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

<ol>
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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.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/63049">Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging</a>. The Discussion&#160;was&#160;updated.
The following paragraph was added to the end of the Discussion:
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 in vivo 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 in vivo imaging. We have performed miniscope in vivo calcium imaging once per week for a few weeks without noticing any impact on mouse health and mouse social behavior12.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/63049">Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging</a>. The Discussion&#160;was&#160;updated.

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

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 ...

stereotaxic-viral-injection-gradient-index-lens-implantation-for-deep

Research

JoVE Journal

Neuroscience

5.6K Views.  University of Wyoming. 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. 

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

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

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 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 ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the &#945;-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

Two-Photon and Wide-Field Calcium Imaging in the Superior Colliculus

Calcium Imaging in Mouse Superior Colliculus

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the emergence of the cerebral cortex. It receives direct inputs from ~30 types of retinal ganglion cells (RGCs), with each encoding a specific visual feature. It remains elusive whether the SC simply inherits retinal features or if additional and potentially de novo processing occurs in the SC. To reveal the neural coding of visual information in the SC, we provide here a detailed protocol to optically record visual responses with two complementary methods in awake mice. One method uses two-photon microscopy to image calcium activity at single-cell resolution without ablating the overlaying cortex, while the other uses wide-field microscopy to image the whole SC of a mutant mouse whose cortex is largely undeveloped. This protocol details these two methods, including animal preparation, viral injection, headplate implantation, plug implantation, data acquisition, and data analysis. The representative results show that the two-photon calcium imaging reveals visually evoked neuronal responses at single-cell resolution, and the wide-field calcium imaging reveals&#160;neural activity across the entire SC. By combining these two methods, one can reveal the neural coding in the SC at different scales, and such combination can also be applied to other brain regions.

Author Spotlight: Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus 

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...

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

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

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal activity in awake animals during behavior tasks. Since the hippocampus is closely associated with episodic and spatial memory, it has become an essential brain region in&#160;this field&#39;s research. In recent research, engram cells and place cells were studied by recording the neural activities in the hippocampal CA1 region using the miniature microscope in mice while performing behavioral tasks including open-field and linear track. Although the dentate gyrus is another important region in the hippocampus, it has rarely been studied with in vivo imaging due to its greater depth and difficulty for imaging. In this protocol, we present in detail a calcium imaging process, including how to inject the virus, implant a GRIN (Gradient-index) lens, and attach a base plate for imaging the dentate gyrus of the hippocampus. We further describe how to preprocess the calcium imaging data using MATLAB. Additionally, studies of other deep brain regions that require imaging may benefit from this method.

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.

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal ...