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In Vivo Calcium Imaging of Granule Cells in the Dentate Gyrus of Hippocampus in Mice
In This Article
Summary
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.
Abstract
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 this field'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.
Introduction
Previous studies found that the hippocampus is a brain structure essential for processing and retrieving memories1,2. Since the 1950s, the neural circuits of the hippocampus in rodents have been a focus in studying memory formation, storage, and retrieval3. The anatomical structures within the hippocampus include the subregions of dentate gyrus (DG), CA1, CA2, CA3, CA4, and subiculum4. Complex bidirectional connections exist among these subregions, of which the DG, CA1, and CA3 form a prominent trisynaptic circuit that consists of granule cells and pyramidal cell....
Protocol
All the animal procedures were approved by the Institutional Animal Care and Use Committee at Fudan University (202109004S). All animals used in this study were 6-month-old C57BL/6J; both sexes were used. Mice were kept on a 12 h light cycle, from 8 AM to 8 PM. We used the following coordinates for virus injection in DG: A/P: -2.2 mm, M/L: 1.5 mm, D/V: 1.7 mm from the brain surface.
1. Virus injection into the dentate gyrus
- Wear protective equipment, including sing.......
Representative Results
Figure 1 shows the schematic of the experimental procedure, including virus injection, GRIN lens implantation, base plate affixation, in vivo calcium imaging via a miniscope, and data processing. Generally, the entire procedure takes 1 month. Figure 2 shows example procedures of virus injection, including the positioning of the drilled hole on the skull and the condition of brain tissue before GRIN lens implantation.
Discussion
Here we described a procedure for in vivo calcium imaging in the DG of mice. We believe that this protocol will be useful for researchers aiming to study DG functions in various cognitive processes, particularly in cases where a genetically identified subpopulation is of interest. Here we explain the advantages of our protocol, emphasizing some key points in surgery, and discuss the limitations of this method.
We have tested various procedures for DG imaging from the available literat.......
Acknowledgements
This work is supported by the Shanghai Pilot Program for Basic Research - Fudan University 21TQ1400100 (22TQ019), Shanghai Municipal Science and Technology Major Project, the Lingang Laboratory (grant no. LG-QS-202203-09) and National Natural Science Foundation of China (32371036).
....Materials
| Name | Company | Catalog Number | Comments |
| 200 μL universal pipette tips | Transcat Pipettes | 1030-260-000-9 | For removing the blood and saline |
| 25 G luer lock blunt needle (Prebent dispensing tips) | iSmile | 20-0105 | For removing the brain tissue |
| 3D printed protective cap | N/A | N/A | To protect the GRIN Lens |
| 75% ethanol | Shanghai Hushi Laboratory Equipment Co.,Ltd | bwsj-230219105303 | For disinfection and cleaning the GRIN lens surface |
| AAV2/9-CaMKIIα-GCaMP6f virus | Brain Case | BC-0083 | For viral injection |
| Adobe Illustrator | Adobe | cc 2018 version 22.1 | To draw figures |
| Anesthesia air pump | RWD Life Science Co.,Ltd | R510-30 | For anesthesia |
| Camera control software | Daheng Imaging | Galaxy Windows SDK_CN (V2) | For recording the behavioral data |
| Cannula/Ceramic Ferrule Holders (GRIN lens holder) | RWD Life Science Co.,Ltd | 68214 | To hold the GRIN lens |
| Carprofen | MedChemExpress | 53716-49-7 | To reduce postoperative pain of the mouse |
| Coax Cable | Open ephys | CW8251 | To connect the miniscope and the miniscope DAQ box |
| Confocal microscope | Olympus Life Science | FV3000 | For observing the brain slices |
| Cotton swab | Nanchang Xiangyi Medical Devices Co.,Ltd | 20202140438 | For disinfection |
| Customized headplate | N/A | N/A | For holding the mouse on the running wheel |
| Customized headplate holder | N/A | N/A | To hold the headplate of the mouse |
| Denture base matierlals (self-curing) | New Centry Dental | 430205 | For attaching the miniscope |
| Depilatory cream | Veet | ASIN : B001DUUPQ0 | For removing the hair of the mouse |
| Desktop digital stereotaxic in strument, SGL M | RWD Life Science Co.,Ltd | 68803 | For viral injection and GRIN lens implantation |
| Dexamethasone | Huachu Co., Ltd. | N/A | To prevent postoperative inflammation of the mouse |
| Dissecting microscope | RWD Life Science Co., Ltd | MZ62-WX | For observing the conditions during surgeries |
| Gas filter canister, large, packge of 6 | RWD Life Science Co.,Ltd | R510-31-6 | For anesthesia |
| GRIN lens | GoFoton | CLHS100GFT003 | For GRIN lens implantation |
| GRIN lens | InFocus Grin Corp | SIH-100-043-550-0D0-NC | For GRIN lens implantation |
| Induction chamber-mouse (15 cm x 10 cm x 10 cm) | RWD Life Science Co.,Ltd | V100 | For anesthesia |
| Industrial camera | Daheng Imaging | MER-231-41U3M-L, VS-0618H1 | For acquiring the behavioral data |
| Iodophor disinfectant | Qingdao Hainuo Innovi Disinfection Technology Co.,Ltd | 8861F6DFC92A | For disinfection |
| Isoflurane | RWD Life Science Co.,Ltd | R510-22-10 | For anesthesia |
| Liquid sample collection tube (Glass Capillaries micropipette for Nanoject III) | Drummond Scientific Company | 3-000-203-G/X | For viral injection |
| MATLAB | MathWorks | R2021b | For analyzing the data |
| Microdrill | RWD Life Science Co.,Ltd | 78001 | For craniotomy |
| Micropipette puller | Narishige International USA | PC-100 | For pulling the liquid sample collection tube |
| Mineral oil | Sigma-Aldrich | M8410 | For viral injection |
| Miniscope DAQ Software | Github (Aharoni-Lab/Miniscope-DAQ-QT-Software) | N/A | For recording the calcium imaging data |
| Miniscope Data Acquisition (DAQ) Box (V3.3) | Open ephys | V3.3 | To acquire the calcium imaging data |
| Miniscope V4 | Open ephys | V4 | For in vivo calcium imaging |
| Miniscope V4 base plate (Variant 2) | Open ephys | Variant 2 | For holding the miniscope |
| nanoject III Programmable Nanoliter Injector | Drummond Scientific Company | 3-000-207 | For viral injection |
| Ophthalmic ointment | Cisen Pharmaceutical Co.,Ltd. | H37022025 | To keep the eyes moist |
| PCR tube | LabServ | 309101009 | For dilue the virus |
| Personal Computer (ThinkPad) | Lenovo | 20W0-005UCD | To record the calcium imaging data and behavioral data |
| Running wheel | Shanghai Edai Pet Products Co.,Ltd | NA-H115 | For holding the mouse when affixing the base plate |
| Screwdriver (M1.6 screws) | Greenery (Yantai Greenery Tools Co.,Ltd) | 60902 | To unscrew the M1.6 screws |
| Screwdriver (set screws) | Greenery (Yantai Greenery Tools Co.,Ltd) | S2 | For unscrew the set screws |
| Set screw | TBD | 2-56 cone point set screw | For fasten the miniscope to its base plate |
| Small animal anesthesia machine | RWD Life Science Co.,Ltd | R500 | For anesthesia |
| Sterile syringe | Jiangsu Great Wall Medical Equipment Co., LTD | 20163140236 | For rinse the blood |
| Surgical scissors | RWD Life Science Co.,Ltd | S14016-13 | For cutting off the hair and scalp |
| ThermoStar temperature controller,69025 pad incl. | RWD Life Science Co.,Ltd | 69027 | To maintain the animal's body temperature |
| Ultra fine forceps | RWD Life Science Co.,Ltd | F11020-11 | For removing the bone debris and dura |
| USB 3.0 cable | Open ephys | N/A | To connect the miniscope DAQ box and the computer |
| UV light | Jinshida | 66105854002 | To fix the GRIN lens on the skull |
| UV resin (light cure adhesive) | Loctite | 32268 | To fix the GRIN lens on the skull |
| Vacuum pump | Kylin-Bell | GL-802B | To remove the blood, saline and the brain tissue |
References
- Scoville, W. B., Milner, B. Loss of recent memory after bilateral hippocampal lesions. J Neurol Neurosurg Psychiatry. 20 (1), 11-21 (1957).
- Spiers, H. J., Burgess, N., Hartley, T., Vargha-Khadem, F., O'keefe, J.
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