female c57bl6j mice aged 4 - 6 weeks were used in both anaesthetised and awake recordings , and all procedures were performed under license from the uk home office in accordance with the animal ( scientific procedures ) act 1986 . anaesthesia was induced with chlorprothixene ( 10 mg kg ip ) and urethane ( 1.5 mg kg , 10% w / v ip ; all data presented in figs.1 - 4 ) , or with chlorprothixene ( as above ) and isoflurane ( 0.25 - 1% ) . atropine sulfate ( 0.07 mg kg sc ) was injected to maintain clear airways , dexamethasone ( 2 mg kg i m ) was used to prevent edema , and rimadyl ( 4 mg kg sc ) was administered as an analgesic and anti - inflammatory . a tracheotomy was performed ( during urethane experiments ) , and animals were passively ventilated with pure o2 . the stimulated eye was protected with a gas permeable contact lens . was implanted over monocular regions of visual cortex ( 0.5 mm a , 2 mm l from lambda suture ) . two small ( < 0.5 mm ) craniotomies were drilled ( within 200 - 400 m of each other ) for the separate entry of lfp and patch pipettes . a third craniotomy was drilled over anterior retrosplenial cortex for insertion of an ag / agcl reference wire . warm hepes buffered artificial cerebrospinal fluid ( acsf , in mm : 135 nacl , 5.4 kcl , 5 hepes , 1 mgcl2 , 1.8 cacl2 ; ph 7.3 ) filled the chamber to prevent desiccation and maintain ionic balance . awake mice were implanted with a lightweight headplate with chamber under isoflurane anaesthesia , and allowed to recover for two days . on the third day , animals were acclimated to head fixation for between 10 - 60 minutes , with sweet liquid reward given at the start and end of training . the duration of training was increased over 4 - 5 days until mice were sitting comfortably for 3 hours . on the day of recording , craniotomies as described above were performed under isoflurane anaesthesia , the animal was allowed to recover for 3 hours , and a single 3 hour recording session commenced . they were also given sweet liquid reward every 30 minutes to keep them comfortable and hydrated . patch pipettes ( 4 - 7 m ) were pulled on a narishige pc-10 and filled with ( in mm ) : 135 k gluconate , 6 kcl , 10 hepes , 4 mgatp , 0.3 na2atp , 0.1 egta , 8 phosphocreatine , ph adjusted to 7.3 with koh , 290 - 295 mosm . for voltage clamp recordings , k gluconate was substituted with 140 cs methanesulfonate . 0.5 qx-314 and 5 tea were included to block voltage gated na and k conductances . while searching for neurons , diffusion of blockers was limited by using low pressures ( 20 - 30 mbar ) ; more importantly , the lfp electrode served as an internal control for visual responsiveness . whole cell recordings were obtained with a multiclamp 700b in voltage clamp mode using standard techniques . average seal resistances for anaesthetised and awake voltage clamp recordings were 3.8  1.7 g and 6.3  1.3 g , respectively . seal resistances for anaesthetised and awake current clamp recordings were 4.5  1.2 g and 5.3  1.1 g , respectively . initial access resistance ranged from 9 - 30 m. this value generally increased within a few minutes and then stabilized . if initial access was greater than 50m the recording was not included for further analysis . access resistance during visual stimulation was not different between anaesthetised and awake recordings in current clamp ( anaesth : 36  6 m ; awake : 39  3 m ) or voltage clamp ( anaesth : 38  5 m ; awake : 38  4 m ) . series compensation averaged 63  1% , yielding an effective series resistance of 22  3 m and 24  3 m during anaesthetised and awake voltage clamp recordings , respectively . this method limits distortion of the somatically recorded current by ongoing voltage fluctuations and membrane capacitance ; however , due to the necessity of intrinsic current blockade , it also masks potential contributions of active dendritic conductances . this method also requires multiple trials and holding potentials ; it thus can not assess the simultaneous relationship of excitation and inhibition within single trials . as in previous studies , to better estimate excitatory conductance we used a holding potential of about + 20 mv rather than 0 mv , as this mitigates the voltage decay across the dendrites . input resistance and series resistance were monitored between protocols with current pulse trains or voltage steps . firing rate adaptation to supra - threshold pulses and broad spike - widths confirmed that our recordings were from regular spiking pyramidal neurons . across all anaesthetised ( n=22 ; 305  27 m ) and awake ( n = 30 ; 279  27 m ) whole - cell recordings , there was no significant difference in laminar depth ( p = 0.94 ) , as estimated from the micromanipulator reading after it was zeroed upon contact with the cortical surface . vm was low - pass filtered at 20 khz ; during voltage clamp , i m was low - pass filtered at 2 khz ; lfp was filtered from 0.1 - 100 hz . data was acquired via a national instruments board and was synchronized to stimulus onset by a photodiode signal . the lcd monitor was positioned 24 cm away from the mouse and at 0 elevation and 30 - 45 azimuth . the azimuthal position was adjusted so that the centre of the lfp receptive field was roughly centred on the screen . stimuli were presented on a grey background , and randomly interleaved across space , with 8  20 repetitions per stimulus location . a blank screen ( the grey background ) was randomly presented every 11 stimulus presentations , for the same duration as the stimulus trials . the unstimulated eye was gently sutured ( anaesthetised ) or shielded by black aluminium foil ( awake ) . throughout the entire recording session , this was to ensure that spontaneous cortical state was not affected by transitions to and from complete darkness of the monitor . in awake recordings , upon establishing stable whole - cell configuration , 1 - 2 minutes of spontaneous activity was recorded before the presentation of any flashed bars . 1,4 ) ; there were no systematic differences with the interleaved blank data . in some awake protocols we shortened the interstimulus interval ( isi ) to 300 there was no difference between the magnitude or duration of evoked conductances with longer ( 1.5 s ) vs. shorter isi ( n = 3 ) . for measurements of vm ( figs . 3 ) any spikes were median filtered ( 7 ms ) and the resulting trace smoothed . in a subset of experiments , we optimized the orientation of flashed bars for each individual neuron ( fig . s10 ) by first presenting full screen drifting gratings ( 50% contrast ; spatial frequency 0.03 cycles / deg ; temporal frequency 2 hz ; 2s duration ) varying randomly in orientation ( 30 degree steps ) . the preferred orientation was designated as the one that evoked the largest number of spikes ( or largest depolarization of vm ) , while the orthogonal orientation was 90 degrees away from preferred . bars identical to all other experiments ( 9 degree width ; 100 ms duration , 1.5 s or 0.3 s isi ) were then presented randomly across space along the two axes defined by the preferred and orthogonal orientations . to obtain robust estimates of vrev and g during the responses , we considered the response onset as the time when g was 5 - 10% above baseline for 10 consecutive ms . across the population of neurons , this time point occurred 30 - 40 ms after stimulus onset , so vrev , g , ge , and gi were averaged starting 40 ms after stimulus onset , across the population ( fig . 4 ; n = 5 urethane anaesthesia , n = 8 awake ; both groups tested identically with vertically oriented bars ) . additional recordings of conductances under isoflurane anaesthesia ( n = 1 ) , and in awake mice presented bars varying in orientation ( n = 4 ) were not included in these plots to maintain equivalent group comparison ( but see supplementary fig . 2 ) was analysed by sorting trials ( within neurons ) by pre - stimulus vm level . the upper- and lower - most quartiles ( that is , the 25% most depolarized and 25% most hyperpolarized non - overlapping trials ) were then averaged across the population , and largely correspond to trials when stimuli where delivered in up versus down states , respectively . blank stimuli were also sorted in this manner in order to estimate the spontaneous state transitions from down to up , and vice versa , in the absence of sensory stimulation . anaesthesia was induced with chlorprothixene ( 10 mg kg ip ) and urethane ( 1.5 mg kg , 10% w / v ip ; all data presented in figs.1 - 4 ) , or with chlorprothixene ( as above ) and isoflurane ( 0.25 - 1% ) . atropine sulfate ( 0.07 mg kg sc ) was injected to maintain clear airways , dexamethasone ( 2 mg kg i m ) was used to prevent edema , and rimadyl ( 4 mg kg sc ) was administered as an analgesic and anti - inflammatory . body temperature was maintained at 37.1 with a feedback regulated heating pad . a tracheotomy was performed ( during urethane experiments ) , and animals were passively ventilated with pure o2 . the stimulated eye was protected with a gas permeable contact lens . a small headplate with chamber was implanted over monocular regions of visual cortex ( 0.5 mm a , 2 mm l from lambda suture ) . two small ( < 0.5 mm ) craniotomies were drilled ( within 200 - 400 m of each other ) for the separate entry of lfp and patch pipettes . a third craniotomy was drilled over anterior retrosplenial cortex for insertion of an ag / agcl reference wire . warm hepes buffered artificial cerebrospinal fluid ( acsf , in mm : 135 nacl , 5.4 kcl , 5 hepes , 1 mgcl2 , 1.8 cacl2 ; ph 7.3 ) filled the chamber to prevent desiccation and maintain ionic balance . awake mice were implanted with a lightweight headplate with chamber under isoflurane anaesthesia , and allowed to recover for two days . on the third day , animals were acclimated to head fixation for between 10 - 60 minutes , with sweet liquid reward given at the start and end of training . the duration of training was increased over 4 - 5 days until mice were sitting comfortably for 3 hours . on the day of recording , craniotomies as described above were performed under isoflurane anaesthesia , the animal was allowed to recover for 3 hours , and a single 3 hour recording session commenced . they were also given sweet liquid reward every 30 minutes to keep them comfortable and hydrated . patch pipettes ( 4 - 7 m ) were pulled on a narishige pc-10 and filled with ( in mm ) : 135 k gluconate , 6 kcl , 10 hepes , 4 mgatp , 0.3 na2atp , 0.1 egta , 8 phosphocreatine , ph adjusted to 7.3 with koh , 290 - 295 mosm . for voltage clamp recordings , k gluconate was substituted with 140 cs methanesulfonate . 0.5 qx-314 and 5 tea were included to block voltage gated na and k conductances . while searching for neurons , diffusion of blockers was limited by using low pressures ( 20 - 30 mbar ) ; more importantly , the lfp electrode served as an internal control for visual responsiveness . whole cell recordings were obtained with a multiclamp 700b in voltage clamp mode using standard techniques . average seal resistances for anaesthetised and awake voltage clamp recordings were 3.8  1.7 g and 6.3  1.3 g , respectively . seal resistances for anaesthetised and awake current clamp recordings were 4.5  1.2 g and 5.3  1.1 g , respectively . initial access resistance ranged from 9 - 30 m. this value generally increased within a few minutes and then stabilized . if initial access was greater than 50m the recording was not included for further analysis . access resistance during visual stimulation was not different between anaesthetised and awake recordings in current clamp ( anaesth : 36  6 m ; awake : 39  3 m ) or voltage clamp ( anaesth : 38  5 m ; awake : 38  4 m ) . access resistance was compensated online during voltage clamp recordings and optimized manually . series compensation averaged 63  1% , yielding an effective series resistance of 22  3 m and 24  3 m during anaesthetised and awake voltage clamp recordings , respectively . this method limits distortion of the somatically recorded current by ongoing voltage fluctuations and membrane capacitance ; however , due to the necessity of intrinsic current blockade , it also masks potential contributions of active dendritic conductances . this method also requires multiple trials and holding potentials ; it thus can not assess the simultaneous relationship of excitation and inhibition within single trials . as in previous studies , to better estimate excitatory conductance we used a holding potential of about + 20 mv rather than 0 mv , as this mitigates the voltage decay across the dendrites . input resistance and series resistance were monitored between protocols with current pulse trains or voltage steps . firing rate adaptation to supra - threshold pulses and broad spike - widths confirmed that our recordings were from regular spiking pyramidal neurons . across all anaesthetised ( n=22 ; 305  27 m ) and awake ( n = 30 ; 279  27 m ) whole - cell recordings , there was no significant difference in laminar depth ( p = 0.94 ) , as estimated from the micromanipulator reading after it was zeroed upon contact with the cortical surface . vm was low - pass filtered at 20 khz ; during voltage clamp , i m was low - pass filtered at 2 khz ; lfp was filtered from 0.1 - 100 hz . data was acquired via a national instruments board and was synchronized to stimulus onset by a photodiode signal . the lcd monitor was positioned 24 cm away from the mouse and at 0 elevation and 30 - 45 azimuth . the azimuthal position was adjusted so that the centre of the lfp receptive field was roughly centred on the screen . stimuli were presented on a grey background , and randomly interleaved across space , with 8  20 repetitions per stimulus location . a blank screen ( the grey background ) was randomly presented every 11 stimulus presentations , for the same duration as the stimulus trials . the unstimulated eye was gently sutured ( anaesthetised ) or shielded by black aluminium foil ( awake ) . throughout the entire recording session , this was to ensure that spontaneous cortical state was not affected by transitions to and from complete darkness of the monitor . in awake recordings , upon establishing stable whole - cell configuration , 1 - 2 minutes of spontaneous activity was recorded before the presentation of any flashed bars . 1,4 ) ; there were no systematic differences with the interleaved blank data . in some awake protocols we shortened the interstimulus interval ( isi ) to 300 there was no difference between the magnitude or duration of evoked conductances with longer ( 1.5 s ) vs. shorter isi ( n = 3 ) . for measurements of vm ( figs . 3 ) any spikes were median filtered ( 7 ms ) and the resulting trace smoothed . in a subset of experiments , we optimized the orientation of flashed bars for each individual neuron ( fig . s10 ) by first presenting full screen drifting gratings ( 50% contrast ; spatial frequency 0.03 cycles / deg ; temporal frequency 2 hz ; 2s duration ) varying randomly in orientation ( 30 degree steps ) . the preferred orientation was designated as the one that evoked the largest number of spikes ( or largest depolarization of vm ) , while the orthogonal orientation was 90 degrees away from preferred . bars identical to all other experiments ( 9 degree width ; 100 ms duration , 1.5 s or 0.3 s isi ) were then presented randomly across space along the two axes defined by the preferred and orthogonal orientations . to obtain robust estimates of vrev and g during the responses , we considered the response onset as the time when g was 5 - 10% above baseline for 10 consecutive ms . across the population of neurons , this time point occurred 30 - 40 ms after stimulus onset , so vrev , g , ge , and gi were averaged starting 40 ms after stimulus onset , across the population ( fig . 4 ; n = 5 urethane anaesthesia , n = 8 awake ; both groups tested identically with vertically oriented bars ) . additional recordings of conductances under isoflurane anaesthesia ( n = 1 ) , and in awake mice presented bars varying in orientation ( n = 4 ) were not included in these plots to maintain equivalent group comparison ( but see supplementary fig . 2 ) was analysed by sorting trials ( within neurons ) by pre - stimulus vm level . the upper- and lower - most quartiles ( that is , the 25% most depolarized and 25% most hyperpolarized non - overlapping trials ) were then averaged across the population , and largely correspond to trials when stimuli where delivered in up versus down states , respectively . blank stimuli were also sorted in this manner in order to estimate the spontaneous state transitions from down to up , and vice versa , in the absence of sensory stimulation .
the activity of the cerebral cortex is thought to depend on the precise relationship between synaptic excitation and inhibition1 - 4 . in visual cortex , in particular , 
 intracellular measurements have related response selectivity to coordinated increases in excitation and inhibition5 - 9 . 
 these measurements , however , have all been performed during anaesthesia , which strongly influences cortical state10 and therefore sensory processing7,11 - 15 . 
 the synaptic activity evoked by visual stimulation during wakefulness is unknown . here , we measured visually evoked responses  and the underlying synaptic conductances  in the visual cortex of anaesthetised and awake mice . under anaesthesia 
 , responses could be elicited from a large region of visual space16 and were prolonged in time . during wakefulness responses were more spatially selective and much briefer . 
 whole - cell patch - clamp recordings of synaptic conductances5,17 revealed a surprising difference in synaptic inhibition during the two conditions . 
 whereas under anaesthesia inhibition tracked excitation in amplitude and spatial selectivity , during wakefulness it was much stronger than excitation and exhibited extremely broad spatial selectivity . 
 we conclude that during wakefulness cortical responses to visual stimulation are dominated by synaptic inhibition , restricting their spatial spread and temporal persistence . 
 these results provide the first direct glimpse of synaptic mechanisms that control visual responses in the awake cortex .