This study was supported by the National Institute on Drug Abuse Grants DA 006886, and DA 032270.

<ol>
	<li>Wire the bipolar stepper motors by screwing the wires into the connectors supplied with the driver board. Wire colors on bipolar stepper motors are standardized (Figure 1). 
	Note: The described stepper motors have a resolution of 1.8&#176;/step, geared to 0.346&#176;/step. A standard stereotax has 3 mm/360&#176; of travel. The final resolution is 2.88 &#956;m/step. The motors are also capable of fractional stepping.
	<ol>
		<li>Connect the green wire to A+, connect the black wire to A-, connect the red wire to B+ and connect the blue wire to B-.</li>
	</ol>
	</li>
	<li>Slide the couplers over the stepper motors, being careful to align the mounting holes, and secure them using 12x M3 Socket Head Screws (20 mm) (Figure 2).
	<ol>
		<li>Ensure that the couplers are firmly attached to the motors. 
		Note: The 3D models do not include threads. The parts are labeled with thread size, but they must be tapped after they are manufactured.</li>
	</ol>
	</li>
	<li>Remove the set screws from the thumb grips on all 3 axes of the stereotaxic instrument using a small hex key. The thumb grips are threaded, so turn them counter-clockwise for removal. Keep the PTFE washers in place on the arm (Figure 3).</li>
	<li>Screw the threaded end of the collars onto the threaded rods of the stereotaxic instrument&#39;s arms (Figure 3).
	<ol>
		<li>Ensure there is no gap between the collars and PTFE O-rings. This guarantees that coordinates are maintained when the robot changes directions.</li>
		<li>Secure the collars onto the threads of the stereotaxic arms using 3x NF10-32 (1/4 inch) cup point set screws.</li>
		<li>Slide each motor and coupler over the collars &#38; stereotaxic arms. Ensure that the motors sit flush with the arms, and the set screw holes on the collars line up with the flat portion of the motor shafts (Figure 4).</li>
		<li>Secure the couplers to the stereotax using the mounting holes and 6x NF10-32 (1/2 inch) cup point set screws (Figure 4).</li>
		<li>Secure the collars to the motor shafts using 3x NF10-32 (1/4 inch) set screws (Figure 4).</li>
	</ol>
	</li>
	<li>Prepare the CNC driver board by setting each of the controller pins to half stepping. 
	Note: This stepper motor driver comes as an exposed circuit board. A case may be built, although it is not necessary. Also, a number of different bipolar stepper motor drivers may be used. If so, ensure that the setup instructions are followed for the specific board purchased.
	<ol>
		<li>Align all 6 pins per stepper motor in the same way. Half-stepping allows for double the step resolution in Degrees/step (Figure 5).</li>
		<li>Flip pin 1 to the on position, pin 2 to the off position, pin 3 to the on position, pin 4 to the off position, pin 5 to the on position, and pin 6 to the off&#160;position (Figure 5).</li>
	</ol>
	</li>
	<li>Plug the motors (X - Y - Z) into the stepper motor driver, along with the 12 V power supply. The correct placement is marked on the driver. Also, attach the stepper driver to a computer&#39;s serial port using a DB25cable (Figure 6).</li>
	<li>Install CNC milling software onto a personal computer (this will need to be located in a surgical area) following the default instructions. Once installed, open the software to begin configuration.
	<ol>
		<li>Configure the software to communicate with the stepper motors. 
		Note: The following instructions are intended for use only with the TB6560 stepper motor driver.</li>
		<li>Click through the software&#39;s menus as follows. Open &#8594;Config&#8594; Ports and Pins&#8594;Output Signals. Fill in the prompt to match Figure 7 and hit apply.</li>
		<li>Click through the software&#39;s menus as follows. Open&#8594;Config&#8594;Ports and Pins&#8594;Input Signals. Fill in the prompt to match Figure 8 and hit apply.</li>
		<li>Click through the software&#39;s menus as follows. Open&#8594;Config&#8594;Ports and Pins&#8594;Motor Outputs. Fill in the prompt to match Figure 9 and hit apply.</li>
		<li>Click through the software&#39;s menus as follows. Open&#8594;Config&#8594;Motor tuning. Fill in the prompt to match&#160;Figure 10 and click&#160;Save Axis Settings. Repeat the previous step for all 3 axes using the same values.</li>
	</ol>
	</li>
	<li>Calibrate the stereotax to the scale of the CNC software. 
	Note: The software is designed for standard milling machines, so its unit of measure will not be proportional to the travel of a stereotaxic instrument.
	<ol>
		<li>Set the motors&#39; velocity to 1 inch per minute, and &#34;jog&#34; the stereotaxic instrument&#39;s Z-axis with PgUp/PgDn to the nearest millimeter. 
		Note: The maximum recommended jogging speed is 3,500 &#181;m/sec and the maximum recommended cutting speed is 500 &#181;m/sec.</li>
		<li>Zero the Z-axis, and Jog the stereotaxic instrument 1 mm. The distance traveled on the Z-axis in Mach3 is the &#34;Scaling Constant&#34;. Machine coordinates are determined by multiplying skull coordinates (mm) by the &#34;Scaling Constant&#34;.</li>
		<li>Perform random tests of all 3 axes by programming them to travel some known distances, and ensure the movements are accurate. If the stereotax travel is too far or short, modify the scaling constant accordingly. 
		Note: Once scaling is complete, the included custom scripts may be used to generate g-code for surgeries. However, it is highly recommended that users become familiar with g-code before attempting to auto-generate surgeries. This is imperative for troubleshooting and modifying automated surgeries.</li>
	</ol>
	</li>
	<li>Attach the micro motor drill to the stereotaxic instrument using the extra large probe holder. Note: The minimum recommended drill bit speed is 40,000 rpm.</li>
	<li>Auto-generate G-Code for a sharp edge craniotomy with 3 skull screw holes.
	<ol>
		<li>Place all of the custom scripts from the software table into a single folder on a PC.</li>
		<li>Open the script &#34;SharpEdgeCraniotomy.m&#34;and run the code.</li>
		<li>Select Both&#160;to the prompt &#34;What Type of Surgery Will You be Performing?&#34; (Figure 11).</li>
		<li>Select&#160;Custom, to define the corners of the skull window. Fill in each prompt to match Figure 12.</li>
		<li>Define the X and Y positions of the craniotomy corners. Each coordinate must be entered in correct order, according the example in Figure 13.</li>
		<li>Enter 3 in the prompt to produce 3 skull holes (Figure 14).</li>
		<li>Select Define Using Coordinates, and enter the coordinates of each hole from the template in Figure 15. 
		Note: If precise coordinates are not important, there is an option to point and click the holes&#39; positions onto an image of a rat skull. Positions will be automatically generated.</li>
		<li>Define the drilling parameters. For the first tests surgery, accept the default values. 
		Note: These values are dependent on the stepper motors, and the animal&#39;s skull. Every rat breed and target location has a slightly different skull thickness. For the initial few surgeries using this device, be prepared to test the drilling depths and remove any remaining skull pieces manually. The values can then be modified for future surgeries (Figure 16).</li>
		<li>Name the g-code; it will be automatically generated and saved to the working directory.</li>
	</ol>
	</li>
	<li>Load the g-code into the CNC milling software and a test skull into the stereotaxic instruments ear bars.
	<ol>
		<li>Manually jog the drill bit to Bregma using the arrow keys. Use a slow jog speed (~5 inch/m) to ensure accuracy.</li>
		<li>Start the drill bit rotating at greater than 38,000 rpm.</li>
		<li>Press&#160;CycleStart; the stereotax will perform many passes of the same cut, at different depths. Between each pass, the stereotax will pause, so the surgeon may continue or abort cutting. Press Continue Cycle (Alt-R) to continue cutting passes.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Yin, H. H., Knowlton, B. J., Balleine, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lesions+of+dorsolateral+striatum+preserve+outcome+expectancy+but+disrupt+habit+formation+in+instrumental+learning.">Lesions of dorsolateral striatum preserve outcome expectancy but disrupt habit formation in instrumental learning.</a> Eur. J. Neurosci. 19 (1), 181-189 (2004).</li><li>West, M. O., Woodward, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+for+microiontophoretic+study+of+single+neurons+in+the+freely+moving+rat.">A technique for microiontophoretic study of single neurons in the freely moving rat.</a> J. Neurosci. Methods. 11 (3), 179-186 (1984).</li><li>Peoples, L. L., West, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phasic+firing+of+single+neurons+in+the+rat+nucleus+accumbens+correlated+with+the+timing+of+intravenous+cocaine+self-administration.">Phasic firing of single neurons in the rat nucleus accumbens correlated with the timing of intravenous cocaine self-administration.</a> J. Neurosci. 16 (10), 3459-3473 (1996).</li><li>Wolske, M., Rompre, P. P., Wise, R. A., West, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+single+neurons+in+the+rat+nucleus+accumbens+during+self-stimulation+of+the+ventral+tegmental+area.">Activation of single neurons in the rat nucleus accumbens during self-stimulation of the ventral tegmental area.</a> J. Neurosci. 13 (1), 1-12 (1993).</li><li>Bozza, T., McGann, J. P., Mombaerts, P., &amp;Wachowiak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+neuronal+activity+by+targeted+expression+of+a+genetically+encoded+probe+in+the+mouse.">In vivo imaging of neuronal activity by targeted expression of a genetically encoded probe in the mouse.</a> Neuron. 42 (1), 9-21 (2004).</li><li>Pitts, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Office+of+Laboratory+Animal+Welfare.">Office of Laboratory Animal Welfare.</a> Institutional animal care and use committee guidebook. , (2002).</li><li>Yoon, T., Otto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+contributions+of+dorsal+vs.+ventral+hippocampus+to+auditory+trace+fear+conditioning.">Differential contributions of dorsal vs. ventral hippocampus to auditory trace fear conditioning.</a> Neurobiol. Learn. Mem. 87 (4), 464-475 (2007).</li><li>Root, D. H., 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=Differential+roles+of+ventral+pallidum+subregions+during+cocaine+self-administration+behaviors.">Differential roles of ventral pallidum subregions during cocaine self-administration behaviors.</a> J. Comp. Neurol. 521 (3), 558-588 (2012).</li><li>Yang, G., Pan, F., Parkhurst, C. N., Grutzendler, J., Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thinned-skull+cranial+window+technique+for+long-term+imaging+of+the+cortex+in+live+mice.">Thinned-skull cranial window technique for long-term imaging of the cortex in live mice.</a> Nat. Protoc. 5 (2), 201-208 (2010).</li><li>Feng, L., Sametsky, E. A., Gusev, A. G., Uteshev, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responsiveness+to+nicotine+of+neurons+of+the+caudal+nucleus+of+the+solitary+tract+correlates+with+the+neuronal+projection+target.">Responsiveness to nicotine of neurons of the caudal nucleus of the solitary tract correlates with the neuronal projection target.</a> J. Neurophysiol. 108 (7), 1884-1894 (2012).</li><li>Babaei, P., Soltani Tehrani, ., B, A., Alizadeh, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplanted+Bone+Marrow+Mesenchymal+Stem+Cells+Improve+Memory+in+Rat+Models+of+Alzheimer's+Disease.">Transplanted Bone Marrow Mesenchymal Stem Cells Improve Memory in Rat Models of Alzheimer's Disease.</a> Stem Cells Int. 2012, 369417 (2012).</li></ol>

