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).

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
<ol>
	<li>Wear protective equipment, including sing...

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

<ol>
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The authors declare no competing financial interests.

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...

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 cells5. This circuit receives its primary input from the entorhinal cortex (EC)&#160;and has been a classic model for studying synaptic plasticity. Prior in vivo research on the hippocampus function has mostly concentrated on the CA16,7 due to its easier access. While CA1 neurons serve important roles in memory formation, consolidation, and retrieval, particularly in place cells for spatial memory, other subregions of the hippocampus are also vital8,9. In particular, recent studies have highlighted the functions of DG in memory formation. Place cells in DG have been reported to be more stable than those in CA110, and their activities reflect context-specific information11. Further, activity-dependent labeling of DG granule cells can be reactivated to induce memory-related behaviors12. Therefore, to gain a deeper understanding of the information coding in DG, it is crucial to investigate the activities of the DG subregion while the animal is carrying out memory-dependent tasks.
Prior studies of DG activities have mostly used in vivo electrophysiology13. However, this technique has some drawbacks: First, in electrical recordings, it may be difficult to directly identify the various types of cells that are generating the signal. The recorded signals are from both inhibitory and excitatory cells. Therefore, further data processing methods are required to separate these two cell types. Moreover, it is difficult to combine other cell type information, such as projection-specific subgroups or activity-dependent labeling, with electric recordings. In addition, due to the anatomical morphology of DG, the recording electrodes are often implanted in an orthogonal direction, which greatly limits the number of neurons that can be recorded. Thus, it is difficult for electric recordings to achieve monitoring&#160;hundreds of individual neurons from the DG structure in the same animal14.
A complementary technique of recording neuron activities in DG is to use in vivo calcium imaging15. Calcium ions are fundamental to cellular signaling processes in organisms, playing a crucial role in many physiological functions, especially within the mammalian nervous system. When neurons are active, the intracellular calcium concentration increases rapidly, reflecting the dynamic nature of neuronal activity and signal transmission. Therefore, recording the real-time changes in intracellular calcium levels in neurons provides important insights into the neural coding mechanisms.
Calcium imaging technology utilizes specialized fluorescent dyes or genetically engineered calcium indicators (GECIs) to monitor calcium ion concentrations in neurons by detecting changes in fluorescence intensity, which can then be captured through microscopic imaging16. Commonly, the GCaMP family of calcium indicator genes, comprising green fluorescent protein (GFP), calmodulin, and M13 polypeptide sequences, are employed. GCaMP can emit green fluorescence when it binds to calcium ions17, allowing the fluctuations in green fluorescence to be recorded via imaging18. Additionally, to obtain clear images of the target brain region, a Gradient Index Lens (GRIN lens) is typically implanted above the region of interest. The GRIN lens enables imaging of the deep brain region that cannot be accessed directly from the surface.
This technique is relatively easy to combine with other genetic tools to label different cell types. Moreover, as the imaging plane is parallel to the cells&#39; orientation in DG, hundreds of neurons are accessible for imaging with each successful surgery. In this work, we present a complete and detailed surgery protocol for in vivo calcium imaging in the dentate gyrus in mice (Figure 1). The procedure involves two major operations. The first one is to inject the AAV-CaMKII&#945;-GCaMP6f virus into the DG. The second operation is to implant a GRIN lens above the virus injection site. These two procedures are conducted in the same sitting. After recovery from these surgeries, the next step is to check the imaging quality with miniaturized microscopes (miniscopes). If the imaging field has hundreds of active cells, the subsequent procedure is to attach the miniscope base plate onto the mouse skull using dental cement; the mouse can then be used for imaging experiments. We also present a MATLAB-based preprocessing pipeline for streamlining the analysis of the collected calcium data.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>200 &#956;L universal pipette tips</td>
			<td>Transcat Pipettes</td>
			<td>1030-260-000-9</td>
			<td>For removing the blood and saline</td>
		</tr>
		<tr>
			<td>25 G luer lock blunt needle (Prebent dispensing tips)</td>
			<td>iSmile</td>
			<td>20-0105</td>
			<td>For removing the brain tissue</td>
		</tr>
		<tr>
			<td>3D printed protective cap</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>To protect the GRIN Lens</td>
		</tr>
		<tr>
			<td>75% ethanol</td>
			<td>Shanghai Hushi Laboratory Equipment Co.,Ltd</td>
			<td>bwsj-230219105303</td>
			<td>For disinfection and cleaning the GRIN lens surface</td>
		</tr>
		<tr>
			<td>AAV2/9-CaMKII&#945;-GCaMP6f virus</td>
			<td>Brain Case</td>
			<td>BC-0083</td>
			<td>For viral injection</td>
		</tr>
		<tr>
			<td>Adobe Illustrator</td>
			<td>Adobe</td>
			<td>cc 2018 version 22.1</td>
			<td>To draw figures</td>
		</tr>
		<tr>
			<td>Anesthesia air pump</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>R510-30</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Camera control software</td>
			<td>Daheng Imaging</td>
			<td>Galaxy Windows SDK_CN (V2)</td>
			<td>For recording the behavioral data</td>
		</tr>
		<tr>
			<td>Cannula/Ceramic Ferrule Holders (GRIN lens holder)</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>68214</td>
			<td>To hold the GRIN lens</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>MedChemExpress</td>
			<td>53716-49-7</td>
			<td>To reduce postoperative pain of the mouse&#160;</td>
		</tr>
		<tr>
			<td>Coax Cable</td>
			<td>Open ephys</td>
			<td>CW8251</td>
			<td>To connect the miniscope and the miniscope DAQ box</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus Life Science&#160;</td>
			<td>FV3000</td>
			<td>For observing the brain slices</td>
		</tr>
		<tr>
			<td>Cotton swab</td>
			<td>Nanchang Xiangyi Medical Devices Co.,Ltd</td>
			<td>20202140438</td>
			<td>For disinfection</td>
		</tr>
		<tr>
			<td>Customized headplate</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>For holding the mouse on the running wheel</td>
		</tr>
		<tr>
			<td>Customized headplate holder</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>To hold the headplate of the mouse</td>
		</tr>
		<tr>
			<td>Denture base matierlals (self-curing)</td>
			<td>New Centry Dental</td>
			<td>430205</td>
			<td>For attaching the miniscope</td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Veet</td>
			<td>ASIN : B001DUUPQ0</td>
			<td>For removing the hair of the mouse</td>
		</tr>
		<tr>
			<td>Desktop digital stereotaxic in strument, SGL M</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>68803</td>
			<td>For viral injection and GRIN lens implantation</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Huachu Co., Ltd.</td>
			<td>N/A</td>
			<td>To prevent postoperative inflammation of the mouse</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>RWD Life Science Co., Ltd</td>
			<td>MZ62-WX</td>
			<td>For observing the conditions during surgeries</td>
		</tr>
		<tr>
			<td>Gas filter canister, large, packge of 6</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>R510-31-6</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>GRIN lens</td>
			<td>GoFoton</td>
			<td>CLHS100GFT003</td>
			<td>For GRIN lens implantation</td>
		</tr>
		<tr>
			<td>GRIN lens</td>
			<td>InFocus Grin Corp</td>
			<td>SIH-100-043-550-0D0-NC</td>
			<td>For GRIN lens implantation</td>
		</tr>
		<tr>
			<td>Induction chamber-mouse (15 cm x 10 cm x 10 cm)</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>V100</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Industrial camera</td>
			<td>Daheng Imaging</td>
			<td>MER-231-41U3M-L, VS-0618H1</td>
			<td>For acquiring the behavioral data</td>
		</tr>
		<tr>
			<td>Iodophor disinfectant</td>
			<td>Qingdao Hainuo Innovi Disinfection Technology Co.,Ltd</td>
			<td>8861F6DFC92A</td>
			<td>For disinfection</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>R510-22-10</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Liquid sample collection tube (Glass Capillaries micropipette for Nanoject III)</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-203-G/X</td>
			<td>For viral injection</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td>R2021b</td>
			<td>For analyzing the data</td>
		</tr>
		<tr>
			<td>Microdrill</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>78001</td>
			<td>For craniotomy</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Narishige International USA</td>
			<td>PC-100</td>
			<td>For pulling the liquid sample collection tube</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma-Aldrich</td>
			<td>M8410</td>
			<td>For viral injection</td>
		</tr>
		<tr>
			<td>Miniscope DAQ Software</td>
			<td>Github (Aharoni-Lab/Miniscope-DAQ-QT-Software)</td>
			<td>N/A</td>
			<td>For recording the calcium imaging data</td>
		</tr>
		<tr>
			<td>Miniscope Data Acquisition (DAQ) Box (V3.3)</td>
			<td>Open ephys</td>
			<td>V3.3</td>
			<td>To acquire the calcium imaging data</td>
		</tr>
		<tr>
			<td>Miniscope V4</td>
			<td>Open ephys</td>
			<td>V4</td>
			<td>For in vivo calcium imaging</td>
		</tr>
		<tr>
			<td>Miniscope V4 base plate (Variant 2)</td>
			<td>Open ephys</td>
			<td>Variant 2</td>
			<td>For holding the miniscope</td>
		</tr>
		<tr>
			<td>nanoject III Programmable Nanoliter Injector</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-207</td>
			<td>For viral injection</td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Cisen Pharmaceutical Co.,Ltd.</td>
			<td>H37022025</td>
			<td>To keep the eyes moist</td>
		</tr>
		<tr>
			<td>PCR tube</td>
			<td>LabServ</td>
			<td>309101009</td>
			<td>For dilue the virus</td>
		</tr>
		<tr>
			<td>Personal Computer (ThinkPad)</td>
			<td>Lenovo</td>
			<td>20W0-005UCD</td>
			<td>To record the calcium imaging data and behavioral data</td>
		</tr>
		<tr>
			<td>Running wheel</td>
			<td>Shanghai Edai Pet Products Co.,Ltd</td>
			<td>NA-H115</td>
			<td>For holding the mouse when affixing the base plate</td>
		</tr>
		<tr>
			<td>Screwdriver (M1.6 screws)</td>
			<td>Greenery (Yantai Greenery Tools Co.,Ltd)</td>
			<td>60902</td>
			<td>To unscrew the M1.6 screws</td>
		</tr>
		<tr>
			<td>Screwdriver (set screws)</td>
			<td>Greenery (Yantai Greenery Tools Co.,Ltd)</td>
			<td>S2</td>
			<td>For unscrew the set screws</td>
		</tr>
		<tr>
			<td>Set screw</td>
			<td>TBD</td>
			<td>2-56 cone point set screw</td>
			<td>For fasten the miniscope to its base plate</td>
		</tr>
		<tr>
			<td>Small animal anesthesia machine</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>R500</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Sterile syringe</td>
			<td>Jiangsu Great Wall Medical Equipment Co., LTD</td>
			<td>20163140236</td>
			<td>For rinse the blood</td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>S14016-13</td>
			<td>For cutting off the hair and scalp</td>
		</tr>
		<tr>
			<td>ThermoStar temperature controller,69025 pad incl.</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>69027</td>
			<td>To maintain the animal&#39;s body temperature</td>
		</tr>
		<tr>
			<td>Ultra fine forceps</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>F11020-11</td>
			<td>For removing the bone debris and dura</td>
		</tr>
		<tr>
			<td>USB 3.0 cable</td>
			<td>Open ephys</td>
			<td>N/A</td>
			<td>To connect the miniscope DAQ box and the computer</td>
		</tr>
		<tr>
			<td>UV light</td>
			<td>Jinshida</td>
			<td>66105854002</td>
			<td>To fix the GRIN lens on the skull</td>
		</tr>
		<tr>
			<td>UV resin (light cure adhesive)</td>
			<td>Loctite</td>
			<td>32268</td>
			<td>To fix the GRIN lens on the skull</td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Kylin-Bell</td>
			<td>GL-802B</td>
			<td>To remove the blood, saline and the brain tissue</td>
		</tr>
	</tbody>
</table>

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

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

Well, in this protocol, we use a miniature microscope to investigate the neural activities in the dentate gyrus of the hippocampus. We aim to investigate neuron coding and spatial representations during the different disease conditions. I believe the key challenge of studying dentate gyrus in vivo involves obtaining high-quality calcium imaging data, whether the virus expression is satisfactory, whether the gradient is implanted at the proper location.And the details like these, the entire surgery are of critical importance for the success of the study. In the traditional calcium imaging surgery, the virus injection and the GRIN lens implantation are typically performed as separate operations. In our protocol, we combined these two steps into a single procedure, which not only reduces the waiting time, but also ensures the accurate placement of the GRIN lens.Our lab uses various microscopy techniques to study the neurodynamics underlying cognitive functions. We hope to understand the neural activity patterns that support cognition. And combining theoretical modeling and causal manipulations, we hope to dissect the processes of information flow and neural computation in the brain.

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.
<strong ...

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 ...

in-vivo-calcium-imaging-granule-cells-dentate-gyrus-hippocampus

Research

JoVE Journal

Neuroscience

Optimized Virus Injection and GRIN Lens Implantation in the Dentate Gyrus of Mice for In Vivo Calcium Imaging

To begin, place the anesthetized mouse on a stereotaxic apparatus. Make a continuous incision along the midline of the scalp using surgical scissors and cut off part of the scalp to expose the skull surface. Make the craniotomy in the specified region with the micro drill and set four points as the border.Stop drilling when brain tissue becomes visible. Use the control panel to set the injector to inject 80 nanoliters of the virus. Click the Inject button on the control panel to inject the virus into the dentate gyrus at a flow rate of 1 nanoliter per second.To suction the brain tissue, revolve the tip of the blunt needle closely above the brain tissue, and press gently to facilitate aspiration. Rinse continuously with cold saline during suctioning to maintain a clear view of the brain. Observe the tissue features closely to monitor the depth of aspiration.The cortical tissue is pale pink. The corpus callosum underneath appears as white fibrous tissue. And the hippocampal CA1 layer is dark red and gray.Stop the suction when the surface of the tissue becomes smooth and a large area of CA1 is exposed. Move the holder attached to the grin lens above the drilled hole. When the grin lens contacts the surface of the brain tissue, set the Z axis to zero.Insert the grin lens into the hole at the dorsal ventral axis minus 1.32 millimeters. Allow the holder to stand for 5 to 10 minutes. Then, raise the holder and rinse the hole with saline.Using a needle, apply the ultraviolet resin around the grin lens. Illuminate the area with ultraviolet light for 15 seconds to ensure that the resin becomes solid. Carefully remove the holder from the grin lens.Cover the entire exposed skull with ultraviolet resin. Place the head plate on the skull in the appropriate position. Illuminate the entire skull and head plate with ultraviolet light for 15 seconds.Cover the exposed grin lens with a 3D printed protective cap. Fix the cap to the head plate using M1.6 screws. Turn on the Miniscope control software and click Select Config File.Then, select vscode, followed by User Configuration Files. Now, click Run. Then, slide the LED power button to adjust the brightness.Slide the focus adjustment button to set the value near zero. Attach the base plate to the mini scope and readjust the position of the mini scope to obtain a field of view with good imaging quality. Carefully apply the first layer of denture base material around the mini scope base plate.Once the first layer of the cement becomes hardened, apply a second layer of cement from the base plate to the head plate. After all denture base material hardens, loosen the set screw and detach the mini scope from the base plate. Put the base plate cap into the base plate to protect the exposed grin lens and tighten the set screw.After imaging, convert the imaging data from AVI to HDF5 format. Apply the non-rigid motion correction and downsample the motion corrected movie. Apply extract on the downsample data to identify single cell signals.Then, apply cell check and manually select the potential cells. Click Save Data before closing the cell check window. Finally, save the data files.In unsuccessful in vivo calcium imaging, no active cells were observed in the imaging field of view. Successful in vivo calcium imaging showed fewer than 10 active cells without obvious blood vessels. More than 50 active cells with visible blood vessels.And hundreds of active cells with clear blood vessels. Extracted individual cells from a successful in vivo calcium imaging recording were superimposed on the field of view with representative calcium traces shown. The position of GCaMP6F expression and the track of the grin lens were verified in the mouse brain slice.

693 Views.  Fudan University.  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 Imaging for Neuron Activity in the Dentate Gyrus Cells of Mice

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