Name: in vivo calcium imaging of granule cells in the dentate gyrus of hippocampus in mice
Uploaded: 2024-08-02T14:30:00.000+00:00
Duration: 7 min
Description: Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal ...

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 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><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

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.
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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

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JoVE Journal

Neuroscience

1.3K Views.  Fudan University. Welnu, in dit protocol gebruiken we een miniatuurmicroscoop om de neurale activiteiten in de dentate gyrus van de hippocampus te onderzoeken. We willen de codering van neuronen en ruimtelijke representaties tijdens de verschillende ziektetoestanden onderzoeken. Ik geloof dat de belangrijkste uitdaging bij het bestuderen van de gyrus dentatus in vivo het verkrijgen van hoogwaardige calciumbeeldvormingsgegevens is, of de virusexpressie bevredigend is, of de gra...

Video: Auteur Spotlight: Onderzoek naar neurale activiteit van getande gyruskorrelcellen met miniatuurmicroscoop

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

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 . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

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 .

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Biology

Successful In vivo Calcium Imaging with a Head-Mount Miniaturized Microscope in the Amygdala of Freely Behaving Mouse

In vivo real-time monitoring of neuronal activities in freely moving animals is one of key approaches to link neuronal activity to behavior. For this purpose, an in vivo imaging technique that detects calcium transients in neurons using genetically encoded calcium indicators (GECIs), a miniaturized fluorescence microscope, and a gradient refractive index (GRIN) lens has been developed and successfully applied to many brain structures1,2,3,4,5,6. This imaging technique is particularly powerful because it enables chronic simultaneous imaging of genetically defined cell populations for a long-term period up to several weeks. Although useful, this imaging technique has not been easily applied to brain structures that locate deep within the brain such as amygdala, an essential brain structure for emotional processing and associative fear memory7. There are several factors that make it difficult to apply the imaging technique to the amygdala. For instance, motion artifacts usually occur more frequently during the imaging conducted in the deeper brain regions because a head-mount microscope implanted deep in the brain is relatively unstable. Another problem is that the lateral ventricle is positioned close to the implanted GRIN lens and its movement during respiration may cause highly irregular motion artifacts that cannot be easily corrected, which makes it difficult to form a stable imaging view. Furthermore, because cells in the amygdala are usually quiet at a resting or anesthetized state, it is hard to find and focus the target cells expressing GECI in the amygdala during baseplating procedure for later imaging. This protocol provides a helpful guideline for how to efficiently target cells expressing GECI in the amygdala with head-mount miniaturized microscope for successful in vivo calcium imaging in such a deeper brain region. It is noted that this protocol is based on a particular system (e.g., Inscopix) but not restricted to it.

In vivo microendoscopic calcium imaging is an invaluable tool that enables real-time monitoring of neuronal activities in freely behaving animals. However, applying this technique to the amygdala has been difficult. This protocol aims to provide a useful guideline for successfully targeting amygdala cells with a miniaturized microscope in mice.

In vivo real-time monitoring of neuronal activities in freely moving animals is one of key approaches to link neuronal activity to behavior. For this ...

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 of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using calcium as a proxy for neuronal activity, these techniques provide a way to monitor the responses of individual cells within large neuronal ensembles to a variety of stimuli in real time. We, and others, have applied these techniques to image the responses of individual geniculate ganglion neurons to taste stimuli applied to the tongues of live anesthetized mice. The geniculate ganglion is comprised of the cell bodies of gustatory neurons innervating the anterior tongue and palate as well as some somatosensory neurons innervating the pinna of the ear. Imaging the taste-evoked responses of individual geniculate ganglion neurons with GCaMP has provided important information about the tuning profiles of these neurons in wild-type mice as well as a way to detect peripheral taste miswiring phenotypes in genetically manipulated mice. Here we demonstrate the surgical procedure to expose the geniculate ganglion, GCaMP fluorescence image acquisition, initial steps for data analysis, and troubleshooting. This technique can be used with transgenically encoded GCaMP, or with AAV-mediated GCaMP expression, and can be modified to image particular genetic subsets of interest (i.e., Cre-mediated GCaMP expression). Overall, in vivo calcium imaging of geniculate ganglion neurons is a powerful technique for monitoring the activity of peripheral gustatory neurons and provides complementary information to more traditional whole-nerve chorda tympani recordings or taste behavior assays.

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.

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using ...

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.

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 ...

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal excitability. Recent studies have revealed the differential gene expression and functional heterogeneity of microglia in different brain regions. The unique functions of the hippocampal neural network in learning and memory may be associated with the active roles of microglia in synapse remodeling. However, inflammatory responses induced by surgical procedures have been problematic in the two-photon microscopic analysis of hippocampal microglia. Here, a method is presented that enables the chronic observation of microglia in all layers of the hippocampal CA1 through an imaging window. This method allows the analysis of morphological changes in microglial processes for more than 1 month. Long-term and high-resolution imaging of the resting microglia requires minimally invasive surgical procedures, appropriate objective lens selection, and optimized imaging techniques. The transient inflammatory response of hippocampal microglia may prevent imaging immediately after surgery, but the microglia restore their quiescent morphology within a few weeks. Furthermore, imaging neurons simultaneously with microglia allows us to analyze the interactions of multiple cell types in the hippocampus. This technique may provide essential information about microglial function in the hippocampus.

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

This paper describes a method for chronic in vivo observation of the resting microglia in the mouse hippocampal CA1 using precisely controlled surgery and two-photon microscopy.

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal ...

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the ...

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

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

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

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.

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

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

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