the cerebellum is essential for the proper coordination of locomotion and posture [ 14 ] . to achieve optimal spatiotemporal control of the multiple motor domains involved in these complex behaviors ,
the cerebellum must orchestrate select repertoires of motor synergies through projections to the spinal cord [ 57 ] and cerebral cortex   via the brainstem and thalamus , respectively .
accordingly , when the integrity of the cerebellar circuit is perturbed , motor output is severely impaired , often resulting in ataxia and dystonia .
the cerebellum receives its main inputs from the inferior olive , which project to the purkinje cells ( pcs ) via climbing fibers ( cfs ) , and from several brainstem nuclei , which provide pcs with mossy fiber ( mf)-parallel fiber ( pf ) afferents via the granule cells .
the interaction between these two orthogonally oriented , afferent systems is critical for cerebellar motor coordination , including that of locomotion and posture . whereas the mf - pf system has a strong impact on the amplitude of the simple spike ( ss ) modulation of pcs ,
the cf system and its afferents determine not only the phase and amplitude of the modulation of the complex spikes ( css ) , but also the phase of the ss modulation .
importantly , cfs are organized in modules projecting to narrow rostro - caudal ( micro)zones of pcs in the cerebellar cortex .
pcs within a ( micro)zone in turn converge on cerebellar nuclei ( cn ) neurons , through which the  cerebellum exerts its excitatory control of premotor nuclei [ 13 , 14 ] and inhibitory feedback to the inferior olive .
the olivocerebellar modules may thus control specific muscle synergies that guide complex movements [ 16 , 17 ] .
indeed , synchronicity of cf inputs , which depends on electrotonic coupling and  intrinsic oscillatory properties of olivary neurons , may dynamically control motor output .
two - photon microscopy has enabled cellular - resolution readout of dozens to hundreds of neighboring pcs in awake behaving mice [ 1922 ] .
however , it remains to be clarified to what extent synchronous pc activity is corrupted in movement disorders .
it is therefore important to resolve whether aberrant patterns of cs activity occur in animal models of ataxia and dystonia , whether potential abnormalities in the delays of ensemble activity can be correlated to deficits in locomotion and posture , and to what extent manipulation of activity patterns can influence limb and trunk movements . here
, we sought to tackle these questions exploiting the direct readout of the behavior of all limbs and tail with a newly developed transparent - disk - based treadmill .
the disk treadmill enabled the stable imaging and optogenetic manipulation of pc ensembles in head - fixed mice not only at rest , but also during self - initiated or sensory perturbation - evoked , reflexive limb and trunk movements .
we applied the disk treadmill to study the behavior of the ataxic and dystonic mouse mutant tottering ( tg / tg ) , which suffer from a p / q - type calcium channel mutation [ 2427 ] .
we find that their aberrant locomotion and posture are correlated with reduced levels and delayed timing of pc cs co - activation within cerebellar microzones .
using optogenetics to evoke ss activity simultaneously in multiple pcs , we show that a variety of movement patterns can be elicited contingent on the behavioral state of the animal . taken together
, our data provide evidence that pc ensembles contribute to on - the - fly control of coordination and posture , with manipulations of these cell assemblies resulting in altered motor repertoires .
a transparent disk treadmill was developed to register movements of trunk , tail , and all limbs in head - fixed awake mice while  allowing stable access for cellular resolution circuit imaging or optogenetic stimulation .
additionally , sensory perturbations could be presented by engaging a magnetic clutch placed along the rotational axis of the disk ( figure  1a ) to trigger spino - cerebellar and other reflexes associated with strong cs co - activation .
paw , body , and tail movements were detected by recording the mouse with an infrared - sensitive charge - coupled device ( ccd ) camera from below via a mirror placed at a 45 angle ( figure  1b ) .
animals were illuminated by two strips of infrared light - emitting diodes ( leds ) , one mounted on the mirror and another one across from it on a custom post . a magnetic encoder placed above the disk in conjunction with a magnet placed in the center of the rotating disk allowed logging of disk position as a voltage signal ( figure  1a ) . for reliable detection of multi - limb gait and posture , camera frames were de - noised and background subtraction was performed prior to paw and tail segmentation .
objects were classified based on position , size , and orientation using a three - layer feedforward neural network ( figure  1c ) .
