through development , the wiring of the cortex is refined to receive and establish both local ( callaway , 1998 ; fino and yuste , 2011 ; ktzel et  al .
, 2011 ; ko et  al . , 2011 ; kozloski et  al . , 2001 ;
yoshimura et  al . , 2005 ) and long - range ( berezovskii et  al . , 2011 ; salin and bullier , 1995 ) projections that convey information for multimodal integration ( iurilli et  al . , 2012 ; mao et  al . , 2011 ) and normal cognitive function ( huang et  al . , 2014 ; reis marques et  al . , 2014 ; zhang et  al . , 2014 )
in many sensory cortical areas , the final organization of the network contains reoccurring features that include dedicated cortico - cortical ( cc ) versus cortico - thalamic ( ct ) projection pathways formed by the principal cells found in deep layer six ( l6 ) ( kumar and ohana , 2008 ; marx and feldmeyer , 2013 ; pichon et  al . , 2012
although the functional importance of these two output pathways is highlighted by their anatomical prominence , their precise physiological role in cortical and cortico - thalamic processing has proven difficult to dissect .
one approach to understanding the function of cortical pathways in general terms has been to chart regional projectivity ( oh et  al . ,
2014 ) with the view that the resultant wiring diagram may be used as a template for understanding the emergent physiological properties of underlying circuits ( douglas and martin , 2007 ; reid , 2012 ) . on the other hand
, while this approach can provide an overview of connection likelihood and strength  both within ( petersen and sakmann , 2000 ) and between ( binzegger et  al . , 2004 ; feldmeyer et  al . , 2013 ; oberlaender et  al . ,
2012 ) cortical layers and regions  such descriptions are often limited by their cellular and functional resolution ( oh et  al . , 2014 ) .
the difficulty in superimposing precisely the function and connectivity of individual elements within the circuit makes it extremely challenging to accurately attribute potential connectivity rules within a functionally heterogeneous population of neurons and prohibits a detailed understanding of network function .
it is well documented that even at a local level , neurons within the same cortical layer can show significant functional heterogeneity ( allman et  al . , 1985 ) .
in visual cortical areas , the diversity of sensory responses of individual neurons is highlighted by their degree of tuning to the orientation ( hofer et  al . , 2011 ; hubel and wiesel , 1968 ; kerlin et  al . , 2010 ; maruyama and ito , 2013 ; nauhaus et  al . , 2008 ; niell and stryker , 2008 ) , velocity ( priebe et  al . , 2006 ; roth et  al . ,
2012 ) , and direction of the motion of alternating bars of different luminance ( gratings ) ( allman et  al . , 1985
; hubel and wiesel , 1968 ; martin and schrder , 2013 ; roth et  al . , 2012 ) .
these functionally diverse populations are also thought to project to , and receive connections from , multiple cortical layers ( angelucci et  al . , 2002 ; bolz and gilbert , 1986 ; olsen et  al . , 2012 ;
thomson and bannister , 2003 ) forming interlaminar pathways for integration of both local and long - range input ( berezovskii et  al .
, 2011 ; de pasquale and sherman , 2011 , 2013 ; glickfeld et  al . , 2013 ;
hup et  al . , 1998 ; salin and bullier , 1995 ; schwarz and  bolz , 1991 ; wang and burkhalter , 2007 ; xu et  al . , 2007 ) .
understanding the functional heterogeneity of cortical networks therefore requires simultaneous analysis of their cellular composition ( helmstaedter et  al . , 2013 ; oberlaender et  al . ,
2012 ) , sensory response properties ( niell and stryker , 2008 ; oberlaender et  al . ,
2012 ) , input connectivity , and output projectivity ( briggman et  al . , 2011 ; denk et  al . ,
v1 we have undertaken an in  vivo single - cell analysis of the sensory response properties and connectivity of the l6 network that is known to contain a functionally heterogeneous population of principal cells ( hirsch et  al . , 1998 ; niell and stryker , 2008 ) that comprise cc- and ct - projecting neurons ( briggs , 2010 ; katz , 1987 ; kumar and ohana , 2008 ; marx and feldmeyer , 2013 ; pichon et  al . , 2012 ; thomson , 2010 ; usrey and fitzpatrick , 1996 ; zhang and deschnes , 1997 ) . by targeting retrograde transsynaptic tracing ( marshel et  al . , 2010 ; rancz et  al . , 2011 ;
wickersham et  al . , 2007 ) to individually recorded cells ( rancz et  al . , 2011 ) and
charting their brain - wide connectivity , we find that cc- versus ct - projecting neurons relay functionally distinct signals and are differentially innervated by higher - order cortical areas .
we performed blind in  vivo whole - cell recordings ( margrie et  al . , 2002 ) in v1 of anesthetized mice at a depth of 600 to 950  m from the pial surface ( n  = 81 cells ) .
on the basis of their recorded intrinsic properties including the initial action potential ( ap ) half - width , the mean frequency of firing , and input resistance , we could distinguish fast spiking cells from regular spiking ( rs ) neurons ( figure  s1 available online ) .
these criteria were used to identify the rs population of l6 cells ( n = 74 ) expected to mediate the cc and ct pathways under investigation in this study . to begin to explore the functional diversity of l6 principal cells , we first recorded the ap tuning of rs neurons in response to moving sinusoidal gratings ( figure  1a ) . the stimulus - evoked instantaneous firing rate of rs cells extended over a large range ( 0400  hz ) and was found to encompass a broad range of selectivity to the orientation and direction of the gratings ( figure  1a ) . in order to attribute these diverse response properties to specific types of l6 projection neurons , we performed morphological reconstructions ( n  = 16 cells ) and identified two distinct anatomical classes ( marx and feldmeyer , 2013 ; thomson , 2010 ; zhang and deschnes , 1997 ) ( figures s2a  s2e ) . the first group ( n  = 6 ) exhibited a large dendritic field ( convex envelope  = 0.0077   0.0011  mm ) and an elongated total basal dendritic length ( 1,884   303 
m ) with dendrites rarely extending beyond layer 4 ( figure  s2b ; figure  1b ) .
the dendritic tree of this neuronal class was morphologically diverse , displaying classical upright , but also inverted and tangential projecting apical dendrites ( figure  s2b ) .
their axonal morphology was strikingly elaborate ( total length 14,152   2,666  m ; figure  1b ) , with an extensive cortical horizontal span ( 1,070   223  m ; figure  s2e ) largely contained within layers 5 and 6 ( 13%   4% and 73%   4% of total length , respectively ; figures s2b and s2e ) .
arborizations often entered the white matter and returned to the cortex and regularly  extended into secondary visual areas .
these dendritic and axonal morphological properties are consistent with previous anatomical descriptions of cc - projecting l6 cells ( figure  1c ) ( pichon et  al . , 2012 ; zhang and deschnes , 1997 ) .
the second group contained cells that extended their apical dendrites beyond the l4-l5 border ( n  = 10 ) and , despite having a similar total dendritic length ( cc : 4,038   1,090  m versus 3,297   738  m [ sd ] ,
p > 0.05 ) , exhibited a significantly less elaborate dendritic morphology ( figures s2c and s2d ; figure  1b ) with a smaller convex envelope ( 0.0046   0.0004  mm , p  < 0.01 ) and a shorter total basal dendritic length ( 1,126   74  m , p  < 0.01 ; figures s2c and s2d ; movie s1 ) . for this class , dendrites exhibited a significantly higher branching frequency compared to the cc cells ( 0.91   0.06 versus cc  = 0.61   0.05 nodes/100  m , p  < 0.01 ) and had a lower mean spine density ( 0.52   0.02 [ n  = 4 cells ] versus cc : 0.64   0.06 [ n  = 3 cells ] spines/m , p  < 0.05 ; figure  s2d ) . despite reconstructing the axons of this cell type beyond the cortical boundaries used for the cc analysis
, axons appeared less elaborate ( figures 1b , s2c , and s2e ) , with a reduced total length ( 4,226   519  m ; p  < 0.01 ) and cortical horizontal span compared to the cc cells ( 299   22  m , p  < 0.001 ; figure  s2e ) . within the cortex , the axons of this group rarely extended beyond
the medial and lateral dendritic boundaries and projected vertically at least to layer 5 ( movie s2 ) .
all of the cells in this morphological class projected one axonal branch into the white matter and then turned laterally into the thalamic tract , reminiscent of l6 ct - projecting pyramidal cells ( kumar and ohana , 2008 ; pichon et  al .
inspection of the intrinsic properties of the reconstructed neurons revealed several biophysical features ( brumberg et  al .
, 2003 ; kumar and ohana , 2008 ) ( initial instantaneous ap firing rate , early accommodation of firing rates , slope of the relationship between the evoked ap firing frequency ( f1 ) and the amplitude of underlying injected current ( i ) , the amount of hyperpolarization - evoked membrane potential depolarization [ membrane sag ] ; see figures s3a and s3b ) that may be used to electrophysiologically distinguish between regular spiking neurons according to their axonal projectivity . using only these intrinsic parameters , we performed a cluster analysis on all regular spiking cells ( n  = 74 cells ; figure  s3c ) that produced two main clusters , whereby one group contained all of the morphologically confirmed cc neurons and the other contained the ct - projecting cells .
these intrinsic parameters therefore reflect biophysical regularities of the cc and ct populations and were used to assign all l6 regular spiking cells recorded in this study ( figure  s3c ) . following this classification procedure
, we could determine that ap firing in cc cells was broadly tuned ( figure  2a ) since spikes could be evoked by gratings moving in several directions and typically included the orientation orthogonal to the preferred ( figures 2a and 2b ) .
in stark contrast , the firing of ct cells was highly orientation tuned , predominantly firing in only one direction ( osioutput cc [ n = 15 ] : median  = 0.6 , q1  = 0.25 , q3  = 1 ; ct [ n = 19 ] : median  = 1 , q1  = 1 , q3  = 1 , p  < 0.01 ; figures 1a and 2c2e ) .
while this cluster - analysis - based classification of cell function reveals differences in tuning and that specific , highly precise direction signals are conveyed to thalamic targets , it also indicates that cc cells have a high overall probability of evoking aps ( figure  2f ) .
this may suggest that differences in cell excitability could explain poor tuning in cc cells that would be consistent with the lower rheobase ( kumar and ohana , 2008 ) and initial ap bursting properties ( kumar and ohana , 2008 ; ledergerber and larkum , 2010 ) observed for this population ( figures s3a and s3b ) .
we therefore next sought to establish the extent to which the underlying evoked input could account for the broad versus narrow range of ap tuning profiles observed in cc and ct cells , respectively .
we first quantified the integral of the evoked postsynaptic potential ( psp ) and determined that cc cells receive significantly more net depolarization than ct cells ( cc  = 3.8   0.5  mv.s [ n  = 17 ] versus ct  = 2.3   0.2  mv.s [ n  = 28 ] , p  < 0.01 ; figures 3a and 3b ) . however , the relative amount of depolarization per grating stimulus revealed little preference for its orientation ( osi pspintegral : cc  = 0.18   0.02 versus ct  = 0.18   0.02 , p > 0.9 ; figures 3c and 3d ) . on the other hand , for both cell types the psp peak amplitude ( figures 3a and 3b ) displayed a significant preference for grating orientation ( osi l6 pspintegral  = 0.18   0.01 versus psppeak  = 0.28   0.03 , p  < 0.01 ) and direction ( dsi l6 pspintegral  = 0.1   0.02 versus psppeak  = 0.23   0.03 , p  < 0.01 ) .
this improved tuning of the psp peak was most striking for ct cells ( osi : ct psppeak  = 0.33   0.03 versus ct pspintegral  = 0.18   0.02 , p  < 0.01 ; figures 3e and 3f ) such that already for gratings presented at 30 from the preferred direction , the average amplitude of the peak depolarization was significantly reduced ( psppeak : pref . 
= 14.6   1.2  mv versus   30  = 9.1   1  mv , n  = 28 , p  < 0.01 ; figure  3e ) .
compared to its integral , the peak amplitude of the psp therefore conveys the most accurate orientation and direction information , whereby the inputs onto ct cells are the most strongly tuned ( osi psppeak : cc  = 0.2   0.04 versus ct  = 0.33   0.03 , p  < 0.01 ; figure  3f ) .
this indicates that the cc population receives comparatively strong yet broadly tuned synaptic drive , while the ct cells receive a highly tuned orientation signal .
although ct cells show sharp synaptic and ap tuning , the orientation selectivity of the input may not necessarily cause the apparent exquisite tuning of ap output .
ct cells are extremely sparse firing ( cc  = 1.2   0.61  hz versus ct  = 0.1   0.04  hz , p  < 0.05 ; figure  2f ) .
for example , over multiple stimulus repetitions cts may only discharge one or two aps .
we therefore looked to establish causality in psp - ap tuning by injecting psp waveforms evoked in both cc and ct cells ( figure  4a ) back into individual neurons in the absence of visual stimulation ( figures 4a and 4b ) .
we found that the injected psps faithfully reproduced the grating - evoked cc and ct cell ap tuning , irrespective of the identity of the injected neuron ( figures 4b and 4c ) .
thus , the ap tuning of these two groups ( figures 4c and 4d ) results directly from the cell - type - specific dynamics of the evoked psp ( cc : ccinject median  = 0.47 , q1  = 0.31 , q3 
= 0.69 versus ctinject median  = 1 , q1  = 0.92 , q3  = 1 , n  = 7 , p  < 0.05 ; ct : ccinject median  = 0.44 , q1  = 0.4 , q3  = 0.56 versus ctinject median  = 1 , q1  = 0.94 , q3  = 1 , n  = 7 , p  < 0.05 ; figure  4d ) .
consistent with these data , we also find that the specificity of the grating - evoked ct spiking is a highly reliable indicator of the orientation preference of the underlying synaptic input ( figure  4e ) .
synaptic signaling onto l6 principal cells therefore produces two functional distinct distributions of tuning profiles , whereby sparse , highly orientation - selective information is relayed to thalamic target areas .
these data show that across the population , different morphological classes of l6 cells relay specific visual information and form unique signaling pathways within and outside the v1 circuit .
one might therefore expect these functionally discrete cc and ct populations to be targeted by specific upstream pathways . to begin examining the functional specificity of cc and ct connectivity , we targeted retrograde transsynaptic tracing from individually recorded neurons using a glycoprotein deficient form of the rabies virus encapsulated with the avian  sarcoma and leucosis virus envelope protein ( rv ) ( wickersham et  al . , 2007 ) . by performing whole - cell recordings with internal solutions containing dna vectors ( rancz et  al . ,
2011 ) , we could drive the  expression of the envelope protein receptor ( tva ) and the  rv glycoprotein ( rvg ) that are required for single - cell targeted infection and monosynaptic retrograde spread of rv ( figure  5a ) . immediately following whole - cell recording with the plasmid - containing internal solution
, we injected rv and up to 12  days later performed whole - brain serial two - photon tomography ( osten and margrie , 2013 ; ragan et  al . , 2012 )
( figure  5b ) to chart the spatial profile of presynaptic cells . to directly assess the regularity of connectivity onto these two classes of l6 neurons ,
the intrinsic , synaptic , and ap tuning response properties were first recorded ( figure  5c ) .
following singe - cell rabies tracing , we found that for cc cells more than 90% of the labeled presynaptic neurons ( 138   21 labeled cells , n  = 3 ; figures 5d and 5e ; movie s3 ) were located locally within v1 .
the majority of these presynaptic cells were observed in layers 5 ( 26%   5.2% ) and 6 ( 38.9%   7.6% ) although input from layer 2/3 ( 17.6%   3.3% ) and to a lesser extent layer 4 ( 9.6%   0.5% ) was also apparent ( figures 5e and 5f ) .
very few long - range projecting cells were observed , though a small fraction of presynaptic neurons were found in areas including thalamus ( 0.5%   0.3% ) , secondary visual ( 2.9%   0.7% ) , and retrosplenial ( 1.4%   0.4% ) cortices .
single - cell rabies tracing in ct cells revealed almost three times the number of presynaptic cells when compared to cc neurons ( 383   70 labeled cells , n  = 4 ; figures 6a and 6b ; movie s3 ) . in relative terms , ct cells received almost the identical fraction of inputs from layer 2/3 ( 18.8%   3.7% ) and layer 4 ( 10.7%   1.7% ) within v1 as that observed for cc cells ( figures  6c and 6d ) .
in contrast , ct neurons received more than 20% of input from secondary visual and retrosplenial cortices ( v2  + rsp : cc  = 4.2%   0.7% versus ct 20.8%   5.2% , p  <  0.05 ; figures 6c and 6d ) .
consequently , ct cells had relatively fewer input cells located in deeper layers 5 and 6 of v1 ( l5  = 17.7%   2.4% and l6  = 29.5%   3.2% ; figure  6d ) .
our electrophysiological and input tracing data indicate that the upstream network connectivity of individual l6 neurons is not random but respects the morphological and hence the functional identity of the target neuron .
if these single - cell - based connectivity maps highlight general rules of monosynaptic connectivity onto these two classes of neurons , one might expect genetically targeted input tracing onto a specific population to show similar connectivity profiles . to examine the correspondence between our single ct cell - based maps and the broader l6 ct population , we took advantage of the fact that in l6 of mouse v1 , ct - projecting cells are known to selectively express neurotensin receptor 1 ( ntsr1 ) ( gong et  al . , 2007 ) . by using a  cre - ntsr1 mouse line , we targeted injections of cre - dependent aav helper viruses to drive expression of the rv glycoprotein and the avian receptor protein across the ct population ( n  = 4 ; figures 6e and 6f ) . when comparing to cc cells , presynaptic connectivity in cre - ntsr1 mice was widespread ( figure  6 g ) , with relatively fewer cells in v1 ( cc  = 92.1%   1.2% versus cre - ntsr1  = 41.3%   2.4% , p  < 0.01 ) and substantially more labeled neurons located in secondary visual and retrosplenial cortices ( v2  + rsp cc  = 4.2%   0.7% versus cre - ntsr1  = 40.8%   1.5% , p  < 0.01 , ct  = 20.8%   5.2% , p  < 0.05 ; figures 6 g and 6h ) . taken together , despite receiving most of their synaptic drive  from neurons located within the v1 circuit , the l6 cc pathway conveys visual motion signals that cover a broad spectrum of orientation selectivity ( figure  7 ) . on the other hand ,
cells relaying motion information to thalamus output exquisitely tuned orientation- and direction - related signals and received substantial widespread innervation from higher - order cortical areas known to convey visual and spatial information ( figure  7 ) .
this study shows that in v1 , the functional diversity of l6 can be attributed to specific populations of output neurons that are embedded in different anatomical microcircuits .
we show that the output tuning of cc and ct cells can be directly attributed to the tuning profile of the somatic depolarization , rather than to their difference in intrinsic properties .
at least for the stimuli used here , the cc and ct cell populations are wired to receive  functionally distinct , direction - related synaptic signals and therefore appear to play distinct roles in visual processing .
we reveal that several intrinsic biophysical parameters of l6 neurons recorded in  vivo may be used to classify cells according to their morphological identity and projection targets .
although we have not directly determined the differential impact of these properties on the integration of signals in the dendrites of cc and ct neurons , it seems likely they will impact psps arriving at the soma ( chadderton et  al . , 2014 ) .
experiments in which we injected previously recorded visually evoked somatic responses back into cc and ct cells indicate that their output tuning can not simply be explained by differential intrinsic membrane properties expressed proximal to the axonal initial segment or soma .
for example , on the basis of their dendritic profile , claustrum - projecting neurons in cat have morphological features similar to that of ct - projecting cells described here ( katz , 1987 ) .
however , previous studies injecting retrograde tracers into the claustrum ( carey and neal , 1985 ) indicate that in rat , claustrum - projecting cells in the visual cortex are  confined to the deep layers of secondary visual areas .
the axonal projection from our population labeling experiments indicated that cre - ntsr1 l6 cells in mouse v1 do not target the claustrum .
also , on the basis of our biocytin reconstructions of cc cell axons , we find no evidence for a direct claustrum projection .
however , since cc cells in v1 can extend their axons into deep layers of secondary visual areas , an indirect primary visual cortical - claustrum pathway may exist .
our data show that the broad stimulus selectivity in l6 can be  attributed specifically to the cc - projecting population .
these cells receive more net depolarization during drifting gratings , yet they are , on average , only modestly tuned to stimulus orientation . in contrast , ct neurons were more selective for stimulus orientation and/or direction . since we have not directly determined the functional identity of the presynaptic cells providing visual information , the precise contribution of intra- versus interlaminar connectivity to the subthreshold tuning of cc and ct neurons remains to be established . in v1 ,
subsets of gaba - ergic interneurons also exhibit broad stimulus selectivity ( sohya et  al . , 2007 ; kerlin et  al . , 2010 ;
their tuning is believed to arise from them receiving input from many local pyramidal cells tuned to different stimulus orientations ( chen et  al .
, 2013 ; hofer et  al . , 2011 ; bock et  al . ,
our data indicate that cc cells receive substantial synaptic input from within l5 and l6 ( mercer et  al . , 2005 ; zarrinpar and callaway , 2006 ) and raise the possibility that such rules of local functional convergence are not specific to inhibitory interneurons but may also apply to this principal cell type .
it also remains a possibility that multiple subclasses of cc cells  differentially tuned to orientation and/or direction  receive input from nonoverlapping constellations of presynaptic cells .
the paucity of information about the influence of cortex on thalamus arises , at least in part , from the functional and morphological diversity of l6 neurons . compared to cc cells , our data show that synaptic drive onto ct cells is highly selective for stimulus orientation and/or direction and that ct cell firing is extremely sparse .
the fact that ct axons project to thalamic structures , including the dorsal lateral geniculate nucleus and the reticular nucleus , indicates that this highly selective feedback signal may be used for stimulus - specific thalamic gain control via excitatory and inhibitory modulation ( mease et  al . , 2014 ; olsen et  al . , 2012 ) .
this highly tuned ct output signal is also expected to directly impact activity within layers 5 and 6 and , via ascending polysynaptic pathways , provide modulation of upper cortical layer activity ( bortone et  al .
the observation that cc and ct cells have a similar total dendritic length ( oberlaender et  al . ,
2012 ) and that cts have a lower spine density appears at odds with our observation that ct cells receive input from more than double the number of presynaptic cells .
this may suggest that the number of contacts per connection is reduced in ct cells such that cts are more densely innervated ( in terms of the number of presynaptic cells ) compared to cc cells .
alternatively , it is possible that cell - specific tropisms may impact or bias the retrograde transmission of the rabies virus in some way .
however , the fact that we find presynaptic cells  located in distant areas , including the thalamus , indicates that long - range inputs can be labeled in both cell types .
the increased connectivity onto ct cells may therefore reflect a dynamic role in integration of information converging from across functionally nonoverlapping upstream networks .
dense innervation of ct cells may also be explained by them receiving input from a larger number of inhibitory neurons that ensure the sharpening of ct responses ( chadderton et  al . , 2014 ) . while our study does not attempt to explain the relative contribution of presynaptic neurons to stimulus selectivity in l6 principal cells , these data do however
show that the sparsely encoded information about stimulus direction is processed by a specific subpopulation of l6 neurons that are biased in receiving input from secondary visual and retrosplenial cortices .
this l6  pathway may therefore mediate thalamic integration of both cortical visual and egocentric information .
integration of self - motion  and head - direction signals ( clark et  al . , 2010 ) within v1 l6 ct cells could optimize object motion detection ( hup et  al . , 1998 ) by providing a contextual influence on thalamic relay neurons .
approaches that combine physiological analysis with dense electron microscopy - based reconstruction are elucidating the circuit organization mediating stimulus motion processing at the very early stages of the visual system ( briggman et  al .
the relation between function and connectivity in large - scale cortical circuits however remains exceedingly challenging ( bock et  al .
the combination of in  vivo single - cell physiology and retrograde monosynaptic tracing enables identification of local and global projections onto individual cells whose subthreshold sensory response properties have been characterized , an approach that permits the generation of cortical wiring diagrams with single - cell and functional resolution .
as recently shown in other sensory systems ( angelo et  al . , 2012 ) , here we find that l6 cc- versus ct - projecting cells have distinct intrinsic and functional properties .
furthermore , these two classes of projection cells are embedded within distinct wiring motifs that indicates top - down , targeted innervation of l6 microcircuits may provide contextual modulation during sensory computation .
adult c57/bl6 mice ( 58  weeks old ) were anaesthetized with a mixture of fentanyl ( 0.05  mg / kg ) , midazolam ( 5.0  mg / kg ) , and medetomidin ( 0.5  mg / kg ) in saline solution ( 0.9% ; intraperitoneal ) and supplemented as necessary ( 20% of initial dose ) .
mice were head fixed using nonpuncture ear bars and a nose clamp ( sg-4n , narishige , japan ) , and their body temperature was maintained at 37c 38c using a rectal probe and a heating blanket ( fhc , bowdoinham , me , usa ) .
an incision was made in the scalp and a small craniotomy was drilled above the primary visual area of the cortex using a dental drill ( osada electric , japan ) and the dura removed .
following recordings , the craniotomy was sealed using a silicone sealant ( kwik - sil , world precision instruments ) and the scalp sutured .
anesthesia was reversed by injection of a  mixture of naxolon ( 1.2 mg / kg ) , flumazenil ( 0.5 mg / kg ) , and atipamezol ( 2.5  mg / kg ) in saline solution ( 0.9% ) .
the wound was infiltrated with lidocaine and an antibiotic ( cicatrin , glaxosmithkline , uk ) topically applied . during initial recovery ,
mice were kept in a climate - controlled chamber ( harvard apparatus , holliston , ma , usa ) for 34  hr under observation .
all procedures were approved by the local ethics panel and the uk home office under the animals ( scientific procedures ) act 1986 . in  vivo whole - cell recordings
data were filtered at 4 khz and digitized at 1020 khz using an itc-18 a / d - d / a  interface ( instrutech , heka elektronik , germany ) and the neuromatic package ( http://www.neuromatic.thinkrandom.com ) under igor pro 5 ( http://www.wavemetrics.com ) .
intracellular solutions for whole - cell recordings were made up in concentrated stock ( two times the final concentration ) to allow for biocytin or plasmid addition .
the final concentrations were ( all from sigma - aldrich or vwr international , uk ) 110  mm k - methanesulphonate , 40  mm hepes , 6  mm nacl , 0.02  mm cacl2 , 3  mm mgcl2 , 0.05  mm egta , 2  mm na2atp , 2  mm mgatp , and 0.5  mm na2gtp ; the ph was adjusted to  7.28 using koh .
the final osmolarity after adding either 0.5% biocytin or suspended plasmids was adjusted to the range of 280294 mosm .
intracellular solutions were filtered through a 0.22  m pore size centrifuge filter ( costar  spin - x ) .
plasmid concentrations were verified by spectrophotometry ( nanodrop 2000 , thermo scientific ) : 200  ng/l rvg plasmid and 40  ng/l tva plasmid . in some cases an xiap plasmid ( 40  ng/l ) was also included . for individual cc and ct tracing experiments ,
only one whole - cell recording was performed in each brain within a maximum of three attempts .
stimuli were presented on a 56  cm lcd monitor positioned 21  cm from the contralateral eye spanning 72 ( in elevation ) and 97 ( in azimuth ) of the animal s visual space .
stimuli consisted of sinusoidal gratings ( spatial frequencies including 0.01 , 0.025 , and 0.04 cycles/ [ niell and stryker , 2008 ] ) drifting in 1 of up to 12 directions at a temporal frequency of 2 cycles / s . in cells where we compared two or more spatial frequencies , we observed no effect on the psp integral , evoked firing rate , or osi . for each trial , gratings were presented in the following manner : stationary ( 1 s)-moving ( 2 s)-stationary ( 1 s ) . gratings were presented in sequences according to their maximal difference ( + 210 ) .
jitter in the onset of the stimulus caused by the refresh rate of  the monitor was compensated for by implementing a small photodiode in front of the screen , which allowed for alignment of the onset of the stimulus .
we excluded direction - nonspecific onset responses by analyzing the membrane voltage during the second half of the 2  s drift .
this analysis time window is expected to include evoked feed - forward and feed - back signals ( ringach et  al . ,
however , when the entire stimulus duration is analyzed , there remains a significant difference between cc and ct tuning for both orientation and direction ( p  < 0.05 ) .
the membrane potential responses for each direction were determined by averaging across stimulus repetitions ( four to six trials ) . for analysis of the subthreshold membrane potential , aps were clipped on each side of the peak at the level where the membrane potential variance ( vm standard deviation ) equaled the mean variance in the absence of spiking .
linear interpolation was then used to join the membrane voltage traces . for the analysis of the peak depolarization ( psppeak , in mv )
, the most depolarized membrane potential value was used for directions in which evoked spiking was recorded .
the analysis of the integral of the responses ( pspintegral , in mv.s ) was performed of average traces for each direction .
the output response to drifting gratings was calculated by detecting action potentials in each trial and averaging the spiking rate for each direction . for a given cell ,
the tuning profile was analyzed by first calculating the vector average of the responses ( for pspintegral , psppeak , or output ) for the 12 directions .
the direction closest to the value of the vector average was then defined as the preferred direction .
the response to the orthogonal direction was calculated as the average of the two sets of responses recorded for the pref  + /2 ( ortho1 ) and pref  /2 ( ortho2 ) .
the null direction response is defined as the response recorded at pref  +  ( null direction ) .
the orientation and direction indexes ( osi and dsi , respectively ) are defined as follows : osi  = ( pref  ortho)/(pref  + ortho ) and dsi  = ( pref  null)/(pref  + null ) . for experiments where the evoked synaptic potentials were injected into cells ,
the last 1  s of the response to four directions was used ( pref , ortho1 , null , ortho2 ) .
the amplitude of the current waveform to be injected was first determined by injecting the same cell type  input  and adjusting the current amplitude such that at least one spike could be evoked .
current - voltage relationships were obtained from each neuron by injecting step currents ranging from 400 to 0  pa ( in  + 50  pa steps of 600  ms ) and depolarizing currents from 0  pa to two times the rheobase ( in  + 25  pa steps of 600  ms ) .
briefly , the initial instantaneous frequency of the first two action potentials ( f1 ) was extracted from rheobase to two times the rheobase , and the slope of the relationship between the initial instantaneous frequency and the current injected was calculated ( f1-i slope ) .
in addition , the initial instantaneous frequency and the instantaneous frequency 200  ms after the onset of the current injection at two times the rheobase were calculated ( f12xrb and f2002xrb , respectively ) in order to assess the early accommodation index of spiking ( eacc index  = ( ( f12xrb  f2002xrb)/f12xrb )  100 ) . for quantification of membrane potential sag ,
hyperpolarizing current steps ( 400  pa , 600  ms ) were injected . to determine the sag potential amplitude , the most negative membrane potential value determined in the first 100  ms ( peak )
was compared to the average membrane potential recorded during the last 200  ms ( steady state ) of the current step period .
the absolute difference in these two values was used as the sag potential amplitude . when comparing these intrinsic properties for morphologically identified cc ( n  = 6 ) and ct cells ( n  = 10 ) , statistically significant differences were observed for all four parameters ( p  < 0.05 ) . on average ,
there was found to be no significant difference in input resistance between cc and ct cells . to classify cells a cluster analysis ( cauli et  al . , 2000
; thorndike , 1953 ; ward , 1963 ) using f12xrb , the f1-i slope , the eacc index and sag potential amplitude was performed .
cells sharing similar parameters are expected to be close to one another in multidimensional euclidean space .
the number of clusters was defined using the thorndike method ( thorndike , 1953 ) , by comparing the within - cluster linkage distances .
student s t test and wilcoxon tests were used to determine the significance of normally and nonnormally distributed data , respectively . for tissue processing - related information ,
see the supplemental information . for neuronal reconstructions , recorded neurons were either ( 1 ) filled with biocytin and mice immediately perfused or ( 2 ) loaded via the patch pipette with a dna vector to drive expression of gfp . in the latter case
, mice were returned to their home cage for up to 72  hr , after which they were anesthetized and transcardially perfused .
for tracing of gfp - labeled cells , the cells were first immunostained as described in the supplemental information .
cells were reconstructed in 3d using neurolucida ( mbf bioscience ) under an olympus bx61 at magnifications ranging from 4 to 100. final 3d reconstructions were analyzed in neurolucida explorer ( mbf bioscience ) .
the field volume of dendrites and axons was calculated by computing the 3d convex hull , which is the convex volume enclosed between the neuronal process ends . to further characterize neuronal processes
, we plotted the number of intersections between processes and concentric spheres of the gradually increased radius ( + 10  m ) centered at the cell body ( sholl , 1953 ) .
cells were first centered according to the location of the soma , then aligned with respect to the pial surface .
dendritic and axonal trees were then separately exported as vectorial models from neurolucida to vrml or wavefront obj files .
the files were opened in a 3d graphic software ( v. 2.68 , the blender foundation , http://www.blender.org ) , and model lines were converted to mesh lines and then to tubes of identical diameter . finally , the meshes were converted to a 3d image stack ( grothausmann et  al .
stacks were then opened in imagej ( fiji , wayne rasband , nih ) using the metaimage reader / writer plugin , converted to 32 bits image stacks and low pass filtered .
stacks of each cell type were averaged and the result projected as an integral in the coronal plane .
finally , images were scaled by the ratio of the integral of the resultant stack and the average total length of processes in the given cell type .
the movies of density map rotations were produced with a custom version of the imagej built - in 3d - projector plugin .
the plugin was modified to work with 32 bit images and produce projections as sums . for spine counting , either biocytin - filled or gfp - expressing cells were imaged using wide - field ( olympus bx61 , 100/1.25 numerical aperture [ na ] ) or confocal ( leica sp5 , 40/1.3 na ) microscopy .
for each cell , spines were manually tagged in between three and seven 80-m - long dendritic segments .
the length of each dendritic segment was extracted on the basis of either neurolucida reconstructions or using a plugin ( simple neurite tracer ) in image j. for each cell , we sampled at least one dendritic segment per cortical layer
. fixed whole brains were embedded in 4% agar and placed under a two - photon microscope containing an integrated vibrating microtome and a motorized x - y - z stage ( osten and margrie , 2013 ; ragan et  al . , 2012 ) .
coronal images were acquired via three optical pathways ( red , green , and blue ) as a set of 6 by 9 tiles with a resolution of 1(x )  1(y ) m obtained every 5  m ( z ) using an olympus 10 objective ( na  = 0.6 ) mounted on a piezoelectric element ( physik instrumente ) . following acquisition ,
image tiles were stitched using fiji and custom routines , including a custom version of the fiji stitching plugin ( preibisch et  al . , 2009 ) allowing the parallel processing of several image planes for higher throughput .
briefly , the illumination profile was computed from the average of all tiles across the brain and used to normalize the individual tiles that were then stitched together using a combination of the readout from the microscope stage and cross - correlations .
the coordinates of each marked cell were then used to position markers ( red spheres ) in the whole - brain image stack .
the brain regions were determined using a standard mouse brain atlas ( franklin and paxinos , 2008 ) . for cc , ct , and cre - ntsr1 rv - labeling , we counted 138   21 , 338   116 , and 4,088   945 cells , respectively . in 2 of the 11 data sets , fixed whole brains
were immediately sliced using a standard vibratome , antibody - stained , and then imaged using a confocal microscope ( 1.8(x )  1.8(y )  5(z ) m , 10 and 20 objective ; leica sp5 ) .
cell counting in these cases were manually performed on individual coronal slices . for combined single - cell physiology and rabies virus tracing experiments from cc neurons , labeled presynaptic cells
were identified and counted for nine different brain regions located in the ipsilateral hemisphere and include v1 , thalamus ( different nuclei pooled ) , hippocampal formation , cortical associational areas ( including temporal associational cortex and parietal cortex ) secondary visual cortex ( including lateral and medial ) , retrosplenial cortex , and auditory cortex ( primary and secondary ) .
for histograms , all of these regions ( excluding v1 , thalamus , v2 , and rsp ) are represented by the  others  category . in the contralateral hemisphere , cells were only found in the secondary visual cortex . for ct experiments , presynaptic cells were found in additional areas and include the hypothalamus , somatosensory , motor , cingulate cortices , and contralateral v1 . for histograms ,
all of these additional regions were included ( excluding v1 , thalamus , v2 , and rsp ) and are represented by the  others  category . for cre - ntsr1 tracing , cells were found in all of the above ct - related brain  areas .
in addition , some cells were found in over 30 other regions ( not  described ) .
all of these regions ( excluding thalamus , v2 , and rsp ) were allocated to the  other 
 summarysensory computations performed in the neocortex involve layer six ( l6 ) cortico - cortical ( cc ) and cortico - thalamic ( ct ) signaling pathways . 
 developing an understanding of the physiological role of these circuits requires dissection of the functional specificity and connectivity of the underlying individual projection neurons . by combining whole - cell recording from identified l6 principal cells in the mouse primary visual cortex ( v1 ) with modified rabies virus - based input mapping 
 , we have determined the sensory response properties and upstream monosynaptic connectivity of cells mediating the cc or ct pathway . 
 we show that cc - projecting cells encompass a broad spectrum of selectivity to stimulus orientation and are predominantly innervated by deep layer v1 neurons . 
 in contrast , ct - projecting cells are ultrasparse firing , exquisitely tuned to orientation and direction information , and receive long - range input from higher cortical areas . 
 this segregation in function and connectivity indicates that l6 microcircuits route specific contextual and stimulus - related information within and outside the cortical network . 
Introduction
Results
Discussion
Experimental Procedures