The authors have no acknowledgements.

All devices used herein are FDA approved and the protocol contained herein constitutes the standard of care at our institution. As such, no IRB approval was needed for the detailing of this protocol.
1. Pre-implantation phase
<ol>
	<li>Create an anatamo-electro-clinical (AEC) hypothesis. 
	NOTE: Creation of the AEC hypothesis relies on the coordination of multiple non-invasive techniques for identifying the potential EZ. A team of experts, including epileptologists, radiologists, and epilepsy surgeons will typically convene a meeting to discuss the clinical data for each patient in order to create the AEC hypothesis, which serves as the initial hypothesis for the patient&#8217;s EZ. The details of how this is accomplished is beyond the scope of this article.</li>
	<li>Identify the best methodology for invasive monitoring depending on the location of the AEC hypothesis. Table 1 lists the different scenarios for which SEEG is preferred over subdural grids (SDG) with or without depth electrodes for invasive monitoring.</li>
	<li>After a patient is deemed a candidate for SEEG evaluation, create an implantation strategy. 
	NOTE: The implantation strategy should adequately cover the area identified as a part of the AEC hypothesis as well as the wider epileptogenic network in general and neighboring areas of eloquent cortex. This monitoring aids the surgeon in defining the borders of the resection.
	<ol>
		<li>Obtain a pre-operative volumetric MRI and CTA.</li>
		<li>Transfer the images in DICOM format to the stereotactic robot&#8217;s native planning software and perform imaging fusion (T1+Gadolinium MRI fused with CTA). 
		NOTE: Imaging fusion is performed automatically by the robot&#8217;s software. One need only select the studies that need to be fused.</li>
		<li>Plan the trajectory of each individual electrode array within the 3D reconstruction of the MRI-CTA fusion, making sure to maximize sampling from a multitude of areas, including superficial, intermediate, and deep cortical and subcortical areas within the AEC hypothesis.
		<ol>
			<li>Define each trajectory by manually selecting the surface entry point and deep target point for each electrode. 
			NOTE: Generally, it is best to initially use a working distance of 150 mm from the drilling platform to the deep target point and then adjust the depth to maximally reduce the working distance in order to improve implantation accuracy.</li>
		</ol>
		</li>
		<li>Verify each implantation trajectory.
		<ol>
			<li>Review each electrode in the 3D MRI-CTA fusion reconstruction individually to make sure that the trajectory does not compromise any vascular structures, adjusting any trajectories as needed.</li>
		</ol>
		</li>
		<li>Review the overall implantation schema in the 3D MRI reconstruction, assessing for any trajectory collisions.</li>
		<li>Verify that the surface entry points are all at least 1.5 cm apart on the skin surface, as anything closer than this would be prohibitive to implantation later.</li>
	</ol>
	</li>
</ol>
2. Operative technique
<ol>
	<li>In the OR, prepare the patient and place them supine while preparing the stereotactic robot for surgery.
	<ol>
		<li>Intubate under general anesthesia according to the recommendations of the anesthesiologist. Use propofol for sufficient anesthesia and verify by adequate electrophysiological recordings as certified by a clinical epileptologist.</li>
		<li>Fix the patient&#8217;s head using a three-point fixation head holder. 
		NOTE: This is a standard 4-point Lexell frame. Occasionally one of the front posts will be removed in order to facilitate registration of the robot to the patient, as described later. Therefore, the fixation is referred to as 3-point.</li>