this network was trained by assigning objects to any of six classes : left hind paw , left front paw , right hind paw , right front paw , tail , or none .
subsequently , paw coordinates were used to extract gait parameters , whereas trunk axis and tail angles were determined to assess parameters pertaining to posture ( figure  1c ; supplemental experimental procedures ) .
we investigated locomotion patterns , posture , and fine paw motor behavior of adult tg / tg mice ( n  = 4 ; weight 20.23   1.5 g ) and wild - type littermates ( n  = 4 ; 23.5   2.9 g ) using the disk treadmill ( figures 2a2e ) .
coordination deficits and ataxia could be readily observed by the naked eye in that stepping of hind limbs onto front limbs occurred in tg / tg mice , but not wild - types ( figure  2a ) . yet
, average locomotion speeds did not differ significantly between genotypes ( 36.4   15.6 per second for wild - type and 35.7   15.6 per second for tg / tg ; p  = 0.70 , two - sample kolmogorov - smirnov [ k - s ] test ) and bouts of locomotion had similar overall duration in wild - type and tg / tg mice ( 16.9   10.3  s and 16.2   10.9 s , respectively ; p  = 0.29 , two - tailed t test ) ( figure  2b ) .
moreover , average duration of stance also did not differ among genotypes ( p  =  0.16 , kruskal - wallis test ) ( figure  2c ) .
in contrast , average duration of swing of tg / tg mice was significantly longer ( p  = 1.3   10 , kruskal - wallis test ) than that in wild - type mice ( table s1 ) , ( figure  2c ) . to quantify to
what extent coordination was affected , we looked at phase coupling between limbs ( figure  2d ) .
phase values were calculated by taking the difference between the stance start of the first and second limb and dividing this by the step cycle duration of the second limb , rendering the value of 0.5 as a close approximation of optimal limb alternation .
a multivariate comparison of deviations from normal limb alternation ( absolute deviation from 0.5 ) revealed that overall limb coupling in wild - type and tg / tg mice was significantly different ( p  = 0.003 , kruskall - wallis test ) .
post hoc individual comparisons indicated that hind limbs ( wild - type , 0.57   0.1 ; tg / tg , 0.81   1.0 ; p  = 0.006 , two - sample k - s test ) , left limbs ( wild - type , 0.62   0.2 ; tg / tg , 0.76   0.5 ; p  = 0.002 ) , and right limbs ( wild - type , 0.61   0.2 ; tg / tg , 0.81   0.6 ; p  = 0.03 ) , but somewhat less so front limbs ( wild - type , 0.54   0.1 ; tg / tg , 0.51   0.1 ; p  = 0.07 ) , were unduly out of phase in tg / tg mice ( figure  2d ) . along the same line ,
during locomotion , body - axis and tail angles in tg / tg mice showed significantly larger deflections ( p  = 7.2   10 and p  = 4.5   10 , respectively ; two - sample k - s test ) than those in wild - types ( tg / tg tail angle 20.5   10.6 and body - axis angle 7.2   3.2 ; wild - type tail angle 14.4   9.9 and body - axis angle 5.7   2.6 ) , ( figures 2e and s2 ) .
thus , coordination between limbs , as well as coordination between trunk and tail , was altered in tg / tg mice .
given the prolonged swing duration and impaired coordination of tg / tg mice during locomotion , we next set out experiments to find correlations between changes in cellular - resolution activity of pc ensembles and differences in motor performance parameters .
we performed two - photon imaging of pcs in vermis lobule v on the disk treadmill during both rest and locomotion ( figure  3a ) .
calcium transients evoked by css in pc dendrites ( field size 266.6   39.7  m ; 23.8   9.7 dendrites / field ) could be readily imaged in wild - type mice ( n  = 4 mice , 630 dendrites ) and tg / tg mice ( n  = 3 mice , 192 dendrites ) . in tg / tg mice , cs - evoked transients had significantly reduced amplitudes compared to those in wild - type littermates ( table s2 ) , possibly reflecting a reduced calcium influx through the mutated p / q - type calcium channels . nevertheless , cs rates in tg / tg mice at rest were higher than those in wild - types ( 1.26   0.4  hz in tg / tg versus 1.13   0.4  hz in wild - type ; p  = 0.001 , two - sample k - s test ) .
likewise , during locomotion , cs rates were moderately yet significantly increased in tg / tg mice ( 1.52   0.3  hz in tg / tg versus 1.43   0.3  hz in wild - type ; p  = 7.9   10 , two - sample k - s test ) . given the longer swing duration in tg / tg mice
, we hypothesized that the timing of cs co - activation may be delayed with respect to their motor behavior .
we applied a custom hyper - acuity algorithm to increase the sampling resolution of cs co - activation events to 1 khz . in a 200-ms window centered around locomotion onset ,
the average peak fraction of co - active pc dendrites in tg / tg mice was significantly reduced ( 0.16   0.0 in tg / tg versus 0.21   0.1 in wild - type ; p  = 0.02 , two - sample k - s test ) , and their peak cs co - activation responses were delayed significantly by 86.9   12.6  ms ( p  = 1.4   10 , tg / tg versus wild - types ; signed - rank test ) . indeed , whereas peak cs co - activation in wild - types preceded the onset of the disk movement , that in tg / tg occurred after it ( figure  3b ) .
in addition , the overall rate of co - active cs events in tg / tg mice during a locomotion bout ( 0.12   0.03 co - active events per second ) was significantly lower ( p  = 0.02 ) than that in wild - type mice ( 0.31   0.27 co - active events per second ) ; in fact , over distances encompassing multiple cerebellar microzones , pairwise correlation distributions in tg / tg mice during locomotion were indistinguishable from those during rest ( for comparison across 150  m , 5  m bin interval , p  = 0.2 , kruskal - wallis test ) , whereas they were significantly increased during locomotion in wild - type mice ( for comparison across 150  m , 5  m bin interval , p  = 1.3   10 , kruskal - wallis test ) ( figure  3c ) .
thus , despite a moderate increase of cs rate during locomotion , cs synchrony was significantly disrupted during self - initiated locomotion in tg / tg mice .
by contrast , tg / tg peak co - activation of pcs in response to sensory perturbations evoked by the magnetic clutch of the disk treadmill showed similar delays ( 53.7   4.1  ms in tg / tg versus 50.4   5.9  ms in wild - type ; p  = 0.9 , two - sample k - s test ) .
moreover , there was no significant difference ( p  = 0.15 , two - sample k - s test ) between the latency periods of peak deviations in the  differentials of the temporal twitch profiles in tg / tg mice ( 51.8   13.6  ms ) and those in wild - types ( 62   12.9  ms ) , while the corresponding peak levels of cs synchrony in tg / tg mice were also similar to those in wild - types during this paradigm ( 0.49   0.2 in tg / tg versus 0.55   0.26 in wild - type ; p  = 0.89 , two - sample k - s test ) .
since granule cell layer activity has also been shown to be increased around locomotion onset , we next explored the impact of ss modulation of pc ensembles on sequences of limb , trunk , and tail movements during periods of rest and locomotion using optogenetic stimulation in vermis lobule v of awake mice .
pcs expressing chr2(h134r)-eyfp driven by pcp2-cre   were stimulated using optical fibers implanted above lobule v ( figures 4a and 4b ) .
we used up to 3 mw output power ( i.e. , 24  mw / mm at the cerebellar surface ) , which in whole - cell recordings in  vivo elicits ss activity without a depolarization block .
optogenetic stimulation ( 13 mw , 250  ms ) resulted in stereotyped behavior that fell into different classes , depending on the state of the animal at the time of stimulation ( figure  4c ) . at rest ,
movements were readily evoked ( 98.7% response , 1.3% no response ) and consisted of whole - body or tail twitches ( 71.6% , 373/521 stimulations , n  = 7 mice ; movie s1 ) or initiation of stepping ( 27.3% , 142/521 , n  = 7 ; movie s2 ) , whereas during locomotion responses were less frequently evoked ( 70% response , 30% no response ) and consisted mainly of halting ( 38.1% , 98/257 , n  = 7 ; movie s3 ) or slowdown of locomotion ( 31.9% , 82/257 , n  = 7 ) .
the whole - body twitches following stimulations at rest could be evoked with stimulus durations as short as 10  ms ( figure  s3 ) , and independent from stimulus duration they occurred at a rather fixed latency of approximately 75  ms after stimulus offset ( e.g. , 74.9   38.7  ms after offset for 372 stimuli at 250  ms duration , n  = 4 mice ; see ) ( figure  s3 ) . by contrast ,
initiation of stepping following stimulations at rest ( i.e. , 250  ms ) usually ( 77% ) started before stimulus offset ( figures 4d ) ; treadmill speed began to increase at 209.9   49.3  ms from stimulus onset ( range 95311  ms , 100  onsets , n  = 3 mice ) and peaked bimodally at 315 and 445  ms ( p  = 0.02 , hartigan s dip test ) .
most of the step - inducing optogenetic stimulations ( 114 , n  = 3 mice ) resulted in  four distinct limb movements in succession ( 78.1% , 89/114 ; as opposed to 12.3% and 9.6% for three and two consecutive displacements , respectively ) .
considering the stimulations with  four consecutive displacements , two particular sequences of displacements occurred in a predominant fashion ( figure  4h ) : lf - rh - rf - lh ( 36.0% , 32/89 ) and rf - lh - lf - rh ( 20.2% , 18/89 ) ( l , r , f , and h indicate left , right , front , and hind limb , respectively ) .
the preferred sequences of these light - evoked stepping patterns were similar to those initiated spontaneously ( n  = 4 mice ) in that the lf - rh - rf - lh ( 16% ) and rf - lh - lf - rh ( 25% ) sequences were also the most frequent ones during self - initiated locomotion , suggesting that the optogenetic stimulation induced a natural type of motor behavior .
stimuli of shorter durations ( e.g. , 50  ms ) could trigger locomotion as well , but here movements started on average 132.7   20.4  ms after stimulus onset ( 52 stimuli , n  = 3 mice ) ( figure  s3 ) ; yet again , the main preferred patterns consisted of lf - rh - rf - lh ( 35% , 17/52 ) and rf - lh - lf - rh ( 29% , 15/52 ) . instead , optogenetic stimulation during locomotion usually caused mice to counteract locomotion affecting gait patterns of all four limbs ( figures 4d4 g ) . in cases of a standstill , disk speed began to decline at 226.5   125.2  ms ( 44 terminations , n  = 5 mice ) after onset of a 250-ms stimulus and  came to a complete standstill after 479.9   142.7  ms ( range 233904  ms ) .
the jitter ( i.e. , 142.7  ms ) around the termination point was not correlated with average locomotion speed in the  500  ms preceding stimulus onset ( r  = 0.003 , f statistic  = 0.16 , p  = 0.69 ) . in cases of slowdown of locomotion ,
here speed began to decline at 205.8   151.6  ms ( 27 slowdowns , n  = 5 mice ) .
the speed prior to either a complete halt or  slowdown of locomotion was not significantly different ( halt  versus slowdown 63.3   27.1 per second and 68.2   23.7 per second ; p  = 0.42 , two - tailed t test ) and is therefore unlikely to determine the time point of movement cessation when ensembles of pcs were stimulated .
in contrast , optogenetic stimulation ( for up to 2 s ) of pcs of the floculus of the vestibulocerebellum or the lateral cerebellar hemispheres did not evoke complex locomotion patterns ( data not shown ) .
thus , using the disk  treadmill , we deciphered complex walking patterns with preferred sequences of limb placements following optogenetic induction of ss activity of ensembles of pcs at rest , and , likewise , we monitored various gait - inhibition patterns when pcs were activated during locomotion .
the transparent disk treadmill allowed us to improve readout of  various aspects of cerebellum - dependent motor behavior including gait , posture , and small limb movements , advancing upon previous treadmill systems that also permit concurrent readout of neural circuits and behavioral state , but not more detailed aspects of complex movements . by imaging ensembles of pcs in awake behaving mice on this disk treadmill , we could study microcircuit level changes of spatiotemporal cs  activity and their behavioral correlates in a mouse model of  ataxia and dystonia .
abnormalities of cs activity of pc ensembles in cerebellar microzones of tg / tg mice co - occurred with deficits in timing and coupling of limb coordination and posture . in addition , we showed that optogenetically evoked ss activity in ensembles of pcs can influence the sequence and timing of limb and trunk movements both at rest and during  locomotion .
thus , the output of cerebellar ( micro)zones , in concert with other downstream regions , contributes to coordination of multi - joint- and multi - limb - mediated movements .
cs co - activity in vermis lobule v  preceded self - initiated movements as detected by disk speed , in tg / tg mice it occurred after locomotion initiation .
more specifically , we found in tg / tg mice that in a 200-ms window centered around onset of locomotion the average peak fraction of co - active pc dendrites was significantly reduced and that their peak cs co - activation responses were delayed by approximately 87  ms . when considering the complete period of a locomotion bout , throughout which phase coupling of limbs was affected continuously ,
the overall rate of co - active cs events in tg / tg mice was not even significantly different from that at rest .
a similar phenotype occurs in knockouts of connexin36 , in which cs co - activation is attenuated concomitantly with altered limb and tail reflex responses following perturbations .
together , these data suggest that attenuation and delay of cs co - activation within cerebellar microzones can contribute to changes in the timing and execution of both complex and reflex movements .
how can the abnormal cs patterns during locomotion in tg / tg mice be explained ? given that synchronous activation of pcs in  response to sensory perturbations evoked by the treadmill magnetic clutch showed similar delays to peak co - activation in tg / tg and wild - type mice , our data suggest that it is not a difference in the sensory inputs to the olive or related phase resetting [ 3436 ] , but rather a result of altered dynamic processing in the olivocerebellar modules
first , there was a general increase in cs firing during both rest and locomotion in tg / tg mice , which causes a reduction of ss activity   and thereby activation of cn neurons that inhibit the inferior olive .
this effect will even be enhanced by the increased noise level in ss activity of tg / tg mice , reducing the impact of ss activity on cns .
second , activity increases in cns that inhibit  olivary neurons are in line with a reduced potential for cs co - activation , because this inhibitory input , which is strategically located next to the gap junctions in the olivary glomeruli , can decouple olivary cells presumably through shunting mechanisms [ 39 , 40 ] .
thus , when ss activity and cn activity are taken into account , all findings on cs activity in tg / tg mice  during locomotion can be explained by mechanisms that  control  activity within the olivocerebellar loop .
the finding that movement onset in tg / tg occurred before co - activation of css supports the possibility that these self - initiated movements occurred through concerted action of other brain regions without initial involvement of the cerebellum .
indeed , optogenetic stimulation of pc ensembles in vermis lobule v , which drives their ss activity , triggered behavior of limbs , trunk , and tail depending on the state of the  animal . at rest , responses consisted of posture twitches or complex walking patterns , whereas during locomotion , various forms of gait inhibition occurred .
given that the walking patterns elicited by optogenetic stimulation produced natural sequences of limb movements that also occurred during spontaneous locomotion , it is likely that the zonal stimulation of ensembles in vermis lobule v triggered motor programs embedded downstream in brainstem and spinal cord .
the locomotion patterns we observed after optogenetic stimulation of pc ensembles are distinct from those generated by stimulation of striatum or cerebral cortex using comparable stimulus durations [ 41 , 42 ] .
optogenetic activation of the direct  and indirect striatal pathways to the thalamus increase and decrease velocity of limb movements , respectively . likewise
, optogenetic stimulation of limb regions in the motor cortex induces relatively simple , time - locked movements that allow mapping of distributions of neurons associated with individual limb movements .
thus , although the numbers of neurons stimulated in these latter studies probably differed , it should be noted that an ss firing increase in an ensemble of pcs after optogenetic stimulation for only a few tens of milliseconds at moderate stimulation intensity can be sufficient to elicit quite elaborate locomotion patterns .
future experiments should address to what extent ss activity of ensembles of pcs interacts with that of cs activity to facilitate or inhibit movements , e.g. , by using optogenetic stimuli timed to a particular phase of the olivary oscillation [ 35 , 36 , 43 ] and/or by sequentially stimulating different cf zones along the medio - lateral extent of the cerebellum . in  addition
, it will be interesting to find out how the output of the cerebellum , i.e. , the cerebellar nuclei , can exactly tweak the downstream regions to control the coordination of complex multi - joint movements .
all experiments adhered to the european guidelines for the care and use of laboratory animals ( council directive 86/6009/eec ) and were approved by the animal experiment committee of the royal netherlands academy of arts and sciences ( dec - knaw ) . for imaging experiments , data were collected from tg / tg mice and wild - type littermates ( c57bl/6j background ; jackson laboratory ) .
a custom stainless - steel plate was attached to the mouse skull ( midline , 2  mm posterior of lambda ) , after a small skin incision , using dental cement ( super bond c&b , sun medical ) .
this plate had a circular cutout of 8-mm and a recessed rim of 1-mm width , allowing a round 8-mm diameter cover glass ( thickness 0.15  mm ; warner instruments , catalog number cs-8r ) to lie flush with the plate surface .
a custom clamp was used to exert additional pressure on the cover glass after 2% agarose ( type iii - a ; sigma - aldrich ) or kwik - sil ( world precision instruments ) application to stabilize the brain during imaging . with these measures ,
axial motion artifacts were kept to a minimum during imaging sessions ( figure  s4 ) .
surgeries were performed under anesthesia with isoflurane ( 2% , isoflo ; abbott ) in a stereotactic frame with mice placed on a temperature - controlled ( tc-1000 ; cwe ) heating mouse pad with thermistor ( ysi-451 ; cwe ) to maintain a constant body temperature of 37c . eye ointment ( terra - cortril ; pfizer ) was applied to keep the eyes moist .
xylocaine ( lidocaine - hcl , 10% ; astra zeneca ) was applied to the incision site and wound areas . the disk treadmill consisted of a transparent disk ( thickness 2  mm , diameter 260  mm ) made of poly ( methyl methacrylate ) secured on an axis with low - friction ball bearings ( skf explorer bearing 608 ) .
rotation was measured with a magnetic encoder ( rfc 4801 - 636 - 111 - 201 , 0.025 angular resolution , 5 khz polling ; novotechnik ) used in conjunction with a magnet ( z - rfc - p04 ; novotechnik ) embedded in the center of the rotating disk .
a mirror ( width 200  mm , height 140  mm ) was placed underneath the disk at a 45 angle to reflect an image of the underside of a head - fixed mouse onto a high - speed ccd camera ( rm-6740cl ; jai ) .
the camera was equipped with a wide - angle lens ( xenoplan 1.4/8 ; schneider - kreuznach ) and set to acquire frames ( width 512 pixels , height 256 pixels ) at a rate of 100  hz using a custom - written labview ( national instruments ) acquisition program .
two flexible led strips ( solarox led strip 50  cm ir 940  nm , catalog number 500083094001 ; connector plug , catalog number 7090 ; powered by a 12v , 500 ma stabilized adaptor ) , one attached to the top edge of the mirror and the other on a post  facing the disk edge , were used to illuminate the mouse from below . a  shaft - mounted computer - controlled electromagnetic clutch ( magneta ; catalog number 14.100.01.30 ) allowed presentation of sensory perturbations to the mouse when briefly engaged . for descriptions of two - photon microscopy , optogenetics , and behavioral tracking , as well as analysis of gait
t.m.h . conceived the disk treadmill , performed the experiments , and analyzed the data using custom - written scripts ; j.r.g . developed custom acquisition and behavioral analysis software and the hyperacuity algorithm and analyzed the data ; l.w . and
 summaryit is a longstanding question in neuroscience how elaborate multi - joint movements are coordinated coherently . 
 microzones of cerebellar purkinje cells ( pcs ) are thought to mediate this coordination by controlling the timing of particular motor domains . 
 however , it remains to be elucidated to what extent motor coordination deficits can be correlated with abnormalities in coherent activity within these microzones and to what extent artificially evoked synchronous activity within pc ensembles can elicit multi - joint motor behavior . to study pc ensemble correlates of limb , trunk , and tail movements , we developed a transparent disk treadmill that allows quantitative readout of locomotion and posture parameters in head - fixed mice and simultaneous cellular - resolution imaging and/or optogenetic manipulation . 
 we show that pc ensembles in the ataxic and dystonic mouse mutant tottering have a reduced level of complex spike co - activation , which is delayed relative to movement onset and co - occurs with prolonged swing duration and reduced phase coupling of limb movements as well as with enlarged deflections of body - axis and tail movements . using optogenetics to increase simple spike rate in pc ensembles 
 , we find that preferred locomotion and posture patterns can be elicited or  perturbed depending on the behavioral state . at rest , 
 preferred sequences of limb movements can be elicited , whereas during locomotion , preferred gait - inhibition patterns are evoked . 
 our findings indicate that synchronous activation of pc ensembles can facilitate initiation and coordination of limb and trunk movements , presumably by tuning downstream systems involved in the execution of behavioral patterns . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions