presented odors Fluorescent signals from head- understanding of active sensory sampling behaviors in humans. passively and changes in fixed B6 mouse injected with fluorescence can be tracked via iGluSnFR under two-photon ESTABLISHED OLFACTORY SEARCH TASK AND UNDERLYING Mathlab software microscopy 1. BEHAVIORAL MOTIFS USED TO LOCATE ODOR C. A. A . Olfactory Search Task Mice are presented with a two- choice olfactory task. Mice initiate trials by sticking their noise into initiation port and Figure 3. Reading and writing glomerular input move toward side with higher patterns. A. Schematic of the miniscope. B. An example iGluSnFR Expression in Mitral Cells of the Olfactory Bulb (OB) miniscope (provided by Davison lab) odor concentration. Nose, Image taken from Wachowaik Lab (University of Utah) In vivo expression of J. Miniscope Design and Future Directions head, and body movements iGluSnFR in OB. Change in fluorescent (ΔF) across presentation of three are monitored by camera Future experiments will record iGluSnFR in real odors: methyl tiglate, 2-hexanone, and ethyl butyrate (Moran, Eiting, above in real-time. Sniffing time through a miniscope mounted to the Wachowiak 2019) behavior is recorded by an mouse’s head. (Figures JA,JB) In addition, implanted thermistor. tracking will be done from underneath and in a iGluSnFR INJECTION PROCEDURE larger arena for more accurate tracking. (Figure JC) (Graphic by Teresa Findley) B. C. F. G. REFERENCES Pause Run Run Left Run Right Straight Ford, T. (8, March 2018). New Neuroscience Tool: The SF-iGluSnFr Glutamate Sensor. Retrieved August 19, 2019, from https://blog.addgene.org/new-neuroscience-tool-the-sf-iglusnfr-glutamate-sensor J.White, J., M.Brown, A., P.Lackey, E., & V.Sillitoe, R. (2016, January 14). An optimized surgical approach for obtaining 4. stable extracellular single-unit recordings from the cerebellum of head-fixed behaving mice. Retrieved from https://www.sciencedirect.com/science/article/pii/S0165027016000212?via=ihub. Moran, A. K., Eiting, T. P., & Wachowiak, M. (2019, January 01). Diverse dynamics of glutamatergic input underlie B. heterogeneous response patterns of olfactory bulb mitral and tufted cells in vivo. Retrieved August 19, 2019, from Veer Left Veer Right Turn Left Turn Right 3. https://www.biorxiv.org/content/10.1101/692574v1?rss=1 2. 1. ACKNOWLEDGEMENTS C. Behavioral Motifs across Trials F. Surgery Image of Viral G. Figure of Olfactory Bulb cell layers B. Behavioral Motifs All trials for two example mice Mentorship Injection 1)Glomeruli Thank you to Dr. Matt Smear, Teresa Findley, Morgan Brown, Jared Acosta-King, and Blake Holcomb for their amazing mentorship Heatmap of each behavioral motif that separated by correct side and sorted by Image adapted (White et al. 2016). and guidance this summer. 2)Periglomerular/Tufted Cell Layer appears frequently in our olfactory trial length. Each color represents a Techniques & InstrumentationInjection of GCamp7f into Left 3)Mitral Cell Layer McAfee et al. 2016 for sniff measurement technique; Lopes et al. 2015 for real-time Bonsai tracking software; Mathis et al. 2018 for search task averaged across multiple discrete behavioral motif. Behavioral offline DeepLabCut tracking programOlfactory Bulb. Image altered to 4)Interneurons structure is similar across different mice. Behavioral Assay Design & Experimental Collaborationmice. Data extracted using Deeplab show suture lines and injection Zachary Mainen, Avinash Singh, Marike Reimer, Sarah Stednitz Cut Data Collectionareas. Jennifer Lauren Cramer, Dorian Yeh, Eric Monasevitch, Robin Attey, Nelly Nouboussi