the following genetically modified mice and crosses between them were used for electrophysiological analysis : nestin - gfp ( cb57bl/6 background ) , pv - cre ( jax laboratory ; stock number : 008069 ; stock name : b6;129p2-pvalb / j ) , sst - cre ( jax laboratory ; stock number : 013044 ; stock name : sst / j ) , vip - cre ( jax laboratory , stock number : 010908 ; stock name : vip / j ) .
the following mice were used for neurogenesis analysis : wild - type ( c57bl/6 ) , nestin - creer;z / eg;2 ( ref ; c57bl/6 ) and nestin - creer;z / eg ( c57bl/6 ) , pv - cre ( b6;129 ) , sst - cre ( b6;129 ) , and vip - cre ( b6;129 ) .
socially isolated animals were individually housed right after weaning for at least 6 weeks before tamoxifen or edu injection , and had free access to food and water .
a single dose of tamoxifen ( tmx ; 62 mg / kg ) was i.p .
injected into 6- to 10-week - old mice as previously described . for optogenetic manipulations ,
cre - dependent recombinant aav vectors were used based on a dna cassette carrying two pairs of incompatible loxp sites with the opsin genes ( chr2-h134-mcherry , chr2-h134-yfp , or enphr3.0-yfp ) inserted between lox sites in the reverse orientation as described previously ( supplementary fig .
the recombinant aav vectors were serotyped with aav2/9 for chr2 ( packaged at the upenn vector core ) , and aav9 for enphr3.0 ( packaged at university of north carolina vector core ) .
the following final viral concentrations were used for aav viruses ( x10 particles / ml ) : 7.4 ( chr2-yfp ) , 36 ( chr2-mcherry ) , and 8 ( enphr3.0-yfp ) , respectively .
aav was stereotaxically delivered into the dentate gyrus using the following coordinates ( in mm ) : anterioposterior = 2 from bregma ; lateral = 1.5 ; ventral = 2.2 .
were implanted at the same injection sites right after aav injection with the dorsal - ventral depth of 1.6 mm from the skull .
animals were then allowed to recover for at least 3 weeks from surgery . for analysis of rgl activation at the population level after optogenetic manipulations , littermates of animals were used and an in vivo light paradigm was administered 8 hrs per day for 5 consecutive days ( supplementary fig .
5a , 6e and 7b ) . for chr2-yfp stimulation , blue light flashes ( 472 nm ; 5 ms at 8 hz ) through the dpssl laser system ( laser century co. ltd . ,
shanghai , china ) were delivered in vivo every 5 minutes for 30 s / per trial . for enphr - yfp stimulation , continuous yellow light ( 593 nm )
, animals were injected with edu ( 41.1 mg / kg body weight ) 6 times with an interval of 2 hrs .
animals were sacrificed 2 hrs after the last edu injection and processed for immunostaining as previously described .
briefly , brains were quickly removed into the ice - cold solution ( in mm : 110 choline chloride , 2.5 kcl , 1.3 kh2po4 , 25.0 nahco3 , 0.5 cacl2 , 7 mgso4 , 20 dextrose , 1.3 sodium l - ascorbate , 0.6 sodium pyruvate , 5.0 kynurenic acid ) .
slices ( 300 m thick ) were sectioned using a vibratome ( leica vt1000s ) and transferred to a chamber containing the external solution ( in mm : 125.0 nacl , 2.5 kcl , 1.3 kh2po4 , 1.3 mgso4 , 25.0 nahco3 , 2 cacl2 , 1.3 sodium l - ascorbate , 0.6 sodium pyruvate , 10 dextrose , ph 7.4 , 320 mosm ) , bubbled with 95% o2/5% co2 .
gfp rgls located within the sgz in adult nestin - gfp mice were visualized by dic and fluorescence microscopy .
a whole - cell patch - clamp configuration was employed in the voltage - clamp mode ( vm = 65 mv ) or current - clamp mode .
microelectrodes ( 46 m ) were pulled from borosilicate glass capillaries and filled with the internal solution containing ( in mm ) : 135 cscl gluconate , 15 kcl , 4 mgcl2 , 0.1 egta , 10.0 hepes , 4 atp ( magnesium salt ) , 0.3 gtp ( sodium salt ) , 7 phosphocreatine ( ph 7.4 , 300 mosm ) .
all rgl recordings were carried out in the presence of kynurenic acid ( 5 mm ) .
data was collected using an axon 200b amplifier and acquired with a digidata 1322a ( axon instruments ) at 10 khz . for measuring gaba - induced responses from gfp rgls ,
focal pressure ejection of 200 mm gaba or muscimol through a puffer pipette controlled by a picospritze ( 2s puff at 35 psi ) was used to activate gabaars under the whole - cell voltage - clamp . a bipolar electrode ( world precision instruments )
low frequency ( 0.1 hz ) and theta bursts ( 8 hz with a train of 100 stimuli ) were delivered . the stimulus intensity ( 50 a )
the following pharmacological agents were used : diazepam ( 1 m ) , no-711 ( 10 m ) , flumazenil ( 10 m ) , midazolam ( 10 m ) , etmd ( 10 m ) , l-655708 ( 50 m ) , and vigabatrin ( 100 m ) .
all drugs were purchased from sigma except bicuculline ( 50 or 100 m ; tocris ) .
rgl recordings under optogenetic manipulation in acute brain slices were performed at least 4 weeks after aav injection . to stimulate chr2 in labelled interneurons , light flashes ( 5 ms at 1 , 8 or 100 hz ) generated by a lambda dg-4 plus high - speed optical switch with a 300w xenon lamp and a 472 nm filter set ( chroma ) were delivered to coronal sections through a 40x objective ( carl zeiss ) .
to stimulate enphr in labelled interneurons , continuous yellow light generated by dg-4 plus system with a 593 nm filter set were delivered to coronal sections across a full high - power ( 40x ) field . for immunostaining with anti - nestin and anti - mcm2 ,
an antigen retrieval protocol was performed by microwaving sections in boiled citric buffer for 7 minutes as previously described . for 2 immunostaining ,
a weak fixation protocol using live tissues was adopted as previously described . for characterization of different interneuron subtypes ,
following antibodies were used : anti - pv ( swant ; mouse or rabbit ; 1:500 ) , anti - gad-67 ( millipore ; mouse or rabbit ; 1:500 ) , ant - sst ( millipore ; rat ; 1:200 ) , and anti - vip ( immunostar ; rabbit ; 1:200 ) . for clonal analysis , coronal brain sections ( 40 m ) through the entire dentate gyrus
were collected in a serial order , and immunostaining was performed using following primary antibodies as previously described : anti - gfp ( rockland ; goat ; 1:500 ) , anti - nestin ( aves ; chick ; 1:500 ) , anti - mcm2 ( bd ; mouse ; 1:500 ) , anti - gfap ( millipore ; mouse or rabbit ; 1:1000 ) , anti - psa - ncam ( millipore , mouse lgm ; 1:500 ) . for quantification of gfp clones at 2 and 7 dpi , a single gfp rgl was scored as a quiescent clone .
clonal analysis at 30 dpi was carried out exactly as previously described . for experiments with diazepam ( 5 mg / kg body weight
; once daily for 5 days ) , coronal brain sections ( 40 m ) through the entire dentate gyrus were collected in a serial order .
for optogenetic manipulations , sections within a distance of 1.0 mm anterior and 1.0 mm posterior to injection sites were used for quantification , given the estimated light spread in vivo .
edu labelling was performed using click - it edu alexa fluor imaging kit ( invitrogen ) .
images were acquired on a zeiss lsm 710 confocal system ( carl zeiss , thornwood , ny , usa ) with a 40x objective using a multi - track configuration .
stereological quantification of cells positive for various molecular markers was assessed in the dentate gyrus using a modified optical fractionator technique .
for quantification of edu or mcm2 rgls , an inverted  y  shape from anti - nestin staining superimposed onto edu or mcm2 nucleus was scored double positive for nestin and edu or mcm2 .
statistic analysis was performed with student t - test . for generation of movie files ,
images were serially reconstructed in reconstruct ( john c. fiala , the national institutes of health ) , normalized , and deconvolved with autoquant x2 ( media cybernetics ) .
images were then segmented in matlab ( the mathworks ) using custom code and imported into imaris ( bitplane ) .
surface renderings and movies were made using the surface and animation functions , respectively , in imaris ( supplementary movies 13 ) .
the following genetically modified mice and crosses between them were used for electrophysiological analysis : nestin - gfp ( cb57bl/6 background ) , pv - cre ( jax laboratory ; stock number : 008069 ; stock name : b6;129p2-pvalb / j ) , sst - cre ( jax laboratory ; stock number : 013044 ; stock name : sst / j ) , vip - cre ( jax laboratory , stock number : 010908 ; stock name : vip / j ) .
the following mice were used for neurogenesis analysis : wild - type ( c57bl/6 ) , nestin - creer;z / eg;2 ( ref ; c57bl/6 ) and nestin - creer;z / eg ( c57bl/6 ) , pv - cre ( b6;129 ) , sst - cre ( b6;129 ) , and vip - cre ( b6;129 ) .
socially isolated animals were individually housed right after weaning for at least 6 weeks before tamoxifen or edu injection , and had free access to food and water .
a single dose of tamoxifen ( tmx ; 62 mg / kg ) was i.p .
injected into 6- to 10-week - old mice as previously described . for optogenetic manipulations ,
cre - dependent recombinant aav vectors were used based on a dna cassette carrying two pairs of incompatible loxp sites with the opsin genes ( chr2-h134-mcherry , chr2-h134-yfp , or enphr3.0-yfp ) inserted between lox sites in the reverse orientation as described previously ( supplementary fig .
the recombinant aav vectors were serotyped with aav2/9 for chr2 ( packaged at the upenn vector core ) , and aav9 for enphr3.0 ( packaged at university of north carolina vector core ) .
the following final viral concentrations were used for aav viruses ( x10 particles / ml ) : 7.4 ( chr2-yfp ) , 36 ( chr2-mcherry ) , and 8 ( enphr3.0-yfp ) , respectively .
aav was stereotaxically delivered into the dentate gyrus using the following coordinates ( in mm ) : anterioposterior = 2 from bregma ; lateral = 1.5 ; ventral = 2.2 .
were implanted at the same injection sites right after aav injection with the dorsal - ventral depth of 1.6 mm from the skull .
animals were then allowed to recover for at least 3 weeks from surgery . for analysis of rgl activation at the population level after optogenetic manipulations , littermates of animals were used and an in vivo light paradigm was administered 8 hrs per day for 5 consecutive days ( supplementary fig .
5a , 6e and 7b ) . for chr2-yfp stimulation , blue light flashes ( 472 nm ; 5 ms at 8 hz ) through the dpssl laser system ( laser century co. ltd . ,
shanghai , china ) were delivered in vivo every 5 minutes for 30 s / per trial . for enphr - yfp stimulation , continuous yellow light ( 593 nm )
, animals were injected with edu ( 41.1 mg / kg body weight ) 6 times with an interval of 2 hrs .
animals were sacrificed 2 hrs after the last edu injection and processed for immunostaining as previously described .
, brains were quickly removed into the ice - cold solution ( in mm : 110 choline chloride , 2.5 kcl , 1.3 kh2po4 , 25.0 nahco3 , 0.5 cacl2 , 7 mgso4 , 20 dextrose , 1.3 sodium l - ascorbate , 0.6 sodium pyruvate , 5.0 kynurenic acid ) .
slices ( 300 m thick ) were sectioned using a vibratome ( leica vt1000s ) and transferred to a chamber containing the external solution ( in mm : 125.0 nacl , 2.5 kcl , 1.3 kh2po4 , 1.3 mgso4 , 25.0 nahco3 , 2 cacl2 , 1.3 sodium l - ascorbate , 0.6 sodium pyruvate , 10 dextrose , ph 7.4 , 320 mosm ) , bubbled with 95% o2/5% co2 .
gfp rgls located within the sgz in adult nestin - gfp mice were visualized by dic and fluorescence microscopy .
a whole - cell patch - clamp configuration was employed in the voltage - clamp mode ( vm = 65 mv ) or current - clamp mode .
microelectrodes ( 46 m ) were pulled from borosilicate glass capillaries and filled with the internal solution containing ( in mm ) : 135 cscl gluconate , 15 kcl , 4 mgcl2 , 0.1 egta , 10.0 hepes , 4 atp ( magnesium salt ) , 0.3 gtp ( sodium salt ) , 7 phosphocreatine ( ph 7.4 , 300 mosm ) .
all rgl recordings were carried out in the presence of kynurenic acid ( 5 mm ) . data was collected using an axon 200b amplifier and acquired with a digidata 1322a ( axon instruments ) at 10 khz . for measuring gaba - induced responses from gfp rgls , focal pressure ejection of 200 mm gaba or muscimol through a puffer pipette controlled by a picospritze ( 2s puff at 35 psi ) was used to activate gabaars under the whole - cell voltage - clamp . a bipolar electrode ( world precision instruments )
low frequency ( 0.1 hz ) and theta bursts ( 8 hz with a train of 100 stimuli ) were delivered . the stimulus intensity ( 50 a )
the following pharmacological agents were used : diazepam ( 1 m ) , no-711 ( 10 m ) , flumazenil ( 10 m ) , midazolam ( 10 m ) , etmd ( 10 m ) , l-655708 ( 50 m ) , and vigabatrin ( 100 m ) .
all drugs were purchased from sigma except bicuculline ( 50 or 100 m ; tocris ) .
rgl recordings under optogenetic manipulation in acute brain slices were performed at least 4 weeks after aav injection . to stimulate chr2 in labelled interneurons , light flashes ( 5 ms at 1 , 8 or 100 hz ) generated by a lambda dg-4 plus high - speed optical switch with a 300w xenon lamp and a 472 nm filter set ( chroma )
were delivered to coronal sections through a 40x objective ( carl zeiss ) . to stimulate enphr in labelled interneurons ,
continuous yellow light generated by dg-4 plus system with a 593 nm filter set were delivered to coronal sections across a full high - power ( 40x ) field .
for immunostaining with anti - nestin and anti - mcm2 , an antigen retrieval protocol was performed by microwaving sections in boiled citric buffer for 7 minutes as previously described . for 2 immunostaining ,
a weak fixation protocol using live tissues was adopted as previously described . for characterization of different interneuron subtypes ,
following antibodies were used : anti - pv ( swant ; mouse or rabbit ; 1:500 ) , anti - gad-67 ( millipore ; mouse or rabbit ; 1:500 ) , ant - sst ( millipore ; rat ; 1:200 ) , and anti - vip ( immunostar ; rabbit ; 1:200 ) . for clonal analysis ,
coronal brain sections ( 40 m ) through the entire dentate gyrus were collected in a serial order , and immunostaining was performed using following primary antibodies as previously described : anti - gfp ( rockland ; goat ; 1:500 ) , anti - nestin ( aves ; chick ; 1:500 ) , anti - mcm2 ( bd ; mouse ; 1:500 ) , anti - gfap ( millipore ; mouse or rabbit ; 1:1000 ) , anti - psa - ncam ( millipore , mouse lgm ; 1:500 ) . for quantification of gfp clones at 2 and 7 dpi ,
clonal analysis at 30 dpi was carried out exactly as previously described . for experiments with diazepam ( 5 mg / kg body weight
; once daily for 5 days ) , coronal brain sections ( 40 m ) through the entire dentate gyrus were collected in a serial order . for optogenetic manipulations , sections within a distance of 1.0 mm anterior and 1.0 mm posterior to injection sites
edu labelling was performed using click - it edu alexa fluor imaging kit ( invitrogen ) .
images were acquired on a zeiss lsm 710 confocal system ( carl zeiss , thornwood , ny , usa ) with a 40x objective using a multi - track configuration .
stereological quantification of cells positive for various molecular markers was assessed in the dentate gyrus using a modified optical fractionator technique .
for quantification of edu or mcm2 rgls , an inverted  y  shape from anti - nestin staining superimposed onto edu or mcm2 nucleus was scored double positive for nestin and edu or mcm2 .
statistic analysis was performed with student t - test . for generation of movie files ,
images were serially reconstructed in reconstruct ( john c. fiala , the national institutes of health ) , normalized , and deconvolved with autoquant x2 ( media cybernetics ) .
images were then segmented in matlab ( the mathworks ) using custom code and imported into imaris ( bitplane ) .
surface renderings and movies were made using the surface and animation functions , respectively , in imaris ( supplementary movies 13 ) .
 adult neurogenesis arises from neural stem cells within specialized niches13 . 
 neuronal activity and experience , presumably acting upon this local niche , regulate multiple stages of adult neurogenesis , from neural progenitor proliferation to new neuron maturation , synaptic integration and survival1 , 3 . 
 whether local neuronal circuitry has a direct impact on adult neural stem cells is unknown . 
 here we show that in the adult hippocampus nestin - expressing radial glia - like quiescent neural stem cells49 ( rgls ) respond tonically to the neurotransmitter gaba via 2 subunit - containing gabaa rs . 
 clonal analysis9 of individual rgls revealed a rapid exit from quiescence and enhanced symmetric self - renewal after conditional 2 deletion . 
 rgls are in close proximity to gad67 + terminals of parvalbumin - expressing ( pv+ ) interneurons and respond tonically to gaba released from these neurons . 
 functionally , optogenetic control of dentate pv+ , but not somatostatin- or vasoactive intestinal polypeptide ( vip)-expressing , interneuron activity can dictate the rgl choice between quiescence and activation . 
 furthermore , pv+ interneuron activation restores rgl quiescence following social isolation , an experience that induces rgl activation and symmetric division8 . 
 our study identifies a niche cell - signal - receptor trio and a local circuitry mechanism that control the activation and self - renewal mode of quiescent adult neural stem cells in response to neuronal activity and experience . 
Methods
Animals, housing, administration of tamoxifen, EdU and AAV, and optogenetic manipulations
Electrophysiology
Immunohistochemistry, Confocal Imaging, Processing and Quantification
Supplementary Material