ESB acknowledges funding by the NIH Director's New Innovator Award (DP2 OD002002-01), NIH Challenge Grant 1RC1MH088182-01, NIH Grand Opportunities Grant 1RC2DE020919-01, NIH 1R01NS067199-01, NSF (0835878 and 0848804), the McGovern Institute Neurotechnology Award Program, the Department of Defense, NARSAD, the Alfred P. Sloan Foundation, Jerry and Marge Burnett, the SFN Research Award for Innovation in Neuroscience, the MIT Media Lab, the Benesse Foundation, and the Wallace H. Coulter Foundation.

1. Constructing Stereotaxic Clamp
<ol>
 <li>The stereotaxic clamp is used for attaching the injector array to the stereotax, so that the injectors are parallel to the stereotaxic arm.</li>
 <li>Each lab needs only one clamp, unless it is anticipated that more than one surgery may take place simultaneously. In this case, make the same number as the anticipated maximum number of simultaneous surgeries. It might also be advisable to make an extra, in case of damage to the first.</li>
 <li>To make the stereotaxic clamp, firstly, a 1.5 mm outer diameter (OD) steel cannula is cut to 2 inches in length, and one of the ends is Dremeled down until flat.</li>
 <li>Next, a small piece (approximately 0.5 x 0.5 cm) is cut out of PCB proto-board, to be used as a spacer.</li>
 <li>Using a laser cutter to ensure that any cuts made are perpendicular to the surface of the material, two identical rectangles (1/2" x 3/8") are cut from 1/8" acrylic sheeting, each rectangle having two circular holes in opposite corners of the rectangle. The first hole has diameter 1.5mm, and is just large enough to hold the metal cannula tightly. The second hole has diameter 1/16" (Figure 1C).</li>
 <li>The metal cannula is inserted into the 1.5 mm hole of one rectangle, then through the 1.5 mm hole of the second. The bottom end of the cannula is aligned with the bottom face of the bottom rectangle, forming the shape of a hockey stick. </li>
 <li>While keeping the spacer tightly held between the two rectangles, this structure is cemented together by using hot glue around the cannula and 1.5mm holes, being careful to avoid gluing the spacer to the rectangles. Modeling clay may be helpful to hold things together during this process.</li>
 <li>After the glue has dried, a 1-72 screw is inserted into the 1/16 hole from the top side of the rectangles. Then a 1-72 hex nut is screwed onto the end of the 1-72 screw, and tightened. The screw and nut serve to hold the clamp together.</li>
 <li>To attach the hex nut to the bottom face of the bottom rectangle, small amounts of 5-minute epoxy are dropped around the edge of the hex nut, without allowing the epoxy to get into the threads of the hex nut or screw. Any modeling clay is removed, and it is confirmed that the spacer can be held firmly in place by tightening the screw.</li>
</ol>
2. Preparing the system for the customized injector array: Hamilton pump and stereotax
<ol>
 <li>The injector array system can be customized to virtually any set of coordinates in the brain. </li>
 <li>The user first locates coordinates of desired injection sites in a mouse (or other species) brain atlas, converting to the appropriate coordinates used by the stereotax (here, X-, Y-, and Z-coordinates). The number of injection sites (three in Figures 1A and 1B) will hereafter be called k.</li>
 <li>k number of 10 &mu;l Hamilton syringes are placed in an injection/withdrawal syringe pump such as this one from Harvard apparatus.</li>
 <li>Next, 3-foot-long pieces of polyethylene tubing are securely attached to the needle of each Hamilton syringe.</li>
 <li>For each piece of polyethylene tubing, an F-252 tubing sleeve from Upchurch Scientific is slid over the open end and attached to a P-627 tubing adapter using the included nut and ferrule, also from Upchurch Scientific.</li>
</ol>
3. Constructing the Customized Injector Array
<ol>
 <li>Each injector array is customized to the set of coordinates that the user chooses. However, for sets of coordinates that differ only by a translation and/or rotation, the same injector array can be used.</li>
 <li>Considering only the relative X and Y coordinates of the injection sites, k holes are drilled into a PCB proto-board of thickness 1/32", with the same relative spacing. These holes are drilled using a Modela mini mill and a .011" diameter drill bit from Mcmaster-Carr. Be sure to use a slow rate of drilling (Z-speed) to avoid breaking or wearing out the drill bit. Before removing the board from the mill, a rectangle is drilled around the small holes, using a larger drill bit of diameter 1/32", to avoid wearing out the smaller one. Code for the mini mill can be generated easily using MATLAB; sample code is provided in the following files.
 <ol class="lroman">
 <li>generate.m - MATLAB code for generating Modela code from desired coordinates</li>
 <li>holes_ex.rml - Modela code for drilling ring pattern (eight holes) into Proto-board</li>
 <li>outline_ex.rml - Modela code for drilling rectangle around holes</li>
 </ol></li>
 <li>245 &mu;m OD/100 &mu;m inner diameter (ID) fused silica capillary tubing, available from Polymicro Technologies, is cut into k number of 3 inch pieces. These comprise the individual injectors. A disposable piece of capillary tubing is poked through each of the holes, to clear away debris without clogging the actual injectors. The injectors are then inserted halfway into each hole, so that they are held tightly and parallel to one another. The injectors are epoxied to the board, forming the structure of the injector array. All that remains is to trim the injectors to the correct length.</li>
 <li>The injector array is attached to the stereotaxic clamp by placing one corner of the proto-board between the acrylic rectangles, and then by tightening the screw of the stereotaxic clamp. Next, the metal cannula of the clamp is attached to the stereotax, using the attachment mechanism of the stereotax.</li>
 <li>All of the following is done under a microscope. For one of the outer injectors (that is, one that can be approached from the side with a straight edge, without bumping into any of the other injectors), the tip extending beyond the bottom of the proto-board is cut with scissors. For cortical injections, it is appropriate to have the injector extending approximately 5mm beyond the surface of the proto-board. For deeper injections, this number can be increased by the corresponding increase in depth. The tip is flattened with a Dremel tool. Injector tips can also be ground at an angle as an extra precaution against clogging, if precision in depth is less important. </li>
 <li>Next, a stable reference point is chosen within range of the stereotaxic arm, and the injector array is moved so that the flattened tip of the outer injector, is at this reference point. </li>
 <li>A second injector is chosen, along with its corresponding coordinates. Considered is the relative difference in height (z-direction) between the first and second injector coordinates. The injector array is moved along the Z axis by this relative distance. The second injector is trimmed and Dremeled to the correct length, so that the tip of the second injector is now flat and at the height of the reference point. The injector array can be moved in the X and Y directions to facilitate comparison of the injector tip with the reference point. </li>
 <li>This process of trimming is repeated for the remaining injectors.</li>
</ol>
4. Assembling the entire system
<ol>
 <li>The stereotaxic clamp and customized injector array, both already constructed, are required for this part.</li>
 <li>The back end of each injector (the end that has not been trimmed) is inserted into a blue F-240 tubing sleeve and, using a P-235 nut and P-200 ferrule from Upchurch Scientific, is attached to the threaded adaptor already connected to the Hamilton pump by polyethylene tubing. Manufacturer&#x2019;s instructions can be consulted for details.</li>
 <li>Using a 27-gauge needle, silicone oil is injected into the back of the Hamilton syringes so that the entire system is filled from the Hamilton syringe to the injector tip, with no air bubbles at all.</li>
 <li>As the Hamilton syringes are re-placed in the Hamilton pump, the greatest possible volume of silicone oil is maintained in the syringes.</li>
 <li>If the experiment requires more syringes than the pump is designed for (two in this case), the syringes can be spaced with small pieces of plastic (e.g., needle caps) to keep them parallel to one another, and to ensure that all pieces are pushed by the pump to the same extent. Or, a multi-rack upgrade kit can be purchased from Harvard Apparatus to hold up to 10 syringes at once.</li>
</ol>
5. Injections/surgery
<ol>
 <li>The process of injecting with a parallel injector is very similar to that of injecting with a single pipette.</li>
 <li>It is essential to use slow refill/infuse rates throughout this process, because fast pumping might put stress on the joint between large and small tubing. Recommended max rate: 2 &mu;l/min for loading virus, and 0.1 &mu;l/min for infusing virus.</li>
 <li>An anesthetized mouse is placed in the stereotax, and remains anesthetized throughout the experiment.</li>
 <li>Prepare the mouse as needed. For example, with a scalpel, a single cut is made down the midline of the skin, from between the eyes to between the ears. The skin is pulled back to expose the skull, and the fascia is cleaned off. A pulled glass pipette is attached to the stereotax. The positions of the ear bars are adjusted until bregma and lambda are aligned to the same height, and so that the line connecting them is parallel to the Y-axis of the stereotax. The axes of the stereotax are oriented according to the coordinate system in which the injection coordinates have been calculated. The stereotaxic is zeroed with the tip of the glass pipette at bregma, and then the tip is positioned slightly above the skull at the X- and Y-coordinates of one of the injection sites. </li>
 <li>Using a dental drill, the skull below the tip is carefully pared away until there remains an extremely thin layer of bone. The glass pipette can be lowered and raised to check that the hole being drilled is centered at the correct position. Using a 30-gauge needle, a tiny piece of the layer is gently picked off, at the correct X- and Y- coordinates, so that the dura mater is exposed. This hole should be just large enough to fit one of the injectors (0.25mm wide). See Figure 1D for diagram. In this way, small, 0.25mm wide holes are made in the skull, at the X- and Y- coordinates corresponding to each injection site.</li>
 <li>The glass pipette is discarded in a sharps container, and the customized injector array is attached to the stereotax using the stereotaxic clamp.</li>
 <li>In order to correctly set the angle of the injector array, two outer injectors are chosen to be calibrated to a given reference point, as follows. After one of the injectors is matched with the reference point, consider the relative X- and Y- distances between the injection sites relative to this injector and one other. The axes of the stereotax are adjusted so that entire injector array is moved in the X- and Y-directions by these relative distances. If the second injector is not now aligned with the reference point, the metal cannula is loosened and rotated. This process is repeated iteratively until the injector array is angled correctly.</li>
 <li>Before filling the injectors with virus, the injectors are filled with silicone oil until the syringes are at the 2 &mu;l point (or greater). This provides a buffer zone, so that any air bubbles or clogging in the tip can be easily removed by pushing oil forward using the Hamilton pump, without having to refill the entire system with silicone oil from the back of the syringes, as describe previously.</li>
 <li>A sterile piece of Parafilm is placed on the skull, and the injectors are gently lowered onto the Parafilm. For coordinates with greatly varying depths, a custom-milled part (e.g. a staircase-shaped object) could facilitate loading.</li>
 <li>The following parts assume that 1 &mu;l of virus is to be loaded at each site (the following quantities should be scaled down if smaller quantities are desired). In order to guarantee that &gt;1 &mu;l of virus is injected at each site, 1.5 &mu;l of virus is pipetted onto the Parafilm or staircase at the tip of each injector.</li>
 <li>With the refill rate of the Hamilton pump set to 1 &mu;l/min, 1.2 &mu;l of the virus is refilled into each injector.</li>
 <li>The tip of the longest injector is zeroed at bregma, and then the injector array is shifted to the X- and Y- coordinates of that injector. If clogging is an issue, say if many deep targets are involved: even before inserting the injector tips into the brain, it is sometimes advisable to start the pump infusing, until virus can be seen emerging from all the injector tips. This removes any air bubbles at the injector tips, and also ensures that there is no clogging. In the case of clogging, the Hamilton pump is set to infuse a brief pulse at a higher speed of 2 &mu;l/min, to gently clear out clogging.</li>
 <li>The injector array is then lowered, through the small holes made with the 30-gauge needles, to the correct Z-depth.</li>
 <li>1 &mu;l of virus is injected through each injector, at 0.1 &mu;l /min. </li>
 <li>The injections are left alone for 30 minutes.</li>
 <li>After the injector array is slowly extracted from the brain, the Hamilton pump is set to infuse at the same rate of 0.1 &mu;l /min, in order to check for clogging in each injector.</li>
 <li>Then the injectors are cleaned by refilling and infusing 1.5 &mu;l of ethanol at 2 &mu;l/min.</li>
 <li>Finally, the injectors are refilled with silicone oil to maintain the 2 &mu;l buffer zone in each Hamilton syringe.</li>
<li>All equipment should be sterilized before and after the procedure, according to your institution's biosafety and animal use protocols.</li>
</ol>
6. Representative Results
The parallel injector array speeds up a surgery roughly by a factor equal to the number of injectors, not counting setup and recovery time, although individual times will depend on the skill of the practitioner. For a 1 microliter injection, we typically saw lentivirus expression in a sphere of approximately diameter 1mm (Fig. 1E). The precision of the injection was such that the variability in tip positioning, from trial to trial, was about 45 microns (standard deviation of the distance from the tip position to the intended tip position).
<img src="/files/ftp_upload/1489/1489fig1.jpg" alt="Figure 1" /> Figure 1. Design, implementation, and use of a parallel virus injector array. A, schematic of the parallel injector array system, showing a triple injector configuration, for three simultaneous injections. B, photograph of a triple parallel injector array as diagrammed in A. C, stereotaxic clamp, shown in outline from the top. D, illustration of technique for efficient, damage-minimizing, opening of holes in skull for injector insertion into brain: with a dental drill, thin the skull down to ~50 microns thickness, then use the tip of a sharp needle to open a small craniotomy. E, fluorescence image showing channelrhodopsin-2 (ChR2)-GFP-labeled cells in three mouse cortical regions, as targeted by the triple injector array shown in B.

