We thank Mr. John Crowell and Mr. Terry Upshall for their technical expertise and assistance with the photography and videography. This work was supported by operating grants to JLV from the Canadian Institutes of Health Research and the Research and Development Corporation of Newfoundland and a Heart &#38; Stroke Foundation of Canada Canadian Partnership for Stroke Recovery Catalyst grant. RBR was a recipient of a Keith Griffiths Memorial Heart &#38; Stroke Foundation Graduate Scholarship.

Ethics statement: All experiments were approved by Memorial University of Newfoundland&#39;s Animal Care Ethics Committee according to the guidelines of the Canadian Council on Animal Care.
1. Mice
<ol>
	<li>Use adult mice. In this study, adult male FVBN mice (n=10) between 2-4 months were used. House mice on a 12:12 hr reverse light-dark cycle and provide standard rodent chow and water ad libitum.</li>
</ol>
2. Materials Required for the Cylinder Test
<ol>
	<li>Obtain a table with a transparent top to film the cylinder test. The dimensions of the table are irrelevant, the top must be plexiglass or glass, and there must be enough room to position a mirror below the table. This allows for the mouse to be videotaped from below. Use a mirror below the table to reflect the image through the cylinder. As an alternative, use an upside-down camera if available. The dimensions of the table used in this protocol are 54 x 56 x 66.5cm (w x l x h) with a 51 x 51cm top (w x l).</li>
	<li>Obtain a mirror. The dimensions of the mirror used in this protocol are 34 x 58cm (w x l).</li>
	<li>Obtain a transparent/plexiglass cylinder for the mouse to perform in. The dimensions of the cylinder used in this protocol are 17.5cm high, 8.8cm I.D, 9.5cm O.D with a wall thickness of 0.35cm. A taller cylinder may be required for more active mouse strains.</li>
	<li>Place the cylinder on the tabletop and film the reflection in the mirror below.</li>
	<li>A videocamera and tripod are required. Record videos at approximately 650Kb/s, which is approximately 190Mb per 5 min of cylinder video. Ensure that the camera has a zoom functionality to ensure that the cylinder encompasses the entire field of view. 
	NOTE: The videocamera used in this protocol is a Sony DCR-SR42, 40x optical zoom, 2,000x digital zoom, 680kpix which uses standard definition, NTSC interlaced video.)</li>
	<li>Obtain software for analysis &#8211; a media player with video support and playback speed modulation. The media player used in this protocol is the VLC Media Player v2.1.2.</li>
	<li>Obtain a computer with an operating system capable of running the media player and a monitor.</li>
	<li>Electronic storage for videos is required. Download videos to an external hard drive or copy it onto DVDs for long-term storage. 
	NOTE: At 190Mb per video, 84 sessions will fit on a 16 GB SD card and 168 sessions will fit on a 32GB SD card. Due to the relative inexpensiveness of SD media, and the uncertainty in how much time some mice require to complete 20 rears, a 32 GB card is recommended. In the current study, the videos were copied from the camera to a 2TB external hard drive and then recopied onto DVDs as backup.</li>
</ol>
3. Experimental Setup of the Cylinder Test
<ol>
	<li>Fasten mirror below the table at a 45 degree angle to the tabletop. Do this using two support brackets attached to the table legs to support the top and bottom of the mirror, respectively. Bracket locations are noted in a side-view of the table (Figure 1A) and a face-on view of the table (Figure 1B).</li>
	<li>Place cylinder on the center of the table. Draw four equidistant lines where the cylinder sits on the table with a black marker (Sharpie) so that the cylinder can be lifted and returned to the same position (Figure 2). Drawing them on the underside of the transparent tabletop allows the tabletop to be cleaned between animal tests without dissolving the marker ink.</li>
	<li>Attach the camera and tripod. Aim the camera at the mirror so that the picture is looking directly through the barrel of the cylinder. Ensure that the full inner wall of the cylinder is visible and unobstructed by the base (Figure 3). See a side-on view of the setup, including the relative angle of the camera and mirror setup, from above (Figure 4A) and from the level of the table, including a mouse rearing (Figure 4B).</li>
	<li>Prepare cue cards to identify each mouse prior to filming. Ensure that the cards typically include an identification number for each mouse, the time-point of the test (e.g., 3 days after treatment) and the date of the filming session. Do not include the treatment group on the cue card to ensure the experimenter is blinded.</li>
	<li>Film in standard indoor lighting conditions as this level of light is required to clearly see the mouse movements around the cylinder. 
	NOTE: If available filming in the dark with a red light camera may suffice, however one would first need to test whether paw touches and drags are clearly visible for quantification.</li>
</ol>
4. Execution
<ol>
	<li>Start filming. Display the appropriate mouse cue card in front of camera lens.</li>
	<li>Lower the mouse into the cylinder from the open top immediately after filming the cue card.</li>
	<li>Begin filming the mouse. Minimize noise during this time, as mice may lose interest in exploring if startled.</li>
	<li>Observe the mice rear to explore the cylinder. Capture the video until the mouse performs a minimum of twenty rears. 
	NOTE: A rear occurs when both forepaws lose contact with the floor and the mouse stands on its hind legs.</li>
	<li>Wipe tabletop and cylinder with an appropriate cleaning solution between each mouse to sanitize and remove mouse scents.</li>
	<li>Freezing is when mice stop exploring and remain quietly sitting on all fours for approximately 5 min. If mice freeze before twenty rears occur, it may be necessary to remove them from the cylinder for 10-20 min before resuming the test. If mice do not perform twenty rears, they are removed from the study. 
	NOTE: In our experience, mice have never needed to be excluded due to failure to explore the cylinder.</li>
</ol>
5. Evaluation of the Cylinder Test using Paw-Dragging Analysis
<ol>
	<li>Play back the video at a rate of between 0.25x and 0.67x regular speed depending on how quickly the mouse explores the cylinder. Use a media player that offers slower playback speeds.</li>
	<li>Quantify the total number of paw touches. Paw touches occur when the mouse rears (Figure 5A), touches the side of the cylinder (Figure 5B), subsequently dismounts with both paws simultaneously (Figure 5C) and lands (Figure 5D). The paw may or may not contact the cylinder wall with a full palm, but some contact with the cylinder wall must occur. Assess paw touches by counting the number of times the mouse makes contact (no matter how brief) with the cylinder wall with either or both forepaws while standing on its hind limbs during a rear. 
	Note that contact with the cylinder wall is only counted as a &#8220;paw touch&#8221; or &#8220;paw-drag&#8221; if the mouse is in a rear position &#8211; standing on its hind limbs with both forepaws off of the tabletop. If the mouse remains in a 3-point stance &#8211; both hind limbs and one forepaw on the tabletop and proceeds to touch the wall with the free paw &#8211; this is not counted as a paw touch. Mice may rear and touch the cylinder wall with a single forepaw and this is counted as a paw touch. Note: a mouse may also move its body around the cylinder during a rear, making more than two contacts. These contacts are tallied &#8211; one for each left forepaw touch and one for each right forepaw touch.</li>
	<li>Quantify the number of paw-drags. Paw-dragging behaviour is distinct from normal paw touches.
	<ol>
		<li>If the paw contacts the cylinder wall with a full open palm (Figure 6B), it will slowly fall away from the wall, often with a slight tremor. The movement begins with the digits dragging against the cylinder wall either in a medial or downward direction, (Figure 6C) before falling away completely (Figure 6D). The mouse will then dismount with its unaffected paw (Figure 6E) before landing on all fours (Figure 6F). This is considered a paw-drag and should be counted in a tally.</li>
		<li>If the paw does not contact the cylinder wall with a fully open palm, it will graze the cylinder wall with its digits before falling away from the cylinder wall. Similarly, a mouse may drag its paw against the cylinder wall but not release it entirely before dismounting. These are both considered paw-drags as well as touches and should be counted as both in a tally.</li>
		<li>The paw may also drag along the cylinder wall while a mouse explores the cylinder. In this case, the paw will follow the twisting of the mouse&#8217;s torso as it explores left or right of its original position (Figure 7A-D) before dismounting (Figure 7E). This is not considered a paw-drag, as it depends on the mouse randomly choosing a direction to explore and does not depend on which cortical hemisphere was damaged.</li>
	</ol>
	</li>
	<li>Paw-drags are expressed as a percentage of paw-drags per total number of paw touches during a session. Express the number of paw-drags as a percentage of total paw contacts for each forelimb separately.</li>
	<li>Touches resulting in a paw-drag count as a paw-drag and a touch simultaneously. Thus, if a mouse drags its paw each time its paw contacts the cylinder wall, the paw-dragging percentage is expressed as 100%.</li>
</ol>
6. Additional Experimental Design Suggestions
<ol>
	<li>To minimize extraneous variables:
	<ol>
		<li>Test the mice at the same time on each testing day. Test the mice during their wake cycle. Keeping mice on a 12 hr reverse light cycle facilitates performance.</li>
		<li>Mice may be reluctant to explore the cylinder if stressed either by noise or a novel environment. Testing mice in their animal holding room or a room they have been familiarized with reduces stress. This can occur if the room is noisy, if the mouse has been jostled prior to entering the cylinder or due to habituation. 
		NOTE: Testing should be performed once before experimental manipulation to serve as a baseline reading. After manipulation, testing days are at the experimenter&#8217;s discretion, though it is advised to avoid an excessive number of exposures to the cylinder over a short period of time.</li>
		<li>Mice may become reluctant to rear after 6-7 exposures to the cylinder. For the current study, mice were tested in the cylinder for a total of seven times, prior to ischemia and on days 1, 3, 7, 14, 21 and 28 post-surgery. 
		NOTE: In this study we used the FVBN mouse strain. We have previously tested C57Bl/6 mice in the cylinder and observed paw-dragging behaviour following an ET-1 ischemic injury (data not shown). C57BL/6 mice were more active than FVBN mice when rearing and frequently jumped onto the rim of the cylinder before climbing out. Taller cylinders should be used if mice attempt to escape by jumping.</li>
	</ol>
	</li>
</ol>
7. Endothelin-1 Surgery and Infarct Volume Measurements
<ol>
	<li>Perform Endothelin-1 surgery and infarct volume measurements according to previously published protocols4 . To target the anterior forelimb motor cortex, each mouse should receive three ET-1 injections at the following coordinates: (i) +0.7 anteriorposterior (AP)/1.5 medial-lateral (ml)/-1.2 dorsal-ventral (DV), (ii) +0.4 AP/1.25 ml/- 1.2 DV and (iii) +0.1 AP/1.75 ml/-1.2 DV4 .</li>
</ol>
8. Statistical Analysis
<ol>
	<li>A two-way repeated measures analysis of variance (ANOVA) is recommended to analyze the percent of paw-dragging for the affected and unaffected paws across different time points.</li>
</ol>

<ol>
	<li>Schallert, T., Fleming, S. M., Leasure, J. L., Tillerson, J. L., Bland, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS+plasticity+and+assessment+of+forelimb+sensorimotor+outcome+in+unilateral+rat+models+of+stroke,+cortical+ablation,+parkinsonism+and+spinal+cord+injury.">CNS plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury.</a> Neuropharmacology. 39, 777-787 (2000).</li><li>Kolb, B., 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=Growth+factor-stimulated+generation+of+new+cortical+tissue+and+functional+recovery+after+stroke+damage+to+the+motor+cortex+of+rats.">Growth factor-stimulated generation of new cortical tissue and functional recovery after stroke damage to the motor cortex of rats.</a> J Cereb Blood Flow Metab. 27 (5), 983-997 (2007).</li><li>Windle, V., 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=An+analysis+of+four+different+methods+of+producing+focal+cerebral+ischemia+with+endothelin-1+in+the+rat.">An analysis of four different methods of producing focal cerebral ischemia with endothelin-1 in the rat.</a> Exp Neurol. 201 (2), 324-334 (2006).</li><li>Roome, R. B., 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+reproducible+Endothelin-1+model+of+forelimb+motor+cortex+stroke+in+the+mouse.">A reproducible Endothelin-1 model of forelimb motor cortex stroke in the mouse.</a> J Neurosci Methods. 233, 34-44 (2014).</li><li>Tennant, K. A., Jones, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensorimotor+behavioral+effects+of+endothelin-1+induced+small+cortical+infarcts+in+C57BL/6+mice.">Sensorimotor behavioral effects of endothelin-1 induced small cortical infarcts in C57BL/6 mice.</a> J Neurosci Methods. 181 (1), 18-26 (2009).</li><li>Wang, Y., Jin, K., Greenberg, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+associated+with+endothelin-induced+cortical+infarction+in+the+mouse.">Neurogenesis associated with endothelin-induced cortical infarction in the mouse.</a> Brain Res. 1167, 118-122 (2007).</li><li>Baskin, Y. K., Dietrich, W. D., Green, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+effective+behavioral+tasks+for+evaluating+sensorimotor+dysfunction+following+traumatic+brain+injury+in+mice.">Two effective behavioral tasks for evaluating sensorimotor dysfunction following traumatic brain injury in mice.</a> J Neurosci Methods. 129 (1), 87-93 (2003).</li><li>Li, X., 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=Chronic+behavioral+testing+after+focal+ischemia+in+the+mouse:+functional+recovery+and+the+effects+of+gender.">Chronic behavioral testing after focal ischemia in the mouse: functional recovery and the effects of gender.</a> Exp Neurol. 187 (1), 94-104 (2004).</li><li>Clarke, J., Ploughman, M., Corbett, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+qualitative+and+quantitative+analysis+of+skilled+forelimb+reaching+impairment+following+intracerebral+hemorrhage+in+rats.">A qualitative and quantitative analysis of skilled forelimb reaching impairment following intracerebral hemorrhage in rats.</a> Brain Res. , 204-212 (2007).</li><li>Montoya, C. P., Campbell-Hope, L. J., Pemberton, K. D., Dunnett, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+'staircase+test':+a+measure+of+independent+forelimb+reaching+and+grasping+abilities+in+rats.">The 'staircase test': a measure of independent forelimb reaching and grasping abilities in rats.</a> J Neurosci Methods. 36 (2-3), 219-228 (1991).</li><li>Farr, T. D., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+qualitative+impairments+in+skilled+reaching+in+the+mouse+(Mus+musculus)+after+a+focal+motor+cortex+stroke.">Quantitative and qualitative impairments in skilled reaching in the mouse (Mus musculus) after a focal motor cortex stroke.</a> Stroke. 33 (7), 1869-1875 (2002).</li><li>Tennant, K. A., 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+vermicelli+and+capellini+handling+tests:+simple+quantitative+measures+of+dexterous+forepaw+function+in+rats+and+mice.">The vermicelli and capellini handling tests: simple quantitative measures of dexterous forepaw function in rats and mice.</a> J Vis Exp. (41), (2010).</li><li>Clarkson, A. N., 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=AMPA+receptor-induced+local+brain-derived+neurotrophic+factor+signaling+mediates+motor+recovery+after+stroke.">AMPA receptor-induced local brain-derived neurotrophic factor signaling mediates motor recovery after stroke.</a> J Neurosci. 31 (10), 3766-3775 (2011).</li><li>Clarkson, A. N., Huang, B. S., Macisaac, S. E., Mody, I., Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+excessive+GABA-mediated+tonic+inhibition+promotes+functional+recovery+after+stroke.">Reducing excessive GABA-mediated tonic inhibition promotes functional recovery after stroke.</a> Nature. 468 (7321), 305-309 (2010).</li></ol>

The authors have no competing financial interests.

The key points to establish when quantifying paw-dragging behaviour in the cylinder test are the following: i) quantify the number of paw-drags versus total paw touches for each paw before brain injury to establish a baseline; ii) quantify the number of paw-drags versus total paw touches for each paw following the ischemic injury; and iii) discriminate between a paw-drag and the lateral motion of the paw along the cylinder wall during lateral rotation of the mouse&#8217;s torso.
Paw-dragging i...

The goal of neural regeneration strategies is to demonstrate both tissue repair and functional recovery. Functional recovery is typically evaluated with behavioural tests that measure functional deficits, in this case involving motor skills that are associated with damage to the specific brain regions. Traumatic brain injury or ischemic damage to the sensorimotor forelimb area of the cortex can be demonstrated by a number of behavioural tests. One such test, the cylinder test is used extensively in rats to assess functional deficits in forelimb activity1. The test has a low set-up cost requiring only a cylinder, camera and table with a transparent top. It is easy to administer as it is based on the natural exploratory behaviour of rodents, so pre-training and food deprivation or rewards are not required. Despite these numerous advantages, the cylinder test is under-utilized to assess forelimb deficits in mice following focal injuries to the forelimb sensorimotor cortex, which we attribute to the analysis of mouse behaviour in the cylinder test. Forelimb asymmetry is the classical measure of analysis for the cylinder test. When placed in the cylinder, rodents naturally explore the walls of the cylinder by rearing onto their hind limbs and touching the cylinder walls with their forelimb paws for postural balance. The number of paw touches with the wall with each forelimb is easily quantified by filming rodents during this exploration of the cylinder. Forelimb asymmetry occurs when the affected forelimb paw makes fewer touches with the wall than the unaffected forelimb paw and is indicative of damage to the contralateral sensorimotor cortex. In rats, intra-cortical injections of the vasoconstrictive agent, Endothelin (ET-1), into the forelimb sensorimotor cortex causes a focal ischemic lesion which results in behavioural deficits in the contralateral forelimb. Deficits in contralateral forelimb use are readily detected as changes in forelimb asymmetry in the cylinder test in rats1-3. In contrast to rats however, changes in forelimb asymmetry are variable and less consistent in mice following comparable ET-1 injections4-6. Here we demonstrate a novel analysis of forelimb behaviour in the cylinder test &#8211; analysis of paw-dragging behaviour. We have previously shown that paw-dragging analysis is a more sensitive measure of damage to the forelimb sensorimotor cortex in mice than the classical forelimb asymmetry analysis and therefore is applicable to a variety of focal cortical injury models.
Examination of how the forepaw contacts the cylinder wall following ischemic damage to the forelimb sensorimotor cortex revealed a novel behaviour in mice - paw-dragging4. A paw-drag occurs when a mouse stands on its rear legs to explore the cylinder wall then drags its affected (contra-lesional) paw along the cylinder wall towards its midline or down the wall while its unaffected forepaw provides postural support against the wall. Paw-drags rarely occur in uninjured mice therefore the appearance of a paw-drag is a positive indicator of injury to the forelimb sensorimotor cortex4. We have previously quantified paw-dragging behaviour in mice following ET-1 ischemic damage to the forelimb sensorimotor cortex and have shown sustained paw-dragging behaviour in mice up to two weeks post-stroke4. Here we show that paw-dragging behaviour is sustained up to four weeks post-stroke. Analysis of paw-dragging behaviour provides a novel and sensitive tool for assessing focal ischemic damage to the forelimb sensorimotor cortex in mice. Its inexpensive set-up, ease of administration and scoring make this a simple, yet useful tool to rapidly assess forelimb behavioural deficits in mice.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Plexi-glass cylinder</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>17.5cm high, 9.5cm outer diameter, 8.8cm inner diameter, wall thickness 0.35cm (or 3.5 mm)</td>
		</tr>
		<tr>
			<td>viewing table</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>54x56x66.5cm (width x length x height), top of table is a 51x51cm sheet of plexiglass.</td>
		</tr>
		<tr>
			<td>mirror</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>34x58cm mirror</td>
		</tr>
		<tr>
			<td>video camera</td>
			<td>Sony</td>
			<td>DCR-SR42</td>
			<td>Video camera with onboard storage, SD functionality, 40x optical zoom</td>
		</tr>
		<tr>
			<td>computer</td>
			<td>Dell</td>
			<td>Optiplex 760</td>
			<td>Processor: Intel, 3.0 GHz, Memory 4.00GB (RAM)&#160;</td>
		</tr>
		<tr>
			<td>computer monitor</td>
			<td>Samsung</td>
			<td>S22C350H</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel (Microsoft Office Professional Plus)</td>
			<td>Microsoft</td>
			<td>v14.0.7106.5003</td>
			<td></td>
		</tr>
		<tr>
			<td>VLC Media Player</td>
			<td>Video LAN</td>
			<td>v2.1.2</td>
			<td>Media player with playback speed modulation and video support</td>
		</tr>
		<tr>
			<td>External Hard Drive</td>
			<td>Western Digital</td>
			<td>WDBAAU0020HBK-01</td>
			<td>2 TB</td>
		</tr>
	</tbody>
</table>

paw-dragging: a novel, sensitive analysis of the mouse cylinder test

The cylinder test is routinely used to predict focal ischemic damage to the forelimb motor cortex in rodents. When placed in the cylinder, rodents explore by rearing and touching the walls of the cylinder with their forelimb paws for postural support. Following ischemic injury to the forelimb sensorimotor cortex, rats rely more heavily on their unaffected forelimb paw for postural support resulting in fewer touches with their affected paw which is termed forelimb asymmetry. In contrast, focal ischemic damage in the mouse brain fails to result in comparable consistent deficits in forelimb asymmetry. While forelimb asymmetry deficits are infrequently observed, mice do demonstrate a novel behaviour post stroke termed &#8220;paw-dragging&#8221;. Paw-dragging is the tendency for a mouse to drag its affected paw along the cylinder wall rather than directly push off from the wall when dismounting from a rear to a four-legged stance. We have previously demonstrated that paw-dragging behaviour is highly sensitive to small cortical ischemic injuries to the forelimb motor cortex. Here we provide a detailed protocol for paw-dragging analysis. We define what a paw-drag is and demonstrate how to quantify paw-dragging behaviour. The cylinder test is a simple and inexpensive test to administer and does not require pre-training or food deprivation strategies. In using paw-dragging analysis with the cylinder test, it fills a niche for predicting cortical ischemic injuries such as photothrombosis and Endothelin-1 (ET-1)-induced ischemia &#8211; two models that are ever-increasing in popularity and produce smaller focal injuries than middle cerebral artery occlusion. Finally, measuring paw-dragging behaviour in the cylinder test will allow studies of functional recovery after cortical injury using a wide cohort of transgenic mouse strains where previous forelimb asymmetry analysis has failed to detect consistent deficits.

We have previously demonstrated that paw-dragging behaviour appears following a focal ischemic injury to the forelimb sensorimotor cortex and is a positive indicator of damage4. Intra-cortical injections of ET-1 into the forelimb sensorimotor cortex were used to induce an ischemic lesion (Figure&#160;8A,B). This study examined whether paw-dragging behaviour extended for longer than 14 days post-injury for its potential use to assess functional recovery. Mice were tested in the cylinder test on...

Watch this Scientific Journal Video about Paw-Dragging: a Novel, Sensitive Analysis of the Mouse Cylinder Test at JoVE.com

Paw-Dragging: a Novel, Sensitive Analysis of the Mouse Cylinder Test

Classical forelimb asymmetry analysis of the cylinder test is routinely used to assess behavioural deficits in rats following brain injury or stroke; however, it fails to detect consistent deficits in mice. This study demonstrates that quantifying paw-dragging behaviour is a more sensitive analysis of brain injury in mice.

The cylinder test is routinely used to predict focal ischemic damage to the forelimb motor cortex in rodents. When placed in the cylinder, rodents explore ...

paw-dragging-a-novel-sensitive-analysis-of-the-mouse-cylinder-test

Research

JoVE Journal

Behavior

23.2K Views.  Memorial University of Newfoundland.  Classica analisi forelimb asimmetria del test del cilindro viene normalmente utilizzato per valutare deficit comportamentali nei ratti seguenti lesione cerebrale o ictus; tuttavia, esso non riesce a rilevare deficit consistenti in topi. Questo studio dimostra che quantificare il comportamento zampa-trascinamento è un'analisi più sensibile di lesioni cerebrali nei topi. 

Video: Paw-Dragging: a Novel, Sensitive Analysis of the Mouse Cylinder Test

Inchworming: A Novel Motor Stereotypy in the BTBR T+ Itpr3tf/J Mouse Model of Autism

Autism Spectrum Disorder (ASD) is a behaviorally defined neurodevelopmental disorder characterized by decreased reciprocal social interaction, abnormal communication, and repetitive behaviors with restricted interest. As diagnosis is based on clinical criteria, any potentially relevant rodent models of this heterogeneous disorder should ideally recapitulate these diverse behavioral traits. The BTBR T+ Itpr3tf/J (BTBR) mouse is an established animal model of ASD, displaying repetitive behaviors such as increased grooming, as well as cognitive inflexibility. With respect to social interaction and interest, the juvenile play test has been employed in multiple rodent models of ASD. Here, we show that when BTBR mice are tested in a juvenile social interaction enclosure containing sawdust bedding, they display a repetitive synchronous digging motion. This repetitive motor behavior, referred to as &#34;inchworming,&#34; was named because of the stereotypic nature of the movements exhibited by the mice while moving horizontally across the floor. Inchworming mice must use their fore- and hind-limbs in synchrony to displace the bedding, performing a minimum of one inward and one outward motion. Although both BTBR and C56BL/6J (B6) mice exhibit this behavior, BTBR mice demonstrate a significantly higher duration and frequency of inchworming and a decreased latency to initiate inchworming when placed in a bedded enclosure. We conclude that this newly described behavior provides a measure of a repetitive motor stereotypy that can be easily measured in animal models of ASD.

Inchworming: A Novel Motor Stereotypy in the BTBR T+ Itpr3tf/J Mouse Model of Autism

Inchworming is a highly repetitive synchronous digging motion displayed by BTBR T+ Itpr3tf/J (BTBR) mice when placed in a testing cage with sufficient sawdust bedding. The procedure is a modification of the juvenile social interaction protocol and is used here to assess repetitive motor stereotypies relevant to Autism Spectrum Disorder.

Autism Spectrum Disorder (ASD) is a behaviorally defined neurodevelopmental disorder characterized by decreased reciprocal social interaction, abnormal ...

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 ...

The Forced Swim Test as a Model of Depressive-like Behavior

The goal of the present protocol is to describe the forced swim test (FST), which is one of the most commonly used assays for the study of depressive-like behavior in rodents. The FST is based on the assumption that when placing an animal in a container filled with water, it will first make efforts to escape but eventually will exhibit immobility that may be considered to reflect a measure of behavioral despair. This test has been extensively used because it involves the exposure of the animals to stress, which was shown to have a role in the tendency for major depression. Additionally, the FST has been shown to share some of the factors that are influenced or altered by depression in humans, including changes in food consumption, sleep abnormalities and drug-withdrawal-induced anhedonia. The main advantages of this procedure are that it is relatively easy to perform and that its results are easily and quickly analyzed. Moreover, its sensitivity to a broad range of antidepressant drugs that makes it a suitable screening test is one of the most important features leading to its high predictive validity. Despite its appeal, this model has a number of disadvantages. First, the issue of chronic augmentation is problematic in this test because in real life patients need to be treated for at least several weeks before they experience any relief from their symptoms. Last, due to the aversiveness of the FST, it is important to take into account possible influences it might have on brain structure/function if brain analyses are to be carried out following this procedure.

This protocol describes the forced swim test, which is used for the study of depressive-like behavior in rodents. This procedure involves placing an animal in a container filled with water that eventually will lead to the exhibition of immobility behavior, which is considered to reflect behavioral despair.

The goal of the present protocol is to describe the forced swim test (FST), which is one of the most commonly used assays for the study of depressive-like ...

Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment

The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, including neurocognitive disorders such as Alzheimer&#8217;s disease. However, the background strain of the mouse model used can have a substantial effect on the observed behavioral phenotype, with some strains exhibiting superior learning ability relative to others. To ensure differences between transgene negative and transgene positive mice can be detected, identification of a training procedure sensitive to the background strain is essential. Failure to tailor the MWM protocol to the background strain of the mouse model may lead to under- or over- training, thereby masking group differences in probe trials. Here, a MWM protocol tailored for use with the F1 FVB/N x 129S6 background is described. This is a frequently used background strain to study the age-dependent effects of mutant P301L tau (rTg(TauP301L)4510 mice) on the memory deficits associated with Alzheimer&#8217;s disease. Also described is a strategy to re-optimize, as dictated by the particular testing environment utilized.

This manuscript describes a Morris water maze (MWM) protocol tailored for use with a commonly used mouse model of Alzheimer's disease. The MWM is widely used in transgenic mouse models. Implementation of a procedure sensitive to the background strain of the mouse model is essential for detecting group differences.

 The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, ...

A Novel Approach to Assess Motor Outcome of Deep Brain Stimulation Effects in the Hemiparkinsonian Rat: Staircase and Cylinder Test

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to screen different neurostimulation patterns in hemiparkinsonian (unilateral lesioned) rats. It consists of creating a unilateral Parkinson&#39;s lesion by injecting 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle, implanting chronic stimulation electrodes into the subthalamic nucleus and evaluating motor outcomes at the end of 24 hr periods of cable-bound external neurostimulation. The stimulation was conducted with constant current stimulation. The amplitude was set 20% below the individual threshold for side effects. The motor outcome evaluation was done by the assessment of spontaneous paw use in the cylinder test according to Shallert and by the assessment of skilled reaching in the staircase test according to Montoya. This protocol describes in detail the training in the staircase box, the cylinder test, as well as the use of both in hemiparkinsonian rats. The use of both tests is necessary, because the staircase test seems to be more sensitive for fine motor skill impairment and exhibits greater sensitivity to change during neurostimulation. The combination of the unilateral Parkinson model and the two behavioral tests allows the assessment of different stimulation parameters in a standardized way.

Deep brain stimulation (DBS) is an effective treatment option for Parkinson's disease. We established a study design to screen novel stimulation paradigms in rats. The protocol describes the use of the staircase test and cylinder test for motor outcome assessment in DBS treated hemiparkinsonian rats.

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to ...

The Treadmill Fatigue Test: A Simple, High-throughput Assay of Fatigue-like Behavior for the Mouse

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of current interventions to adequately treat fatigue in all patients leaves a need for new treatment options. Despite the therapeutic need and importance of preclinical research in helping identify promising novel treatments, few preclinical assays of fatigue are available. Moreover, the most common preclinical assay used to assess fatigue-like behavior, voluntary wheel running, is not suitable for use with some strains of mice, may not be sensitive to drugs that reduce fatigue, and has relatively low throughput. The current protocol describes a novel, non-voluntary preclinical assay of fatigue-like behavior, the treadmill fatigue test, and provides evidence of its efficacy in detecting fatigue-like behavior in mice treated with a chemotherapy drug known to cause fatigue in humans and fatigue-like behavior in animals. This assay may be a beneficial alternative to wheel running, as fatigue-like behavior and potential interventions can be assessed in a greater number of mice over a shorter time frame, thus permitting faster discovery of new therapeutic options.

Fatigue is a common, undertreated and frequently poorly-understood symptom in many diseases and disorders. New preclinical assays of fatigue may help to improve current understanding and future treatment of fatigue. To that end, the current protocol provides a novel means of measuring fatigue-like behavior in the mouse.

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of ...

The 4 Mountains Test: A Short Test of Spatial Memory with High Sensitivity for the Diagnosis of Pre-dementia Alzheimer's Disease

This protocol describes the administration of the 4 Mountains Test (4MT), a short test of spatial memory, in which memory for the topographical layout of four mountains within a computer-generated landscape is tested using a delayed match-to-sample paradigm. Allocentric spatial memory is assessed by altering the viewpoint, colors and textures between the initially presented and target images.
Allocentric spatial memory is a key function of the hippocampus, one of the earliest brain regions to be affected in Alzheimer&#39;s disease (AD) and impairment of hippocampal function predates the onset of dementia. It was hypothesized that performance on the 4MT would aid the diagnosis of predementia AD, which manifests clinically as Mild Cognitive Impairment (MCI).
The 4MT was applied to patients with MCI, stratified further based on cerebrospinal fluid (CSF) AD biomarker status (10 MCI biomarker positive, 9 MCI biomarker negative), and with mild AD dementia, as well as healthy controls. Comparator tests included tests of episodic memory and attention widely accepted as sensitive measures of early AD. Behavioral data were correlated with quantitative MRI measures of the hippocampus, precuneus and posterior cingulate gyrus.
4MT scores were significantly different between the two MCI groups (p = 0.001), with a test score of &#8804;8/15 associated with 100% sensitivity and 78% specificity for the classification of MCI with positive AD biomarkers, i.e., predementia AD. 4MT test scores correlated with hippocampal volume (r = 0.42) and cortical thickness of the precuneus (r = 0.55).
In conclusion, the 4MT is effective in identifying the early stages of AD. The short duration, easy application and scoring, and favorable psychometric properties of the 4MT fulfil the need for a simple but accurate diagnostic test for predementia AD.

The 4 Mountains Test: A Short Test of Spatial Memory with High Sensitivity for the Diagnosis of Pre-dementia Alzheimer&#039;s Disease

This article describes the 4 Mountains Test (4MT), a hippocampus-dependent test of working allocentric spatial memory. The hippocampus is affected early in Alzheimer's disease (AD) and this article outlines the 4MT methodology and results of patient testing, which demonstrates the value of the 4MT in the diagnosis of pre-dementia AD.

This protocol describes the administration of the 4 Mountains Test (4MT), a short test of spatial memory, in which memory for the topographical layout of ...

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors remains poorly understood. In Drosophila melanogaster, environmental cues, such as food availability, affect mating activity offering a tractable system to investigate the mechanisms modulating sexual behavior. In D. melanogaster, environmental cues are often sensed via the chemosensory gustatory and olfactory systems. Here, we present a method to test the effect of environmental chemical cues on mating behavior. The assay consists of a small mating arena containing food medium and a mating couple. The mating frequency for each couple is continuously monitored for 24 h. Here we present the applicability of this assay to test environmental compounds from an external source through a pressurized air system as well as manipulation of the environmental components directly in the mating arena. The use of a pressurized air system is especially useful to test the effect of very volatile compounds, while manipulating components directly in the mating arena can be of value to ascertain a compound's presence. This assay can be adapted to answer questions about the influence of genetic and environmental cues on mating behavior and fecundity as well as other male and female reproductive behaviors.

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

We demonstrate an assay to analyze the environmental and genetic cues that influence mating behavior in the fruit fly Drosophila melanogaster.

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors ...

Novel Object Recognition Test for the Investigation of Learning and Memory in Mice

The object recognition test (ORT) is a commonly used behavioral assay for the investigation of various aspects of learning and memory in mice. The ORT is fairly simple and can be completed over 3 days: habituation day, training day, and testing day. During training, the mouse is allowed to explore 2 identical objects. On test day, one of the training objects is replaced with a novel object. Because mice have an innate preference for novelty, if the mouse recognizes the familiar object, it will spend most of its time at the novel object. Due to this innate preference, there is no need for positive or negative reinforcement or long training schedules. Additionally, the ORT can also be modified for numerous applications. The retention interval can be shortened to examine short-term memory, or lengthened to probe long-term memory. Pharmacological intervention can be used at various times prior to training, after training, or prior to recall to investigate different phases of learning (i.e., acquisition, early or late consolidation, or recall). Overall, the ORT is a relatively low-stress, efficient test for memory in mice, and is appropriate for the detection of neuropsychological changes following pharmacological, biological, or genetic manipulations.

The object recognition test (ORT) is a simple and efficient assay for evaluating learning and memory in mice. The methodology is described below.

The object recognition test (ORT) is a commonly used behavioral assay for the investigation of various aspects of learning and memory in mice. The ORT is ...

Measuring Active and Passive Tameness Separately in Mice

Domesticated animals such as dogs and laboratory mice show a high level of tameness, which is important for humans to handle them easily. Tameness has two behavioral components: a reluctance to avoid humans (passive tameness) and a motivation to approach humans (active tameness). To quantify these components in mice, we previously developed behavioral tests for active tameness, passive tameness, and the willingness to stay on a human hand, each designed to be completed within 3 min. The data obtained were used for selective breeding, with a large number of mice analyzed per generation. The active tameness test measures the movement of the mouse toward a human hand and the contact it engages in. The passive tameness test measures the duration of time that a mouse tolerates human touch. In the stay-on-hand test, a mouse is placed on a human hand and touched slowly using the thumb of that hand; the duration of time that the animal remains on the hand is measured. Here, we describe the test set-up and apparatus, explain the procedures, and discuss the appropriate data analysis. Finally, we explain how to interpret the results.

Tameness in animals includes a reduction of avoidance responses to humans (passive tameness) and an increase in active approaches to humans (active tameness). Here, we describe detailed protocols for three behavioral tests (active tameness, passive tameness, and stay-on-hand tests) to separately measure the active and passive tameness of mice.

Domesticated animals such as dogs and laboratory mice show a high level of tameness, which is important for humans to handle them easily. Tameness has two ...