We thank members of the Wilson lab for their helpful advice on the fabrication method.
This work was supported by the Simons Foundation, a NIH pathway to independence career award from the NINDS and a NARSAD Young Investigator Award (to M.M.H.) as well as grants from the NIH (to M.A.W.).

1. Design Intent
<ol>
	<li>Identify the brain region of choice (lateral geniculate nucleus (LGN, visual thalamus)) by scrolling through the sagittal sections of the electronic mouse brain atlas.</li>
	<li>At A/P coordinates (-2.3 &#8211; -2.7 mm), the LGN is widest. Use this region to design the drive bottom (bottom pieces). 
	NOTE: A total of 8 independently movable electrodes can be used to target LGN (4-6 electrodes will make it to the LGN, 2-4 electrodes are added to offset implantation errors, Figure 1A).</li>
	<li>In Solidworks, draw a sketch of the design body (Figure 1B) in the front plane. Click sketch, and then use combination of lines and curves to draw a sketch which will include the contours for the drive base, handles and polyamide half slots, as shown. Ensure that the contour does not contain any open gaps. Then click Exit Sketch.</li>
	<li>Next, select both the front and right planes, and click &#8220;Create Axis&#8221;. Then, create the 3D design body model by rotating the highlighted blue sketch contour (Figure 1B) 360&#176;. In the features menu, click &#8220;Revolved Boss/Base&#8221;. Choose the midline as the Axis of revolution. In the parameters section, under direction 1 click Blind, and under angle select 360.00 deg. In the selected contours section, ensure that the blue highlighted contour is the one selected.</li>
	<li>Create one polyimide half-slot by revolving the red highlighted contours 13&#176; (Figure 1C, top left). Steps are identical to 1.4 above except for the angle specification</li>
	<li>Create one drive handles by revolving the green contour 15&#176; (Figure 1C, top right).</li>
	<li>Create the second drive handle using the circular pattern function (Figure 1C, bottom left). In the features menu, click &#8220;Circular Pattern&#8221;. In parameters, choose the midline as axis of revolution. Select 180.00 deg as the angle, and 2 as the number of instances. Ensure that the first handle is selected under &#8220;Features to Pattern&#8221;.</li>
	<li>Create sixteen polyimide half-slots using the circular pattern function (Figure 1C, bottom left). Perform similar motions to 1.7, but select the first polyimide half-slot as the &#8220;Features to Pattern&#8221;. The angle is 22.5&#176; and the number of instances are 16 (Note: this is just 360&#176;&#160;divided by the number of times you want to pattern the feature)</li>
	<li>Create a new plane on which to draw the polyimide receptacle. Achieve this by clicking &#8220;insert&#8221; on the main menu. Click &#8220;Reference Geometry&#8221;, Select the two sides of polyimide half slots, and then click &#8220;Create New Plane&#8221;; (Figure 1D, top)</li>
	<li>Create the microdrive receptacle (the screw hole, polyimides hole and anti-torque rail (Figure 1D, bottom). Achieve this by creating a sketch that encompasses all these features on the new plane created in 1.9. Note that for the anti-torque rails, define a centerline in between the two sides of the polyamide top slots. Then, draw the anti-torque rails by creating two circles perpendicular to the centerline, whose centers are 1 radius apart, and then trimming the middle contour.</li>
	<li>In the Features menu, click on &#8220;Extrude Boss/Base&#8221; to create the antitorque rail and choose a blind extrude of 10 mm going upwards and 2 mm going downwards. For the screw hole and polyimide hole, click on &#8220;Extrude cut&#8221;, and choose blind 6mm , and a few mms going upwards for both (Figure 1E, left).</li>
	<li>Pattern the micro-drive receptacle 16x, using the center as the axis of revolution (22.5&#176;, 16 instances, equal spacing), (Figure 1E, right)</li>
	<li>On the top of the handle, draw a 3 mm x 3 mm box starting at the center tip of the drive handle, facing the central axis. Extrude this 2 mm upwards using the &#8220;Extrude Boss&#8221; function. Draw circles of 1mm diameter at the locations in which the EIB screws will go on. Afterward, make 1.5 mm &#8220;Extrude Cut&#8221; to make a hole. Then, pattern the box and hole twice using the Circular Pattern function (Text overlay: 180&#176;, 2 instances, equal spacing, about central axis).</li>
	<li>Use the dimensions (in millimeters) in Figure 1F to draw a top piece sketch. Use the &#8220;Extrude Boss/Base&#8221; to make a 3D model of it. 
	NOTE: After these steps the drive design is complete. The physical drive body is created through the process of stereolithography. There are a number of companies that offer stereolithography printing based on STL files. We recommend services that can print in hard plastic (such as Accura&#174; 55), with a minimum resolution of at least 0.1 mm.</li>
</ol>
2. Preparation of Hyperdrive Components
<ol>
	<li>Lay out a small piece of double-sided tape on a flat surface and cut the necessary number of 31 G&#160;polyimide tubes to approximately 8 centimeters (ID/OD:.0071&#39;&#39;/.0116&#39;&#39;; Wall: .00225) (Figures 2A - 2B).</li>
	<li>Lay out the first layer of guide tubes on the double-sided tape, taking care to place guide tubes as close as possible to one another on the tape. Dab a small amount of thin, cyanoacrylate glue over the layer of polyimides. (Figure 2C)</li>
	<li>Quickly lay out a second layer of polyimides&#160;(Figure 2D).</li>
	<li>Create a fiber optic placeholder using a 26 G&#160;cannula. Ensure that this is lubricated using a Teflon based lubricant prior to being incorporated in the assembly (Figure 2E).</li>
	<li>Apply a line of epoxy 4-5 mm in length perpendicular to the polyimide bundle (Figure 2F). Once the epoxy has hardened (2-3 hr), remove the tape from the bottom layer and reepoxy the other side. After the epoxy has hardened again, the 26 G&#160;cannula can be removed and the construct cut in the middle using a razor blade (Figure 2G), resulting in two polyimide matrices, each of which can be used for one hyperdrive (Figure 2H).</li>
	<li>Print out the cone template on a sheet of transparency paper and cut a corresponding sheet of heavy duty aluminum foil&#160;(Figures 3A - 3C).</li>
	<li>Apply a layer of epoxy to the aluminum foil and quickly apply the transparency paper. Using a heavy object or a wooden dowel, smooth out the epoxy so that it is evenly distributed&#160;(Figure 3D).</li>
	<li>Cut out the cone template and clamp together using an alligator clip. Finally, use another dab of epoxy to permanently affix the pieces&#160;(Figure 3E).</li>
</ol>
3. Final Assembly of the Microdrive
<ol>
	<li>Attach the EIB to the drive body, and reinsert the 26 G&#160;cannula through the polyimide guide tube matrix. Align the polyimide matrix with the drive body using the fiber optic hole in the EIB to ensure that the guide tubes are perpendicular to the EIB and epoxy the matrix to the drive body taking care to ensure that no epoxy flows into the guide tubes or into the drive body&#160;(Figures 4A - 4C).</li>
	<li>Map each guide tube in the polyimide matrix to a corresponding bracket on the inner wall of the drive body. Slide a small ring of 33 G&#160;polyimide over each guide tube and into the bracket and apply a small amount of cyanoacrylate glue to affix each guide tube. (Figures 4D - 4E) Finally, epoxy the whole apparatus to the inner wall of the drive body and cut the polyimides so that they protrude just above the inner lip&#160;(Figures 4F - 4G).</li>
	<li>Build a microdrive assembly by putting one of the custom-built screws through the center hole of a top piece followed by one of the 5 mm springs. Slide the outer hole of the top piece over one of the rails, and gently drive the screw. Drive the screw until the spring reaches it&#39;s minimum compressed length. (Figures 4H - 4I) Repeat this process for each rail/microdrive&#160;(Figure 4J).</li>
	<li>Turn the drive array upside down and take a picture of the guide tube matrix. This picture will be used to map the location of the guide tube corresponding to each microdrive&#160;(Figure 4K).</li>
	<li>Insert a polyimide tube (0.005&#39;) into each guide tube from the bottom of the drive base. Let the carrier tube extend 1-2 mm from the top of the fully lowered microdrive and record on the photograph the identity of the corresponding micro-drive. (Figures 4L - 4M)</li>
	<li>Epoxy the polyimide tube to the microdrive support, taking care not to let epoxy run through the microdrive onto the spring or screw&#160;(Figures 4N, 4P - 4Q).</li>
	<li>Fully lower all the microdrives. Cut all the polyimide tubes off flush at the bottom of the polyimide matrix&#160;(Figure 4O).</li>
	<li>Mount the electrode interface board to the drive base using two #00-90 x 3/16&#8217;&#8217; screws (Figure 4R). 
	NOTE: At this point the drive array is ready to be loaded with stereotrodes or tetrodes. For details on tetrode construction and loading, please see 10. The printed drive base and the microdrives have been designed in SolidWorks 2011 3D CAD software: please <a href="/files/ftp_upload/51675/MouseDriveDoublePost.STL">click here</a> to download the SolidWorks file.</li>
	<li>After loading, invert the drive and carefully lower the shielding cone over the drive so that only the bottom piece protrudes. Affix the shielding cone by epoxying the cone to the drive body.</li>
	<li>After the cone is attached, strip a small length of stainless steel wire (.008&#39;&#39; Bare, .011&#39;&#39; Coated) and pin to the EIB. Scratch the inner, aluminum part of the cone with a needle and ground the steel wire to the cone using silver paint. Once the silver paint has dried, reinforce with a dab of epoxy. Alternatively, the steel wire can directly be attached to the cone with a dab of conductive epoxy (MG Chemicals, Surrey, Canada).</li>
</ol>

<ol>
	<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, 1263-1268 (2005).</li><li>Fenno, L., Yizhar, O., 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=The+development+and+application+of+optogenetics.">The development and application of optogenetics.</a> Annu Rev Neurosci. 34, 389-412 (2011).</li><li>Alexander, G. M., et al. <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+in+transgenic+mice+expressing+evolved+G+protein-coupled+receptors.">Remote control of neuronal activity in transgenic mice expressing evolved G protein-coupled receptors.</a> Neuron. 63, 27-39 (2009).</li><li>Halassa, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+optical+drive+of+thalamic+reticular+nucleus+generates+thalamic+bursts+and+cortical+spindles.">Selective optical drive of thalamic reticular nucleus generates thalamic bursts and cortical spindles.</a> Nat Neurosci. 14, 1118-1120 (2011).</li><li>Tsien, J. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subregion-+and+cell+type-restricted+gene+knockout+in+mouse+brain.">Subregion- and cell type-restricted gene knockout in mouse brain.</a> Cell. 87, 1317-1326 (1996).</li><li>Nestler, E. J., Hyman, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+neuropsychiatric+disorders.">Animal models of neuropsychiatric disorders.</a> Nat Neurosci. 13, 1161-1169 (2010).</li><li>Collins, P. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grand+challenges+in+global+mental+health.">Grand challenges in global mental health.</a> Nature. 475, 27-30 (2011).</li><li>Wilson, M. A., McNaughton, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+hippocampal+ensemble+code+for+space.">Dynamics of the hippocampal ensemble code for space.</a> Science. 261, 1055-1058 (1993).</li><li>Wilson, M. A., McNaughton, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactivation+of+hippocampal+ensemble+memories+during+sleep.">Reactivation of hippocampal ensemble memories during sleep.</a> Science. 265, 676-679 (1994).</li><li>Nguyen, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-drive+array+for+chronic+in+vivo+recording:+tetrode+assembly.">Micro-drive array for chronic in vivo recording: tetrode assembly.</a> J Vis Exp. (26), (2009).</li></ol>

The authors have nothing to disclose.

This protocol outlines the process of constructing an ultra-lightweight microdrive array for targeting a single or multiple brain regions in the mouse. After the final steps of construction, the hyperdrive is ready to be implanted using standard surgical implantation techniques and affixed to the mouse&#39;s skull with dental cement. Post implantation, the electrodes can each be advanced independently using a small screwdriver, while the mouse is restrained by hand. The distance per turn that each electrode advances is determined by the pitch of the screw. Using the screws referenced here advances each electrode approximately 150 mm per turn, though half and quarter-turns may be used for greater resolution.
The dimensions of the sketch in Figure 1B determine the overall size of the implant, therefore, an obvious way to scale implants bidirectionally is to change the dimensions on that critical sketch. Additionally, the length of the screws can be extended to target deeper brain structures. We recommend custom made titanium screws, as those are light and less brittle than steel. Note that the antitorque rails need to scale linearly with the screw length, and at this point we have not determined the maximal length at which these structures can be printed. To target multiple brain regions, the shape of the bottom piece can be modified. The addition of known sized washers (thickness 200 &#181;m), could provide spacers needed between polyimides targeting separate brain structures (for example, hippocampus and prefrontal cortex). These could be included in the bottom piece assembly steps, and later cut off after the epoxy hardens.
A big limitation of this design is its dependence on proprietary software (Solidworks in this case). Future development of open source programs that provide user friendly interfaces conducive to designing such equipment with minimal engineering background would be of tremendous benefit to the neuroscience community.
This method provides several advantages over existing methods. First, the design is simple, dependent on very few sketches (Figure 1). Second, it is ultra-light, requiring no dental cement or heavy material to go into its assembly. Overall, it weighs around 1.7 g &#8211; almost a third of the weight of commercially available implants of similar functionality. Third, it requires no specialized equipment to make &#8211; the implant body can be 3D printed from multiple sources (for example approto.com, but there are several others); the screws can be custom made (for example antrinonline.com); the springs are commercially available (for example leesprings.com); and as a result the entire assembly process can happen in a day. Finally, these implants have been used to record from multiple brain regions during natural foraging, structured behavioral tasks and sleep (Figure 5).
Future applications of this method include implementing its scalability. It is likely that the implant can be bidirectionally scaled simply by changing 1) the size of the sketch in Figure 1B and, 2) the number of microdrive receptacles (Figure 1D) patterned. For example, it can be scaled downwards to record from freely behaving mice early in development, and scaled upwards to record from rats, rabbits, ferrets and perhaps nonhuman primates.
A final word is to remind the reader that critical to successful implementation of the outlined method is to prototype any modifications they implement to .stl design files attached. The reader will notice, for example, that the attached design contains a &#8220;figure 8&#8221; antitorque rail. This was the best design possible given the limitation of 3D printing, as it is often required that we drill these holes. Having it be a circle, would compromise stability, but having it be a square or an angled shape would limit the ability to fix 3D printing imperfections by drilling.

Mus musculus has, due to its genetic tractability, quickly become the animal model of choice for physiologists interested in microcircuit-level dissection of genetically-identified neurons and in investigating mouse models of human disease. For example, the recent introduction of causal genetic tools, such as optogenetic and chemical genetic actuators has allowed experimentalists to test the necessity and sufficiency of identified neural circuits in behavior1-4. The wide availability of recombinant transgenic mouse driver lines (Cre-lines), has amplified the experimental ease by which neuron subtypes are targeted, adding to the value of the mouse for these experiments5.
Likewise, genetic screens and genome wide associations of common neurological and psychiatric disorders have facilitated the identification of genetic risk factors for brain illness6,7. These advances, combined with the growing toolbox for genetic manipulation and genome engineering in mice, have made it the organism of choice for modeling human disease. The combination of disease models and causal genetic tools provides an unprecedented opportunity for understanding brain illness and identifying circuit-level targets for interventions.
To fully capitalize on these molecular tools and gain insight into microcircuit function in health and disease, it is essential to couple them with physiological readouts of brain activity. Ideally, the experimenter would be able to monitor a large number of neurons while maintaining the single cell resolution. Extracellular, multi-electrode recordings in freely behaving animals provide such opportunity; however, the use of this technology in the mouse has been limited. To record from small targets (e.g., CA1 layer in the hippocampus), the use of adjustable electrodes is necessary as small movements in recording electrodes following surgical implantation make it impossible to maintain recording stability8,9. Traditionally, the methods that have been employed to move electrodes within the brain impose weight limitations when used in the mouse, making it challenging to couple recording of a large number of neurons with behavior in this organism.
Here, methods are introduced for fabricating miniature, ultra-lightweight, microelectrode arrays that are individually customizable to the brain region being targeted, optogenetics-compatible, and easily assembled from commercially available parts. Each &#8220;microdrive&#8221; within the multi-electrode &#8220;hyperdrive&#8221; utilizes a spring-and-screw mechanism to advance the electrode and a plastic rail, built into the hyperdrive body, to counteract torque from the screw. First, the process of designing the hyperdrive bodies and microdrives in a CAD program for 3D printing is described. By designing hyperdrive bodies that are customized for specific structures, it is possible to increase the precision of targeting and to further increase the yield of the preparation. Second, the fabrication process is described in detail, wherein the multi-electrode array is assembled by hand from parts that are commercially available. This technique has been used, successfully, to record from ensembles of neurons in hippocampus, thalamus and cortex in the freely behaving animal during natural foraging and operant tasks.

<table border="0" cellpadding="0" cellspacing="0" width="618">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:169px;">Part Name</td>
			<td style="width:131px;">Manufacturer&#160;</td>
			<td style="width:143px;">Catalogue # (if applicable)</td>
			<td style="width:176px;">Part Description</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:169px;">Microdrive screws</td>
			<td style="width:131px;">Antrin</td>
			<td style="width:143px;"></td>
			<td style="width:176px;">Half Circle .6UNM Titanium Screws. 8mm thread. 9mm length from under head.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Tap-ease</td>
			<td style="width:131px;">AGS CO.</td>
			<td style="width:143px;">#TA2</td>
			<td style="width:176px;">Tapping Grease</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:169px;">Microdrives</td>
			<td style="width:131px;"></td>
			<td style="width:143px;"></td>
			<td style="width:176px;">See .STL file</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:169px;">Drive Body</td>
			<td style="width:131px;"></td>
			<td style="width:143px;"></td>
			<td style="width:176px;">See .STL file</td>
		</tr>
		<tr height="20">
			<td height="81" rowspan="4" style="height:81px;width:169px;">Outer Polyimide Guide Tube</td>
			<td rowspan="4" style="width:131px;">Minvasive Components&#160;&#160;</td>
			<td rowspan="4" style="width:143px;">IWG Item # 72113300022-012</td>
			<td style="width:176px;">Length:12&#8217;&#8217;,&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">ID:.0071&#8217;&#8217;,&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">OD:.0116&#8217;&#8217;,&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">WALL:.00225&#8217;&#8217;</td>
		</tr>
		<tr height="20">
			<td height="81" rowspan="4" style="height:81px;width:169px;">Inner Polyimide Guide Tube</td>
			<td rowspan="4" style="width:131px;">Minvasive Components&#160;</td>
			<td rowspan="4" style="width:143px;">IWG Item # 72113900001-012</td>
			<td style="width:176px;">Length: 12&#8217;&#8217;,&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">ID:.0035&#8217;&#8217;,&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">OD:.0055&#8217;&#8217;,&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">WALL:.001&#8217;&#8217;</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:169px;">Grounding Wire</td>
			<td style="width:131px;">A-M Systems, Inc.</td>
			<td style="width:143px;">&#160;Catalog # 791900</td>
			<td style="width:176px;">.008&#39;&#39; Bare, .011&#39;&#39; Coated</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:169px;"></td>
			<td style="width:131px;"></td>
			<td style="width:143px;"></td>
			<td style="width:176px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:169px;">Tri-Flow&#160;</td>
			<td style="width:131px;"></td>
			<td style="width:143px;"></td>
			<td style="width:176px;">Teflon based lubricant - Aerosol</td>
		</tr>
		<tr height="40">
			<td height="181" rowspan="5" style="height:181px;width:169px;">Microdrive Springs</td>
			<td rowspan="5" style="width:131px;">Lee Spring</td>
			<td rowspan="5" style="width:143px;">Part # CB0050B 07 E</td>
			<td style="width:176px;">Outside Diameter: 1.016 mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:176px;">Hole Diameter: 1.193 mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:176px;">Wire Diameter: 0.127 mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:176px;">Free Length 10.160 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Solid Length 3.581 mm</td>
		</tr>
		<tr height="59">
			<td height="80" rowspan="2" style="height:80px;width:169px;"><a name="RANGE!A24">Z-poxy 5 Minute</a></td>
			<td rowspan="2" style="width:131px;">Pacer Technology (Zap)</td>
			<td rowspan="2" style="width:143px;">PT37</td>
			<td rowspan="2" style="width:176px;"></td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">&#160;Silver Paint</td>
			<td style="width:131px;">&#160;GC Electronics</td>
			<td style="width:143px;">&#160;Part #: 22-023</td>
			<td style="width:176px;">Silver Print II</td>
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:169px;"></td>
			<td rowspan="2" style="width:131px;">Tri-Flow&#160;</td>
			<td align="right" rowspan="2" style="width:143px;">20009</td>
			<td rowspan="2" style="width:176px;"></td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="59">
			<td height="100" rowspan="3" style="height:100px;width:169px;">26 Gauge Hypodermic Tube - Stainless Steel</td>
			<td rowspan="3" style="width:131px;">Small Parts&#160;</td>
			<td rowspan="3" style="width:143px;">HTXX-26T-12-10</td>
			<td style="width:176px;">Length: 12&#8217;&#8217;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">ID: .012&#8217;&#8217;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">OD: .018&#8217;&#8217;</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:169px;">EIB screws</td>
			<td style="width:131px;">Component Supply Co.</td>
			<td style="width:143px;">MX-0090-03SP</td>
			<td style="width:176px;">#00-90 x 3/16&#8217;&#8217;</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:169px;">Fine Scissors - Toughcut</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:143px;">14058-09</td>
			<td style="width:176px;">22mm</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:169px;">Transparency Paper</td>
			<td style="width:131px;">3M</td>
			<td style="width:143px;">PP2500</td>
			<td style="width:176px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:169px;">Aluminum Foil</td>
			<td style="width:131px;">Reynold&#39;s Wrap Heavy Duty</td>
			<td style="width:143px;"></td>
			<td style="width:176px;">Extra Thick</td>
		</tr>
	</tbody>
</table>

design and fabrication of ultralight weight, adjustable multi-electrode probes for electrophysiological recordings in mice

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic targeting for microcircuit dissection and disease modeling. The introduction of optogenetics, for example, has allowed for bidirectional manipulation of genetically-identified neurons, at an unprecedented temporal resolution. To capitalize on these tools and gain insight into dynamic interactions among brain microcircuits, it is essential that one has the ability to record from ensembles of neurons deep within the brain of this small rodent, in both head-fixed and freely behaving preparations. To record from deep structures and distinct cell layers requires a preparation that allows precise advancement of electrodes towards desired brain regions. To record neural ensembles, it is necessary that each electrode be independently movable, allowing the experimenter to resolve individual cells while leaving neighboring electrodes undisturbed. To do both in a freely behaving mouse requires an electrode drive that is lightweight, resilient, and highly customizable for targeting specific brain structures.
A technique for designing and fabricating miniature, ultralight weight, microdrive electrode arrays that are individually customizable and easily assembled from commercially available parts is presented. These devices are easily scalable and can be customized to the structure being targeted; it has been used successfully to record from thalamic and cortical regions in a freely behaving animal during natural behavior.

Implant construction is a process that starts with the design of the 3D printed hyperdrive (Figure 1), proceeding to the construction of the bottom piece (Figure 2), the shielding cone (Figure 3), and the final assembly of the hyperdrive, by individual construction of the microdrives (Figure 4). These steps are followed by loading the microdrives with electrodes (please see 10). Following this step, it is possible to use these devices to record from multiple brain regions. In Figure 5, example traces from a simultaneous recording of the lateral geniculate nucleus (LGN) and Hippocampus (HPC) are shown. The stability of the single units shown in Figure 5B has been remarkable, showing consistent waveforms over a course of several days. These neurons were confirmed to be LGN neurons by being responsive to light-emitting diode stimulation, as shown by the peristimulus time histogram (PSTH) in Figure 5C. In this same preparation, HPC local field potential was recorded as a proxy for behavioral state. These traces showed sharp wave ripples (Figure 5D), during behavioral quiescence, consistent with their hippocampal origin.
<img alt="Figure 1" src="/files/ftp_upload/51675/51675fig1highres.jpg" /> 
Figure 1.&#160;Designing the hyperdrive in Solidworks. A. Schematic of a coronal section of a mouse brain at A/P coordinates -2.3 &#8211; -2.7 mm from bregma. Four individual polyimides (300 &#181;m) are drawn above the cortex, illustrating the targeting of the LGN region (red) with electrodes. B. Sketch of the design body. Revolving the blue contour 180&#176; results in a 3D design body model (inset). C. Addition of polyimide slots and drive handles to the design body. Revolving the red highlighted contours in B by 13&#176; results in a polyimide half-slot (top left). One drive handle is added by revolving the green contour in B by 15&#176; (top right). The second handle is added by using the circular pattern function (bottom left). The same function can be used to create the 16 polyimide half slots (bottom right). D. A new plane is added to the design (top), allowing to create a new sketch for the microdrive receptacle, comprised of the screw hole, polyimides hole and antitorque rail (bottom). E. These features will be implemented into the design using the cut and extrude functions and revolved 360&#176; to create 16 receptacles. F. Dimensions of the top piece sketch (left) and the 3D model (right). <a href="https://www.jove.com/files/ftp_upload/51675/51675fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/51675/51675fig2highres.jpg" /> 
Figure 2.&#160;Preparing the bottom piece of the hyperdrive. A. The first polyimide tube is placed onto double-sided tape. B. Subsequent tubes are placed individually, taking care to minimize space between tubes. C. After the first layer is laid out, a thin layer of cyanoacrylate glue is applied D. A second layer of polyimides is added quickly before the glue is dried. E. On top of the polyimides bundle, a 26 G&#160;cannula is added as a place holder for the optic fiber. F. The whole construct is securely fixed with a drop of epoxy. G. After removal of the cannula, the construct can be cut in the middle with a razor blade, yielding two identical bottom pieces. H. View on the cut surface of a finished bottom piece, illustrating the two double rows of four polyimides and the hole for the optic fiber. <a href="https://www.jove.com/files/ftp_upload/51675/51675fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/51675/51675fig3highres.jpg" /> 
Figure 3. Assembling the hyperdrive. A. The polyimide matrix is inserted into the drive body, and aligned with the electronic interface board (EIB) using the 26 G&#160;cannula. B. A small amount of epoxy is used to affix the polyimid matrix to the drive body. C. A second application of epoxy may be necessary, after which excess epoxy should be dremeled away D. Top view on the drive body with the matrix inserted. E. Using a small piece of 33 G&#160;polyimide tubing, the outer guide tubes are attached in the corresponding slots of the drive body. F. All outer guide tubes should be mapped to a rail, taking care to minimize tension on the tubes. G. After all outer guide tubes are mapped, they should be secured with epoxy and cut just above the inner lip. H. A microdrive assembly, consisting of a custom-built screw, a 5 mm spring and a top piece should be assembled and placed over a rail corresponding to one of the guide tubes. I. Each microdrive assembly should be carefully screwed into the drive body. J. After assembly, each guide tube should have a corresponding microdrive. K. Bottom view of the polyimide matrix. L - M. Polyimide tubes (0.005&#39;) are inserted into each outer guide tube. N. Each inner guide tube should fit snugly into the fork of it&#39;s corresponding microdrive. O.&#160;The inner polyimide tubes are attached with epoxy to the corresponding microdrive and cut as short as possible. After all inner guide tubes are epoxied, the inner guide tubes protruding from the polyimide-matrix should be cut flush with the matrix lip. P. Inverted macro view of the drive during the inner guide tube loading. Q. Top macro view of the drive during inner guide tube loading. R. Fully assembled hyperdrive with the EIB attached, ready to be loaded with electrodes. <a href="https://www.jove.com/files/ftp_upload/51675/51675fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/51675/51675fig4highres.jpg" /> 
Figure 4.&#160;Preparing the shielding cone. A. Cone template printed on transparency paper. B - D. A sheet of aluminum foil is glued to the template using a thin layer of epoxy. E. After cutting out the template, the cone is formed and glued together with epoxy.
<img alt="Figure 5" src="/files/ftp_upload/51675/51675fig5highres.jpg" /> 
Figure 5. Multi-site recordings using the ultralight-weight hyperdrive. A. Image of a freely behaving mouse with the hyperdrive implanted. B. Examples of two single unit waveforms recordings from this mouse. C. Left, Coronal section of the mouse brain highlighting the lateral geniculate nucleus, where some of the electrodes were lowered. Right, example peristimulus time histograms (PSTHs) of two LGN neurons aligned to visual stimulation (yellow bar). D. Right, coronal section highlighting the hippocampus (HPC), where another set of electrodes were lowered. Right, Example of local field potential recording of a hippocampal ripple (red highlight).
<img alt="Figure 6" src="/files/ftp_upload/51675/51675fig6highres.jpg" /> 
Figure 6. Overview of drive components. (Left) Comprehensive overview of hyperdrive components. (Right) Illustration of na individual microdrive assembly.

Watch this Scientific Journal Video about Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice at JoVE.com

Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse.

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic ...

design-fabrication-ultralight-weight-adjustable-multi-electrode

Research

JoVE Journal

Neuroscience

17.5K Views.  New York University Langone Medical Center. Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse.

Video: Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the stimulation parameters (e.g., orientation, field strength, and stimulation duration) need to be examined in mice brain slice preparations. Testing and arranging the orientation of the electrode relative to the position of the mice brain slice are feasible. The present method preserves the thalamocingulate pathway to evaluate the effect of DCS on anterior cingulate cortex seizure-like activities. The results of the multichannel array recordings indicated that cathodal DCS significantly decreased the amplitude of the stimulation-evoked responses and duration of 4-aminopyridine and bicuculline-induced seizure-like activity. This study also found that cathodal DCS applications at 15 min caused long-term depression in the thalamocingulate pathway. The present study investigates the effects of DCS on thalamocingulate synaptic plasticity and acute seizure-like activities. The current procedure can test the optimal stimulation parameters including orientation, field strength, and stimulation duration in an in vitro mouse model. Also, the method can evaluate the effects of DCS on cortical seizure-like activities at both the cellular and network levels.

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the ...

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for ...

Open&amp;#45;Source Implant for Multi&amp;#45;Region Electrophysiological Monitoring in Rats

The TD Drive: A Parametric, Open-Source Implant for Multi&#45;Area Electrophysiological Recordings in Behaving and Sleeping Rats

Intricate interactions between multiple brain areas underlie most functions attributed to the brain. The process of learning, as well as the formation and consolidation of memories, are two examples that rely heavily on functional connectivity across the brain. In addition, investigating hemispheric similarities and/or differences goes hand in hand with these multi-area interactions. Electrophysiological studies trying to further elucidate these complex processes thus depend on recording brain activity at multiple locations simultaneously and often in a bilateral fashion. Presented here is a 3D-printable implant for rats, named TD Drive, capable of symmetric, bilateral wire electrode recordings, currently in up to ten distributed brain areas simultaneously. The open-source design was created employing parametric design principles, allowing prospective users to easily adapt the drive design to their needs by simply adjusting high-level parameters, such as anterior-posterior and mediolateral coordinates of the recording electrode locations. The implant design was validated in n = 20 Lister Hooded rats that performed different tasks. The implant was compatible with tethered sleep recordings and open field recordings (Object Exploration) as well as wireless recording in a large maze using two different commercial recording systems and headstages. Thus, presented here is the adaptable design and assembly of a new electrophysiological implant, facilitating fast preparation and implantation.

Author Spotlight: Deciphering Memory and Learning Through Neural Implants for Multi&amp;#45;Region Brain Studies

Here, we present a unique, 3D-printable implant for rats, named TD Drive, capable of symmetric, bilateral wire electrode recordings, currently in up to ten distributed brain areas simultaneously.

Intricate interactions between multiple brain areas underlie most functions attributed to the brain. The process of learning, as well as the formation and ...

Recording Network Activity in Brain Assembloids: Multi-Electrode Array Approach

A Multi-Electrode Array Platform for Modeling Epilepsy Using Human Pluripotent Stem Cell-Derived Brain Assembloids

Human brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that recapitulate&#160;aspects of fetal brain development. The fusion of dorsal with ventral regionally specified brain organoids in vitro generates assembloids, which have functionally integrated microcircuits with excitatory and inhibitory neurons. Due to their structural complexity and diverse population of neurons, assembloids have become a useful in vitro tool for studying aberrant network activity. Multi-electrode array (MEA) recordings serve as a method for capturing electrical field potentials, spikes, and longitudinal network dynamics from a population of neurons without compromising cell membrane integrity. However, adhering assembloids onto the electrodes for long-term recordings can be challenging due to their large size and limited contact surface area with the electrodes. Here, we demonstrate a method to plate assembloids onto MEA plates for recording electrophysiological activity over a 2-month span. Although the current protocol utilizes human cortical organoids, it can be broadly adapted to organoids differentiated to model other brain regions. This protocol establishes a robust, longitudinal, electrophysiological assay for studying the development of a neuronal network, and this platform has the potential to be used in drug screening for therapeutic development in epilepsy.

Author Spotlight: Advancing Genetic Epilepsy Studies with Multi-Electrode Array-Based Long-Term Electrophysiological Monitoring of Human Brain Assembloids

This protocol aims to stably plate dorsal-ventral fused assembloids on multi-electrode arrays for modeling epilepsy in vitro.

Human brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that recapitulate&#160;aspects of fetal ...

Fabrication of TD Drive Implants for Electrophysiological Recordings of Sleeping and Live Rats 

To begin, create the 3D models. Click on upload to open the drive body design in the Autodesk Fusion software. Under the Modify tab, click on Change parameters.Adjust the coordinates for the first recording location by entering the anterior posterior coordinate and anterior posterior site one and the medial-lateral coordinate in medial-lateral site one and press OK.On the 3D printed model, use an M1 tap to tap the guide holes for the shuttle screws, which are the extension of the counter sink holes. Then slide a 3D-printed shuttle onto an M1 by 16 screw and use an M1 brass insert to hold the 3D printed shuttle in place. Using a small amount of soldering paste, solder the brass insert to the screw.After the shuttle assembly is cooled down, gently rotate the 3D-printed shuttle multiple times around the screw. Next, to assemble the drive, cut commercially available polyimide tubes to approximately 25 millimeters in length. Insert the polyimide guide tubes into the drive body and finalize the insertion using tweezers.Using a thin needle or toothpick, apply a small amount of liquid cyanoacrylate glue to the holes at the top of the drive body to fix the guide tubes in place. Also apply cyanoacrylate glue to the interface between the guide tubes and the guide stencil at the bottom of the drive body. Cut the polyimide guide tubes on the bottom such that they extend approximately one millimeter beyond the center pedestals of the drive body.Insert two shuttle assemblies in the drive body. Screw the electrode interface board to the drive body with M-2.5 by five polyamide screws. Then insert a stainless steel M2 nut into the extrusion on the left cap half and fix it with cyanoacrylate glue.Tap the hole in the front of the right cap half with an M1 tap. Next, prepare two metal plates as the surface for creating the electrode wire bundles. Attach plotting paper to the first plate and not-too-sticky painters tape onto the second plate with the sticky surface pointing up.Draw a clear line with an angle of 60 degrees on the plotting paper. For each electrode bundle, cut the electrode wire into the required number of pieces with the specified length. Gently pick up the four wires by touching them with a fingertip and place them as close as possible next to each other on the painters tape.Under a microscope, use fingers or forceps to place the wires together as close as possible. Apply a thin layer of liquid cyanoacrylate glue to the first two centimeters of the top of the bundle. After the setup dries fully, remove the wire bundle from the tape and transfer it to the plate with the plotting paper.For the retrosplenial cortex bundles, make a straight cut at the bottom of the array. For the hippocampus electrode bundles, place the array on the plotting paper such that it intersects the 60 degree line. Then use the line as a guide to make a cut at an angle of 60 degrees to the direction of the wires.Then using a razor or scalpel blade, carefully split the shortest of the four wires, cutting perpendicular to the wire direction. For the prefrontal cortex bundles, split the bottom of the array into two two-wire bundles. Shorten one of the two-wire bundles by one millimeter by cutting it perpendicular to the wire direction.Then cut two pieces of six centimeter length for the ground wire. Also cut eight pieces of six centimeters length for the electroencephalogram wire. Next, place an M1 by three stainless steel screw in a third hand.Wrap the de-insulated side of the ground or electroencephalogram wire around the shank of the screw. Apply a small amount of solder flux and solder the wire to the screw. Place a SIP DIP pin in the third hand so that the female side is accessible.Insert the de-insulated part of the opposite side of the wire into the SIP DIP pin and solder the wire to the pin. Next, place another SIP DIP pin in the holder with the male side accessible. Solder the de-insulated side of the other wire to the male side of the pin.Use a multimeter to verify that there is a continuous connection between the screw and the de-insulated wire end of the wire pin assembly when both assemblies are connected. To load the wire bundles into the drive, attach the drive to a holder. Once the drive body is stable, carefully slide one of the wire bundles into the respective polyimide tube.Use thin forceps to grab one of the wires and carefully bend it towards the desired hole and insert it. Once inserted, use a gold pin to pin it into the electrocorticography hole. Next, when the fixed wire arrays are aligned, apply a small amount of strong epoxy glue to the top of the guide tubes to fix the bundles in place.To fix the movable hippocampus wire arrays, first move the shuttle to the highest required position. Then push the wire bundles into the U-shaped opening of the shuttle and glue them in place with a small amount of a strong epoxy glue. Carefully insert the open end of the wire pin assembly of a ground wire through one of the through-holes marked ground and fix it using a gold pin.Remove the drive from the holder. Take care not to bend any the wire assemblies. To recover the electrode interface board after experimentation, gently push soft tweezers between the board and the drive body, or carefully lift the EIB by hand to release the remaining cyanoacrylate bond.The TD drive was implanted into eight rats for a pilot run, and implant surgeries were carried out two weeks post-arrival. Post-surgery, animals showed adaptability to the implants with fixed electrodes in frontal and retrosplenial regions and adjustable hippocampal bundles to enhance signal coverage. Sleep recordings were performed on tethered rats in a recording box, while wake data was recorded as wireless recordings in a larger maze.In seven out of eight animals, all target sites were reached on at least one hemisphere. While some animals began to lose their implants after two months, most retained them for up to 100 days. During this time, the local field potential remains stable, as observed by Delta oscillations in the hippocampal channel.