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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.

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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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

17.6K 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

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

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 ...

A Computer-assisted Multi-electrode Patch-clamp System

The patch-clamp technique is today the most well-established method for recording electrical activity from individual neurons or their subcellular compartments. Nevertheless, achieving stable recordings, even from individual cells, remains a time-consuming procedure of considerable complexity. Automation of many steps in conjunction with efficient information display can greatly assist experimentalists in performing a larger number of recordings with greater reliability and in less time. In order to achieve large-scale recordings we concluded the most efficient approach is not to fully automatize the process but to simplify the experimental steps and reduce the chances of human error while efficiently incorporating the experimenter's experience and visual feedback. With these goals in mind we developed a computer-assisted system which centralizes all the controls necessary for a multi-electrode patch-clamp experiment in a single interface, a commercially available wireless gamepad, while displaying experiment related information and guidance cues on the computer screen. Here we describe the different components of the system which allowed us to reduce the time required for achieving the recording configuration and substantially increase the chances of successfully recording large numbers of neurons simultaneously.

Multi-electrode patch-clamp recordings constitute a complex task. Here we show how, by automating of many of the experimental steps, it is possible to accelerate the process leading to qualitative improvement in performance and number of recordings.

The patch-clamp technique is today the most  well-established method for recording electrical activity from individual  neurons or their subcellular ...

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 ...

Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in the brain stem. Loss of hair cells is a major cause of sensory hearing loss. Prosthetic devices such as cochlear implants function by bypassing lost hair cells and directly stimulating SGNs electrically, allowing for restoration of hearing in deaf patients. The performance of these devices depends on the functionality of SGNs, the implantation procedure and on the distance between the electrodes and the auditory neurons. 
We hypothesized, that reducing the distance between the SGNs and the electrode array of the implant would allow for improved stimulation and frequency resolution, with the best results in a gapless position. Currently we lack in vitro culture systems to study, modify and optimize the interaction between auditory neurons and electrode arrays and characterize their electrophysiological response. To address these issues, we developed an in vitro bioassay using SGN cultures on a planar multi electrode array (MEA). With this method we were able to perform extracellular recording of the basal and electrically induced activity of a population of spiral ganglion neurons. We were also able to optimize stimulation protocols and analyze the response to electrical stimuli as a function of the electrode distance. This platform could also be used to optimize electrode features such as surface coatings.

We present a protocol to culture primary murine spiral ganglion neuron explants on multi electrode arrays to study neuronal response profiles and optimize stimulation parameters. Such studies aim to improve the neuron-electrode interface of cochlear implants to benefit hearing in patients as well as the energy consumption of the device.

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in ...

A Visual Guide to Sorting Electrophysiological Recordings Using 'SpikeSorter'

Few stand-alone software applications are available for sorting spikes from recordings made with multi-electrode arrays. Ideally, an application should be user friendly with a graphical user interface, able to read data files in a variety of formats, and provide users with a flexible set of tools giving them the ability to detect and sort extracellular voltage waveforms from different units with some degree of reliability. Previously published spike sorting methods are now available in a software program, SpikeSorter, intended to provide electrophysiologists with a complete set of tools for sorting, starting from raw recorded data file and ending with the export of sorted spikes times. Procedures are automated to the extent this is currently possible. The article explains and illustrates the use of the program. A representative data file is opened, extracellular traces are filtered, events are detected and then clustered. A number of problems that commonly occur during sorting are illustrated, including the artefactual over-splitting of units due to the tendency of some units to fire spikes in pairs where the second spike is significantly smaller than the first, and over-splitting caused by slow variation in spike height over time encountered in some units. The accuracy of SpikeSorter's performance has been tested with surrogate ground truth data and found to be comparable to that of other algorithms in current development.

The article shows how to use the program SpikeSorter to detect and sort spikes in extracellular recordings made with multi-electrode arrays.

Few stand-alone software applications are available for sorting spikes from recordings made with multi-electrode arrays. Ideally, an application should be ...

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 ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...