Name: video imaging and spatiotemporal maps to analyze gastrointestinal motility in mice
Uploaded: 2016-02-03T15:00:00
Description: Watch this Scientific Journal Video about Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice at JoVE.com

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 weigh...

<ol>
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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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			<td>Reagents</td>
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			<td>NaCl (MW: 58.44)</td>
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			<td>S7653-250G</td>
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			<td>KCl (MW: 74.55)</td>
			<td>Sigma-Aldrich</td>
			<td>P9333-500G</td>
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			<td>NaH2PO4.2H2O (MW: 156.01)</td>
			<td>Chem Supply</td>
			<td>471-500G</td>
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			<td>MgSO4.7H20 (MW: 246.48)</td>
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			<td>MA048</td>
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			<td>CaCl2.2H2O (MW: 147.02)</td>
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			<td>CA033</td>
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			<td>D-Glucose anhydrous (MW: 180.16)</td>
			<td>Chem Supply</td>
			<td>GA018-500G</td>
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			<td>NaHCO3 (MW: 84.01)</td>
			<td>Chem Supply</td>
			<td>GA018-500G</td>
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			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
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		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Two chambered organ bath 
			Dimentions: 14 cm x 8 cm x 3 cm</td>
			<td>Custom Made</td>
			<td></td>
			<td>Contact Laboratory Directly&#160;</td>
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		<tr>
			<td>&#160;732 MULTI -PURPOSE SEALANT CLEAR</td>
			<td>Dow Corning Australia Pty Ltd</td>
			<td>1890573</td>
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			<td>SYLGARD 184 SILICONE ELASTOMER KIT&#160;</td>
			<td>Dow Corning Australia Pty Ltd</td>
			<td>1064291</td>
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			<td>STOPCOCK 3 WAY FEM-ML L/LOCK S</td>
			<td>Terumo Medical Corporation</td>
			<td>0912-2006</td>
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			<td>SYRINGES with Luer Lock Tips 50mL, 20 mL, 10 mL</td>
			<td>Terumo Medical Corporation</td>
			<td>N/A</td>
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			<td>1.57 mm (ID) x 3.16 mm (OD) - Silastic Tubing</td>
			<td>Masterflex</td>
			<td>508-008</td>
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		<tr>
			<td>1.02 mm (ID) x 2.16 mm (OD) - Silastic Tubing</td>
			<td>Masterflex</td>
			<td>508-005</td>
			<td></td>
		</tr>
		<tr>
			<td>1.50 mm (ID) x 2.50 mm (OD) - Silastic Tubing</td>
			<td>Masterflex</td>
			<td>508-007</td>
			<td></td>
		</tr>
		<tr>
			<td>1.60 mm (ID) - Platinum cured silicone tubing&#160;</td>
			<td>Masterflex</td>
			<td>96410 - 14</td>
			<td></td>
		</tr>
		<tr>
			<td>4.40 mm (ID) - Platinum cured silicone tubing&#160;</td>
			<td>Masterflex</td>
			<td>96410 - 15&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>3.10 mm (ID) - Platinum cured silicone tubing&#160;</td>
			<td>Masterflex</td>
			<td>96410 -16</td>
			<td></td>
		</tr>
		<tr>
			<td>Graduated Laboratory Glass Bottles - 500 ml&#160;&#160;&#160;&#160;&#160;</td>
			<td>Thermofisher Scientific&#160;</td>
			<td>100-400</td>
			<td></td>
		</tr>
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			<td>CHEMICAL RUBBER STOPPER 57 x 65mm&#160;</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>CHEMICAL RUBBER STOPPER 29 x 32mm</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Water heater&#160; (thermo regulator)&#160;</td>
			<td>Ratek&#160;</td>
			<td>TH7000&#160;</td>
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			<td>Logitech Webcam</td>
			<td>Logitech</td>
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			<td></td>
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		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
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			<td>Software</td>
			<td></td>
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			<td></td>
		</tr>
		<tr>
			<td>Virtual Dub - 1.9 11</td>
			<td>virtualdub.org</td>
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			<td></td>
		</tr>
		<tr>
			<td>MATLAB R2012a&#160;</td>
			<td>Graph Pad</td>
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			<td></td>
		</tr>
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			<td>Logitech Webcam Software</td>
			<td>Logitech</td>
			<td></td>
			<td></td>
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</table>

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 ...

The overall goal of this technique is to identify changes in the neural regulation of colonic motility in mice using video imaging. This technique can help answer key questions related to the enteric neuroscience field, such as do subtle genetic mutations in neuronal genes affect colonic motility. The main advantage of this technique is that the tissue is a cyst in the absence of central nervous system input, and as a result the method allows investigation of the enteric nervous system's involvement in the control of gastrointestinal motility.This technique extends towards the therapy of neurodevelopmental diseases such as autism, as gene mutations altering synaptic function in the brain, might also alter gut motility. Begin by using dissecting forceps and scissors to make an incision through the epidermis overlying the lower abdominal muscle layers. Open the abdominal cavity along the midline to the sternum.Moisten the tissue with physiological saline and use the forceps to gently lift the cecum four to five centimeters above the abdomen. Then, using fine dissection scissors, trim the mesentery, taking care not to handle, stretch, or cut the gastrointestinal tissue. Remove the bladder, testes, and any other excess tissue.Then, using coarse dissecting scissors, make two vertical incisions approximately 0.5 centimeters from the midline into the pelvic bone on each side of the colon, and use the fine scissors to separate the rectum from the adjoining pelvic tissue. Trim the mesentery from the colon and rinse the full-length colon tissue in a beaker containing physiological saline. To remove the fecal pellets and other intestinal contents, first trim approximately one centimeter from the end of a 200 microliter pipette and attach the pipette to a five milliliter syringe filled with physiological saline.Then, after removing the cecum and the rectum, place an insect pin in the most proximal end of the colon where the striations in the mucosa are visible to orient the tissue. Now, gently grip the tissue at the proximal end, and insert the pipette tip into the lumen and flush the saline through the colon into a waste container. Next, with the same syringe and pipette tip, flush any debris from the tubing attached to a two-chambered organ bath with fresh physiological saline.Set the organ bath so that the chambers will be continuously superfused with physiological saline bubbled with carbogen and use a temperature probe to regularly confirm that the saline temperature remains between 33 and 37 degrees Celsius. Use a rubber stopper with a five millimeter inside diameter glass tube inserted through the center to maintain the pressure in the inflow reservoir. Then, fully submerge a five to seven centimeter piece of colon into the organ bath chamber and use forceps and standard cotton sewing thread to cannulate the proximal end of the colon to the inlet tube and the distal end of the colon to the outlet tube.After determining the baseline distance between the proximal and distal cannulations of the colon, equilibrate the tissue in physiological saline for 30 minutes. To record the intestinal movements, while the colon is acclimatizing, position a video camera 10 to 15 centimeters above the organ bath on a standard laboratory retort stand, and set up the appropriate video capture software. When the camera is ready, record four 15 minute video segments of the colon under control conditions.Then, superfuse the drug of interest into the organ bath for 30 to 60 minutes, capturing another four 15 minute video segments. Finally, replace the treatment solution with fresh physiological saline and record four more 15 minute video segments during the one-hour wash out period. Spatiotemporal maps of the colon, in response to the different treatment periods can then be generated.To assess whether synaptic mutations alter colonic migrating motor complexes, or CMMC's, when the enteric nervous system is pharmacologically perturbed, in this experiment, the five HT3 and 4 receptor antagonists, tropisetron, was added to the colon preparation organ bath chamber. In the presence of tropisetron, NL3R451C mice exhibited a decrease in CMMC frequency compared to their wild type litter mates. Although no significant differences were observed between the wild type and NL3R451C contractility during controlled conditions, tropisetron significantly reduced the CMMC frequency in both wild type and NL3R451C mice.Moreover, tropisetron imposed a larger effect on the frequency of CMMC's in NL3R451C mice compared to wild type animals. Once mastered, this technique can be completed in four hours if it's performed properly. While attempting this technique, make sure that the organ bath is maintained at a constant temperature, it has a continuous supply of carbogen, and overflow of physiological saline is prevented by a vacuum.Following this procedure, other techniques such as immunocytochemistry can be performed to answer additional questions such as are there changes in neuronal proportions or cell numbers in mice exhibiting altered colonic motility? Since its development by this method has allowed a variety of studies of intestinal motility in guinea pig and rabbit, rat, and other species, and most particularly in this case in the mouse. After watching this video, you should have a good understanding of how to identify changes in the neuronal regulation of colonic motility using video imaging.

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

Research

JoVE Journal

Neuroscience

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 ...

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 ...

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 ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the &#945;-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

Dolichoectasia: Vascular Dementia and Alzheimer&amp;#39;s Disease Models

A Visual Approach for Inducing Dolichoectasia in Mice to Model Large Vessel-Mediated Cerebrovascular Dysfunction

The blood-brain (BBB) is a crucial system that regulates selective brain circulation with the periphery, as an example, allowing necessary nutrients to enter and expel excessive amino acids or toxins from the brain. To model how the BBB can be compromised in diseases like vascular dementia (VaD) or Alzheimer's disease (AD), researchers developed novel methods to model vessel dilatation. A compromised BBB in these disease states can be detrimental and result in the dysregulation of the BBB leading to untoward and pathological consequences impacting brain function. We were able to modify an existing technique that enabled us to inject directly into the Cisterna magna (CM) to induce dilatation of blood vessels using elastase, and disrupt the tight junctions (TJ) of the BBB. With this method, we were able to see various metrics of success over previous techniques, including consistent blood vessel dilatation, reduced mortality or improved recovery, and improving the fill/opacifying agent, a silicone rubber compound, delivery for labeling blood vessels for dilatation analysis. This modified minimally invasive method has had promising results, with a 19%-32% increase in sustained dilatation of large blood vessels in mice from 2 weeks to 3 months post-injection. This improvement contrasts with previous studies, which showed increased dilatation only at the 2 week mark. Additional data suggests sustained expansion even after 9.5 months. This increase was confirmed by comparing the diameter of blood vessels of the elastase and the vehicle-injected group. Overall, this technique is valuable for studying pathological disorders that affect the central nervous system (CNS) using animal models.

Author Spotlight: Modeling Vascular Contributions to Alzheimer&#039;s Disease in Transgenic Mice

We demonstrate chemically inducing large blood vessel dilatation in mice as a model for investigating cerebrovascular dysfunction, which can be used for vascular dementia and Alzheimer's disease modeling. We also demonstrate visualizing the vasculature by injecting silicone rubber compound and providing clear visual guidance for measuring changes in blood vessel size.

The blood-brain (BBB) is a crucial system that regulates selective brain circulation with the periphery, as an example, allowing necessary nutrients to ...

In Vivo Imaging and Data Analysis of Thyroid Hormone Action Indicator (THAI) Mice to Characterize Endocrine Disrupting Chemical Interaction

To begin, launch the software compatible with the in vivo imaging system. Log in and wait for the imaging wizard panel to fully load. Click on the Initialize button on the imaging wizard panel to initiate the cooling of the camera.Then set the temperature of the heating pad between 30 to 37 degrees Celsius to keep the animals warm. Next, gently pinch the foot pads of the anesthetized animals to assess their pedal reflex. Intraperitoneally inject 10 microliters per gram body weight of sodium luciferin into the shaved animals.Now align the animal against the camera's center as denoted by the plus symbol in the imaging instrument. Set the imaging time in the image wizard panel to three minutes for luminescence imaging and select the checkboxes for both photo and luminescence. Click on Acquire to take the first measurement.After all measurements have been recorded, convert the image scale to radiance. Now determine the optimal binning and color scale settings for the images and keep it uniform for all images. Then click on ROI tool on the tool palette panel and use the Place ROI option to select the areas of interest on the image.Lastly, click on Measure ROIs and copy the resulting data for exporting into the organizing or statistical software of choice. In vivo imaging of the THAI mice showed that areas that lacked fur, such as foot pads, tail, and nose, had higher basal signals. Luciferase signal was influenced by the thyroid hormone status.Robust signals were observed in the testicular region. The thyroid hormone action was significantly heightened in the brown adipose tissue of cold stressed THAI mice. The treatment did not impact the luciferase signals from the foot pads and tail.Imaging studies also captured the accumulation and clearance of diclazuril, an endocrine-disrupting compound, over a three-week period.