it integrates signals from cortical areas and subserves important functions like motor control ( tecuapetla et  al . , 2014 , kravitz et  al . , 2010 ) and reinforcement / punishment coding ( kravitz et  al . , 2012 ) . striatal neurons comprise gabaergic spiny projection neurons ( spns , 95% ) and interneurons ( 5% ) . spns are classified into direct pathway spns ( dspns ) , which project to the substantia nigra reticulata and external and internal segments of the globus pallidus ( gpe and gpi , respectively ) , and indirect pathway spns ( ispns ) , which project to gpe ( bolam et  al . , 2000 , wu et  al . , 2000 striatal interneurons include large aspiny cholinergic neurons and different populations of gabaergic interneurons ( kawaguchi et  al . , 1995 ) . excitatory glutamatergic neurons from virtually all cortical areas send projections to the striatum ( mcgeorge and faull , 1989 ) , and several studies suggest their recruitment during action selection ( xiong et  al . , 2015 , znamenskiy and zador , 2013 , koralek et  al . , 2012 ) . in contrast , cortical gabaergic neurons projecting to the  striatum were not considered to be a component of the canonical corticostriatal network because they were identified only in the prefrontal , somatosensory , and retrosplenial cortices ( lee et  al . , 2014 , jinno and kosaka , 2004 ) . only recently were direct gabaergic neurons projecting to the striatum also described in the motor and auditory cortices ( rock et  al . , 2016 ) . the authors identified the long - range projecting neurons as somatostatin - positive ( som ) and , furthermore , reported that inhibition conveyed by these neurons was onto both dspns and  ispns . we previously showed that long - range gabaergic neurons connecting several brain structures comprise different molecular subtypes . for instance , connectivity between the hippocampus and medial entorhinal cortex is supported by parvalbumin - positive ( pv ) and som neurons ( melzer et  al . , 2012 ) . moreover , projections from the septum to the medial entorhinal cortex are pv and calbindin , and they inhibit specific interneurons differentially ( fuchs et  al . , 2016 ) . hence , we wondered whether long - range gabaergic projecting neurons from the motor cortex to the striatum are diverse with respect to their molecular identity , target specificity , and function at the behavioral level . based on virus - mediated tracing , optogenetics , patch - clamp  recordings in  vitro , and behavioral essays , we identified two distinct populations of long - range projecting gabaergic neurons in the primary ( m1 ) and secondary ( m2 ) motor cortex targeting the dorsal striatum and established that these two populations exhibit target cell preference in the striatum and affect locomotion differentially . som cells are a major source of intracortical and corticofugal long - range gabaergic projections ( tomioka et  al . , 2005 , rock et  al . , 2016 ) . to substantiate and extend these studies focusing on long - range gabaergic neurons connecting the motor cortex  and the striatum , we injected adeno - associated virus ( aav ) double - floxed inverse open reading frame ( dio ) chr2-mcherry into the m1 and m2 area of som mice . this resulted in labeling of a subpopulation of gabaergic neurons ( figures 1a and 1b ; figure  s1a ) and revealed projections in several ipsilateral cortical and subcortical areas and , to a lesser extent , in contralateral cortices ( table s1 ) . there was consistent innervation of the ipsilateral dorsal striatum ( figure  1b ; table s1 ) . motor cortex som neuron projections traversed the dorsal striatum and branched preferentially ventro - laterally , sparing the most rostral and caudal part of the dorsal striatum ( figure  1b).figure  1motor cortex som gabaergic neurons innervate the striatum(a ) schematic drawing of the injection site and the location of long - range projections in the striatum shown in ( b ) . viral constructs encoding chr2-mcherry were injected into the motor cortex of som mice.(b ) bright - field images of dab - stained sections showing the injection site in the motor cortex ( left ) and mcherry - labeled axons in the striatum ( center ) following aav dio chr2-mcherry injection into the motor cortex of som mice . a higher magnification of the boxed area is shown on the right.(c and d ) confocal images showing a retrogradely labeled area ( c ) following injection of the retrograde tracer ctb647 into the striatum and a retrogradely labeled gabaergic som neuron in the motor cortex , visualized via fish for sst and gad1/2 ( d).(e  h ) som projecting neurons were identified by retrograde tracing with sadg - egfp(enva ) rabies virus . tcb was expressed cre - dependently in the motor cortex of som mice , and rabies virus was injected into the striatum . ( e ) shows differential interference contrast ( dic ) and epifluorescent images of a retrogradely labeled tcb neuron in the motor cortex with the corresponding firing pattern shown in ( f ) . ( g ) shows a confocal image of a retrogradely labeled tcb neuron in m1 immunostained for egfp and som with the corresponding morphological reconstruction shown in ( h).str , striatum . motor cortex som gabaergic neurons innervate the striatum ( a ) schematic drawing of the injection site and the location of long - range projections in the striatum shown in ( b ) . ( b ) bright - field images of dab - stained sections showing the injection site in the motor cortex ( left ) and mcherry - labeled axons in the striatum ( center ) following aav dio chr2-mcherry injection into the motor cortex of som mice . ( c and d ) confocal images showing a retrogradely labeled area ( c ) following injection of the retrograde tracer ctb647 into the striatum and a retrogradely labeled gabaergic som neuron in the motor cortex , visualized via fish for sst and gad1/2 ( d ) . ( e h ) som projecting neurons were identified by retrograde tracing with sadg - egfp(enva ) rabies virus . tcb was expressed cre - dependently in the motor cortex of som mice , and rabies virus was injected into the striatum . ( e ) shows differential interference contrast ( dic ) and epifluorescent images of a retrogradely labeled tcb neuron in the motor cortex with the