<ol>
	<li>Chow, B., Han, X., Qian, X., Boyden, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-performance+halorhodopsin+variants+for+improved+genetically-targetable+optical+neural+silencing.">High-performance halorhodopsin variants for improved genetically-targetable optical neural silencing.</a> Frontiers in Systems Neuroscience. , (2009).</li><li>Bi, A., Cui, J., Ma, Y. P., Olshevskaya, E., Pu, M., Dizhoor, A. M., Pan, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+expression+of+a+microbial-type+rhodopsin+restores+visual+responses+in+mice+with+photoreceptor+degeneration.">Ectopic expression of a microbial-type rhodopsin restores visual responses in mice with photoreceptor degeneration.</a> Neuron. 50, 23-33 (2006).</li><li>Han, X., Qian, X., Bernstein, J., Zhou, H., Franzesi, G., Stern, P., Bronson, R., Graybiel, A., Desimone, R., Boyden, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millisecond-Timescale+Optical+Control+of+Neural+Dynamics+in+the+Nonhuman+Primate+Brain.">Millisecond-Timescale Optical Control of Neural Dynamics in the Nonhuman Primate Brain.</a> Neuron. 62 (2), 191-198 (2009).</li><li>Zhang, F., Wang, L. P., Brauner, M., Liewald, J. F., Kay, K., Watzke, N., Wood, P. G., Bamberg, E., Nagel, G., Gottschalk, A., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+fast+optical+interrogation+of+neural+circuitry.">Multimodal fast optical interrogation of neural circuitry.</a> Nature. 446 (7136), 633-639 (2007).</li><li>Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millisecond-timescale+genetically+targeted+optical+control+of+neural+activity.">Millisecond-timescale genetically targeted optical control of neural activity.</a> Nat Neurosci. 8 (9), 1263-1268 (2005).</li><li>Han, X., Boyden, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple-color+optical+activation,+silencing,+and+desynchronization+of+neural+activity,+with+single-spike+temporal+resolution.">Multiple-color optical activation, silencing, and desynchronization of neural activity, with single-spike temporal resolution.</a> PLoS ONE. 2, e299-e299 (2007).</li><li>Szobota, S., Gorostiza, P., Del Bene, F., Wyart, C., Fortin, D. L., Kolstad, K. D., Tulyathan, O., Volgraf, M., Numano, R., Aaron, H. L., Scott, E. K., Kramer, R. H., Flannery, J., Baier, H., Trauner, D., Isacoff, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+control+of+neuronal+activity+with+a+light-gated+glutamate+receptor.">Remote control of neuronal activity with a light-gated glutamate receptor.</a> Neuron. 54 (4), 535-545 (2007).</li><li>Lima, S. Q., Miesenbock, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+control+of+behavior+through+genetically+targeted+photostimulation+of+neurons.">Remote control of behavior through genetically targeted photostimulation of neurons.</a> Cell. 121 (1), 141-152 (2005).</li><li>Zhang, F., Wang, L. P., Boyden, E. S., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Channelrhodopsin-2+and+optical+control+of+excitable+cells.">Channelrhodopsin-2 and optical control of excitable cells.</a> Nat. Methods. 3, 785-792 (2006).</li><li>Ishizuka, T., Kakuda, M., Araki, R., Yawo, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+evaluation+of+photosensitivity+in+genetically+engineered+neurons+expressing+green+algae+light-gated+channels.">Kinetic evaluation of photosensitivity in genetically engineered neurons expressing green algae light-gated channels.</a> Neurosci Res. 54 (2), 85-94 (2006).</li><li>Luo, L., Callaway, E. M., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+neural+circuits.">Genetic dissection of neural circuits.</a> Neuron. 57 (5), 634-660 (2008).</li></ol>

All procedures were in accordance with the NIH Guide for Laboratory Animals and approved by the MIT Animal Care and Use and Biosafety Committees.

In recent years, a number of genetically-encoded optical sensitizers have enabled neurons to be activated and silenced in vivo in a temporally-precise fashion, in response to brief pulses of light (e.g., 1,4,5,6,7,8,11). A key method with which neurons have been sensitized to light in the mammalian brain, is via viruses such as lentiviruses and adeno-associated viruses (AAV), which can deliver genes encoding for opsins to brains of animals ranging from mice to monkeys, in a safe and enduring fashion (e.g., 2,9,10). Viruses allow faster turnaround time than do transgenics, especially for organisms that are not genetic model organisms such as rats and monkeys, and for opsins may enable high expression levels that may not be possible in transgenic scenarios. Here we demonstrate a parallel injector array capable of creating, in a rapid timescale, "circuit-level transgenics," enabling entire 3-dimensional brain structures to be virally targeted with a gene, in a single surgical step. The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 &mu;L/min). 
This technology will enable a wide variety of new kinds of experiments, such as millisecond-timescale shutdown of complexly-shaped structures (such as the hippocampus) at precise times during behavior, temporally-precise inactivation of bilateral structures that may act redundantly (such as the left and right amygdala), and the perturbation of multiple discrete brain regions (e.g., driving two connected regions out of phase to study how cross-region synchrony depends upon activity within each region, or stimulating inputs to a region while silencing a subset of the targets in order to understand which of the several targets are critical for mediating the effects of those inputs). For large brains like those in the primate, in which we have recently demonstrated optical cell-type specific neural activation3, perturbing activity in a behaviorally-relevant area may require viral labeling of large, complex structures. We note that parallel injector arrays may be used to inject almost any payload &#x2013; drugs, neuromodulators, neurotransmitters, or even cells &#x2013; in complex 3-D patterns in the brain, in a temporally-precise manner. Finally, from a translational standpoint, it is possible that rapid, patient-customized gene therapy or drug delivery devices may be rapidly custom-designed and fabricated to match individual brain geometries, supporting new treatments for a variety of pathologies, potentially through the use of optical control molecules. 
The injector arrays are designed to be precise, both spatially and volumetrically. In the X- and Y-directions, this is accomplished by drilling very accurately placed holes using an inexpensive mini-mill, with the holes just large enough to fit the injectors, so that injectors are held parallel to one another, and in a precise location. In the Z-direction, the injectors are trimmed using a stereotaxic apparatus, allowing a level of precision equivalent to that of the stereotaxic surgery itself. The volumetric precision arises from the precision of the Hamilton pump, as well as the near-zero dead-volume connectors, adapted from the high-pressure liquid chromatography (HPLC) field. The injectors are made from fused silica capillary tubing, which is strong and rigid enough that it maintains precise shape and spacing under pressure, without the larger wall thickness of alternatives such as steel cannulas. Small modifications can easily be made to adapt the parallel injector array to a variety of experiments. For example, if a smaller volume of virus or finer spacing is required, smaller capillary tubing can be employed, along with a corresponding smaller drill bit. Future devices may utilize microfluidic channels and pumps, to increase the number of parallel injectors, to minimize the size (perhaps enabling such devices to be mounted on the heads of freely-moving animals).

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Dremel Tool</td>
<td>Tool</td>
<td>Dremel</td>
<td>3956-02</td>
<td>&nbsp;</td>
<tr>
<td>Laser Cutter</td>
<td>Tool</td>
<td>Universal Laser Systems</td>
<td>VLS2.30</td>
<td>&nbsp;</td>
<tr>
<td>Hot glue gun</td>
<td>Tool</td>
<td>Stanley Bostitch</td>
<td>GR20</td>
<td>&nbsp;</td>
<tr>
<td>Injection/withdrawal syringe pump (Hamilton pump)</td>
<td>Tool</td>
<td>Harvard Apparatus</td>
<td>702001</td>
<td>&nbsp;</td>
<tr>
<td>10 &mu;l Hamilton syringes</td>
<td>Tool</td>
<td>Hamilton Company</td>
<td>701N</td>
<td>Need one per injection site</td>
</tr>
<tr>
<td>Mouse stereotax</td>
<td>Tool</td>
<td>Stoelting</td>
<td>51725D</td>
<td>&nbsp;</td>
<tr>
<td>Modela mini-mill</td>
<td>Tool</td>
<td>Roland</td>
<td>MDX-15</td>
<td>&nbsp;</td>
<tr>
<td>0.011" diameter drill bit for mini mill</td>
<td>Tool</td>
<td>Mcmaster-Carr</td>
<td>8915A12</td>
<td>&nbsp;</td>
<tr>
<td>1/32" diameter drill bit For mini-mill</td>
<td>Tool</td>
<td>McMaster-Carr</td>
<td>8848A35</td>
<td>&nbsp;</td>
<tr>
<td>High speed dental drill</td>
<td>Tool</td>
<td>Lynx</td>
<td>333</td>
<td>&nbsp;</td>
<tr>
<td>Dental drill accessories</td>
<td>Tool</td>
<td>Pearson</td>
<td>F 35-08-25 F 35-07-10 P 86-02-38</td>
<td>&nbsp;</td>
<tr>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr>
<td>1.5mm outer diameter (OD) stainless steel cannula</td>
<td>Material</td>
<td>Small-Parts</td>
<td>HTX-15R</td>
<td>&nbsp;</td>
<tr>
<td>1-72 binding slotted machine screw</td>
<td>Material</td>
<td>Small-Parts</td>
<td>MX-0172B</td>
<td>&nbsp;</td>
<tr>
<td>1-72 hex nut</td>
<td>Material</td>
<td>Small-Parts</td>
<td>HNX-0172</td>
<td>&nbsp;</td>
<tr>
<td>PCB proto-board, 1/32" thick</td>
<td>Material</td>
<td>Digi-Key</td>
<td>PC57-T-ND</td>
<td>&nbsp;</td>
<tr>
<td>Acrylic Sheet, 1/8" thick</td>
<td>Material</td>
<td>Mcmaster-Carr</td>
<td>8560K239</td>
<td>&nbsp;</td>
<tr>
<td>HPLC connectors</td>
<td>Material</td>
<td>Upchurch Scientific</td>
<td>F-252, P-627, P-200, P-235, F-240 (some of these can be bought in 10-packs; simply add an &#x2018;x&#x2019; to the end of the part number)</td>
<td>Need one per injection per site, except F-252, P-627, and P-235, which can be reused</td>
</tr>
<tr>
<td>Fused silica capillary tubing, OD: 245 &mu;m, ID: 100 &mu;m</td>
<td>Material</td>
<td>Polymicro Technologies</td>
<td>2000022-10M</td>
<td>&nbsp;</td>
<tr>
<td>5-minute general purpose epoxy</td>
<td>Material</td>
<td>Permatex</td>
<td>84101</td>
<td>5-minute general purpose epoxy</td>
</tr>
<tr>
<td>Polyethylene tubing .066 x .095 inch</td>
<td>Material</td>
<td>VWR</td>
<td>63018-827</td>
<td>&nbsp;</td>		</tbody>
		</table>
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scalable fluidic injector arrays for viral targeting of intact 3-d brain circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Watch this Scientific Journal Video about Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits at JoVE.com

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

scalable-fluidic-injector-arrays-for-viral-targeting-intact-3-d-brain

Research

JoVE Journal

Neuroscience

14.2K Views.  Massachusetts Institute of Technology. Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

Video: Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an injecting needle produces during its trajectory is a major concern especially in the mouse brain for not only the brain is small but also sometimes multiple injections are needed.  We show here a method to produce glass capillary needles with a 50-&mu;m lumen which significantly reduces the brain damage and allows a precise targeting into the rodent brain. This method allows a delivery of small volumes (from 20 to 100 nl), reduces bleeding risks, and minimizes passive diffusion of drugs into the brain parenchyma. By using different size of capillary glass tubes, or changing the needle lumen, several types of substances and cells can be injected. Microinjections with a glass capillary tube represent a significant improvement in injection techniques and deep brain targeting with minimal collateral damage in small rodents.

In this article, we show a method to make glass capillary needles with a 50-&mu;m lumen. This technique significantly reduces the brain damage, minimizes passive diffusion of drugs and allows a precise targeting into the rodent brain.

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an ...

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

Research into the design and utilization of brain-implanted microdevices, such as microelectrode arrays, aims to produce clinically relevant devices that interface chronically with surrounding brain tissue. Tissue surrounding these implants is thought to react to the presence of the devices over time, which includes the formation of an insulating "glial scar" around the devices. However, histological analysis of these tissue changes is typically performed after explanting the device, in a process that can disrupt the morphology of the tissue of interest.
Here we demonstrate a protocol in which cortical-implanted devices are collected intact in surrounding rodent brain tissue. We describe how, once perfused with fixative, brains are removed and sliced in such a way as to avoid explanting devices. We outline fluorescent antibody labeling and optical clearing methods useful for producing an informative, yet thick tissue section. Finally, we demonstrate the mounting and imaging of these tissue sections in order to investigate the biological interface around brain-implanted devices.

Here we present a histological method for capturing, labeling, optically clearing, and imaging the intact brain tissue interface around chronically implanted microdevices in rodent brain tissue. Results from the techniques comprising this method are useful for understanding the impact of various penetrating brain-implants on their surrounding tissue.

