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Me Mory Jen

Me Mory Jen

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Published by: Roberto Pineda on Apr 05, 2014
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LETTER
The activity-dependent transcription factor NPAS4regulates domain-specific inhibition
Brenda L. Bloodgood
1,2
*
, Nikhil Sharma
1,3
*
, Heidi Adlman Browne
1
{
, Alissa Z. Trepman
1
{
 & Michael E. Greenberg
1
A heterogeneous population of inhibitory neurons controls theflowofinformationthroughaneuralcircuit
1–3
.Inhibitorysynapsesthatformonpyramidalneurondendritesmodulatethesummationof excitatory synaptic potentials
4–6
and prevent the generation of dendritic calcium spikes
7,8
. Precisely timed somatic inhibition lim-its both the number of action potentials and the time window dur-ing which firing can occur 
8,9
. The activity-dependent transcriptionfactorNPAS4regulatesinhibitorysynapsenumberandfunctionincellculture
10
,buthowthistranscriptionfactoraffectstheinhibitory inputsthatformondistinctdomainsofaneuron
invivo 
wasunclear.Hereweshowthatinthemousehippocampusbehaviourallydrivenexpression of NPAS4 coordinates the redistribution of inhibitory synapsesmadeontoaCA1pyramidalneuron,simultaneouslyincreas-inginhibitorysynapsenumberonthecellbodywhiledecreasingthenumberofinhibitorysynapsesontheapicaldendrites.Thisrearran-gement of inhibition is mediated in part by the NPAS4 target genebrain derived neurotrophic factor (
Bdnf  
), which specifically regu-latessomatic,andnotdendritic,inhibition.Thesefindingsindicatethat sensory stimuli, by inducing NPAS4 and its target genes, dif-ferentially control spatial features of neuronal inhibition in a way that restricts the output of the neuron while creating a dendriticenvironment that is permissive for plasticity.
We assessed NPAS4 expression in the hippocampus by immuno-fluorescence microscopy. We found that in hippocampi from micemaintained in standard housing, NPAS4 protein is undetectable by immunostaining(Fig.1a).Bycontrast,exposureofmicetoanenrichedenvironment induces NPAS4 protein expression in neurons in thepyramidal layer, whereas no NPAS4-positive nuclei are observed intheneuropil(Fig.1b).ToobtainmorewidespreadinductionofNPAS4,mice were injected intraperitoneally with kainic acid to trigger seizureactivity.ThistreatmentledtoNPAS4expressioninnearlyallexcitatory neuronsinthepyramidallayerandaminorityofinhibitoryneuronsinthe neuropil (Fig. 1c).WenextaskedwhetherthelossofNPAS4affectsinhibitorysynapsefunction
 in vivo
. Adeno-associated virus encoding a Cre–GFP fusionprotein (AAV-Cre–GFP)
11
was injected into the CA1 region of thehippocampus in
 Npas4
 conditional mice (
Npas4
f/f 
) (Fig. 1d, ExtendedData Fig. 1). Post-operative animals were transferred to an enrichedenvironment or maintained in standard housing for 7 to 12days.Subsequentlyacutehippocampalsliceswereprepared,whole-cellvolt-ageclamprecordingsweremadefromneighbouringNPAS4knockoutand wild-type neurons (referred to as NPAS4-KO and NPAS4-WT,respectively) and pharmacologically isolated miniature inhibitory postsynaptic currents (mIPSCs) were recorded (Fig. 1e).WefoundthatwhenmicewereexposedtoanenrichedenvironmentsignificantlylessfrequentandslightlysmalleramplitudemIPSCswererecorded from NPAS4-KO neurons compared to the neighbouring NPAS4-WT neurons (Fig. 1g, Supplementary Table 1). Similar resultswere obtained from mice injected with kainic acid to induce NPAS4(Fig. 1h). By contrast, no significant difference in mIPSC frequency or amplitude was observed between neighbouring NPAS4-WT andNPAS4-KOneuronsinslicesacquiredfrommicemaintainedinstand-ardhousing(Fig.1f).NextwemeasuredmIPSCsinwild-typemiceanddetected nodifferences between AAV-Cre–GFP infected and uninfec-ted neurons (Extended Data Fig. 2a, b) indicating that the observedchanges in mIPSCs in
 Npas4
f/f 
neurons are due to
 Npas4
 excision.Finally, we crossed EMX-Cre and
 Npas4
f/f 
mice to generate animalsthat lack NPAS4 postnatally and specifically in excitatory neurons(ExtendedDataFig.2c,d).BothmIPSCfrequencyandamplitudeweresignificantly reduced in NPAS4-KO neurons from animals injectedwith kainic acid compared to mice maintained in standard housing (ExtendedData Fig.2e).Fromtheseanalysesweconcludethatdisrup-tion ofbehaviourallyinduced NPAS4expressionleads toa decrease inmIPSC frequency recorded from CA1 pyramidal neurons.ThisdecreaseinmIPSCfrequencycouldbeaconsequenceofhomeo-staticregulationofallinhibitorysynapsesorthemodificationofinhib-itory synapses that form on discrete regions of the pyramidal neuron.Local inhibitory neuron subtypes have axons that elaborate preferen-tiallyinoneormorelayersofthehippocampus
2,3
.Thus,stimulationof axons in the different laminar domains of the hippocampus and ana-lysis of the resulting inhibitory currents has the potential to revealwhether the NPAS4-dependent effects on inhibition are due to cell-wide or domain-specific regulation of inhibitory synapses.We sparsely excised
 Npas4
 and made direct comparisons of layer-specific, pharmacologically isolated, monosynaptic evoked inhibitory postsynaptic currents (eIPSCs) recorded simultaneously from neigh-bouring NPAS4-KO and NPAS4-WT CA1 neurons (Fig. 2a). As pre-dicted by cable properties, eIPSC rise times correlated with distanceof the stimulating electrode from the recording electrode (ExtendedData Fig. 3, Supplementary Table 2) indicating that stimulation of axons within a given layerispredominately activatingsynapses withinthat layer.We found that in slices obtained from mice housed in a standardenvironment, equivalent eIPSCs were recorded from NPAS4-WT andNPAS4-KO neurons in response to stimulation of axons in any of thehippocampal layers (Fig. 2b–e, Supplementary Table 2). However, inslicesobtainedfrommiceexposedtoanenrichedenvironment,stimu-lation of axons in the pyramidal layer generated smaller eIPSCs inNPAS4-KO neurons than in the neighbouring NPAS4-WT neurons(Fig.2h).Bycontrast,stimulationofaxonsinstratumradiatumgeneratedsignificantly larger eIPSCs in NPAS4-KO neurons than in neighbour-ing NPAS4-WT neurons (Fig.2g), whereas no difference was detectedbetween NPAS4-WT and NPAS4-KO neurons upon stimulation of axons in oriens or lacunosum (Fig. 2f, i). Similar results were obtainedfrom mice injected with kainic acid to induce NPAS4 (Extended DataFig. 4). These findings indicate that NPAS4 expression does not resultinuniform,homeostaticregulationofinhibitorysynapses.Rather,NPAS4functions in an unprecedented manner, simultaneously decreasing 
*
These authors contributed equally to this work.
1
DepartmentofNeurobiology,HarvardMedicalSchool,Boston,Massachusetts02115,USA.
2
DivisionofBiologicalSciences,UniversityofCaliforniaSanDiego,LaJolla,California92093,USA.
3
Departmentof Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
 {
Present addresses: Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA (H.A.B.);Department of Neuroscience, Brown University, National Institutes of Health Graduate Partnership Program, Providence, Rhode Island 02912, USA (A.T.).
7 N O V E M B E R 2 0 1 3 | V O L 5 0 3 | N AT U R E | 1 2 1
Macmillan Publishers Limited. All rights reserved
©201
3
 
inhibitionwithintheproximalapicaldendriteswhileincreasinginhibi-tion at the soma.Extracellularstimulation of axons could resultinthedepolarizationof axons of passage that preferentially form synapses on NPAS4-WTor NPAS4-KO neurons and in regions other than the intended layer.To address this possibility we compared the rise times of the eIPSCsrecorded from NPAS4-WT and NPAS4-KO neurons and found themto be indistinguishable (Extended Data Fig. 3). This indicates that theredistribution of inhibition observed is not due to a bias in synaptictransmission associated with axons of passage onto NPAS4-WT orNPAS4-KO neurons. Additionally, the hippocampus contains severalinterneuron subtypes that form synapses within multiple layers (thatis, bistratified and trilaminar cells). If NPAS4 regulates synapses madeby these cell types, it is probable thatonly boutons that synapse on theapical, and not basal, dendrites are affected by loss of NPAS4.We next investigated the cellular mechanisms by which NPAS4 dif-ferentially regulates inhibition, including possible effects on dendriticgrowth,changesintheprobabilityofinhibitorysynapticvesiclereleaseor changes in inhibitory synapse number. We first asked whether lossofNPAS4resultsinaltereddendriticcomplexitywhichcouldindirectly lead to larger eIPSCs in response to stimulation of axons in radiatum.However, this seems unlikely because Sholl analysis revealed that thedisruption of NPAS4 function has no effect on dendrite number orlength(Fig.3a,b,ExtendedDataFig.5a,b).Inaddition,thecapacitanceofneuronsfrommicehousedunderstandardconditions,inanenrichedenvironment or exposed to kainic acid are equivalent (Supplementary Table 3).We next asked whether disruption of 
 Npas4
 leads to changes in thepresynaptic release of neurotransmitter at axons that synapse ontodifferent regions of the pyramidal neuron. We measured the pairedpulse ratios (PPRs) upon stimulation of axons in each layer, and com-pared NPAS4-WT and NPAS4-KO neurons in slices obtained frommice housed under standard conditions, in an enriched environment,orinjectedwithkainicacid.WefoundnodifferencebetweenthePPRsofNPAS4-WTandNPAS4-KOneuronsunderanyoftheseconditions(Fig.3c–e,ExtendedDataFig.5c–e,SupplementaryTable2)indicating that the NPAS4-dependent changes in eIPSCs are not likely due tochanges in the probability of presynaptic vesicle release.FinallyweaskedwhetherNPAS4differentiallyregulatesthenumberof inhibitory synapses on the soma or apical dendrites. To quantify inhibitory synapses we co-injected AAVs encoding mCherry-IRES-Cre and Cre-dependent GFP–gephyrin fusion protein into the CA1region of the hippocampus in wild-type and
 Npas4
f/f 
mice. GFP–gephyrinfusionproteinswhenexpressedatlowlevelslocalizetoinhib-itory synapses without significantly altering neurotransmission
12,13
.Cre was sparsely expressed and GFP–gephyrin puncta quantified onNPAS4-WTandNPAS4-KOneurons.Thisanalysisrevealedthatwhenmice were kept in standard housing, NPAS4-WT and NPAS4-KOhippocampal pyramidal neurons have equivalent densities of GFP–gephyrin puncta on the soma and apical dendrites. However, after
Standard housingHippocampus
   N   P   A   S   4  c  e   l   l  s  p  e  r  s  e  c   t   i  o  n
00.61.6
Kainic acidStandard housingKainic acidCA1 AAV-Cre–GFP;
Npas4
f/f
Cre–GFPCA1CA3DGNPAS4-WTCre–GFP
+
NPAS4-KOCre–GFP
Npas4
f/f 
Cre–GFP
+
Npas4
f/f 
50 pA 500 msCA1CA3DG
    N   P   A   S   4  c  e   l   l  s   (   %   )
 StrdKA StrdEE
Enriched environment
010050151050151050
6030060300
151050151050 6030060300151050151050
6030060300
   N   P   A   S   4  -   K   O  a  m  p   (  p   A   )
NPAS4-WT amp (pA)
   N   P   A   S   4  -   K   O   f  r  e  q   (   H  z   )
NPAS4-WT freq (Hz)
   N   P   A   S   4  -   K   O  a  m  p   (  p   A   )
NPAS4-WT amp (pA)
   N   P   A   S   4  -   K   O   f  r  e  q   (   H  z   )
NPAS4-WT freq (Hz)
   N   P   A   S   4  -   K   O  a  m  p   (  p   A   )
NPAS4-WT amp (pA)
   N   P   A   S   4  -   K   O   f  r  e  q   (   H  z   )
NPAS4-WT freq (Hz)Enriched environment
a b cd ef g h
200 µm25 µm25 µm25 µm200 µm200 µmNPAS4NeuNPairs AveragePairs AveragePairs Average200 µm Cre–GFP
CA1CA1
**
S  o  m  a  t  i   c   A   p  i   c  a  l   B  a  s  a  l   
Somatic
**
Figure 1
 |
 Exposure of mice toincreased circuit activity reveals anNPAS4-dependent regulation of inhibition
 in vivo 
.
c
, Hippocampal sections fromwild-type mice in standard housing (
), enriched environment (EE)(
b
)orafterkainicacid(KA)injection(
c
).SectionswereimmunostainedforNPAS4 (red) and NeuN (blue)protein. Quantification is shown in
b
 and
 c
. Data are shown asmean
6
s.e.m. For each condition
n
5
3 animals with 3–5 sections peranimal.
 *
,
0.01.
 d
, Wide-field(left) and fluorescence (right) imageof hippocampal slice from an
Npas4
f/f 
mouse injected with AAV-Cre–GFP.
 e
, Experimentalconfiguration.
 f 
h
, mIPSCfrequency (top) and amplitude (bottom)recorded from neighbouring NPAS4-WT and NPAS4-KOneurons from mice maintained instandard housing (
) (
n
5
14 pairs),enriched environment (
) (
n
5
23pairs, freq:
 P 
,
0.05) or after kainicacid injection (
h
) (
n
5
26 pairs, freq:
,
0.01, amp:
 P 
,
0.05). Opencircles represent NPAS4-KO/NPAS4-WT pairs. Red circlesindicate mean
6
s.e.m.
RESEARCH LETTER
1 2 2 | N AT U R E | V O L 5 0 3 | 7 N O V E M B E R 2 0 1 3
Macmillan Publishers Limited. All rights reserved
©201
3
 
exposuretoanenrichedenvironment,NPAS4-KOpyramidalneuronshave significantly fewer somatic and more dendritic GFP–gephyrinpuncta than NPAS4-WT neurons (Fig. 3f–h) consistent with the elec-trophysiology data described above. Strikingly, comparison of thenumber of GFP–gephyrin puncta measured on wild-type neuronsrevealedthatexposuretoanenrichedenvironmentleadstoanincreasein the number of somatic and a decrease in the number of dendriticinhibitory synapses. In both domains of the neuron, the behaviourally triggered changes in inhibition are NPAS4 dependent.NPAS4 is a transcription factor and thus to understand how itmediates sensory-dependent changes in inhibitory synapse numberwe sought to identify NPAS4 target genes and assess their function.We focused our attention on putative NPAS4 targets that satisfy threecriteria: (1) by deep sequencing NPAS4-bound DNA (ChIP-seq),NPAS4isshowntobebound within10kilobasesoftheputativetargetgene; (2) by RNA-seq, the mRNA transcribed near the NPAS4 peak isinduced greater than fivefold (Extended Data Table 1)
14
; (3) uponshort hairpin RNA (shRNA)-mediated knockdown of the putativetarget gene there is a significant change in mIPSC frequency or ampli-tude.Throughthisscreenweidentified16putativeNPAS4targetsthatdisplay a change in mIPSC frequency or amplitude upon knockdownwith gene-specific shRNAs in organotypic hippocampal slices, five of which we further validated (Extended Data Figs 6–8).Of the putative NPAS4 target genes identified by the screen,
 Bdnf 
seemedapossiblecandidatetocontrolthenumberofinhibitorysynapsesthat form on a specific region of an excitatory neuron in response tosensorystimulation.BDNFexpressionisregulatedbyneuronalactivity 
invivo
15–18
anditregulatesinhibitorysynapsenumberandfunction
19–21
.In addition,
 Bdnf 
 mRNAs can be targeted to discrete regions of theneuron
22
,andBDNF-containingvesiclesareknowntobesecretedfromthe axon, soma or dendrites of pyramidal neurons
23,24
. Furthermore,weconfirmedbyChIPandquantitativePCRwithreversetranscription(qRT–PCR) that NPAS4 binds to three sites within the
 Bdnf 
 gene(Fig. 4a, b), and that induction of the transcription of multiple
 Bdnf 
isoforms is significantly reduced in the hippocampus of 
 Npas4
 knock-out (
Npas4
2
/
2
) compared to wild-type (C57BL/6J) mice (Fig. 4c).TodetermineifBDNFisatargetofNPAS4thatselectivelyregulatessomatic and/or dendritic inhibition of pyramidal neurons
 in vivo
, weinjectedAAV-Cre–GFPintoBDNFconditionalknockoutmice(
Bdnf 
f/f 
)miceandcomparedeIPSCsbetweenneighbouring 
Bdnf 
knockoutand
Bdnf 
 wild-type neurons (BDNF-KO and BDNF-WT, respectively) inresponse to stimulation of axons in the somatic or dendritic layers. Inmice housed under standard conditions, we found no differences ineIPSCamplitudemeasuredfromneighbouringBDNF-KOandBDNF-WT neurons upon stimulation of axons in any of the layers of thehippocampus (Fig. 4d–f, Supplementary Table 2).However, when we exposed mice to an enriched environment toinduce NPAS4 as well as BDNF expression and secretion, stimulationofaxons inthe pyramidallayerresults ina significantlysmallerampli-tudeeIPSCinBDNF-KOneuronsrelativetoneighbouringBDNF-WTneurons (Fig. 4h). The disruption of 
 Bdnf 
 function did not have asignificanteffectoneIPSCamplitudewhenaxonsinradiatumororienswere stimulated (Fig. 4g, i). Similar results were observed when BDNFexpressionwasinducedwithkainicacid(ExtendedDataFig.9).Takentogether these findings indicate that the differential effects of NPAS4on pyramidal neuron inhibition are mediated, at least in part, throughthe induction of BDNF that functions selectively and locally to pro-mote inhibition at the soma. Given that the disruption of BDNF func-tiondoesnotaffecteIPSCsinradiatum,theabilityofNPAS4torelieveinhibition on the apical dendrites of CA1 pyramidal neurons is prob-ably mediated by NPAS4 targets other than BDNF.These findings indicate that when a mouse engages with its envir-onment, theneuronalactivity-regulatedtranscriptionfactor NPAS4isexpressed in pyramidal neurons of the hippocampus where it pro-motes an increase in the number of inhibitory synapses on the cellsomaandadecreaseinthenumberofinhibitorysynapsesontheapical
LRPOStandard housing
aecb
80400804003.01.503.01.501.00.501.00.50
   N   P   A   S   4  -   K   O  a  m  p   (  p   A   )
NPAS4-WT amp (pA)
Pairs AveragePairs Average
2.01.002.01.00
Enriched environment
3.01.503.01.50060300603001.20.601.20.603.01.53.01.50
LacunosumRadiatumPyramidalOriensLacunosumRadiatumPyramidalOriens
dihgf
30%25 ms30%25 ms30%25 ms30%25 ms30%25 ms30%25 ms30%25 ms30%25 ms
   N   P   A   S   4  -   K   O  a  m  p   (  n   A   )
NPAS4-WT amp (nA)
   N   P   A   S   4  -   K   O  a  m  p   (  n   A   )
NPAS4-WT amp (nA)
   N   P   A   S   4  -   K   O  a  m  p   (  n   A   )
NPAS4-WT amp (nA)
   N   P   A   S   4  -   K   O  a  m  p   (  p   A   )
NPAS4-WT amp (pA)
   N   P   A   S   4  -   K   O  a  m  p   (  n   A   )
NPAS4-WT amp (nA)
   N   P   A   S   4  -   K   O  a  m  p   (  n   A   )
NPAS4-WT amp (nA)
   N   P   A   S   4  -   K   O  a  m  p   (  n   A   )
NPAS4-WT amp (nA)Cre–GFP
;
 
Npas4
f/f 
Cre–GFP
+
;
 
Npas4
f/f 
Figure 2
 |
 Behaviourally inducedNPAS4 differentially regulatesinhibitory synapse function acrossthe somato-dendritic axis of pyramidalneurons. a 
,Experimentalconfiguration. L, lacunosum; R,radiatum; P, pyramidale; O, oriens.
b
e
, eIPSCs measured from micemaintainedinstandardhousing.Topshows average eIPSC, normalizedpairwise to the wild-type neuron,measured from NPAS4-WT (black)and NPAS4-KO (green) neurons inresponse to stimulation inlacunosum (
b
), radiatum(
c
), pyramidale (
d
) or oriens (
e
).Scale bars indicate per cent changefrom wild type. Bottom shows eIPSCamplitude measured from pairs of neighbouring NPAS4-KO andNPAS4-WT neurons in response tostimulation of axons in lacunosum(
b
) (
n
5
14 pairs), radiatum(
c
) (
n
5
13 pairs), pyramidale(
d
) (
n
5
17 pairs) or oriens(
e
) (
n
5
13 pairs). Open circlesrepresent NPAS4-KO/NPAS4-WTpairs. Red circles indicatemean
6
s.e.m.
 f 
i
, eIPSCs measuredfrom mice exposed to an enrichedenvironment. Data are displayed asin
 b
e
. L,
 n
5
16 pairs; R,
 n
5
16pairs,
 P 
,
0.05; P,
 n
5
14 pairs,
,
0.01; O,
 n
5
18 pairs.
LETTER RESEARCH
7 N O V E M B E R 2 0 1 3 | V O L 5 0 3 | N AT U R E | 1 2 3
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