Name: In Vivo Calcium Imaging of Granule Cells in the Dentate Gyrus of Hippocampus in Mice (Video) | JoVE Journal
Uploaded: 2024-08-02T14:30:00.000+00:00
Duration: 7 min
Description: Learn in vivo calcium imaging of granule cells in the mouse dentate gyrus using a combined virus injection and GRIN lens implantation procedure. This streamlined surgical approach enhances data quality for studying neural coding and spatial representations in cognitive functions.

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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).

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

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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 literature and optimized the experimental procedures as detailed in this protocol. In our opinion, the following three key modifications greatly improved the success rate for our protocol. First, we combined all surgical procedures in one sitting, while many calcium imaging studies for subcortical regions performed virus injection and lens implantation in two separate surgeries. A key advantage of our approach is that virus expression and brain tissue recovery are now happening during the same period, reducing the waiting time for mouse preparation by half, which usually takes months21. Furthermore, separate surgeries require accurate re-positioning of the mouse, as slight tilts with a mismatch in the range of 100 &#181;m will lead to failure of the procedure. To avoid misplacing the lens, some groups developed an approach that attaches the miniscope to the lens and tried to view the neuronal expression of the sensor during the implanting surgery. This workaround requires non-trivial engineering of the commonly used stereotaxic apparatus and is complex to use due to the suppression of neuron activities during anesthesia. Our method does not require re-positioning of the mouse, thereby greatly reducing the time and stress associated with the procedure.
The second key point of our approach is that we repeatedly inject small amounts of the virus into the target area22. This leads to more cells becoming infected. Importantly, compared to increasing the virus load in a single injection site, this approach yielded superior results in that the locations of infected cells spread uniformly across the field of view. Another related key point is that diluting the virus titer improves the quality of calcium imaging. In our experience, an excessive amount of virus is associated with the appearance of wave-like clusters of activities, which is consistent with a previous report23. While we initially suspected that tissue aspiration may lead to premature clearance of the virus in the injection site and insufficient expression, we did not observe such a phenomenon, and our protocol generally yielded robust infection of the target region. Compared to transgenic mice expressing GCaMP, the virus approach is inevitably less consistent but offers more flexible use because it does not require complicated breeding. However, as the expression of the GCaMP may spill into adjacent brain structures or become unevenly distributed in the target brain region, careful examination of the anatomical positions of virus and lens from the fixed tissue is critical for the interpretation of the results. Combining region- or celltype-specific Cre lines with Cre-dependent GCaMP expression could improve the specificity of imaged signals.
The third key point of our protocol is that during the implantation of the lens, we repeated lens insertion and rinsing several times to make sure that there was no leftover blood in the implantation area. This is a key operation to facilitate tissue recovery and improve the success rate of the procedure. While the exact mechanism is not known, we speculate that lens insertion will inevitably cause some damage to the tissue, and the rinsing step would clear the bleeding associated with this damage. Otherwise, the insertion-associated bleeding may take a long time for the brain to clear, which increases the probability of local inflammation and macrophage infiltration, both of which would reduce the optical quality. In addition, at the coordinates we provided in this protocol, we found that placing the lens a little closer to the sagittal suture usually results in an increased number of visible cells, although it is important to avoid piercing into the third ventricle. We have also provided a list of common problems for better troubleshooting of this step (Table 1).
The mouse has to be trained to wear the dummy miniscope prior to behavioral experiments to minimize the impact of added weight on the head. Typically, this training takes place ~3 days, with each session lasting 20 min. During the behavioral experiments, the physical condition of the mouse should be monitored regularly. The behavioral performance of the mouse will be greatly impacted by its health.
This protocol contains several limitations. First, it should be noted that calcium imaging is a surrogate of neural activity, which can be influenced by the expression of the sensors. The one-photon imaging methods using a miniscope may lead to overlapping signals from neighboring cells, which require de-mixing from subsequent analysis. In addition, our protocol involves invasive surgeries. A chunk of brain tissue is removed during the GRIN lens implantation step, usually including part of the CA1 region, retrosplenial cortex, and associative visual cortex. This is necessary due to the limitation of the imaging depth afforded by the one-photon illumination. In our observations, we did not notice any apparent behavioral deficits in the mice following the surgical procedure, neither in their general, unrestricted behaviors nor in the learning of the linear track tasks (Supplemental Figure S1). Alternatively, the use of automated surgical instruments for brain tissue suction can reduce the errors associated with manual operation, thereby enhancing the precision of the surgical procedure24. Moreover, our calcium imaging data in general yielded normal calcium dynamics. The effect on more complex cognitive functions needs further characterization. An alternative method for lens insertion without tissue aspiration has been described before25. This is achieved by making a cut in the brain tissue along the tract of intended insertion and placing the lens directly through the opening. In our experience, this approach leads to folding or other deformations of the brain tissue along the insertion tract, which can still be invasive. The high variability of the deformation makes this approach less reliable than the protocol we described. Another potential caveat of our protocol is that the behavioral performance of the mouse is slightly impacted by the coax cable that connects the miniscope and the miniscope Data Acquisition (DAQ) Box. This is not only due to the wires coiling together after numerous turns, but also because the wire can be a distractor for the mouse during the task. Utilizing a wireless miniscope26 would effectively resolve this problem. 
Despite the existing limitations, we believe this methodology can be highly beneficial for researchers interested in using in vivo calcium imaging to study neuronal activity patterns within the hippocampal dentate gyrus. For example, this in vivo calcium imaging approach can be applied to investigate place cells specifically within the dentate gyrus region. This would enable the exploration of how place cells encode information across different brain areas10,27. Our validation indicates that this protocol is reasonably reproducible, with high rates of success, and can be applied to different brain regions.

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><tbody><tr><td>200 &amp;mu;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&amp;alpha;-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&amp;nbsp;</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&amp;nbsp;</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&#039;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

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.

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.
Figure 3 shows the process of removing the brain tissue, which is crucial for the success of this surgery. As more tissue is suctioned, different tissue layers become visible. This suctioning step can be stopped once a grey, reflective layer is observed.
Figure 4 shows the procedure of the GRIN implantation, including inserting the GRIN lens, adhering the headplate on the skull, and covering the GRIN lens with a customized cap. The imaging quality of in vivo calcium imaging depends on the success of the virus injection and GRIN lens implantation. 
Figure 5 shows four types of results from in vivo calciumimaging. Figure 5A is the unsuccessful result; no active cells were observed in the imaging field of view. Figure 5B-D are successful results. Figure 5B has fewer than 10 active cells, but there are no obvious blood vessels. Figure 5C has more than 50 active cells and visible blood vessels. In Figure 5D, there are hundreds of active cells in the imaging field of view; at the same time, we observed clear blood vessels. High-quality in vivo calcium imaging typically has hundreds of activated cells. Fewer than a dozen activated cells are thought to be suboptimal for imaging results. Usually, the number of cells correlates with the diameter of the GRIN lens as well as the part of the brain region. A larger GRIN lens diameter and a closer brain region to the dorsal side will increase the probability of observing more active cells. It is also vital to note that the larger the diameter of the GRIN lens, the more damage it does to the mouse brain; thus, selecting the appropriate size is crucial. Implanting a GRIN lens of 1 mm diameter does not affect the animal's ability to acquire the linear track behavior (Supplemental Figure S1).
Behavioral data and calcium imaging data are usually processed separately. Mouse behavioral data can be labeled manually or processed using open-source analysis software. Calcium imaging data are processed using Non-Rigid Motion Correction (NoRMCorre) and EXTRACT in MATLAB. Figure 6 shows the extracted individual cells superimposed on the field of view and shows five representative calcium traces from a successful in vivo calcium imaging recording. See Supplemental Video S1 for a representative video of the raw data.
Verifying that the GRIN lens implantation and viral injection take place in the intended brain region is the last step and is also very important for the interpretation of the data. Figure 7 is a mouse brain slice showing the position of GCaMP6f expression (the green fluorescence region) and the track of the GRIN lens.
<img alt="Brain imaging process: viral injection, GRIN lens implantation, behavioral tests, data processing diagram." class="xfigimg" src="/files/ftp_upload/66916/66916fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Diagram of the procedures of virus injection and GRIN lens implantation. (A) Inject the AAV-CaMKIIα-GCaMP6f virus in the dentate gyrus. (B) Implant the GRIN lens above the dentate gyrus. (C) Check the imaging quality after surgery. (D) Affix the miniscope base plate after checking. (E) The timeline and process of the whole protocol. Abbreviation: GRIN = Gradient index. <a href="https://www.jove.com/files/ftp_upload/66916/66916fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Rodent stereotaxic setup and brain positioning with ear bars and micropipette for precise targeting." class="xfigimg" src="/files/ftp_upload/66916/66916fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Representative surgical view during virus injection. (A) Place the mouse on the stereotaxic apparatus. (B) Mark the GRIN lens implantation area (the black region). (C) Craniotomy on the target area. (D) Inject 240 nL of virus into the dentate gyrus. (E) Remove the micropipette after injection. (F) Remove the cortex and part of CA1 brain tissue. <a href="https://www.jove.com/files/ftp_upload/66916/66916fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Tissue biopsy experiment sequence; surgical extraction process; microscopic close-up analysis." class="xfigimg" src="/files/ftp_upload/66916/66916fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: Representative surgical view during brain tissue aspiration. (A) The region enclosed by the black marker is polished using a microdrill, leaving behind (B) partially exposed cortical tissue. The left part shows an exposed cortex and the right part is covered by dura and skull tissue. (C) Representative image during the aspiration of the cortical brain tissue. The tissue remains pale pink. (D) White matter is visible within the field of view, as indicated by the white arrow. (E) Further suctioning revealed a dark grey-red, reflective surface underneath the white matter. The region (black arrow) is thought to be the CA1 surface and the suction should not go deeper. (F) No more bleeding is visible from the region after rinsing with saline several times. In all panels, the asterisks indicate surgical tools: a 30 G blunt needle for saline and a 25 G blunt needle for brain tissue aspiration. Scale bars = 500 µm. <a href="https://www.jove.com/files/ftp_upload/66916/66916fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="GRIN lens installation on rodent for optical studies, showing setup and components like headplate." class="xfigimg" src="/files/ftp_upload/66916/66916fig04.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4: An example procedure for GRIN lens implantation and base plate attachment. (A) Implant the GRIN lens into the brain tissue using a GRIN lens holder. (B) Adhere the GRIN lens in the proper location with UV resin; the reflective part represents the UV resin. (C) Attach the headplate on the mouse skull using UV resin. (D) Cover the GRIN lens with a 3D printed protective cap. (E) Check the surgery quality. The miniscope was fixed by a holder. (F) Apply the denture base materials around the base plate. <a href="https://www.jove.com/files/ftp_upload/66916/66916fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microscopy images of GCaMP6f expression and blood vessels in a biological imaging study." class="xfigimg" src="/files/ftp_upload/66916/66916fig05.jpg" data-altupdatedat="1788304762000" data-alt="Figure 5"> 
Figure 5: Successful and unsuccessful imaging results. (A) Unsuccessful in vivo calcium imaging result. There are no active cells in A and multiple regions have dark blood stains as indicated by the black arrows. (B-D) Successful in vivo calcium imaging results. (B) Less than 10 active cells can be observed but no significant blood vessels in the imaging field. (C) More than 50 active cells and significant blood vessels can be observed. (D) Hundreds of active cells and more significant blood vessels can be observed. The dashed circles represent the imaging field of view. The green arrows represent the cells expressing GCaMP6f. The red arrows represent the blood vessels. Scale bars = 250 µm. <a href="https://www.jove.com/files/ftp_upload/66916/66916fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Neural activity analysis using miniature microscopy in mouse; includes cell imaging, signals, movement path." class="xfigimg" src="/files/ftp_upload/66916/66916fig06.jpg" data-altupdatedat="1788304762000" data-alt="Figure 6"> 
Figure 6: Representative in vivo calcium imaging data from a mouse running on the linear track. (A) The imaging field of view recording by the miniscope, total cell number = 227. (B) Representative calcium traces. The locations of the cell are shown in the same color in A. (C) Behavioral task: Linear track with water rewards at both ends. (D) Representative result of the behavioral analysis of the mouse's activity; total time = 100 s. Blue vertical lines indicate time points of the licking behavior. <a href="https://www.jove.com/files/ftp_upload/66916/66916fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Neural activity analysis, fluorescence microscopy, DAPI and GCaMP6f labeling, brain section." class="xfigimg" src="/files/ftp_upload/66916/66916fig07.jpg" data-altupdatedat="1788304762000" data-alt="Figure 7"> 
Figure 7: Brain tissue sections from mice after surgery. (A,B) Two examples of mouse brain tissues. The dashed rectangular areas indicate the GRIN lens implantation path. The curved lines represent the dentate gyrus in the hippocampus. Scale bars = 500 µm. <a href="https://www.jove.com/files/ftp_upload/66916/66916fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Problem</td>
			<td>Possible cause</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>The imaging field appears dark or black.</td>
			<td>There is a significant amount of residual blood on the underside of the GRIN lens.</td>
			<td>Prior to implanting the GRIN lens, the saline solution is used to continuously flush the surface of the surgical site until there is no longer any observable bleeding.</td>
		</tr>
		<tr>
			<td rowspan="5">There are clearly visible blood vessels present in the imaging field, but no discernible neuronal activity.</td>
			<td>1. The virus expression was suboptimal or poor.</td>
			<td>1. It would be advisable to check the following:</td>
		</tr>
		<tr>
			<td>2. The location of the observed virus expression does not match the position where the GRIN lens was implanted (generally, the implantation position of the GRIN lens is too shallow compared to the desired location).</td>
			<td>a. Ensure the virus storage conditions are appropriate.</td>
		</tr>
		<tr>
			<td></td>
			<td>b. Verify the virus injection location is correct.</td>
		</tr>
		<tr>
			<td></td>
			<td>c. Confirm the virus concentration is suitable.</td>
		</tr>
		<tr>
			<td></td>
			<td>2. During the next surgical procedure, the depth of the GRIN lens implantation can be increased appropriately, and the exact depth should be carefully recorded each time the experiment is conducted.</td>
		</tr>
		<tr>
			<td rowspan="2">The imaging field shows some regional patterns of neuronal activity, but there is no clearly visible activity at the individual cell level.</td>
			<td>1. The volume of injected virus was excessively high.</td>
			<td>1. Dilute the virus or reduce the volume of the virus injection.</td>
		</tr>
		<tr>
			<td>2. The location of the virus expression does not align with the position of the GRIN lens (typically, the GRIN lens is implanted at a shallower depth than the optimal position for the virus expression).</td>
			<td>2. During the next surgical procedure, the depth of the GRIN lens implantation can be increased appropriately, and the exact depth should be carefully recorded each time the experiment is conducted.</td>
		</tr>
		<tr>
			<td>There are certain cells within the imaging field that maintain a consistently bright fluorescence signal at all times, and there is no change in the intensity of this fluorescence.</td>
			<td>The virus is overexpressed within a single cell.</td>
			<td>Dilute the virus or reduce the volume of the virus injection.</td>
		</tr>
	</tbody>
</table>
Table 1: Troubleshooting chart. <a href="https://www.jove.com/files/ftp_upload/66916/66916_Table%201.xlsx" target="_blank">Please click here to download this Table.</a>
Supplemental Figure S1: Normal acquisition of the spatial memory task by mice that have undergone surgery. The graph shows the quantification of the number of completed trials in the 20 min session each day. Data are represented as mean ± SEM. N = 3 mice in with-surgery group and 4 in without-surgery group. Two-way ANOVA was used for statistical comparison. Abbreviation: ns = not significant. <a href="https://www.jove.com/files/ftp_upload/66916/Supplemental%20Figure%20S1.zip" target="_blank">Please click here to download this File.</a>
Supplemental Video S1: Representative raw data of calcium imaging in DG. This video demonstrates calcium activity over a 5 min period. The video is played at 10x of the original speed. <a href="https://www.jove.com/files/ftp_upload/66916/Supplemental%20Video%20S1.zip" target="_blank">Please click here to download this File.</a>
Supplemental File 1: The design of the headplate. This file outlines the 2D drawing of the headplate design. The production of the headplate involves cutting of stainless steel according to the drawing. The two small holes on the sidebar should be tapped with M1.6 threads. <a href="https://www.jove.com/files/ftp_upload/66916/Supplemental%20File%201.zip" target="_blank">Please click here to download this File.</a>
Supplemental File 2: The design of the 3D printed protective cap. This file provides the gcode of the protective cap slices that can be used with a 3D printer. In this protocol, we used polylactic acid plastic as the raw material for the printing. The small holes on the side match the positions of tapped holes on the headplate for installation. <a href="https://www.jove.com/files/ftp_upload/66916/Supplemental%20File%202.zip" target="_blank">Please click here to download this File.</a>
Supplemental File 3: The design of the miniscope holder. This file provides the gcode of the miniscope holder slices that can be used with a 3D printer. The cylindrical hole is used for installation to a stereotaxic holder with a M4 screw. The holes on the side are intended to add screws for tightening, but the holder can be used without this feature. <a href="https://www.jove.com/files/ftp_upload/66916/Supplemental%20File%203.zip" target="_blank">Please click here to download this File.</a>
Supplemental File 4: Arduino control code and scripts for raw calcium imaging video preprocessing. This file contains the scripts for controlling Arduino and the code for pre-processing calcium imaging data. The Arduino code controls the behavior setup in which the mouse learns to lick for water rewards at alternating ends of a linear track. Upon learning the task, the mice will repeatedly run back and forth on the track. The calcium imaging preprocessing code performs file format conversion, motion correction, spatial down-sample, and cell signal extraction from the data collected by the miniscope. <a href="https://www.jove.com/files/ftp_upload/66916/66916_Supplemental%20File%204.zip" target="_blank">Please click here to download this File.</a>

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

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

Source: Shannonhouse, J., et al.In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia. J. Vis. Exp. (2023).The video demonstrates an in vivo calcium imaging technique to monitor neuronal activity in the dorsal root ganglia (DRG) of a transgenic mouse. DRG neurons express a calcium indicator that increases fluorescence intensity upon binding to calcium. Mechanical or thermal stimuli applied to a hind paw generate action potentials that...

In Vivo Calcium Imaging of Neuronal Ensembles in the Intact Dorsal Root Ganglia of a Mouse

Source: Calvo-Rodríguez, et al. Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons. J. Vis. Exp. (2015). This video demonstrates the use of fluorescence imaging to measure cytosolic calcium levels in short-term and long-term rat hippocampal cultures. By comparing young and aged neurons, it highlights how increased L-type voltage-gated calcium channel density and prolonged activation in aged neurons result in calcium overloading, a hallmark of neuronal aging.

In Vitro Calcium Imaging in Short-Term and Long-Term Rat Hippocampal Cultures

Source: Lee, H. et. al., Successful In vivo Calcium Imaging with a Head-Mount Miniaturized Microscope in the Amygdala of Freely Behaving Mouse. J. Vis. Exp. (2020)This video demonstrates in vivo calcium imaging in a mouse using a gradient refractive index, or GRIN lens, and a miniaturized microscope. The setup combines head fixation within a frictionless mobile cage system, stimulating neurons and triggering calcium ion influx. The GRIN lens captures calcium signaling in the brain, enabling...

Real-Time Microendoscopic Calcium Imaging in a Freely Moving Mouse

Here we present how to expose the geniculate ganglion of a live, anesthetized laboratory mouse and how to use calcium imaging to measure the responses of ensembles of these neurons to taste stimuli, allowing for multiple trials with different stimulants. This allows for in depth comparisons of which neurons respond to which tastants.

In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

In vivo Calcium Imaging in Mouse Inferior Olive

Miniscope in vivo calcium imaging is a powerful technique to study neuronal dynamics and microcircuits in freely behaving mice. This protocol describes performing brain surgeries to achieve good in vivo calcium imaging using a miniscope.

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

The present protocol outlines in vivo calcium imaging for measuring the responses of ensembles of lumbar-6 DRG neurons to somatic and visceral stimuli. Thorough comparisons can be made among neurons responding to different stimuli. This protocol is valuable for investigating mechanisms of visceral pain and somatic stimulation, such as acupuncture.

In Vivo Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli

Genetically encoded calcium indicators (GECI) enable a robust, population-level analysis of sensory neuron signaling. Here, we have developed a novel approach that allows for in vivo GECI visualization of rat trigeminal ganglia neuron activity.

In-Vivo Calcium Imaging of Sensory Neurons in the Rat Trigeminal Ganglion

This protocol describes in vivo GCaMP calcium imaging of intact trigeminal ganglion (TG) neurons. This description includes TG exposure surgery, in vivo confocal microscopy imaging of TG neurons, application of somatic stimuli during TG neurons microscopic imaging, and analysis of in vivo GCaMP calcium imaging data.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Trigeminal Ganglia

Learn in vivo calcium imaging of granule cells in the mouse dentate gyrus using a combined virus injection and GRIN lens implantation procedure. This streamlined surgical approach enhances data quality for studying neural coding and spatial representations in cognitive functions.

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

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