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Behavioural Brain Research 259 (2014) 274– 283

Contents lists available at ScienceDirect

Behavioural Brain Research

j ourna l h o mepa ge: www.elsev ier .com/ locate /bbr

esearch report

bnormalities in whisking behaviour are associated with lesions inrain stem nuclei in a mouse model of amyotrophic lateral sclerosis

obyn A. Granta,∗, Paul S. Sharpb,c, Aneurin J. Kennerleyb, Jason Berwickb,ndrew Griersonc, Tennore Rameshc,1, Tony J. Prescottb,1

Division of Biology and Conservation Ecology, Manchester Metropolitan University, Manchester, UKDepartment of Psychology, University of Sheffield, Sheffield, UKDepartment of Neuroscience, University of Sheffield, Sheffield, UK

i g h l i g h t s

Whisking is relatively resistant to neurodegeneration.However, specific whisker movements, especially retraction velocity, are impacted by disease progression at P120.These behaviour changes are correlated to facial nucleus decline.And likely to be caused by degeneration of the type ii, white muscle fibres in the caudal facial muscles.We conclude that studying the facial musculature and whisking behaviours will give new insights in to neurodegenerative diseases.

r t i c l e i n f o

rticle history:eceived 12 August 2013eceived in revised form 30 October 2013ccepted 2 November 2013vailable online 13 November 2013

eywords:acial nucleusctive sensingibrissae

a b s t r a c t

The transgenic SOD1G93A mouse is a model of human amyotrophic lateral sclerosis (ALS) and recapitulatesmany of the pathological hallmarks observed in humans, including motor neuron degeneration in thebrain and the spinal cord. In mice, neurodegeneration particularly impacts on the facial nuclei in thebrainstem. Motor neurons innervating the whisker pad muscles originate in the facial nucleus of thebrain stem, with contractions of these muscles giving rise to “whisking” one of the fastest movementsperformed by mammals.

A longitudinal study was conducted on SOD1G93A mice and wild-type litter mate controls, com-paring: (i) whisker movements using high-speed video recordings and automated whisker tracking,and (ii) facial nucleus degeneration using MRI. Results indicate that while whisking still occurs in

G93A

otor neuron diseaseOD1 mouse

SOD1 mice and is relatively resistant to neurodegeneration, there are significant disruptions tocertain whisking behaviours, which correlate with facial nuclei lesions, and may be as a result ofspecific facial muscle degeneration. We propose that measures of mouse whisker movement couldpotentially be used in tandem with measures of limb dysfunction as biomarkers of disease onsetand progression in ALS mice and offers a novel method for testing the efficacy of novel therapeuticcompounds.

. Introduction

Amyotrophic lateral sclerosis (ALS) is an adult-onset progres-ive neuromuscular disorder that leads to degeneration of motor

eurons in the motor cortex, brain stem and spinal cord, causingrogressive weakness in limbs and difficulties with walking, speak-

ng and swallowing. The extensively used SOD1G93A mouse model

∗ Corresponding author. Tel.: +44 161 247 6210.E-mail addresses: robyn.grant@mmu.ac.uk (R.A. Grant),

.tamesh@sheffield.ac.uk (T. Ramesh), t.j.prescott@sheffield.ac.uk (T.J. Prescott).1 Joint last author.

166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.bbr.2013.11.002

© 2013 Elsevier B.V. All rights reserved.

of ALS, harbours a Gly93 to Ala amino acid substitution, and hasbeen validated using biochemical and behavioural tests; however,the exact mechanism causing selective motor neuron degenera-tion is unknown [1]. The loss of motor neurons results in muscleparalysis; however, several studies have shown the degeneration ofneuromuscular junctions and muscles to occur prior to motor neu-ron degeneration [2,3]. ALS causes significant changes throughout,but not restricted to, the pyramidal motor system, including, thenigrostriatal system, the neocortex, allocortex and the cerebellum

[4]. However, MRI studies have only found evidence for neurode-generation in the motor nuclei of the brainstem, rather than motorcortex [5–7]. In the brainstem, neurodegeneration is particularlyevident in the facial nucleus with reports that, in mice, up to 50%

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f the neurons in this nucleus are lost by the 120th post-natal dayP), compared to ∼10% losses in the oculomotor and hypoglossalucleus [5,7].

