gain- and loss - of - function experiments have shown laminar - specific effects on local cortical processing ( beltramo et  al . , 2013 , olsen et  al . , 2012 ) , but how the layers work together remains unclear .
the synchrony of action potential ( ap ) firing across cortical layers is thought to be a fundamental aspect of translaminar processing and is determined by the strength , sign and timing of the underlying synaptic input . here
, we investigate the synaptic mechanisms of cortical synchrony between excitatory neurons in layers 2/3 and 5 in behaving mice .
measuring translaminar membrane potential ( vm ) synchrony and linking it to sensory processing and behavior require simultaneous vm recordings from different layers in awake animals .
however , the vast majority of vm recordings of cortical neurons in behaving animals have been made from superficial layers ( bennett et  al .
, 2013 , poulet and petersen , 2008 , poulet et  al . , 2012 ,
these studies have shown that internally generated , spontaneous network activity dominates the vm of cortical neurons across cortical regions and is correlated with the behavioral and arousal state .
large - amplitude , slow fluctuations are highly correlated between neighboring layer 2/3 ( l2/3 ) neurons in resting animals but are abolished during movement , resulting in a desynchronized or  active
 cortical state ( harris and thiele , 2011 , poulet and petersen , 2008 ) .
the active state may result from arousal - related effects associated with movement and has been linked to a modulation in sensory responsiveness ( crochet and petersen , 2006 , otazu et  al . , 2009 , pinto et  al . , 2013 ,
, 2013 , reimer et  al . , 2014 , schneider et  al . , 2014 ,
, 2015 , zhou et  al . , 2014 ) , adaptation ( castro - alamancos , 2004 ) , and even perception itself ( bennett et  al . , 2013 , mcginley et  al . , 2015 ) .
few studies have examined the vm activity of deeper layer cortical neurons in behaving animals ( mcginley et  al . , 2015 , schiemann et  al . , 2015 ) .
extracellular recordings , however , have shown higher spontaneous and sensory - evoked firing rates in deeper layer neurons ( de kock et  al .
, 2007 , oconnor et  al . , 2010 ) and , intriguingly , that sensory - evoked and spontaneous spiking have different temporal structures across layers ( sakata and harris , 2009 ) . the rodent forepaw somatosensory system is a relevant and accessible model system to investigate cortical sensory processing during behavior .
the forepaw has five digits ( figure  1a ) that can be used to grasp and manipulate objects as well as discriminate somatosensory stimuli ( milenkovic et  al . , 2014 ) .
we made whole - cell recordings from primary forepaw somatosensory cortex l2/3 and l5 excitatory neurons in awake mice to compare the synchrony and integration of external ( sensory ) and internal ( movement - evoked and spontaneous ) synaptic input .
our data highlight layer - specific membrane properties that underlie differences in ap firing and show that translaminar vm synchrony is dependent both on the behavioral state and the source of synaptic input .
to investigate the intrinsic membrane properties of l2/3 and l5 excitatory neurons , we made blind whole - cell patch clamp recordings targeted to the digit 3 cortical representation in primary somatosensory forepaw cortex ( s1 ) in awake mice .
mice were head - restrained and had their right forepaw tethered to the platform ( figure  1b ) .
the tips of the digits 2 and 4 overhung the edge of the platform while the tip of digit 3 was positioned on a flat , circular head of a combined movement sensor and force - feedback tactile stimulating arm .
the stimulating arm head was held in contact with the glabrous skin of digit  3  throughout all recordings .
whole - cell recordings from 17 l2/3 neurons were made at subpial depths between 121 and 384  m ( 245.30  17.91  m ) and from 28 l5 neurons between 538 and  823  m ( 649.43  14.28  m ) ( figure  1c ) .
all neurons had evoked regular - spiking firing patterns during current injection , and a subset ( l2/3  = 4/17 and l5  = 15/28 neurons ) was confirmed by post hoc biocytin staining to be excitatory pyramidal neurons .
we first examined the intrinsic membrane properties soon after break - in during quiet wakefulness .
l5 neurons generated more aps in response to equivalent current injection amplitudes than l2/3 neurons ( figures 1d and 1e ) .
the increased excitability may be due to the higher resting vm value of l5 neurons during quiet wakefulness ( l2/3mean  = 56.92  1.21  mv , n  = 12 cells versus l5mean  = 50.70  0.65  mv , n  = 19 cells ; p  < 0.001 ) and their higher input resistance ( figure  1f ; l2/3i / r  = 33.46  2.80 m , n  = 11 cells versus l5 
= 51.66  3.35 m , n  = 18 cells ; p  <  0.001 ) .
interestingly , following the termination of a depolarizing current step , l5 neurons showed a larger - amplitude afterhyperpolarization than l2/3 neurons ( figure  1 g ; at 400  pa : l2/3ahp  = 0.56  0.12  mv , n  = 10 cells versus l5ahp 
= 2.42  0.17  mv , n  = 22 cells ; p  < 0.001 ) .
overall , our data show distinct intrinsic membrane properties of forepaw s1 l5 and l2/3 neurons .
we next compared the vm properties and dynamics of l2/3 and l5 neurons during forepaw behavior .
spontaneous digit movements were monitored by the sensing arm and used to define periods of quiet wakefulness ( q ) and digit movement ( m ) ( see experimental procedures ) .
q was characterized by large - amplitude , low - frequency vm fluctuations observed in all recordings from neurons in both layers ( figure  2a ) .
slow fluctuations had a similar mean duration across layers ( l2/3 329.06  27.55  ms , n  = 13 cells versus l5 266.94  11.15  ms , n  = 23 cells ) but lower frequency in l2/3 ( l2/3 2.03  0.08  hz , n  = 13 cells versus l5 2.29  0.04  hz , n  = 23 cell ; p  = 0.005 ) ( figure  s1 ) . in both layers , m was accompanied by a reduction in the amplitude of low - frequency fluctuations , with fast fourier transform analysis highlighting a reduction in the power in the 1 to 5  hz range ( figures 2b and 2c ) , and a reduction in the sd of the vm ( figure  2d ) .
mean ap firing rates were higher in l5 than l2/3 neurons during q ( figure  2e ; l2/3fr , q  = 0.32  0.10  hz , n  = 12 cells versus l5fr , q  = 2.86  0.60  hz , n  = 19 cells ; p  < 0.001 ) .
moreover , a significant increase in mean firing rates during m was observed in l5 neurons ( l5fr q  = 2.86  0.60  hz versus m  = 6.14  1.16  hz , n  = 19 cells , p  = 0.005 ) , but not l2/3
( l2/3fr q  = 0.32  0.10  hz versus m  = 0.45  0.18  hz , n  = 12 cells ; p  = 0.851 ) .
inter - spike intervals ( isis ) showed a skewed distribution in both layers , with 22% of l2/3 aps and 30% of l5 aps having an isi of < 25  ms ( figure  s2 ) .
we next analyzed ap bursting and observed similar burst durations across layers but overall more busts in l5 neurons .
no differences were observed between q and m periods ( burst frequency l2/3bf , q  = 0.02  0.004  hz , m  = 0.03  0.01  hz , n  = 13 cells versus l5bf , q  = 0.14  0.05  hz , m  = 0.25  0.12  hz , n  = 23 cells , l2/3 versus l5 q , p  = 0.019 ; figure  s2 ) . to investigate what drove the laminar differences in mean ap rates , we next examined the ap threshold .
ap threshold in l2/3 and l5 neurons varies dependent on the speed of pre - spike depolarization in vm , with faster depolarizing ramps evoking aps at lower threshold ( figure  s3 ) .
there was , however , no overall difference in ap threshold between l2/3 and l5 neurons in q or m periods ( figure  2f ) .
