acute dorsolateral striatum slices were prepared from p58p87 hemizygous bacterial artificial chromosome ( bac ) d2 dopamine receptor - egfp ( d2-egfp ) or d1 dopamine receptor - tdtomato ( d1-tdtomato ) mice , males and females in the c57bl/6 background . in accordance with northwestern university animal studies committee , mice were deeply anesthetized with ketamine and xylazine , perfused intracardially with ice - cold modified artificial cerebrospinal fluid ( acsf ) ( in mm ) : 125 nacl , 7 glucose , 25 nahco3 , 2.5 kcl , 1.25 nah2po4 , equilibrated with 95% oxygen , 5% co2 plus 0.5 mm cacl2 , 2 mm mgcl2 , 2 mm ascorbic acid . coronal slices , 250 m , were cut in the above modified acsf , then incubated at 32c for 30 min in the above modified acsf containing 2 mm cacl2 , 1 mm mgcl2 , 5 mm l - glutathione and 1 m pyruvate and subsequently stored at room temperature . except when noted , drugs were obtained from sigma , tocris , or invitrogen . slices were perfused at 23 ml per minute with oxygenated modified acsf containing 2 mm cacl2 and 1 mm mgcl2 at 25c . for all experiments , 20 m gabazine and 2 m cgp55845 were added to the perfusion to block gabaa and gabab receptors , respectively . spns restricted to the dorsolateral striatum were identified using infrared differential interference contrast on an upright olympus microscope and a cooled ccd camera ( coolsnap hq ) controlled with metamorph software . in bac d1-tdtomato mice , indirect pathway spns in these mice were identified by the lack of fluorescence in the soma , possessing spines ( visualized with the aid of an alexa fluor hydrazide fluorescent dye ) , and passive membrane properties of spns . in bac d2-egfp mice , direct pathway spns in these mice were identified by the lack of fluorescence , possessing spines ( visualized with the aid of an alexa fluor hydrazide fluorescent dye ) , and passive membrane properties of spns . dual simultaneous somatic whole - cell recordings were made with borosilicate patch pipettes having open tip resistances of 34.5 mohms . the intracellular pipette solution contained ( in mm ) : 115 cesium methylsulfonate , 5 hepes , 5 tetraethyammonium - cl , 2 qx-314 , 0.25 egta , 2 mg - atp , 0.5 na - gtp , 10 sodium phosphocreatine , and 100200 m alexa fluor 488 or 568 hydrazide , ph 7.3 and osmolarity 290 . after dual somatic recordings were established for a randomly selected dspn and ispn pair , no more than 70 m apart , with similar depths from the slice surface ( cell body of each pair had no more than 10 m depth difference between each other ) , the cells were allowed to dialyze with the internal solution for 710 mins . epscs were occluded with bath application of 100 m d-(-)-2-amino-5-phosphonopentanoic acid ( apv ) and 20 m 6-cyano-7-nitroquinoxaline-2,3-dione ( cnqx ) or 1 m tetrodotoxin . data were acquired at 100 khz , filtered at 10 khz using an 8-pole bessel filter , and digitized using a digidata 1440 16-bit a / d converter . somatic access resistance < 25 mohms was monitored and cells with unstable access resistance ( > 30% change ) were discarded . cells were held at 80 mv , not corrected for liquid junction potential ( calculated to be approximately 11 mv ) . stereotaxic guided surgeries were performed on the above mice anesthetized with ketamine and xylazine mixture or isoflurane . after positioning the head to obtain a flat skull between bregma and lamda , a small hole was bored with a micro drill bit , and a glass pipette was slowly inserted at the following coordinates . dorsolateral striatum injection coordinates ( relative to bregma ) were : 2.0 mm posterior , 3.5 mm lateral , and 3.5 mm ventral ; with the manipulator tiled 48 from the z - axis and 72 from the y - axis . pons injection coordinates ( relative to lambda ) were : 2.0 and 2.5 mm posterior , 0.5 mm lateral , and 5.56.5 mm ventral ; with the manipulator tilted 12 from the z - axis , and the nose bar adjusted to 30 from x - y plane . motor cortex injection coordinates ( relative to bregma ) were : 1.6 mm anterior , 1.2 mm lateral and 0.75 mm ventral . to minimize diffusion , over the course of 1 minute the following volumes were slowly injected : 0.2 l of a recombinant rabies virus carrying a channelrhodopsin-2-venus expression construct ( rrabies - chr2-vn ) for pons , 0.1 l rrabies - chr2-vn for striatum , and 0.1 l of an adeno - associated virus carrying a chr2-venus expression construct ( aav2/9-chr2-vn , supplied by u. penn vector core ; addgene 20071 ) for motor cortex . successful targeting produced fluorescence within the pontine nuclei , dorsolateral striatum , or motor cortex , as well as appropriate placement of fluorescent neurons within the cortex . to activate channelrhodopsin containing axons within the dorsolateral striatum , 473 nm light was generated with a crystalaser ( safety protocols for class iiib laser ) , intensity - modulated with a conoptics pockels cell , expanded with concave lens ( thorlabs ) , passed through an adjustable iris , and 5 objective lens ( 0.15 na ) to produce a 700 m diameter column of light at the slice surface . blue light pulses ( 3 ms ) were triggered with a digidata interface that gated a fast uniblitz shutter . light power was calibrated with a fast photo - diode ( thorlabs ) at the laser and sample . light intensity used for maximal activation was approximately 25 mw , and 5 mw for submaximal activation ( producing at minimum a 20% decrease in epsc amplitude compared to the epsc amplitude at 25 mw ) . the amplitudes of epscs were averaged from at least 3 trials ( up to 10 trials ) , with an intersweep interval of 1 minute . images were acquired on an upright olympus or zeiss microscope , qimaging or photometrics camera and with ephus , metamorph , or qcature software . images were adjusted in nih imagej or adobe photoshop for brightness , contrast , and pseudocoloring . axon pclamp data files was imported into igor ( wavemetrics ) and custom written routines were used to analyze the data . pilot studies to determine the variability of outcome measures and the effective size were used to estimate the number of observations necessary to test the null hypothesis . all graphically representations display the ranks of observations within a group using boxplots , illustrating the median ( thick line ) , interquartile range , and minimum / maximum range whiskers . reponses smaller than 34 times the root mean square of the baseline noise average were displayed as 0.1 on the log scale plots , for graphical reasons . data points falling outside of the following range were not included in the analysis : ( the interquartile range multiplied by 3 and then subtracted from the lower quartile value ) and ( the interquartile range multiplied by 3 and then added to the upper quartile value ) . epsc peak amplitudes greater than 2 na were not included in the data set due to changes in voltage control . distribution - free statistical analysis were performed in graphpad prism or originpro , using the nonparametric wilcoxon matched - pairs signed rank two - tailed test or a wilcoxon signed - rank test comparing the ratio medians to 1 . statistical significance ( p < 0.05 ) acute dorsolateral striatum slices were prepared from p58p87 hemizygous bacterial artificial chromosome ( bac ) d2 dopamine receptor - egfp ( d2-egfp ) or d1 dopamine receptor - tdtomato ( d1-tdtomato ) mice , males and females in the c57bl/6 background . in accordance with northwestern university animal studies committee , mice were deeply anesthetized with ketamine and xylazine , perfused intracardially with ice - cold modified artificial cerebrospinal fluid ( acsf ) ( in mm ) : 125 nacl , 7 glucose , 25 nahco3 , 2.5 kcl , 1.25 nah2po4 , equilibrated with 95% oxygen , 5% co2 plus 0.5 mm cacl2 , 2 mm mgcl2 , 2 mm ascorbic acid . coronal slices , 250 m , were cut in the above modified acsf , then incubated at 32c for 30 min in the above modified acsf containing 2 mm cacl2 , 1 mm mgcl2 , 5 mm l - glutathione and 1 m pyruvate and subsequently stored at room temperature . except when noted , drugs were obtained from sigma , tocris , or invitrogen . slices were perfused at 23 ml per minute with oxygenated modified acsf containing 2 mm cacl2 and 1 mm mgcl2 at 25c . for all experiments , 20 m gabazine and 2 m cgp55845 were added to the perfusion to block gabaa and gabab receptors , respectively . spns restricted to the dorsolateral striatum were identified using infrared differential interference contrast on an upright olympus microscope and a cooled ccd camera ( coolsnap hq ) controlled with metamorph software . in bac d1-tdtomato mice , indirect pathway spns in these mice were identified by the lack of fluorescence in the soma , possessing spines ( visualized with the aid of an alexa fluor hydrazide fluorescent dye ) , and passive membrane properties of spns . in bac d2-egfp mice , direct pathway spns in these mice were identified by the lack of fluorescence , possessing spines ( visualized with the aid of an alexa fluor hydrazide fluorescent dye ) , and passive membrane properties of spns . dual simultaneous somatic whole - cell recordings were made with borosilicate patch pipettes having open tip resistances of 34.5 mohms . the intracellular pipette solution contained ( in mm ) : 115 cesium methylsulfonate , 5 hepes , 5 tetraethyammonium - cl , 2 qx-314 , 0.25 egta , 2 mg - atp , 0.5 na - gtp , 10 sodium phosphocreatine , and 100200 m alexa fluor 488 or 568 hydrazide , ph 7.3 and osmolarity 290 . after dual somatic recordings were established for a randomly selected dspn and ispn pair , no more than 70 m apart , with similar depths from the slice surface ( cell body of each pair had no more than 10 m depth difference between each other ) , the cells were allowed to dialyze with the internal solution for 710 mins . epscs were occluded with bath application of 100 m d-(-)-2-amino-5-phosphonopentanoic acid ( apv ) and 20 m 6-cyano-7-nitroquinoxaline-2,3-dione ( cnqx ) or 1 m tetrodotoxin . data were acquired at 100 khz , filtered at 10 khz using an 8-pole bessel filter , and digitized using a digidata 1440 16-bit a / d converter . somatic access resistance < 25 mohms was monitored and cells with unstable access resistance ( > 30% change ) were discarded . cells were held at 80 mv , not corrected for liquid junction potential ( calculated to be approximately 11 mv ) . stereotaxic guided surgeries were performed on the above mice anesthetized with ketamine and xylazine mixture or isoflurane . after positioning the head to obtain a flat skull between bregma and lamda , a small hole was bored with a micro drill bit , and a glass pipette was slowly inserted at the following coordinates . dorsolateral striatum injection coordinates ( relative to bregma ) were : 2.0 mm posterior , 3.5 mm lateral , and 3.5 mm ventral ; with the manipulator tiled 48 from the z - axis and 72 from the y - axis . pons injection coordinates ( relative to lambda ) were : 2.0 and 2.5 mm posterior , 0.5 mm lateral , and 5.56.5 mm ventral ; with the manipulator tilted 12 from the z - axis , and the nose bar adjusted to 30 from x - y plane . motor cortex injection coordinates ( relative to bregma ) were : 1.6 mm anterior , 1.2 mm lateral and 0.75 mm ventral . to minimize diffusion , over the course of 1 minute the following volumes were slowly injected : 0.2 l of a recombinant rabies virus carrying a channelrhodopsin-2-venus expression construct ( rrabies - chr2-vn ) for pons , 0.1 l rrabies - chr2-vn for striatum , and 0.1 l of an adeno - associated virus carrying a chr2-venus expression construct ( aav2/9-chr2-vn , supplied by u. penn vector core ; addgene 20071 ) for motor cortex . successful targeting produced fluorescence within the pontine nuclei , dorsolateral striatum , or motor cortex , as well as appropriate placement of fluorescent neurons within the cortex . to activate channelrhodopsin containing axons within the dorsolateral striatum , 473 nm light was generated with a crystalaser ( safety protocols for class iiib laser ) , intensity - modulated with a conoptics pockels cell , expanded with concave lens ( thorlabs ) , passed through an adjustable iris , and 5 objective lens ( 0.15 na ) to produce a 700 m diameter column of light at the slice surface . blue light pulses ( 3 ms ) were triggered with a digidata interface that gated a fast uniblitz shutter . light power was calibrated with a fast photo - diode ( thorlabs ) at the laser and sample . light intensity used for maximal activation was approximately 25 mw , and 5 mw for submaximal activation ( producing at minimum a 20% decrease in epsc amplitude compared to the epsc amplitude at 25 mw ) . the amplitudes of epscs were averaged from at least 3 trials ( up to 10 trials ) , with an intersweep interval of 1 minute . images were acquired on an upright olympus or zeiss microscope , qimaging or photometrics camera and with ephus , metamorph , or qcature software . images were adjusted in nih imagej or adobe photoshop for brightness , contrast , and pseudocoloring . axon pclamp data files was imported into igor ( wavemetrics ) and custom written routines were used to analyze the data . pilot studies to determine the variability of outcome measures and the effective size were used to estimate the number of observations necessary to test the null hypothesis . all graphically representations display the ranks of observations within a group using boxplots , illustrating the median ( thick line ) , interquartile range , and minimum / maximum range whiskers . reponses smaller than 34 times the root mean square of the baseline noise average were displayed as 0.1 on the log scale plots , for graphical reasons . data points falling outside of the following range were not included in the analysis : ( the interquartile range multiplied by 3 and then subtracted from the lower quartile value ) and ( the interquartile range multiplied by 3 and then added to the upper quartile value ) . epsc peak amplitudes greater than 2 na were not included in the data set due to changes in voltage control . distribution - free statistical analysis were performed in graphpad prism or originpro , using the nonparametric wilcoxon matched - pairs signed rank two - tailed test or a wilcoxon signed - rank test comparing the ratio medians to 1 .
anatomical studies have led to the assertion that intratelencephalic ( it ) and pyramidal tract ( pt ) cortical neurons innervate different striatal projection neurons . to test this hypothesis , the responses of mouse striatal neurons to optogenetic activation of it and pt axons were measured . 
 contrary to expectation , direct and indirect pathway striatal spiny projection neurons ( spns ) responded to both it and pt activation , arguing that these cortical networks innervate both striatal projection neurons .