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 

<ol>
	<li>Scoville, W. B., Milner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+recent+memory+after+bilateral+hippocampal+lesions.">Loss of recent memory after bilateral hippocampal lesions.</a> J Neurol Neurosurg Psychiatry. 20 (1), 11-21 (1957).</li><li>Spiers, H. J., Burgess, N., Hartley, T., Vargha-Khadem, F., O'keefe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bilateral+hippocampal+pathology+impairs+topographical+and+episodic+memory+but+not+visual+pattern+matching.">Bilateral hippocampal pathology impairs topographical and episodic memory but not visual pattern matching.</a> Hippocampus. 11 (6), 715-725 (2001).</li><li>Kandel, E. R., Spencer, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+neurophysiological+approaches+in+the+study+of+learning.">Cellular neurophysiological approaches in the study of learning.</a> Physiol Rev. 48 (1), 65-134 (1968).</li><li>Zemla, R., Basu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+function+in+rodents.">Hippocampal function in rodents.</a> Curr Opin Neurobiol. 43, 187-197 (2017).</li><li>Basu, J., Siegelbaum, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+corticohippocampal+circuit,+synaptic+plasticity,+and+memory.">The corticohippocampal circuit, synaptic plasticity, and memory.</a> Cold Spring Harb Perspect Biol. 7 (11), a021733 (2015).</li><li>Gobbo, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+signature+of+spatial+decision-making+during+navigation+by+freely+moving+rats+by+using+calcium+imaging.">Neuronal signature of spatial decision-making during navigation by freely moving rats by using calcium imaging.</a> Proc Natl Acad Sci U S A. 119 (44), e2212152119 (2022).</li><li>Schuette, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gabaergic+ca1+neurons+are+more+stable+following+context+changes+than+glutamatergic+cells.">Gabaergic ca1 neurons are more stable following context changes than glutamatergic cells.</a> Sci Rep. 12 (1), 10310 (2022).</li><li>Daumas, S., Halley, H., Franc&#233;s, B., Lassalle, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encoding,+consolidation,+and+retrieval+of+contextual+memory:+Differential+involvement+of+dorsal+ca3+and+ca1+hippocampal+subregions.">Encoding, consolidation, and retrieval of contextual memory: Differential involvement of dorsal ca3 and ca1 hippocampal subregions.</a> Learn Mem. 12 (4), 375-382 (2005).</li><li>Ognjanovski, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erratum:+Parvalbumin-expressing+interneurons+coordinate+hippocampal+network+dynamics+required+for+memory+consolidation.">Erratum: Parvalbumin-expressing interneurons coordinate hippocampal network dynamics required for memory consolidation.</a> Nat Commun. 8, 16120 (2017).</li><li>Hainmueller, T., Bartos, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+emergence+of+stable+and+dynamic+memory+engrams+in+the+hippocampus.">Parallel emergence of stable and dynamic memory engrams in the hippocampus.</a> Nature. 558 (7709), 292-296 (2018).</li><li>Yassa, M. A., Stark, C. E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+separation+in+the+hippocampus.">Pattern separation in the hippocampus.</a> Trends Neurosci. 34 (10), 515-525 (2011).</li><li>Ryan, T. J., Roy, D. S., Pignatelli, M., Arons, A., Tonegawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory.+Engram+cells+retain+memory+under+retrograde+amnesia.">Memory. Engram cells retain memory under retrograde amnesia.</a> Science. 348 (6238), 1007-1013 (2015).</li><li>Manahan-Vaughan, D., Reymann, K. G., Brown, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electrophysiological+investigations+into+the+role+of+histamine+in+the+dentate+gyrus+of+the+rat.">In vivo electrophysiological investigations into the role of histamine in the dentate gyrus of the rat.</a> Neuroscience. 84 (3), 783-790 (1998).</li><li>Kim, S., Jung, D., Royer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Place+cell+maps+slowly+develop+via+competitive+learning+and+conjunctive+coding+in+the+dentate+gyrus.">Place cell maps slowly develop via competitive learning and conjunctive coding in the dentate gyrus.</a> Nat Commun. 11 (1), 4550 (2020).</li><li>Danielson, N. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+dentate+gyrus+mossy+cells+in+behaving+mice.">In vivo imaging of dentate gyrus mossy cells in behaving mice.</a> Neuron. 93 (3), 552-559.e4 (2017).</li><li>Chen, T. -. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasensitive+fluorescent+proteins+for+imaging+neuronal+activity.">Ultrasensitive fluorescent proteins for imaging neuronal activity.</a> Nature. 499 (7458), 295-300 (2013).</li><li>Barnett, L. M., Hughes, T. E., Drobizhev, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+the+molecular+mechanism+responsible+for+gcamp6m's+ca2+-dependent+change+in+fluorescence.">Deciphering the molecular mechanism responsible for gcamp6m's ca2+-dependent change in fluorescence.</a> PLoS One. 12 (2), e0170934 (2017).</li><li>Ghosh, K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniaturized+integration+of+a+fluorescence+microscope.">Miniaturized integration of a fluorescence microscope.</a> Nat Methods. 8 (10), 871-878 (2011).</li><li>Pnevmatikakis, E. A., Giovannucci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normcorre:+An+online+algorithm+for+piecewise+rigid+motion+correction+of+calcium+imaging+data.">Normcorre: An online algorithm for piecewise rigid motion correction of calcium imaging data.</a> J Neurosci Methods. 291, 83-94 (2017).</li><li>Inan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+statistically+robust+cell+extraction+from+large-scale+neural+calcium+imaging+datasets.">Fast and statistically robust cell extraction from large-scale neural calcium imaging datasets.</a> bioRxiv. , (2021).</li><li>Thapa, R., Liang, B., Liu, R., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotaxic+viral+injection+and+gradient-index+lens+implantation+for+deep+brain+in+vivo+calcium+imaging.">Stereotaxic viral injection and gradient-index lens implantation for deep brain in vivo calcium imaging.</a> J Vis Exp. (176), (2021).</li><li>Wirtshafter, H. S., Disterhoft, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+multi-day+calcium+imaging+of+ca1+hippocampus+in+freely+moving+rats+reveals+a+high+preponderance+of+place+cells+with+consistent+place+fields.">In vivo multi-day calcium imaging of ca1 hippocampus in freely moving rats reveals a high preponderance of place cells with consistent place fields.</a> J Neurosci. 42 (22), 4538-4554 (2022).</li><li>Masala, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+hippocampal+Ca2++micro-waves+following+synapsin-dependent+adeno-associated+viral+expression+of+Ca2++indicators.">Aberrant hippocampal Ca2+ micro-waves following synapsin-dependent adeno-associated viral expression of Ca2+ indicators.</a> bioRxiv. , (2024).</li><li>Liang, B., Zhang, L., Moffitt, C., Li, Y., Lin, D. -. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+open-source+automated+surgical+instrument+for+microendoscope+implantation.">An open-source automated surgical instrument for microendoscope implantation.</a> J Neurosci Methods. 311, 83-88 (2019).</li><li>Hsiao, Y. -. T., Wang, A. Y. -. C., Lee, T. -. Y., Chang, C. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+baseplating+and+a+miniscope+preanchored+with+an+objective+lens+for+calcium+transient+research+in+mice.">Using baseplating and a miniscope preanchored with an objective lens for calcium transient research in mice.</a> J Vis Exp. (172), e62611 (2021).</li><li>Barbera, G., Liang, B., Zhang, L., Li, Y., Lin, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+wireless+miniscope+for+deep+brain+imaging+in+freely+moving+mice.">A wireless miniscope for deep brain imaging in freely moving mice.</a> J Neurosci Methods. 323, 56-60 (2019).</li><li>Cholvin, T., Bartos, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemisphere-specific+spatial+representation+by+hippocampal+granule+cells.">Hemisphere-specific spatial representation by hippocampal granule cells.</a> Nat Commun. 13 (1), 6227 (2022).</li></ol>

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.

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 ...

Author Spotlight: Investigating Neural Activity of Dentate Gyrus Granule Cells with Miniature Microscope

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.

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 ...

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

Research

JoVE Journal

Neuroscience

1.2K 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.

Video: Author Spotlight: Investigating Neural Activity of Dentate Gyrus Granule Cells with Miniature Microscope

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Preparation of Acute Slices from Dorsal Hippocampus for Whole-Cell Recording and Neuronal Reconstruction in the Dentate Gyrus of Adult Mice

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in molecular, anatomical and functional criteria suggesting a division into different sub-circuits along its rostro-caudal extent. Owing to differential connectivity and function the most fundamental distinction is made between the dorsal and the ventral hippocampus, which are preferentially involved in spatial and emotional processing, respectively. Accordingly, in vivo work regarding spatial memory formation has focused on the dorsal hippocampus.
In contrast, electro-physiological in vitro recordings have been preferentially performed on intermediate-ventral hippocampus, largely motivated by factors like slice viability and circuit integrity. To allow for direct correlation of in vivo data on spatial processing with in vitro data we have adapted previous sectioning methods to obtain highly viable transverse brain slices from the dorsal-intermediate hippocampus for long-term recordings of principal cells and interneurons in the dentate gyrus. As spatial behavior is routinely analyzed in adult mice, we have combined this transversal slicing procedure with the use of protective solutions to enhance viability of brain tissue from mature animals. We use this approach for mice of about 3 months of age. The method offers a good alternative to the coronal preparation which is frequently used for in vitro studies on dorsal hippocampus. We compare these two preparations in terms of quality of recordings and preservation of morphological features of recorded neurons.

We present a protocol to prepare acute-slices from the dorsal-intermediate hippocampus of mice. We compare this transversal preparation with coronal slicing in terms of quality of recordings and preservation of morphological features of recorded neurons.

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

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.