This research was supported by the US National Institutes of Health (NIH UF1 NS116241 and NIH R01 DC000566), and the National Science Foundation (NSF BCS-1926676). The authors thank Andrew Scallon for helping setting up the Odor Arena chamber.

Recording Neuronal Activity During Odor Plume Navigation with Miniscope Imaging

<ol>
	<li>Adoff, M. D., Climer, J. R., Davoudi, H., Marvin, J. S., Looger, L. L., Dombeck, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functional+organization+of+excitatory+synaptic+input+to+place+cells.">The functional organization of excitatory synaptic input to place cells.</a> Nat Commun. 12 (1), 3558 (2021).</li><li>Radvansky, B. A., Oh, J. Y., Climer, J. R., Dombeck, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior+determines+the+hippocampal+spatial+mapping+of+a+multisensory+environment.">Behavior determines the hippocampal spatial mapping of a multisensory environment.</a> Cell Rep. 36 (5), 109444 (2021).</li><li>Lewis, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plume+dynamics+structure+the+spatiotemporal+activity+of+mitral/tufted+cell+networks+in+the+mouse+olfactory+bulb.">Plume dynamics structure the spatiotemporal activity of mitral/tufted cell networks in the mouse olfactory bulb.</a> Front Cell Neurosci. 15, 633757 (2021).</li><li>Findley, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sniff-synchronized,+gradient-guided+olfactory+search+by+freely+moving+mice.">Sniff-synchronized, gradient-guided olfactory search by freely moving mice.</a> ELife. 10, e58523 (2021).</li><li>Gire, D. H., Kapoor, V., Arrighi-Allisan, A., Seminara, A., Murthy, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+develop+efficient+strategies+for+foraging+and+navigation+using+complex+natural+stimuli.">Mice develop efficient strategies for foraging and navigation using complex natural stimuli.</a> Curr Biol. 26 (10), 1261-1273 (2016).</li><li>Liu, A., Papale, A. E., Hengenius, J., Patel, K., Ermentrout, B., Urban, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+navigation+strategies+for+odor+source+localization.">Mouse navigation strategies for odor source localization.</a> Front Neurosci. 14, 218 (2020).</li><li>Tariq, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+head-mounted+ethanol+sensors+to+monitor+olfactory+information+and+determine+behavioral+changes+associated+with+ethanol-plume+contact+during+mouse+odor-guided+navigation.">Using head-mounted ethanol sensors to monitor olfactory information and determine behavioral changes associated with ethanol-plume contact during mouse odor-guided navigation.</a> eNeuro. 8 (1), (2021).</li><li>Rigolli, N., Reddy, G., Seminara, A., Vergassola, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternation+emerges+as+a+multi-modal+strategy+for+turbulent+odor+navigation.">Alternation emerges as a multi-modal strategy for turbulent odor navigation.</a> ELife. (11), e76989 (2022).</li><li>Ma, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+activity+of+CA1+hippocampal+cells+constitutes+a+temporal+memory+map+for+associative+learning+in+mice.">Sequential activity of CA1 hippocampal cells constitutes a temporal memory map for associative learning in mice.</a> Curr Biol. 34 (4), 841-854 (2024).</li><li>Aharoni, D., Khakh, B. S., Silva, A. J., Golshani, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All+the+light+that+we+can+see:+a+new+era+in+miniaturized+microscopy.">All the light that we can see: a new era in miniaturized microscopy.</a> Nat Methods. 16 (1), 11-13 (2019).</li><li>Aharoni, D., Hoogland, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circuit+investigations+with+open-source+miniaturized+microscopes:+Past,+present+and+future.">Circuit investigations with open-source miniaturized microscopes: Past, present and future.