		<li>Position the robot at the head of the patient, such that the distance between the base of the robotic arm and the midpoint of the cranium is 70 cm. Lock the robot into position and secure the three-point head-holder to the robot. 
		NOTE: Do not make any more adjustments to the position of the patient or the robot after this time. Any further adjustment after this point will potentially result in implantation inaccuracies.</li>
		<li>Use the semi-automatic laser based facial recognition system to register the preoperative volumetric MRI with the patient, following all prompts given by the robot.
		<ol>
			<li>Calibrate the laser using the set distance calibration tool.</li>
			<li>Select the preset anatomical facial landmarks manually with the laser. Registration is then completed as the robot automatically scans the facial surface.</li>
			<li>Confirm the accuracy of the registration by correlating additional independent surface landmarks with the registered MRI. 
			NOTE: The planned trajectories are then automatically verified by the robot software.</li>
		</ol>
		</li>
		<li>Prepare and drape the patient in standard sterile fashion.</li>
		<li>Drape the robotic working arm using sterile plastic.</li>
		<li>Attach the drilling platform, with a 2.5 mm working cannula, to the robotic arm.</li>
	</ol>
	</li>
	<li>Implant the bolts along their designated trajectories.
	<ol>
		<li>Select the desired trajectory on the touch screen of the robot.</li>
		<li>Step on the robot pedal to initiate movement of the robotic arm to the correct trajectory. When the correct position is reached, the arm is automatically locked by the robot.</li>
		<li>Insert a 2 mm drill through the working cannula and use it to create a pinhole through the entire thickness of the skull.</li>
		<li>Open the dura with an insulated dural perforator using monopolar cautery at a low setting. 
		NOTE: Opening the dura can be particularly challenging in small children. Because the dura is not completely adherent to the internal layers of the skull, it is very easy to displace rather than open the dura without noticing.</li>
		<li>Screw guide bolt firmly into each pin hole.</li>
		<li>Measure the distance from the drilling platform to the guide bolt using a sterile ruler. 
		NOTE: This is a fixed distance related to the length of the drilling adapter.
		<ol>
			<li>Subtract this measured distance from the value of the distance &#8220;platform to target&#8221; used in planning the trajectory. 
			NOTE: Remember that the recommendation is to always use the standard 150 mm platform to target distance unless a need arises to change this distance. Using this standard will simplify this step in the OR.</li>
			<li>Record and note the result as it will be used later as the final length of the implanted electrode.</li>
		</ol>
		</li>
		<li>Measure and note the final length of the electrode and ensure that it matches the newly calculated length for the bolt. Ensure the electrode and bolt have matching labels to prevent confusion later during electrode implantation.</li>
		<li>Repeat steps 2.2.1 &#8211; 2.2.7 for every bolt (i.e., implant all bolts) and mark all electrodes accordingly.</li>
	</ol>
	</li>
	<li>Change surgical gloves and open a new sterile field.</li>
	<li>Implant all electrodes to the target depth via the implanted bolts.
	<ol>
		<li>Insert a 2 mm diameter stylet through the guide bolt to the intended depth of the final electrode as calculated after implantation of the bolt previously.</li>
		<li>Immediately insert the electrode through the bolt after removing the stylet and screw the electrode into the bolt for fixation.</li>
		<li>Ensure that the electrode is appropriately labeled.</li>
		<li>Repeat steps 2.4.1 &#8211; 2.4.3 for every electrode.</li>
	</ol>
	</li>
	<li>Connect the electrodes to the clinical electrophysiology hardware.</li>
	<li>Wrap the patient&#8217;s head using standard head bandaging technique.</li>
</ol>

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Epilepsy. , (2018).</li><li>Talairach, J., Bancaud, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotaxic+approach+to+epilepsy.">Stereotaxic approach to epilepsy.</a> Progress in neurological surgery. 5, 297-354 (1973).</li><li>Bancaud, J., Talairach, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+organization+of+the+supplementary+motor+area.+Data+obtained+by+stereo-E.E.G.">Functional organization of the supplementary motor area. Data obtained by stereo-E.E.G.</a> Neurochirurgie. 13, 343-356 (1967).</li><li>Jehi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Epileptogenic+Zone:+Concept+and+Definition.">The Epileptogenic Zone: Concept and Definition.</a> Epilepsy Currents. 18 (1), 12-16 (2018).</li><li>Nowell, 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=A+novel+method+for+implementation+of+frameless+StereoEEG+in+epilepsy+surgery.">A novel method for implementation of frameless StereoEEG in epilepsy surgery.</a> Operative Neurosurgery. 10 (4), 525-534 (2014).</li><li>Abel, T. J., 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=Frameless+robot-assisted+stereoelectroencephalography+in+children:+technical+aspects+and+comparison+with+Talairach+frame+technique.">Frameless robot-assisted stereoelectroencephalography in children: technical aspects and comparison with Talairach frame technique.</a> Journal of Neurosurgery: Pediatrics. 1, 1-10 (2018).</li><li>van der Loo, L. E., 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=Methodology,+outcome,+safety+and+in+vivo+accuracy+in+traditional+frame-based+stereoelectroencephalography.">Methodology, outcome, safety and in vivo accuracy in traditional frame-based stereoelectroencephalography.</a> Acta neurochirurgica. 159 (9), 1733-1746 (2017).</li><li>Gonz&#225;lez-Mart&#237;nez, J., 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=Technique,+results,+and+complications+related+to+robot-assisted+stereoelectroencephalography.">Technique, results, and complications related to robot-assisted stereoelectroencephalography.</a> Neurosurgery. 78 (2), 169-180 (2015).</li><li>Mullin, J. P., Smithason, S., Gonzalez-Martinez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereo-electro-encephalo-graphy+(SEEG)+with+robotic+assistance+in+the+presurgical+evaluation+of+medical+refractory+epilepsy:+a+technical+note.">Stereo-electro-encephalo-graphy (SEEG) with robotic assistance in the presurgical evaluation of medical refractory epilepsy: a technical note.</a> Journal of visualized experiments. , 112 (2016).</li><li>Jones, J. C., 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=Techniques+for+placement+of+stereotactic+electroencephalographic+depth+electrodes:+Comparison+of+implantation+and+tracking+accuracies+in+a+cadaveric+human+study.">Techniques for placement of stereotactic electroencephalographic depth electrodes: Comparison of implantation and tracking accuracies in a cadaveric human study.</a> Epilepsia. 59 (9), 1667-1675 (2018).</li><li>Mullin, J. 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=Is+SEEG+safe?+A+systematic+review+and+meta-analysis+of+stereo-electroencephalography-related+complications.">Is SEEG safe? A systematic review and meta-analysis of stereo-electroencephalography-related complications.</a> Epilepsia. 57 (3), 386-401 (2016).</li><li>Serletis, D., 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=The+stereotactic+approach+for+mapping+epileptic+networks:+a+prospective+study+of+200+patients.">The stereotactic approach for mapping epileptic networks: a prospective study of 200 patients.</a> Journal of Neurosurgery. 121, 1239-1246 (2014).</li><li>Taussig, D., 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=Stereo-electroencephalography+(SEEG)+in+65+children:+an+effective+and+safe+diagnostic+method+for+pre-surgical+diagnosis,+independent+of+age.">Stereo-electroencephalography (SEEG) in 65 children: an effective and safe diagnostic method for pre-surgical diagnosis, independent of age.</a> Epileptic Disorders. 16, 280-295 (2014).</li><li>Munyon, C., 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=The+3-dimensional+grid:+a+novel+approach+to+stereoelectroencephalography.">The 3-dimensional grid: a novel approach to stereoelectroencephalography.</a> Neurosurgery. 11, 127-133 (2015).</li><li>Ortler, 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=Frame-based+vs+frameless+placement+of+intrahippocampal+depth+electrodes+in+patients+with+refractory+epilepsy:+a+comparative+in+vivo+(application)+study.">Frame-based vs frameless placement of intrahippocampal depth electrodes in patients with refractory epilepsy: a comparative in vivo (application) study.</a> Neurosurgery. 68, 881-887 (2011).</li></ol>

The authors have nothing to disclose.

Meticulously defining of the AEC hypothesis coupled with particularly detailed attention to the design of the implantation strategy is ultimately what will determine the success of the SEEG methodology for each individual patient. As such, careful pre-surgical planning of the procedure is critical and makes for a relatively simple, low-risk surgery. Generally it is best to orient the trajectories orthogonally to the sagittal midline, thereby facilitating an easier anatomo-electrophysiological correlation in the future an...

Medically refractory epilepsy (MRE) is estimated to afflict fifteen million people world-wide1. Many of these patients, therefore, may well be treated with surgery. Epilepsy surgery relies on the precise localization of the theorized epileptogenic zone (EZ) in order to guide surgical resections. Jean Tailarach and Jean Bancaud developed the stereoelectroencephalography (SEEG) methodology in the 1950s as a method for more accurately localizing the EZ based on the in situ electrophysiology of the epileptic brain in both cortical and deep structures2,3. However, only recently has the SEEG methodology started to gain favor across North America4.
Various techniques and technologies are used throughout the world as part of the SEEG methodology, based on the clinical experience of different professionals and epilepsy centers5,6,7. Recently, however, there has been an evolution of the surgical techniques used to implant SEEG electrodes, beyond the classical use manual headframe based strategies. Specifically, the use of robotic stereotactic guidance systems has been shown to be an accurate alternative for SEEG implantation8. Robotic implantation can be safely and effectively used by those with surgical expertise who are looking for a faster, more automated, approach to electrode implantation.
Herein is discussed the specific steps undertaken when employing the use of a robotic stereotactic guidance system for the implantation of SEEG electrodes. Though the SEEG methodology has previously been described, herein particular attention is given to the surgical technique employed with the use of the robot9.

<table>
	<tbody>
		<tr>
			<td>2 mm drill bit</td>
			<td>DIXI</td>
			<td>KIP-ACS-510</td>
			<td>For opening the cranium</td>
		</tr>
		<tr>
			<td>Coagulation Electrode Dura</td>
			<td>DIXI</td>
			<td>KIP-ACS-600</td>
			<td>for opening and coagulating the dura</td>
		</tr>
		<tr>
			<td>Cordless driver</td>
			<td>Stryker</td>
			<td>4405-000-000</td>
			<td>to drive the drill bit</td>
		</tr>
		<tr>
			<td>Leksell Coordinate Frame G</td>
			<td>Elekta</td>
			<td>14611</td>
			<td>For head fixation</td>
		</tr>
		<tr>
			<td>Microdeep Depth Electrode</td>
			<td>DIXI</td>
			<td>D08-**AM</td>
			<td>SEEG electrodes that are implanted, complete with: guide bolt and stylet, as described in manuscript.</td>
		</tr>
		<tr>
			<td>ROSA</td>
			<td>Medtech</td>
			<td>n/a</td>
			<td>stereotactic guidance system with robotic arm, complete with: robotic arm, calibration tool, registration laser, head frame attachment, and software, as described in the manuscript.</td>
		</tr>
		<tr>
			<td>Stylet</td>
			<td>DIXI</td>
			<td>ACS-770S-10</td>
			<td>for creating a path through the parenchyma for the electrode</td>
		</tr>
	</tbody>
</table>

operative technique and nuances for the stereoelectroencephalographic (seeg) methodology utilizing a robotic stereotactic guidance system

The SEEG methodology has gained favor in North America over the last decade as a means of localizing the epileptogenic zone (EZ) prior to epilepsy surgery. Recently, the application of a robotic stereotactic guidance system for implantation of SEEG electrodes has become more popular in many epilepsy centers. The technique for the use of the robot requires extreme precision in the pre-surgical planning phase and then the technique is streamlined during the operative portion of the methodology, as the robot and surgeon work in concert to implant the electrodes. Herein is detailed precise operative methodology of using the robot to guide implantation of SEEG electrodes. A major limitation of the procedure, namely its heavy reliance on the ability to register the patient to a preoperative volumetric magnetic resonance image (MRI), is also discussed. Overall, this procedure has been shown to have a low morbidity rate and an extremely low mortality rate. The use of a robotic stereotactic guidance system for the implantation of SEEG electrodes is an efficient, fast, safe, and accurate alternative to conventional manual implantation strategies.

The absolute indicator of success following use of the SEEG methodology is seizure freedom for the patient, which ultimately follows successful electrode implantations, successful electrophysiological recordings, as well as successful resection of the EZ. Such a case is shown in Figure 1. Panels A and B of Figure 1 show two tests (single positron emission computed tomography (SPECT) and magnetoelectroencephalography (MEG), respec...

Watch this Scientific Journal Video about Operative Technique and Nuances for the Stereoelectroencephalographic SEEG Methodology Utilizing a Robotic Stereotactic Guidance System at JoVE.com

Operative Technique and Nuances for the Stereoelectroencephalographic SEEG Methodology Utilizing a Robotic Stereotactic Guidance System

The SEEG methodology is simplified and made faster with a stereotactic robot. Careful attention must be paid to the registration of the preoperative volumetric MRI to the patient prior to use of the robot in the OR. The robot streamlines the procedure, leading to decreased operative times and accurate implantations.

Operative Technique and Nuances for the Stereoelectroencephalographic (SEEG) Methodology Utilizing a Robotic Stereotactic Guidance System

The SEEG methodology has gained favor in North America over the last decade as a means of localizing the epileptogenic zone (EZ) prior to epilepsy ...

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3.4K Views.  Houston Methodist. The SEEG methodology is simplified and made faster with a stereotactic robot. Careful attention must be paid to the registration of the preoperative volumetric MRI to the patient prior to use of the robot in the OR. The robot streamlines the procedure, leading to decreased operative times and accurate implantations.

Video: Operative Technique and Nuances for the Stereoelectroencephalographic SEEG Methodology Utilizing a Robotic Stereotactic Guidance System

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an entire gene expression pathway by inducing destruction and/or inhibiting translation of these targets. Several miRNAs play key roles in maintaining neuronal structure and function and in higher-level brain functions, and methods are sought for manipulating their levels for exploring these functions. Here, we present a direct in vivo method for examining the cognitive consequences of enforced miRNAs excess in mice by stereotactic injection of miRNA-encoding virus particles. Specifically, the current protocol involves injection into the hippocampal CA1 region, which contributes to mammalian memory consolidation, learning, and stress responses, and offers a convenient injection site. The coordinates are measured according to the mouse bregma and virus perfusion is digitally controlled and kept very slow. After injection, the surgery wound is sealed and the animals recover. Lentiviruses encoding silencers of the corresponding mRNA targets serve to implicate the specific miRNA/target interaction responsible for the observed effect, with na&iuml;ve mice, mice injected with saline and mice injected with "empty" lentivirus vectors as controls. One month post-injection, the animals are examined in the Morris Water Maze (MWM) for assessing their navigation learning and memory abilities. The MWM is a round tank filled with colored water with a small platform submerged 1 cm below the water surface. Steady visual cues around the tank allow for spatial navigation (sound and the earth's magnetic field may also assist the animals in navigating). Video camera monitoring enables measuring the route of swim and the time to find and amount the platform. The mouse is first taught that mounting the hidden platform offers an escape from the enforced swimming; it is then tested for using this escape and finally, the platform is removed and probe tests examine if the mouse remembers its previous location. Repeated tests over several consecutive days highlight improved performance of tested mice at shorter latencies to find and mount the platform, and as more direct routes to reach the platform or its location. Failure to show such improvement represents impaired learning and memory and/or anxiety, which may then be tested specifically (e.g. in the elevated plus maze). This approach enables validation of specific miRNAs and target transcripts in the studied cognitive and/or stress-related processes.

MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain regions.

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an ...

Construction of Microdrive Arrays for Chronic Neural Recordings in Awake Behaving Mice

State-of-the-art electrophysiological recordings from the brains of freely behaving animals allow researchers to simultaneously examine local field potentials (LFPs) from populations of neurons and action potentials from individual cells, as the animal engages in experimentally relevant tasks. Chronically implanted microdrives allow for brain recordings to last over periods of several weeks. Miniaturized drives and lightweight components allow for these long-term recordings to occur in small mammals, such as mice. By using tetrodes, which consist of tightly braided bundles of four electrodes in which each wire has a diameter of 12.5 &mu;m, it is possible to isolate physiologically active neurons in superficial brain regions such as the cerebral cortex, dorsal hippocampus, and subiculum, as well as deeper regions such as the striatum and the amygdala. Moreover, this technique insures stable, high-fidelity neural recordings as the animal is challenged with a variety of behavioral tasks. This manuscript describes several techniques that have been optimized to record from the mouse brain. First, we show how to fabricate tetrodes, load them into driveable tubes, and gold-plate their tips in order to reduce their impedance from M&Omega; to K&Omega; range. Second, we show how to construct a custom microdrive assembly for carrying and moving the tetrodes vertically, with the use of inexpensive materials. Third, we show the steps for assembling a commercially available microdrive (Neuralynx VersaDrive) that is designed to carry independently movable tetrodes. Finally, we present representative results of local field potentials and single-unit signals obtained in the dorsal subiculum of mice. These techniques can be easily modified to accommodate different types of electrode arrays and recording schemes in the mouse brain.

The design and assembly of microdrives for in vivo electrophysiological recordings of brain signals from the mouse is described. By attaching microelectrode bundles to sturdy driveable carriers, these techniques allow for long-term and stable neural recordings. The lightweight design allows for unrestricted behavioral performance by the animal following drive implantation.

State-of-the-art electrophysiological recordings from the brains of freely behaving animals allow researchers to simultaneously examine local field ...

Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association between environmental cues and aversive experiences. In this test, mice are placed into a conditioning chamber and are given parings of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric footshock). After a delay time, the mice are exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the test is measured as an index of fear memory. To analyze the behavior automatically, we have developed a video analyzing system using the ImageFZ application software program, which is available as a free download at http://www.mouse-phenotype.org/. Here, to show the details of our protocol, we demonstrate our procedure for the contextual and cued fear conditioning test in C57BL/6J mice using the ImageFZ system. In addition, we validated our protocol and the video analyzing system performance by comparing freezing time measured by the ImageFZ system or a photobeam-based computer measurement system with that scored by a human observer. As shown in our representative results, the data obtained by ImageFZ were similar to those analyzed by a human observer, indicating that the behavioral analysis using the ImageFZ system is highly reliable. The present movie article provides detailed information regarding the test procedures and will promote understanding of the experimental situation.

This article presents a protocol for a contextual and cued fear conditioning test using a video analyzing system to assess fear learning and memory in mice.

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association ...

Assessment of Murine Exercise Endurance Without the Use of a Shock Grid: An Alternative to Forced Exercise

Using laboratory mouse models, the molecular pathways responsible for the metabolic benefits of endurance exercise are beginning to be defined. The most common method for assessing exercise endurance in mice utilizes forced running on a motorized treadmill equipped with a shock grid. Animals who quit running are pushed by the moving treadmill belt onto a grid that delivers an electric foot shock; to escape the negative stimulus, the mice return to running on the belt. However, avoidance behavior and psychological stress due to use of a shock apparatus can interfere with quantitation of running endurance, as well as confound measurements of postexercise serum hormone and cytokine levels. Here, we demonstrate and validate a refined method to measure running endurance in na&#239;ve C57BL/6 laboratory mice on a motorized treadmill without utilizing a shock grid. When mice are preacclimated to the treadmill, they run voluntarily with gait speeds specific to each mouse. Use of the shock grid is replaced by gentle encouragement by a human operator using a tongue depressor, coupled with sensitivity to the voluntary willingness to run on the part of the mouse. Clear endpoints for quantifying running time-to-exhaustion for each mouse are defined and reflected in behavioral signs of exhaustion such as splayed posture and labored breathing. This method is a humane refinement which also decreases the confounding effects of stress on experimental parameters.

A method to assess exercise endurance in laboratory mice without the use of a shock grid is demonstrated. This method is a humane refinement that can decrease the confounding effects of stress on experimental parameters.

Using laboratory mouse models, the molecular pathways responsible for the metabolic benefits of endurance exercise are beginning to be defined. The most ...

Practical Methodology of Cognitive Tasks Within a Navigational Assessment

This paper describes an approach for measuring navigation accuracy relative to cognitive skills. The methodology behind the assessment will thus be clearly outlined in a step-by-step manner. Navigational skills are important when trying to find symbols within a speech-generating device (SGD) that has a dynamic screen and taxonomical organization. The following skills have been found to impact children&#8217;s ability to find symbols when navigating within the levels of an SGD: sustained attention, categorization, cognitive flexibility, and fluid reasoning1,2. According to past studies, working memory was not correlated with navigation1,2.
The materials needed for this method include a computerized tablet, an augmentative and alternative communication application, a booklet of symbols, and the Leiter International Performance Scale-Revised (Leiter-R)3. This method has been used in two previous studies. Robillard, Mayer-Crittenden, Roy-Charland, Minor-Corriveau and B&#233;langer1 assessed typically developing children, while Rondeau, Robillard and Roy-Charland2 assessed children and adolescents with a diagnosis of Autism Spectrum Disorder. The direct observation of this method will facilitate the replication of this study for researchers. It will also help clinicians that work with children who have complex communication needs to determine the children&#8217;s ability to navigate an SGD with taxonomical categorization.

Children who have complex communication needs can benefit from the use of a speech-generating device (SGD). Cognitive skills were identified as having an impact on the ability to navigate between levels of SGDs. This protocol describes the steps needed for Speech-language Pathologists to assess cognitive skills and navigational abilities.

This paper describes an approach for measuring navigation accuracy relative to cognitive skills. The methodology behind the assessment will thus be ...

Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or knockdown gene expression in a relatively focused region without significant damage to the brain tissue. After recovery, the injected mouse can be tested on various behavioral tasks such as the Open Field Test (OFT) and the Forced Swim Test (FST). The OFT is designed to assess locomotion and the anxious phenotype in mice by measuring the amount of time that a mouse spends in the center of a novel open field. A more anxious mouse will spend significantly less time in the center of the novel field compared to controls. The FST assesses the anti-depressive phenotype by quantifying the amount of time that mice spend immobile when placed into a bucket of water. A mouse with an anti-depressive phenotype will spend significantly less time immobile compared to control animals. The goal of this protocol is to use the stereotactic injection of a lentivirus in conjunction with behavioral tests to assess how genetic factors modulate animal behaviors.

Stereotactic injection of lentiviruses expressing cDNAs or shRNAs can modulate gene expression in specific brain areas of mice. Here, we present a protocol to combine stereotactic injections with behavioral tasks, such as the Open Field Test (OFT) and the Forced Swim Test (FST).

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or ...

Investigating Motor Skill Learning Processes with a Robotic Manipulandum

Skilled reaching tasks are commonly used in studies of motor skill learning and motor function under healthy and pathological conditions, but can be time-intensive and ambiguous to quantify beyond simple success rates. Here, we describe the training procedure for reach-and-pull tasks with ETH Pattus, a robotic platform for automated forelimb reaching training that records pulling and hand rotation movements in rats. Kinematic quantification of the performed pulling attempts reveals the presence of distinct temporal profiles of movement parameters such as pulling velocity, spatial variability of the pulling trajectory, deviation from midline, as well as pulling success. We show how minor adjustments in the training paradigm result in alterations in these parameters, revealing their relation to task difficulty, general motor function or skilled task execution. Combined with electrophysiological, pharmacological and optogenetic techniques, this paradigm can be used to explore the mechanisms underlying motor learning and memory formation, as well as loss and recovery of function (e.g. after stroke).

A paradigm is presented for training and analysis of an automated skilled reaching task in rats. Analysis of pulling attempts reveals distinct subprocesses of motor learning.

Skilled reaching tasks are commonly used in studies of motor skill learning and motor function under healthy and pathological conditions, but can be ...

Utilizing the Modified T-Maze to Assess Functional Memory Outcomes After Cardiac Arrest

Background: Evaluating mild to moderate cognitive impairment in a global cerebral ischemia (i.e. cardiac arrest) model can be difficult due to poor locomotion after surgery. For example, rats who undergo surgical procedures and are subjected to the Morris water maze may not be able to swim, thus voiding the experiment.
New Method: We established a modified behavioral spontaneous alternation T-maze test. The major advantage of the modified T-maze protocol is its relatively simple design that is powerful enough to assess functional learning/memory after ischemia. Additionally, the data analysis is simple and straightforward. We used the T-maze to determine the rats&#39; learning/memory deficits both in the presence or absence of mild to moderate (6 min) asphyxial cardiac arrest (ACA). Rats have a natural tendency for exploration and will explore the alternate arms in the T-maze, whereas hippocampal-lesioned rats tend to adopt a side-preference resulting in decreased spontaneous alternation ratios, revealing the hippocampal-related functional learning/memory in the presence or absence of ACA.
Results: ACA groups have higher side-preference ratios and lower alternations as compared to control.
Comparison with Existing Method(s): The Morris water and Barnes maze are more prominent for assessing learning/memory function. However, the Morris water maze is more stressful than other mazes. The Barnes maze is widely used to measure reference (long-term) memory, while ACA-induced neurocognitive deficits are more closely related to working (short-term) memory.
Conclusions: We have developed a simple, yet effective strategy to delineate working (short-term) memory via the T-maze in our global cerebral ischemia model (ACA).

This protocol describes the use of a modified T-maze to evaluate functional learning/memory in asphyxia cardiac arrest-induced cerebral ischemia.

Background: Evaluating mild to moderate cognitive impairment in a global cerebral ischemia (i.e. cardiac arrest) model can be difficult due to poor ...

Methodology for Establishing a Community-Wide Life Laboratory for Capturing Unobtrusive and Continuous Remote Activity and Health Data

An end-to-end suite of technologies has been established for the unobtrusive and continuous monitoring of health and activity changes occurring in the daily life of older adults over extended periods of time. The technology is aggregated into a system that incorporates the principles of being minimally obtrusive, while generating secure, privacy protected, continuous objective data in real-world (home-based) settings for months to years. The system includes passive infrared presence sensors placed throughout the home, door contact sensors installed on exterior doors, connected physiological monitoring devices (such as scales), medication boxes, and wearable actigraphs. Driving sensors are also installed in participants' cars and computer (PC, tablet or smartphone) use is tracked. Data is annotated via frequent online self-report options that provide vital information with regard to the data that is difficult to infer via sensors such as internal states (e.g., pain, mood, loneliness), as well as data referent to activity pattern interpretation (e.g., visitors, rearranged furniture). Algorithms have been developed using the data obtained to identify functional domains key to health or disease activity monitoring, including mobility (e.g., room transitions, steps, gait speed), physiologic function (e.g., weight, body mass index, pulse), sleep behaviors (e.g., sleep time, trips to the bathroom at night), medication adherence (e.g., missed doses), social engagement (e.g., time spent out of home, time couples spend together), and cognitive function (e.g., time on computer, mouse movements, characteristics of online form completion, driving ability). Change detection of these functions provides a sensitive marker for the application in health surveillance of acute illnesses (e.g., viral epidemic) to the early detection of prodromal dementia syndromes. The system is particularly suitable for monitoring the efficacy of clinical interventions in natural history studies of geriatric syndromes and in clinical trials.

Unobtrusive sensors and pervasive computing technology incorporated into the daily home life of older adults enables meaningful health and activity changes to be recorded continuously for months to years, providing ecologically valid, high frequency, multi-domain data for research or clinical use.

An end-to-end suite of technologies has been established for the unobtrusive and continuous monitoring of health and activity changes occurring in the ...

Tickling, a Technique for Inducing Positive Affect When Handling Rats

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk for both the animals and the hands of their handlers, decreased animal welfare, and less valid research data. To minimize negative effects on experimental results and human-animal relationships, research animals are often habituated to being handled. However, the methods of habituation are highly variable and often of limited effectiveness. More potently, it is possible for humans to mimic aspects of the animals&#39; playful rough-and-tumble behavior during handling. When applied to laboratory rats in a systematic manner, this playful handling, referred to as tickling, consistently gives rise to positive behavioral responses. This article provides a detailed description of a standardized rat tickling technique. This method can contribute to future investigations into positive affective states in animals, make it easier to handle rats for common husbandry activities such as cage changing or medical/research procedures such as injection, and be implemented as a source of social enrichment. It is concluded that this method can be used to efficiently and practicably reduce rats&#39; fearfulness of humans and improve their welfare, as well as reliably model positive affective states.

This article demonstrates the standardized application of playful handling, a tickling technique designed to mimic rat rough-and-tumble play. This technique is effective at reducing fearful reactions to humans and generating positive affect when rats are handled for common husbandry activities and medical and research procedures such as injection.

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk ...