Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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

  1. 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

NameCompanyCatalog NumberComments
200 μL universal pipette tipsTranscat Pipettes1030-260-000-9For removing the blood and saline
25 G luer lock blunt needle (Prebent dispensing tips)iSmile20-0105For removing the brain tissue
3D printed protective capN/AN/ATo protect the GRIN Lens
75% ethanolShanghai Hushi Laboratory Equipment Co.,Ltdbwsj-230219105303For disinfection and cleaning the GRIN lens surface
AAV2/9-CaMKIIα-GCaMP6f virusBrain CaseBC-0083For viral injection
Adobe IllustratorAdobecc 2018 version 22.1To draw figures
Anesthesia air pumpRWD Life Science Co.,LtdR510-30For anesthesia
Camera control softwareDaheng ImagingGalaxy Windows SDK_CN (V2)For recording the behavioral data
Cannula/Ceramic Ferrule Holders (GRIN lens holder)RWD Life Science Co.,Ltd68214To hold the GRIN lens
CarprofenMedChemExpress53716-49-7To reduce postoperative pain of the mouse 
Coax CableOpen ephysCW8251To connect the miniscope and the miniscope DAQ box
Confocal microscopeOlympus Life Science FV3000For observing the brain slices
Cotton swabNanchang Xiangyi Medical Devices Co.,Ltd20202140438For disinfection
Customized headplateN/AN/AFor holding the mouse on the running wheel
Customized headplate holderN/AN/ATo hold the headplate of the mouse
Denture base matierlals (self-curing)New Centry Dental430205For attaching the miniscope
Depilatory creamVeetASIN : B001DUUPQ0For removing the hair of the mouse
Desktop digital stereotaxic in strument, SGL MRWD Life Science Co.,Ltd68803For viral injection and GRIN lens implantation
DexamethasoneHuachu Co., Ltd.N/ATo prevent postoperative inflammation of the mouse
Dissecting microscopeRWD Life Science Co., LtdMZ62-WXFor observing the conditions during surgeries
Gas filter canister, large, packge of 6RWD Life Science Co.,LtdR510-31-6For anesthesia
GRIN lensGoFotonCLHS100GFT003For GRIN lens implantation
GRIN lensInFocus Grin CorpSIH-100-043-550-0D0-NCFor GRIN lens implantation
Induction chamber-mouse (15 cm x 10 cm x 10 cm)RWD Life Science Co.,LtdV100For anesthesia
Industrial cameraDaheng ImagingMER-231-41U3M-L, VS-0618H1For acquiring the behavioral data
Iodophor disinfectantQingdao Hainuo Innovi Disinfection Technology Co.,Ltd8861F6DFC92AFor disinfection
IsofluraneRWD Life Science Co.,LtdR510-22-10For anesthesia
Liquid sample collection tube (Glass Capillaries micropipette for Nanoject III)Drummond Scientific Company3-000-203-G/XFor viral injection
MATLABMathWorksR2021bFor analyzing the data
MicrodrillRWD Life Science Co.,Ltd78001For craniotomy
Micropipette pullerNarishige International USAPC-100For pulling the liquid sample collection tube
Mineral oilSigma-AldrichM8410For viral injection
Miniscope DAQ SoftwareGithub (Aharoni-Lab/Miniscope-DAQ-QT-Software)N/AFor recording the calcium imaging data
Miniscope Data Acquisition (DAQ) Box (V3.3)Open ephysV3.3To acquire the calcium imaging data
Miniscope V4Open ephysV4For in vivo calcium imaging
Miniscope V4 base plate (Variant 2)Open ephysVariant 2For holding the miniscope
nanoject III Programmable Nanoliter InjectorDrummond Scientific Company3-000-207For viral injection
Ophthalmic ointmentCisen Pharmaceutical Co.,Ltd.H37022025To keep the eyes moist
PCR tubeLabServ309101009For dilue the virus
Personal Computer (ThinkPad)Lenovo20W0-005UCDTo record the calcium imaging data and behavioral data
Running wheelShanghai Edai Pet Products Co.,LtdNA-H115For holding the mouse when affixing the base plate
Screwdriver (M1.6 screws)Greenery (Yantai Greenery Tools Co.,Ltd)60902To unscrew the M1.6 screws
Screwdriver (set screws)Greenery (Yantai Greenery Tools Co.,Ltd)S2For unscrew the set screws
Set screwTBD2-56 cone point set screwFor fasten the miniscope to its base plate
Small animal anesthesia machineRWD Life Science Co.,LtdR500For anesthesia
Sterile syringeJiangsu Great Wall Medical Equipment Co., LTD20163140236For rinse the blood
Surgical scissorsRWD Life Science Co.,LtdS14016-13For cutting off the hair and scalp
ThermoStar temperature controller,69025 pad incl.RWD Life Science Co.,Ltd69027To maintain the animal's body temperature
Ultra fine forcepsRWD Life Science Co.,LtdF11020-11For removing the bone debris and dura
USB 3.0 cableOpen ephysN/ATo connect the miniscope DAQ box and the computer
UV lightJinshida66105854002To fix the GRIN lens on the skull
UV resin (light cure adhesive)Loctite32268To fix the GRIN lens on the skull
Vacuum pumpKylin-BellGL-802BTo remove the blood, saline and the brain tissue

References

  1. Scoville, W. B., Milner, B. Loss of recent memory after bilateral hippocampal lesions. J Neurol Neurosurg Psychiatry. 20 (1), 11-21 (1957).
  2. Spiers, H. J., Burgess, N., Hartley, T., Vargha-Khadem, F., O'keefe, J.

Explore More Articles

In Vivo Calcium ImagingGranule CellsDentate GyrusHippocampusNeuronal ActivityLearning And MemoryBehavioral TasksEngram CellsPlace CellsMiniature MicroscopeCalcium Imaging ProtocolGRIN LensData PreprocessingMATLABDeep Brain Regions

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved