Name: craniotomy procedure for visualizing neuronal activities in hippocampus of behaving mice
Uploaded: 2021-07-24T14:00:00
Duration: 12 min 16 s
Description: Watch this Scientific Journal Video about Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice at JoVE.com

We would like to thank all the members of the Molecular BioEngineering Group at Westlake University for all the help and useful discussion. We also thank Jinze Li and Jie-Min Jia from Westlake University for the help with filming the surgical procedure.
This work was supported by start-up funding from the Foundation of Westlake University, 2020 BBRF Young Investigator Grant, and National Natural Science Foundation of China grant 32050410298 all to K.D.P.

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The authors have nothing to disclose.

Here we describe a method for long-term imaging of the hippocampus CA1 region in behaving mice. The method is based on the chronic implantation of a custom-made imaging window, which also enables the targeted administration of viruses or drugs directly to the neurons of interest. The present protocol consists of four major parts: i) assembling of imaging implant; ii) installation of imaging implant; iii) virus injection via imaging implant; iv) functional imaging in behaving mice. Below we describe and discuss critical steps in the protocol, troubleshooting, modifications, and limitations of the method. We also discuss the significance of the method and its potential alternative applications.
There are several critical steps in the described protocol that are rather important for successful surgery: (i) preparation of a high-quality imaging implant; (ii) sterile surgical conditions; (iii) aspiration of the cortex; (iv) precise placing of the imaging implant; (v) viral injection. As Step 1.6 indicates, excess soldering tin would require a larger craniotomy and thus increases the risk of inflammation. It is also very important to use an appropriate amount of the adhesive optical glue when fixing the cover glass to the imaging cannula, as indicated in Step 1.11, as an insufficient amount may result in leakage of cerebrospinal fluid into the imaging cannula and making it opaque. On the other hand, excess optical adhesive can result in the decreased transparency of the glass window. Possible contamination of the imaging implant can cause an active proliferation of connective tissue under the optical window and/or severe inflammation, which will lead to early termination of the experiment. Therefore, imaging implant assembling and preparation before the surgery is almost as important as the surgical procedure itself.
During the surgery, the part of the cortex under craniotomy is ablated by gentle aspiration, which results in inevitable bleeding. Blood at the surgical site significantly reduces the visibility of the brain tissue that must be removed. This complicates the precise assessment of the required depth of tissue ablation. Careful flushing of the surgical site with PBS every time before applying suction to remove the next portion of tissue provides better control of depth. The brain tissue should always be removed in small portions step-by-step confirming the depth of ablated tissue before proceeding with more suction. Finer control of suction can also be achieved with a thinner blunt needle. We suggest using a 26 G needle, however, smaller than 26 G diameter is more prone to clogging. Furthermore, it usually takes lots of practice to determine the precise depth of aspiration required for each animal, as the color of the cortex, corpus callosum, and hippocampus may vary from mouse to mouse (Figure 3).
Insertion and securing of the imaging implant should be done very precisely to ensure the closest possible position of the imaging window to the dorsal surface of the hippocampus. The size of the prepared craniotomy should closely match the implant and allow its insertion without significant resistance. At the same time, there should be no visible gap between the skull and the implant to ensure proper sealing and avoid brain tissue exposure. Gentle and stable pressure should be applied to the top of the implant during its sealing to the skull. It is almost unavoidable to have blood under the imaging window during the implant installation. If the surgical procedure is done properly, the window should clear out in 3-7 days, and brain vasculature becomes clearly visible. It is also important to ensure that virus is properly injected under the window. In the case of failed expression, virus can be reinjected multiple times.
The major complication we encountered in some cases is reduced visibility of the imaging window. There are several possible reasons for poor imaging quality: i) ongoing inflammation; ii) outgrowth of connective tissue on the glass; iii) big gap between the window and hippocampus. Inflammation is usually caused by contamination during surgery or by not properly sterilized imaging implant. We suggest autoclaving the surgical instruments before and after each surgery, disinfect the surgery area right before the procedure, and wear clean personal protective equipment during surgery. Imaging implants should be cleaned after assembling, sterilized, and stored in sterile conditions. The outgrowth of connective tissue on the glass of imaging implant can be due to mechanical contamination on the surface of the glass or excessive trauma of the brain tissue during cortex ablation. After assembling the implant, it is important to confirm the glass surface is clean and smooth. Also, all pieces of damaged brain tissue must be carefully removed before inserting imaging implant into the craniotomy. In certain cases, the gap between the glass window and hippocampus results in the accumulation of cerebrospinal fluid, reducing the quality of imaging. Therefore, during implant installation, it is crucial to insert it all the way in ensuring good contact between hippocampus and glass window. Sometimes, it is difficult to identify the exact reason for opaque imaging window. We suggest performing post-mortem analysis to reveal conditions under the optical window and correspondingly adjust subsequent surgeries.
The method has several fundamental and technical limitations that should be taken into account before and during in vivo imaging. One of the major limitations is cortex ablation. Part of the visual and sensory cortex is removed during the surgery. While it is hard to precisely evaluate the impact of cortex ablation, as removed brain tissue does not directly project onto hippocampus, several studies demonstrated no noticeable impairment of hippocampal-dependent learning or other relevant hippocampus functions15,16. The optical limitations should also be considered, especially when high NA objective lenses are used. For example, in this study, we used a 1.75 mm long cannula with a 1.9 mm inner diameter. The geometry of this cannula will not preserve the full NA of air objective with NA more than ~0.5 or water objective with NA more than ~0.6 as it will clip some of light. Another limitation, common for all brain imaging implants, is that part of the brain is getting exposed, thus promoting heat loss17,18. However, physiological brain temperature can be easily restored during imaging by perfusion of a warm buffer.
The described method can be easily modified or adjusted for other applications. For example, the preparation can be adapted for imaging of striatum7. As the striatum lays slightly deeper than hippocampus, the longer imaging cannula should be used for assembling imaging implant. We suggest using 2.0 mm imaging cannula. The coordinates of the craniotomy should be adjusted correspondingly (AP: +0.8 mm, ML: &#8722;1.8 mm). In addition, virus injection via infusion cannula allows achieving the expression of a transgene in a thin layer of neurons when using AAV serotype with restricted spread19,20. It is particularly beneficial for one-photon imaging due to reduced out-of-focus fluorescence from deeper layers and, as a result, improved single-cell resolution imaging. Furthermore, injection cannula can also be used during functional imaging for the administration of drugs or other chemicals directly onto the neurons in FOV (Figure 5B). Overall infusion cannula adds useful functionalities to the imaging implant, improving imaging quality due to the targeted viral expression and enabling pharmacological stimulation of neurons in FOV. The used head plate provides extraordinary stability of imaging implant minimizing motion artifacts even in actively moving animals on a treadmill. The head plate is small and light, causing minimal discomfort to animals, and remains stable for several months after installation. The imaging implant is also compatible with multiphoton imaging15,16,21 and can be combined with micro-endoscopes22,23. A similar imaging implant was also used for multiphoton imaging of the deeper hippocampus structures, including stratum radiatum, stratum lacunose, and dentate gyrus16,24,25,26,27. However, targeting the deeper hippocampus structures with AAVs via infusion cannula may require further optimization of the AAV serotype and volume19.
We believe that the described protocol will facilitate studies that aim to investigate neuronal activity with high spatiotemporal resolution in the hippocampus of behaving mice using simple and affordable one-photon imaging setups.

The hippocampus is a key brain region responsible for learning and memory1 as well as for spatial navigation2. Hippocampal atrophy is associated with neurological and psychiatric disorders involving memory loss and cognitive decline3,4,5. In mice, the hippocampus is a very well-established model to study spatial, contextual, and associative learning and memory formation on the cellular and network levels4,5. The mechanistic studies of learning and memory require longitudinal interrogation of neuronal structure and function in behaving mice. Fluorescence imaging in combination with genetically encoded probes6 provides unprecedented capabilities to record membrane voltage dynamics7,8, calcium transients9, and structural changes10 over large subsets of neurons intravitally. However, optical access to the hippocampus in mice is obstructed by the cortex, which can reach over 1 mm in thickness. Here, we described a procedure for assembling a custom-made imaging device and its chronic implantation into the mouse head for long-term optical access to the CA1 subregion of the dorsal hippocampus in behaving mice. Infusion cannula integrated into the imaging implant allows administration of viruses or drugs directly onto the neurons in the field of view. The described preparation in combination with wide-field microscopy enables recurrent imaging of the large subsets of neurons in behaving mice over long periods of time. We utilized this preparation to express calcium and voltage genetically encoded indicators in hippocampal CA1 region via targeted injection of recombinant adeno-associated virus (rAAV) for neuronal activity recordings at single-cell resolution. We also performed longitudinal calcium imaging of the corresponding neuronal subsets at high spatiotemporal resolution in behaving animals. In addition, this preparation is compatible with multiphoton microscopy and microendoscopy, thus further expanding the toolbox of imaging techniques to study neuronal networks at cellular and subcellular levels in behaving mice. We described critical steps and troubleshooting of the protocol. We also discussed the possible pitfalls and limitations of the method.

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	<tbody>
		<tr>
			<td>Cover glass</td>
			<td>Deckgl&#228;ser</td>
			<td>72296-03</td>
			<td></td>
		</tr>
		<tr>
			<td>denture&#160;base resin</td>
			<td>ShangHai New Centery Dentel Material</td>
			<td>N/A</td>
			<td>Type I, self-solidifying</td>
		</tr>
		<tr>
			<td>Dummy Cannula</td>
			<td>RWD Life Science Co.,LTD</td>
			<td>62102</td>
			<td>OD 0.30mm</td>
		</tr>
		<tr>
			<td>Guide Cannula</td>
			<td>RWD Life Science Co.,LTD</td>
			<td>62003</td>
			<td>26G</td>
		</tr>
		<tr>
			<td>Head Fork</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Custom made</td>
		</tr>
		<tr>
			<td>Head Plate</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Custom made</td>
		</tr>
		<tr>
			<td>Imaging Cannula</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Custom Made; OD 3mm, ID 2.7mm, Height 1.8mm, #108 stainless steel</td>
		</tr>
		<tr>
			<td>Internal Cannula</td>
			<td>RWD Life Science Co.,LTD</td>
			<td>62203</td>
			<td>&#160;0.30*0.14 (OD*ID, mm)</td>
		</tr>
		<tr>
			<td>Kwik Sil</td>
			<td>World Precision Instruments</td>
			<td>KWIK-SIL</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperBond C&#38;B</td>
			<td>SUN MEDICAL</td>
			<td>N/A</td>
			<td>SuperBond C&#38;B kit</td>
		</tr>
		<tr>
			<td>Treadmill Kit</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Custom made</td>
		</tr>
		<tr>
			<td>UV-cured Adhesive</td>
			<td>NORLAND PRODUCTS</td>
			<td>NOA 60</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In vivo imaging of neuronal activity using a genetically encoded calcium indicator. On average, in vivo imaging starts 3-4 weeks after implantation if a sufficient level of transgene expression is achieved. By this time, cerebral edema and hemorrhage are usually completely resolved, and brain vasculature can be easily observed through the optical window. Here we utilized the described preparation to perform the repeated recordings of neuronal activity in the dorsal hippocampal CA1 region in behaving mice under fluorescence wide-field microscope. To record neuronal activity, we used a bright genetically encoded calcium indicator, named NCaMP713, which exhibits similar calcium sensitivity and temporal resolution to that of GCaMP6s14. To express the NCaMP7 indicator in the hippocampus, we injected the rAAV/DJ-CAG-NCaMP7 virus using an infusion cannula and initiated longitude imaging at 14 days post-injection. To record neuronal activity, we used a 10x NA 0.3 air objective lens and Hamamatsu OrcaFusion sCMOS camera that allowed imaging at ~1.5x1.5 mm FOV at up to 100 Hz frequency. Green fluorescence was excited by a commercially available 470 nm LED using a standard GFP filter set. The average depth of imaging achieved in green channel is about 50-120 &#181;m, which allows recording of neuronal activity mainly in stratum oriens and stratum pyramidale. Imaging depth in near-infrared channels can be up to 200 &#181;m reaching the deeper layers of hippocampus8. The average recording time per FOV was 6-12 min, although much longer imaging sessions are possible as NCaMP7 is characterized by extremely high photostability and no detectable phototoxicity was observed (Figure 6).
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62266/62266fig06.jpg" /> 
Figure 6: Recording of the neuronal activity in the hippocampal neurons using a green fluorescence genetically encoded calcium indicator. (A) A selected FOV imaged under a wide-field fluorescence microscope in green channel. (B) The 15 ROIs corresponding to the single neurons shown in A and selected using the standard deviation projection of the whole recording. (C) Representative fluorescence single-trial traces of the 15 selected neurons in B. (D) A representative zoom-in view of 2 calcium traces shown in corresponding color boxes shown in C. Scale bar, 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/62266/62266fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To obtain fluorescence traces, the regions of interest (ROIs) corresponding to neuronal somas were segmented manually and analyzed by ImageJ software. Prior to the image analysis motion correction, common post-recording procedures in awake animals were not required as the acquired datasets did not exhibit motion artifacts due to the high stability of imaging implant. A representative single-trial optical recording of neuronal activities from the hippocampus in an awake behaving mouse is presented in Figure 6. 15 ROIs corresponding to neuronal somas were selected manually from the same FOV shown in Figure 6B, and the single-trial fluorescence traces within each ROI are shown in Figure 6C. Figure 6D shows two representative parts of fluorescence traces from two different ROIs. We performed 4 consecutive imaging sessions for the same FOV with 3-day intervals. It was possible to identify and image the same neurons in certain FOVs for at least two weeks (the longer imaging sessions were not performed for this study, however, the same preparation has been used for up to 6 month-long imaging study in mice previously7; Figure 7). In this study, we used AAV/DJ-CAG vector, which was driving a strong expression of the gene of interest even 21 days after virus delivery (Supplementary Figure 1). The continuous expression complicated long-term identification of the same neurons due to increased fluorescence background and appearance of new neurons expressing the calcium indicator. Therefore, selection of the AAV serotype and promoter to drive target gene expression should be one of the important considerations during experimental design in particular if longitudinal imaging of the same subset of neurons is required. The imaging quality allowed to resolve proximal dendrites as well as visualize blood vessels.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62266/62266fig07v2.jpg" /> 