</a> Front Cell Neurosci. 13, 141 (2019).</li><li>Flusberg, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-speed,+miniaturized+fluorescence+microscopy+in+freely+moving+mice.">High-speed, miniaturized fluorescence microscopy in freely moving mice.</a> Nat Methods. 5 (11), 935-938 (2008).</li><li>Connor, E. G., McHugh, M. K., Crimaldi, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+airborne+odor+plumes+using+planar+laser-induced+fluorescence.">Quantification of airborne odor plumes using planar laser-induced fluorescence.</a> Exp Fluids. 59 (9), 137 (2018).</li><li>Gumaste, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comparison+between+mouse,+in+silico,+and+robot+odor+plume+navigation+reveals+advantages+of+mouse+odor+tracking.">A Comparison between mouse, in silico, and robot odor plume navigation reveals advantages of mouse odor tracking.</a> eNeuro. 7 (1), (2020).</li><li>Gumaste, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+discrimination+and+olfactory+bulb+encoding+of+odor+plume+intermittency.">Behavioral discrimination and olfactory bulb encoding of odor plume intermittency.</a> ELife. 13, e85303 (2024).</li><li>Marin, A. C., Schaefer, A. T., Ackels, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+information+from+the+odour+environment+in+mammalian+olfaction.">Spatial information from the odour environment in mammalian olfaction.</a> Cell Tissue Res. 383 (1), 473-483 (2021).</li><li>Eichenbaum, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+hippocampus+in+navigation+is+memory.">The role of the hippocampus in navigation is memory.</a> J Neurophysiol. 117 (4), 1785-1796 (2017).</li><li>Dombeck, D. A., Harvey, C. D., Tian, L., Looger, L. L., Tank, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+imaging+of+hippocampal+place+cells+at+cellular+resolution+during+virtual+navigation.">Functional imaging of hippocampal place cells at cellular resolution during virtual navigation.</a> Nat Neurosci. 13 (11), 1433-1440 (2010).</li><li>Wirtshafter, H. S., Disterhoft, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+multi-day+calcium+imaging+of+CA1+hippocampus+in+freely+moving+rats+reveals+a+high+preponderance+of+place+cells+with+consistent+place+fields.">In vivo multi-day calcium imaging of CA1 hippocampus in freely moving rats reveals a high preponderance of place cells with consistent place fields.</a> J Neurosci. 42 (22), 4538-4554 (2022).</li><li>Mizuta, K., Sato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphoton+imaging+of+hippocampal+neural+circuits:+techniques+and+biological+insights+into+region-,+cell-type-,+and+pathway-specific+functions.">Multiphoton imaging of hippocampal neural circuits: techniques and biological insights into region-, cell-type-, and pathway-specific functions.</a> Neurophotonics. 11 (3), 033406 (2024).</li><li>Radvansky, B. A., Dombeck, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+olfactory+virtual+reality+system+for+mice.">An olfactory virtual reality system for mice.</a> Nat Commun. 9 (1), 839 (2018).</li><li>Almeida-Santos, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+isolation+impairs+the+persistence+of+social+recognition+memory+by+disturbing+the+glutamatergic+tonus+and+the+olfactory+bulb-dorsal+hippocampus+coupling.">Social isolation impairs the persistence of social recognition memory by disturbing the glutamatergic tonus and the olfactory bulb-dorsal hippocampus coupling.</a> Sci Rep. 9 (1), 473 (2019).</li><li>Dana, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thy1-GCaMP6+transgenic+mice+for+neuronal+population+imaging+in+vivo.">Thy1-GCaMP6 transgenic mice for neuronal population imaging in vivo.</a> PLoS One. 9 (9), e108697 (2014).</li><li>Thapa, R., Liang, B., Liu, R., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotaxic+viral+injection+and+gradient-index+lens+implantation+for+deep+brain+in+vivo+calcium+imaging.">Stereotaxic viral injection and gradient-index lens implantation for deep brain in vivo calcium imaging.</a> J Vis Exp. (176), e63049 (2021).</li><li>Hsiao, Y. -. T., Wang, A. Y. -. C., Lee, T. -. Y., Chang, C. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+baseplating+and+a+miniscope+preanchored+with+an+objective+lens+for+calcium+transient+research+in+mice.">Using baseplating and a miniscope preanchored with an objective lens for calcium transient research in mice.</a> J Vis Exp. (172), e62611 (2021).</li><li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniscope+GRIN+lens+system+for+calcium+imaging+of+neuronal+activity+from+deep+brain+structures+in+behaving+animals.">Miniscope GRIN lens system for calcium imaging of neuronal activity from deep brain structures in behaving animals.</a> Curr Protoc Neurosci. 86 (1), e56 (2019).</li><li>Guo, D., G&#252;rkan &#214;zer, A., Uusisaari, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+calcium+imaging+in+mouse+inferior+olive.">In vivo calcium imaging in mouse inferior olive.</a> J Vis Exp. (172), e62222 (2021).</li><li>Yun, M., Shin, J., Jung, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+calcium+imaging+of+dorsal+and+ventral+CA1+neurons+in+head-fixed+mice.">Protocol for calcium imaging of dorsal and ventral CA1 neurons in head-fixed mice.</a> STAR Protoc. 4 (3), 102439 (2023).</li><li>Yun, M., Hwang, J. Y., Jung, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Septotemporal+variations+in+hippocampal+value+and+outcome+processing.">Septotemporal variations in hippocampal value and outcome processing.</a> Cell Rep. 42 (2), 112094 (2023).</li><li>Sheffield, M. E. J., Dombeck, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+transient+prevalence+across+the+dendritic+arbour+predicts+place+field+properties.">Calcium transient prevalence across the dendritic arbour predicts place field properties.</a> Nature. 517 (7533), 200-204 (2015).</li><li>Pnevmatikakis, E. A., Giovannucci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NoRMCorre:+An+online+algorithm+for+piecewise+rigid+motion+correction+of+calcium+imaging+data.">NoRMCorre: An online algorithm for piecewise rigid motion correction of calcium imaging data.</a> J Neurosci Methods. 291, 83-94 (2017).</li><li>Inan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+statistically+robust+cell+extraction+from+large-scale+neural+calcium+imaging+datasets.">Fast and statistically robust cell extraction from large-scale neural calcium imaging datasets.</a> bioRxiv. , (2021).</li><li>Segalin, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+action+recognition+system+(MARS)+software+pipeline+for+automated+analysis+of+social+behaviors+in+mice.">The mouse action recognition system (MARS) software pipeline for automated analysis of social behaviors in mice.</a> ELife. 10, e63720 (2021).</li><li>Chen, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hardware+system+for+real-time+decoding+of+in+vivo+calcium+imaging+data.">A hardware system for real-time decoding of in vivo calcium imaging data.</a> ELife. (12), e78344 (2023).</li><li>Xue, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-region+calcium+imaging+in+freely+behaving+mice+with+ultra-compact+head-mounted+fluorescence+microscopes.">Multi-region calcium imaging in freely behaving mice with ultra-compact head-mounted fluorescence microscopes.</a> Natl Sci Rev. 11 (1), nwad294 (2024).</li><li>Tabourin, L., Bretzner, F., Galstian, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+mini-endoscope+design+with+spatially+selective+excitation+and+imaging.">Towards a mini-endoscope design with spatially selective excitation and imaging.</a> Biomed Opt Express. 15 (3), 1750-1760 (2024).</li></ol>

The authors declare no conflict of interest.

This protocol meticulously outlines the steps to record place-cells and odor-responsive cells in the dCA1 area of the hippocampus of mice navigating an odor plume. The critical steps in the protocol include stereotaxic surgery, placement of the miniscope baseplate, construction of the odor area, checking the plume in the odor arena, behavioral training, miniscope recording of the freely moving mouse, data preprocessing, and data analysis. Additionally, the protocol explains the process of decoding the mouse trajectory fr...

Although significant progress has been made in understanding neural circuits involved in olfactory navigation in head-fixed mice1,2,3 and navigation strategies in freely moving mice4,5,6,7,8, the role of neural circuits in ethologically relevant freely moving navigation of turbulent odor plumes is still unknown. This article describes monitoring neural activity by imaging Ca2+ transients in cells expressing the genetically encoded calcium sensor GCaMP6f in Thy1-GCaMP6f mice9 to study whether sequential neural dynamics of dorsal CA1 stratum pyramidale neurons in the hippocampus (dCA1) plays a role in odorant plume navigation. The methods provide information on imaged GCaMP6f fluorescence through a miniature epifluorescence microscope focused through a GRIN lens on dCA110,11,12. The methods explain how to monitor simultaneously spatial navigation and dCA1 neuron GCaMP6f calcium transients in mice performing an odor-plume navigation task where they received a water reward when they reached the spout delivering an odorant into an odor arena with a background laminar air flow13,14. This article describes the methods required to achieve this task (Figure 1), including the stereotaxic surgery for the implantation of gradient-index (GRIN) lenses, the placement of a baseplate to secure the miniscope to the skull in a freely moving mouse, imaging with the miniature microscope and monitoring mouse movement with a high-speed digital camera, data preprocessing for removing motion artifacts and finding the regions of interest (ROIs), and preparation of datasets and artificial neural network training and prediction for decoding the X and Y positions of the mouse in the odor arena from changes in fluorescence in dCA1 ROIs7.
Miniscope recording of calcium signals in the CA1 region of the hippocampus of mice navigating an odor plume is relevant for understanding the computation of neural circuits involved with olfaction and spatial information in the complex behavior task of odor-plume navigation2,14,15,16. The CA1 region of the hippocampus plays a role in spatial navigation and is crucial for creating a cognitive map of the environment for efficient navigation17,18. Recording calcium signals with a miniscope is a valuable way to investigate the CA1 neurons that encode spatial information during odor plume navigation.
This technique combines the advantages of miniscope technology for recording GCaMP calcium signals with the well-established role of the CA1 hippocampus in spatial navigation to understand better how neural circuits drive complex behaviors19. Alternatively, approaches using 2-photon microscopy can record CA1 neurons9,20, which requires a head-fixed mouse and restrains the possibility of freely moving to navigate an odor plume21. Local-field electrophysiological recordings of CA1 neurons allow the investigation of freely-moving mice navigating odor plumes22. Still, local field electrical signals impose limitations to estimating intracellular firing by isolating single-unit signals through spike sorting techniques. Miniscope signals allow the identification of ROIs associated directly with intracellular calcium signals in a reliable way10,11&#160;to investigate neural computations at single-cell resolution precisely. Miniscope technology provides a unique opportunity to better understand how the CA1 region encodes spatial information based on odor cues.
Furthermore, this technique investigates how specific neuronal populations process odor information for navigation and the relationship between neuronal activity patterns and decision-making during odor plume tracking. This method can contribute to a better understanding of how the brain processes odor and spatial information. While miniscopes offer a single-cell resolution for recording a freely moving mouse&#39;s brain, they require specialized surgery and data analysis expertise. In this paper, we provide a comprehensive protocol for helping researchers go through each step to investigate the neural mechanisms of odor-plume navigation.
The odor navigating task is a promising framework for studying neural coding and spatial odor cue memory in mice. The article&#39;s findings indicate that it is possible to decode the trajectory of the mouse navigating an odor plume based on neuronal ensemble calcium signals in dCA1. Understanding the role of dCA1 calcium signals in odor plume navigation is a crucial step to crack the neural circuit basis for odor-guided navigation in realistic environments13,14.

<table><tbody><tr><td>Arduino Micro</td><td>Arduino</td><td>Micro</td><td></td></tr><tr><td>Biocompatible Methacrylate Resin</td><td>Parkell</td><td>S380</td><td>C&amp;amp;B-Metabond Adhesive Luting Cement</td></tr><tr><td>Decoding Brain Signals Software</td><td>CU Anschutz</td><td></td><td>https://github.com/restrepd/drgMiniscope</td></tr><tr><td>Dental Drill</td><td>Osada</td><td>LHP-6</td><td>AZ210015</td></tr><tr><td>Dental Drill Box</td><td>Osada</td><td>XL-230</td><td>30000 rotations per minute</td></tr><tr><td>Digital stereotaxic instrument</td><td>Stoelting</td><td>51730D</td><td>Mouse Stereotaxic Instument, #51904 Digital Manipulator Arm, 3-Axes, Add-On, LEFT&amp;nbsp;</td></tr><tr><td>Drill Bit</td><td>FST Fine Science Tools</td><td>19007-05</td><td>Tip diameter 0.5 mm</td></tr><tr><td>Fast Digital Camera</td><td>Edmund Optics</td><td>BFS-U3-63S4C</td><td>FLIR Blackfly S</td></tr><tr><td>Focal Lens</td><td>Edmund Optics</td><td>C-Series</td><td>3.5 mm</td></tr><tr><td>GRIN lens</td><td>Inscopix</td><td>1050-004595</td><td>1 mm diameter and 4 mm length</td></tr><tr><td>GRIN lens Holder</td><td>UCLA</td><td></td><td>http://miniscope.org/index.php/Surgery_Protocol</td></tr><tr><td>Liquid Tissue Adhesive</td><td>3M</td><td>1469C</td><td>Vetbond Tissue Adhesive</td></tr><tr><td>Low-Flow Anesthesia System for Mice</td><td>Kent Scientific Corporation</td><td>SomnoSuite</td><td>https://www.kentscientific.com/products/somnosuite/</td></tr><tr><td>Low Toxicity Silicone Adhesive</td><td>WPI &amp;ndash; World Precision Instruments</td><td>Kwik-sil</td><td></td></tr><tr><td>miniPID Controller</td><td>ASI &amp;ndash; Aurora Scientific Inc.</td><td>Model 200B</td><td>Fast-Response Miniature Photo-Ionization Detector</td></tr><tr><td>Miniscope V4 Holder</td><td>UCLA</td><td>NA</td><td>https://github.com/Aharoni-Lab/Miniscope-v4/tree/master/Miniscope-v4-Holder</td></tr><tr><td>Miniscope V4</td><td>Labmaker</td><td>NA</td><td>https://www.labmaker.org/products/miniscope-v4</td></tr><tr><td>Miniscope Base Plate V2</td><td>Labmaker</td><td>NA</td><td>https://www.labmaker.org/products/miniscope-v4-base-plates-variant-2-pack-of-10</td></tr><tr><td>Miniscope DAQ-QT software</td><td>UCLA</td><td></td><td>https://github.com/Aharoni-Lab/Miniscope-DAQ-QT-Software/wiki</td></tr><tr><td>Motion Correction Software</td><td>CU Anschutz</td><td></td><td>https://github.com/restrepd/drgMiniscope</td></tr><tr><td>Odor Arena Hardware</td><td>Custom Made</td><td>3D Model</td><td>https://www.dropbox.com/scl/fo/lwtpqysnpzis32mhrx3cd/ADomsxyhxu42sqDmTBl2O6k?rlkey=b3l4809eradundt5l3iz0gq74&amp;amp;&lt;br/&gt;dl=0</td></tr><tr><td>Odor Arena Software</td><td>CUAnschutz</td><td></td><td>https://github.com/wryanw/odorarena</td></tr><tr><td>Odorant Isoamyl Acetate</td><td>Aldrich Chemical Co</td><td>06422AX</td><td>Diluted at 1% in odorless mineral oil</td></tr><tr><td>RHD USB Interface Board</td><td>Intan Technologies</td><td>C3100</td><td>Product discontinued. Alternatively use another equivalent board.</td></tr><tr><td>ROI Extraction Software</td><td>CU Anschutz</td><td></td><td>https://github.com/restrepd/drgMiniscope</td></tr><tr><td>Sutter Micromanipulator</td><td>Sutter Instrument Company</td><td>MP-285</td><td></td></tr><tr><td>Synchronization Software</td><td>CU Anschutz</td><td></td><td>https://github.com/fsimoesdesouza/Synchronization</td></tr><tr><td>Thy1-GCaMP6f mice</td><td>Jackson Laboratory</td><td>IMSR_JAX 028281</td><td>C57BL/6J-Tg(Thy1-GCaMP6f)GP5.12Dkim/J)</td></tr></tbody></table>

miniscope recording calcium signals at hippocampus of mice navigating an odor plume