In mice, the facial nerve originates within the facial nucleus andnnervates both the intrinsic and extrinsic muscles of the mysta-ial pad. In particular, motor neurons that innervate the mystacialad muscles are mainly found in the lateral subnucleus of the facialucleus [29]. Intrinsic muscles form a sling around each whisker

ollicle and actuate individual vibrissae from within the mystacialad [8]. Small units of motorneurons are thought to innervate the

ntrinsic muscles, with about 25–30 motorneurons per whisker fol-icle (Klein and Rhoades, 1985). The extrinsic muscles are externalo the pad and generate whole pad movements, such as the m.asolabialis and the m. Maxillolabialis that cause retraction move-ents of the vibrissae [32,8,9,20]. Through contractions of the

ntrinsic and extrinsic muscles, the whiskers show rhythmic boutsf protractions and retractions, referred to as whisking, that aremong the fastest movements performed by mammals [10–12].he vibrissal motor neurons in the facial nucleus receive input fromultiple brain regions, including motor cortex, and thus constitutes

he final common motor path for whisker control.Behavioural studies in ALS mice have been extensively used

or the development of drug treatments for ALS, but have mainlyocussed on locomotion thus far, such as gait analyses and rotorodests [13–15,36,16]. However, locomotion can be hard to quan-ify and often requires training on a particular task. In light ofhe impact of neurodegeneration on the facial nucleus, this paperims to address whether measurements of whisker movementsight provide a reliable quantification of disease onset and pro-

ression. In addition, assessing the impact of neurodegenerationn vibrissal movement in the SOD1G93A mouse could lead to anmproved understanding of rodent whisking motor control, whichs an important model system for understanding the control ofhythmic movements in mammals. An understanding of the effectsf neurodegeneration on whisker movement in these mice couldherefore lead to advances in both fundamental motor neuro-cience and in our understanding of ALS.

This study examines the relationship between facial nucleiesions and whisker behaviour. We provide an analysis of whisker

ovements in SOD1G93A mice and of age-matched, non-transgenicNtg) littermates using high-speed video recordings and automatichisker tracking. Histology of the mystacial pads qualitatively

ompares the musculature between SOD1G93A and Ntg mice bytaining for cytochrome oxidase, and the facial nucleus is imagedsing MRI. Our results indicate that lesions in the facial nucleiesults in a significant disruption of whisking behaviour, whichrovides an early behavioural biomarker of disease that has notreviously been reported.

. Materials and methods

.1. Animals

Mice were originally obtained from the Jackson Laboratory,6SJL-Tg (SOD1-G93A)1Gur/J (stock number 002726), and wereubsequently backcrossed onto the C57Bl/6 background (HarlanK, C57Bl/6 J OlaHsd) for >20 generations [15]. Our model, on aefined inbred genetic background, shows no effect of sex or lit-er of origin on survival [15]. These transgenics have an averageurvival of 140 days, slightly longer than the outbred B6SJL-TgSOD1-G93A)1Gur/J (stock number 002726) strain [15]. In the first

tudy, five SOD1G93A mice and five age-matched wild-type litter-ate control mice (Ntg) were filmed roaming in an open arena at

ostnatal day (P) 60, P90 and P120 (±5 days). At P120 two SOD1G93A

ice and their littermate controls were sacrificed to examine their

esearch 259 (2014) 274– 283 275

facial muscles further. In the second study, at P30, P60, P90 andP120 (±3 days), five SOD1G93A mice and five control mice (Ntg)were imaged in a 7 T magnet, to measure loss of pelvic muscle vol-ume and to assess the development of facial nuclei lesions. Theywere also filmed in an open arena three days post imaging, as inStudy 1. All the animals were female kept on a 12:12 light sched-ule at 22 ◦C, with water and food ad libitum. All procedures wereapproved by the local Ethics Committee and UK Home Office, underthe terms of the UK Animals (Scientific Procedures) Act, 1986.

2.2. Behaviour recordings

High-speed digital video recordings were made using a PhotronFastcam PCI camera, recording at 500 frames per second, shutter-speed of 0.5 ms, and resolution of 1024 × 1024. The camera wassuspended from the ceiling, above a custom-built rectangular(40 cm × 40 cm) viewing arena with a glass floor, ceiling, and end-wall.

The mice were recorded at P30 (for the second study only),P60, P90 and P120 on two consecutive days. Video data was col-lected in near darkness using an infrared light box (for Study 1) ornormal spectrum lightbox (for Study 2) for illumination. Multiple1.6 second video clips were collected opportunistically (by manualtrigger) when the animal moved beneath the field of view of thecamera. Approximately 24 clips were collected from each animalper day. One to two clips from each day were selected per mouse(giving around n = 80 clips per day in total) according to a selec-tion criteria. These clips were selected when the mouse was clearlyin frame, both sides of the face were visible, the head was levelwith the floor (no extreme pitch or yaw), the whiskers were notin contact with a vertical wall and the mouse was clearly mov-ing forward. In each selected clip the mouse snout and whiskerswere tracked, using the BIOTACT Whisker Tracking Tool [17]. Thetracker semi-automatically finds the orientation and position ofthe snout, and the angular position (relative to the midline of thehead), of each identified whisker. Tracking was validated by man-ually inspecting the tracking overlaid on to the video frames (seeFig. 1a and c). Clips from Study 1 were more successfully trackedthan in Study 2, owing to a greater contrast of the video footageby using an infrared lightbox. This gave rise to a larger behaviouralsample size in Study 1 (P120:n = 87, P90:n = 69, P60:n = 72) thanStudy 2 (P120:n = 34, P90:n = 37, P60:n = 32, P30:n = 39), althoughthe results agree between the two studies (see Section 3).

Analysis focussed on using the movement of the entire whiskerfield on each side of the snout using a measure of mean angu-lar position calculated as the unsmoothed mean of all the trackedwhisker angular positions on each side, in each frame (see [18],and the two examples shown in Fig. 1b and d). Whisker offsetwas calculated as the mean angular position for each clip. Meanangular retraction and protraction velocities were calculated fromthe angular position, as the average velocity of all the backward(negative) whisker movements, and forward (positive) whiskermovements, respectively. These measures were all averaged togive a mean value per clip, and each side (left and right) wasalso averaged together. Whisk frequency was estimated using anautocorrelogram of the tracked angular position. Specifically, thetime series of whisker angles for each side was first smoothedusing a zero-phase low-pass filter (boxcar) with a cut-off frequencyof 16 Hz which is well above the highest expected whisking fre-quency and removes the high-frequency information. The first peak(maximum) in the autocorrelation of this smoothed time serieswas then identified automatically to give a first estimate of sig-

nal period. This estimate was then refined by gradient ascent onthe unsmoothed autocorrelation series to locate the nearest peakto that found automatically in the smoothed series. To estimatethe amplitude the mean value was removed from the whisking

276 R.A. Grant et al. / Behavioural Brain Research 259 (2014) 274– 283

Fig. 1. Tracking and whisker trace examples for one SOD1G93A mouse and one non-transgenic (Ntg) mouse. (a) An example of whisker and head tracking for a SOD1G93A

mouse; (b) the corresponding whisker traces (mean angular position, MAP), red is left and blue is right; (c) an example of whisker and head tracking for a Ntg mouse; (d)t is rigr

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he corresponding whisker traces (mean angular position, MAP), red is left and blueeferred to the web version of this article.)

ngle time series and the root mean square value was computed toive the root-mean-square (RMS) whisking amplitude. These timeeries were approximately sinusoidal, so the “peak-to-peak whisk-ng amplitude” was estimated by multiplying the RMS whiskingmplitude by 2

√2 [19]. This estimate of amplitude is reasonably

obust to departures from a purely sinusoidal pattern. Locomo-ion velocity was calculated as the speed of the movement forwardy the animal, as a mean over the clip, using the pixel informa-ion from the nose tracking (see the yellow head outline in Fig. 1and c) and calibrated, using a calibrator tool measured using thepen source video tracking toolbox ‘Tracker’ (Tracker 4.80, Douglasrown 2013, www.cabrillo.edu). All the whisker and locomotionata was distributed normally so parametric statistical tests werearried out throughout, more information can be found in Section.

.3. Muscle staining

In study 1, two SOD1G93A and two Ntg mice were also sacri-ced for qualitative examination of their facial musculature. The

ht. (For interpretation of the references to colour in this figure legend, the reader is

mystacial pads were removed bilaterally, by cutting down thesagittal plane and cutting around each pad (about 2 mm each sideof the pad). Any pieces of bone were removed from the pads andthey were placed flat between stainless steel grids in perforatedplastic histology cases (Medex Supply) to prevent curling. Thehistology cases were then put in a solution of 100 ml of phosphatebuffer (PB) pH = 7.4 followed by a mixture of 2.5% glutaraldehydeand 0.5% paraformaldehyde and refrigerated for 2 days. They weretransferred into a solution of 20% sucrose in 0.1 M Phosphate bufferpH 7.4 for 2 days.

After fixing, each of the pads was sectioned with a micro-cosm cryostar cryostat into 40 �m slices. The eight pads (fromfour animals) were sliced tangentially. All slices were stainedfor CCO activity (see [20]) as follows. The slices were floated ina solution of 10 ml of 0.1 M PB containing 0.75 mg cytochromec, 40 �l catalase solution and 5 mg diaminobenzidine (DAB) and

0.5 ml distilled water. They were then placed in an incubator at37 ◦C on a shaking platform for 2 h until the stain developed. Theslices were then rinsed in 0.05 M PB, mounted and left to air drybriefly before coverslipping with Entellan. Figures of the stained

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usculature were prepared from digital images. A Zeiss Lumar V12icroscope was used to obtain the images, at magnification 12× to

mage the whole pad, and 150× in close-up photographs. Imagesere collected in Axiovision and exported as .jpg images. Only

mall adjustments in contrast and brightness were made to thegures.

.4. Magnetic resonance imaging

Five female transgenic C57Bl/6 SOD1G93A and five age matchedon-transgenic littermates were blind imaged in a 7 T magnetBruker BioSpecAVANCE, 310 mm bore, MRI system B/C 70/30),ith pre-installed 12 channel RT-shim system (B-S30) and fit-

ed with an actively shielded, 116 mm inner diameter, waterooled, 3 coil gradient system (Bruker BioSpin MRI GmbH-GA12. 400 mT/m maximum strength per axis with 80 �samps) to assess pelvic muscle volumes and MR signal inten-ity of the facial nucelus at the 30, 60, 90 and 120 day timeoints.

Animals were placed in a custom built Perspex magnet cap-ule and imaged under gaseous anaesthesia (1–1.5%, flow rate.8–1.0 L/min continuous inhalation through a nose cone). Anaes-hetic level was controlled on the basis of respiratory parameters;

onitored using a pressure sensitive pad placed under the sub-ect’s chest (SAII Model 1025 monitoring and gating system). Insidehe capsule, a non-magnetic ceramic heated hot air system (SAII –

R-compatible Heater System for Small Animals) and rectal probe,ntegrated into the physiological monitoring system maintainedhe temperature of the animal. All animals were euthanized at the20 day time point.

A 1H birdcage volume resonator (Bruker, 300 MHz, 1 kWax, outer diameter 114 mm/inner diameter 72 mm), placed

t the iso-centre of the magnet was used for both RF trans-ission and reception. A workstation configured for use with

araVisionTM 4.0 software operated the spectrometer. Fol-owing field shimming, off-resonance correction and RF gainetting a tri-plane FLASH sequence (TR = 100 ms, TE = 6 ms,lip angle = 30◦, Av = 1, FOV = 40 mm × 40 mm, Slice thk = 2 mm,atrix = 128 × 128) was used for subject localisation. From this

fast (∼5 min) 3D FISP sequence (TR = 8/1200 ms, TE = 4 ms,OV = 40 mm × 40 mm × 40 mm, Matrix = 256 × 256 × 128) allowedow SNR visualisation of the hind limb area and thus planning of 21xial high SNR single Spin Echo images (TR = 3200 ms, TE = 7.5 ms,v = 1, FOV = 40 mm × 40 mm, Slice thk = 1 mm, Matrix = 256 × 256)overing the entire lower hind limb and pelvic region. No fat sup-ression was used to maximise muscle/fat contrast difference forasy segmentation.

For data processing, a macro built into ParaVision 4.0 was usedor manual segmentation of muscle from fat and bone across alllices in 3 regions (the left and right hind limb and the pelvic region).sing the scan FOV setting and the slice thickness allowed for vol-me conversion of segmented data. The mean and standard erroror each group (Sod1 and Ntg) at each time point was calculated inraphPad Prism.

Signal intensity within the facial nucleus was assessed using aagittal high SNR single Spin Echo image (TR = 3200 ms, TE = 7.5 ms,v = 1, FOV = 40 mm × 40 mm, Slice thk = 1 mm, Matrix = 256 × 256).acial nucleus images in the MR data were confirmed using theouse brain in stereotactic coordinates [35]. Once subjects had

een sub-divided into control and SOD1 groups two Region of

nterests (ROIs) were selected and applied to all images acrosshe time points; the first over the facial nucleus and the sec-nd within the white matter of the brainstem. The latter ROIas used to normalise the signal within the facial nucleus across

ime.

esearch 259 (2014) 274– 283 277

3. Results

3.1. Study 1: Time course of changes to whisking behaviour inSOD1G93A mice

3.1.1. Locomotion and whisking behaviours are affected inSOD1G93A mice

Locomotion and whisking behaviours are affected during dis-ease progression. All the whisker variables that showed significantchanges by P120 can be seen in Fig. 2. In particular, by P120,the SOD1G93A mouse whiskers are held further back (have loweroffset values), move more (have larger amplitude whisks), moveslower (have lower frequencies) and retract faster (have largerretraction velocities). In addition, locomotion speeds are muchslower throughout in the SOD1G93A mice. Protraction velocity wasnot significantly affected and, therefore, will not be included infurther analyses. Between-ANOVA tests compared the locomo-tion and whisking behaviours between the SOD1G93A and Ntgmice at each time point to confirm these observations, significantresults (p < 0.05) are indicated in Fig. 2 with asterisks (*), and canalso be seen in Table 1 for the P120 time point. While whisk-ing behaviour is impacted most, on more whisker variables, atP120, whisker offset and retraction velocity are both significantlyaffected at two time points, and locomotion is impacted through-out, from P60. The analysis was also conducted with the mouseidentity as a covariate, to see if the differences between individ-uals could account for the differences in variables. Table 1 showsthe results of this analysis at P120 and confirms earlier statistics,implying that the differences in locomotion and whisker variablesare as a result of the disease rather than differences in individuals.Results at the P120 time-point for all of the variables can also beseen graphically for each animal in the Supplementary material(Supplement 2).

As whisking and locomotion are believed to be closely coupled[18], a further analysis was conducted to determine whether itis the declining locomotion abilities or disease progression thatis driving the differences in whisking behaviour. A multivariateANOVA was conducted with locomotion speed introduced as acovariate to control for its effects on whisking behaviour. Table 1shows the results from this analysis. Statistics confirm that thereare still differences in whisking behaviour between the SOD1G93A

and control mice, indicating that whisker variables do changeirrespective of locomotion speed. This suggests that it is likelyto be the disease progression, rather than simply the declin-ing locomotion levels that account for the changes in whiskingvariables. To further assess whether disease progression is affect-ing whisking behaviour, the muscle staining results will also beconsidered.

3.1.2. Muscle staining is affected in SOD1G93A miceMuscle staining results are purely qualitative in this instance

and applied to confirm our behavioural findings. At a firstglance, the musculature does not look significantly affected in theSOD1G93A mice. However, on closer inspection, the both the morecaudal areas of the muscle pad and the intrinsic muscles seemslightly reduced and darker in the SOD1G93A mouse (compare, espe-cially the more caudal areas in Fig. 3a, with equivalent areas inFig. 3d). In particular, these are the caudal extrinsic muscles (M.nasolabialis and maxillolabialis), named the superficial retractingmuscles as they control the retraction movements of the whiskers.A close-up look of the superficial retraction muscles in Fig. 3c andf, shows that while the Ntg mice show striated muscle fibres of red,

pink and white (Fig. 3c), the SOD1G93A mice only show the darkermuscles fibres, red and pink (Fig. 3f). This suggests that there is adecrease of Type ii, white muscle fibres in SOD1G93A mice, in thecaudal superficial retracting muscles and can be seen even clearer

278 R.A. Grant et al. / Behavioural Brain Research 259 (2014) 274– 283

Fig. 2. Locomotion and whisking behaviours at P60, P90 and P120 for SOD1G93A (solid black line) and Ntg (dashed line) mice. (a) locomotion velocity; (b) whisker offset; (c)whisking amplitude; (d) whisking frequency; (e) whisker retraction velocity. Significant differences (p < 0.05) between SOD1G93A and Ntg are denoted by the asterisk (*). Allw ougho

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hisker variables are affected in SOD1G93A mice by P120, locomotion is affected thr

n Fig. 4. Reduction of these fibres might well explain the changesn retraction velocity behaviour that have been observed in theOD1G93A mice.

Overall, the whisker muscles of the SOD1G93A pad seem slightlyeduced and darker than that of the Ntg mice (Fig. 3a and d); how-ver, close-ups of the intrinsic muscles (Fig. 3b and e) do not showny clear differences between the SOD1G93A and Ntg mice. There

ight be a tendency for the SOD1G93A mice to have less lighteruscle fibres throughout the pad, but slices from these four ani-als only clearly shows a decline in the white muscles of the caudal

uperficial retracting extrinsic muscles.

able 1NOVA analysis of the locomotion and whisking variables at P120, with disease presenc

dentity and locomotion as covariates can also be seen.

Loco. speed Offset Protraction velocity

Sod1 mean 0.46 ± 0.13 89.90 ± 8.45 1.52 ± 0.178

Ntg mean 0.77 ± 0.23 99.35 ± 7.04 1.56 ± 0.21

F(1, 86) 63.422 31.140 0.796

p-Value <0.001** <0.001** 0.375 n.s.

Mouse covar.a ** ** n.s.Loco. covar.b n.a. ** n.s.

.s. indicates not significant (p > 0.05).

.a. indicates analysis not applicable.a Mouse identity is used as a covariate in the ANOVA analysis, significance is indicatedb Locomotion is used as a covariate in the ANOVA analysis, significance is indicated wit* Indicates where p < 0.05.

** Indicates where p < 0.001.

ut.

3.2. Study 2: Disease progression effects on whisking behaviourand facial nucleus

3.2.1. Validating whisker behaviours in SOD1G93A miceAs different mice were used in the second study, the behaviour

findings were confirmed as before (compare Fig. 2 to Fig. 5). Thevisual lightbox used in these behavioural recordings did not give

as clear images, causing the tracking programme to fail on more ofthe clips, leading to a reduction in tracked clips. The behaviouralfindings were confirmed again in these mice and showed similarpatterns, although results were slightly weaker than those in Study

e (Sod1 or Ntg) as the between-factor. Additional analyses conducted with mouse

Retraction velocity Frequency Amplitude

0.32 ± 0.16 12.18 ± 6.93 42.42 ± 9.140.26 ± 0.13 17.97 ± 7.92 35.97 ± 6.464.320 13.196 13.7770.041* <0.001** <0.001**

** ** **

n.s. ** **

with the asterisks.h the asterisks.

R.A. Grant et al. / Behavioural Brain Research 259 (2014) 274– 283 279

Fig. 3. Staining for cytochrome oxidase in Ntg (top) and SOD1G93A (bottom) mice. (a) Entire pad staining in Ntg mouse; (b) intrinsic sling muscle staining in Ntg mouse (seecorresponding box in a); (c) a close-up of the superficial retracting muscles (m. Nasolabialis) in Ntg mouse (see corresponding box in a); (d) entire pad staining in SOD1G93A

mouse; (e) intrinsic sling muscle staining in SOD1G93A mouse (see corresponding box in a) c; (f) a close-up of the superficial retracting muscles (m. Nasolabialis) in SOD1G93A

mouse (see corresponding box in a); (g) an additional close-up of the superficial retracting muscles in SOD1G93A mouse. Photos suggest that SOD1G93A mice have decreasedwhite muscle fibres in the superficial retracting muscles, compared to the Ntg mice.

Fig. 4. superficial retracting muscles (m. Nasolabialis) in Ntg (a) and Sod1 (b) mice. The SOD1G93A mice show a decline in the white muscle fibres.

280 R.A. Grant et al. / Behavioural Brain Research 259 (2014) 274– 283

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ig. 5. Locomotion and whisking behaviours at P60, P90 and P120 for Sod1 (solidhisking amplitude; (d) whisking frequency; (e) Whisker retraction velocity. All wh

hese results confirm earlier findings in Fig. 2.

due to lower sample numbers (Fig. 5). By P120 SOD1G93A miceere similarly slower, with their whisker held further back, movingore, at a slower frequency, with faster retractions.

.2.2. Pelvic muscle volume and facial nucleus MRI signalsecline in SOD1G93A mice

SOD1G93A mice show a decline in pelvic muscle volume through-ut the testing period from P30 to P120. This can be clearly seen inhe MR images in Fig. 6a. They also have a large percentage changen the difference in MR signal of the Facial Nucleus compared tohe surrounding area. This difference can be seen from P60 to P120Fig. 6b). Fig. 6b shows MR images of SOD1G93A and Ntg mouserains, with the Facial Nucleus region of interest (ROI) clearly paler

n the SOD1G93A mouse.

.2.3. Whisker variables correlate well with declines in the facialucleus

To examine further whether it is the decline of motor neurons inhe Facial Nucleus, or the declining locomotion (pelvic muscle) thatauses the changes in locomotion and whisker variables, Spear-ans Rank correlations were run on the SOD1G93A mouse dataset

nly, with all the age points included together. Scattergraphs ofhese correlations are presented in Fig. 7. Fig. 7 shows that off-et and retraction velocity are both significantly correlated withhe Facial Nucleus decline (p < 0.05). Only locomotion is correlatedignificantly with the declining pelvic volume (p < 0.05). Resultsrom these correlation analyses can be found in the Supplemen-ary material (Supplement 3). When the same analysis was run on

he non-transgenic (Ntg) mice in the same way, the Facial Nucleusyperintensity was not correlated to any of the whisker variablesnd the Pelvic Muscle volume was not correlated to locomotionelocity (Supplement 3).

line) and Ntg (dashed line) mice. (a) Locomotion velocity; (b) whisker offset; (c)variables are affected in SOD1G93A mice by P120, locomotion is affected throughout,

While correlations cannot imply causation, retraction velocity,in particular, looks to be affected by neurodegeneration; it is corre-lated to hyperintensity of the facial nucleus (Fig. 7i), but not to thepelvic muscle decline (Fig. 7j). In addition, the muscles responsiblefor retraction movements seem to be most affected by the disease(Figs. 3 and 4). Therefore, retraction velocity and offset, are likelyto be directly impacted by changes in the Facial Nucleus and facialmusculature in the SOD1G93A mouse. Other important behaviouralobservations can also seen in terms of locomotion speed, whiskfrequency and whisk movement.

4. Discussion

Here, we present compelling evidence that while whisking isstill present in SOD1G93A mice, ALS does affect certain whiskingbehaviours. SOD1G93A mice have longer whisks with larger ampli-tudes; their whiskers are held further back and retract quicker.However, that the whiskers are still actively whisking indicatesthat the facial areas are relatively robust to ALS. Changes in whiskerbehaviour are likely to be caused by degeneration of very specificmuscles, in particular the caudal, superficial extrinsic muscles (m.Nasolabialis and m. Maxillolabialis) that control retraction move-ments. In addition, both whisker offset and retraction velocity arecorrelated to the hyperintensity of the Facial Nucleus in SOD1G93A

mice. Other areas do change significantly throughout the progres-sion of disease in the SOD1G93A mouse, including the cerebralcortex and cerebellum. Therefore, although behavioural deficits arestrongly correlated to the hyperintensity of the Facial Nucleus, thisis likely to be due to the neurodegeneration of a combination of

brain structures, of which the Facial Nucleus is the final commonmotor path for whisker control.

Conversely, other brain areas may be able to compensate forthe changes in the Facial Nucleus. Certainly, brain areas such as the

R.A. Grant et al. / Behavioural Brain Research 259 (2014) 274– 283 281

Fig. 6. Changes in pelvic muscle volume and Facial Nucleur MR signal in SOD1G93A mice, at P30, P60, P90 and P120. (a) Pelvic muscle volume decreases in SOD1G93A mice,which can be seen in the graph (P30–P120) and by comparing the MR image on the right between Ntg and SOD1G93A mice, which was taken at P120. (b) % MR signal increasesi undino learly

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n SOD1G93A mice, between the Facial Nucleus (ROI: region of interest) and the surron the right between Ntg and SOD1G93A mice, which was taken at P120. The ROI is c

asal ganglia [21–23], superior colliculus [24] and cortex [25,26]ave all been found to significantly alter in structure followinghisker removal or ablation of the sensory nerve. These structures

re all connected to the Facial Nucleus, either directly or via othertructures, and therefore, could realistically compensate for somef the effects of neurodegeneration, and contribute to the relativeesistance of the facial area to neurodegeneration.

That the pelvis and locomotion behaviour are affected before,nd perhaps more than the facial nucleus and whisking behaviour isn interesting finding. It has been observed previously in a limitedumber of muscle studies that facial muscles are more resistanthan locomotion muscles [3,27]; however, most research has notonsidered facial muscles at all. Here it can be seen that pelvicuscles have a more marked decline (Fig. 6), compared to the

acial muscles (Fig. 4). Valdez et al. [3], who used SOD1G93A mices a model of ageing, suggested that muscles innervated by longerotor axons, such as the hindlimbs, might be more affected by

eurodegeneration than shorter axons, such as the facial muscles.t might also be that rostral areas (facial muscles) are more diseaseesistant than caudal areas (pelvic muscles). Indeed, Valdez et al.3] found that neuromuscular junctions in rostral muscles showess disease-related changes than caudal neuromuscular junctions.his might also explain why the caudal muscles of the mystacialad (superficial extrinsic muscles) are affected more than otherore rostral facial muscles, such as the deep retracting muscles

nd extrinsic protracting muscles [20].Not only do the limb and facial areas differ in terms of their dis-

ance from the brain, and their rostro-caudal coordinates, but theacial musculature of the mouse has a very specific architecture that

ight make it more resistant to muscular degeneration. The intrin-ic muscles are coupled from whisker-to-whisker in the same row,

g region. This can be seen in the graph (P30–P120) and by comparing the MR image seen in the SOD1G93A mouse example.

like a chain [8]. Therefore, degeneration of some intrinsic musclesmight be compensated by others in the same row, and passivelymoved as part of the chain. The intrinsic muscles are those that areresponsible for protraction movements, which were least affectedin the SOD1G93A mice in this study. In addition, protraction move-ments are thought to be more actively controlled than retractionmovements [28] as they are slower and more variable. Intrinsicmuscles also have proportionally more motorneurons innervatingthem in the facial nucleus, than extrinsic muscles [29]. The super-ficial extrinsic muscles are, therefore, more likely to be affectedby degeneration than the intrinsics as they are caudal to the pad,innervated by fewer motorneurons and not in a chain architecture.Decline of the superficial extrinsic muscles as we observed here,could cause less resistance to forward movements, hence account-ing for the increase in amplitude. The animals would also have lesscontrol over retraction movements, hence causing quicker retrac-tions, as we have seen here.

Previous studies have also found that disease progressionimpacts fast muscles more than slow muscles in SOD1G93A mice[2] with fast fibres being more prone to dystrophy, denervation,and ischaemia [2,3]. This agrees with the qualitative muscle imageshere (Figs. 3 and 4) that show a decline in the fast, type ii musclefibres of the superficial retracting muscles. All the muscles in themystacial pad contain type i and type ii muscle fibres [20], althoughthe intrinsic muscles are thought to contain the highest percentageof the fast, type ii fibres [30]. However, for the reasons discussedabove, these muscles might be fairly resistant to degeneration.

Future work exploring whether it is the facial muscle architec-ture or its reduced neurodegeneration that explains the robustnessof whisking protraction movements to disease progression, wouldbe an important addition to this study. However, significant

282 R.A. Grant et al. / Behavioural Brain Research 259 (2014) 274– 283

Fig. 7. Correlating whisker variables with MRI variables, % change in facial nucleus MR signal (left) and Pelvic area muscle volume (right). For variables locomotion (a, b),offset (c, d), amplitude (e, f), frequency (g, h) and retraction velocity (i, j), respectively. Locomotion, offset and retraction velocity are all significantly correlated with decliningFacial Nucleus signal, while only locomotion is significantly correlated to Pelvic area volume. Significant results (p < 0.05) are indicated by an asterisk (*). The four age pointsare also denoted, P30 (yellow), P60 (red), P90 (blue) and P120 (red). (For interpretation of the references to colour in this figure legend, the reader is referred to the webv

cfwmnubtt

ersion of this article.)

hanges in whisker retractions especially, and offset, amplitude andrequency have been identified for the first time in this study. Usinghisking and locomotion behaviours together could be a usefulodel to study disease progression and treatment. The method is

on-invasive, quick, and does not require the animal to be trained,

nlike other behavioural tasks, such as the rotorod test. It is worthearing in mind that behaviour data is relatively variable, indeedhere were earlier behavioural locomotion deficits observed inhe SOD1G93A mice in study 1 (Fig. 2), which had larger sample

numbers. This agrees with other observations of behavioural exper-iments in SOD1G93A mice [33] and it is recommended to have largeanimal numbers for all preclinical animal research in ALS, accordingto updated guidelines [31].

5. Conclusions

Whisking is conserved during disease progression in theSOD1G93A mouse; however, specific whisker movements are

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ffected by neurodegeneration. While the facial area is fairly resis-ant to ALS in the SOD1G93A mouse overall, we observe someeductions in the caudal fast muscle fibres of the mystacial pad,hat affects the retraction movements of the whiskers at P120. Thebserved changes in whisker behaviours are strongly correlatedo neurodegeneration of the Facial Nucleus. We present here thathisker analyses could be used as a new behavioural model of ALS.hile this model is still in an early phase of development, we think

hat further exploring the relationship between muscle degener-tion and behaviour is a useful and important step in quantifyinglinical aspects of neurodegenerative disorders, for better charac-erisation and treatment.

cknowledgments

The authors would like to thank Dr Ben Mitchinson and Kendrarkley, for their help and advice with data collection and whisker

racking. We are grateful to Dr Andy Grierson and Dr Richard Meador their support. Many thanks also to Natalie Kennerley for super-ising the histology work, and Graham Tinsley for microscope tips.e also acknowledge the continued support and advice from all

he members of the ABRG. Video analysis was performed usinghe BIOTACT Whisker Tracking Tool which was jointly created byhe International School of Advanced Studies, the University ofheffield, and the Weizmann Institute of Science under the aus-ices of the FET Proactive project FP7 BIOTACT project (ICT 215910),hich also partly funded the study. The MRI work was funded by an

PSRC/BBSRC collaborative project grant (EP/G062137/1) awardedo Dr. G. Battaglia.

ppendix A. Supplementary data

Supplementary material related to this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.bbr.2013.11.002.

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