Research  into the design and utilization of brain-implanted microdevices, such as  microelectrode arrays, aims to produce clinically relevant devices ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma establishes a lifelong persistent infection in the brain. While this brain infection is asymptomatic in most immunocompetent people, in the developing fetus or immunocompromised individuals such as acquired immune deficiency syndrome (AIDS) patients, this predilection for and persistence in the brain can lead to devastating neurologic disease. Thus, it is clear that the brain-Toxoplasma interaction is critical to the symptomatic disease produced by Toxoplasma, yet we have little understanding of the cellular or molecular interaction between cells of the central nervous system (CNS) and the parasite. In the mouse model of CNS toxoplasmosis it has been known for over 30 years that neurons are the cells in which the parasite persists, but little information is available about which part of the neuron is generally infected (soma, dendrite, axon) and if this cellular relationship changes between strains. In part, this lack is secondary to the difficulty of imaging and visualizing whole infected neurons from an animal. Such images would typically require serial sectioning and stitching of tissue imaged by electron microscopy or confocal microscopy after immunostaining. By combining several techniques, the method described here enables the use of thick sections (160 &#181;m) to identify and image whole cells that contain cysts, allowing three-dimensional visualization and analysis of individual, chronically infected neurons without the need for immunostaining, electron microscopy, or serial sectioning and stitching. Using this technique, we can begin to understand the cellular relationship between the parasite and the infected neuron.

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Using this protocol, we were able to image 160 &#181;m thick brain sections from mice infected with the parasite Toxoplasma gondii, which enables visualization and analysis of the spatial relationship between the encysting parasite and the infected neuron.

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma ...

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

Using multiple angles to cut the mouse pup brain, we improve upon a previously-described acute brain slice which captures the connections between most of the major auditory midbrain and forebrain structures.

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays

Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not represent the underlying cytoarchitectural and functional complexity of the affected individuals carrying suspected disease variants. Conventional 2-D models remain incomplete representations of in vivo-like structures and do not adequately capture the complexity of the brain. Thus, there is an emerging need for more 3-D hiPSC-based models that can better recapitulate the cellular interactions and functions seen in an in vivo system.
Here we report a protocol to develop a 3-D system from undifferentiated hiPSCs based on the serum free embryoid body (SFEB). This 3-D model mirrors aspects of a developing ventralized neocortex and allows for studies into functions integral to living neural cells and intact tissue such as migration, connectivity, communication, and maturation. Specifically, we demonstrate that the SFEBs using our protocol can be interrogated using physiologically relevant and high-content cell based assays such as calcium imaging, and multi-electrode array (MEA) recordings without cryosectioning. In the case of MEA recordings, we demonstrate that SFEBs increase both spike activity and network-level bursting activity during long-term culturing. This SFEB protocol provides a robust and scalable system for the study of developing network formation in a 3-D model that captures aspects of early cortical development.

Here we report a protocol to develop a three-dimensional (3-D) system from human induced-pluripotent stem cells (hiPSCs) called the serum free embryoid body (SFEB). This 3-D model can be used like an organotypic slice culture to model human cortical development and for the physiological interrogation of developing neural circuits.

Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and smaller functional subgroups, it becomes paramount to record from projections and/or genetically-defined subpopulations of neurons. Fiber photometry is an accessible and powerful approach that can overcome these challenges. By combining optical and genetic methodologies, neural activity can be measured in deep brain structures by expressing genetically-encoded calcium indicators, which translate neural activity into an optical signal that can be easily measured. The current protocol details the components of a multi-fiber photometry system, how to access deep brain structures to deliver and collect light, a method to account for motion artifacts, and how to process and analyze fluorescent signals. The protocol details experimental considerations when performing single and dual color imaging, from either single or multiple implanted optic fibers.

This protocol details how to implement and perform multi-fiber photometry recordings, how to correct for calcium-independent artifacts, and important considerations for dual-color photometry imaging.

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and ...