The authors have no competing financial interests to disclose.

The use of automated surgery equipment helps to eliminate some of the most common problems in neuroscience research. First, the tool paths are 100% reproducible. Every cut is guaranteed to be in the same location relative to Bregma. Second, it should reduce experimenter error. Although many researchers are highly skilled surgeons, it takes an exceptional amount of practice to become even a competent surgeon. This device will allow new students to quickly and easily perform highly accurate surgeries. Third, motorized surg...

Stereotactic rodent surgery is used in a wide variety of neuroscience applications, including lesioning1, iontophoresis2, microwire implantation3, stimulation4, and thin skull imaging5. However, there are major hurdles facing those who wish to apply these techniques, including the steep learning curve for performing accurate stereotactic surgery and the high probability of human error. Human errors include measurement and calculation failures, as well as the low accuracy and replicability of human movements. In an effort to reduce these confounding errors, stereotactic surgeons would benefit from a system that ensures that all surgical procedures are performed identically across subjects. The reduction of errors is also one method by which investigators can minimize the use of animal subjects, a primary goal of the National Institutes of Health for animal experimentation6. In an ideal world, all stereotactic surgeries would be perfectly replicable within experiments, and between labs. To address this issue, companies have developed new ultra-precise stereotaxics, and digital displays for reading measurements. To remove human movement errors, motorized micro manipulators and stereotaxics were produced commercially, but their high cost can be prohibitive to a laboratory with a limited budget. Also, their software is fully proprietary, and cannot be modified by the researcher to accommodate a new type of surgery.
An affordable solution to the human error problem is to build a robotic stereotax from a lab&#39;s existing model, using industry standard CNC equipment. Because of a burgeoning CNC hobbyist community, the materials are significantly less expensive than scientific equipment. This allows one to build an accurate CNC stereotaxic instrument, which is also highly flexible and inexpensive. With a basic knowledge of CNC machining and G-Code, individuals can program any stereotactic surgery that they imagine, without the limitations of proprietary software. And, in order to expedite the production of g-code for simple surgeries, this protocol includes software that allows the user to design surgeries (sharp edge craniotomy, thin skull windowing, hole drilling &#38; implant lowering) within point and click menus. These programs output a completed g-code that may be run directly from CNC software.
In all, a motorized stereotaxic upgrade is ideal for those who have an interest in increasing the accuracy and replicability of surgeries, while retaining the flexibility and low cost of an open source platform.

<table border="0" cellpadding="0" cellspacing="0" width="749">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="44">
			<td height="44" style="height:44px;width:141px;">1x Standard U Frame Stereotax</td>
			<td style="width:137px;">Kopf</td>
			<td style="width:117px;"><a href="http://www.kopfinstruments.com/Stereotaxic/900.html">Kopf</a></td>
			<td style="width:355px;">This protocol should work with most existing stereotaxic devices.</td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:141px;">3x 12 V, 1.6 A, 233 oz-inch Geared Bipolar Stepper Motor</td>
			<td style="width:137px;">Phidgets</td>
			<td style="width:117px;"><a href="http://www.robotshop.com/geared-bipolar-stepper-motor-3v-233-oz-in.html">Robot Shop</a></td>
			<td style="width:355px;">Any high torque geared stepper motor should do.&#160;</td>
		</tr>
		<tr height="73">
			<td height="73" style="height:73px;width:141px;">1x 3 Axis CNC Stepper Motor Driver Board Controller</td>
			<td style="width:137px;">Toshiba</td>
			<td style="width:117px;"><a href="http://www.ebay.com/itm/CNC-Router-3-Axis-TB6560-3-5A-Stepper-Motor-Driver-Board-For-Engraving-Machine-/300981888819">Ebay</a></td>
			<td style="width:355px;">Any 3 Axis CNC driver should do. Linked Item includes Mach3 CNC software.&#160;</td>
		</tr>
		<tr height="94">
			<td height="94" style="height:94px;width:141px;">2x Arm Couplers: medial-lateral (ML) &#38; dorsal-ventral (DV)</td>
			<td style="width:137px;">custom machined</td>
			<td style="width:117px;"><a href="http://eden.rutgers.edu/~mrcoffey/images/3D%20Models%20-%20CNC%20Stereotax%20Parts.zip">Part Drawings</a></td>
			<td style="width:355px;">These must be machined by your local machine shop. (costs will vary)</td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:141px;">1x anterior-posterior (AP) Coupler</td>
			<td style="width:137px;">custom machined</td>
			<td style="width:117px;"><a href="http://eden.rutgers.edu/~mrcoffey/images/3D%20Models%20-%20CNC%20Stereotax%20Parts.zip">Part Drawings</a></td>
			<td style="width:355px;">These must be machined by your local machine shop. (costs will vary)</td>
		</tr>
		<tr height="127">
			<td height="172" rowspan="3" style="height:172px;width:141px;">3x Motor to Stereotax Collar</td>
			<td rowspan="3" style="width:137px;">custom machined</td>
			<td style="width:117px;"><a href="http://eden.rutgers.edu/~mrcoffey/images/3D%20Models%20-%20CNC%20Stereotax%20Parts.zip">Part Drawings</a></td>
			<td rowspan="3" style="width:355px;">These must be machined by your local machine shop. (costs will vary)</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:117px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:117px;"><a href="http://eden.rutgers.edu/~mrcoffey/images/Collar.html">View in Browser</a></td>
		</tr>
		<tr height="43">
			<td height="65" rowspan="2" style="height:65px;width:141px;">12x NF10-32 Cup Point Set Screws</td>
			<td rowspan="2" style="width:137px;">McMaster Carr</td>
			<td style="width:117px;"><a href="http://www.mcmaster.com/#92313A829">&#189;&#8221; Length</a></td>
			<td rowspan="2" style="width:355px;">You will need 6 of each.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:117px;"><a href="http://www.mcmaster.com/#92313A825">&#188;&#8221; Length</a></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:141px;">12x M3 Socket Head Screws (20 mm)</td>
			<td style="width:137px;">McMaster Carr</td>
			<td style="width:117px;"><a href="http://www.mcmaster.com/#92855A319">20mm Length</a></td>
			<td style="width:355px;">You will need 4 for each motor</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:141px;">1x Micro-Motor Drill&#160;</td>
			<td style="width:137px;">Buffalo Dental</td>
			<td style="width:117px;"><a href="http://www.buffalodental.com/pdfnew/x50.pdf">X50</a></td>
			<td style="width:355px;">Any Micromotor drill will work.&#160; At least 38,000 rpm recommended</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:141px;">1x 12 V DC Power Supply</td>
			<td style="width:137px;">12 Volt Adapters</td>
			<td style="width:117px;"><a href="http://www.12vadapters.com/adapter/power-supply/12v/7-amp-7a.html">12v DC &#8211; 7 Amp</a></td>
			<td style="width:355px;">Any 12 V DC PSU should work (ensure amperage rating is higher than the sum of the motors&#8217; amperage).</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:141px;">1x Extra Large Probe Holder</td>
			<td style="width:137px;">Stoelting</td>
			<td><a href="http://www.stoeltingco.com/neuroscience/accessories/stereotaxic-probe-holders/extra-large-probe-holder-551.html">Stoelting</a></td>
			<td style="width:355px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:141px;">1x Grade B Rat Skull</td>
			<td style="width:137px;">Skulls Unlimited</td>
			<td><a href="http://www.skullsunlimited.com/record_variant.php?id=4009">Skulls Unlimited</a></td>
			<td style="width:355px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Mach 3 Mill</td>
			<td style="width:137px;">ArtSoft USA</td>
			<td style="width:117px;"><a href="http://www.machsupport.com/ccount/click.php?id=1">Trial Download</a></td>
			<td style="width:355px;">Any Standard CNC controlling software should work.</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:141px;">Surgery Designer</td>
			<td style="width:137px;">Kevin Coffey &#38; David Barker</td>
			<td style="width:117px;"><a href="http://www.mathworks.com/matlabcentral/fileexchange/40282-robot-assisted-stereotactic-surgery">MATLAB File Exchange</a></td>
			<td style="width:355px;">These codes are available to modify. We accept no responsibility for your use or modification of code.</td>
		</tr>
	</tbody>
</table>

building an open-source robotic stereotaxic instrument

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (CNC) stereotaxic instrument for around $1,000 (excluding a drill), using industry standard stepper motors and CNC controlling software. Each axis has variable speed control and may be operated simultaneously or independently. The robot&#39;s flexibility and open coding system (g-code) make it capable of performing custom tasks that are not supported by commercial systems. Its applications include, but are not limited to, drilling holes, sharp edge craniotomies, skull thinning, and lowering electrodes or cannula. In order to expedite the writing of g-coding for simple surgeries, we have developed custom scripts that allow individuals to design a surgery with no knowledge of programming. However, for users to get the most out of the motorized stereotax, it would be beneficial to be knowledgeable in mathematical programming and G-Coding (simple programming for CNC machining).
The recommended drill speed is greater than 40,000 rpm. The stepper motor resolution is 1.8&#176;/Step, geared to 0.346&#176;/Step. A standard stereotax has a resolution of 2.88 &#956;m/step. The maximum recommended cutting speed is 500 &#956;m/sec. The maximum recommended jogging speed is 3,500 &#956;m/sec. The maximum recommended drill bit size is HP 2.

The end result of the surgery designed in the methods will be a rat skull with a sharp edge craniotomy, and 3 skull holes (Figure 17). Note that the skull used to demonstrate the surgery was much wider than the prototypical rat skull. The sharp edge craniotomy may be used to insert a microwire array into the brain, for high density neural recordings. The CNC stereotax may also be used to lower the array with great precision. Software is included in this protocol that allows the surgeon to define the para...

Watch this Scientific Journal Video about Building An Open-source Robotic Stereotaxic Instrument at JoVE.com

Building An Open-source Robotic Stereotaxic Instrument

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (computer numeric controlled; CNC) stereotaxic instrument for around $1,000 (excluding a drill).

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (CNC) stereotaxic instrument for ...

building-an-open-source-robotic-stereotaxic-instrument

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

14.8K Views.  Rutgers, The State University of New Jersey. This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (computer numeric controlled; CNC) stereotaxic instrument for around $1,000 (excluding a drill).

Video: Building An Open-source Robotic Stereotaxic Instrument

Rodent Stereotaxic Surgery and Animal Welfare Outcome Improvements for Behavioral Neuroscience

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to investigate the effects of locally manipulated neurotransmitter and signaling pathways in awake, behaving animals. Moreover, the stereotaxic implantation of electrodes for electrophysiological stimulation and recording studies has been instrumental to our current understanding of neuroplasticity and brain networks in behaving animals. Ever-increasing knowledge about optimizing surgical techniques in rodents1-4, public awareness concerning animal welfare issues and stringent legislation (e.g., the 2010 European Union Directive on the use of laboratory animals5) prompted us to refine these surgical procedures, particularly with respect to implementing new procedures for oxygen supplementation and the continuous monitoring of blood oxygenation and heart rate levels during the surgery as well as introducing a standardized protocol for post-surgical care. Our observations indicate that these modifications resulted in an increased survival rate and an improvement in the general condition of the animals after surgery (e.g. less weight loss and a more active animal). This video presentation will show the general procedures involved in this type of stereotaxic surgery with special attention to our several modifications. We will illustrate these surgical procedures in rats, but it is also possible to perform this type of surgery in mice or other small laboratory animals by using special adaptors for the stereotaxic apparatus6.

Stereotaxic surgery on rodents allows for targeted administration of drugs or electrical stimulation and recordings in awake, behaving animals. In this video presentation we will demonstrate recent procedural refinements to this long-standing procedure that successfully improved survival rate and reduced post-surgical weight loss.

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Non-restraining EEG Radiotelemetry: Epidural and Deep Intracerebral Stereotaxic EEG Electrode Placement

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and neurodegenerative diseases as well as epilepsies. This powerful technique does not only provide valuable insights into the underlying pathophysiological mechanisms, i.e., the etiopathogenesis of CNS related diseases, it also facilitates the development of new translational, i.e., therapeutic approaches. Whereas competing techniques that make use of recorder systems used in jackets or tethered systems suffer from their unphysiological restraining to semi-restraining character, radiotelemetric EEG recordings overcome these disadvantages. Technically, implantable EEG radiotelemetry allows for precise and highly sensitive measurement of epidural and deep, intracerebral EEGs under various physiological and pathophysiological conditions. First, we present a detailed protocol of a straight forward, successful, quick and efficient technique for epidural (surface) EEG recordings resulting in high-quality electrocorticograms. Second, we demonstrate how to implant deep, intracerebral EEG electrodes, e.g., in the hippocampus (electrohippocampogram). For both approaches, a computerized 3D stereotaxic electrode implantation system is used. The radiofrequency transmitter itself is implanted into a subcutaneous pouch in both mice and rats. Special attention also has to be paid to pre-, peri- and postoperative treatment of the experimental animals. Preoperative preparation of mice and rats, suitable anesthesia as well as postoperative treatment and pain management are described in detail.

Non-restraining EEG radiotelemetry is a valuable methodological approach to record in vivo long-term electroencephalograms from freely moving rodents. This detailed protocol describes stereotaxic epidural and deep intracerebral electrode placement in different brain regions in order to obtain reliable recordings of CNS rhythmicity and CNS related behavioral stages.

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and ...

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has proven to be an invaluable model organism in helping to tackle this challenge, from inspiring technological approaches,&#160;building the human brain connectome, to actually shedding light on the specific molecular drivers of basic functional patterns. The bulk of the behavioral studies in C. elegans have been performed on solid substrates. In liquid, animals exhibit behavioral patterns that include movement at a range of speeds in 3D, as well as partial body movements, such as a posterior curl without anterior shape change, which introduce new challenges for quantitation. The steps of a simple procedure, and use of a software that enables high-resolution analysis of C. elegans swim behavior, are presented here. The software, named CeleST, uses a specialized computer program that tracks multiple animals simultaneously and provides novel measures of C. elegans locomotion in liquid (swimming). The measures are mostly grounded in animal posture and based on mathematics used in computer vision and pattern recognition, without computational requirements for threshold cut-offs. The software tool can be used to both assess overall swimming prowess in hundreds of animals from combined small batch trials and to reveal novel phenotypes even in well-characterized genetic mutants. The preparation of specimens for analysis with CeleST is simple and low-tech, enabling wide adaptation by the scientific community. Use of the computational approach described here should therefore contribute to the greater understanding of behavior and behavioral circuits in the C. elegans model.

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

An efficient and simple methodology for computer-based analysis of nematode swimming behavior in liquid is described. The method requires little to no investment for C. elegans laboratories. The hardware used is standard, and the computer software for the behavioral analysis (CeleST) is an open source one.

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has ...

High-density Electroencephalographic Acquisition in a Rodent Model Using Low-cost and Open-source Resources

Advanced electroencephalographic analysis techniques requiring high spatial resolution, including electrical source imaging and measures of network connectivity, are applicable to an expanding variety of questions in neuroscience. Performing these kinds of analyses in a rodent model requires higher electrode density than traditional screw electrodes can accomplish. While higher-density electroencephalographic montages for rodents exist, they are of limited availability to most researchers, are not robust enough for repeated experiments over an extended period of time, or are limited to use in anesthetized rodents.1-3 A proposed low-cost method for constructing a durable, high-count, transcranial electrode array, consisting of bilaterally implantable headpieces is investigated as a means to perform advanced electroencephalogram analyses in mice or rats.

Procedures for headpiece fabrication and surgical implantation necessary to produce high signal to noise, low-impedance electroencephalographic and electromyographic signals are presented. While the methodology is useful in both rats and mice, this manuscript focuses on the more challenging implementation for the smaller mouse skull. Freely moving mice are only tethered to cables via a common adapter during recording. One version of this electrode system that includes 26 electroencephalographic channels and 4 electromyographic channels is described below.

Instructions for the low-cost construction and surgical implantation of a chronic transcranial high-density electroencephalographic montage into mice are provided. Signal recording, extraction, and processing techniques are also described.

Advanced electroencephalographic analysis techniques requiring high spatial resolution, including electrical source imaging and measures of network ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset (Callithrix jacchus)

Marmosets (Callithrix jacchus) are small non-human primates that are gaining popularity in biomedical and preclinical research, including the neurosciences. Phylogenetically, these animals are much closer to humans than rodents. They also display complex behaviors, including a wide range of vocalizations and social interactions. Here, an effective stereotaxic neurosurgical procedure for implantation of recording electrode arrays in the common marmoset is described. This protocol also details the pre- and postoperative steps of animal care that are required to successfully perform such a surgery. Finally, this protocol shows an example of local field potential and spike activity recordings in a freely behaving marmoset 1 week after the surgical procedure. Overall, this method provides an opportunity to study the brain function in awake and freely behaving marmosets. The same protocol can be readily used by researchers working with other small primates. In addition, it can be easily modified to allow other studies requiring implants, such as stimulating electrodes, microinjections, implantation of optrodes or guide cannulas, or ablation of discrete tissue regions.

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset Callithrix jacchus

This work presents a protocol to perform a stereotaxic, neurosurgical implantation of&#160;microelectrode arrays in the common marmoset. This method specifically enables electrophysiological recordings in freely behaving animals but can be easily adapted to any other similar neurosurgical intervention in this species (e.g., cannula for drug administration or electrodes for brain stimulation).

Building An Open-source Robotic Stereotaxic Instrument

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Rodent Stereotaxic Surgery and Animal Welfare Outcome Improvements for Behavioral Neuroscience

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Non-restraining EEG Radiotelemetry: Epidural and Deep Intracerebral Stereotaxic EEG Electrode Placement

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

High-density Electroencephalographic Acquisition in a Rodent Model Using Low-cost and Open-source Resources

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset (Callithrix jacchus)