corresponding firing pattern shown in ( f ) . ( g ) shows a confocal image of a retrogradely labeled tcb neuron in m1 immunostained for egfp and som with the corresponding morphological reconstruction shown in ( h ) . see also figure  s1 and tables s1 and s2 . to further substantiate the presence of gabaergic corticostriatal projections , we performed retrograde labeling . we injected cholera toxin b ( ctb ) subunit 647 into the ventro - lateral part of the dorsal striatum and analyzed retrogradely labeled cells in the m1 region ( figures s1b and s1c ) . as expected , a dense band of retrogradely labeled cells became visible in cortical l5 ( figure  1c ) ; i.e. , in the layer that is the major source of corticostriatal excitatory projections ( wilson , 1987 , cowan and wilson , 1994 ) . to visualize gabaergic cells among the m1 retrogradely labeled cells , we performed multi - fluorescence in  situ hybridization ( fish ) for sst ( encoding som ) and gad1/2 ( encoding gad67/65 ) . we found 13 retrogradely labeled cells in m1 that were clearly positive for gad1/2 ( n  = 3,582 ctb cells and 5,064 gad1/2 cells , cell counts across all layers in 35 slices from 4 hemispheres in 4 mice ) , 8 of which co - labeled for sst ( figure  1d ) . to confirm a direct long - range gabaergic connection between the motor cortex ( m1/m2 ) and the dorsal striatum , we performed retrograde monosynaptic tracing with rabies virus ( wickersham et  al . , 2007 ) . we injected aavs encoding cre - dependent avian virus receptor ( avian tumor virus receptor a mcherry [ tcb ] ; weissbourd et  al . , 2014 ) and rabies glycoprotein ( rg ) into the striatum of a2a - cre mice that express cre recombinase specifically in ispns ( gong et  al . , 2003 ) . subsequent injection of rg - deleted envelope protein  from avian aslv type a ( enva)-pseudotyped rabies virus ( sadg - egfp(enva ) ) into the striatum resulted in transsynaptically retrogradely labeled cells in the cortex ( figures s1e and s1f ) . fish for rabies virus - specific mrna ( rabv - gp1 ) and gad1/2 revealed double - positive neurons in the motor cortex ( 7  cells in 34 slices from 4 hemispheres in 4 mice ; figure  s1 the average number of labeled cells per slice was lower than after ctb647 injections , suggesting that ispns were not the only striatal target cells of gabaergic projecting neurons and/or reflecting lower efficiency of transsynaptic tracing ( marshel et  al . , , we expressed tcb cre - dependently in the motor cortex ( m1/m2 ) of som mice and injected sadg - egfp(enva ) rabies virus into the striatum . tcb retrogradely labeled cells in the motor cortex had a classical or burst accommodating firing pattern ( n  = 11 cells from 5 hemispheres in 4 mice ; figures 1e and 1f ; figure  s1h ) similar to non - retrogradely labeled tcb cells ( table s2 ) . reconstructed cells had a martinotti cell - like morphology ( wang et  al . , 2004 ) with axonal projections extending over all cortical layers ( three reconstructions from three hemispheres in two mice ; figures 1 g and 1h ; figures s1i we next tested whether som projecting neurons form functional synapses onto striatal neurons and whether the connectivity exhibits target specificity . we injected aav dio chr2-mcherry into m1/m2 of som mice and combined optogenetic stimulation of long - range projections with patch - clamp recordings of putative postsynaptic cells in the striatum ( figure  2a ) . all injections ( n  = 36 hemispheres ) resulted in labeled axons that projected to the dorsal striatum . of 305 patched neurons ( in 27 mice ) , 50 responded with short - latency postsynaptic currents ( pscs ) to 5-ms photostimulation of motor  cortex som neuron projections ( figure  2b ) . as a specificity control , we repeated the experiment in wild - type mice injected with aav dio chr2-mcherry and found neither mcherry fibers in the dorsal striatum nor a response after photostimulation ( n  = 58 cells in 2 mice ) . responses in som mice could not be blocked with 6-cyano-2,3-dihydroxy-7-nitro - quinoxaline ( cnqx ) and d-2-amino-5-phosphonovaleric acid ( d - ap5 ) ( 165.6  32.7  pa baseline versus 169.5  31.1  pa with drugs [ mean  sem ] , paired t test , t(14 ) = 0.49 , p  = 1 , n  = 15 cells in 11 mice ; figure  s2a ) but with gabazine ( 117.8 [ 134.3 ] pa versus 1.6 [ 1.6 ] pa [ median interquartile range ( iqr ) ] , wilcoxon signed - rank test , w  = 210 , p  = 0.0002 , n  = 20 cells in 15 mice ; figures s2a and s2b ) . responses reversed around the reversal potential of gabaergic receptors ( n  = 14 cells in 9 mice ; figure  2b ) , thus confirming the gabaergic nature of motor cortex som neuron projections.figure  2motor cortex som neuron projections form functional synapses on striatal output and cholinergic neurons(a ) schematic drawing indicating the injection site ( left ) and location of an exemplary patched neuron in the striatum ( right ) . aav dio chr2-mcherry was injected into the motor cortex of som mice , and target cells were patched in the striatum ( dic image).(b ) pscs recorded in a striatal neuron at the indicated holding potentials following 5-ms photostimulation ( blue ticks ) of motor cortex som neuron projections . responses were blocked with gabazine but not d - ap5/cnqx.(c ) firing pattern ( membrane potential upon 50-pa current injection and at the action potential [ ap ] threshold ) of a representative spn that was responsive to photostimulation of motor cortex som neuron projections.(d ) firing pattern ( spontaneous activity and maximal firing frequency ) and dic image of a representative cholinergic interneuron ( arrow ) that was responsive to photostimulation of motor cortex som neuron projections.(e ) firing pattern of a striatal gabaergic interneuron that was responsive to photostimulation of motor cortex som neuron projections ( from top to bottom : maximal firing frequency , ap threshold , and 50-pa current injection).(f ) percentage of striatal neurons responding to photostimulation of motor cortex som neuron projections . number of mice : spns , 19 ; cholinergic , 11 ; gabaergic interneurons , 19.(g ) schematic drawing indicating injection sites of aav dio chr2-mcherry in som / drd1a - egfp and som / drd2-egfp mice.(h ) confocal image of egfp - immunostained sagittal sections of som / drd1a - egfp ( left ) and som / drd2-egfp ( right ) mice exhibiting differential egfp expression in the globus pallidus ( arrow ) and substantia nigra ( arrowhead).(i ) exemplary traces of dspn and ispn responses to photostimulation ( blue ticks ) of m1 and m2 som neuron projections using cs - based low cl intracellular solution ( from top to bottom : 0  mv , reversal potential , and 95  mv holding potential).(j ) percentage of striatal neurons responding to photostimulation of m1 and m2 som neuron projections . number of mice : m1-dspns , 7 ; m1-ispns , 16 ; m2-dspns , 8 ; m2-ispns , 17 ; m1-cholinergic , 19 ; m2-cholinergic , 13.(k ) schematic drawing indicating the localization of responding cells in a coronal ( left ) and sagittal ( right ) cross - section . color code : orange , m1 to dspn ; yellow , m2 to dspn ; dark green , m1 to ispn ; light green , m2 to ispn ; purple , m1 to cholinergic interneuron ; red , m2 to cholinergic interneuron.hp , hippocampus ; ach , cholinergic interneuron . som neuron projections form functional synapses on striatal output and cholinergic neurons ( a ) schematic drawing indicating the injection site ( left ) and location of an exemplary patched neuron in the striatum ( right ) . aav dio chr2-mcherry was injected into the motor cortex of som mice , and target cells were patched in the striatum ( dic image ) . ( b ) pscs recorded in a striatal neuron at the indicated holding potentials following 5-ms photostimulation ( blue ticks ) of motor cortex som neuron projections . ( c ) firing pattern ( membrane potential upon 50-pa current injection and at the action potential [ ap ] threshold ) of a representative spn that was responsive to photostimulation of motor cortex som neuron projections . ( d ) firing pattern ( spontaneous activity and maximal firing frequency ) and dic image of a representative cholinergic interneuron ( arrow ) that was responsive to photostimulation of motor cortex som neuron projections . ( e ) firing pattern of a striatal gabaergic interneuron that was responsive to photostimulation of motor cortex som neuron projections ( from top to bottom : maximal firing frequency , ap threshold , and 50-pa current injection ) . ( f ) percentage of striatal neurons responding to photostimulation of motor cortex som neuron projections . number of mice : spns , 19 ; cholinergic , 11 ; gabaergic interneurons , 19 . ( g ) schematic drawing indicating injection sites of aav dio chr2-mcherry in som / drd1a - egfp and som / drd2-egfp mice . ( h ) confocal image of egfp - immunostained sagittal sections of som / drd1a - egfp ( left ) and som / drd2-egfp ( right ) mice exhibiting differential egfp expression in the globus pallidus ( arrow ) and substantia nigra ( arrowhead ) . ( i ) exemplary traces of dspn and ispn responses to photostimulation ( blue ticks ) of m1 and m2 som neuron projections using cs - based low cl intracellular solution ( from top to bottom : 0  mv , reversal potential , and 95  mv holding potential ) . ( j ) percentage of striatal neurons responding to photostimulation of m1 and m2 som neuron projections . number of mice : m1-dspns , 7 ; m1-ispns , 16 ; m2-dspns , 8 ; m2-ispns , 17 ; m1-cholinergic , 19 ; m2-cholinergic , 13 . ( k ) schematic drawing indicating the localization of responding cells in a coronal ( left ) and sagittal ( right ) cross - section . color code : orange , m1 to dspn ; yellow , m2 to dspn ; dark green , m1 to ispn ; light green , m2 to ispn ; purple , m1 to cholinergic interneuron ; red , m2 to cholinergic interneuron . hp , hippocampus ; ach , cholinergic interneuron . see also figures s2 and s3 and tables s3 and s4 . to scrutinize target specificity , striatal neurons were sorted into spns and cholinergic and gabaergic interneurons based on their electrophysiological properties and cell soma shape ( planert et  al . , 2013 , gertler et  al . , 2008 , kawaguchi , 1992 , bennett and wilson , 1999 , kawaguchi et  al . , 1995 ; experimental procedures ; figures 2c2e ; table s3 ) . we found that 22.6% of spns , 33.3% of cholinergic cells , and only 2% of gabaergic interneurons responded to 5-ms photostimulation of motor cortex som neuron projections ( figure  2f ; figures s2c and s2d ; table s3 ) . together , these data indicate that spns and cholinergic cells are the main target of motor cortex som neuron projections . to answer whether dspns and ispns are differentially targeted by motor cortex som projecting neurons , we cross - bred som mice to drd1a - egfp and drd2-egfp mice in which dspns and ispns , respectively , are selectively labeled ( gong et  al . , 2003 ; we injected aav dio chr2-mcherry into m1 or m2 of som / drd1a - egfp or som / drd2-egfp mice ( figure  2 g ; figure  s2e ) and combined photostimulation of m1 or m2 som neuron projections with patch - clamp recordings of striatal neurons ( figure  s2f ) . we found that m1 som projecting neurons innervated a comparable proportion of dspns ( 29% ) , ispns ( 22% ) , and cholinergic cells ( 40% ) ( figures 2i and 2j ; fisher s exact test , p  = 0.19 ) . m2 som projecting neurons targeted dspns ( 25% ) and ispns ( 16% ) to a similar extent , whereas cholinergic cells tended to be innervated less frequently ( 4% ) ( figure  2j ; fisher s exact test , p  = 0.08 ) . inhibition of cholinergic cells by m2 was significantly less frequent than by m1 ( figure  2j ; fisher s exact test , m1 dspns versus m2 dspns : p  = 1 ; m1 ispns versus m2 ispns : p  = 1 ; m1 cholinergic versus m2 cholinergic : p  = 0.006 ) . responses had a latency of 2.5 ( 1.3 ) ms ( median [ iqr ] ; n  = 47 responding cells ; see figure  s3a and table s4 for more details ) and a reversal potential of 58.0  1.4  mv ( mean  sem ; n  = 22 responding cells ; see figure  s3b and table s4 for more details ) . the strength of m1 and m2 som neuron connections to spns was similar ( table s4 ) . however , when comparing inputs with dspns and ispns both from m1 and m2 , the response amplitudes were significantly larger for ispns ( amplitudes at 0  mv with cesium [ cs ] internal solution : 15.7   pa versus 44.0 [ 36.5 ] pa [ median ( iqr ) ] in dspns and ispns respectively ; mann - whitney u test , u  = 102 , p  = 0.02 , n  = 14 and 26 responding cells , respectively ; figure  s3c ) . detected target cells were located preferentially in the ventro - lateral part of the dorsal striatum ( figure  2k ; figures s3d targeted dspns and ispns were intermingled and localized 2.4 ( 0.7 ) mm lateral , 0.1 ( 0.7 ) mm posterior to the bregma and 4 ( 0.7 ) mm deep ( median [ iqr ] ) . spns targeted by m1 projections were more lateral than spns targeted by m2 projections ( mann - whitney u test , u  = 142 , p  = 0.04 ; table s4 ) . the retrograde labeling experiments suggested that som neurons are not the only m1 gabaergic population projecting to the  striatum . pv neurons appeared to be attractive candidates because we identified them before as a major class of neurons providing long - range inhibition in the entorhinal cortex - hippocampal formation ( melzer et  al . , 2012 ) . testing for the presence of gabaergic long - range projecting pv cells based on virus tracing in pv mice may be complicated by the fact that a fraction of cortical pv cells are glutamatergic ( jinno and kosaka , 2004 ) . hence , we first tested the gabaergic nature of pv cells in m1/m2 by counting the number of double - positive cells in injected pv / gad67 mice . 99.5% of pv cells were gad67 ( 759 of 763 pv cells from 9 slices in 3 mice ) . this result is in agreement with previous evidence showing the absence of pv glutamatergic neurons in the motor cortex ( jinno and kosaka , 2004 ) . in the mouse neocortex , pv and som neurons constitute two non - overlapping neuronal entities ( xu et  al . , 2010 , tasic et  al . , 2016 ) . thus , we injected aav dio chr2-mcherry into m1 of pv mice , and , indeed , we detected labeled axons in the dorsal striatum that branched preferentially ventro - laterally ( in 12 of 17 injected hemispheres ) . in contrast , injections into m2 of pv mice were less likely to result in labeled striatal projections ( 5 of 12 injections ) , and projections  when present  were sparser than the ones detected after m1 injections . thus , for a more detailed analysis of innervation patterns , we focused on projections from m1 . we injected aav dio chr2-mcherry into m1 of pv / drd1a - egfp or pv / drd2-egfp mice ( figure  3a ) and found that 31% of dspns , 6% of ispns , and 6% of cholinergic and 0% of gabaergic interneurons responded to photostimulation of m1 pv neuron projections ( figures 3b and 3c ) . thus , pv projecting neurons preferentially innervated dspns ( fisher s exact test ; dspns versus ispns : p  = 0.009 , dspns versus cholinergic : p  = 0.04 , ispns versus cholinergic : p  = 1 ; figure  3b ) . spns targeted by m1 pv projecting neurons had a response amplitude comparable with that following stimulation of m1 som projections ( figure  s3c ; table  s4 ) . although responses could not be blocked by cnqx / d - ap5 ( although one of five cells showed a clear decrease in amplitude ) , subsequent application of gabazine led to a significant amplitude decrease ( 68.9 [ 147.3 ] pa baseline versus 70.3 [ 86.1 ] pa with cnqx / d - ap5 versus 0.5 [ 3.2 ] pa with gabazine , friedman test , p  = 0.02 , post hoc conover s test with bonferroni correction , baseline versus cnqx / d - ap5 : p  = 1 , baseline versus gabazine : p  = 0.004 , cnqx / d - ap5 versus gabazine : p  = 0.009 , n  = 5 cells in 4 mice ; figure  3d ; figures s3 g and s3h ) . the reversal potential of the responses was 60.3  2.5  mv ( mean  sem , n  = 4 cells in 4  mice ; figure  s3b ; table s4 ) , reconfirming the gabaergic nature . spns targeted by m1 pv projecting neurons were located 0.6 ( 0.7 ) mm posterior to the bregma , 3.0 ( 0.6 ) mm lateral , and 3.7  ( 0.8 ) deep ( median [ iqr ] ) ( figure  3e ; figures s3d s3f ; table  s4).figure  3m1 pv neuron projections preferentially target dspns in the striatum(a ) schematic drawing indicating the injection site of aav dio chr2-mcherry in pv / drd1a - egfp and pv / drd2-egfp mice.(b ) percentage of striatal neurons responding to photostimulation of m1 pv neuron projections . number of  mice : dspns , 8 ; ispns , 7 ; cholinergic , 13 ; gabaergic interneurons , 9.(c ) firing pattern ( upon 50  pa current injection and at the ap threshold ) of a representative dspn that was responsive to photostimulation of m1 pv neuron projections.(d ) responses of the dspn shown in ( c ) at 70  mv holding potential using high  cl intracellular solution . responses were blocked with gabazine but not d - ap5/cnqx.(e ) schematic drawing indicating the localization of responding cells in a coronal ( left ) and sagittal ( right ) cross - section . color code : orange , m1 to dspn ; yellow , m1 to ispns ; gray , unidentified responding neurons.(f and g ) epifluorescent images of a retrogradely labeled tcb neuron in the  motor cortex ( arrow ) ( f ) with the corresponding firing pattern ( g ) identified by retrograde tracing with sadg - egfp(enva ) rabies virus . tcb was expressed cre - dependently in the motor cortex of pv mice , and rabies virus was injected into the striatum.(h ) dot plot of the action potential half - width for retrogradely labeled som ( n  =  11 cells from 5 hemispheres in 4 mice ) and pv neurons ( n  = 3 cells from 3  hemispheres in 2 mice).see also figure  s3 and tables s2 and s4 . m1 pv neuron projections preferentially target dspns in the striatum ( a ) schematic drawing indicating the injection site of aav dio chr2-mcherry in pv / drd1a - egfp and pv / drd2-egfp mice . ( b ) percentage of striatal neurons responding to photostimulation of m1 pv neuron projections . number of  mice : dspns , 8 ; ispns , 7 ; cholinergic , 13 ; gabaergic interneurons , 9 . ( c ) firing pattern ( upon 50  pa current injection and at the ap threshold ) of a representative dspn that was responsive to photostimulation of m1 pv neuron projections . ( d ) responses of the dspn shown in ( c ) at 70  mv holding potential using high  cl intracellular solution . ( e ) schematic drawing indicating the localization of responding cells in a coronal ( left ) and sagittal ( right ) cross - section . color code : orange , m1 to dspn ; yellow , m1 to ispns ; gray , unidentified responding neurons . ( f and g ) epifluorescent images of a retrogradely labeled tcb neuron in the  motor cortex ( arrow ) ( f ) with the corresponding firing pattern ( g ) identified by retrograde tracing with sadg - egfp(enva ) rabies virus . tcb was expressed cre - dependently in the motor cortex of pv mice , and rabies virus was injected into the striatum . ( h ) dot plot of the action potential half - width for retrogradely labeled som ( n  =  11 cells from 5 hemispheres in 4 mice ) and pv neurons ( n  = 3 cells from 3  hemispheres in 2 mice ) . the different innervation patterns of som and pv projecting neurons suggested that the two cell types represent distinct subpopulations in the motor cortex . we hence analyzed whether the firing pattern of pv projecting neurons differed from that of som projecting neurons and resembled that of  classical  pv interneurons . the detection of projecting neurons was assisted by retrograde tracing with sadg - egfp(enva ) rabies virus injections into the striatum of pv mice expressing cre - dependent tcb in motor cortex neurons . indeed , all tcb / rbv - egfp non - pyramidal neurons in the motor cortex exhibited a fast spiking firing pattern with faster action potentials than som projecting neurons ( figures 3f3h ; figure  s3i ; table s2 ) . to investigate the behavioral effect of activating the newly discovered motor cortex som and pv projecting neurons ( striatal targeting is summarized in figure  4a ) , we implanted optic fibers bilaterally into the dorsal striatum of pv and som mice that were injected with aav dio chr2-mcherry into the motor cortex ( figures 4b4d ) . we compared the performance of four groups of animals : pv mice injected in m1 ( pv - m1 ) , som mice injected in m1 ( som - m1 ) , som mice injected in m2 ( som - m2 ) , and control mice ( pv and som mice injected in m1 or m2 with aav dio eyfp and wild - type mice injected in m1 or m2 with aav tomato ) . we first asked whether  activation of motor cortex pv and som neuron projections in  the striatum modulated spontaneous locomotion . mice were allowed to explore a circular arena , and locomotion was measured before and during light stimulation of corticostriatal projections ( 5-ms pulses at 20  hz ; power , 3 mw ) . we next calculated the difference between motion ( defined as the distance moved in 5 s ) during and before light stimulation on four epochs of different durations ; i.e. , 10 , 30 , 60 , and 120 s , starting at light stimulation onset . the performance of control mice was similar before and during light stimulation ( figures 4e4 g ) ( the slight increase observed during the 10-s epoch was not significantly different from zero ; one - sample t test ( null hypothesis [ h0 ]  = 0 ) , t(12 )  = 2.1 , p  = 0.06 ) . conversely , the performance of som - m1 , pv - m1 , and som - m2 changed upon photostimulation ( figures 4e and 4f ) . during the 10- , 30- , and 60-s epochs , som - m1 mice showed a significant motion increase , whereas pv - m1 and som - m2 mice showed a significant motion decrease with respect to control mice ( figures 4e and 4f ; one - way anova followed by post hoc comparisons , 10 s : f(3 , 31 )  = 7.71 , p  = 0.0005 , control versus pv - m1 : p  = 0.03 , control versus som - m1 : p  = 0.02 , control versus som - m2 : p  = 0.04 ; 30 s : f(3 ,  31 )  = 10.48 , p  = 0.0001 , control versus pv - m1 : p  = 0.04 , control versus som - m1 : p  = 0.002 , control versus som - m2 : p  = 0.05 ; 60 s : f(3 , 31 )  = 12.52 , p  = 0.0001 , control versus pv - m1 : p  = 0.002 , control versus som - m1 : p  = 0.01 , control versus som - m2 : p  = 0.008 ) . for the 120-s epoch , only pv - m1 mice still showed a motion decrease with respect to control mice , whereas som - m1 and som - m2 performance could not be distinguished from that of control mice ( figures 4e and 4f ; one - way anova followed by post hoc comparisons , f(3 , 31 )  = 3.96 , p  = 0.01 , control versus pv - m1 : p  = 0.01 , control versus som - m1 : p  = 0.42 , control versus som - m2 : p  = 0.13).figure  4motor cortex som and pv projecting neurons mediate locomotion change(a ) schematic drawing summarizing the newly identified corticostriatal gabaergic projections . m1 som neurons innervate dspns , ispns , and cholinergic interneurons ( blue ) ; m2 som neurons preferentially innervate dspns and ispns ( green ) ; m1 pv neurons preferentially innervate dspns ( pink ) ; m2 pv neuron projections are scarce ( purple ) . d1 , dspns ; d2 , ispns ; in , gabaergic interneuron.(b ) schematic drawing indicating sites of aav dio chr2-mcherry injection in som and pv mice ( top ) and of optic fiber implantation in the striatum ( bottom).(c and d ) bright - field images of dab - stained sections showing m1 and m2 injection sites ( c ) , optic fiber position , and mcherry - labeled axons in the striatum ( d).(e ) exemplary locomotion traces ( in cm/40  ms ) of control , som - m1 , pv - m1 , and som - m2 mice before ( black ) and during light stimulation ( blue ) ( 5-ms pulses delivered at 20  hz ; light power , 3 mw).(f ) mean  sem difference between motion levels ( in cm/5 s ) during and before light stimulation for 10- , 30- , 60- , and 120-s epochs , starting at light stimulation onset.(g j ) mean  sem cumulative relative frequency of running speed ( top ) , and mean  sem number , speed ( in cm / s ) , and duration ( in seconds ) of mobility and immobility bouts 60  s before ( black lines and white bars ) and during 60-s light stimulation ( blue lines and bars ) for control mice ( g ) and som - m1 ( h ) , pv - m1 ( i ) , and som - m2 ( j ) mice . control , n  = 13 mice ; som - m1 , n  = 7 mice ; pv - m1 , n  = 9 mice ; som - m2 , n  = 6 mice . motor cortex som and pv projecting neurons mediate locomotion change ( a ) schematic drawing summarizing the newly identified corticostriatal gabaergic projections . m1 som neurons innervate dspns , ispns , and cholinergic interneurons ( blue ) ; m2 som neurons preferentially innervate dspns and ispns ( green ) ; m1 pv neurons preferentially innervate dspns ( pink ) ; m2 pv neuron projections are scarce ( purple ) . ( b ) schematic drawing indicating sites of aav dio chr2-mcherry injection in som and pv mice ( top ) and of optic fiber implantation in the striatum ( bottom ) . ( c and d ) bright - field images of dab - stained sections showing m1 and m2 injection sites ( c ) , optic fiber position , and mcherry - labeled axons in the striatum ( d ) . ( e ) exemplary locomotion traces ( in cm/40  ms ) of control , som - m1 , pv - m1 , and som - m2 mice before ( black ) and during light stimulation ( blue ) ( 5-ms pulses delivered at 20  hz ; light power , 3 mw ) . ( f ) mean  sem difference between motion levels ( in cm/5 s ) during and before light stimulation for 10- , 30- , 60- , and 120-s epochs , starting at light stimulation onset . ( g  j ) mean  sem cumulative relative frequency of running speed ( top ) , and mean  sem number , speed ( in cm / s ) , and duration ( in seconds ) of mobility and immobility bouts 60  s before ( black lines and white bars ) and during 60-s light stimulation ( blue lines and bars ) for control mice ( g ) and som - m1 ( h ) , pv - m1 ( i ) , and som - m2 ( j ) mice . control , n  = 13 mice ; som - m1 , n  = 7 mice ; pv - m1 , n  = 9 mice ; som - m2 , n  = 6 mice . we characterized the photostimulation - induced motion changes occurring during the 60-s epoch in more detail . we first  analyzed the cumulative frequency distribution of running speed 60  s before and for 60  s during photostimulation ( figures 4g4j ) . as expected , in control mice , no difference was found between the cumulative distribution curves before and during photostimulation ( figure  4 g , top ; wilcoxon matched - pairs signed - rank test , w  = 193 , p  = 0.15 ) . in som - m1 mice , the curve during photostimulation was shifted to the right , and , accordingly , the median running speed increased significantly upon photostimulation ( figure  4h , top ; wilcoxon matched - pairs signed - rank test , w  = 215 , p  = 0.01 ) . in pv - m1 and som - m2 mice , the cumulative distribution curve during photostimulation was shifted to the left , and the median running speed was significantly lower during than before photostimulation ( figures 4i and  4j , top ; wilcoxon matched - pairs signed - rank test , pv - m1 : w  = 113 , p  = 0.005 ; som - m2 : w  = 90 , p  = 0.02 ) . finally , based on running speed , we defined mobility and immobility bouts ( experimental procedures ) and measured their number , speed , and duration before and during photostimulation . in control mice , the properties of mobility and immobility bouts did not change upon photostimulation ( figure  4 g ; paired t test , number of bouts ( nr ) mobility : t(12 )  = 0.76 , p  = 0.46 ; speed mobility : t(12 )  = 1.33 , p  = 0.21 ; nr immobility : t(12 )  = 0.51 , p  = 0.62 ; speed immobility : t(12 )  = 0.64 , p  = 0.53 ; wilcoxon matched - pairs signed - rank test : duration mobility : w  = 19 , p  = 0.54 ; duration immobility : w  = 6 , p  = 0.83 ) . in som - m1 mice , photostimulation elicited significant changes in mobility bouts : the number decreased , whereas the speed and duration increased ( figure  4h ; paired t  test : nr : t(6 )  = 3.17 , p  = 0.02 ; speed : t(6 )  = 2.97 , p  = 0.02 ; wilcoxon matched - pairs signed - rank test , duration : w  = 21 , p  = 0.03 ) . immobility bouts remained unaffected ( figure  4h ; paired t test : nr : t(6 )  = 1.46 , p  = 0.19 ; speed : t(6 )  = 0.97 , p  = 0.37 ; wilcoxon matched - pairs signed - rank test , duration : w  = 9 , p  = 0.44 ) . in  pv - m1 mice , the duration of mobility bouts decreased significantly , and the duration of immobility bouts increased significantly upon photostimulation ( figure  4i ; wilcoxon matched - pairs signed - rank test , duration mobility : w  = 35 , p  = 0.04 , duration immobility : w  = 39 , p  = 0.02 ) , whereas their number and speed remained unchanged ( figure  4i ; paired t test : nr mobility : t(8 )  = 0.97 , p  = 0.36 ; speed mobility : t(8 )  = 1.57 , p  = 0.14 ; nr immobility : t(8 )  = 1.62 , p  = 0.19 ; speed immobility : t(6 )  = 0.78 , p  = 0.46 ) . in som - m2 mice , the speed of mobility bouts was significantly reduced upon photostimulation ( figure  4j ; paired t test : t(5 )  = 3.48 , p  = 0.02 ) , whereas all other variables remained unchanged ( paired t test : nr mobility : t(5 )  = 0.14 , p  = 0.89 ; nr immobility : t(5 )  = 0.03 , p  = 0.97 ; speed immobility : t(5 )  = 0.26 , p  = 0.8 ; wilcoxon matched - pairs signed - rank test , duration mobility : w  = 1 , p  = 0.99 ; duration immobility : w  = 11 , p  = 0.31 ) . in sum , we conclude that stimulation of striatal long - range projections of m1 som neurons increased locomotion by increasing the duration and speed of mobility bouts , that stimulation of striatal long - range projections of m1 pv neurons reduced locomotion by increasing the duration of immobility bouts , and that stimulation of striatal long - range projections of m2 som neurons reduced locomotion by decreasing the speed of mobility bouts . it has been proposed that movement control and reinforcement coding are mediated by common corticostriatal circuits ( kravitz and kreitzer , 2012 ) . thus , we next asked whether stimulation of motor cortex pv and som neuron projections in the striatum also affect reinforcement / punishment coding . we  tested the mice in a place preference task ( figure  s4 ) . the task lasted 3  days , during which we recorded the time mice spent in each compartment . during the first and second days ( habituation and baseline , respectively ) , place preference was measured without photostimulation . during the third day ( test ) , one of the compartments ( stimulation side ) was paired with photostimulation ( 5-ms pulses at 20  hz ; power , 3 mw ) , and place preference was measured . we calculated a difference score as the percentage of time spent on the  stimulation  side during baseline minus the percentage of time spent on the same side during the test . we found that the difference score obtained for pv - m1 , som - m1 , and som - m2 mice was similar to that of control mice ( figure  s4b ; one - way anova : f(3,21 )  = 0.67 , p  = 0.58 ) . hence , stimulation of striatal long - range projections of motor cortex pv and som neurons did not elicit place preference by employing this paradigm . here we show that distinct long - range gabaergic neurons connect m1 and m2 with the dorsal striatum . the newly identified long - range gabaergic neurons express either som or pv and differ with respect to target cell preference and the modulatory