Name: Video: Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice
Uploaded: 2016-02-03T15:00:00.000+00:00
Duration: 7 min 41 s
Description: 13.9K Views. The University of Melbourne. This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

JCB and ELH-Y were supported by the US Department of Defense CDMRP Autism Research Program (AR11034). NHMRC (1047674) to ELH-Y.The May Stewart Bursary-University of Melbourne trust funded scholarship to MS. We thank Ali Taher, F&#225;tima Ramalhosa and Gracia Seger for technical contributions.

Animal handling and cervical dislocation of animals prior to all experiments were performed strictly according to protocols approved by the Animal Experimentation Committee for the University of Melbourne (Ethics ID: 1212494.7)
1. Tissue Collection and Dissection
<ol>
	<li>Euthanize adult mice by cervical dislocation. If possible avoid anesthesia to prevent influences on gut function via receptors located on neuronal populations of interest.</li>
	<li>Record the animal&#39;s total body weight, pin the body (via the four paws, ventral side exposed to experimenter) firmly to a dissection board using hypodermic needles (Size: 20 G).</li>
	<li>Using dissecting forceps and scissors; make an incision through the epidermis overlaying the lower abdominal muscle layers. Continue to use the forceps and scissors to open the abdominal cavity along the midline of the abdomen to the sternum.</li>
	<li>To prevent the tissue dehydrating, pour physiological saline (Krebs solution: 118 NaCl, 4.6 KCl, 2.5 CaCl2, 1.2 MgSO4, 1 NaH2PO4, 25 NaHCO3, 11 D-glucose in mM - bubbled at RT with carbogen gas; 95% O2 and 5% CO2 for a minimum of 20 min) onto abdominal contents at regular intervals (i.e., every 30-60 sec) during the dissection process.</li>
	<li>To isolate the colon while attached to the cecum and rectum, hold the cecum (located adjacent to the proximal end of colon) gently using dissecting forceps approximately 4-5 cm above the body of the animal while trimming the mesentery using fine dissection scissors.</li>
	<li>Take care not to handle, stretch or cut the gastrointestinal tissue while removing the adjoining mesentery.</li>
	<li>Remove excess tissue (i.e., urinary bladder and testes). Using coarse dissecting scissors, make two vertical incisions (i.e.,&#160;approximately 0.5 cm from the midline) to cut the pelvic bone on each side of the colon.</li>
	<li>Use fine scissors to separate the rectum from adjoining pelvic tissue and trim surrounding muscle from the colon.</li>
	<li>Place the full-length colon (from cecum to rectum) into a beaker containing physiological saline (previously oxygenated with carbogen) and continue to oxygenate at RT. Remove the caecum and rectum using fine dissecting scissors and replace in beaker.</li>
	<li>Empty fecal pellets/intestinal content from colonic preparations by applying gentle positive pressure at the oral end using a 5 ml syringe attached to a 200 &#181;l pipette tip filled with physiological saline. In order to orientate the tissue in future, use an insect pin to mark the most proximal end of the colon, where striations in the mucosa are visible.
	<ol>
		<li>Modify the plastic pipette tip to fit the 5 ml syringe by removing approximately 1 cm of the widest/proximal end using a razor blade. To increase the flow rate, widen the distal/smaller end by cutting on an angle (approximately 45 &#176;C) using a razor blade.</li>
	</ol>
	</li>
	<li>Grip the colon tissue gently at its proximal end with dissecting forceps, insert the pipette tip into the lumen of the colon and flush physiological saline through the lumen.</li>
</ol>
2. Preparation of Colonic Tissue and Experimental Set Up for Video Imaging
<ol>
	<li>Flush physiological saline through all tubing attached to a two chambered organ bath set up (Figure 1 and Figure 3), using a syringe attached to a 200 &#181;l pipette tip. This step removes any debris within the tubing that could block the flow of solution. For tubing specifications refer to the materials table.</li>
	<li>Ensure that the chambers of the organ bath are continuously superfused with physiological saline bubbled with carbogen (95% O2 and 5% CO2, flow rate of 5 ml min-1). Using a temperature probe at regular intervals, ensure that the temperature of the bath is maintained between 33 &#176;C -37 &#176;C.</li>
	<li>Fill the inflow reservoir connected via a 3-way stopcock to the inlet tube with approximately 20-30 ml physiological saline using a 50 ml syringe.</li>
	<li>During the experiment, maintain the pressure in the inflow reservoir using arubber stopper with a glass tube (5 mm inside diameter) inserted through its center. Make sure that the upper opening of the inflow reservoir is securely sealed (with the inner glass tube remaining unsealed).</li>
	<li>Place the colon (length 5-7 cm) into the organ bath chamber, making sure that it is fully submerged in physiological saline.</li>
	<li>Cannulate each end of the colonic segment to the two submerged inlet/outlet tubes (inlet/outlet tubes of different diameter can be changed according to the size of the tissue lumen eg. postnatal tissue, adult mice and guinea pig) in the bath using standard cotton sewing thread. Attach the proximal end of the colon to the inlet tube and the distal end of the colon to the outlet tube (connected via a 3-way stopcock to a vertical outflow tube; 6 cm in height). Make sure that the tissue is not overstretched or too relaxed after cannulation to avoid interference with future measurements and analysis.</li>
	<li>Record the length of the colon by measuring the distance between the proximal and distal cannulations using a 15 cm ruler. This is important for interpreting changes in contraction length, propagation speed and contraction initiation points.</li>
	<li>Ensure a stable baseline luminal pressure (cm H2O) is established before beginning the experiment. Calculate luminal pressure by measuring the vertical distance from the tissue to the meniscus of physiological saline within the glass tube of inflow reservoir (oral) and the vertical outflow tube (anal).</li>
	<li>Maintain tissue preparations at a constant intraluminal pressure (e.g., 1-2 cm H2O) by ensuring that the inflow stopper seal pressure is kept constant and that no blockages are present in the set up.</li>
	<li>Leave the colon to equilibrate in physiological saline for 30 min before recording intestinal movements. Check for any blockages arising in the in- or outflow tubes and remove them by applying pressure through the inflow reservoir or by re-cannulating the oral and anal ends of the colon.</li>
</ol>
3. Image Capture and Experimental Protocol
<ol>
	<li>Record intestinal movements as video files using a video camera (30 frames/sec, 640 x 840 pixels) positioned 10-15 cm above the organ bath on a standard laboratory retort stand (Figure 1).</li>
	<li>Open Virtual Dub (video capture software) from the icon on the desktop. From the &#39;File&#39; tab, select &#39;capture AVI&#39; to view the camera image. On the &#39;Audio&#39; tab deselect &#39;enable audio capture&#39; to disable sound.
	<ol>
		<li>In the video tab select &#39;compression&#39; and &#39;DivX6.9.2. Codec&#39; to compress video files to minimize usage of storage space. This function will become available upon the installation of the compression software &#39;DivX&#39;.</li>
	</ol>
	</li>
	<li>Manually adjust the camera location so that the entire cannulated colon segment is visible (with cannulated regions immediately adjacent to the vertical edges of the video frame) via the video capture software window (see Figure 1). In order to improve image quality and minimize reflections, attach a paper/cardboard shield to the camera support to block extraneous light onto the organ bath. Optimize video quality by adjusting the brightness, contrast and exposure settings using the camera software interface.
	<ol>
		<li>Before recording, set a specific file name; in the &#39;File&#39; tab, select &#39;Set capture file&#39; and specify a unique name for the video.</li>
		<li>Begin capturing video by pressing &#39;Capture file&#39; from the &#39;Capture&#39; tool bar. To stop recording select the &#39;Stop capture&#39; button or the &#39;Esc&#39; key on the keyboard.</li>
	</ol>
	</li>
	<li>Replace the physiological saline contained within the inflow reservoir with fresh physiological saline every 30 min.
	<ol>
		<li>Record luminal pressure (see 2.9) and using a temperature probe, take note of the organ bath temperature. Repeat these steps throughout the experiment (i.e., every 30 min) to ensure that these variables are constant.</li>
	</ol>
	</li>
	<li>Record colonic activity in total for 3 hr (including drug application and washout duration). For data storage purposes, record data in 15 min video segments. A full experiment will typically be composed of 12 x 15 min video recordings.
	<ol>
		<li>Record activity for 1 hr under control conditions (physiological saline).</li>
		<li>Apply the drug (either superfused into the bath or administered into the lumen via the inflow reservoir) for 30 min to 1 hr. 
		Note: Different routes of administration can yield different effects23.</li>
		<li>Apply fresh physiological saline to the bath or lumen during a 1 hr washout period.</li>
	</ol>
	</li>
</ol>
4. Data Processing and Generation of Spatiotemporal Maps
<ol>
	<li>Process each video recording offline using in-house software written in MATLAB (R2012a, Version 7.4; available on request) in order to generate spatiotemporal maps illustrating colonic motility.
	<ol>
		<li>Open analysis software from the icon on the desktop.</li>
		<li>In the command window, enter &#34;Analyse2&#34; in order to open the analysis control panel.</li>
	</ol>
	</li>
	<li>Utilize the edge detection function to generate spatiotemporal maps by clicking &#34;Edge detection for .avi files&#34; in the control panel window. This function will enable the analysis software to read the recoded videos in Audio Video Interleaved (AVI) format.</li>
	<li>Carry out the following steps sequentially to generate spatiotemporal maps using an adaptive edge detection algorithm.
	<ol>
		<li>Open recorded video files by selecting the &#34;Add files&#34; tab, select &#39;Open video&#39; and select the location of the video files.</li>
		<li>From the video files listed within the analysis software, select the file of interest.</li>
		<li>Select a rectangular region of interest within the frame of the video, e.g., the entire length of the colon, from the point at which the colon is cannulated orally to the anal cannulation. Ensure that the proximal and distal cannulation points are adjacent to the vertical boundaries of the region of interest. Edge detection lines (red and green) will automatically appear superimposed on the real time image.</li>
		<li>Ensure that the edge detection threshold lines are immediately adjacent to the colon on both the upper and lower boundaries of the colon tissue (this can be optimized for each video by altering detection threshold values on the control panel). Ensure that the edge detection lines are continuous along the length of the colon image by adjusting the contrast and brightness using the control panel.</li>
		<li>Enter the full length of the colon (mm) into the width dialog box (as recorded in step 2.7). Then select &#34;Generate heat map&#34;.</li>
		<li>In the pop up window, select a location for the file to be saved and specify a file name for the spatiotemporal map.</li>
		<li>Select the &#39;.su2&#39; format and select &#39;Add to queue&#39;. 
		Note: .su2 format compresses data to a smaller file size and multiple video files can be added to the queue.</li>
		<li>Select &#39;Begin detection&#39; to generate spatiotemporal maps.</li>
	</ol>
	</li>
</ol>
5. Analysis of Spatiotemporal Maps
<ol>
	<li>Utilize spatiotemporal maps to estimate parameters such as frequency, propagation speed, length and duration of CMMCs, gut diameter and the point of initiation and direction of contractions.
	<ol>
		<li>To start analyzing spatiotemporal maps, type &#39;analyse2&#39; in the command window as described in the previous section. Instead of &#34;Edge detection&#34;, select the &#34;Heat map analysis&#34; button.</li>
		<li>Open spatiotemporal map files (.su2 files) by selecting the &#39;Add files&#39; tab and specifying the location of previously obtained .su2 files.</li>
		<li>Once the spatiotemporal map is visible on the screen, specify a color range on the control panel, ensuring that the minimum is set to 1. 
		Note that the maximum can range from 5-10 according to the contractility of the tissue.</li>
		<li>Select &#39;Lock color range&#39; to ensure all the maps from a single experiment are analyzed under the same conditions.</li>
		<li>Ensure that the time frame settings are constant for all spatiotemporal maps from a given experiment.</li>
	</ol>
	</li>
	<li>In order to determine CMMC frequency, manually count contractions that traverse more than 50% of the length of the colon. These contractions can be visualized as red/orange streaks (standard HSV color index) on the spatiotemporal map (see Figure 2 for an example). Other contractions that are shorter or do not propagate can also be identified and quantified.</li>
	<li>If desired, conduct further analysis at a higher resolution from these maps. For example, detailed properties including the speed and duration of each contraction can be examined.
	<ol>
		<li>In order to measure the speed and duration, select the &#34;Annotate contraction waves&#34; button on the heat map analysis control panel.</li>
		<li>Next, zoom in to each CMMC by clicking on the &#34;Zoom&#34; button and selecting the area of interest. Then select the &#34;Manually annotate&#34; button to annotate each contraction by using the mouse to draw a line from the most initial position to the end of each contraction. This method can be used to measure the apparent propagation speed and duration of dilations that appear to propagate (for e.g., see anal end of maps in Figure 2A, 2B). 
		Note: The data for speed and duration will appear under the results window. These values can be transferred to a spreadsheet of interest by selecting the &#34;Export data&#34; tab. Unwanted annotations can be removed by clicking on the &#34;Remove selected annotations&#34; button.</li>
	</ol>
	</li>
	<li>If desired, utilize the x and y coordinates of the spatiotemporal maps (time and position along the colon, respectively) to determine the position of initiation of small contractions or CMMCs.</li>
	<li>Similarly, in order to determine intervals between contractions, use the &#34;Annotate&#34; function to measure the duration between contractions. As previously, these results can be exported to a spreadsheet by selecting the &#34;Export data&#34; tab.</li>
	<li>In order to measure the resting gut width, select the &#34;Take cross section&#34; button on the Heat map analysis panel.
	<ol>
		<li>Select &#39;Add&#39; on the temporal cross sections panel. Double click at any location within the map to generate a horizontal line superimposed on the heat map. Gut diameter data will now be displayed in a new window.</li>
		<li>To measure gut diameter, place the cursor on a peak and adjacent trough to obtain average gut width for a specific experimental condition. Changes in gut diameter can be compared between experimental conditions.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Powell, A. K., O'Brien, S. D., Fida, R., Bywater, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+integrity+is+essential+for+the+propagation+of+colonic+migrating+motor+complexes+in+the+mouse.">Neural integrity is essential for the propagation of colonic migrating motor complexes in the mouse.</a> Neurogastroenterol Motil. 14, 495-504 (2002).</li><li>Furness, J. 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+enteric+nervous+system+and+neurogastroenterology.">The enteric nervous system and neurogastroenterology.</a> Nat Rev Gastroenterol Hepatol. 9, 286-294 (2012).</li><li>Gwynne, R. M., Bornstein, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+underlying+nutrient-induced+segmentation+in+isolated+guinea+pig+small+intestine.">Mechanisms underlying nutrient-induced segmentation in isolated guinea pig small intestine.</a> Am J Physiol Gastrointest Liver Physiol. 292, G1162-G1172 (2007).</li><li>Bush, T. G., Spencer, N. J., Watters, N., Sanders, K. M., Smith, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+migrating+motor+complexes+occur+in+both+the+terminal+ileum+and+colon+of+the+C57BL/6+mouse+in+vitro.">Spontaneous migrating motor complexes occur in both the terminal ileum and colon of the C57BL/6 mouse in vitro.</a> Auton Neurosci. 84, 162-168 (2000).</li><li>Fida, R., Lyster, D. J., Bywater, R. A., Taylor, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonic+migrating+motor+complexes+(CMMCs)+in+the+isolated+mouse+colon.">Colonic migrating motor complexes (CMMCs) in the isolated mouse colon.</a> Neurogastroenterol Motil. 9, 99-107 (1997).</li><li>Neal, K. B., Parry, L. J., Bornstein, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-specific+genetics,+anatomy+and+function+of+enteric+neural+serotonergic+pathways+in+inbred+mice.">Strain-specific genetics, anatomy and function of enteric neural serotonergic pathways in inbred mice.</a> J Physiol. 587, 567-586 (2009).</li><li>Roberts, R. R., Murphy, J. F., Young, H. M., Bornstein, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+colonic+motility+in+the+neonatal+mouse-studies+using+spatiotemporal+maps.">Development of colonic motility in the neonatal mouse-studies using spatiotemporal maps.</a> Am J Physiol Gastrointest Liver Physiol. 292, G930-G938 (2007).</li><li>Spencer, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+migrating+motor+activity+in+the+colon.">Control of migrating motor activity in the colon.</a> Curr Opin Pharmacol. 1, 604-610 (2001).</li><li>Spencer, N. J., Bywater, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+nerve+stimulation+evokes+a+premature+colonic+migrating+motor+complex+in+mouse.">Enteric nerve stimulation evokes a premature colonic migrating motor complex in mouse.</a> Neurogastroenterol Motil. 14, 657-665 (2002).</li><li>Roberts, R. R., Bornstein, J. C., Bergner, A. J., Young, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disturbances+of+colonic+motility+in+mouse+models+of+Hirschsprung's+disease.">Disturbances of colonic motility in mouse models of Hirschsprung's disease.</a> Am J Physiol Gastrointest Liver Physiol. 294, G996-G1008 (2008).</li><li>Tough, I. R., 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=Endogenous+peptide+YY+and+neuropeptide+Y+inhibit+colonic+ion+transport,+contractility+and+transit+differentially+via+Y(1)+and+Y(2)+receptors.">Endogenous peptide YY and neuropeptide Y inhibit colonic ion transport, contractility and transit differentially via Y(1) and Y(2) receptors.</a> Br J Pharmacol. 164, 471-484 (2011).</li><li>Hennig, G. W., Costa, M., Chen, B. N., Brookes, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+peristalsis+in+the+guinea-pig+small+intestine+using+spatio-temporal+maps.">Quantitative analysis of peristalsis in the guinea-pig small intestine using spatio-temporal maps.</a> J Physiol. 517 (Pt 2), 575-590 (1999).</li><li>Hoffman, J. M., Brooks, E. M., Mawe, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+Motility+Monitor+(GIMM).">Gastrointestinal Motility Monitor (GIMM).</a> J Vis Exp. , (2010).</li><li>Smith, T. K., Gershon, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rebuttal+from+Terence+K.+Smith+and+Michael+D.+Gershon.">Rebuttal from Terence K. Smith and Michael D. Gershon.</a> J Physiol. 593, 3233 (2015).</li><li>Spencer, N. J., Sia, T. C., Brookes, S. J., Costa, M., Keating, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CrossTalk+opposing+view:+5-HT+is+not+necessary+for+peristalsis.">CrossTalk opposing view: 5-HT is not necessary for peristalsis.</a> J Physiol. 593, 3229-3231 (2015).</li><li>Tabuchi, K., 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+neuroligin-3+mutation+implicated+in+autism+increases+inhibitory+synaptic+transmission+in+mice.">A neuroligin-3 mutation implicated in autism increases inhibitory synaptic transmission in mice.</a> Science. 318, 71-76 (2007).</li><li>Jamain, S., 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=Mutations+of+the+X-linked+genes+encoding+neuroligins+NLGN3+and+NLGN4+are+associated+with+autism.">Mutations of the X-linked genes encoding neuroligins NLGN3 and NLGN4 are associated with autism.</a> Nat Genet. 34, 27-29 (2003).</li><li>Chaidez, V., Hansen, R. L., Hertz-Picciotto, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+problems+in+children+with+autism,+developmental+delays+or+typical+development.">Gastrointestinal problems in children with autism, developmental delays or typical development.</a> J Autism Dev Disord. 44, 1117-1127 (2014).</li><li>Ibrahim, S. H., Voigt, R. G., Katusic, S. K., Weaver, A. L., Barbaresi, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+gastrointestinal+symptoms+in+children+with+autism:+a+population-based+study.">Incidence of gastrointestinal symptoms in children with autism: a population-based study.</a> Pediatrics. 124, 680-686 (2009).</li><li>Kohane, I. S., 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+co-morbidity+burden+of+children+and+young+adults+with+autism+spectrum+disorders.">The co-morbidity burden of children and young adults with autism spectrum disorders.</a> PloS One. 7, e33224 (2012).</li><li>McElhanon, B. O., McCracken, C., Karpen, S., Sharp, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+symptoms+in+autism+spectrum+disorder:+a+meta-analysis.">Gastrointestinal symptoms in autism spectrum disorder: a meta-analysis.</a> Pediatrics. 133, 872-883 (2014).</li><li>Peters, 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=Rigid-compulsive+behaviors+are+associated+with+mixed+bowel+symptoms+in+autism+spectrum+disorder.">Rigid-compulsive behaviors are associated with mixed bowel symptoms in autism spectrum disorder.</a> J Autism Dev Disord. 44, 1425-1432 (2014).</li><li>Ellis, M., Chambers, J. D., Gwynne, R. M., Bornstein, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+and+cholecystokinin+mediate+nutrient-induced+segmentation+in+guinea+pig+small+intestine.">Serotonin and cholecystokinin mediate nutrient-induced segmentation in guinea pig small intestine.</a> Am J Physiol Gastrointest Liver Physiol. 304, G749-G761 (2013).</li><li>Parracho, H. M., Bingham, M. O., Gibson, G. R., McCartney, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+between+the+gut+microflora+of+children+with+autistic+spectrum+disorders+and+that+of+healthy+children.">Differences between the gut microflora of children with autistic spectrum disorders and that of healthy children.</a> J Med Microbiol. 54, 987-991 (2005).</li><li>Buie, T., 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=Evaluation,+diagnosis,+and+treatment+of+gastrointestinal+disorders+in+individuals+with+ASDs:+a+consensus+report.">Evaluation, diagnosis, and treatment of gastrointestinal disorders in individuals with ASDs: a consensus report.</a> Pediatrics. 125, S1-S18 (2010).</li><li>Etherton, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autism-linked+neuroligin-3+R451C+mutation+differentially+alters+hippocampal+and+cortical+synaptic+function.">Autism-linked neuroligin-3 R451C mutation differentially alters hippocampal and cortical synaptic function.</a> Proc Natl Acad Sci U S A. 108, 13764-13769 (2011).</li><li>Etherton, M. R., Tabuchi, K., Sharma, M., Ko, J., Sudhof, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+autism-associated+point+mutation+in+the+neuroligin+cytoplasmic+tail+selectively+impairs+AMPA+receptor-mediated+synaptic+transmission+in+hippocampus.">An autism-associated point mutation in the neuroligin cytoplasmic tail selectively impairs AMPA receptor-mediated synaptic transmission in hippocampus.</a> EMBO J. 30, 2908-2919 (2011).</li><li>Zhang, Q., 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=Expression+of+neurexin+and+neuroligin+in+the+enteric+nervous+system+and+their+down-regulated+expression+levels+in+Hirschsprung+disease.">Expression of neurexin and neuroligin in the enteric nervous system and their down-regulated expression levels in Hirschsprung disease.</a> Mol Biol Rep. 40, 2969-2975 (2013).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+significance+of+neuroligins+in+myenteric+cells+of+Cajal+in+Hirschsprung's+disease.">Expression and significance of neuroligins in myenteric cells of Cajal in Hirschsprung's disease.</a> PloS One. 8, e67205 (2013).</li><li>Yang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+down-regulation+of+neuroligin-2+and+the+correlative+clinical+significance+of+serum+GABA+over-expression+in+Hirschsprung's+disease.">The down-regulation of neuroligin-2 and the correlative clinical significance of serum GABA over-expression in Hirschsprung's disease.</a> Neurochem Res. 39, 1451-1457 (2014).</li><li>Roberts, R. R., 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+first+intestinal+motility+patterns+in+fetal+mice+are+not+mediated+by+neurons+or+interstitial+cells+of+Cajal.">The first intestinal motility patterns in fetal mice are not mediated by neurons or interstitial cells of Cajal.</a> J Physiol. 588, 1153-1169 (2010).</li><li>Barnes, K. J., Spencer, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+colonic+migrating+motor+complexes+occur+in+mice+lacking+the+endothelin-3+gene?.">Can colonic migrating motor complexes occur in mice lacking the endothelin-3 gene?.</a> Clin Exp Pharmacol Physiol. 42, 485-495 (2015).</li><li>Chambers, J. D., Bornstein, J. C., Thomas, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+neural+oscillators+and+muscle+feedback+are+required+for+the+intestinal+fed+state+motor+program.">Multiple neural oscillators and muscle feedback are required for the intestinal fed state motor program.</a> PloS One. 6, e19597 (2011).</li><li>Heredia, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Important+role+of+mucosal+serotonin+in+colonic+propulsion+and+peristaltic+reflexes:+in+vitro+analyses+in+mice+lacking+tryptophan+hydroxylase+1.">Important role of mucosal serotonin in colonic propulsion and peristaltic reflexes: in vitro analyses in mice lacking tryptophan hydroxylase 1.</a> J Physiol. 591, 5939-5957 (2013).</li><li>Chambers, J. D., Bornstein, J. C., Thomas, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+mechanisms+of+intestinal+segmentation+in+guinea+pigs:+a+combined+computational+modeling+and+in+vitro+study.">Insights into mechanisms of intestinal segmentation in guinea pigs: a combined computational modeling and in vitro study.</a> Am J Physiol Gastrointest Liver Physiol. 295, G534-G541 (2008).</li><li>Huizinga, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+segmentation+motor+activity+in+the+intestine.">The origin of segmentation motor activity in the intestine.</a> Nat Commun. 5, 3326 (2014).</li><li>Neild, T. O., Shen, K. Z., Surprenant, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vasodilatation+of+arterioles+by+acetylcholine+released+from+single+neurones+in+the+guinea-pig+submucosal+plexus.">Vasodilatation of arterioles by acetylcholine released from single neurones in the guinea-pig submucosal plexus.</a> J Physiol. 420, 247-265 (1990).</li></ol>

The authors have nothing to disclose

Using this video imaging technique, CMMC frequency was measured as an indication of colonic motility in wild type and NL3R451C mice, a mouse model of autism spectrum disorder17. Our results indicate a reduction in the number of CMMCs in mutant NL3R451C mice compared to wild type mice in the presence of the 5HT3/4 receptor antagonist Tropisetron suggesting that NL3R451C mice exhibit an increased sensitivity to Tropisetron. Accordingly, we propose that the neuroligin-3...

The enteric nervous system (ENS) is the intrinsic neuronal network of the gastrointestinal tract and modulates various functions such as digestion of intestinal content, absorption of nutrients and the secretion and reabsorption of fluid. Neurons of the ENS are located in the myenteric and submucosal plexuses. The myenteric plexus plays a major role in regulating gastrointestinal motility1 whereas the submucosal plexus is primarily involved in the control of secretion2,3. The myenteric plexus is situated between the longitudinal and circular muscle layers of the gastrointestinal wall. The contractile activity of the smooth muscle layers of the intestinal wall facilitates the primary functions of the gastrointestinal tract by mixing and propelling intestinal content along the length of the intestine3. Although the extrinsic nerve supply to the gastrointestinal tract from the CNS contributes to gastrointestinal function in vivo, the ENS is capable of regulating gastrointestinal function independently. This unique characteristic enables the functional investigation of enteric neuronal circuits and their contribution to gastrointestinal motility ex vivo.
Colonic migrating motor complexes (CMMCs) are spontaneous, neurogenic events that are the predominant motor pattern observed in isolated mouse colon in the absence of fecal pellets4-9. CMMCs are defined as rhythmic contractions that propagate along a horizontal distance that is at least half the total length of the colon (i.e.,&#160;from the cecum to the rectum)10. The relationship between CMMCs and the contractile patterns that propel fecal pellets is yet to be clearly established, however some pharmacological differences have been reported11. Nevertheless, the ability of the ENS to function independently of the CNS and the existence of neural-mediated motor patterns in the isolated colon provides an ideal assay system to examine disturbances in motility resulting from underlying ENS dysfunction. The spontaneity of gastrointestinal motor patterns allows functional changes in response to pharmacological stimuli to be evaluated.
The use of video imaging and spatiotemporal mapping was first developed to quantitatively examine small intestinal peristalsis in guinea pigs12. Here, an ex vivo technique is described that enables the study of mouse colonic motility patterns using video imaging and analysis of these recordings to construct high-resolution (~100 &#181;m, 33 msec) maps of colonic diameter as a function of position along the colon and of time (spatiotemporal maps). Using in-house edge detection software (Analyse2; available on request), data from full length colonic segments contracting in real time are processed to generate spatiotemporal maps for each experiment. In this step, video (AVI) files are summarized and converted to spatiotemporal maps using Analyse2. Spatiotemporal maps (Figure 2) depict contractility over time and enable the measurement of multiple parameters including propagation speed, magnitude, length and duration. Gut diameter is also recorded throughout the duration of the experiment as a measure of the overall contractility of the tissue segment. This method can be applied to identify differences in the point of initiation of contractile complexes which could indicate altered enteric neural connectivity.
A similar video imaging protocol designed to assess pellet propulsion in guinea pigs has been reported13 however here we outline the application of the video imaging approach for quantification of spontaneous colonic motility (i.e., in the absence of pellets). We also provide detailed information to assist in the dissection and preparation of gastrointestinal tissue for the video imaging approach. This protocol provides researchers with an accessible and easily replicated tool for analyzing enteric neural control of gastrointestinal function in animal models of disease including genetic mouse models.
The video imaging technique enables the analysis of colonic motility in response to various pharmacological agents. Drugs can be administered via the gut lumen or the organ bath external to the colonic preparation. Different regions of the mouse gastrointestinal tract exhibit specific motility patterns such as small intestinal segmentation and CMMCs in the colon.
This technique has been used to identify strain differences in small intestinal function; differential sensitivity to 5-HT3 and 5-HT4 antagonists were observed in the jejunum of Balb/c and C57/Bl6 mice due to the polymorphic nature of the tph2 gene expressed in the two strains6. The effect of 5-HT inhibition on motility remains controversial, as conflicting data has been reported on the importance of endogenous 5-HT on colonic peristalsis and CMMCs14,15. Alterations in motility pre- and postnatally during development7, and the effects of gene mutations on gastrointestinal motility in animal models of disease10 can also be examined by utilizing video imaging. Here we illustrate use of the method for a study of colonic motility in the NL3R451C mouse model of autism, which expresses a missense mutation in the Nlgn3 gene encoding the synaptic adhesion protein Neuroligin-316. This mutation was first identified in patients diagnosed with Autism spectrum disorder (ASD)17, which is strongly associated with GI dysfunction18-22. We investigated whether the NL3 R451C synaptic mutation affects neural outputs in the ENS using the video imaging technique. We present data characterizing CMMCs at baseline and in response to the serotonergic 5HT3/4 receptor antagonist tropisetron in the NL3R451C mouse model of autism.

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video imaging and spatiotemporal maps to analyze gastrointestinal motility in mice

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3R451C mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.

Up to 90% of patients with ASD experience an array of gastrointestinal disorders, including diarrhea and constipation18,24,25. However, the underlying causes of these gastrointestinal issues are unknown. Many mutations identified in patients with ASD are associated with synaptic proteins contributing to alterations and disturbances in synaptic transmission or function. One such mutation, in the gene coding for the cell adhesion molecule neuroligin-3 (NL3 R451C), was identified ...

Watch this Scientific Journal Video about Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice at JoVE.com

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...

video-imaging-spatiotemporal-maps-to-analyze-gastrointestinal

Research

JoVE Journal

Neuroscience

13.9K Views.  The University of Melbourne. This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

Video: Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Video-oculography in Mice

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the mouse, have a limited capacity to lock their eyes onto a target. In contrast to these target directed eye movements, compensatory ocular eye movements are easily elicited in afoveate animals1,2,3,4. Compensatory ocular movements are generated by processing vestibular and optokinetic information into a command signal that will drive the eye muscles. The processing of the vestibular and optokinetic information can be investigated separately and together, allowing the specification of a deficit in the oculomotor system. The oculomotor system can be tested by evoking an optokinetic reflex (OKR), vestibulo-ocular reflex (VOR) or a visually-enhanced vestibulo-ocular reflex (VVOR). The OKR is a reflex movement that compensates for "full-field" image movements on the retina, whereas the VOR is a reflex eye movement that compensates head movements. The VVOR is a reflex eye movement that uses both vestibular as well as optokinetic information to make the appropriate compensation. The cerebellum monitors and is able to adjust these compensatory eye movements. Therefore, oculography is a very powerful tool to investigate brain-behavior relationship under normal as well as under pathological conditions (f.e. of vestibular, ocular and/or cerebellar origin).
Testing the oculomotor system, as a behavioral paradigm, is interesting for several reasons. First, the oculomotor system is a well understood neural system5. Second, the oculomotor system is relative simple6; the amount of possible eye movement is limited by its ball-in-socket architecture ("single joint") and the three pairs of extra-ocular muscles7. Third, the behavioral output and sensory input can easily be measured, which makes this a highly accessible system for quantitative analysis8. Many behavioral tests lack this high level of quantitative power. And finally, both performance as well as plasticity of the oculomotor system can be tested, allowing research on learning and memory processes9.
Genetically modified mice are nowadays widely available and they form an important source for the exploration of brain functions at various levels10. In addition, they can be used as models to mimic human diseases. Applying oculography on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors under normal and pathological conditions. Here, we describe how to measure video-oculography in mice8.

Video-oculography is a very quantitative method to investigate ocular motor performance as well as motor learning. Here, we describe how to measure video-oculography in mice. Applying this technique on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors.

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 &#181;m)3 can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl2 leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn2+ transport completely arrests at the lesion site. Conversely, active Mn2+ transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn2+ transport kinetics along the visual path in a transgenic mouse model (NF-&#954;B p50KO) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn2+ transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-&#954;B mutations.
In summary, MEMRI conveniently bridges in vivo assays and post mortem histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

This video illustrates a method, using a clinical 3 T scanner, for contrast-enhanced MR imaging of the na&#239;ve mouse visual projection and for repetitive and longitudinal in vivo studies of optic nerve degeneration associated with acute optic nerve crush injury and chronic optic nerve degeneration in knock-out mice (p50KO).

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice

Hypoxia ischemia is the most common cause of neonatal seizures. Animal models are crucial for understanding the mechanisms and physiology underlying neonatal seizures and hypoxia ischemia. This manuscript describes a method for continuous video electroencephalogram (EEG) monitoring in neonatal mice to detect seizures and analyze EEG background during hypoxia ischemia. Use of video and EEG in conjunction allows description of seizure semiology and confirmation of seizures. This method also allows analysis of power spectrograms and EEG background pattern trends over the experimental time period. In this hypoxia ischemia model, the method allows EEG recording prior to injury to obtain a normative baseline and during injury and recovery. Total monitoring time is limited by the inability to separate pups from the mother for longer than four hours. Although, we have used a model of hypoxic-ischemic seizures in this manuscript, this method for neonatal video EEG monitoring could be applied to diverse disease and seizure models in rodents.

This manuscript describes a method for continuous video EEG recordings using multiple depth electrodes in neonatal mice undergoing hypoxia-ischemia.

Hypoxia ischemia is the most common cause of neonatal seizures. Animal models are crucial for understanding the mechanisms and physiology underlying ...

Whole-Brain 3D Activation and Functional Connectivity Mapping in Mice using Transcranial Functional Ultrasound Imaging

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ultrafast doppler angiography. As brain perfusion is strongly linked to local neuronal activity, this technique allows the whole-brain 3D mapping of task-induced regional activation as well as resting-state functional connectivity, non-invasively, with unmatched spatio-temporal resolution and operational simplicity. In comparison with fMRI (functional magnetic resonance imaging), a main advantage of fUS imaging consists in enabling a complete compatibility with awake and behaving animal experiments. Moreover, fMRI brain mapping in mice, the most used preclinical model in Neuroscience, remains technically challenging due to the small size of the brain and the difficulty to maintain stable physiological conditions. Here we present a simple, reliable and robust protocol for whole-brain fUS imaging in anesthetized and awake mice using an off-the-shelf commercial fUS system with a motorized linear transducer, yielding significant cortical activation following sensory stimulation as well as reproducible 3D functional connectivity pattern for network identification.

This protocol describes the quantification of volumetric cerebral hemodynamic variations in the mouse brain using functional ultrasound (fUS). Procedures for 3D functional activation map following sensory stimulation as well as resting-state functional connectivity are provided as illustrative examples, in anesthetized and awake mice.

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ...