Name: stereotaxic viral injection and gradient-index lens implantation for deep brain in vivo calcium imaging
Uploaded: 2021-10-08T14:00:00
Duration: 11 min 11 s
Description: Watch this Scientific Journal Video about Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging at JoVE.com

This work is supported by grants from the National Institute of Health (NIH) 5P20GM121310, R61NS115161, and UG3NS115608.

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The authors report no competing financial interests.

A central question in neuroscience is understanding how neural dynamics and circuits encode and store information, and how they are altered in brain diseases. Using a miniscope in vivo Ca2+ imaging system, individual neural activity from several hundreds of neurons within a local microcircuit can be simultaneously monitored from a freely behaving animal. Here, a detailed surgery protocol for viral injection and GRIN lens implantation is described to prepare rodents for a deep brain in vivo Ca2+ imaging via a custom-developed miniscope recording system. Table 1 shows the comparisons of our miniscope system with other commercially available and custom-built miniscope systems7,8,9,10,11,13,25,26. It is worth noting that GRIN lens implantation using the present surgical protocol is compatible with any commercial or custom-built single-photon and two-photon imaging systems for a deep brain in vivo calcium imaging.
From viral injection to data acquisition of miniscope in vivo calcium imaging, the entire experimental procedure takes at least 2 months to complete. It is a complicated and labor-intensive process. The ultimate success of the experiment depends on multiple factors, including proper choice of GECIs, injection of virus accurately in the targeted brain area, sufficient viral expression in the desired neural population, implantation of GRIN lens precisely in the desired location, adequate recovery from surgeries, as well as, whether severe inflammation occurs post-surgery, and whether animal&#39;s behavior is severely affected by surgeries, and so on.
Two critical steps include stereotaxic injection of virus and GRIN lens implantation. For the demonstration purpose, the stereotaxic microinjection was performed in the mouse mPFC, with adeno-associated virus (AAV1) encoding GCaMP6f under the control of CaMKII promoter that selectively labels pyramidal neurons in the mPFC. GCaMP6f was chosen as it is one of the fastest and most sensitive calcium indicators with a half-decay time of 71 ms15. In addition, AAV viral expression of GCaMP6f is long-lasting (i.e., several months), making it ideal for performing repetitive in vivo Ca2+ imaging over a long period for longitudinal studies in mouse models of neurodegenerative diseases27. The current surgery protocol can be adapted for targeting different cell populations in any other brain region. Various available viral tools allow selective labeling of specific neural populations in the desired brain region at the desired age. In addition, researchers can take advantage of the Cre-LoxP recombination system and various available transgenic mouse models to carry out genetic modifications and study the behavioral and neural circuitry outcome28,29.
One unique feature of the presented protocol is that the automated layer-by-layer brain tissue aspiration was performed before the GRIN lens (1 mm in diameter) implantation. This is achieved through a 27 G needle connected to a vacuum system, controlled by a custom-built robotic arm and software23. Based on our experience, this method generates a uniform surface for the GRIN lens to contact and causes less damage to the neighboring tissue than manual tissue aspiration23. For this reason, this procedure brings an obvious advantage for GRIN lenses with a relatively wider diameter (e.g., 1 mm). However, tissue aspiration may not be necessary for implanting a GRIN lens with a smaller diameter (0.5 mm or 0.25 mm). Instead, it can be directly planted along the leading track made with a 30 G needle21.
Besides the two critical steps discussed above, many other factors must be carefully considered for a successful operation. (1) All the instruments that contact the brain should be sterilized to prevent infection. (2) All surgery steps need to be performed to minimize damage to the brain to prevent further inflammation and excessive scar tissue formation. (3) The anesthesia doses given initially and maintained during the surgery, especially those administered intraperitoneally, need to be carefully considered. The anesthesia doses may be modified according to different mouse strains, as some may be more susceptible. (4) The condition of the mouse needs to be constantly monitored during surgery. Lastly, (5) the mice need to be regularly monitored post-surgery, as many complications may occur after the surgery.
Although a chunk of brain tissue is removed unilaterally during the GRIN lens implantation step, we did not observe any obvious behavior deficits7,12. The weight of the miniscope is around 2 grams and the cable is custom-designed to make it light and to ensure that the mouse can easily carry it. The miniscope and cable are only attached to the animal prior to&#160;in vivo&#160;imaging and detached after imaging. The entire imaging process usually takes no longer than 30 minutes. Therefore, these instrumentations do not prevent the mouse from freely behaving. The miniscope installation and deinstallation steps need a brief anesthesia (less than 2 minutes) with isoflurane for the purpose of animal restraining. We typically let the mouse recover from the brief exposure of isoflurane for 30 minutes before performing&#160;in vivo&#160;imaging. We have performed miniscope&#160;in vivo&#160;calcium imaging once per week for a few weeks without noticing any impact on mouse health and mouse social behavior12.
One major limitation of the current miniscope recording system is the need to connect the microscope to a cable for data acquisition. The presence of the cable sometimes restricts the mouse task performance and limits the recording of one animal at a time. Recently, a wireless miniscope has been developed25,26. This will broaden the task performance and allow simultaneous in vivo imaging from multiple animals in a group. Moreover, developing more sensitive GECIs with spectrally separable wavelengths combined with a dual-color miniscope will offer more exciting possibilities for neuroscience research.

Intracellular Ca2+ signaling is an essential regulator of cell growth, proliferation, differentiation, migration, gene transcription, secretion, and apoptosis1. In neurons, Ca2+ signaling is precisely controlled since its spatio-temporal pattern is related to crucial functions such as membrane excitability, neurotransmitter release, and synaptic plasticity2.
In vivo calcium imaging is a powerful technique that can be utilized to decode neural circuit representation elemental to normal animal behaviors, identify aberrant neuronal activities in animal models of brain disorders, and unravel potential therapeutic targets that may normalize these altered circuitries. The two common in vivo calcium imaging systems are two-photon laser scanning fluorescence microscopy3,4,5,6 and head-mounted miniaturized microendoscopy (miniscope)7,8,9,10,11,12,13. The conventional two-photon microscopy offers commanding advantages such as better resolution, lower noise, and lower photobleaching; however, the experimental animals are required to be head-fixed, limiting the behavior studies that can be performed3,4,5,6. By contrast, the head-mounted miniscope system is small and portable, making it possible to study a wide variety of behavior tests using freely behaving animals7,8,9,10,11,12,13.
There are two leading Ca2+ indicators, chemical indicators5,14 and genetically encoded calcium indicators (GECIs)15,16. Ca2+ imaging has been facilitated by using highly sensitive GECIs delivered with viral vectors that allow specific labeling of neurons in the targeted circuit. The continuous effort to enhance the sensitivity, longevity, and ability to label even the subcellular compartments, makes GECIs ideal for various in vivo calcium imaging studies17,18,19.
The scattering of light in brain tissue during imaging limits the optical penetration in-depth, even with the two-photon microscopy. However, the Gradient Index (GRIN) lens overcomes this issue as the GRIN lens can be directly embedded into the biological tissues and relay images from the deep brain region to the microscope objective. Unlike conventional lens made of optically homogenous material and requires a complicated shaped surface to focus and create images, GRIN lens performance is based on a gradual refractive index change within the lens material that achieves focus with a plane surface20. GRIN lens can be fabricated down to 0.2 mm in diameter. Therefore, a miniaturized GRIN lens can be implanted into the deep brain without causing too much damage.
In this article, a complete surgery protocol is presented for the deep brain in vivo calcium imaging. For the demonstration purpose, we describe brain surgeries specifically targeting the medial prefrontal cortex (mPFC) of the mouse brain and in vivo calcium imaging recording via a custom-built miniscope system developed by Dr. Lin&#39;s group at the National Institute on Drug Abuse (NIDA/IRP)7,12. The experimental procedure involves two major brain surgeries. The first surgery is stereotaxically injecting a viral vector expressing GCaMP6f (a GECI) in the mPFC. The second surgery is to implant the GRIN lens into the same brain region. After recovery from these brain surgeries, the subsequent procedure is to affix the miniscope holder (base) on the mouse skull using dental cement. In vivo Ca2+ imaging can be performed any time after mounting the miniscope onto its base. The surgery protocol for viral injection and GRIN lens implantation is compatible with any commercial or custom-built single-photon and two-photon imaging systems for the deep brain in vivo calcium imaging.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.6mm and 1.2mm drill burrs</td>
			<td>KF technology</td>
			<td>8000037800</td>
			<td>For craniotomy</td>
		</tr>
		<tr>
			<td>27-G and 30-G needle</td>
			<td>BD PrecisionGlide Needle</td>
			<td>REF 305109 and REF305106</td>
			<td>For both surgeries</td>
		</tr>
		<tr>
			<td>45 angled forceps</td>
			<td>Fine Science tools</td>
			<td>11251-35</td>
			<td>For surgeries</td>
		</tr>
		<tr>
			<td>7.5% povidone-iodine solution (Betadine)</td>
			<td>Purdue Products L.P.</td>
			<td>NDC 67618-151-17</td>
			<td>Surface disinfectant</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>179124-1L</td>
			<td>GRIN lens cleaner</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539-25G</td>
			<td>For GRIN lens implantation</td>
		</tr>
		<tr>
			<td>Antibiotic ointment</td>
			<td>HeliDerm Technology</td>
			<td>81073087</td>
			<td>For virus injection</td>
		</tr>
		<tr>
			<td>Anti-inflamatory drug (Ibuprofen)</td>
			<td>Johnson &#38; Johnson Consumer Inc</td>
			<td>30043308</td>
			<td>Acts as pain killer after surgeries</td>
		</tr>
		<tr>
			<td>AutoStereota</td>
			<td>NIDA/IRP</td>
			<td>github.com/liang-bo/autostereota</td>
			<td>For GRIN lens implantation</td>
		</tr>
		<tr>
			<td>Behavior Recoding Software (Point Grey FlyCap2)</td>
			<td>Point Grey</td>
			<td>Point Grey Research Blackfly BFLY-PGE-12A2C</td>
			<td>For recording behavior</td>
		</tr>
		<tr>
			<td>Brass hex nut</td>
			<td>McMASTER-CARR</td>
			<td>92736A112</td>
			<td>For GRIN lens implantation</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Par Pharmaceuticals</td>
			<td>NDC 4202317905</td>
			<td>For GRIN lens implantation</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma</td>
			<td>10043-52-4</td>
			<td>For preparing aCSF</td>
		</tr>
		<tr>
			<td>Commutator</td>
			<td>NIDA/IRP</td>
			<td>Custom-designed</td>
			<td>Component of image acquisition system</td>
		</tr>
		<tr>
			<td>Compressed Oxygen and Caxbondioxide tank</td>
			<td>Rocky Mountain Air Solutions</td>
			<td>BI-OX-CD5C-K</td>
			<td>For GRIN lens implantation</td>
		</tr>
		<tr>
			<td>Compressed Oxygen tank</td>
			<td>Rocky Mountain Air Solutions</td>
			<td>OX-M-K</td>
			<td>For virus injection</td>
		</tr>
		<tr>
			<td>Cordless Microdrill</td>
			<td>KF technology</td>
			<td>8000037800</td>
			<td>For craniotomy</td>
		</tr>
		<tr>
			<td>Cyanoacrylate</td>
			<td>Henkel Coorporation</td>
			<td># 1811182</td>
			<td>For GRIN lens implantation</td>
		</tr>
		<tr>
			<td>Data acquisition controller</td>
			<td>NIDA/IRP</td>
			<td>Custom-designed</td>
			<td>Component of image acquisition system</td>
		</tr>
		<tr>
			<td>Data transmission cable</td>
			<td>NIDA/IRP</td>
			<td>Custom-designed</td>
			<td>Component of image acquisition system</td>
		</tr>
		<tr>
			<td>Dental cement set</td>
			<td>C&#38;B Metabond and Catalyst</td>
			<td>A00253revA306 and A00168revB306</td>
			<td>For GRIN lens implantation</td>
		</tr>
		<tr>
			<td>Dental cement set</td>
			<td>Duralay</td>
			<td>2249D</td>
			<td>For GRIN lens implantation</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>VETone</td>
			<td>NDC 1398503702</td>
			<td>For GRIN lens implantation</td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Sigma</td>
			<td>50-99-7</td>
			<td>For preparing aCSF</td>
		</tr>
		<tr>
			<td>Diet gel</td>
			<td>Clear H20</td>
			<td>72-06-5022</td>
			<td>Diet Supplement for mouse</td>
		</tr>
		<tr>
			<td>GRIN lens</td>
			<td>GRINTECH</td>
			<td>NEM-100-25-10-860-S</td>
			<td>For GRIN lens implantation</td>
		</tr>
		<tr>
			<td>Heating Pad</td>
			<td>Physitemp Instruments LLC.</td>
			<td>#10023</td>
			<td>To keep the mouse body warm during surgeries</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>VETone</td>
			<td>V1 502017</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>VETone</td>
			<td>V1 501072</td>
			<td>For GRIN lens implantation</td>
		</tr>
		<tr>
			<td>Lidocaine</td>
			<td>WEST-WARD</td>
			<td>NDC 0143-9575-01</td>
			<td>Local anesthesia</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>Sigma</td>
			<td>7791-18-6</td>
			<td>For preparing aCSF</td>
		</tr>
		<tr>
			<td>Microliter syringe (Hamilton)</td>
			<td>Hamilton</td>
			<td>7653-01</td>
			<td>For virus injection</td>
		</tr>
		<tr>
			<td>MicroSyringe Pump Controller</td>
			<td>World Precision Instrument</td>
			<td>#178647</td>
			<td>For virus injection</td>
		</tr>
		<tr>
			<td>Miniscope</td>
			<td>NIDA/IRP</td>
			<td>Custom-designed</td>
			<td>For imaging</td>
		</tr>
		<tr>
			<td>Miniscope base</td>
			<td>Protolabs</td>
			<td>Custom-designed</td>
			<td>For mounting the base</td>
		</tr>
		<tr>
			<td>Miniscope holding arm</td>
			<td>NIDA/IRP</td>
			<td>Custom-designed</td>
			<td>For mounting the base</td>
		</tr>
		<tr>
			<td>Miniscope protection cap</td>
			<td>Protolabs</td>
			<td>Custom-designed</td>
			<td>For protecting the miniscope</td>
		</tr>
		<tr>
			<td>Motorized controller</td>
			<td>Thorlabs</td>
			<td>KMTS50E</td>
			<td>For mounting the base</td>
		</tr>
		<tr>
			<td>NeuView</td>
			<td>NIDA/IRP</td>
			<td>https://github.com/giovannibarbera/miniscope_v1.0</td>
			<td>For in vivo imaging</td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Puralube Vet Ointment</td>
			<td>NDC 17033-211-38</td>
			<td>Ophthalmic</td>
		</tr>
		<tr>
			<td>PCR tube</td>
			<td>Thermo Scientific</td>
			<td>AB-0622</td>
			<td>For GRIN lens implantation</td>
		</tr>
		<tr>
			<td>Pinch Clamp</td>
			<td>World Precision Instrument</td>
			<td>14040</td>
			<td>For clamping the tubing</td>
		</tr>
		<tr>
			<td>Polytetrafluoroethylene (PTFE) tape</td>
			<td>TegaSeal PTFE Tape</td>
			<td>A-A-58092</td>
			<td>For fastening miniScope to the base</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma</td>
			<td>7447-40-7</td>
			<td>For preparing aCSF</td>
		</tr>
		<tr>
			<td>Robotic arm</td>
			<td>NIDA/IRP</td>
			<td>Custom-designed</td>
			<td>For GRIN lens implantation</td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>Hospira</td>
			<td>RL 7302</td>
			<td>For both surgeries</td>
		</tr>
		<tr>
			<td>Set screw</td>
			<td>DECORAH LLC.</td>
			<td>3BT-P9005-00-0025</td>
			<td>For screwing the brass hex nut in miniscope base</td>
		</tr>
		<tr>
			<td>&#160;Silicone Rubber tubing, 0.062&#8221;ID, 1/8&#8221;OD</td>
			<td>McMaster</td>
			<td>2124T3</td>
			<td>For irrigation of aCSF</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma</td>
			<td>144-55-8</td>
			<td>For preparing aCSF</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma</td>
			<td>7647-14-5</td>
			<td>For preparing aCSF</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>Sigma</td>
			<td>7558-80-7</td>
			<td>For preparing aCSF</td>
		</tr>
		<tr>
			<td>Stereotaxic stage</td>
			<td>KOPF</td>
			<td>Model 962 Dual Ultra Precise Small Animal Stereotaxic</td>
			<td>For both surgeries</td>
		</tr>
		<tr>
			<td>Sterile cotton swab</td>
			<td>Puritan</td>
			<td>REF 806-WC</td>
			<td>For both surgeries</td>
		</tr>
		<tr>
			<td>Surgical tools</td>
			<td>Fine Science tools</td>
			<td>11251-35</td>
			<td>For surgeries</td>
		</tr>
		<tr>
			<td>Suture</td>
			<td>Sofsilk</td>
			<td>REF SS683</td>
			<td>For virus injection</td>
		</tr>
		<tr>
			<td>&#160;Syringe filter (0.22 &#181;m)</td>
			<td>Millex</td>
			<td>SLGVR33RS</td>
			<td>For filtering aCSF during GRIN lens implantation</td>
		</tr>
		<tr>
			<td>Viral suspension (AAV1-CamKII-GCamp6f)</td>
			<td>Addgene</td>
			<td>100834-AAV1</td>
			<td>For virus injection</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Titre: 2.8 X 10^13 GC/ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>VETone</td>
			<td>V1 510650</td>
			<td>For GRIN lens implantation</td>
		</tr>
	</tbody>
</table>

stereotaxic viral injection and gradient-index lens implantation for deep brain in vivo calcium imaging

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages over conventional multi-photon calcium imaging systems: (1) compact; (2) light-weighted; (3) affordable; and (4) allows recording from freely behaving animals. This protocol describes brain surgeries for deep brain in vivo calcium imaging using a custom-developed miniscope recording system. The preparation procedure consists of three steps, including (1) stereotaxically injecting the virus at the desired brain region of a mouse brain to label a specific subgroup of neurons with genetically encoded calcium sensor; (2) implantation of gradient-index (GRIN) lens that can relay calcium image from deep brain region to the miniscope system; and (3) affixing the miniscope holder over the mouse skull where miniscope can be attached later. To perform in vivo calcium imaging, the miniscope is fastened onto the holder, and neuronal calcium images are collected along with simultaneous behavior recordings. The present surgery protocol is compatible with any commercial or custom-built single-photon and two-photon imaging systems for deep brain in vivo calcium imaging.

Figure 1 shows the schematic experimental procedure, including viral injection, GRIN lens implantation, affixation of the miniscope base to the mouse skull, and in vivo calcium imaging via a miniscope. The entire procedure takes ~2 months. Figure 2 shows the major components described in the protocol for miniscope in vivo calcium imaging. Figure 3 displays the interfaces of AutoStereota software during GRIN lens implantation. Figure 4 displays the interfaces of NeuView and behavior recording software during in vivo calcium imaging.
The outcome of in vivo calcium imaging is dependent on the success of both viral injection and GRIN lens implantation surgeries. Figure 5 shows a range of outcomes (i.e., unsuccessful, suboptimal, and good) from in vivo calcium imaging recordings. In unsuccessful cases, the calcium image could appear either dark or bright but usually reveals no or very few active neurons. We typically do not pursue in vivo calcium recording experiments if there are fewer than five active neurons. A good in vivo calcium imaging typically reveals several hundreds of active neurons. If a recording contains less than a hundred active neurons, we consider it a suboptimal recording.
In both suboptimal and good recordings in vivo calcium imaging experiments were pursued, and the subsequent data analysis was performed. Movie 1 shows a representative in vivo calcium imaging recording from the mouse mPFC. Behavior videos and calcium imaging data are usually processed separately. Mouse behavior videos can be manually scored. Calcium imaging files are processed using the CaImAn calcium image processing toolbox24. Figure 6 shows a representative cell map and several calcium traces from a good in vivo calcium imaging recording.
After completing in vivo calcium imaging, the final step is to confirm whether the viral injection and GRIN lens implantation has occurred in the desired brain region. For this purpose, the mouse was perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA). The mouse brain was harvested, postfixed in 4% PFA for 12 h, and stored in PBS at 4 &#176;C. The mouse brain was then sectioned in 50 &#181;m thick slices with a vibratome. The brain slices were stained with DAPI and observed under the microscope (not described in the protocol)12. Figure 7 is a mouse brain slice ~1.94 mm anterior to bregma from an experimental mouse, showing the track where the GRIN lens was implanted. The green fluorescence region beneath and around the GRIN lens track indicates the expression of GCaMP6f in the mPFC region.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63049/63049fig01.jpg" /> 
Figure 1: Schematic overview of the experimental procedure. (A) Stereotaxic injection of virus in the mPFC. (B) GRIN lens implantation in the mPFC. (C) Affixing miniscope base to the mouse skull. (D) Miniscope mounting and in vivo calcium imaging. <a href="https://www.jove.com/files/ftp_upload/63049/63049fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63049/63049fig02.jpg" /> 
Figure 2: Main components needed for in vivo calcium imaging. (A) A GRIN lens with 1 mm diameter and 4.38 mm length. (B) A 27 G manually polished blunt-end needle used for brain tissue aspiration. (C) The robotic arm coupled to the needle holder. (D) A custom-made cap from a PCR tube to protect the exposed GRIN lens until the affixation of miniscope base on the mouse skull. (E) The miniscope holder (base) with a hex nut. (F) A miniscope whose thread part is wrapped with the PTFE tape. (G) A miniscope fastened to its base with a locking screw. (H) The miniscope holding arm. (I) A custom-built 3D motorized controller used to facilitate the movement of miniscope in XYZ positions. (J) A protective cap fastened onto the base to protect the exposed GRIN lens while the mouse is not performing in vivo calcium imaging. (K) The miniscope connected to the cable. (L) The Data Acquisition System for in vivo Ca2+ imaging. <a href="https://www.jove.com/files/ftp_upload/63049/63049fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63049/63049fig03.jpg" /> 
Figure 3: Interfaces of AutoStereota software during layer-by-layer brain tissue aspiration. (A) The interface corresponding to steps 2.17.1 to 2.17.3. (B) The interface corresponding to steps 2.17.4 to 2.17.6. (C) The interface corresponding to steps 2.17.7 to 2.17.9. (D) The interface corresponding to steps 2.17.10 to 2.17.12. Red boxes highlight the input values. <a href="https://www.jove.com/files/ftp_upload/63049/63049fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63049/63049fig04.jpg" /> 
Figure 4: Interfaces of NeuView software and the behavior recording software during in vivo calcium imaging. (A) The interface of NeuView. (B,C) The interfaces of the behavior recording software. Red boxes highlight buttons that need to be clicked. <a href="https://www.jove.com/files/ftp_upload/63049/63049fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63049/63049fig05.jpg" /> 
Figure 5: Maximum projection fluorescence cell maps to show the range of possible outcomes. (A,B) Unsuccessful in vivo calcium imaging that is not acceptable for subsequent data analysis. (A) is dark and contains less than 5 active neurons. (B) is bright but has no active neurons. (C) The cell map from a suboptimal in vivo calcium imaging that contains some active neurons. (D) The cell map from a good in vivo calcium imaging that includes several hundreds of active neurons. Scale bar: 100 &#956;m. <a href="https://www.jove.com/files/ftp_upload/63049/63049fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63049/63049fig06.jpg" /> 
Figure 6: Representative cell map and calcium transients from a successful&#160;in vivo calcium imaging. The left panel is the maximum projection fluorescence cell map from an in vivo calcium imaging recording in the mPFC during an open field test. The recording lasts for 5 min. The right panel shows calcium transients from 15 regions of interest (color-matched). Scale bar: 100 &#956;m. <a href="https://www.jove.com/files/ftp_upload/63049/63049fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63049/63049fig07.jpg" /> 
Figure 7: Postmortem assessment of an experimental mouse. The postmortem assessment for GCaMP6f expression and GRIN lens implantation in the mPFC of an experimental mouse. The rectangular area indicates the path for GRIN lens implantation. The green area under the GRIN lens implanted region confirms that GCaMP6f was expressed, and the GRIN lens was implanted precisely in the desired brain region. Cg, cingulate cortex; PrL, prelimbic cortex; IL, infralimbic cortex. Scale bar: 400 &#956;m. <a href="https://www.jove.com/files/ftp_upload/63049/63049fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Comparisons of the custom-built miniscope system at the NIDA with other miniscope systems 7,8,9,10,11,13,25,26. <a href="https://www.jove.com/files/ftp_upload/63049/63049_Table 1_1.xlsx" target="_blank">Please click here to download this Table.</a>
Movie 1: An in vivo calcium imaging recording from the mouse mPFC during an open field test. For demonstration purposes, this video shows only 1 min recording. The original recording frame rate is 10 frames/s. The video is 6 times faster than the original recording. <a href="https://www.jove.com/files/ftp_upload/63049/movie.mp4" target="_blank">Please click here to download this Movie.</a>

Watch this Scientific Journal Video about Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging at JoVE.com

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

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

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

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages ...

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