The authors acknowledge the funding of the Breeding Program of Shanghai Tenth People&#39;s Hospital (YNCR2C022).

This study was conducted at the Sports Medicine and Rehabilitation Centre, Shanghai University of Sport, and has been approved by the ethics committee of the Shanghai University of Sport (No. 102772020RT001). Before the test, the participants were given details about the experimental purpose and procedures; all participants signed the informed consent.
1. Participant selection
<ol>
	<li>Include participants who (1) are aged over 60 years old; (2) can maintain standing position alone; (3) can walk independently, without help from others, prosthesis, or mobility aids; (4) can display normal cognitive function and can understand the procedures and instructions of the test. Exclude participants who (1) were diagnosed with severe cardiopulmonary disease; (2) diagnosed with motor neuron disorders, such as Alzheimer&#39;s disease and Parkinson&#39;s disease; and (3) had a history of lower limb trauma in the past year were excluded. 
	NOTE: To evaluate the function of the foot core system, 42 elderly participants and 42 young participants whose demographic data matched with the old group (control group) were recruited for this study. The sample size was calculated for t-test with setting of &#945; = 0.05, power (1 &#8722; &#946;) = 0.95, and effect size = 0.8. The result shows that 42 participants in each group should be included in this study.</li>
</ol>
2. Active subsystem
NOTE: The morphology and strength tests of intrinsic and extrinsic foot muscles are used to evaluate the active subsystem.
<ol>
	<li>Muscle morphology
	<ol>
		<li>Turn on the musculoskeletal ultrasound system, and then click on the Freeze button. Plug the probe connector into the connection port on the rear side of the host and lock the Probe Lock button. Click on the iStation button, and then click on New Patient. Input the ID, name, gender, and date of birth of each participant. 
		NOTE: The probe cable should be arranged properly and placed in a location where it will not be easily trampled to ensure that the cable is not entangled with the other objects. Place the probe in a safe location to avoid collision and damage.</li>
		<li>Abductor hallucis (AbH): Apply the ultrasound coupling gel at the middle of the scanning line of tuberosity and navicular tuberosity. Place the probe at the medial calcaneal tuberosity toward the navicular tuberosity. Move the probe sightly to capture the thickest part of the AbH, and then click on the Save button to save the still image.
		<ol>
			<li>Then, rotate the probe 90&#176; to obtain the cross-sectional image of the AbH and save the image. 
			NOTE: Maintain good contact between the probe and the skin without applying excessive pressure in muscle morphology measurements.</li>
		</ol>
		</li>
		<li>Flexor digitorum brevis (FDB): Align the probe longitudinally on the line from the medial tubercle of the calcaneus to the third toe and scan the muscle to measure the thickness. Rotate the probe 90&#176; to obtain the cross-sectional image.</li>
		<li>Quadratus plantae (QP): Align the probe longitudinally along the muscle fibers at the talocalcaneonavicular joint. Move the probe sightly to locate the thickest part of QP. Capture three images for thickness measurement. Rotate the probe 90&#176; to obtain cross-sectional images. 
		NOTE: QP lies deep in the FDB.</li>
		<li>Flexor hallucis brevis (FHB): Mark the first metatarsal, apply the ultrasound coupling gel, and then place the probe longitudinally along the shaft. Move the probe sightly to capture the thickest part of the FHB, and then rotate the probe 90&#176; to obtain the cross-sectional image.</li>
		<li>Peroneus longus and brevis (PER): Instruct the participants to lie in the supine position. Mark the fibular head and the inferior border of the lateral malleolus, and mark 50% of the line connecting the two points. Apply the coupling gel and place the probe to capture the thickness. To obtain the cross-sectional image, rotate the probe 90&#176; at the point where the thickness measurement was taken.</li>
		<li>Tibialis anterior (TA): Apply the coupling gel in front of the calf over 20% of the distance between the fibular head and the inferior border of the lateral malleolus. Place the probe longitudinally along the TA to obtain a thickness measurement. 
		NOTE: Due to the scanning range of the probe, the CSA of the TA cannot be captured completely.</li>
		<li>Image measurement: Look for the previously captured images on the right side of the screen. Use the trackball to move the cursor, select one image, and click on the Set button. Then, click on the Measure button. The measurement items appear on the left side of the screen.
		<ol>
			<li>Thickness: Use the trackball to move the cursor, select the distance measurement, and click on the Set button. Mark the two points of the thickest part of the muscle in the image (Figure 1 and&#160;Figure 2). Record the distance for the thickness.</li>
			<li>Cross-sectional area (CSA): Use the trackball to move the cursor to trace the periphery of the muscle in the image. After tracing the cross-section of the entire muscle, click on the Set button (Figure 1 and Figure 2). Record the area for the CSA.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Muscle strength
	<ol>
		<li>Insert dynamometer Bluetooth stick into the USB interface of the computer. Open the dynamometer and FET Data Collection Software and click on the Start Gauge button to wait for automatic pairing.</li>
		<li>Toe flexion strength test (FT1)
		<ol>
			<li>Instruct the participant to sit in a chair with 90&#176; flexion of the knee and ankle joint. Fix the dynamometer to the front side of the wooden frame. Connect the great toe to the dynamometer by carabiner (Figure 3B). 
			NOTE: Adjust the appropriate bars to avoid pain during the test.</li>
			<li>Interchange the panels behind the foot to ensure that the heel to the head of the first metatarsal is supported while still allowing for unimpaired toe flexion. Adjust the carabiner so that the toe produces a steady baseline force, and then click on the Reset button to zero out the dynamometer.</li>
			<li>Click on the Start Gauge button in the software. Instruct the participant to remain stable until instructed to flex the big toe, pull as hard as possible for 3 s, and then relax the grip. Click on the Stop Gauge button, and save the data collected.</li>
		</ol>
		</li>
		<li>Toe flexion strength test (FT2-3 and FT2-5)
		<ol>
			<li>Use the T-shaped metal bars to attach to the dynamometer. Instruct the participant to flex the 2nd-3rd toes or 2nd-5th toes. Perform a similar test procedure as the FT1 test (Figure 3C,D).</li>
		</ol>
		</li>
		<li>Doming test
		<ol>
			<li>Place the dynamometer against the scaphoid tubercle. Instruct the participant to slide the forefoot toward the heel or raise the arch as much as possible without lifting or curling the toes, which would result in &#34;shortening&#34; of the foot and a raised medial longitudinal arch (Figure 3A).</li>
			<li>Then, ask the participant to do maximum voluntary contraction for 3 s. Perform data collection like previous toe flexion tests (steps 2.2.2 and 2.2.3). 
			&#8203;NOTE: Record three successful trials for the data process and provide sufficient rest time between trials to avoid fatigue.</li>
		</ol>
		</li>
		<li>Open the program software processing window and import the CSV files of the original strength data.
		<ol>
			<li>Toe flexion force (FT1, FT2-3, FT2-5): Click on the Run button, select the Automatic Calculation option in the calculation list, and then click on the Calculation button. The software will actively calculate the peak strength of the toe grip (Figure 4).</li>
			<li>Doming force data: Import the original data into the software and click on the Run button. Select the Manual Calculation option in the calculation list. Then, drag the movable 0.5 s window manually, where the force curve is in the shape of a plateau, and the software will automatically calculate the average force in the window (Figure 5).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63479/63479fig01.jpg" /> 
Figure 1: Representative ultrasound images of three intrinsic muscles. (A) Thickness Image of the abductor hallucis; (B) cross-sectional area of the abductor hallucis; (C) thickness image of the flexor digitorum brevis; (D) cross-sectional area of the flexor digitorum brevis; (E) thickness image of the quadratus plantae; and (F) cross-sectional area of the quadratus plantae. <a href="https://www.jove.com/files/ftp_upload/63479/63479fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63479/63479fig02.jpg" /> 
Figure 2: Representative ultrasound images of three extrinsic muscles. (A) Thickness image of the flexor hallucis brevis; (B) cross-sectional area of the flexor hallucis brevis; (C) thickness image of peroneus longus and brevis muscles; (D) cross-sectional area of peroneus longus and brevis muscles; and (E) thickness image of the tibialis anterior. <a href="https://www.jove.com/files/ftp_upload/63479/63479fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63479/63479fig03.jpg" /> 
Figure 3: Foot muscle strength test. (A) Doming test; (B) toe flexion strength test (FT1); (C) toe flexion strength test (FT2-3); (D) toe flexion strength test (FT2-5). <a href="https://www.jove.com/files/ftp_upload/63479/63479fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63479/63479fig04.jpg" /> 
Figure 4: Representative toe flexion strength plot. The peak force of toe flexion is calculated as the average value of six data points around the selected peak point. In the custom software, it is programmed that 10 points, including peak force remain relatively stable to avoid false peaks, which means that the remaining nine points do not exceed &#177;0.5 of the peak value. <a href="https://www.jove.com/files/ftp_upload/63479/63479fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63479/63479fig05.jpg" /> 
Figure 5: Representative doming strength plot. The force of maximum voluntary contraction is calculated for the doming strength. A movable 0.5 s window is present to determine where the force curve is in the shape of a plateau, which could be dragged manually. The strength of doming is programmed to calculate the average value of the selection window (0.5 ms). <a href="https://www.jove.com/files/ftp_upload/63479/63479fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Passive subsystem
NOTE: The ND and foot posture index-6 (FPI-6) tests were applied to evaluate the foot structure (passive subsystem).
<ol>
	<li>Navicular drop (ND) test
	<ol>
		<li>Assemble the height vernier caliper with the base, fixture block, and scribing claw. To specify the navicular tuberosity, extend the scribing claw through a stick. Place the height vernier caliper on the horizontal platform. 
		NOTE: The ND test is performed on the same horizontal platform.</li>
		<li>Instruct the participants to sit on a height-adjustable chair and turn sideways to allow visualization of the medial longitudinal arch. Palpate the navicular tuberosity and mark its location. Instruct the participants to sit in a position where the knee, hip, and ankle joints make a 90&#176; angle.</li>
		<li>Palpate the medial and lateral aspects of the participant&#39;s talus head. Supinate and pronate the subtalar joint until the medial and lateral sides of the talus are equally positioned.</li>
		<li>Align the head of the scribing claw with the marked navicular tuberosity. Read and record the height at this non-weight-bearing position (height 1).</li>
		<li>Instruct the participants to stand and keep the normal, bilateral, weight-bearing stance. Consistently, record the height (height 2).</li>
		<li>Define the vertical movement of the navicular tuberosity (i.e., height 1-height 2) in the sagittal plane as ND. 
		NOTE: In the process of the ND test, the participants must keep straight and look straight ahead.</li>
	</ol>
	</li>
	<li>Foot posture index-6 (FPI-6)
	<ol>
		<li>Perform the FPI-6 test on the horizontal platform as in the ND test (step 3.1.1).</li>
		<li>Instruct the participants to take several steps, marching on the spot, and then stand in their relaxed stance position with double limb support. Inform them to stand still for approximately 2 min during the assessment.</li>
		<li>Palpate the talar head and rate its position on the lateral and medial sides.</li>
		<li>Palpate the lateral malleolar and score the supra- and infra-lateral malleolar curvature.</li>
		<li>Observe the calcaneal frontal plane position and score the angle between the posterior aspect of the calcaneus and the long axis of the foot.</li>
		<li>Palate the talonavicular joint (TNJ) and score the bulge or concave in this area.</li>
		<li>Palate and observe the curve of the medial longitudinal arch and score its height and congruence.</li>
		<li>Observe the forefoot directly behind and in line with the long axis of the heel and score the relative position of the forefoot on the rearfoot (abduction/adduction). 
		&#8203;NOTE: In this test, each item is scored as -2, -1, 0, 1, and 2 (see Supplementary File 1).</li>
	</ol>
	</li>
</ol>
4. Neural subsystem
NOTE: In the assessment of the neural subsystem, the plantar light touch threshold, and a two-point discriminator (TPD) were applied to evaluate the plantar sensitivity.
<ol>
	<li>Plantar light touch threshold
	<ol>
		<li>Prepare Semmes-Weinstein monofilament (SWM) kit, consisting of 20 pieces. Each SWM kit has an index number ranging from 1.65 to 6.65 (1.65, 2.36, 2.44, 2.83, 3.22, 3.61, 3.84, 4.08, 4.17, 4.31, 4.56, 4.74, 4.93, 5.07, 5.18, 5.46, 5.88, 6.10, 6.45, and 6.65), which is related to a calibrated breaking force (i.e., index 1.65 is the equivalent of 0.008 g of force). 
		NOTE: The higher the index value, the stiffer and harder it is to bend.</li>
		<li>Mark the test regions in the plantar sole, including the first toe (T1), first metatarsal head (MT1), third metatarsal head (MT3), fifth metatarsal head (MT5), midfoot (M), and heel (H).</li>
		<li>Apply 4.74 SWM to the participants&#39; thenar eminences to feel the stimulus, which they will receive on the plantar sole in the formal test. Instruct participants to say &#34;yes&#34; and inform the examiner of the accurate site clearly and loudly every time the participants perceive the sensory stimulus of SWM at any tested sites. 
		NOTE: Every marked region can be replaced by one specific number in the convenience of memory.</li>
		<li>Place each participant in the prone position on a standard treatment table facing away from the examiner with the foot hanging on the edge of the table. Instruct them to close their eyes and wear headphones to avoid the assistance of vision and minimize distraction, respectively.</li>
		<li>Apply SWM perpendicularly to the skin at the target region. Pressure is appropriate until the nylon SWM is bent to form a &#34;C&#34; shape. Then, hold it for 1 s before removal. 4.74 SWM is first applied over the marked region, and a 4-2-1 stepping algorithm is utilized to standardize the assessment21. Test six plantar regions at random. 
		NOTE: Provide a few seconds for rest in the interval of trails in case of sensory disturbance between marked regions. The last detected SWM is regarded as the threshold for that site.</li>
	</ol>
	</li>
	<li>Two-point discriminator (TPD)
	<ol>
		<li>Prepare the two-point discriminator device. The adjustable device has different distances, ranging from 1 mm to 15 mm. 
		NOTE: One side of the dial ranges from 1 mm to 8 mm, and revolving the dial to the other side ranges from 9 mm to 15 mm.</li>
		<li>Mark the six test regions in the plantar sole, which are the same as those in the case of the plantar light touch threshold test (step 4.1.2).</li>
		<li>To make participants familiar with the testing process, apply the two-point discriminator in the participants&#39; tip of the middle finger. Inform them to say &#34;one&#34; if they perceived one point or &#34;two&#34; if they perceived two points. 
		NOTE: The test position is the same as that in the plantar light touch threshold test. The participants should keep their eyes closed.</li>
		<li>Start the test from the greatest distance (8 mm), and then decrease the width distance by 5 mm until the participants report one point. Move the device in 1 mm increments applying randomization of one or two points until the participants can consistently identify two points at a test width. 
		NOTE: Three times of correctly identifying two-point touch out of five touches is defined as positive. The last two-point value is recorded as the TPD threshold value.</li>
	</ol>
	</li>
</ol>

The authors have no conflicts of interest.

The presented protocol is used to measure the characteristics of the foot in the elderly, which provides a comprehensive assessment to investigate foot core stability, including the passive, active, and neural subsystems. This new paradigm illuminates the foot function that interacts to stabilize the foot and sustain sensorimotor function in daily activities33. In previous studies, the researchers paid more attention to exploring foot deformity; toe flexion strength; diminished plantar sensory; an...

The human foot is a highly complex structure, consisting of bones, muscles, and tendons that attach to the foot. As a segment of the lower extremity, the foot constantly provides direct source contact with the supporting surface and hence contributes to weight-bearing tasks1. Based on the complex biomechanical interplay between muscles and passive structures, the foot contributes to shock absorption, adjusts for irregular surfaces, and generates momentum. Evidence shows that the foot contributes meaningfully to postural stability, walking, and running2,3,4.
According to a new paradigm proposed by McKeon5 in 2015, the foot core is rooted in the functional interdependence of the passive, active, and neural subsystems, which combine into the foot core system controlling foot motion and stability. In this paradigm, the foot bony anatomy forms the functional half dome, which includes the longitudinal arches and transverse metatarsal arches and flexibly adapts to load changes6. This half dome and passive structures, including the ligaments and joint capsules, constitute the passive subsystem. Additionally, the active subsystem consists of foot intrinsic muscles, extrinsic muscles, and tendons. The intrinsic muscles act as local stabilizers responsible for supporting the foot arches, load dependence, and modulation7,8, while the extrinsic muscles generate foot motion as global movers. For the neural subsystem, several kinds of sensory receptors (e.g., capsuloligamentous and cutaneous receptors) in the plantar fascia, ligaments, joint capsules, muscles, and tendons contribute to foot dome deformation, gait, and balance9,10.
Several researchers have speculated that the foot contributes to daily activities in two main ways. One is by mechanical support via the functional arch and the modulation among lower limb muscles. The other is the input of plantar sensory information about the position11. Based on the foot core system, deficits in this system, including foot posture, the strength of intrinsic and extrinsic foot muscles, and sensation sensibility, may predispose to the weakness of mobility and balance9,11,12,13.
However, with advancing age, alterations to the aspect, biomechanics, structure, and function of the foot commonly occur, including foot or toe deformities, weakness of foot or toe strength, plantar pressure distribution, and reduced plantar tactile sensitivity14,15,16,17. The presence of toe deformity and the severity of hallux valgus are associated with mobility and fall risk in the elderly11,18. Moreover, the strength of toe flexor muscles, which used to be overlooked, contributes to balance in elderly people19. Meanwhile, the elderly are also at higher risks to have foot conditions associated with pathologies such as diabetes, peripheral arterial disease, neuropathy, and osteoarthritis20,21.
The assessment, examination, and health care of the foot, especially in the elderly, have attracted increasing attention14,21. However, there is a limited study to explore the comprehensive evaluation for the function of the foot core system. Numerous studies aimed to explore foot pathological problems in the elderly, such as pain and nail, skin, bone/joint, and neurovascular disorders21,22,23. The role of the foot in mechanical support and sensory input during daily activities and as a functional core system needs to be recognized and evaluated, which was ignored in previous studies. Especially, the foot active components, including the intrinsic and extrinsic muscles, works as the local stabilizers and global movers and contribute to the foot stability and behavior in static posture and dynamic movement5.
The toe flexion strength is singularly reported to represent foot muscle strength, and it&#39;s also utilized to explore the relationship between foot function and other health situations, such as balance, and mobility24,25,26. Inherently, the foot muscle strength is limited to distinguishing the action of intrinsic and extrinsic muscles. Moreover, several tests, including the paper grip test and an intrinsic positive test, were criticized as non-quantitative tests that have poor reliability and validity7,27. Recently, a new evaluation of foot doming strength was reported to quantify the intrinsic foot muscle strength and it has been shown to have a good validity28. By measuring the doming (short-foot movement) strength, it contributes to directly quantifying the function of intrinsic muscle.
Therefore, a protocol is proposed here aiming to explore the characteristics of the foot in the elderly based on the foot core system, especially the function of the active subsystem. This protocol provides a comprehensive assessment to investigate foot core stability, including the passive, active, and neural subsystem, in the elderly. Moreover, alterations in foot core function have been reported in several health situations, such as plantar fasciitis, flat foot, and diabetes24,29,30. In the future studies, it might help to evaluate the foot function among different populations in a multidimensional measurement.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Diagnostic Ultrasound System</td>
			<td>Mindray</td>
			<td></td>
			<td>It is used in clinical ultrasonic diagnostic examination.</td>
		</tr>
		<tr>
			<td>ergoFet dynamometer</td>
			<td>ergoFet</td>
			<td></td>
			<td>It is an accurate, portable, push/pull force gauge, which is designed to be a stand-alone gauge for capturing individual force measurements under any 
			job condition.</td>
		</tr>
		<tr>
			<td>Height vernier caliper</td>
			<td></td>
			<td></td>
			<td>It is an accurate measure tool for height.</td>
		</tr>
		<tr>
			<td>LabVIEW</td>
			<td></td>
			<td></td>
			<td>It is a customed program software for strength analysis.</td>
		</tr>
		<tr>
			<td>Semmes-Weinstein monofilaments</td>
			<td>Baseline</td>
			<td></td>
			<td>It consists of 20 pieces, and each SWM haves an index number ranging from 1.65 to 6.65, that is related with a calibrated breaking force.</td>
		</tr>
		<tr>
			<td>Two-Point Discriminator</td>
			<td>Touch Test</td>
			<td></td>
			<td>It is a set of two aluminum discs, each containing a series of prongs spaced between 1 to 15 mm apart.</td>
		</tr>
	</tbody>
</table>

evaluating the function of the foot core system in the elderly

As a complex structure to link the body and the ground, the foot contributes to postural control in human static and dynamic activities. The foot core is rooted in the functional interdependence of the passive, active, and neural subsystems, which combine into the foot core system controlling foot motion and stability. The foot arch (passive subsystem), responsible for load, is considered the functional core of the foot, and its stability is necessary for normal foot functions. The functional abnormalities of the foot have been widely reported in the elderly, such as weakness of toe flexor muscles, abnormal foot postures, and decreased plantar sensory sensitivity. In this paper, a comprehensive approach is introduced for evaluating the foot function based on foot core subsystems. The strength and morphology of the foot intrinsic and extrinsic muscles were used to evaluate the foot muscle (active subsystem) function. The doming strength test was applied to determine the function of foot intrinsic muscles, while the toe flexion strength test focused more on the function of extrinsic muscles. The navicular drop test and foot posture index were applied to evaluate foot arch (passive subsystem) function. For the neural subsystem, the plantar light touch threshold test and two-point discrimination test were used to assess plantar tactile sensitivity at nine regions of the foot. This study provides new insights into the foot core function in the elderly and other populations.

In this study, 84 participants were included for measurement. The young&#160;group included 42 university students with an average age of 22.4 &#177; 2.9 years and height of 1.60 &#177; 0.05 m. The elderly group included 42 community-dwelling elderly with an average age of 68.9 &#177; 3.3 years and height of 1.59 &#177; 0.05 m.
Representative active subsystem results 
The morphology and strength of foot muscles are used to determine the function of the active subsystem. M...

Watch this Scientific Journal Video about Evaluating the Function of the Foot Core System in the Elderly at JoVE.com

Evaluating the Function of the Foot Core System in the Elderly

The functional core stability of the foot contributes to the human static posture and dynamic activities. This paper proposes a comprehensive evaluation for the function of the foot core system, which combines three subsystems. It may provide increased awareness and multifaceted protocol to explore the foot function among different populations.

As a complex structure to link the body and the ground, the foot contributes to postural control in human static and dynamic activities. The foot core is ...

evaluating-the-function-of-the-foot-core-system-in-the-elderly

Research

JoVE Journal

Neuroscience

2.6K Views.  Shanghai University of Sport. The functional core stability of the foot contributes to the human static posture and dynamic activities. This paper proposes a comprehensive evaluation for the function of the foot core system, which combines three subsystems. It may provide increased awareness and multifaceted protocol to explore the foot function among different populations.

Video: Evaluating the Function of the Foot Core System in the Elderly

A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebella ataxia. It is derived from previously published phenotype assessments in several disease models, including spinocerebellar ataxias, Huntington s disease and spinobulbar muscular atrophy. Measures include hind limb clasping, ledge test, gait and kyphosis. Each measure is recorded on a scale of 0-3, with a combined total of 0-12 for all four measures. The results effectively discriminate between affected and non-affected individuals, while also quantifying the temporal progression of neurodegenerative disease phenotypes. Measures may be analyzed individually or combined into a composite phenotype score for greater statistical power. The ideal combination of the four described measures will depend upon the disorder in question. We present an example of the protocol used to assess disease severity in a transgenic mouse model of spinocerebellar ataxia type 7 (SCA7).
Albert R. La Spada and Gwenn A. Garden contributed to this manuscript equally.

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebellar ataxia. Measures include hind limb clasping, ledge test, gait and kyphosis. This protocol effectively discriminates between affected and non-affected individuals, and detects the progression of affected individuals over time.

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebella ataxia. It is derived from previously ...

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be investigated in a single task sequence. The unique feature of this system is a custom-made, acoustically transparent chamber that eliminates many of the issues associated with auditory cue control in most commercially available chambers. The ease with which operant devices can be added or replaced makes this system quite versatile, allowing for the implementation of a variety of auditory, visual, and olfactory behavioral tasks. Automation of the system allows fine temporal (10 ms) control and precise time-stamping of each event in a predesigned behavioral sequence. When combined with a multi-channel electrophysiology recording system, multiple cognitive brain functions, such as motivation, attention, decision-making, patience, and rewards, can be examined sequentially or independently.

In this report, we present a fully automated and highly versatile system capable of simultaneously testing multi-cognitive behaviors and recording neuronal activities for rodents.

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types that comprise the BBB; these cells have a very high-energy demand, which requires optimal mitochondrial function. In the case of disease or injury, the mitochondrial function in these cells can be altered, resulting in disease or&#160;the opening of the BBB. In this manuscript, we introduce a method to measure mitochondrial function in CVE cells by using whole, intact cells and a bioanalyzer. A mito-stress assay is used to challenge the cells that have been perturbed, either physically or chemically, and evaluate their bioenergetic function. Additionally, this method also provides a useful way to screen new therapeutics that have direct effects on mitochondrial function. We have optimized the cell density necessary to yield oxygen consumption rates that allow for the calculation of a variety of mitochondrial parameters, including ATP production, maximal respiration, and spare capacity. We also show the sensitivity of the assay by demonstrating that the introduction of the&#160;microRNA, miR-34a, leads to a pronounced and detectable decrease in mitochondrial activity. While the data shown in this paper is optimized for the bEnd.3 cell line, we have also optimized the protocol for primary CVE cells, further suggesting the utility in preclinical and clinical models.

Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...