The authors wish to appreciate Christopher Neville, Girolamo Mammolito, and F. Jerome Pabulayan for their vital contributions to developing this protocol and data collection.

The Institutional Review Board (IRB) of SUNY Upstate Medical University approved this protocol.
1. Participant screening
<ol>
	<li>Perform all research methods with IRB approval by the Declaration of Helsinki.</li>
	<li>Recruit non-disabled adults who do not have any neurological or musculoskeletal issues that prevent upper extremity motor task performance.</li>
	<li>Recruit chronic stroke survivors whose stroke onset is at least six months before study participation and who have mild-to-moderate upper extremity motor impairment, indicated by Fugl-Meyer Assessment of upper extremity score of 19 to 60 out of 66, and can extend hemiparetic wrist and fingers at least 10 degrees voluntarily.</li>
	<li>Schedule potential participants to attend a data collection session.</li>
	<li>Obtain written informed consent from all research participants before initiating any experimental procedures.</li>
	<li>Screen all participants for study participation eligibility using questionnaires regarding their demographics, previous arm injury history, hand dominance, and confidence in specific fine hand motor skill tasks.</li>
</ol>
2. Upper Extremity Motor Outcome Measures
<ol>
	<li>Perform the Perdue Pegboard Test with the standard procedure10.</li>
	<li>Perform the Fugl-Meyer Assessment of Upper Extremity Motor (FMA-UE) using the standard procedure11,12.</li>
</ol>
3. Psychosocial and cognitive-behavioral assessments
<ol>
	<li>Have participants complete the following questionnaires using the online survey platform: Edinburg Handedness Inventory; a questionnaire for previous experience on the use of chopsticks.; and a self-efficacy questionnaire for the use of chopsticks.</li>
</ol>
4. Preparation of Goal-directed Arm Reaching Tasks
<ol>
	<li>Prepare the motion capture camera system for kinematic recording.
	<ol>
		<li>Calibrate the motion capture camera using the motion capture workstation software.</li>
		<li>Set the origin of the world coordinate using the motion capture workstation software.</li>
		<li>Place all marker triads on a table in the field of view of motion capture cameras and check if all marker triads are within the field of view.</li>
	</ol>
	</li>
	<li>Prepare the motion capture data acquisition software to build the skeletal model.
	<ol>
		<li>Import the marker sets from the motion capture workstation software to the motion capture data acquisition software.</li>
		<li>Activate virtual sensors (i.e., marker triads).</li>
		<li>Set world axes.</li>
		<li>Assign virtual sensors to body segments of the skeleton model.</li>
	</ol>
	</li>
	<li>Set up goal-directed arm reaching task conditions.
	<ol>
		<li>Place a table at the center of the motion capture cameras field of view.</li>
		<li>Put the laminated goal-directed arm reaching template paper at the designated area on the table.</li>
		<li>Prepare a pair of chopsticks on the table.</li>
		<li>Prepare to play the auditory cue audio file.</li>
		<li>Prepare the task instruction scripts.</li>
		<li>Test the motion capture system to ensure it is working appropriately.</li>
	</ol>
	</li>
	<li>Set up the participant.
	<ol>
		<li>Attach the reflective marker triads to the skin of the participant&#39;s arms, hands, and trunk. Use the following description for the marker triad locations: 
		A marker triad for the trunk: between medial borders of the scapulae 
		A marker triad for each upper arm: in the middle of the lateral surface of the upper arm 
		A marker triad for each forearm: in the middle of the dorsal surface of the forearm 
		&#8203;A marker triad for each hand: in the middle of the dorsal surface of the 3rd metacarpal bone</li>
		<li>Prepare a chopstick with a marker triad.</li>
		<li>Place a marker triad on a table located center of the field of view of motion capture cameras.</li>
		<li>Digitize the participant&#39;s body segments using an upper extremity joints and trunk skeleton model that includes following landmarks using the motion capture data acquisition software: 
		Upper trunk: a spot between C7 and T1 vertebrae 
		Lower trunk: a spot between T12 and L1 vertebrae 
		Shoulder (glenohumeral joint), two spots equidistance from center of the head of humerus 
		Elbow: two spots on the medial and lateral elbow that are equidistant from the joint center 
		Wrist: two spots on the medial and lateral wrist that are equidistant from the joint center 
		&#8203;Hand: the tip of the third phalanx of each hand</li>
		<li>Digitize the tip of the chopstick with a marker triad using the motion capture data acquisition software.</li>
		<li>Digitize the home position and target position using the marker triad located on the table using the motion capture data acquisition software.</li>
	</ol>
	</li>
</ol>
5. Performance of Goal-directed Arm Reaching Tasks
<ol>
	<li>Position the participant in a sitting position.</li>
	<li>Ask the participant to reach forward without trunk movement, then locate the table to position the target at approximately 80% of the participant&#39;s maximum arm reaching distance.</li>
	<li>Instruct the participant to maintain the upright trunk posture at the beginning of each task performance. There will be no restriction to the trunk movements during the task performance.</li>
	<li>Instruct the participant how to use chopsticks using a Youtube video (https://youtu.be/2Bns2m5Bg4M) to standardize the way to use the chopsticks.</li>
	<li>Perform the task condition 1 - Reaching and Pointing to a large target.
	<ol>
		<li>Instruct the participant to hold a chopstick with the dominant hand (non-disabled adults) or the paretic hand (stroke participants). The participant will place the tip of a chopstick touching at the center of the home position. Instruct the participant to maintain the upright trunk posture at the beginning.</li>
		<li>Fixate the task condition template paper at the designated area on the table. The template paper includes two squares with a cross at the center of each square: one for the home position and the other one for the target area. For this task, the target square size is 1 x 1 cm2. The target position is located 20 in front of the home position.</li>
		<li>Describe the task instructions.
		<ol>
			<li>State the following: &#34;The goal of this task is to reach and tap the target area with the chopstick&#39;s tip as quickly and accurately as possible. You will hold a chopstick with your right (or left) [indicate the performing hand]. Place the chopstick&#39;s tip on the home position [indicate the home position]. When you hear a &#39;GO&#39; signal, reach and tap the target [indicate the target] with the chopstick&#39;s tip as quickly as possible. Try to tap the center of the target as much as you can. You will have three seconds to tap the target. I will give you a &#39;STOP&#39; signal 3 seconds after the &#39;GO&#39; signal. If you did not tap the target within 3 seconds, bring the chopstick&#39;s tip to the home position and wait for the next trial. You will perform ten trials with a 10-second break between trials. Do you have any questions? [Address any questions that the participant has, then proceed to familiarization trial] You will have three trials as a practice. [After the practice trials, proceed to the actual trials] Now, we will perform actual trials. Try to reach and tap as quickly as you can.&#34;</li>
		</ol>
		</li>
		<li>Play the auditory cue signal audio file with a computer to familiarize the participant with the cue.</li>
		<li>Perform three familiarization trials.</li>
		<li>Instruct the participant to be ready for the task performance. Ensure the participant fully understands the task performance procedure.</li>
		<li>Start motion capture recording with the motion capture data acquisition software.</li>
		<li>Play the auditory cue audio file with a computer.</li>
		<li>Perform 10 trials.</li>
		<li>Stop motion capture recording.</li>
		<li>Take a 2-minute break.</li>
	</ol>
	</li>
	<li>Perform the task condition 2 - Reaching and Pointing to a small target.
	<ol>
		<li>Instruct the participant to hold a chopstick with the dominant hand (non-disabled adults) or the paretic hand (stroke participants). The participant will place the tip of a chopstick touching at the center of the home position. Ask the participant to maintain the upright trunk posture at the beginning.</li>
		<li>Fixate the task condition template paper at the designated area on the table. For this task, the target square size is 0.3 X 0.3 cm2. The target position is located 20 in front of the home position.</li>
		<li>Describe the task instruction.
		<ol>
			<li>State the following: &#34;The goal of this task is the same as the previous task: reach and tap with the chopstick&#39;s tip the target as quickly and accurately as you can. We will use a smaller target [indicate the target]. The instruction is the same as the previous task. When you hear a &#39;GO&#39; signal, reach and tap the target [indicate the target] with the chopstick&#39;s tip as quickly as possible. Try to tap the center of the target as much as you can. Do you have any questions? [Address any questions that the participant has, then proceed to familiarization trial] You will have three trials as a practice. [After the familiarization trials, proceed to the actual trials] Now, we will perform actual trials. Try to reach and tap as quickly as you can.&#34;</li>
		</ol>
		</li>
		<li>Play the auditory cue signal audio file with a computer to familiarize the participant with the cue.</li>
		<li>Perform three familiarization trials.</li>
		<li>Instruct participant to be ready for the task performance. Ensure the participant fully understands the task performance procedure.</li>
		<li>Start motion capture recording with the motion capture data acquisition software.</li>
		<li>Play the auditory cue audio file with a computer.</li>
		<li>Perform 10 trials.</li>
		<li>Stop motion capture recording.</li>
		<li>Take a 2-minute break.</li>
	</ol>
	</li>
	<li>Perform the task condition 3 - Reaching and Picking up a large target object.
	<ol>
		<li>Instruct the participant to hold a pair of chopsticks with the dominant hand (non-disabled adults) or the paretic hand (stroke participants). The participant will place the tips of chopsticks touching at the center of the home position. Ask the participant to maintain upright trunk posture at the beginning.</li>
		<li>Fixate the task condition template paper at the designated area on the table. For this task, the target object is a plastic cube 1 cm on edge. The target object is located 20 in front of the home position.</li>
		<li>Place the target object on the target area.</li>
		<li>Describe the task instructions.
		<ol>
			<li>State the following: &#34;The goal of this task is to reach and pick up a plastic cube [indicate the cube] with a pair of chopsticks as quickly as possible, about an inch in height without dropping. You will hold a pair of chopsticks with your right (or left) [indicate the performing hand]. Place the chopsticks&#39; tips on the home position [indicate the home position]. When you hear a &#39;GO&#39; signal, reach and pick up the cube [indicate the target] with the chopsticks as quickly as you can, about an inch in height. You will have three seconds to pick up the target. I will give you a &#39;STOP&#39; signal 3 seconds after the &#39;GO&#39; signal. If you did not pick up the target within 3 seconds, bring the chopsticks&#39; tips to the home position and wait for the next trial. You will perform ten trials with a 10-second break between trials. Do you have any questions? [Address any questions that the participant has, then proceed to familiarization trial] You will have three trials as a practice. [After the familiarization trials, proceed to the actual trials] Now, we will perform actual trials. Try to reach and pick up as quickly as you can.&#34;</li>
		</ol>
		</li>
		<li>Play the auditory cue signal audio file with a computer to familiarize the participant with the cue.</li>
		<li>Perform three familiarization trials.</li>
		<li>Instruct participant to be ready for the task performance. Ensure the participant fully understands the task performance procedure.</li>
		<li>Start motion capture recording with the motion capture data acquisition software.</li>
		<li>Play the auditory cue audio file with a computer.</li>
		<li>Perform 10 trials.</li>
		<li>Stop motion capture recording.</li>
		<li>Take a 2-minute break.</li>
	</ol>
	</li>
	<li>Perform the task condition 4 - Reaching and Picking up a small target object.
	<ol>
		<li>Instruct the participant to hold a pair of chopsticks with the dominant hand (non-disabled adults) or the paretic hand (stroke participants). The participant will place the tips of chopsticks touching at the center of the home position. Ask the participant to maintain upright trunk posture at the beginning.</li>
		<li>Fixate the task condition template paper at the designated area on the table. For this task, the target object is a plastic cube 0.3 cm on edge. The target object is located 20 in front of the home position.</li>
		<li>Place the target object on the target area.</li>
		<li>Describe the task instructions.
		<ol>
			<li>State the following: &#34;The goal of this task is the same as the previous task: reach and pick up a plastic cube with a pair of chopsticks as quickly as you can. We will use a smaller plastic cube [indicate the target]. The instruction is the same as the previous task. When you hear a &#39;GO&#39; signal, reach and pick up the cube [indicate the target] with chopsticks as quickly as possible. Do you have any questions? [Address any questions that the participant has, then proceed to familiarization trial] You will have three trials as a practice. [After the familiarization trials, proceed to the actual trials] Now, we will perform actual trials. Try to reach and tap as quickly as you can.&#34;</li>
		</ol>
		</li>
		<li>Play the auditory cue signal audio file with a computer to familiarize the participant with the cue.</li>
		<li>Perform three familiarization trials.</li>
		<li>Ask participant to be ready for the task performance. Make sure the participant fully understands the task performance procedure.</li>
		<li>Start motion capture recording with the motion capture data acquisition software.</li>
		<li>Play the auditory cue audio file with a computer.</li>
		<li>Perform the actual 10 trials.</li>
		<li>Stop motion capture recording.</li>
		<li>Take a 2-minute break.</li>
	</ol>
	</li>
	<li>Perform the Intrinsic motivation inventory (IMI) for the use of chopsticks using online survey platform.</li>
</ol>
6. Kinematic data analysis
<ol>
	<li>Export the data of the following landmarks from the motion capture data acquisition software. Export position data in the x-, y-, and z-axes as a text file for each task condition. 
	Tip of a chopstick 
	Home position on the table 
	Target position on the table 
	Hands 
	Elbow joints 
	Shoulder joints (glenohumeral joints) 
	Trunk (at C7)</li>
	<li>Preprocess the kinematic data.
	<ol>
		<li>Use custom programming script to process the kinematic data.</li>
		<li>Filter and smooth the raw position data using a 3rd order Butterworth low pass filter with a 3 Hz cutoff.</li>
		<li>Calculate the resultant of x-, y-, and z-direction position of the performing hand.</li>
	</ol>
	</li>
	<li>Determine movement onset and offset of each goal-directed arm reach.
	<ol>
		<li>To determine the reaching movement onset and offset, use the tangential velocity (the first derivative of the position data) from the resultant of the 3-dimensional position of the performing hand.</li>
		<li>Define movement onset as the first frame of the reach, where the tangential velocity is above 0.01 m/s.</li>
		<li>Define movement offset as the last frame of the reach, where the tangential velocity is above 0.01 m/s.</li>
		<li>Inspect the individual reaching movement onset and offset visually to ensure the onset and offset are correctly labeled.</li>
	</ol>
	</li>
	<li>Determine the peak velocity. The peak velocity is defined as the maximum tangential velocity amplitude of the trial that exceeds the amplitude of 0.2 m/s, and the time interval between 2 peaks must be at least 2 seconds13.</li>
	<li>Calculate kinematic variables of reaching movements.
	<ol>
		<li>Calculate movement duration (MD). Calculate the time difference between movement onset and offset13.</li>
		<li>Calculate peak velocity (PV). Calculate the highest velocity during each of the reaches.</li>
		<li>Calculate absolute and relative time to peak velocity (TTPV and TTPV % of movement duration)13.
		<ol>
			<li>Calculate the time difference between movement onset and peak velocity (absolute TTPV).</li>
			<li>Calculate the percentage of TTPV relative to movement duration (relative TTPV).</li>
		</ol>
		</li>
		<li>Calculate log dimensionless jerk.
		<ol>
			<li>Calculate the third derivative from the resultant of the 3-dimensional position of the performing hand, then calculate the log dimensionless jerk of each arm reaching movement.</li>
		</ol>
		</li>
		<li>Calculate trunk displacement during goal-directed arm reaching movement9,14.
		<ol>
			<li>Calculate the trunk displacement.
			<ol>
				<li>Calculate the distance difference of the trunk landmark between movement onset and offset. Use the following equation. 
				<img alt="Equation 1" src="/files/ftp_upload/61940/61940eq01v4.jpg" /> 
				Where X, Y, and Z are the trunk landmark positions in x-, y-, and z-axis, respectively; 1 is the time frame at the reaching movement onset; k is the time frame at the reaching movement offset.</li>
			</ol>
			</li>
			<li>Calculate shoulder trajectory length.
			<ol>
				<li>Calculate the shoulder landmark&#39;s travel distance between arm reaching movement onset and offset. The shoulder landmark is a virtual landmark digitized from the motion capture data acquisition software using the upper extremity skeleton model. Use the following equation for the shoulder trajectory length calculation. 
				<img alt="Equation 2" src="/files/ftp_upload/61940/61940eq2b.jpg" /> 
				Where X, Y,and Z are the positions of the shoulder landmark in x-, y-, and z-axis, respectively; t is the time frame; t=1 is the time frame at the reaching movement onset; t=k is the time frame at the reaching movement offset</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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A., Robinson-Podolski, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effect+of+Context+on+Skill+Acquisition+and+Transfer.">The Effect of Context on Skill Acquisition and Transfer.</a> American Journal of Occupational Therapy. 53 (2), 138-144 (1999).</li><li>Rosenbaum, D. A., Engelbrecht, S. E., Bushe, M. M., Loukopoulos, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knowledge+Model+for+Selecting+and+Producing+Reaching+Movements.">Knowledge Model for Selecting and Producing Reaching Movements.</a> Journal of Motor Behavior. 25 (3), 217-227 (1993).</li></ol>

No disclosure.

Preliminary results support that this protocol may be appropriate to investigate the impact of task complexity on trunk compensation and goal-directed arm reaching kinematics in both non-disabled adults and chronic stroke survivors.
These representative results also support that this protocol may be appropriate to determine the kinematic differences in goal-directed arm reaches between non-disabled adults and chronic stroke survivors. These findings are consistent with previous studies that ch...

Trunk movement is the most common strategy to compensate for limited degrees of freedom in the elbow and shoulder in individuals with post-stroke upper extremity (UE) motor deficits1,2. Previous studies have shown that post-stroke individuals employ different movement strategies in different motor task environments3,4,5. Dynamic systems motor control theory explains that movements emerge from internal individual factors and external factors, such as task conditions and environment6. Further, Fitt&#39;s law explains the relationship between task difficulty and movement speed, with a tendency to perform more difficult tasks with slower speeds7. In terms of goal-directed arm reaching tasks, Gentilucci reported that people slow down their reaching movements when they reach and grasp a smaller object compared to a larger object8. However, the impact of task complexity on goal-directed arm reaching kinematics and compensatory movement strategies in chronic stroke survivors is not well understood. A previous study that examined pointing and grasping tasks in chronic stroke survivors demonstrated that differences in kinematic variables between two different tasks explained differences in UE motor impairment as measured by the Fugl-Meyer Upper Extremity Score9. However, this study did not directly compare how movement strategies are different in terms of kinematic variables between pointing and grasping tasks. A better understanding of the impact of task conditions on compensatory movement strategies in consideration of individual motor impairment level is crucial to design effective treatment sessions to minimize compensatory movements and maximize restitution of motor impairment. Therefore, it is imperative to investigate how task conditions, specifically task complexity, impact movement strategies in individuals with post-stroke motor impairment. This proposed study protocol will investigate the impact of task conditions on goal-directed arm reaching kinematics in non-disabled adults and stroke survivors. The aims of this protocol are two-fold: 1) to investigate whether the task complexity influences trunk compensation and goal-directed arm reaching kinematics in chronic stroke survivors; 2) to determine if this protocol can differentiate the kinematics of goal-directed arm reaches between non-disabled adults and chronic stroke survivors.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A pair of chopsticks</td>
			<td>NA</td>
			<td>NA</td>
			<td>20 cm length, one chopstick had the passive motion capture markers (custom made)</td>
		</tr>
		<tr>
			<td>Auditory cues for motor tasks</td>
			<td>NA</td>
			<td>NA</td>
			<td>Custom made audio file are played on a smart phone</td>
		</tr>
		<tr>
			<td>Matlab R2018b software</td>
			<td>Mathworks</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MotionMonitor v 8.52 Software</td>
			<td>Innovative Sports Training, Inc., Chicago, IL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Perdue Pegboard Test</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic cubes (0.3 cm on edge)</td>
			<td>NA</td>
			<td>NA</td>
			<td>Custom made plastic cubes with 0.3 cm on edge. These were made using 3D printer</td>
		</tr>
		<tr>
			<td>Plastic cubes (1cm on edge)</td>
			<td>NA</td>
			<td>NA</td>
			<td>Custom made plastic cubes with 1 cm on edge. These were made using 3D printer</td>
		</tr>
		<tr>
			<td>Template print</td>
			<td>NA</td>
			<td>NA</td>
			<td>Custom made templates of the motor tasks, including home position, outlines of target positions.</td>
		</tr>
		<tr>
			<td>Vicon 512 Motion-analysis System and Work station v5.2 software</td>
			<td>OMG plc, Oxford, UK</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

the impact of motor task conditions on goal-directed arm reaching kinematics and trunk compensation in chronic stroke survivors

Trunk compensation is the most common movement strategy to substitute for upper extremity (UE) motor deficits in chronic stroke survivors. There is a lack of evidence examining how task conditions impact trunk compensation and goal-directed arm reaching kinematics. This protocol aims to investigate the impact of task conditions, including task difficulty and complexity, on goal-directed arm reaching kinematics. Two non-disabled young adults and two chronic stroke survivors with mild UE motor impairment were recruited for testing the protocol. Each participant performed goal-directed arm reaches with four different task conditions (2 task difficulties [large vs. small targets] X 2 task complexities [pointing vs. picking up]). The task goal was to reach and point at a target or pick up an object located 20 cm in front of the home position as quickly as possible with a stylus or a pair of chopsticks, respectively, in response to an auditory cue. Participants performed ten reaches per task condition. A 3-dimensional motion capture camera system was used to record trunk and arm kinematics. Representative results showed that there was a significant increase in movement duration, movement jerkiness, and trunk compensation as a function of task complexity, but not task difficulty in all participants. Chronic stroke survivors showed significantly slower, jerkier, and more feedback-dependent arm reaches and significantly more compensatory trunk movements than non-disabled adults. Our representative results support that this protocol can be used to investigate the impact of task conditions on motor control strategies in chronic stroke survivors with mild UE motor impairment.

These results are preliminary data from two non-disabled young adults and two chronic stroke survivors with mild motor impairment (Fugl-Meyer Scores of these two participants were above 60 out of 66). Non-disabled participants were right-handed and performed the tasks with their right hand. Stroke participants were also right-handed before the stroke and both had right hemiparesis. They also performed the task with their right hand. These kinematic variables between populations and between target conditions were compared...

Watch this Scientific Journal Video about The Impact of Motor Task Conditions on Goal-Directed Arm Reaching Kinematics and Trunk Compensation in Chronic Stroke Survivors at JoVE.com

The Impact of Motor Task Conditions on Goal-Directed Arm Reaching Kinematics and Trunk Compensation in Chronic Stroke Survivors

This protocol is intended to investigate the impact of task conditions on movement strategies in chronic stroke survivors. Further, this protocol can be used to examine if a restriction in elbow extension induced by neuromuscular electrical stimulation causes trunk compensation during goal-directed arm reaches in non-disabled adults.

Trunk compensation is the most common movement strategy to substitute for upper extremity (UE) motor deficits in chronic stroke survivors. There is a lack ...

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3.4K Views.  SUNY Upstate Medical University. This protocol is intended to investigate the impact of task conditions on movement strategies in chronic stroke survivors. Further, this protocol can be used to examine if a restriction in elbow extension induced by neuromuscular electrical stimulation causes trunk compensation during goal-directed arm reaches in non-disabled adults.

Video: The Impact of Motor Task Conditions on Goal-Directed Arm Reaching Kinematics and Trunk Compensation in Chronic Stroke Survivors

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. 
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options.  We seek to define how neural immune ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of motor and cognitive disabilities. Its high prevalence poses a serious health burden, as stroke is among the principal causes of long-term disability and death worldwide1. Recovery of neuronal function is, in most cases, only partial. So far, treatment options are very limited, in particular due to the narrow time window for thrombolysis2,3. Determining methods to accelerate recovery from stroke remains a prime medical goal; however, this has been hampered by insufficient mechanistic insights into the recovery process. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. Beyond the acute phase, stroke research is increasingly focused on the sub-acute and chronic phase following cerebral ischemia. Most stroke researchers apply permanent or transient occlusion of the MCA in mice or rats. In patients, occlusions of the MCA are among the most frequent causes of ischemic stroke4. Besides proximal occlusion of the MCA using the filament model, surgical occlusion of the distal MCA is probably the most frequently used model in experimental stroke research5. Occlusion of a distal (to the branching of the lenticulo-striate arteries) MCA branch typically spares the striatum and primarily affects the neocortex. Vessel occlusion can be permanent or transient. High reproducibility of lesion volume and very low mortality rates with respect to the long-term outcome are the main advantages of this model. Here, we demonstrate how to perform a chronic cranial window (CW) preparation lateral to the sagittal sinus, and afterwards how to surgically induce a distal stroke underneath the window using a craniotomy approach. This approach can be applied for sequential imaging of acute and chronic changes following ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice.

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when compared to warm-blooded vertebrates. Because of this, in vitro experiments in turtles can be performed for extended periods of time to investigate the neural signals and control of their target actions. Using an isolated head preparation, we measured the kinematics of eye movements in turtles, and their modulation by electrical signals carried by cranial nerves. After the brain was removed from the skull, leaving the cranial nerves intact, the dissected head was placed in a gimbal to calibrate eye movements. Glass electrodes were attached to cranial nerves (oculomotor, trochlear, and abducens) and stimulated with currents to evoke eye movements. We monitored eye movements with an infrared video tracking system and quantified rotations of the eyes. Current pulses with a range of amplitudes, frequencies, and train durations were used to observe effects on responses. Because the preparation is separated from the brain, the efferent pathway going to muscle targets can be examined in isolation to investigate neural signaling in the absence of centrally processed sensory information.

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

This protocol describes how to use an in vitro isolated turtle head preparation to measure the kinematics of their eye movements. After removal of the brain from the cranium, cranial nerves can be stimulated with currents to quantify rotations of the eye and changes in pupil sizes.

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

Non-invasive Strategies for Chronic Manipulation of DREADD-controlled Neuronal Activity

Chemogenetic strategies have emerged as reliable tools for remote control of neuronal activity. Among these, designer receptors exclusively activated by designer drugs (DREADDs) have become the most popular chemogenetic approach used in modern neuroscience. Most studies deliver the ligand clozapine-N-oxide (CNO) using a single intraperitoneal injection, which is suitable for the acute activation/inhibition of the targeted neuronal population. There are, however, only a few examples of strategies for chronic modulation of DREADD-controlled neurons, the majority of which rely on the use of delivery systems that require surgical intervention. Here, we expand on two non-invasive strategies for delivering the ligand CNO to chronically manipulate neural population in mice. CNO was administered either by using repetitive (daily) eye-drops, or chronically through the animal&#39;s drinking water. These non-invasive paradigms result in robust activation of the designer receptors that persisted throughout the CNO treatments. The methods described here offer alternatives for the chronic DREADD-mediated control of neuronal activity and may be useful for experiments designed to evaluate behavior in freely moving animals, focusing on less-invasive CNO delivery methods.

Here we describe two non-invasive methods to chronically control neuronal activity using chemogenetics in mice. Eye-drops were used to deliver clozapine-N-oxide (CNO) daily. We also describe two methods for prolonged administration of CNO in drinking water. These strategies for chronic neuronal control require minimal intervention reducing animals&#8217; stress.

Chemogenetic strategies have emerged as reliable tools for remote control of neuronal activity. Among these, designer receptors exclusively activated by ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Laser-Induced Brain Injury in the Motor Cortex of Rats

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) occlusion of the middle cerebral artery (MCA) using a catheter. This generally accepted technique, however, has some limitations, thereby limiting its extensive use. Stroke induction by this method is often characterized by high variability in the localization and size of the ischemic area, periodical occurrences of hemorrhage, and high death rates. Also, the successful completion of any of the transient or permanent procedures requires expertise and often lasts for about 30 minutes. In this protocol, a laser irradiation technique is presented that can serve as an alternative method for inducing and studying brain injury in rodent models.
When compared to rats in the control and MCAO groups, the brain injury by laser induction showed reduced variability in body temperature, infarct volume, brain edema, intracranial hemorrhage, and mortality. Furthermore, the use of a laser-induced injury caused damage to the brain tissues only in the motor cortex unlike in the MCAO experiments where destruction of both the motor cortex and striatal tissues is observed.
Findings from this investigation suggest that laser irradiation could serve as an alternative and effective technique for inducing brain injury in the motor cortex. The method also shortens the time for completing the procedure and does not require expert handlers.

The protocol presented here shows a technique to create a rodent model of brain injury. The method described here uses laser irradiation and targets motor cortex.

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) ...

Motor Dual-Tasks for Gait Analysis and Evaluation in Post-Stroke Patients

Eighteen stroke patients were recruited for this study involving the evaluation of cognition and walking ability and multitask gait analysis. Multitask gait analysis consisted of a single walking task (Task 0), a simple motor dual-task (water-holding, Task 1), and a complex motor dual-task (crossing obstacles, Task 2). The task of crossing obstacles was considered to be equivalent to the combination of a simple walking task and a complex motor task as it involved more nervous system, skeletal movement, and cognitive resources. To eliminate heterogeneity in the results of the gait analysis of the stroke patients, the dual-task gait cost values were calculated for various kinematic parameters. The major differences were observed in the proximal joint angles, especially in the angles of the trunk, pelvis, and hip joints, which were significantly larger in the dual motor tasks than in the single walking task. This research protocol aims to provide a basis for the clinical diagnosis of gait function and an in-depth study of motor control in stroke patients with motor control deficits through the analyses of dual-motor walking tasks.

This paper presents a protocol specifically for dual motor task gait analysis in stroke patients with motor control deficits.

Eighteen stroke patients were recruited for this study involving the evaluation of cognition and walking ability and multitask gait analysis. Multitask ...

Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings

Multichannel electrode arrays offer insight into the working brain and serve to elucidate neural processes at the single-cell and circuit levels. Development of these tools is crucial for understanding complex behaviors and cognition and for advancing clinical applications. However, it remains a challenge to densely record from cell populations stably and continuously over long time periods. Many popular electrodes, such as tetrodes and silicon arrays, feature large cross-diameters that produce damage upon insertion and elicit chronic reactive tissue responses associated with neuronal death, hindering the recording of stable, continuous neural activity. In addition, most wire bundles exhibit broad spacing between channels, precluding simultaneous recording from a large number of cells clustered in a small area. The carbon fiber microelectrode arrays described in this protocol offer an accessible solution to these concerns. The study provides a detailed method for fabricating carbon fiber microelectrode arrays that can be used for both acute and chronic recordings in vivo. The physical properties of these electrodes make them ideal for stable and continuous long-term recordings at high cell densities, enabling the researcher to make robust, unambiguous recordings from single units across months.

Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings

This protocol describes a procedure for constructing carbon fiber microelectrode arrays for chronic and acute in vivo electrophysiological recordings in mouse (Mus musculus) and ferret (Mustela putorius furo) from multiple brain regions. Each step, following the purchase of raw carbon fibers to microelectrode array implantation, is described in detail, with emphasis on microelectrode array construction.

The Impact of Motor Task Conditions on Goal-Directed Arm Reaching Kinematics and Trunk Compensation in Chronic Stroke Survivors

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Chapters in this video

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Non-invasive Strategies for Chronic Manipulation of DREADD-controlled Neuronal Activity

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Laser-Induced Brain Injury in the Motor Cortex of Rats

Motor Dual-Tasks for Gait Analysis and Evaluation in Post-Stroke Patients

Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings