The authors acknowledge the funding of National Natural Science Fund of China (11572202, 11772201, and 31700815).

Prior to tests, the participants signed an informed consent after receiving information about the experimental process. This experiment has been approved by the ethics committee of Shanghai University of Sports.
1. Equipment setup
<ol>
	<li>Turn on the CDP system, complete self-calibration, and ensure that the instrument operates normally at 100 Hz sampling frequency. 
	NOTE: Each of the two installed independent force plates measures three forces (Fx, Fy, and Fz) and three moments (Mx, My, and Mz). The x-axis is in the left&#8211;right direction and is perpendicular to the sagittal plane. The y-axis is in the forward&#8211;backward direction and is perpendicular to the coronal plane. The z-axis is perpendicular to the horizontal plane. The origins are located at the centers of the force plates.</li>
	<li>Double-click Balance Manager System | Clinical Module, and then click New Patient and establish the patient ID. Input an accurate height, weight, and age. Select Sensory Organization Test, Unilateral Stance, Limits of Stability, Motor Control Test, and Adaption Test. 
	NOTE: Such demographic data are also used for age-matched normative diagnostic analysis.</li>
	<li>Turn on the surface electromyography (sEMG) system, and double click the EMG Motion Tools icon. Specify the trigger signal as Trigger In (Manual Stop), establish the participant ID, and match the measured muscles with the wireless electrode. The muscles of unstable lower limb are vastus medialis (VM), vastus lateralis (VL), biceps femoris (BF), tibialis anterior (TA), peroneal longus (PL), gastrocnemius medialis (GM), and gastrocnemius lateralis (GL). 
	NOTE: The phrase Trigger In (Manual Stop) indicates that CDP triggers the sEMG system to capture EMG data during tests, but the &#34;end&#34; flag requires manual clicking to stop the acquisition.</li>
	<li>Connect sEMG system with CDP system through the synchronization line. Adjust the camera of sEMG system to capture the signal indicator light of the CDP system. 
	NOTE: The video of the indicator light is collected synchronously with the CDP system and sEMG to cut the corresponding cycle of the EMG in accordance with the CDP tests. &#8220;Light on&#8221; indicates that the test is in progress, and &#8220;light off&#8221; indicates that the test is paused/stopped.</li>
</ol>
2. Participant selection and preparation
<ol>
	<li>Use the following inclusion criteria for CAI participants: (1) 35 male participants with regular daily activity, excluding professional athletes or sedentary participants; (2) 20&#8211;29 years old; (3) history of at least one significant ankle sprain, and the initial sprain must have occurred at least 12 months before enrollment in the study; (4) feelings of &#8220;giving away&#8221; of the injured ankle joint and/or recurrent sprain and/or &#8220;feeling of instability;&#8221; and (5) a Cumberland Ankle Instability Tool questionnaire score of less than 24 points16.
	<ol>
		<li>Exclude participants with a history of bilateral sprains, lower limb fracture, operation, nervous and vestibular system diseases, or allergy to taping. Additionally, recruit 35 male participants without CAI, whose demographic data matched with the CAI group, as the control group.</li>
	</ol>
	</li>
	<li>For preparation, fix the electrode piece on the belly of the measured muscles. Instruct the participants to wear a safety harness and stand barefoot on the force plates to face the visual surround.
	<ol>
		<li>Adjust the alignment of the feet on the force plates. Align the malleolus medialis with the horizontal line and lateral edge of foot with the corresponding computer-generated height line (S, M, and T lines). Turn off the screen embedded in the visual surround (Figure 1). 
		NOTE: These guidelines are based on the following heights. &#8220;S&#8221; means &#8220;small&#8221; and includes heights ranging from 76 cm to 140 cm. &#8220;M&#8221; means &#8220;medium&#8221; and includes heights ranging from 141 cm to 165 cm. &#8220;T&#8221; means &#8220;tall&#8221; and includes heights ranging from 166 cm to 203 cm. The screen may produce learning effects, because it can provide real-time visual feedback. Thus, the screen should remain closed during the test, except during the limit of stability (LOS) test17.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61592/61592fig01.jpg" /> 
Figure 1: Participant preparation for measurement.&#160;The participants stand upright barefoot to face the visual surround, wear safety harness, correctly align their feet with the force plates, and fix the wireless EMG electrodes on their legs. <a href="https://www.jove.com/files/ftp_upload/61592/61592fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Measurement procedures
<ol>
	<li>CDP measurement
	<ol>
		<li>Sensory organization test
		<ol>
			<li>Instruct the participants to stand upright and to keep their COG as stable as possible to cope with the interference from vision, somatosensory, and vestibular sensation (singly or combined) (Table 1). Complete the measurements of conditions 1&#8211;6. Each test lasts for 20 s. Repeat the procedure thrice for each condition.</li>
		</ol>
		</li>
		<li>Unilateral stance
		<ol>
