Name: dosage-adjusted resistance training in mice with a reduced risk of muscle damage
Uploaded: 2022-08-31T14:20:00
Description: Watch this Scientific Journal Video about Dosage-Adjusted Resistance Training in Mice with a Reduced Risk of Muscle Damage at JoVE.com

This study was funded by grants from the Jain Foundation Inc., R03HD091648 from NICHD, a Pilot Grant from AR3T under NIH P2CHD086843, a FRAP Award from EACPHS at Wayne State University, a Faculty Startup Package from Wayne State University, and a subcontract from 1R01AR079884-01 (Peter L. Jones PI) to JAR. This study was also funded by an American Physical Therapy Association - Michigan (APTA-MI) research grant to JMB, MEP, and JAR. The authors acknowledge Dr. Renuka Roche (Associate Professor, Eastern Michigan University, MI) for critically reading the manuscript and providing feedback. The authors acknowledge Mr. Anselm D. Motha for advice on 3D printing. The authors thank the patients with dysferlinopathies who have shared their stories on the Jain Foundation website at https://www.jain-foundation.org/patient-physician-resources/patient-stories,&#160;particularly their experiences with exercise.

The experiments described in this article were approved by the Institutional Animal Care and Use Committee (IACUC) at Wayne State University, Detroit, Michigan, USA, in accordance with the Guide for the Care and Use of Laboratory Animals (1996, published by National Academy Press, 2101 Constitution Ave. NW, Washington, DC 20055, USA). B6.A-Dysfprmd/GeneJ mice (a.k.a. BLAJ mice, males, ~1.5 years old) that model LGMD2B/R2 were used for the present study. The mice were obtained from a commercial source ...

This article presents step-by-step instructions on how to construct a device to perform a type of precision rehabilitation training called dosage-adjusted resistance training (DART). The work also describes the application of the DART device and methodology in a training study to compare muscle damage 3 days after a single bout of DART (DART group) with damage 3 days after a comparable bout of isometric training (ISOM group).
The critical steps in the protocol are the proper construction of th...

Exercise confers numerous health benefits on skeletal muscle (reviewed in Vina et al.1). Specifically, progressive resistance training (PRT), which involves performing muscle contractions against progressively greater external loads (e.g., barbells, dumbbells, cable-pulley-weight circuits), is known to help increase muscle mass and strength in both healthy individuals and patient populations (reviewed in previous publications2,3). PRT is based on the overload principle, which states that, when the muscle contracts against progressively greater external loads, it adapts by increasing its physiological cross-sectional area as well as force-producing capacity4. Existing models of PRT in rodents include ladder climbing with resistance applied to the tail, co-contraction of agonist muscles against resistance from antagonists, running with a weighted harness, a squatting exercise elicited by an electrical shock, and resisted wheel-running5,6,7,8,9,10 (reviewed in previous publications11,12). However, there are currently no research tools to perform precisely muscle-targeted, dosage-adjusted PRT in mice that closely resemble the PRT methods and devices used in human clinical research and practice12,13. This limits the ability of investigators to study the safety and effectiveness of precisely dosed PRT in basic and preclinical studies in mice.
To overcome this barrier, a PRT methodology and device are developed in this study based on the cable-pulley-weight circuit designs employed in resistance training equipment in modern gymnasiums14,15,16. This method of PRT is referred to as dosage-adjusted resistance training (DART), and the device is called the DART device. In addition to its functionality as a precision rehabilitation training tool, the DART device can also be used as a standalone instrument to objectively assess the maximum concentric contractile torque that can be generated by the tibialis anterior (TA) muscle in a mouse, similar to how the one-repetition maximum (1RM, the maximum load that can be successfully lifted/moved/pressed/squatted just one time while maintaining good form) is assessed in humans17,18. The DART device can also be coupled with a custom-built or commercial isokinetic dynamometer to measure the peak isometric tetanic force produced by the TA muscle in a mouse (comparable to maximum voluntary contraction [MVC] in humans) and then perform dosage-adjusted PRT with a resistance that is based on the peak tetanic force (e.g., 50% of the peak force).
This article describes the construction of the DART device and explains how it can be coupled with a custom-built dynamometer, which has been described in prior publications19,20,21,22, to assess contractile torque and perform DART. The study also describes how the DART device was used to compare exercise-induced muscle damage caused by a single bout of DART (4 sets of 10 concentrically biased contractions with 50% 1RM) to damage caused by a comparable bout of isometric contractions (4 sets of 10 isometric contractions) in a mouse model of limb-girdle muscular dystrophy type 2B (LGMD2B, or LGMDR2)23,24. The mouse model that was studied lacks a protein called dysferlin, which plays an important role in protecting skeletal muscle against delayed-onset muscle damage following injurious eccentric contractions22,25,26,27,28,29,30. It has also been demonstrated in dysferlin-deficient male mice that concentrically biased forced exercise is not as damaging as eccentrically biased forced exercise and that prior exposure to concentrically biased training offers protection against injury from a subsequent bout of eccentrically biased contractions22. Since the current study was conducted to test the feasibility of the present DART methodology and device in performing dosage-adjusted, concentrically biased resistance training, male dysferlin-deficient mice were chosen for the investigation to compare new data from the DART device with previous data. In future studies, female BLAJ mice will be included to study the effect of sex as a biological variable in relation to the response to DART. Mice that were ~1.5 years old were studied since they already have dystrophic changes in many muscle groups and, therefore, model the pathophysiological state in which muscles might be in patients who already have muscle weakness and wasting and are seeking rehabilitative care to maintain muscle mass and strength26.

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			<td>AnMiao Star 608 Ceramic Ball Bearing</td>
			<td>Anmiao Star (N/A)</td>
			<td>AMS127</td>
			<td>High precision, low friction wheel bearing.&#160; If make and model is not commercially available, an alternative version of a 608 low-friction wheel bearing, 8 mm bore diameter,&#160; 22 mm outside diameter, with silicon nitride ceramic balls in 420 stainless steel housing should suffice.&#160; Excess friction in the wheel bearing will adversely impact performance of the DART device and will increase overall resistance to muscle contractions.</td>
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			<td>Axio Scope.A1 microscope</td>
			<td>Carl Zeiss (Peabody, MA)</td>
			<td>Product #Axio Scope.A1</td>
			<td>Light and fluorescence microscope</td>
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			<td>B6.A-Dysfprmd/GeneJ (a.k.a. BLAJ mice)</td>
			<td>The Jackson Laboratory (Bar Harbor, ME).&#160; Special colony maintained by The Jain Foundation Inc. for collaborators who study dysferlin.</td>
			<td>Stock #012767</td>
			<td>Dysferlin deficient mice that model human limb girdle muscular dystrophy type 2B/R2.</td>
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			<td>Bipolar, transcutaneous, neuromuscular electrical stimulation (NMES) electrode</td>
			<td>Harvard Apparatus, Holliston, MA</td>
			<td>BS4 50&#8211;6824</td>
			<td>Electrode for NMES.&#160; If this electrode is not commercially available, please contact corresponding author for alternatives.</td>
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			<td>Coplin Staining Dish</td>
			<td>ThermoFisher (Waltham, MA)</td>
			<td>Catalog No. S17495</td>
			<td>Staining dish/jar for hematoxylin and eosin (H&#38;E) staining of sections</td>
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			<td>Ultimaker, Utrecht, Netherlands</td>
			<td>Ultimaker Cura 4.4.1.</td>
			<td>Slicing software to convert stereolithography files into G-CODE files</td>
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			<td>Deltaphase isothermal gel heating pad</td>
			<td>Braintree Scientific (Braintree, MA)</td>
			<td>Item #39DP</td>
			<td>Heating pad to provide thermal support to animals while under anesthesia</td>
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			<td>Eosin Y</td>
			<td>Millipore Sigma (Burlington, MA)</td>
			<td>HT110132-1L</td>
			<td>Pink cytoplasmic stain</td>
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			<td>Gorilla Super Glue</td>
			<td>The Gorilla Glue Company (Cincinnati, OH)</td>
			<td>Gorilla Super Glue Micro Precise</td>
			<td>Cyanoacrylate adhesive to bond PLA components</td>
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			<td>Hematoxylin solution, Gill No.3</td>
			<td>Millipore Sigma (Burlington, MA)</td>
			<td>GHS332-1L</td>
			<td>Dark blue stain for nuclei</td>
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			<td>HM525NX cryostat</td>
			<td>ThermoFisher (Waltham, MA)</td>
			<td>Catalog #HM525NX</td>
			<td>Cryostat to make frozen sections of muscle</td>
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			<td>Lab Wipes.&#160; Kimberly-Clark Professional Kimtech Science Kimwipes Delicate Task Wipers, 1-Ply</td>
			<td>ThermoFisher (Waltham, MA)</td>
			<td>Catalog No. 06-666.&#160; Manufacturer #34120</td>
			<td>Laboratory wipes to blot mineral oil from muscle tissue before snap freezing and for other purposes.</td>
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			<td>Labview 2014</td>
			<td>National Instruments, Austin, Texas, USA</td>
			<td>Labview 2014</td>
			<td>Software for custom-written programs/routines that operate the dynamometer and trigger the NMES stimulator.</td>
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			<td>Liquid nitrogen HDPE Dewar Flasks</td>
			<td>ThermoFisher (Waltham, MA)</td>
			<td>S34074B.&#160; Thermo Scientific 41502000/EMD</td>
			<td>Flask to hold liquid nitrogen for snap freezing muscle or other tissue</td>
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			<td>Magic depilatory cream</td>
			<td>Softsheen Carson (New York, NY)</td>
			<td>N/A</td>
			<td>Razorless hair removal cream</td>
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			<td>Metal alligator clip</td>
			<td>JINSHANGTOPK (web-based business)</td>
			<td>24Pcs 51mm Metal Alligator Clip Spring Clamps</td>
			<td>Spring clamp to hold tibial pin</td>
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			<td>Micrscope slides</td>
			<td>Globe Scientific (Mahwah, NJ)</td>
			<td>1354W. Diamond White Glass Slides</td>
			<td>Charged microscope slides</td>
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			<td>Mineral Oil</td>
			<td>ThermoFisher (Waltham, MA)</td>
			<td>BP26291</td>
			<td>Mineral oil to cryoprotect muscle tissue before snap freezing</td>
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			<td>Monoprice Premium 3D Printer Filament PLA</td>
			<td>Monoprice (Rancho Cucamonga, CA)</td>
			<td>#11778</td>
			<td>Premium 3D Printer Filament PLA 1.75mm 1 kg/spool, Gray.&#160; This is the material used to 3D print device components.</td>
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			<td>Monoprice Select Mini V2 3D printer</td>
			<td>Monoprice (Rancho Cucamonga, CA)</td>
			<td>Mini V2 3D</td>
			<td>3D printer for computer-aided fabrication of device components.</td>
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			<td>NIH Image software</td>
			<td>National Instritues of Health (NIH, Bethesda, MD)</td>
			<td>NIH Image for Windows</td>
			<td>Image processing and analysis software used to quantify area of muscle damage.&#160; NIH Image is also known as Image J.</td>
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			<td>Photoshop CS4</td>
			<td>Adobe (San Jose, CA)</td>
			<td>Creative Suite (CS4). 64 bit version for Windows</td>
			<td>Image processing and analysis software used to generate tiled/stiched images of entire muscle cross-section from images of indvidual overlapping fields</td>
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			<td>PSIU6 stimulation isolation unit</td>
			<td>Grass Instruments (West Warwick, RI)</td>
			<td>PSIU6 isolation unit</td>
			<td>Isolation unit for NMES.&#160; Stimulators, such as Model 4100 from A-M come with a built in stimulation isoloation unit</td>
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			<td>Roboz 4-0 silk black braided suture material</td>
			<td>Roboz Surgical (Gaithersburg, MD)</td>
			<td>Roboz Surgical SUT152</td>
			<td>Suture material to connect DART device footplate to dynamometer footplate or resistance for resistance training</td>
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			<td>S48 square pulse stimulator</td>
			<td>Grass Instruments (West Warwick, RI)</td>
			<td>S48 Stimulator</td>
			<td>Laboratory electrical stimulator for NMES .&#160; If this stimulator is not commercially available, Model 4100 Isolated High Power Stimulator from A-M systems could be an alternative.&#160; Please contact co-author Jones for more information.</td>
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			<td>Scott&#8217;s bluing reagent</td>
			<td>Ricca Chemical Company (Arlington, TX)</td>
			<td>6697-32</td>
			<td>Bluing solution that intensifies hematoxylin nuclear staining</td>
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			<td>SigmaStat version 3.5</td>
			<td>Systat Software (San Jose, CA)</td>
			<td>SigmaStat version 3.5</td>
			<td>Statistical software package for statistical analyses</td>
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			<td>Tabletop isoflurane vaporizer</td>
			<td>VetEquip (Livermore, CA)</td>
			<td>Item #901801</td>
			<td>Inhaled tabletop anesthesia system</td>
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			<td>Triple antibiotic first aid ointment</td>
			<td>Global Health Products (wed-based business)</td>
			<td>Globe Triple Antibiotic First Aid Ointment, 1 oz (2-Pack) First Aid Antibiotic Ointment</td>
			<td>Antibiotic ointment applied on tibial pin as part of post-procedural care</td>
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dosage-adjusted resistance training in mice with a reduced risk of muscle damage

Progressive resistance training (PRT), which involves performing muscle contractions against progressively greater external loads, can increase muscle mass and strength in healthy individuals and in patient populations. There is a need for precision rehabilitation tools to test the safety and effectiveness of PRT to maintain and/or restore muscle mass and strength in preclinical studies on small and large animal models. The PRT methodology and device described in this article can be used to perform dosage-adjusted resistance training (DART). The DART device can be used as a standalone dynamometer to objectively assess the concentric contractile torque generated by the ankle dorsiflexors in mice or can be added to a pre-existing isokinetic dynamometry system. The DART device can be fabricated with a standard 3D printer based on the instructions and open-source 3D print files provided in this work. The article also describes the workflow for a study to compare contraction-induced muscle damage caused by a single bout of DART to muscle damage caused by a comparable bout of isometric contractions (ISOM) in a mouse model of limb-girdle muscular dystrophy type 2B/R2 (BLAJ mice). The data from eight BLAJ mice (four animals for each condition) suggest that less than 10% of the tibialis anterior (TA) muscle was damaged from a single bout of DART or ISOM, with DART being less damaging than ISOM.

BLAJ male mice, which were ~1.5 years in age, were studied. BLAJ mice model the human muscle disease, LGMD2B/R2. These mice are particularly susceptible to delayed onset muscle damage from a single bout of eccentric muscle contractions22,29. BLAJ mice were, therefore, chosen for these studies to learn if DART could be performed in a non-injurious manner by precisely adjusting the resistance against which the TA muscle has to work in a concentrically biased manner...

Watch this Scientific Journal Video about Dosage-Adjusted Resistance Training in Mice with a Reduced Risk of Muscle Damage at JoVE.com

Dosage-Adjusted Resistance Training in Mice with a Reduced Risk of Muscle Damage

The present protocol describes a unique technique called dosage-adjusted resistance training (DART), which can be incorporated into precision rehabilitation studies performed in small animals, such as mice.

Progressive resistance training (PRT), which involves performing muscle contractions against progressively greater external loads, can increase muscle ...

This protocol is one of the first studies that enables scientists to perform dosage-adjusted resistance training in mice similar to how it's performed in humans. The main advantage of this technique is that it allows investigators to precisely adjust resistance against which the mouse's anterior tibial muscles have to work during resistance training. Furthermore, since muscle contractions are eccentrically biased, there is a lower chance of contraction-induced muscle injury.Our dosage-adjusted resistance training technique can be incorporated into basic and preclinical studies in mice to develop interventions for the improvement or maintenance of muscle mass and strength across a diverse array of disease conditions. The most challenging aspect of the protocol is to precisely stimulate the fibular branch of the sciatic nerve, which innervates the anterior tibial muscles. To reduce inaccurate electrical stimulation, adjust the electrode position and stimulation amplitude until maximum twitch torque is recorded by a dynamometer.To begin, provide thermal support to the mouse by using an isothermal gel heating pad and place a heat lamp 1 meter above the mouse. To prepare the skin for DART or ISOM, remove the fur from the left hind limb by applying a depilatory cream. After 2 minutes, clean the leg with wipes soaked in distilled water to remove fur and all residual cream from the skin.Disinfect the skin using a povidone iodine scrubbing solution and 70%ethanol, then use a clean cotton swab to apply a protectant over the eyes and the depilated skin to prevent drying. Next, apply 5%lidocaine cream over the tibia to numb the area. Pass a 1/2-inch 26 gauge sterile hypodermic needle through the widest part of the proximal portion of the tibial bone.Once the stabilizing pin is secured, hold the needle with a sterile hemostat and bend the plastic portion until it breaks off, then lay the mouse in a supine position. Ensure that the mouse is still securely connected to the nose cone to maintain anesthesia. With a pair of sterile tipped tweezers, feed the tibial pin into a metal alligator clip, such that the end of the tibial pin are held by the alligator clamp.Move the adjustable arm of the alligator clamp to ensure that the foot of the mouse is placed on the footplate of the DART device. Strap the foot of the mouse onto the DART device footplate with adhesive laboratory tape. Ensure that the foot is placed at a 90-degree angle in relation to the long axis of the mouse tibial bone.Place an 18 gauge 1.5=inch-long hypodermic needle through the pre-drilled holes on the protractor of the DART device to create a plantarflexion stop, then rest the footplate on the plantarflexion stop. To optimize the electrode placement, place a bipolar, transcutaneous, and NMES electrode on the inferolateral aspect of the mouse knee joint. Use a laboratory electrical stimulator to apply single pulses of 1 hertz to stimulate the fibular branch of the sciatic nerve.Observe the tibialis anterior, or TA belly muscle and tendon for evidence of electrically-elicited twitch contractions. Now, strap the suture to the dynamometer floor plate, then optimize the amplitude of the voltage output from the NMES stimulator so that NMES is confined to the common fibular nerve and TA muscle. To set the stimulator to produce repeated pulse trains, adjust the dials for pulse frequency to 125 hertz, train duration to 500 milliseconds, and trains per second to 1.Turn on the toggle switch for repeating pulse trains. Set the stimulator to produce pulse trains that are 500 milliseconds in duration interspersed with 500 millisecond rest between pulse trains. Move the plantarflexion stop to the hole on the protractor that corresponds to 160 degrees on the long axis of the tibia.In DART, for the TA muscle to work concentrically, apply resistance by hanging a suitable weight such as 5 grams with a non-elastic silk suture tied to the DART device footplate. Adjust the resistance by applying 50%weight of the 1-repetition maximum. Ensure that the foot pulls through at least half of the available active range of dorsiflexion.Perform a single bout of DART training that involves 1 set of 10 repetitions of concentric contractions and 2-minute rest between the sets. For ISOM training, place the foot of the mouse at 160 degrees to the long axis of the tibia. Maintain the static position by taping the silk suture to the footplate of the robotic dynamometer.Perform a single bout of ISOM training that involves 4 sets of 10 repetitions to isometric contractions and 2-minute rest between sets. As post-procedural care for mice, take precautions to maintain the hygiene of the exercised hind limb and reduced needle site pain. Histological changes in the TA muscle were studied after 3 days of DART or ISOM training.Hematoxylin and eosin staining indicated that the extent of muscle damage was low in both DART and ISOM groups, but muscle damage was slightly more obvious in the ISOM group. It is important to specifically stimulate contractions in the anterior tibial muscles and precisely adjust the resistance against which these muscles have to work during dosage-adjusted resistance training. Following this procedure, investigators will be able to assess exercise tolerance in the form of susceptibility to fatigue and injury from repeated muscle contractions against resistance.This technique can be incorporated in a wide variety of basic and preclinical research applications, such as studies on mouse models, of neuromuscular diseases like muscular dystrophies and mouse models of sports injuries.

dosage-adjusted-resistance-training-mice-with-reduced-risk-muscle

Research

JoVE Journal

Neuroscience

Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo pharmacological studies of the signals essential for the formation and maintenance of mammalian NMJs. However, drug penetration into these muscles after subcutaneous or intramuscular administration is often incomplete or uneven and many NMJs can remain unaffected. Although systemic administration with devices such as mini-pumps can improve the spatiotemporal effects, the invasive nature of this approach can cause confounding inflammatory responses and/or direct muscle damage. Moreover, complete analysis of the NMJs in a hind limb muscle is challenging because it requires time-consuming serial sectioning and extensive immunostaining. 
The mouse LAL is a thin, flat sheet of muscle located superficially on the dorsum of the neck. It is a fast-twitch muscle that functions to move the pinna. It contains rostral and caudal portions that originate from the midline of the cranium and extend laterally to the cartilaginous portion of each pinna. The muscle is supplied by a branch of the facial nerve that projects caudally as it exits the stylomastoid foramen. We and others have found LAL to be a convenient preparation that offers advantages for the investigation of both short and long-term in vivo effects of drugs on NMJs and muscles. First, its superficial location facilitates multiple local applications of drugs under light anesthesia. Second, its thinness (2-3 layers of muscle fibers) permits visualization and analysis of almost all the NMJs within the muscle. Third, the ease of dissecting it with its nerve intact together with the pattern of its innervation permits supplementary electrophysiological analysis in vitro9,5. Last, and perhaps most importantly, a small applied volume (&tilde;50&mu;l) easily covers the entire muscle surface, provides a uniform and prolonged exposure of all its NMJs to the drug and eliminates the need for a systemic approach1,8.

We describe procedures for repeated administration of inhibitors of muscarinic signaling to the levator auris longus (LAL) muscle of young adult mice and for subsequent immunostaining of its neuromuscular junctions (NMJs) in wholemounts. The LAL muscle has unique advantages for revealing in vivo pharmacological effects on NMJs.

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo  pharmacological studies of the signals ...

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, photoreceptor degeneration triggers M&#252;ller glial cells to re-enter the cell cycle and produce transient-amplifying progenitors. These progenitors continue to proliferate as they migrate to the damaged area, where they ultimately give rise to new photoreceptors. Currently, there are two widely-used LIRD paradigms, each of which results in varying degrees of photoreceptor loss and corresponding differences in the regeneration response. As more genetic and pharmacological tools are available to test the role of individual genes of interest during regeneration, there is a need to develop a robust LIRD paradigm. Here we describe a LIRD protocol that results in widespread and consistent loss of both rod and cone photoreceptors in which we have combined the use of two previously established LIRD techniques. Furthermore, this protocol can be extended for use in pigmented animals, which eliminates the need to maintain transgenic lines of interest on the albino background for LIRD studies.

Multiple light damage protocols have been described to damage photoreceptors and consequently induce a retinal regeneration response in adult zebrafish. This protocol describes an improved method that can be used in pigmented animals and that damages the vast majority of rod and cone photoreceptors across the entire retina.

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, ...

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons are involved in proprioceptor signaling and also report on metabolic state and injury related events. We describe an adult mouse in vitro muscle-nerve preparation for studies on stretch-activated muscle afferents.

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

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 ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain.

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and ...

Optogenetics Identification of a Neuronal Type with a Glass Optrode in Awake Mice

It is a major concern in neuroscience how different types of neurons work in neural circuits. Recent advances in optogenetics have enabled the identification of the neuronal type in in vivo electrophysiological experiments in broad brain regions. In optogenetics experiments, it is critical to deliver the light to the recording site. However, it is often hard to deliver the stimulation light to the deep brain regions from the brain&#39;s surface. Especially, it is difficult for the stimulation light to reach the deep brain regions when the optical transparency of the brain surface is low, as is often the case with recordings from awake animals. Here, we describe a method to record spike responses to the light from an awake mouse using a custom-made glass optrode. In this method, the light is delivered through the recording glass electrode so that it is possible to reliably stimulate the recorded neuron with light in the deep brain regions. This custom-made optrode system consists of accessible and inexpensive materials and is easy to assemble.

This work introduces a method to perform an optogenetic single-unit recording reliably from an awake mouse using a custom-made glass optrode.

It is a major concern in neuroscience how different types of neurons work in neural circuits. Recent advances in optogenetics have enabled the ...

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Our current goal is to determine the mechanisms underlying ABT-induced improvements in urinary, bowel, and sexual function in SCI rats after a moderate to severe level of contusion. After securing each individual animal in a custom-made adjustable vest, they are secured to a versatile body weight support mechanism, lowered to a modified three-lane treadmill and assisted in step-training for 58 minutes, once a day for 10 weeks. This setup allows for the training of both quadrupedal and forelimb-only animals, alongside two different non-trained groups. Quadrupedal-trained animals with body weight support are aided by a technician present to assist in stepping with proper hind limb placement as necessary, while forelimb-only trained animals are raised at the caudal end to ensure no hind limb contact with the treadmill and no weight-bearing. One non-trained SCI group of animals is placed in a harness and rests next to the treadmill, while the other control SCI group remains in its home cage in the training room nearby. This paradigm allows for the training of multiple SCI animals at once, thus making it more time-efficient in addition to ensuring that our pre-clinical animal model mimics the clinical representation as close as possible, particularly with respect to the body weight support with manual assistance.

This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) ...

Simulator Training for Endovascular Neurosurgery

Simulation-based training has become common practice across medical specialties, especially for learning complex skills performed in high-risk environments. In the field of endovascular neurosurgery, the demand for consequence- and risk-free learning environments led to the development of simulation devices valuable for medical trainees. The goal of this protocol is to provide instructive guidelines for the use of an endovascular neurosurgery simulator in an academic setting. The simulator provides trainees with the opportunity to receive realistic feedback on their knowledge of anatomy, as well as haptic feedback indicative of their success in handling the catheter-based systems without negative consequences. The utility of this specific protocol in relation to other neuroendovascular training modalities is also discussed.

Simulation of complex, high-risk procedures is critical to the education of medical trainees. A protocol for simulator-based endovascular neurosurgery training in a controlled academic environment is described. The protocol includes stepwise guidelines for trainees of varying levels, with a discussion of the advantages and limitations of this model.

Simulation-based training has become common practice across medical specialties, especially for learning complex skills performed in high-risk ...

Assessment of Audio-Tactile Sensory Substitution Training in Participants with Profound Deafness Using the Event-Related Potential Technique

This paper examines the application of electroencephalogram-based methods to assess the effects of audio-tactile substitution training in young, profoundly deaf (PD) participants, with the aim of analyzing the neural mechanisms associated with vibrotactile complex sound discrimination. Electrical brain activity reflects dynamic neural changes, and the temporal precision of event-related potentials (ERPs) has proven to be key in studying time-locked processes while performing behavioral tasks that involve attention and working memory. 
The current protocol was designed to study electrophysiological activity in PD subjects while they performed a continuous performance task (CPT) using complex-sound stimuli, consisting of five different animal sounds delivered through a portable stimulator system worn on the right index finger. As a repeated-measures design, electroencephalogram (EEG) recordings in standard conditions were performed before and after a brief training program (five 1 h sessions over 15 days), followed by offline artifact correction and epoch averaging, to obtain individual and grand-mean waveforms. Behavioral results show a significant improvement in discrimination and a more robust P3-like centroparietal positive waveform for the target stimuli after training. In this protocol, ERPs contribute to the further understanding of learning-related neural changes in PD subjects associated with audio-tactile discrimination of complex sounds.

This protocol is designed to explore underlying learning-related electrophysiological changes in subjects with profound deafness after a short training period in audio-tactile sensory substitution by applying the event-related potential technique.