Summary

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

Published: September 20, 2024
doi:

Summary

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.

Abstract

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.

Introduction

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 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'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'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.

Protocol

Studies were carried out in 3-6-month-old male and femaleThy1-GCaMP6f transgenic mice23. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Colorado Anschutz Medical Campus in accordance with National Institutes of Health guidelines. The surgical procedures for GRIN lens implantation (Section 1) and the baseplate placement (Section 2) were adapted from previous works9,24,</…

Representative Results

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 δF/F0 signals from the ROIs with…

Discussion

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…

Declarações

The authors have nothing to disclose.

Acknowledgements

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.

Materials

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

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Simoes de Souza, F. M., Williamson, R., McCullough, C., Teel, A., Futia, G., Ma, M., True, A., Crimaldi, J. P., Gibson, E., Restrepo, D. Miniscope Recording Calcium Signals at Hippocampus of Mice Navigating an Odor Plume. J. Vis. Exp. (211), e67039, doi:10.3791/67039 (2024).

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