male young adult ( 4 - 6 weeks of age ) c57bl/6 ( charles river ) , drd1a  tdtomato bac transgenic ( d1-tdtomato , gift of n. calakos , duke university , durham , nc ) , drd2egfp bac transgenic ( d2-egfp ) , oxtrtm1.1wsy homozygous ( cotr ko , jackson laboratory ) , or otr venus neo/+ ( otr - venus , gift of l.j .
young , emory university , atlanta , ga ) mice backcrossed to c57bl/6 were used for all experiments .
all procedures complied with the animal care standards set forth by the national institutes of health and were approved by stanford university s administrative panel on laboratory animal care .
experimenters were blind to the treatment condition when subjective criteria were used as a component of data analysis , and control and test conditions were interleaved for all experiments .
the protocol for social conditioned place preference ( scpp ) was shortened to 2 days of conditioning ( fig .
animals were weaned ( or delivered from charles river ) at 3 weeks of age into  home  cages containing 3 - 5 cage - mates , and housed on corncob bedding ( bed - ocobs , 1/8  , pharmaserv ) .
one to two weeks later , animals were subjected to experimental manipulations and returned to their home cage ( all cage - mates were of the same genotype and received the same experimental manipulation ) .
animals were then placed in open field activity chamber ( env-510 , med associates ) equipped with infrared beams and a software interface ( activity monitor , med associates ) that monitors the position of the mouse .
the apparatus was divided into two equally sized zones using a clear plastic wall , with a 5 cm diameter circular opening at the base ; each zone contained one type of novel bedding ( alpha - dri , pharmaserv , alpha chip , pharmaserv ; bed - ocobs , 1/4  , pharmaserv ; or kaytee soft granule , petco ) .
the amount of time spent freely exploring each zone was recorded during 30-minute test sessions .
after an initial test ( pre - conditioning trial ) to establish baseline preference for the two sets of bedding cues , mice were assigned to receive social conditioning ( with cage - mates ) for 24 hours on one type of bedding , followed by 24 hours on the isolate bedding cue ( without cage - mates ) on the other type of bedding .
24 hours later , animals received a 30-minute post - conditioning trial to establish preference for the two conditioned cues .
animals were excluded ( pre - established criteria ) if they exhibited a pre - conditioning preference score > 1.5 or < 0.5 ( for an unbiased procedure ) ; pre - conditioning versus post - conditioning social preference scores were considered significant if paired student s t - test p values were < 0.05 .
comparisons between experimental conditions were made using both normalized social preference scores ( time spent in social zone post over pre ) , and subtracted social preference scores ( time spent in social zone post minus pre ) ; these were considered significant if unpaired student s t - test ( two conditions ) , or anova ( three conditions , supplementary fig .
6 ) p values were < 0.05 . for cocaine - conditioned place preference ( ccpp ) ,
the apparatus was divided into two equally sized zones using plastic floor tiles with distinct visual and tactile cues ( grey & smooth or white & rough ) .
after 5 days b.i.d . saline injections for habituation in the home cage , the amount of time spent freely exploring each zone was recorded during 30-minute test sessions .
after an initial test to establish baseline preference for the two sets of cues , mice in each of the two treatment groups ( i.p .
otr - a ) were randomly assigned in a counterbalanced fashion to receive cocaine ( 20 mg / kg ) or saline in the presence of one set of cues ( i.e. , an unbiased design ) .
the second conditioning session was conducted 24 hours later in the presence of the other set of cues .
the post - conditioning test session was conducted 24 hours after the second conditioning session to determine time spent in the presence of the cocaine versus saline associated cue . isolation and socially housed animals were not different in terms of ccpp so they were pooled for further analysis .
pre - conditioning , post - conditioning , subtracted , and normalized cocaine preference scores were calculated as in scpp .
briefly , probes consisted of a reservoir ( polypropylene luer hub ) attached to a double cannula guide ( c235gs , 26ga , c / c distance 2.0 mm , 5 mm pedestal , cut 4 mm below pedestal , custom specified for mouse bilateral nac coordinates , plastics one inc ) . polyimide tubing ( 40 awg , 0.0031 " i d , 0.0046 " od , 0.00075 " wall , small parts ) was threaded through the stainless steel tubing of the cannula guide on one end , and out of a hole drilled into the luer hub to act as a flush outlet ( outflow tube ) on the other end .
the dialysis membrane ( spectra / por , 13 kd molecular weight cut - off , spectrum labs ) was then threaded over the outflow tube and through the cannula guide ; ends were cut such that  500 m of dialysis membrane was exposed below the cannula guide and above the sealed end .
junctions were sealed with bio - compatible epoxies ( epo - tek 730 , epo - tek 301 , epoxy technologies ) . in this design
, a pharmacological agent could be intracranially delivered rapidly , continually , and concurrently to all members of the social group , without anesthesia . at postnatal day 35 - 40 , probes were implanted into the nac of male mice following bilateral craniotomy ( bregma 1.54 mm ; lateral 1.0 mm ) and attached to the skull using dental acrylic .
previous reports indicate that for complete pharmacological effect , drug concentration in the reservoir must be 500 times the dose used for direct injections , thus otr and 5htr1b antagonists were applied at 10 mm ( l-368,899 ) and 85 mm ( nas-181 ) in a volume of 25 l saline .
probe placement and competency was verified by post - hoc application of concentrated fluorescein sodium salt ( sigma - aldrich ) to reservoir prior to intracardial pfa perfusion and histology ( supplementary fig .
1f ) . rabies virus ( rbv ) was generated from a full length cdna plasmid containing all components of rbv ( sad l16 ; gift from dr .
we replaced the rabies virus glycoprotein with egfp ( rbv - egfp ) , tdtomato ( rbv - tdtomato ) or cre - egfp to generate rbv expressing cre - egfp ( rbv - cre - egfp ) , egfp ( rbv - egfp ) or tdtomato ( rbv - tdtomato ) .
to rescue rbv from this cdna we used a modified version of a published protocol .
briefly , hek293 t cells were transfected with a total of 6 plasmids ; 4 plasmids expressing the rbv components ptit - n , ptit - p , ptit - g , and ptit - l ; one plasmid expressing t7 rna polymerase ( pcaggs - t7 ) , and the aforementioned glycoprotein - deleted rbv cdna plasmid expressing cre - egfp , egfp or tdtomato . for the amplification of rbv ,
the media bathing these hek293 t ( atcc ) cells was collected 3 - 4 days posttransfection and moved to baby hamster kidney ( bhk ) cells stably expressing rbv glycoprotein ( bhk - b19 g ) .
after three days , the media from bhk - b19 g cells was collected , centrifuged for 5 min at 3,000 x g to remove cell debris , and concentrated by ultracentrifugation ( 55,000 x g for 2 hr ) .
the adeno - associated viruses ( aavs ) used in this study were produced by the stanford neuroscience gene vector and virus core .
briefly , aav - dj was produced by transfection of aav 293 cells ( agilent , inc ) with three plasmids : an aav vector expressing cre - egfp , aav helper plasmid ( phelper , agilent , inc ) , and aav rep - cap helper plasmid ( prc - dj , gift from mark kay , stanford ) . at 72 h after transfection ,
the iodixanol was diluted and the aav was concentrated using a 100 kda molecular weight cutoff ultrafiltration device .
stereotaxic injection of viruses into nac was performed under general ketamine - medetomidine anesthesia using a stereotaxic instrument ( david kopf ) .
a small volume ( 1 l ) of concentrated virus solution was injected bilaterally into nac core ( bregma 1.54 mm ; lateral 1.0 mm ; ventral 4.0 mm ) , unilaterally into the dorsal raphe ( drph ; bregma -3.3 mm ; lateral 0.0 mm ; ventral 3.35 mm ) , bilaterally into the ventral subiculum ( vsub ; bregma -2.95 mm ; lateral 3.1 mm ; ventral 4.35 mm ) , or bilaterally anterior cingulate ( acc ; bregma 1.0 mm ; lateral 0.3 mm ; ventral 1.25 mm ) at a slow rate ( 100 nl / min ) using a syringe pump ( harvard apparatus , ma ) .
injection sites and viral infectivity were confirmed in all animals post - hoc by preparing sections ( 50 m ) containing the relevant brain region ( supplementary fig .
briefly , after intracardial perfusion with 4% paraformaldehyde in pbs ( ph 7.4 ) , the brains were post fixed overnight in this same solution and the following day 50 m coronal , sagittal , or horizontal sections were prepared .
primary antibodies were used at the following concentrations : mouse anti - oxytocin - neurophysin ( ot - np , 1:50 ; gift of harold gainer , nih bethesda , md ) ; rat anti - green fluorescent protein ( gfp , 1:1000 ; nacalai ) ; rabbit anti - parvalbumin ( parv , 1:750 ; swant ) ; rabbit anti - neuronal nitric oxide synthase ( nnos , 1:100 ; bd transduction laboratories ) ; rabbit anti - glial fibrillary protein ( gfap , 1:80 ; sigma - aldrich ) ; rabbit anti - choline acetyltransferase ( chat , 1:100 ; millipore ) ; rabbit anti - dopamine receptor protein ( darp , 1:100 , millipore ) ; sheep anti - tryptophan hydroxylase ( tryph , 1:100 millipore ) diluted in a solution containing 1% horse serum , 0.2% bsa , and 0.5% triton x-100 in pbs . after overnight incubation in primary antibody ( rt , shaker )
, slices were washed four times in pbs and then incubated with appropriate secondary antibody diluted at 1:750 for 2 h in pbs containing 0.5% triton x-100 .
subsequently , slices were washed 5 times and mounted using vectashield mounting medium ( vector laboratories ) . to identify cells expressing gfp or tdtomato due to the injection of rbv - egfp or rbv - tdtomato into the nac ,
image acquisition was performed with a confocal microscope ( zeiss lsm510 ) using a 10x/0.30 plan neofluar and a 40x/1.3 oil dic plan apochromat objective .
parasagittal slices ( 250 m ) containing the nac core were prepared from c57bl/6 and d1-tdtomato / d2-egfp bac transgenic mice on a c57bl/6 background using standard procedures .
briefly , after mice were anesthetized with isoflurane and decapitated , brains were quickly removed and placed in ice - cold low sodium , high sucrose dissecting solution .
slices were cut by adhering the two sagittal hemispheres brain containing the nac core to the stage of a leica vibroslicer .
slices were allowed to recover for a minimum of 60 min in a submerged holding chamber ( 25c ) containing artificial cerebrospinal fluid ( acsf ) consisting of 119 mm nacl , 2.5 mm kcl , 2.5 mm cacl2 , 1.3 mm mgso4 , 1 mm nah2po4 , 11 mm glucose and 26.2 mm nahco3 .
slices were then removed from the holding chamber and placed in the recording chamber where they were continuously perfused with oxygenated ( 95% o2 , 5% co2 ) acsf at a rate of 2 ml per min at 26  2c . for epsc recordings , bicuculline ( 20 m )
for ipsc recordings , dl-2-amino-5-phosphonovalerate ( dapv , 10 m ) and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide ( nbqx , 5 m ) dissolved in dmso were added to block nmda and ampa receptors respectively .
whole - cell voltage - clamp recordings from msns were obtained under visual control using a 40x objective .
d1- and d2 msns in the nac core were identified by the presence of tdtomato and egfp , respectively , that were excited with uv light using bandpass filters ( hq545/30x ex for tdtomato ; hq470/40x ex for egfp ) .
recordings were made with electrodes ( 3.56.5 m ) filled with 115 mm csmeso4 , 20 mm cscl , 10 mm hepes , 0.6 mm egta , 2.5 mm mgcl , 10 mm na - phosphocreatine , 4 mm na - atp , 0.3 mm na - gtp , and 1 mm qx-314 .
excitatory and inhibitory afferents were stimulated with a bipolar nichrome wire electrode placed at the border between the nac core and cortex dorsal to the anterior commissure .
recordings were performed using a multiclamp 700b ( molecular devices ) , filtered at 2 khz and digitized at 10 khz .
epscs were evoked at a frequency of 0.1 hz while msns were voltage - clamped at 70 mv .
input resistance and access resistance were monitored continuously throughout each experiment ; experiments were terminated if these changed by > 15% .
summary ltd graphs were generated by averaging the peak amplitudes of individual epscs in 1 min bins ( 6 consecutive sweeps ) and normalizing these to the mean value of epscs collected during the 10 min baseline immediately before the ltd - induction protocol .
oxytocin ( ot , tocris biosciences , 1 m , 10 minute ) was bath applied following the collection of baseline for induction of ot - ltd . for experiments examining the blockade of ot - ltd , slices were pre - incubated in antagonist ( otr - a , 1 m l-368,899 hydrochloride or 5htr1b - a , 20 m nas-181 ; tocris biosciences ) for at least 30 minutes prior to recording . for experiments examining the reversal of ot - ltd , 30 - 40 minutes
following the collection of stable baseline epscs , 5ht1b - ltd was induced by 10 minute bath application of 2 m cp-93129 dihydrochloride ( tocris biosciences ) as described previously . for experiments examining the occlusion of ot - ltd , after stabilization of 5ht1b - ltd ( at 30 - 40 minutes post induction ) ,
miniature epscs were collected at a holding potential of 70 mv in the presence of ttx ( 0.5 m ) .
2 minutes after break - in ( sweep number 5 , 30s sweeps ) , thirty second blocks of events ( total of 200 events per cell ) were acquired and analyzed using mini - analysis software ( synaptosoft ) with threshold parameters set at 5 pa amplitude and <3 ms rise time .
slices were incubated in the appropriate drug ( dissolved in acsf - bicuculline ) for 10 minutes prior to recording , and cross - cell comparisons were made .
paired - pulse ratios ( ppr ) were acquired by applying a second afferent stimulus of equal intensity , 50 ms after the first stimulus , and then calculating the ratio of epsc2/epsc1 .
coefficient of variance ( cv ) was calculated from the standard deviation divided by the average ( stdev / avg ) of 10-minute blocks ( minutes 0 - 10 , pre ; minutes 40 - 50 , post ) .
comparisons between different experimental manipulations were made using a two- tailed , students t - test ( paired or unpaired , as appropriate ) with p < 0.05 considered significant .
all statements in the text regarding differences between grouped data indicate that statistical significance was achieved , assuming normal distribution and equal variance .
male young adult ( 4 - 6 weeks of age ) c57bl/6 ( charles river ) , drd1a  tdtomato bac transgenic ( d1-tdtomato , gift of n. calakos , duke university , durham , nc ) , drd2egfp bac transgenic ( d2-egfp ) , oxtrtm1.1wsy homozygous ( cotr ko , jackson laboratory ) , or otr venus neo/+ ( otr - venus , gift of l.j .
young , emory university , atlanta , ga ) mice backcrossed to c57bl/6 were used for all experiments .
all procedures complied with the animal care standards set forth by the national institutes of health and were approved by stanford university s administrative panel on laboratory animal care .
experimenters were blind to the treatment condition when subjective criteria were used as a component of data analysis , and control and test conditions were interleaved for all experiments .
the protocol for social conditioned place preference ( scpp ) was shortened to 2 days of conditioning ( fig .
animals were weaned ( or delivered from charles river ) at 3 weeks of age into  home  cages containing 3 - 5 cage - mates , and housed on corncob bedding ( bed - ocobs , 1/8  , pharmaserv ) .
one to two weeks later , animals were subjected to experimental manipulations and returned to their home cage ( all cage - mates were of the same genotype and received the same experimental manipulation ) .
animals were then placed in open field activity chamber ( env-510 , med associates ) equipped with infrared beams and a software interface ( activity monitor , med associates ) that monitors the position of the mouse .
the apparatus was divided into two equally sized zones using a clear plastic wall , with a 5 cm diameter circular opening at the base ; each zone contained one type of novel bedding ( alpha - dri , pharmaserv , alpha chip , pharmaserv ; bed - ocobs , 1/4  , pharmaserv ; or kaytee soft granule , petco ) .
the amount of time spent freely exploring each zone was recorded during 30-minute test sessions .
after an initial test ( pre - conditioning trial ) to establish baseline preference for the two sets of bedding cues , mice were assigned to receive social conditioning ( with cage - mates ) for 24 hours on one type of bedding , followed by 24 hours on the isolate bedding cue ( without cage - mates ) on the other type of bedding .
24 hours later , animals received a 30-minute post - conditioning trial to establish preference for the two conditioned cues .
animals were excluded ( pre - established criteria ) if they exhibited a pre - conditioning preference score > 1.5 or < 0.5 ( for an unbiased procedure ) ; pre - conditioning versus post - conditioning social preference scores were considered significant if paired student s t - test p values were < 0.05 .
comparisons between experimental conditions were made using both normalized social preference scores ( time spent in social zone post over pre ) , and subtracted social preference scores ( time spent in social zone post minus pre ) ; these were considered significant if unpaired student s t - test ( two conditions ) , or anova ( three conditions , supplementary fig .
6 ) p values were < 0.05 . for cocaine - conditioned place preference ( ccpp ) ,
the apparatus was divided into two equally sized zones using plastic floor tiles with distinct visual and tactile cues ( grey & smooth or white & rough ) .
after 5 days b.i.d . saline injections for habituation in the home cage , the amount of time spent freely exploring each zone was recorded during 30-minute test sessions .
after an initial test to establish baseline preference for the two sets of cues , mice in each of the two treatment groups ( i.p .
otr - a ) were randomly assigned in a counterbalanced fashion to receive cocaine ( 20 mg / kg ) or saline in the presence of one set of cues ( i.e. , an unbiased design ) .
the second conditioning session was conducted 24 hours later in the presence of the other set of cues .
the post - conditioning test session was conducted 24 hours after the second conditioning session to determine time spent in the presence of the cocaine versus saline associated cue . isolation and socially housed animals were not different in terms of ccpp so they were pooled for further analysis .
pre - conditioning , post - conditioning , subtracted , and normalized cocaine preference scores were calculated as in scpp .
briefly , probes consisted of a reservoir ( polypropylene luer hub ) attached to a double cannula guide ( c235gs , 26ga , c / c distance 2.0 mm , 5 mm pedestal , cut 4 mm below pedestal , custom specified for mouse bilateral nac coordinates , plastics one inc ) . polyimide tubing
( 40 awg , 0.0031 " i d , 0.0046 " od , 0.00075 " wall , small parts ) was threaded through the stainless steel tubing of the cannula guide on one end , and out of a hole drilled into the luer hub to act as a flush outlet ( outflow tube ) on the other end . the dialysis membrane ( spectra / por , 13 kd molecular weight cut - off , spectrum labs ) was then threaded over the outflow tube and through the cannula guide ; ends were cut such that  500 m of dialysis membrane was exposed below the cannula guide and above the sealed end .
junctions were sealed with bio - compatible epoxies ( epo - tek 730 , epo - tek 301 , epoxy technologies ) . in this design
, a pharmacological agent could be intracranially delivered rapidly , continually , and concurrently to all members of the social group , without anesthesia . at postnatal day 35 - 40 , probes were implanted into the nac of male mice following bilateral craniotomy ( bregma 1.54 mm ; lateral 1.0 mm ) and attached to the skull using dental acrylic .
previous reports indicate that for complete pharmacological effect , drug concentration in the reservoir must be 500 times the dose used for direct injections , thus otr and 5htr1b antagonists were applied at 10 mm ( l-368,899 ) and 85 mm ( nas-181 ) in a volume of 25 l saline .
probe placement and competency was verified by post - hoc application of concentrated fluorescein sodium salt ( sigma - aldrich ) to reservoir prior to intracardial pfa perfusion and histology ( supplementary fig .
rabies virus ( rbv ) was generated from a full length cdna plasmid containing all components of rbv ( sad l16 ; gift from dr .
we replaced the rabies virus glycoprotein with egfp ( rbv - egfp ) , tdtomato ( rbv - tdtomato ) or cre - egfp to generate rbv expressing cre - egfp ( rbv - cre - egfp ) , egfp ( rbv - egfp ) or tdtomato ( rbv - tdtomato ) .
to rescue rbv from this cdna we used a modified version of a published protocol .
briefly , hek293 t cells were transfected with a total of 6 plasmids ; 4 plasmids expressing the rbv components ptit - n , ptit - p , ptit - g , and ptit - l ; one plasmid expressing t7 rna polymerase ( pcaggs - t7 ) , and the aforementioned glycoprotein - deleted rbv cdna plasmid expressing cre - egfp , egfp or tdtomato . for the amplification of rbv ,
the media bathing these hek293 t ( atcc ) cells was collected 3 - 4 days posttransfection and moved to baby hamster kidney ( bhk ) cells stably expressing rbv glycoprotein ( bhk - b19 g ) .
after three days , the media from bhk - b19 g cells was collected , centrifuged for 5 min at 3,000 x g to remove cell debris , and concentrated by ultracentrifugation ( 55,000 x g for 2 hr ) .
the adeno - associated viruses ( aavs ) used in this study were produced by the stanford neuroscience gene vector and virus core .
briefly , aav - dj was produced by transfection of aav 293 cells ( agilent , inc ) with three plasmids : an aav vector expressing cre - egfp , aav helper plasmid ( phelper , agilent , inc ) , and aav rep - cap helper plasmid ( prc - dj , gift from mark kay , stanford ) . at 72 h after transfection ,
the iodixanol was diluted and the aav was concentrated using a 100 kda molecular weight cutoff ultrafiltration device .
stereotaxic injection of viruses into nac was performed under general ketamine - medetomidine anesthesia using a stereotaxic instrument ( david kopf ) .
a small volume ( 1 l ) of concentrated virus solution was injected bilaterally into nac core ( bregma 1.54 mm ; lateral 1.0 mm ; ventral 4.0 mm ) , unilaterally into the dorsal raphe ( drph ; bregma -3.3 mm ; lateral 0.0 mm ; ventral 3.35 mm ) , bilaterally into the ventral subiculum ( vsub ; bregma -2.95 mm ; lateral 3.1 mm ; ventral 4.35 mm ) , or bilaterally anterior cingulate ( acc ; bregma 1.0 mm ; lateral 0.3 mm ; ventral 1.25 mm ) at a slow rate ( 100 nl / min ) using a syringe pump ( harvard apparatus , ma ) .
injection sites and viral infectivity were confirmed in all animals post - hoc by preparing sections ( 50 m ) containing the relevant brain region ( supplementary fig .
briefly , after intracardial perfusion with 4% paraformaldehyde in pbs ( ph 7.4 ) , the brains were post fixed overnight in this same solution and the following day 50 m coronal , sagittal , or horizontal sections were prepared .
primary antibodies were used at the following concentrations : mouse anti - oxytocin - neurophysin ( ot - np , 1:50 ; gift of harold gainer , nih bethesda , md ) ; rat anti - green fluorescent protein ( gfp , 1:1000 ; nacalai ) ; rabbit anti - parvalbumin ( parv , 1:750 ; swant ) ; rabbit anti - neuronal nitric oxide synthase ( nnos , 1:100 ; bd transduction laboratories ) ; rabbit anti - glial fibrillary protein ( gfap , 1:80 ; sigma - aldrich ) ; rabbit anti - choline acetyltransferase ( chat , 1:100 ; millipore ) ; rabbit anti - dopamine receptor protein ( darp , 1:100 , millipore ) ; sheep anti - tryptophan hydroxylase ( tryph , 1:100 millipore ) diluted in a solution containing 1% horse serum , 0.2% bsa , and 0.5% triton x-100 in pbs . after overnight incubation in primary antibody ( rt , shaker )
, slices were washed four times in pbs and then incubated with appropriate secondary antibody diluted at 1:750 for 2 h in pbs containing 0.5% triton x-100 .
subsequently , slices were washed 5 times and mounted using vectashield mounting medium ( vector laboratories ) . to identify cells expressing gfp or tdtomato due to the injection of rbv - egfp or rbv - tdtomato into the nac ,
image acquisition was performed with a confocal microscope ( zeiss lsm510 ) using a 10x/0.30 plan neofluar and a 40x/1.3 oil dic plan apochromat objective .
parasagittal slices ( 250 m ) containing the nac core were prepared from c57bl/6 and d1-tdtomato / d2-egfp bac transgenic mice on a c57bl/6 background using standard procedures . briefly , after mice were anesthetized with isoflurane and decapitated , brains were quickly removed and placed in ice - cold low sodium , high sucrose dissecting solution .
slices were cut by adhering the two sagittal hemispheres brain containing the nac core to the stage of a leica vibroslicer .
slices were allowed to recover for a minimum of 60 min in a submerged holding chamber ( 25c ) containing artificial cerebrospinal fluid ( acsf ) consisting of 119 mm nacl , 2.5 mm kcl , 2.5 mm cacl2 , 1.3 mm mgso4 , 1 mm nah2po4 , 11 mm glucose and 26.2 mm nahco3 .
slices were then removed from the holding chamber and placed in the recording chamber where they were continuously perfused with oxygenated ( 95% o2 , 5% co2 ) acsf at a rate of 2 ml per min at 26  2c . for epsc recordings , bicuculline ( 20 m ) was added to the acsf to block gabaa receptor  mediated inhibitory synaptic currents . for ipsc recordings , dl-2-amino-5-phosphonovalerate ( dapv , 10 m ) and
2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide ( nbqx , 5 m ) dissolved in dmso were added to block nmda and ampa receptors respectively .
whole - cell voltage - clamp recordings from msns were obtained under visual control using a 40x objective .
d1- and d2 msns in the nac core were identified by the presence of tdtomato and egfp , respectively , that were excited with uv light using bandpass filters ( hq545/30x ex for tdtomato ; hq470/40x ex for egfp ) .
recordings were made with electrodes ( 3.56.5 m ) filled with 115 mm csmeso4 , 20 mm cscl , 10 mm hepes , 0.6 mm egta , 2.5 mm mgcl , 10 mm na - phosphocreatine , 4 mm na - atp , 0.3 mm na - gtp , and 1 mm qx-314 .
excitatory and inhibitory afferents were stimulated with a bipolar nichrome wire electrode placed at the border between the nac core and cortex dorsal to the anterior commissure .
recordings were performed using a multiclamp 700b ( molecular devices ) , filtered at 2 khz and digitized at 10 khz .
epscs were evoked at a frequency of 0.1 hz while msns were voltage - clamped at 70 mv .
input resistance and access resistance were monitored continuously throughout each experiment ; experiments were terminated if these changed by > 15% .
summary ltd graphs were generated by averaging the peak amplitudes of individual epscs in 1 min bins ( 6 consecutive sweeps ) and normalizing these to the mean value of epscs collected during the 10 min baseline immediately before the ltd - induction protocol .
oxytocin ( ot , tocris biosciences , 1 m , 10 minute ) was bath applied following the collection of baseline for induction of ot - ltd . for experiments examining the blockade of ot - ltd ,
slices were pre - incubated in antagonist ( otr - a , 1 m l-368,899 hydrochloride or 5htr1b - a , 20 m nas-181 ; tocris biosciences ) for at least 30 minutes prior to recording . for experiments examining the reversal of ot - ltd , 30 - 40 minutes
post induction , otr - a was bath applied for 10 minutes . following the collection of stable baseline epscs , 5ht1b - ltd was induced by 10 minute bath application of 2 m cp-93129 dihydrochloride ( tocris biosciences ) as described previously . for experiments examining the occlusion of ot - ltd , after stabilization of 5ht1b - ltd ( at 30 - 40 minutes post induction ) ,
miniature epscs were collected at a holding potential of 70 mv in the presence of ttx ( 0.5 m ) .
2 minutes after break - in ( sweep number 5 , 30s sweeps ) , thirty second blocks of events ( total of 200 events per cell ) were acquired and analyzed using mini - analysis software ( synaptosoft ) with threshold parameters set at 5 pa amplitude and <3 ms rise time .
slices were incubated in the appropriate drug ( dissolved in acsf - bicuculline ) for 10 minutes prior to recording , and cross - cell comparisons were made .
paired - pulse ratios ( ppr ) were acquired by applying a second afferent stimulus of equal intensity , 50 ms after the first stimulus , and then calculating the ratio of epsc2/epsc1 .
coefficient of variance ( cv ) was calculated from the standard deviation divided by the average ( stdev / avg ) of 10-minute blocks ( minutes 0 - 10 , pre ; minutes 40 - 50 , post ) .
comparisons between different experimental manipulations were made using a two- tailed , students t - test ( paired or unpaired , as appropriate ) with p < 0.05 considered significant .
all statements in the text regarding differences between grouped data indicate that statistical significance was achieved , assuming normal distribution and equal variance .
 social behaviors in species as diverse as honey bees and humans promote group survival but often come at some cost to the individual . 
 although reinforcement of adaptive social interactions is ostensibly required for the evolutionary persistence of these behaviors , the neural mechanisms by which social reward is encoded by the brain are largely unknown . here 
 we demonstrate that in mice oxytocin ( ot ) acts as a social reinforcement signal within the nucleus accumbens ( nac ) core , where it elicits a presynaptically expressed long - term depression of excitatory synaptic transmission in medium spiny neurons . 
 although the nac receives ot receptor - containing inputs from several brain regions , genetic deletion of these receptors specifically from dorsal raphe nucleus , which provides serotonergic ( 5-ht ) innervation to the nac , abolishes the reinforcing properties of social interaction . 
 furthermore , ot - induced synaptic plasticity requires activation of nac 5-ht1b receptors , the blockade of which prevents social reward . 
 these results demonstrate that the rewarding properties of social interaction in mice require the coordinated activity of ot and 5-ht in the nac , a mechanistic insight with implications for understanding the pathogenesis of social dysfunction in neuropsychiatric disorders such as autism . 
METHODS
Animals
Behavioral Assays
Andalman probes
Virus Generation
Stereotaxic injections
Immunohistochemistry
Electrophysiology
Supplementary Material