Figure 7: Image sequence of four different FOVs from the hippocampal area tracked over 12 days. The animal was implanted with the window on Day 0 and was injected with the rAAV/DJ-CAG-NCaMP7 virus on Day 7. Arrowheads indicate the neuron tracked within the FOV. Scale bars: 80 &#181;m. <a href="https://www.jove.com/files/ftp_upload/62266/62266fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In vivo imaging of neuronal activity using a genetically encoded voltage sensor. 
In this study, we also used a novel genetically encoded voltage sensor, named SomArchon7, which allows for voltage imaging with single-cell single-spike resolution in behaving animals7,8. To express SomArchon, we injected rAAV/DJ-CAG-SomArchon virus using an infusion cannula and performed voltage imaging in a head-fixed behaving mouse several days post-injection. To record neuronal activity, we used a 40x NA 0.8 objective lens and Hamamatsu OrcaFusion sCMOS camera that allowed us to image 150x40 &#181;m FOV at up to 830 Hz acquisition rate. The GFP protein, which a part of SomArchon construct to facilitate visualization of expression in the visible range of the spectrum, can be easily imaged in the green channel (LED excitation at 470/20 nm, emission 525/50 nm) to locate cells of interest for voltage imaging. Optical voltage recordings were performed in the near-infrared channel (laser excitation 637 nm at 3.4 W/mm2, emission 665 nm long pass) with 4 x 4 binning at 830 Hz acquisition rate. We recorded the spontaneous activity of a hippocampal neuron in an awake mouse with average SNR of 7 per action potential (Figure 8). 
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/62266/62266fig08.jpg" /> 
Figure 8: Recording of the neuronal activity in the hippocampal neurons using a near-infrared fluorescence genetically encoded voltage indicator SomArchon. (A) A selected FOV imaged under wide-field fluorescence microscope in the near-infrared channel. (B) Single-trial fluorescence trace of the neuron in A. (C) A representative zoom-in view of the voltage trace in the corresponding box shown in B. Scale bar: 25 &#181;m. <a href="https://www.jove.com/files/ftp_upload/62266/62266fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Histology 
After functional imaging study is done, post-mortem analysis is used to confirm the correct placement of the implant, the area of virus expression, and localization of a protein of interest in imaged neurons. For histological verification of virus expression and placement of the implant, coronal sections of the PFA fixed brain were examined under a fluorescence wide-field microscope (Figure 9A, C). A confocal microscope was used to acquire high-resolution images of individual neurons expressing the calcium indicator, as well as the voltage indicator (Figure 9B, D). DAPI staining was used to visualize the overall morphology of the brain slice. In addition, the brain slices can be assessed using immunohistochemistry to verify astrogliosis or gliosis caused by window implantation and viral expression. Our previous studies demonstrated that the procedure did not induce noticeable gliosis7.
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/62266/62266fig09.jpg" /> 
Figure 9:&#160;Histological verification of the optical window position and virus expression. (A) A representative fluorescent image of the coronal section brain slice showing the placement of the optical window from an NCaMP-expressing mouse. Scale bar: 1 mm. (B) A representative confocal image of neurons expressing the NCaMP7 indicators. Scale bar: 25 &#181;m. (C) A representative fluorescence image of the coronal section brain slice showing the placement of the optical window from a SomArchon-expressing mouse. Scale bar: 1 mm. (D) A representative confocal image of neurons expressing the SomArchon indicators. Scale bar: 25 &#181;m. <a href="https://www.jove.com/files/ftp_upload/62266/62266fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Quantitive analysis of relative fluorescence intensity along with expression time. <a href="https://www.jove.com/files/ftp_upload/62266/Supplementary Figure 1.pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice at JoVE.com

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

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 ...

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