Mice navigate an odor plume with a complex spatiotemporal structure in the dark to find the source of odorants. This article describes a protocol to monitor behavior and record Ca2+ transients in dorsal CA1 stratum pyramidale neurons in the hippocampus (dCA1) in mice navigating an odor plume in a 50 cm x 50 cm x 25 cm odor arena. An epifluorescence miniscope focused through a gradient-index (GRIN) lens imaged Ca2+ transients in dCA1 neurons expressing the calcium sensor GCaMP6f in Thy1-GCaMP6f mice. The paper describes the behavioral protocol to train the mice to perform this odor plume navigation task in an automated odor arena. The methods include a step-by-step procedure for the surgery for GRIN lens implantation and baseplate placement for imaging GCaMP6f in CA1. The article provides information on real-time tracking of the mouse position to automate the start of the trials and delivery of a water reward. In addition, the protocol includes information on using an interface board to synchronize metadata describing the automation of the odor navigation task and frame times for the miniscope and a digital camera tracking mouse position. Moreover, the methods delineate the pipeline used to process GCaMP6f fluorescence movies by motion correction using the NorMCorre algorithm followed by identification of regions of interest with EXTRACT. Finally, the paper describes an artificial neural network approach to decode spatial paths from CA1 neural ensemble activity to predict mouse navigation of the odor plume.

Using this procedure allows for visualizing and recording dCA1 GCaMP6f fluorescence transients in mice navigating the odor arena to find the source of odorants (Figure 6A,B, Supplementary Movie 1, and Supplementary Movie 2). The fluorescence images are motion-corrected with NoRMCorre, and EXTRACT is used to extract the ROIs. In addition, recording with an interface board allows for synchronization of the &#948;F/F0 signals from the ROIs with...

Author Spotlight: Understanding Processing of Olfactory and Spatial Information by Brain with Real-Time Behavioral Analysis

This protocol investigates the brain-behavior relationship in hippocampal CA1 in mice navigating an odor plume. We provide a step-by-step protocol, including surgery to access imaging of the hippocampus, behavioral training, miniscope GCaMP6f recording and processing of the brain, and behavioral data to decode the mouse position from ROI neural activity.

Miniscope Recording Calcium Signals at Hippocampus of Mice Navigating an Odor Plume

Mice navigate an odor plume with a complex spatiotemporal structure in the dark to find the source of odorants. This article describes a protocol to ...

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Research

JoVE Journal

Neuroscience

703 Views.  University of Colorado Anschutz Medical Campus. Mijn onderzoek is gericht op het begrijpen hoe de hersenen olfactorische en ruimtelijke informatie verwerken. En we proberen de rol van hippocampale CA1-neuronen bij de navigatie van geurpluimen te begrijpen. Momenteel wordt de technologie van vrij bewegende opnames van neuronen met miniatuurmicroscopen gebruikt om onderzoek in het veld vooruit te helpen.We ontdekten dat het mogelijk is om het traject van de muis te decoderen die door de geurpluim navi...

Video: Auteur Spotlight: Inzicht in de verwerking van olfactorische en ruimtelijke informatie door de hersenen met realtime gedragsanalyse

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical neurons with two-photon microscopy has provided unique insights into cortical structure, function and plasticity. However, these studies are limited to head fixed animals, greatly reducing the behavioral complexity available for study. In this paper, we describe a procedure for performing chronic fluorescence microscopy with cellular-resolution across multiple cortical layers in freely behaving mice. We used an integrated miniaturized fluorescence microscope paired with an implanted prism probe to simultaneously visualize and record the calcium dynamics of hundreds of neurons across multiple layers of the somatosensory cortex as the mouse engaged in a novel object exploration task, over several days. This technique can be adapted to other brain regions in different animal species for other behavioral paradigms.

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

Here, we present a procedure for performing large-scale Ca2+ imaging with cellular-resolution across multiple cortical layers in freely moving mice. Hundreds of active cells can be observed simultaneously using a miniature, head-mounted microscope coupled with an implanted prism probe.

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical ...

Behavior

Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from subcellular to network levels and at temporal scales from milliseconds to weeks. In addition, two-photon imaging can be combined with a variety of behavioral tasks to explore the causal relationships between brain function and behavior. However, in mammals, limited penetration and scattering of light have limited two-photon intravital imaging mostly to superficial brain regions, thus precluding longitudinal investigation of deep-brain areas such as the hippocampus. The hippocampus is involved in spatial navigation and episodic memory and is a long-standing model used to study cellular as well as cognitive processes important for learning and recall, both in health and disease. Here, a preparation that enables chronic optical access to the dorsal hippocampus in living mice is detailed. This preparation can be combined with two-photon optical imaging at cellular and subcellular resolution in head fixed, anesthetized live mice over several weeks. These techniques enable repeated imaging of neuronal structure or activity-evoked plasticity in tens to hundreds of neurons in the dorsal hippocampal CA1. Furthermore, this chronic preparation can be used in combination with other techniques such as micro-endoscopy, head-mounted wide field microscopy or three-photon microscopy, thus greatly expanding the toolbox to study cellular and network processes involved in learning and memory.

This method describes a chronic preparation that allows optical access to the hippocampus of living mice. This preparation can be used to perform longitudinal optical imaging of neuronal structural plasticity and activity-evoked cellular plasticity over a period of several weeks.

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes. In brain areas with laminar cytoarchitecture such as the hippocampus, one-dimensional CSD can be obtained by estimating the second spatial derivative of the LFP. Here, we describe a method to record multilaminar LFPs using linear silicon probes implanted into the dorsal hippocampus. CSD traces are computed along individual shanks of the probe. This protocol thus describes a procedure to resolve spatially restricted neuronal network oscillations in the hippocampus of freely moving mice.

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

Successful In vivo Calcium Imaging with a Head-Mount Miniaturized Microscope in the Amygdala of Freely Behaving Mouse

In vivo real-time monitoring of neuronal activities in freely moving animals is one of key approaches to link neuronal activity to behavior. For this purpose, an in vivo imaging technique that detects calcium transients in neurons using genetically encoded calcium indicators (GECIs), a miniaturized fluorescence microscope, and a gradient refractive index (GRIN) lens has been developed and successfully applied to many brain structures1,2,3,4,5,6. This imaging technique is particularly powerful because it enables chronic simultaneous imaging of genetically defined cell populations for a long-term period up to several weeks. Although useful, this imaging technique has not been easily applied to brain structures that locate deep within the brain such as amygdala, an essential brain structure for emotional processing and associative fear memory7. There are several factors that make it difficult to apply the imaging technique to the amygdala. For instance, motion artifacts usually occur more frequently during the imaging conducted in the deeper brain regions because a head-mount microscope implanted deep in the brain is relatively unstable. Another problem is that the lateral ventricle is positioned close to the implanted GRIN lens and its movement during respiration may cause highly irregular motion artifacts that cannot be easily corrected, which makes it difficult to form a stable imaging view. Furthermore, because cells in the amygdala are usually quiet at a resting or anesthetized state, it is hard to find and focus the target cells expressing GECI in the amygdala during baseplating procedure for later imaging. This protocol provides a helpful guideline for how to efficiently target cells expressing GECI in the amygdala with head-mount miniaturized microscope for successful in vivo calcium imaging in such a deeper brain region. It is noted that this protocol is based on a particular system (e.g., Inscopix) but not restricted to it.

In vivo microendoscopic calcium imaging is an invaluable tool that enables real-time monitoring of neuronal activities in freely behaving animals. However, applying this technique to the amygdala has been difficult. This protocol aims to provide a useful guideline for successfully targeting amygdala cells with a miniaturized microscope in mice.

In vivo real-time monitoring of neuronal activities in freely moving animals is one of key approaches to link neuronal activity to behavior. For this ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

Using Baseplating and a Miniscope Preanchored with an Objective Lens for Calcium Transient Research in Mice

Neuroscientists use miniature microscopes (miniscopes) to observe neuronal activity in freely behaving animals. The University of California, Los Angeles (UCLA) Miniscope team provides open resources for researchers to build miniscopes themselves. The V3 UCLA Miniscope is one of the most popular open-source miniscopes currently in use. It permits imaging of the fluorescence transients emitted from genetically modified neurons through an objective lens implanted on the superficial cortex (a one-lens system), or in deep brain areas through a combination of a relay lens implanted in the deep brain and an objective lens that is preanchored in the miniscope to observe the relayed image (a two-lens system). Even under optimal conditions (when neurons express fluorescence indicators and the relay lens has been properly implanted), a volume change of the dental cement between the baseplate and its attachment to the skull upon cement curing can cause misalignment with an altered distance between the objective and relay lenses, resulting in the poor image quality. A baseplate is a plate that helps mount the miniscope onto the skull and fixes the working distance between the objective and relay lenses. Thus, changes in the volume of the dental cement around the baseplate alter the distance between the lenses. The present protocol aims to minimize the misalignment problem caused by volume changes in the dental cement. The protocol reduces the misalignment by building an initial foundation of dental cement during relay lens implantation. The convalescence time after implantation is sufficient for the foundation of dental cement to cure the baseplate completely, so the baseplate can be cemented on this scaffold using as little new cement as possible. In the present article, we describe strategies for baseplating in mice to enable imaging of neuronal activity with an objective lens anchored in the miniscope.

The shrinkage of dental cement during curing displaces the baseplate. This protocol minimizes the problem by creating an initial foundation of the dental cement that leaves space to cement the baseplate. Weeks later, the baseplate can be cemented in position on this scaffold using little new cement, therebyreducing shrinkage.

Neuroscientists use miniature microscopes (miniscopes) to observe neuronal activity in freely behaving animals. The University of California, Los Angeles ...

Real-Time Imaging for Neuron Activity in the Dentate Gyrus Cells of Mice 

In Vivo Calcium Imaging of Granule Cells in the Dentate Gyrus of Hippocampus in Mice

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal activity in awake animals during behavior tasks. Since the hippocampus is closely associated with episodic and spatial memory, it has become an essential brain region in&#160;this field&#39;s research. In recent research, engram cells and place cells were studied by recording the neural activities in the hippocampal CA1 region using the miniature microscope in mice while performing behavioral tasks including open-field and linear track. Although the dentate gyrus is another important region in the hippocampus, it has rarely been studied with in vivo imaging due to its greater depth and difficulty for imaging. In this protocol, we present in detail a calcium imaging process, including how to inject the virus, implant a GRIN (Gradient-index) lens, and attach a base plate for imaging the dentate gyrus of the hippocampus. We further describe how to preprocess the calcium imaging data using MATLAB. Additionally, studies of other deep brain regions that require imaging may benefit from this method.

Author Spotlight: Investigating Neural Activity of Dentate Gyrus Granule Cells with Miniature Microscope

The dentate gyrus of the hippocampus carries out essential and distinct functions in learning and memory. This protocol describes a set of robust and efficient procedures for in vivo calcium imaging of granule cells in the dentate gyrus in freely moving mice.

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal ...

Miniscope Recording Calcium Signals at Hippocampus of Mice Navigating an Odor Plume

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