measurement of the mean vm showed a depolarization during m in both l2/3 ( figure  2 g ; l2/3mean q  = 56.92  1.21  mv versus m  = 51.45  1.73  mv , n  = 12 cells ; p  < 0.001 ) and l5 neurons ( l5mean q  = 50.70  0.65  mv versus m  = 45.89  0.80  mv ,
0.001 ) , with l5 neurons significantly more depolarized in both behavioral states ( q l2/3mean versus l5mean p  <
notably , plotting the mean spontaneous ap rates as a function of the difference between ap threshold and the mean value of the top 10% of the vm distribution ( max vm ) revealed an exponential decay ( figure  2h ) .
l5 neurons were distributed on the falling slope and l2/3 on the tail of the slope ( figure  2h ) , suggesting that the simplest explanation for higher firing rates in l5 neurons is their more depolarized vm .
most likely , this works in combination with the higher input resistance of l5 neurons ( figure  1 ) to push excitatory postsynaptic potentials over ap threshold and trigger more aps in l5 than l2/3 neurons in behaving mice .
synchrony of cortical activity across layers is thought to be an important feature of cortical coding .
to examine translaminar subthreshold and spiking synchrony , we next inserted two pipettes through neighboring craniotomies to target recording sites in the same vertical axis and made nine simultaneous , dual whole - cell recordings from l2/3 and l5 neurons ( figure  3a ) .
we went on to measure vm synchrony at different timescales using cross - correlation , coherence analysis , and spike - triggered averaging .
visual inspection of dual vm recordings and cross - correlation analysis shows that large - amplitude slow fluctuations in resting mice are highly correlated across layers ( figures 3a3e ) . as mice went from q to m
, however , the correlation of the vm between l2/3 and l5 neurons was reduced in all pairs of cells ( figures 3d and 3e ) .
unexpectedly , the peak time of the cross - correlation showed a significant time lag , indicating that l5 subthreshold activity preceded that in l2/3 by 8.04  1.40  ms in resting mice and by 4.35  1.85  ms during m ( figure  3f ) .
coherence measurements suggested that the drop in correlation during m was due mostly to the reduction in low - frequency ( 15  hz ) coherence ( figures 3 g and 3h ) .
thus , dual whole - cell recordings showed that subthreshold activity of cortical neurons in awake animals is under dynamic control , with cortical layers becoming more independent during active brain states . to quantify ap synchrony from dual recordings , we made spike - triggered peri - stimulus time histograms ( psths ) of spontaneous aps .
the chance of observing an ap in a l5 neuron in a 10-ms window around a l2/3 ap is 6.1% during q and 5.6% during m , and the chance of observing an ap in a l2/3 neuron around a l5 ap is 1.0% during q and 0.4% during m. thus , while the chance of observing an ap is dependent upon the firing rate , ap firing appears asynchronous at fast timescales across cortical l2/3 and l5 in awake mice .
, aps were triggered by large - amplitude and cell - specific depolarizing synaptic input ( figures 3i3k ) . at the time of the ap , a slow
, small - amplitude depolarization was observed in the simultaneously recorded cell in resting mice , necessarily induced by the slow network fluctuations .
a similar picture was present during movement : aps were triggered by large - amplitude , depolarizing inputs in the spiking cell that were absent in the simultaneously recorded neuron .
these observations provide a synaptic basis for independent laminar spontaneous firing and suggest that spontaneous ap firing in l2/3 and l5 excitatory neurons is driven by sparsely connected excitatory networks
. the time lag of the cross - correlation analysis and independent synaptic input during spiking suggested that there might be laminar differences in the fine temporal structure of spontaneous subthreshold fluctuations .
we therefore first compared the onset timing of the slow depolarizing fluctuations in resting mice from dual l2/3 and l5 recordings ( figures 4a and 4b ) .
we defined a slow depolarizing event ( sde ) as depolarizing epochs in q periods whose average vm level between onset and offset was > 60% of the vm range between the most hyperpolarized and depolarized values for at least 100  ms . plotting the duration and vm of isolated sdes across seven dual recordings as a heatmap
( figures 4b and  s4 ) revealed a range of durations with a mean of 300  ms ( figure  s1b for single and dual recordings , l2/3  = 329.06  27.55  ms , n  = 13 cells , l5  = 266.94  11.15  ms ,
normally a sde in one neuron was accompanied by a sde in the simultaneously recorded neuron , but a minority of small - amplitude , short - duration sdes were observed in one layer only ( figure  s5 ) , likely accounting for the slightly shorter durations of sdes in l5 . in those sdes with a measureable vm onset in both layers , plotting the trial - by - trial onset times ( figure  4c ; supplemental experimental procedures ) , the population averaged vm ( figure  4d ) and population spiking rates ( figure  4e ) revealed that l5 sdes started earlier than those in l2/3 ( figure  4f ; l5 leading l2/3  = 9.07  2.19  ms , n  = 7 pairs , p  = 0.031 ) .
in contrast , the hyperpolarizing offset time was not significantly different across layers when sdes were triggered on l2/3 onset ( figures 4a and 4f ) .
sdes thus have an earlier onset in higher - firing l5 neurons , supporting the proposal that l5 neurons are important drivers of supragranular slow network activity ( beltramo et  al .
, 2013 , sanchez - vives and mccormick , 2000 ) . is the laminar - specific synaptic input observed during ap firing ( figure  3 ) and spontaneous activity ( figure  4 ) a general feature of subthreshold processing in cortical neurons , or are there other sources of more synchronized synaptic input ?
movement triggers an active , desynchronized cortical state , which can be driven by thalamic input ( poulet et  al .
to examine the fast dynamics of movement related synaptic input across layers , we next analyzed the vm dynamics at m onset ( detected by thresholding the first derivative of the digit movement signal ; supplemental experimental procedures ) .
averaging the vm showed that m onset is accompanied by simultaneous depolarizing synaptic input to l2/3 and l5 neurons ( figures 5a and 5b ) .
close inspection of the log - scale digit movement trace indicated that the depolarization in both layers was tightly coupled to the tiny initial movements of the digits and did not appear before movement . to quantify the timing of the depolarization , we performed cross - correlation analysis between the averaged digit movement and the averaged vm response at m onset .
the peak of the cross - correlation showed no significant time lag ( figure  5b ) , suggesting an internal , non - sensory origin for synaptic input at movement onset .
analysis of 2-s epochs of vm activity showed an overall reduction in the sd of the vm during extended m periods ( figure  2 ) , but how fast this occurs was unclear .
we therefore quantified the variance of the l2/3 and l5 subthreshold activity around all recorded m onsets , including short - duration movements ( figures 5e and 5f ) . in all cells across both layers
, we observed a rapid and robust reduction in variance within the first 200  ms of movement onset .
thus , early movement - evoked input acts to reduce subthreshold variability simultaneously across cortical layers .
the reduction in variance , however , does not result in synchronous ap firing . instead , l5 neurons showed higher firing rates after movement onset than l2/3 neurons ( figures 5c and 5d ) .
thus , unlike the laminar - specific vm dynamics during sdes and spontaneous aps , movement onset triggers simultaneous vm depolarization across layers , which results in an increase in firing only in l5 neurons .
thalamo - cortical axons are unequally distributed across the cortical layers , with some l5 neurons receiving direct  thalamic input ( bureau et  al .
we therefore next examined whether there is laminar specificity in sensory processing across layers in behaving animals .
we delivered brief ( 2  ms ) , light ( 10 mn ) tactile stimuli via the stimulating / sensing arm to digit 3 during single and dual whole - cell recordings . the sensing arm was in constant contact with the glabrous skin of digit 3 .
while the first spike latency was similar for both behavioral conditions and layers ( figure  s6b ) , the amplitude of the vm response was strongly dependent on the behavioral state ( figures 6a6d ) . in q periods , stimuli evoked a large - amplitude depolarizing response in both layers , whereas the amplitude of the response was reduced during m ( figures 6c and 6e ; l2/3amp q  = 10.80  0.65  mv , m  = 4.84  1.24  mv , n  = 10 cells , p  = 0.037 ; l5amp q  = 6.19  0.69  mv , m  = 2.41  0.60  mv , n  = 20 cells , p  = 0.001 ; q l2/3amp versus l5amp p  < 0.001 , and m l2/3amp versus l5amp p  =  0.005 ) . despite the difference in subthreshold response amplitude , tactile stimulation evoked few extra aps over the background rate in both layers in both behavioral conditions ( figures 6d , 6f , and s6a ; l2/3ap q  = 1.59  0.62  hz , m  = 1.95  1.36  hz , n  = 10 cells , p  = 0.375 ; l5ap q  = 2.44  1.40  hz , m  = 1.55  1.16  hz , n  = 20 cells , p  = 0.370 ; q l2/3ap versus l5ap p  = 0.613 , m l2/3ap versus l5ap p  = 0.523 ) . to examine why l5 neurons do not fire more aps in response to tactile stimulation despite having a more depolarized vm , we measured the tactile responses as a function of the vm just prior to stimulus onset .
as neurons became more depolarized , the sensory response reduced in amplitude until eventually showing a hyperpolarizing response ( figure  s7 ) .
the vm level for the tactile - evoked reversal potential was similar during q and m periods and hyperpolarized relative to glutamate reversal potential ( figure  7 g , l2/3rev q  = 46.67  1.60  mv , m  = 47.09  1.59  mv , n  = 10 cells , p  = 0.492 ; l5rev q  = 46.17  0.75  mv , m  = 46.61  0.86  mv , n  = 20 cells , p  = 0.412 ; q l2/3rev versus l5rev p  = 0.644 , m l2/3rev versus l5rev p  = 0.775 ) .
notably , a minority of cells with higher mean firing rates showed a smaller difference between ap threshold and the sensory reversal potential ( figure  6h ) .
these data provide an explanation for why the evoked rates are similar across layers and suggest that sensory - evoked gaba - ergic inhibition plays a critical role in defining the sensory reversal potential in both layers ( crochet et  al . , 2011 ,
we next examined correlations between the timing and amplitude of sensory - evoked synaptic responses across layers during  dual recordings .
while the onset latencies of the synaptic and spiking responses to tactile stimulation in l5 neurons were  more broadly distributed than in l2/3 , there was no significant difference across layers ( figure  6i , l2/3psplat  = 11.07  0.58  ms , n  = 13 versus l5psplat  = 11.34  0.76 ms , n  = 21 , p  = 0.972 ; figure  s6b , l2/31staplat , q  = 33.78  4.73  ms , n  = 8 cells , m  = 44.67  8.30  ms , n  = 3 cells ; l51staplat , q  = 39.52  4.72  ms , n  = 19 cells , m  = 31.10  5.90  ms , n  = 9 cells ) .
moreover , trial - by - trial analysis of the tactile - evoked postsynaptic potential ( psp ) showed that subthreshold response amplitudes were highly correlated between l2/3 and l5 neurons ( figures 6j and 6k ) .
together , these data indicate that , in contrast to sdes and spontaneous aps , tactile stimulation , like movement onset , triggers synchronized subthreshold input across layers . following 75.2% of stimuli delivered in q periods ,
the mouse s forepaw remained stationary ( quiet - quiet [ qq ] trials ) .
however , 24.8% of stimuli in q periods evoked short - latency ( < 100  ms ) forepaw digit movements ( figure  7 ) , termed quiet - move ( qm ) trials .
we next compared sub- and supra - threshold responses in qq with qm trials ( figures 7a7c ) .
tactile stimulation evoked a short - latency , large - amplitude subthreshold response in qq and qm trials in both layers ( figures 7a and 7b ) .
notably , the amplitude of the subthreshold response was larger in qm than qq trials in both layers ( figure  7d ; l2/3 qq  = 10.80  0.65  mv versus qm  = 11.83  0.79  mv , n  = 10 cells , p  = 0.037 ; l5 qq  = 6.19  0.69  mv versus qm  = 8.16  0.78  mv , n  = 20 cells , p  = 0.002 ; and qq l2/3 versus l5  p  < 0.001 , qm l2/3 versus l5  p  = 0.005 ) .
moreover , we observed that the vm prior to stimulus onset was significantly more hyperpolarized in qm trials than qq  trials in l5 , but not in l2/3 neurons ( figures 7e ; l2/3 qq  =  57.37  1.36  mv , qm  = 58.19  1.58  mv , n  = 10 cells , p  = 0.232 ; l5 qq  = 52.53  0.69  mv , qm  = 54.24  0.61  mv , n  = 20 cells , p  = 0.009 ; and qq l2/3 versus l5  p  = 0.006 , qm l2/3 versus l5  p  = 0.033 ) . in qq trials , the peak response was then followed by a brief hyperpolarization and subsequent depolarization peaking at 350  ms ( figure  7b ) .
this secondary depolarization was significantly larger in both layers during qm than qq trials ( figure  7f ) and evoked aps in l5 , but not l2/3 , neurons ( figures 7c and 7 g ;
l2/3 qq  = 0.18  0.13  hz , qm  = 0.12  0.34  hz , n  = 10 cells , p  = 0.910 ; l5 qq  = 0.01  0.24  hz , qm  = 2.66  0.81  hz , n  = 20 cells , p  = 0.003 ; qq l2/3 versus l5  p  = 0.613 , qm l2/3 versus l5  p  = 0.011 ) .
thus , movements evoked by a tactile input are linked to a more hyperpolarized pre - stimulus vm , a larger subthreshold early response , and a higher late increase in mean firing rate of l5 neurons .
the coordinated activity of six layers of primary sensory cortical neurons underlies sensory perception . using whole - cell recordings in awake mice
, we investigated synaptic mechanisms of translaminar synchronous activity . we show that laminar - specific differences in membrane properties drive distinct firing rates , that translaminar synchrony is dependent both on the behavioral state and the source of synaptic input ( spontaneous , sensory , and movement evoked ) , and that l5 neurons signal spontaneous and tactile - triggered movement .
prior work has observed low firing rates of l2/3 excitatory neurons during synchronized states in resting and anesthetized animals ( barth and poulet , 2012 , jouhanneau et  al . , 2014 ) and no change ( crochet and petersen , 2006 , polack et  al .
, 2013 , poulet and petersen , 2008 , zhou et  al . , 2014 ) or a reduction ( bennett et  al . , 2013 ,
we observed that l5 neurons have a higher mean spontaneous rate of aps ( 2.86  0.60  hz ) than l2/3 neurons ( 0.32  0.09  hz ) in resting mice , supporting previous reports in anesthetized and awake animals ( brecht et  al .
, 2003 , constantinople and bruno , 2011 , de kock et  al . , 2007 , de kock and sakmann , 2009 , manns et  al . , 2004 ,
oconnor et  al . , 2010 , sakata and harris , 2009 ) , and , in contrast to l2/3 , a significant increase in mean firing rates during movement ( figure  2 ) .
previous studies of deeper layers have observed both increases and decreases in firing rates of somatosensory ( curtis and kleinfeld , 2009 , de kock and sakmann , 2009 ) and motor ( carvell et  al . , 1996 , schiemann et  al . , 2015 , zagha et  al . , 2015 )
our whole - cell recordings indicate that higher l5 rates were not the result of an intrinsic difference in ap threshold .
moreover , ap triggered averaging showed that the depolarizing synaptic inputs driving aps had similar amplitudes and dynamics in l2/3 and l5 neurons ( figure  3 ) .
instead , higher mean firing rates in l5 neurons appeared to result from a larger input resistance and a more depolarized vm , as observed in subsets of l5 pyramidal neurons in  vitro ( lefort et  al .
, 2009 , mason and larkman , 1990 ) , which likely combine to push excitatory input over ap threshold more often than in l2/3 neurons . because most excitatory connections to pyramidal neurons are small in amplitude ( jouhanneau et  al .
, 2015 , lefort et  al . , 2009 ) and the rise time of the depolarization prior to an ap is negatively correlated with ap threshold ( figure  s3 ) ( azouz and gray , 2000 , poulet and petersen , 2008 ) , it is likely that coincident synaptic input is required to drive spontaneous ap firing in both layers . in resting mice , the absolute tactile - evoked firing rate was higher in l5 ( 5.03  1.74  hz ) than l2/3 ( 1.96  0.75  hz ) neurons , but this was superimposed on different background firing rates .
in fact , a brief tactile stimulus added similarly few additional aps to the background rate in both layers ( evoked rate : l2/3 1.59  0.62  hz and l5 2.44  1.40  hz ) .
our observation of low evoked rates across layers is similar to prior studies of s1 in anesthetized animals ( barth and poulet , 2012 , brecht et  al . , 2003 ,
l5 pyramidal neurons ( de kock et  al . , 2007 , sakata and harris , 2009 ) .
we did not differentiate between subtypes of cortical excitatory l5 neurons , but we did observe a minority ( 3/20 ) of l5 neurons with sensory - evoked rates > 10  hz ( figure  6f ) .
future work targeting whole - cell recordings to within - layer excitatory neuron subtypes in sensory cortex will be of great importance .
cortical sensory responses are modulated by behavioral state in different sensory systems ( bennett et  al .
, 2013 , crochet and petersen , 2006 , niell and stryker , 2010 , otazu et  al .
, 2009 , schneider et  al . , 2014 , zhou et  al . ,
we report a reduction in subthreshold response amplitude in l2/3 neurons to brief tactile stimuli during movement .
we go on to show that l5 neurons also have a reduced subthreshold response during movement ( figure  6 ) . despite this
, the numbers of evoked aps remained the same in both layers during quiet and moving periods . in both layers ,
subthreshold responses were reduced in amplitude as the baseline vm values became more depolarized and exhibited a reversal potential more hyperpolarized than ap threshold .
interestingly , in the cells with higher sensory - evoked firing rates , the sensory reversal potential was closer to threshold .
together , this suggests that strong , local gabaergic inhibition plays a significant role in clamping the subthreshold sensory response below ap threshold and regulating ap firing during behavior in both l2/3 ( crochet et  al . , 2011 ) and l5 neurons .
synchronous activity in cortical networks is thought to be fundamental to sensory processing and perception .
prior work has shown that spontaneous vm activity in l2/3 neurons during resting states is more correlated than in activated states in behaving or attentive animals ( okun et  al .
neurons showed large - amplitude , highly correlated fluctuations of the vm during resting periods and an active state with low sd , as well as a reduction in slow fluctuation amplitude and translaminar vm synchrony ( figures 2 and 3 ) .
however , close inspection of the fine timing of subthreshold inputs evoked at different time points revealed differences in the timing of synaptic input across layers .
spontaneous aps were driven by cell - specific , depolarizing inputs ( figure  3 ) .
in contrast , movement- ( figure  5 ) and sensory - driven ( figure  6 ) synaptic input had a similar timing across layers .
interestingly , slow subthreshold fluctuations in resting mice had an earlier onset in l5 , reminiscent of the earlier timing of  upstate  onsets in deeper layers observed in anesthetized and sleeping animals ( chauvette et  al . , 2010 , sakata and harris , 2009 ) , cortical slices ( sanchez - vives and mccormick , 2000 ) , and extracellular recordings in awake rats ( sakata and harris , 2009 ) .
thus , the type of input ( spontaneous , sensory , or movement evoked ) determines the timing of synaptic input across layers .
one possibility is that they result from differences in the wiring supporting spontaneous , sensory , and motor events . in support of this suggestion ,
a recent anatomical study showed laminar differences in local and long - range cortico - cortical inputs to s1 excitatory neurons ( denardo et  al . , 2015 ) .
l2/3 neurons receive proportionally more input from distant l2/3 neurons than l5 neurons , which receive a greater proportion from distant l5 neurons .
moreover , locally , l2/3 neurons receive a greater proportion of inhibitory inputs than l5 neurons .
luczak et  al . , 2007 ) and slow fluctuations in awake resting animals ( ferezou et  al . , 2007 )
are thought to propagate as waves of activity across cortex supported by long - range cortico - cortical connections .
thus , a later onset in l2/3 neurons could result from a combination of lower firing rates , increased inhibitory input , and laminar - specific cortico - cortical wiring .
in contrast , because sensory thalamic neurons are driven both by sensory stimulation and movement ( poulet et  al . , 2012 ) , we suggest that punctate thalamic input drives the synchronized synaptic input following tactile stimuli or movement onset . subthreshold sensory responses are correlated not only with the  behavioral and cortical state but also with the behavioral outcome .
both l2/3 and l5 neurons showed a larger - amplitude subthreshold sensory response in trials that lead to short - latency forepaw movements ( qm trials ) compared to trials with no movement before or after the stimulus ( qq trials ) .
in l5 neurons , a difference was also observed in the pre - stimulus vm , with qm trials having a more hyperpolarized value than qq trials .
this suggests that the link between subthreshold response amplitude and behavioral output is due to the prestimulus cortical state rather than delivery of different amplitude stimuli .
our findings resemble recent vm recordings in auditory cortex during an auditory discrimination go / no - go task , where neurons from l4 and l5 showed more hyperpolarized vm value in hit trials than in false - positive trials in mice performing an auditory perception task ( mcginley et  al . , 2015 ) .
future experiments could address whether this is the result of a higher signal to noise ratio in a phase of low network activity resulting in an enhanced probability of signal transmission to downstream motor centers . during evoked movement trials , neurons in both layers showed a prominent late depolarization ( figure  7 ) , with l5 neurons also showing an increase in late spiking .
because forepaw movements necessarily occurred soon after tactile stimulation in qm trials , it was difficult to assess whether the late activity is causally related to the perception of the stimulus ( sachidhanandam et  al . , 2013 ) , an intrinsic part of the transformation of sensory input to motor output and/or the start of the active cortical state associated with attention or arousal .
examining mice trained to delay sensory - triggered movements will help link late activity to perception and movement .
we observed higher background firing rates in l5 neurons but no difference in the numbers of additional , sensory - evoked spikes across layers , suggesting that l2/3 and l5 have distinct sensory coding strategies ( sakata and harris , 2009 ) .
the fine timing differences of synaptic input may be important in the processing of dynamic sensory stimuli and for changes of synaptic strength under spike - time - dependent plasticity rules .
fast , laminar - specific manipulations of synchronized activity in trained mice are now required to define the causal role of translaminar synchrony in perception .
all experiments were approved by the berlin animal ethics committee and carried out in accordance with european animal welfare law .
head - restrained 6- to 9-week - old c57bl6j mice were paw - tethered , and digit movements were monitored by a force - feedback sensing and stimulating arm .
the sensing arm ending was a 4.7-mm - diameter flat disk with one edge pressed up against the glabrous skin of digit 3 .
blind whole - cell patch - clamp recordings were made from primary somatosensory forepaw cortex located by intrinsic optical imaging .
all data were statistically analyzed using non - parametric tests , paired data with a wilcoxon signed rank test and unpaired data with a wilcoxon rank sum test .
 summarythe synchronized activity of six layers of cortical neurons is critical for sensory perception and the control of voluntary behavior , but little is known about the synaptic mechanisms of cortical synchrony across layers in behaving animals . 
 we made single and dual whole - cell recordings from the primary somatosensory forepaw cortex in awake mice and show that l2/3 and l5 excitatory neurons have layer - specific intrinsic properties and membrane potential dynamics that shape laminar - specific firing rates and subthreshold synchrony . 
 first , while sensory and movement - evoked synaptic input was tightly correlated across layers , spontaneous action potentials and slow spontaneous subthreshold fluctuations had laminar - specific timing ; second , longer duration forepaw movement was associated with a decorrelation of subthreshold activity ; third , spontaneous and sensory - evoked forepaw movements were signaled more strongly by l5 than l2/3 neurons . 
 together , our data suggest that the degree of translaminar synchrony is dependent upon the origin ( sensory , spontaneous , and movement ) of the synaptic input . 
Introduction
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Experimental Procedures
Author Contributions