			<li>Instruct the participants to place their hands on the anterior superior iliac spine with their eyes open/closed. Consider the unstable ankle side as the support leg. Fully extend their knee joint, and bend the knee of their non-supporting leg by approximately 30&#176;. Allow the participants to remain standing stably for 10 s. Repeat the procedure thrice for each visual condition.</li>
		</ol>
		</li>
		<li>Limit of stability
		<ol>
			<li>Instruct the participants to maintain their COG in the central area. Upon hearing the ring, lean their body and shift their COG quickly into the targeted frame in the screen. Instruct the participants to remain steady for 10 s. Complete the eight directional shifting of their COG (forward, forward-right, right, right-backward, backward, backward-left, left, and left-forward). 
			NOTE: In the process of COG shifting, the body is kept straight, the heel or toes are not far from the force plates, and the hip joint is not bent.</li>
		</ol>
		</li>
		<li>Motor control test
		<ol>
			<li>Instruct the participants to respond effectively to restore body stability and to cope with the unexpected slipping of the force plates. Repeat the procedure thrice for each slip condition. 
			NOTE: The force plates are slipped with small/medium/large amplitude in the anterior/posterior direction. According to the participant&#8217;s height, the slip amplitude of the force plates is automatically adjusted. Standard procedures must be followed to align the foot position on the force plates. Random delay exists between trials.</li>
		</ol>
		</li>
		<li>Adaption test
		<ol>
			<li>Instruct the participants to respond effectively to restore body stability and to cope with five consecutive unexpected rotations at a velocity of 20&#176;/s. Direct the toes upward or downward.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Condition</td>
			<td>Eyes</td>
			<td>Force plates</td>
			<td>Visual surround</td>
			<td>Interference</td>
			<td>Anticipated Response</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Open</td>
			<td>Fix</td>
			<td>Fix</td>
			<td></td>
			<td>Somatosensory</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Close</td>
			<td>Fix</td>
			<td>Fix</td>
			<td>Vision</td>
			<td>Somatosensory</td>
		</tr>
		<tr>
			<td>3</td>
			<td>Open</td>
			<td>Fix</td>
			<td>Sway-reference</td>
			<td>Vision</td>
			<td>Somatosensory</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Open</td>
			<td>Sway-reference</td>
			<td>Fix</td>
			<td>Somatosensory</td>
			<td>Vision, vestibular</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Close</td>
			<td>Sway-reference</td>
			<td>Fix</td>
			<td>Somatosensory, vision</td>
			<td>Vestibular</td>
		</tr>
		<tr>
			<td>6</td>
			<td>Open</td>
			<td>Sway-reference</td>
			<td>Sway-reference</td>
			<td>Somatosensory, vision</td>
			<td>Vestibular</td>
		</tr>
	</tbody>
</table>
Table 1: Different interference and corresponding anticipated response in sensory organization test. The term &#8220;sway-referenced&#8221; means that the movement of the force plates and visual surround follows the participant&#8217;s COG sway.
<ol start="2">
	<li>sEMG measurement and data process
	<ol>
		<li>After triggering by CDP system during SOT, US, LOS, MCT and ADT, start the automatic acquisition of lower-limb muscle activity raw data. Manually stop the acquisition during the sEMG system when the light is off. The sample size is 1000 Hz.</li>
		<li>Enter the processing window of the sEMG software. Import the C3d file of the EMG raw data and mp4 file of the light video. Cut the trial cycle when the light is on.</li>
		<li>In the &#8220;processing pipeline&#8221; operations, include the following options in the run pipeline: Butterworth filter with low-pass (450 Hz, 2. Order) and high-pass (20 Hz, 2. Order); notch filter at 50 Hz; and root mean square smoothing window of 100 ms. 
		NOTE: Choose the Butterworth filter with low-pass (450 Hz, 2. Order) and high-pass (20 Hz, 2. Order) to filter out unwanted low and high-frequency components. Set the notch filter at 50 Hz to remove 50 Hz interference from the main power. Use the root mean square smoothing window of 100 ms to smooth the noisy signal.</li>
		<li>In the Generate Events options, include the following events in the run pipeline. &#8220;muscle on&#8221; is defined as &#8220;all channels go above 5x baseline noise standard deviations for at least 50 ms&#8221;. &#8220;muscle off&#8221; is defined as &#8220;all channels drop below 5x standard deviations over baseline for at least 50 ms &#8221;.</li>
		<li>In the Generate Parameters options, include the following parameters in the run pipeline: integral electromyography (iEMG); root mean square (RMS); mean power frequency (MPF); medium frequency (MDF); and co-activation ratio. 
		NOTE: The following are the referenced calculation formulas for the above parameters (Equations 1&#8211;4): 
		<img alt="Equation 1" src="/files/ftp_upload/61592/61592equ01v2.jpg" /> 
		<img alt="Equation 2" src="/files/ftp_upload/61592/61592equ02v2.jpg" /> 
		<img alt="Equation 3" src="/files/ftp_upload/61592/61592equ03v2.jpg" /> 
		<img alt="Equation 4" src="/files/ftp_upload/61592/61592equ04v2.jpg" /></li>
		<li>Normalize the RMS values of the SOT, US, LOS, MCT and ADT trials with the RMS values of maximal voluntary isometric contraction (MVIC) for each muscle (Equation 5). 
		<img alt="Equation 5" src="/files/ftp_upload/61592/61592eq5v3.png" /> 
		NOTE: MVIC indicates the maximum force contraction of each muscle for participants in the standard posture for 5 s (Supplementary file 1)18.</li>
	</ol>
	</li>
</ol>

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A., Hertel, J., Plisky, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+Star+Excursion+Balance+Test+to+assess+dynamic+postural-control+deficits+and+outcomes+in+lower+extremity+injury:+a+literature+and+systematic+review.">Using the Star Excursion Balance Test to assess dynamic postural-control deficits and outcomes in lower extremity injury: a literature and systematic review.</a> Journal of Athletic Training. 47 (3), 339-357 (2012).</li></ol>

The authors have nothing to disclose.

The presented protocol is used to measure dynamic postural control and related muscle activity in individuals with CAI by synchronizing CDP with sEMG. CDP traces the trajectory of the COP and COG and provides insight into the interaction between sensory information (visual, somatosensory, and vestibular sensation) input and the external environment8,21,22. It is an effective tool for the diagnosis of the functional activity limi...

Computerized dynamic posturography (CDP) is an objective technique for the evaluation of postural stability under static and dynamic conditions and perturbation. CDP is based on the inverted pendulum model that traces the interrelationship between the center of pressure (COP) and the center of gravity (COG). COG is the vertical projection of the center of mass (COM), whereas COM is the point equivalent of the total body mass in the global reference system. COP is the point location of the vertical ground reaction force vector. It represents a weighted average of all the pressures over the surface of the contact area with the ground1. Postural stability is the ability to maintain the COM within the base of support in a given sensory environment. It reflects neuromuscular control ability that coordinates the central nervous system with the afferent sensory system (vision, proprioception, and vestibular sensation) and motor command output2.
Previous evaluation methods for postural control, such as the time for a single-leg stance and the reach distance for Y-balance tests, are results-oriented and cannot be used to objectively evaluate the coordination between sensory systems and motor control3. In addition, some studies used portable computerized wobble board, which quantified dynamic balance performances out of laboratory settings4,5,6. CDP differs from the abovementioned test methods, because it can be applied to the analysis of the proportion of vision, proprioception, and vestibular sensation in postural stability maintenance and to the evaluation of the proportion of motor strategy, such as ankle or hip dominant strategy. It has been viewed as a gold standard for postural control measurement7 because of its accuracy, reliability, and validity8.
Chronic ankle instability (CAI) is characterized by persistent ankle pain, swelling, and feeling of &#8220;giving way&#8221;; it is one of the most common sports injuries9. CAI originates mostly from lateral ankle sprains, which destroy the integrity and stability of the lateral ankle ligament complex. The proprioception, fibular muscle strength, and normal trajectory of talus are impaired10,11. The deficiencies of the weak ankle segment can result in deficient postural control and muscle activation in individuals with CAI12. However, few studies have investigated the postural stability of individuals with CAI by using CDP3,13. Current measurements could rarely analyze the posture control deficiency of CAI from the perspective of sensory analysis. Therefore, the ability of sensory organization and postural strategy of CAI to maintain postural stability needs further exploration.
Muscle activity is an important component of neuromuscular control that affects the regulation of postural stability14,15. However, CDP only monitors the interrelationship between COP and COG through force plates, and its application to the observation of the specific activation level of lower limb muscles in individuals with CAI is difficult. Currently, few studies have evaluated the postural stability of individuals with CAI through a method that combines CDP with electromyography (EMG).
Therefore, the developed protocol aims to explore postural control and related muscle activity by combining CDP and surface electromyography system (sEMG). This protocol provides a novel approach to investigate neuromuscular control, including sensory organization, postural control, and related muscle activity, for participants with CAI.

<table>
	<tbody>
		<tr>
			<td>NeuroCom Balance Manager SMART EquiTest</td>
			<td>Natus Medical Incorporated, USA</td>
			<td></td>
			<td>Its major components include: NeuroCom Balance Manager Software Suite, dynamic dual force plate (rotate &#38; translate), moveable visual surround with 15&#8221; LCD display (it could provide a real time display of the subject&#8217;s center of gravity shown as a cursor during the task) and illumination, overhead support bar with patient harness, computer and other parts.</td>
		</tr>
		<tr>
			<td>wireless Myon 320 sEMG system</td>
			<td>Myon AG</td>
			<td></td>
			<td>The system consists of 16 parallel channels of transmitter signals, receiver, &#34;EMG motion Tools&#34; and &#34;ProEMG&#34; software,computer and other parts.</td>
		</tr>
	</tbody>
</table>

evaluating postural control and lower-extremity muscle activation in individuals with chronic ankle instability

Computerized dynamic posturography (CDP) is an objective technique for the evaluation of postural stability under static and dynamic conditions and perturbation. CDP is based on the inverted pendulum model that traces the interrelationship between the center of pressure and the center of gravity. CDP can be used to analyze the proportions of vision, proprioception, and vestibular sensation to maintain postural stability. The following characters define chronic ankle instability (CAI): persistent ankle pain, swelling, the feeling of &#8220;giving way,&#8221; and self-reported disability. Postural stability and fibular muscle activation level in individuals with CAI decreased due to lateral ankle ligament complex injuries. Few studies have used CDP to explore the postural stability of individuals with CAI. Studies that investigate postural stability and related muscle activation by using synchronized CDP with surface electromyography are lacking. This CDP protocol includes a sensory organization test (SOT), a motor control test (MCT), and an adaption test (ADT), as well as tests that measure unilateral stance (US) and limit of stability (LOS). The surface electromyography system is synchronized with CDP to collect data on lower limb muscle activation during measurement. This protocol presents a novel approach for evaluating the coordination of the visual, somatosensory, and vestibular systems and related muscle activation to maintain postural stability. Moreover, it provides new insights into the neuromuscular control of individuals with CAI when coping with real complex environments.

Representative CDP Results 
Sensory organization test 
The system evaluates the participant&#8217;s ability to maintain COG in the predetermined target area, when the environment changes as the peripheral signal input. Equilibrium score (ES) is the score under conditions 1&#8211;6 that reflects the ability to coordinate the sensory system to maintain postural stability (Equation 6). The composite score (COMP) is the weighted average score of al...

Watch this Scientific Journal Video about Evaluating Postural Control and Lower-extremity Muscle Activation in Individuals with Chronic Ankle Instability at JoVE.com

Evaluating Postural Control and Lower-extremity Muscle Activation in Individuals with Chronic Ankle Instability

Individuals with chronic ankle instability (CAI) exhibit postural control deficiency and delayed muscle activation of lower extremities. Computerized dynamic posturography combined with surface electromyography provides insights into the coordination of the visual, somatosensory, and vestibular systems with muscle activation regulation to maintain postural stability in individuals with CAI.

Computerized dynamic posturography (CDP) is an objective technique for the evaluation of postural stability under static and dynamic conditions and ...

evaluating-postural-control-lower-extremity-muscle-activation

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Neuroscience

8.2K Views.  Tongji University. Individuals with chronic ankle instability (CAI) exhibit postural control deficiency and delayed muscle activation of lower extremities. Computerized dynamic posturography combined with surface electromyography provides insights into the coordination of the visual, somatosensory, and vestibular systems with muscle activation regulation to maintain postural stability in individuals with CAI.

Video: Evaluating Postural Control and Lower-extremity Muscle Activation in Individuals with Chronic Ankle Instability

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain1. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue1-3. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates4-6.

Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons. 

Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology7.By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo7-10. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using F&ouml;rster Resonance Energy Transfer (FRET) in transfected cultured cells11.More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged &alpha;4 nAChR subunits (&alpha;4YFP), enabling precise quantification of the receptor ex vivo at submicrometer resolution in CNS neurons via spectral confocal microscopy12. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS.

In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from &alpha;4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue.We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain &alpha;4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of &alpha;4YFP receptors in the medial perforant path of the hippocampus.

We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.

Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons ...

Automated Gait Analysis in Mice with Chronic Constriction Injury

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some disadvantages such as subjective data and the requirement of a skilled, experienced experimenter. To date, a variety of modifications have improved the von Frey method, but it still has a few limitations. Recent reports have suggested that gait analysis produces more accurate and objective data from the neuropathic animals. This protocol demonstrates how to perform the automated gait analysis to determine the degree of neuropathic pain in mice. After several days of acclimation, the mice were allowed to walk freely on the glass floor to illuminate footprints. Then, quantification of the footprints and gait were performed through video clips with automatic analysis of various walking parameters, such as area of paw print, swing time, angle of paw, etc.
The main purpose of this study is to describe the methodology of automated gait analysis and briefly compare it with data from the classical sensory test using von Frey filament.

The precise assessment of pain response in a neuropathic animal model is critical to investigate the pathophysiology of pain diseases and develop new analgesics. We present a sensitive and objective method to determine the sensory function of the rodent hind paw by an automated gait analysis system.

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Functional MRI in Conjunction with a Novel MRI-compatible Hand-induced Robotic Device to Evaluate Rehabilitation of Individuals Recovering from Hand Grip Deficits

Functional magnetic resonance imaging (fMRI) is a non-invasive magnetic resonance imaging technique that images brain activation in vivo, using endogenous deoxyhemoglobin as an endogenous contrast agent to detect changes in blood-level-dependent oxygenation (BOLD effect). We combined fMRI with a novel robotic device (MR-compatible hand-induced robotic device [MR_CHIROD]) so that a person in the scanner can execute a controlled motor task, hand-squeezing, which is a very important hand movement to study in neurological motor disease. We employed parallel imaging (generalized auto-calibrating partially parallel acquisitions [GRAPPA]), which allowed higher spatial resolution resulting in increased sensitivity to BOLD. The combination of fMRI with the hand-induced robotic device allowed precise control and monitoring of the task that was executed while a participant was in the scanner; this may prove to be of utility in rehabilitation of hand motor function in patients recovering from neurological deficits (e.g., stroke). Here we outline the protocol for using the current prototype of the MR_CHIROD during an fMRI scan.

We performed functional MRI using a novel MRI-compatible hand-induced robotic device to evaluate its utility for monitoring hand motor function in individuals recovering from neurological deficits.

Functional magnetic resonance imaging (fMRI) is a non-invasive magnetic resonance imaging technique that images brain activation in vivo, using endogenous ...

Comprehensive Autopsy Program for Individuals with Multiple Sclerosis

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 days a year. Participants consent to donate their brain and spinal cord. Most patients were followed by neurologists at the Cleveland Clinic Mellen Center for MS Treatment and Research. Their clinical courses and neurological disabilities are well-characterized. Soon after death, the body is transported to the MS Imaging Center, where the brain is scanned in situ by 3 T magnetic resonance imaging (MRI). The body is then transferred to the autopsy room, where the brain and spinal cord are removed. The brain is divided into two hemispheres. One hemisphere is immediately placed in a slicing box and alternate 1 cm-thick slices are either fixed in 4% paraformaldehyde for two days or rapidly frozen in dry ice and 2-methylbutane. The short-fixed brain slices are stored in a cryopreservation solution and used for histological analyses and immunocytochemical detection of sensitive antigens. Frozen slices are stored at -80 &#176;C and used for molecular, immunocytochemical, and in situ hybridization/RNA scope studies. The other hemisphere is placed in 4% paraformaldehyde for several months, placed in the slicing box, re-scanned in the 3 T magnetic resonance (MR) scanner and sliced into centimeter-thick slices. Postmortem in situ MR images (MRIs) are co-registered with 1 cm-thick brain slices to facilitate MRI-pathology correlations. All brain slices are photographed and brain white-matter lesions are identified. The spinal cord is cut into 2 cm segments. Alternate segments are fixed in 4% paraformaldehyde or rapidly frozen. The rapid procurement of postmortem MS tissues allows pathological and molecular analyses of MS brains and spinal cords and pathological correlations of brain MRI abnormalities. The quality of these rapidly-processed postmortem tissues (usually within 6 h of death) is of great value to MS research and has resulted in many high-impact discoveries.

Multiple sclerosis is an inflammatory demyelinating disease with no cure. Analysis of brain tissue provides important clues to understanding the pathogenesis of the disease. Here we discuss the methodology and downstream analysis of MS brain tissue collected through a unique rapid autopsy program in operation at the Cleveland Clinic.

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 ...