effect on motor activity . our results indicate that both m1 and m2 harbor long - range gabaergic neurons that target the dorsal striatum and thus extend recent findings by rock et  al . furthermore , we report the following new findings . first , m1 and m2 contribute differentially to gabaergic corticostriatal projections . second , we identified and characterized an additional projection formed by pv neurons that differs significantly from that formed by som neurons . multiple reasons can explain why these connections have not been noticed until recently ( rock et  al . , 2016 ) . first , the scarcity of long - range gabaergic neurons constitutes a challenge as to their detection by anterograde or retrograde labeling , considering the high number of excitatory neurons that are also labeled in the same area with their axons extending along a similar path . second , most studies focused on more dorso - anterior areas of the striatum . hence , retrograde tracer injections are unlikely to reveal gabaergic projecting neurons in motor cortices because their axons target preferentially more lateral , posterior , and ventral parts of the dorsal striatum . however , jinno and kosaka ( 2004 ) did not detect motor cortical long - range gabaergic neurons even though injections included target areas that were innervated in our study . a possible reason may be low uptake efficiency and transport of the tracer fluorogold in gabaergic neurons . first , there was robust axon labeling in the striatum even with regionally restricted minimal anterograde injections . second , long - range gabaergic neurons were retrogradely labeled with ctb from the striatum . third , long - range gabaergic neurons were transsynaptically retrogradely labeled with rabies virus that infected only striatal starter cells . finally , the electrophysiological and pharmacological results provide strong evidence for the gabaergic nature of pv and som projecting neurons . although we have no indication for any glutamatergic inputs deriving from som cells , based on our immunocytochemistry , pharmacology , and rabies virus tracing , we can not exclude that glutamatergic transmission has a minor contribution to the behavioral effects seen upon stimulation of striatal long - range projections from m1 pv neurons . it is important to note that the number of long - range gabaergic neurons presented in this study remains an underestimation because quantitative evaluations are currently hampered by a number of technical constraints . first , experiments entail  conservative / limited  virus injection to prevent viral spread beyond the target area . second , labeling by retrograde tracing is strongly dependent on the tracer and cell type ; e.g. , pv neurons could be detected following transsynaptic virus - mediated tracing but not by ctb labeling . for quantitative studies , it would be highly desirable to identify markers / promoters for long - range gabaergic neurons . characterization of motor cortex gabaergic projecting neurons revealed that m1 and m2 som and pv cells differentially innervate striatal neurons . moreover , bilateral stimulation of corticostriatal long - range gabaergic projections modulates motor activity in spite of the scarcity of gabaergic corticostriatal neurons and the relatively small amplitude responses of targeted striatal neurons . thus , stimulation of m1 som neuron projections , targeting dspns , ispns , and cholinergic cells , leads to  an increase in locomotion . in contrast , stimulation of m2 som  neuron projections , targeting preferentially dspns and ispns , and of m1 pv neuron projections , targeting preferentially dspns , leads to a decrease in locomotion . decreased locomotion upon preferential inhibition of dspns ( pv - m1 ) is in line with previous studies showing either bradykinesia upon deletion of dspns ( drago et  al . , 1998 ) or increased locomotion upon stimulation of dspns ( kravitz et  al . notably , there was a similar effect on locomotion upon preferential inhibition of dspns ( pv - m1 ) or of both dspns and ispns ( som - m2 ) . comparable effects were also reported when optogenetically silencing either dspn or both dspns and ispns ( tecuapetla et  al . , 2014 ) . increased locomotion upon stimulation of m1 som neuron projections most likely reflects the participation of a larger fraction of cholinergic cells . these striatal interneurons , constituting 1%3% of all striatal neurons , are tonically active and provide powerful feedforward inhibition to spns ( english et  al . , 2011 , nelson et  al . , 2009 ) , and enhance dopamine release ( threlfell et  al . , 2012 ) . direct activation or inhibition of cholinergic striatal interneurons in the dorsal anterior striatum had no effect on locomotor activity ( maurice et  al . , 2015 ) . however , based on our results , it is tempting to speculate that cholinergic cells in more ventral and posterior striatal areas receiving input from m1 som neurons are involved in motor control . at present , we can not resolve whether the observed change in locomotor activity results from long - range axon activation in the striatum only or whether activation of putative collaterals via back - propagating action potentials also plays a role . in either case , our results show that the activity of som and pv projecting neurons in the motor cortex differentially modulates locomotor activity . this study is also relevant when interpreting data regarding silencing of cortical areas by manipulating gabaergic neurons because the effects may also involve long - distance targets . it has been proposed that motor control and reward coding are mediated by common corticostriatal circuits ( kravitz and kreitzer , 2012 ) . our data indicate that , although activation of motor cortex pv and som neuron projections in the dorsal striatum affected locomotor activity , it did not affect place preference , although we can not exclude their implication in reward coding more generally . on the other hand , stimulation of gabaergic projections from the prefrontal cortex to the ventral striatum induces avoidance behavior , suggesting that they are involved in the coding of punishment ( lee et  al . , 2014 ) . further experiments will be required to elucidate whether , and , if so , which , corticostriatal gabaergic projections mediate both locomotion and reward coding . this study adds to the increasing evidence that long - range gabaergic neurons are more frequent than previously thought . the heterogeneity of long - range gabaergic neurons described here is in line with previous studies indicating neurochemical diversity of long - range gabaergic neurons in the cortex and hippocampus ( jinno et  al . , 2007 , higo et  al . , 2007 , lee et  al . , 2014 , notably , we demonstrate that distinct long - range gabaergic neurons exhibit specific functional properties and differential connectivity . finally , it will be of interest to study long - range gabaergic neurons in the context of movement disorders that are thought to be caused by an imbalance of dspn and ispn activity . thus , parkinsonian - like movements can be reproduced by increased ispn activity ( kravitz et  al . , 2010 ) and can be reduced by selective inhibition of striatal cholinergic interneurons ( maurice et  al . , huntington s disease is marked by an early degeneration of ispns ( vonsattel et  al . , 1985 , mitchell et  al . , 1999 ) and an imbalance of excitation and inhibition of dspns and ispns ( andr et  al . , 2011 ) . in light of our findings , it is tempting to speculate that motor cortex gabaergic projections  to the striatum might be a potential target for restoring the balance of striatal output . all experiments were performed in 8- to 20-week - old male mice and were approved by the regierungsprsidium karlsruhe in compliance with the european guidelines for the care and use of laboratory animals ( licenses g74/13 and g248/14 ) . for anterograde tracing experiments , in  vitro patch - clamp recordings , and behavioral experiments , aav dio chr2-mcherry was injected into the primary and/or secondary motor cortex of som ( melzer et  al . , 2012 ) , pv ( hippenmeyer et  al . , 2005 ) , pv / gad67-egfp ( tamamaki et  al . , 2003 ) , som / drd1a - egfp , pv / drd1a - egfp , som / drd2-egfp ( gong et  al . , 2003 ) , and pv / drd2-egfp mice with a c57bl/6 background . for retrograde tracing experiments , ctb 647 was injected into the dorsal striatum of wild - type mice . for retrograde transsynaptic rabies virus tracing , aav - cag - flex - tcb , aav - cag - flex - rg , and sadg - egfp(enva ) were injected into the dorsal striatum of a2a - cre mice . for monosynaptic retrograde rabies virus tracing , aav - cag - flex - tcb was injected into the motor cortex of som ( sst , jackson laboratory ) and pv mice , followed by sadg - egfp(enva ) injection into the dorsal striatum . in all cases , anesthesia was induced and maintained with isoflurane ( 1%2.5% ) , and the virus was delivered through a small craniotomy at the appropriate coordinates by a glass micropipette . for behavioral experiments , immunofluorescence and dab staining were performed on sagittal and coronal brain sections using standard protocols . fresh - frozen 20-m sections were stained with fish using the rnascope fluorescent multiplex kit ( advanced cell diagnostics ) . mice were deeply anesthetized with isoflurane , transcardially perfused with 30  ml ice - cold sucrose solution , and 300-m - thick brain sections were cut . chr2-expressing long - range axonal fibers were stimulated with 5-ms photostimulation ( 473  nm , 120  mw / mm laser intensity ) . pscs were measured at 0-mv holding potential ( using cs - based intracellular solution ) or at 70-mv holding potential ( using k - based , high cl intracellular solution ) . for firing pattern analysis , incrementally increasing currents of 1-s duration were injected in current clamp mode starting at 50 or 200  pa . series resistances of 37 megohm were accepted for analysis of pscs . stimulus delivery and data acquisition were performed using pulse software . mice were video - tracked at 25 frames / s , and their movements were subsequently analyzed using a position tracking system ( ethovision xt9 , noldus ) . the implanted optic fiber cannulas were connected to two optic fibers attached to a rotary joint ( doric lenses ) . a patch cord connected the optic fibers to a  diode - pumped , solid - state , 473-nm laser ( crystalaser ) . we used a pulse generator ( master 8) and a transistor - transistor logic ( ttl ) control box ( universal serial bus input / output [ usb - io ] box , noldus ) to automatically control the photostimulation ( 5-ms pulses delivered at 20  hz ; laser power , 3 mw ) . evaluation of locomotor activity was performed in a circular arena ( 40   40  cm ) placed in a dimly lit room where mice were allowed to run freely for 21  min . it lasted 2  min and was repeated three times with an inter - stimulation period of 4  min . brown - forsythe and f tests were used to test the homogeneity of variances . for non - pairwise  comparisons , unpaired t tests ( either for equal or unequal variance ) or mann - whitney u tests were used . for pairwise comparisons , paired t tests or wilcoxon matched - pairs signed - rank tests were used . one - way anova followed by tukey s multiple comparisons tests was used to compare motion differences . performed in  vitro electrophysiology , analysis , immunohistochemistry , reconstructions , and retrograde tracing studies .
summarythe motor cortico - basal ganglion loop is critical for motor planning , execution , and learning . 
 balanced excitation and inhibition in this loop is crucial for proper motor output . 
 excitatory neurons have been thought to be the only source of motor cortical input  to the striatum . here , we identify long - range projecting gabaergic neurons in the primary ( m1 ) and secondary ( m2 ) motor cortex that target the  dorsal striatum . 
 this  population of projecting gabaergic neurons comprises both somatostatin - positive ( som+ ) and parvalbumin - positive ( pv+ ) neurons that target direct and indirect pathway striatal output neurons as well as cholinergic interneurons differentially . 
 notably , optogenetic stimulation of m1 pv+ and m2 som+ projecting neurons reduced locomotion , whereas stimulation of m1 som+ projecting neurons enhanced locomotion . 
 thus , corticostriatal gabaergic projections modulate striatal output and motor activity .