Research ArticleNpas4 Expression in Two Experimental Models ofthe Barrel Cortex Plasticity
Aleksandra Kaliszewska and Malgorzata Kossut
Department of Molecular and Cellular Neurobiology Nencki Institute 3 Pasteur Street 02-093 Warsaw Poland
Correspondence should be addressed to Malgorzata Kossut kossutnenckigovpl
Received 25 December 2014 Accepted 29 January 2015
Academic Editor George W Huntley
Copyright copy 2015 A Kaliszewska and M Kossut This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited
Npas4 has recently been identified as an important factor in brain plasticity particularly in mechanisms of inhibitory control Littleis known about Npas4 expression in terms of cortical plasticity In the present study expressions of Npas4 and the archetypalimmediate early gene (IEG) c-Fos were investigated in the barrel cortex of mice after sensory deprivation (sparing one rowof whiskers for 7 days) or sensory conditioning (pairing stimulation of one row of whiskers with aversive stimulus) Lasermicrodissection of individual barrel rows allowed for analysis of IEGs expression precisely in deprived and nondeprived barrels (indeprivation study) or stimulated and nonstimulated barrels (in conditioning study) Cortex activation by sensory conditioning wasfound to upregulate the expression of bothNpas4 and c-Fos Reorganization of cortical circuits triggered by removal of selected rowsof whiskers strongly affected c-Fos but not Npas4 expression We hypothesize that increased inhibitory synaptogenesis observedpreviously after conditioning may be mediated by Npas4 expression
1 Introduction
Npas4 has been recognized as a brain-specific transcriptionfactor [1 2] important for structural and functional neuronalplasticity Lin et al [3] identified it as an element of theprogram controlling inhibitory synapse development andplasticity They postulated that Npas4 induction in responseto increased excitatory input acts to reduce activity levelsand therefore may serve to maintain the homeostatic balancebetween excitation and inhibition In accordance with thishypothesis Sim et al [4] found that Npas4 is required foractivity-dependent increases in GABAergic input on dentategyrus granule cells and thatNpas4 signalingwithin individualneurons in the dentate gyrus is necessary to trigger activity-dependent changes in dendritic morphology Findings ofBloodgood et al [5] indicate that in pyramidal neurons ofhippocampus of mice kept in enriched environment Npas4promotes an increase in the number of inhibitory synapseson the cell soma and a decrease in the number of inhibitorysynapses on the apical dendrites Spiegel et al [6] demon-strated that Npas4 promotes the development of excitatorybut not inhibitory synapses onto somatostatin interneurons
This opposite impact of Npas4 on connectivity of excitatoryand inhibitory neurons is explained by its ability to activatecell-type specific transcriptional programs It is hypothesizedthat Npas4 works as a part of homeostatic mechanism torestrict excitation within neuronal circuits Npas4 mediatesstructural plasticity by controlling neurite outgrowth in vitro[7] dendrite polarization within the barrel cortex duringdevelopment [8] and sensory-driven changes in density ofspines in olfactory bulb granule cells [9] Npas4 is capable ofregulating transcription of drebrin [2] which is involved instructural changes in dendritic spines [10]
Studies of Npas4 expression in learning and memorymainly concern limbic structures It was implicated in con-text learning and long term-contextual memory [11] andamygdala-dependent fear conditioning [12] The role ofNpas4 in cortical plasticity has just recently started to beelucidated Npas4 was reported to play a role in mediatingthe reinstatement of ocular dominance plasticity in the visualcortex of adult rats following fluoxetine treatment [13] Herewe characterized Npas4 expression in adult mouse barrelcortex using two models of plasticity sensory deprivationand sensory conditioning
Hindawi Publishing CorporationNeural PlasticityVolume 2015 Article ID 175701 9 pageshttpdxdoiorg1011552015175701
2 Neural Plasticity
Local elimination of excitatory input to deprived corticalbarrels disrupts the equilibrium between excitation and inhi-bition which leads to modifications in functional activationand anatomical rewiring of cortical sensory areas Functionalrepresentation of spared inputs starts to expand to deprivedareas [14ndash19] This is accompanied by large-sale structuralchanges such as axonal retraction and sprouting [20ndash23]and dendritic reorganization [24 25] and also by moresubtle changes involving spines and synapses with decreaseddensity of inhibitory synapse in deprived barrels [26ndash31]Classical conditioning involving unilateral stimulation ofrow B vibrissae paired with tail shock results in behavioralchanges (increased immobility) expansion of functional cor-tical representation of the row of vibrissae stimulated duringconditioning and increased density of inhibitory synapseson spines in barrels that represent the stimulated vibrissae[32 33] As Npas4 is implicated in structural plasticity in thepresent study we aimed to determine how Npas4 expressionin the barrel cortex is influenced by sensory conditioning andsensory deprivation
2 Methods
21 Deprivation and Somatosensory Stimulation Six maleC57BL6 mice aged 8-9 weeks were used in the deprivationexperiment The mice were reared in a 12 12 lightdark cyclein standard cages and had ad libitum access to water andfood All experimental procedures were approved by the FirstEthical Commission in Warsaw Poland and were in accor-dance with the European Communities Council Directiveof 24 November 1986 (86609EEC) Mice were deprivedunder short (2-3 minutes) isoflurane anesthesia by pluckingout all whiskers except row C on one side of the snout(referred to as experimental side later in the text Figure 1(a))This experimental model of sensory deprivation leaves thecentrally situated row C of vibrissae intact with symmetricalspace for remodeling of the cortex in the medial and lateraldirection Regrowing vibrissae were removed every secondday On the 6th day (24 hours before the experiment) theother side of the snout (control side) was subjected to thesame deprivation procedure so thatmice were leftwith intactrow C whiskers on both sides Animals were deprived 24hours prior to exploration of the stimulatory cage to avoidincrease in Npas4 and c-Fos expression induced by whiskerplucking Mice were allowed to explore the stimulatory cageon the 7th day The walls and floor were made of bars andthe cage was equipped with objects of different shapes andtextures mouse wheels plastic toys maze-like constructionsand pieces of Styrofoam Animals were placed in the cageand left for 30min in a room without illumination to pro-mote sensory stimulation Next they were killed by cervicaldislocation The brains were dissected out and cortices wereflattened [34] and frozen in minus70∘C isopentane
22 Training Procedure 29 male Swiss albino mice aged 8-9weeks were used in training procedure Prior to behavioraltraining mice were habituated to a neck restraint for 10mina day 5 days a week for 2-3 weeks Animals were placed inseparate home cages a few days before onset of the training
Experimentalrow C
Controlrow C
Experimentalrow C
30min
6 days 24h
Enriched environment
(a) Deprivation
05 s
Training session 40x CS + UCS 10min1 session daily on 3 consecutive days
UCSmdash05mA
CSmdashstroking row B
(b) Training
Figure 1 (a) Scheme of the deprivation procedure All whiskers onone side of the snout except for row C are removed After 6 days thesame deprivation procedure is applied to the other side of the snoutand the mouse is left with both C rows intact After 24 h the animalexplores a stimulatory cage for 30 minutes and is then immediatelykilled (b) Scheme of the training procedure Row B of whiskers onthe left side of the snout is stroked (CS) The CS lasts for 9 s Duringthe last second an electric shock (05mA for 05 s UCS) is deliveredand coterminates with the CS A single training session lasts for 10minutes and encompasses 40 CS and UCS pairings The animal issubjected to one session daily for three consecutive days Followingthe last training session the mouse is placed in its home cage for 20minutes to allow increase in Npas4 expression and is then killed
During training row B whiskers on the left side of the snoutwere stroked in the posterior to anterior direction (CS) witha fine handheld brush (Figure 1(b)) The CS lasted for 9 sDuring the last second an aversiveUCS (amild electric shockof 05mA for 05 s applied to the tail) was delivered andcoterminated with the CS This trial was repeated after a 6 sinterval and the routine was continued for 10min Trainingencompassed three training sessions on three consecutivedays Following the last training session animals were placedin their home cages for 20 minutes to allow for increase inNpas4 expression and then killed by cervical dislocationThebrainswere dissected out and corticeswere flattened [34] andfrozen in minus70∘C isopentane
Figure 2 (a) Single rows of barrels are microdissected from Nissl-stained sections RNA from dissected tissue is extracted and processed forRT-PCR (b) Nissl-stained section with row B dissected out Neighbouring row C is left intact ((c) (d)) Rows of barrels microdissected indeprivation (C) and sensory conditioning (D) experiment Microdissected barrels are colored in green and red Legend below the schemeexplains terminology used in the text for description of particular rows
(ii) PSEUDO (pseudoconditioned 119899 = 7) unpaired appli-cation of the same number of CS and UCS as duringconditioning
(iii) CS only (119899 = 8) sessions of whisker stroking(iv) naıve ndash (119899 = 5) unstimulated controls in neck restrain-
ing apparatus
23 Laser Microdissection Flattened cortices were cryosec-tioned (16 120583m) tangentially to the surface Slices from layerIV were mounted on membrane slides (MembraneSlide 10PEN Carl Zeiss MicroImaging GmbH) and stored at minus70∘Cuntil further usage Immediately prior to themicrodissectionslices were subjected to modified Nissl staining using Arc-turus HistoGene Staining Solution (KIT0401 Life Technolo-gies) which preserves nucleic acid integrity Sections fromboth hemispheres of the animal were processed togetherIsolation of tissue was performed using the ArcturusXTLCMSystem equipped with a Nikon Eclipse Ti-Emicroscope
(Figures 2(a) and 2(b)) Microdissected tissue was collectedon CapSureMacro LCMCaps (LCM0211 Life Technologies)In slices frommice used in the conditioning experiment rowsB and D from the right hemisphere were dissected and fromthe left hemisphere only row B was dissected (Figure 2(d))Row B from the right hemisphere in the CS + UCS groupis referred to as the trained row of whiskers In slices fromdeprived mice rows B C and D from both hemisphereswere microdissected (Figure 2(c)) Row C was collected ona separate membrane and rows B and Dwere collected on thesame membrane In further steps tissue from rows B and Dfrom the same hemisphere was pooled Samples from differ-ent animals were not pooled In summation three sampleswere collected from animals in the conditioning experiment(row B right hemisphere row D right hemisphere and rowB left hemisphere) and four samples were collected fromdeprived animals (rowC experimental hemisphere rows B +D experimental hemisphere row C control hemisphere androws B + D control hemisphere) RNA was recovered using
4 Neural Plasticity
Table 1 Sequences of primers used in real-time PCR
a PicoPure RNA Isolation Kit (KIT0204 Life Technologies)with concurrent genomic DNA elimination using DNase(RNase-Free DNase Set 79254 Qiagen)
24 Real-Time PCR Reversed transcription was performedusing aMaxima First Strand cDNASynthesisKit (K1641ThermoScientific-Fermentas) Real-time PCR was conducted usingPower SYBR Green PCR Master Mix (4368702 Life Tech-nologies) In real-time PCR experiment each sample wasrun in triplicate Amplification was carried out with a 7500real-time PCR System (Applied Biosystems) using PowerSYBR Green PCR Master Mix specific primers (Table 1)and cDNA for each sample The glyceraldehyde 3-phosphatedehydrogenase (GAPDH) gene was used as a housekeepinggene The amplification reaction was cycled 40 times witha 95∘C denaturation step for 15 s and a 60∘C annealing stepfor 1 minute A dissociation stage was performed to assessspecificity of primers Results were calculated using standardcurve method
25 Data Analysis and Statistics Ratios of a target gene andhousekeeping gene levels were used for analysis Statisticalanalysis was performed using GraphPad Prism 5 software(GraphPad Software Inc) Gene expression levels in differ-ent barrel rows were analyzed using ANOVA followed byNewman-Keuls post hoc tests Studentrsquos 119905-test was usedwhereapplicable
3 Results
31 Sensory Deprivation
311 Npas4 and c-Fos Mice were subjected to one week ofsensory deprivationmdashall vibrissae on one side of the snoutwere removed except for row C while vibrissae on the otherside were left intact On the day preceding stimulation theother side of the snout was subjected to the same procedureso that mice were left with two C rows intact Animalswere placed in the stimulatory cage for 30 minutesmdasha timeinterval demonstrated to be appropriate to observe increasein Npas4 expression in the barrel cortex after explorationof an enriched environment [35] Regions of interest weremicrodissected from slices of layer IV Real-timePCRmethodwas used to assess Npas4 mRNA level in individual barrel
rows Npas4 level was evaluated in spared rows C and indeprived rows B and D in the experimental and controlhemisphere
Elimination of sensory input to selected rows of vibrissaeevoked differences in Npas4 expression between spared anddeprived barrels (ANOVA 119865(318) = 1004 119875 = 00004Figure 3(a)) Post hoc analysis demonstrated that the levelof Npas4 transcript in the control hemisphere was 484lower in deprived rows B and D than in the spared row C(035 plusmn 005 versus 067 plusmn 006 119875 lt 001) There were nodifferences in Npas4 transcript levels between spared rowsC in both hemispheres (119875 gt 005) Also no differenceswere observed between deprived regions in both hemispheres(119875 gt 005) which shows that duration of deprivation (7days versus 24 hours) had no impact on Npas4 expressionin deprived barrel rows We have previously shown that thisdeprivation procedure followed by two hours of explorationof stimulatory cage resulted in an increased number of cellsexpressing c-Fos in deprived rows B and D after 7 daysof deprivation in comparison with 24 hours of deprivation[18] We also observed this phenomenon regarding the otheractivity-regulated genes Arc and Zif268 [36] As Npas4 isalso an activity-regulated gene we expected a similar patternof its expression As this was not the case we decided toassess c-Fos mRNA in the same samples to confirm thatthe Npas4 pattern of expression is atypical of other activity-regulated genes in this model of plasticity The deprivationprocedure produced differences in c-Fos expression amonganalyzed barrel rows (ANOVA 119865(316) = 8028 119875 = 00022Figure 3(b)) In the experimental hemisphere the level ofc-Fos mRNA was 742 higher in deprived rows B and D(138 plusmn 020 versus 079 plusmn 009 119875 lt 005) than in homotypicregions in the control hemisphere
32 Sensory Conditioning The training procedure encom-passed three training sessions on three consecutive dayseach lasting for 10 minutes and consisting of 40 CS (strokingrow B of vibrissae) and UCS (electric shock to the tail)pairings This paradigm evokes freezing-like behavior whichindicates that association of CS and UCS occurred [37] InCS + UCS animals Npas4 expression was on average 529higher in trained row B barrels than in the contralateral rowB (024 plusmn 003 versus 015 plusmn 003 119905-test 119875 lt 0001) We foundno differences in Npas4 expression between hemispheresin naıve animals and for further analysis we pooled datafrom both B rows Npas4 expression in the trained row Bfor the CS + UCS group was 481 higher than in naıvegroup B rows (119905-test 119875 lt 005) There were no differencesbetween B rows in the naıve group and left (control) rowB in the CS + UCS group (119905-test 119875 gt 005) Analysis ofratios of Npas4 expression in right row B (trained row inCS + UCS group) and left row B revealed significant effectof the training procedure (ANOVA 119865(325) = 6780 119875 =00017 Figure 4) Ratio of Npas4 expression in right row Band left row B was higher in CS + UCS group in comparisonwith other experimental groups (119875 lt 001) indicating thatinterhemispheric difference in Npas4 mRNA level is not justan effect of stimulation of the row B of vibrissae Npas4
Neural Plasticity 5
00
02
04
06
08
Npa
s4 re
lativ
e exp
ress
ion
leve
l
Con
trolC
Exp
C
Con
trolB
D
Exp
BD
lowastlowastlowastlowast
lowastlowast
(a)
0
1
2
3
c-Fo
s rela
tive e
xpre
ssio
n le
vel
Con
trolC
Exp
C
Con
trolB
D
Exp
BD
lowastlowastlowast
lowastlowast
lowastlowast
lowast
lowast
(b)
Figure 3 (a) Npas4 expression in spared C rows and in deprived regions (averaged rows B and D) Control C spared row C on controlside (deprived for 24 hours) Exp C spared row C on experimental side (deprived 7 days) Control BD deprived B and D rows on controlside Exp BD deprived B and D rows on experimental side mean plusmn SEM lowastlowast
119875
lt 001 Duration of deprivation had no impact on Npas4expression (b) In deprived B and D rows deprivation lasting 7 days induced increase in c-Fos expression mean plusmn SEM lowast
119875
lt 005 lowastlowast119875
lt 001lowastlowastlowast
119875
lt 0001
00
05
10
15
20
relat
ive e
xpre
ssio
n le
vel
Righ
t Ble
ft B
Npa
s4
Naıve CS + UCS PSEUDO CS
lowastlowastlowastlowast
lowastlowast
Figure 4 Changes in Npas4 expression induced by training Intrained (CS + UCS) animals expression of Npas4 in the right(trained) row B of barrels is elevated in comparison with left(control unstimulated) row B Sole stimulation of whiskers (CSonly) and application of unpaired CS and UCS (PSEUDO) do notproduce increase in Npas4 expression in comparison with controlside Data are presented as ratio of Npas4 expression level in rightand left row B of barrels mean plusmn SEM lowastlowast
119875
lt 001
expression was increased in the ldquotrainedrdquo row in every singleconditioned animal (Figure 5)
000
005
010
015
020
025
030
035
040
045
050
Npa
s4 re
lativ
e exp
ress
ion
leve
l
Trained 1
Trained 2
Trained 3
Trained 4
Trained 5
Trained 6
Trained 7
Trained 8
Trained 9
L R
Figure 5 Changes of Npas4 mRNA expression in individualconditionedmicemdashcomparison between trained rowB of barrels (Rright) and control row B (L left)
6 Neural Plasticity
00
01
02
03
Npa
s4 re
lativ
e exp
ress
ion
leve
l
Left B Right D Right B
lowastlowast
(a)
00
05
10
15
c-Fo
s rela
tive e
xpre
ssio
n le
vel
Left B Right D Right B
lowast
(b)
Figure 6 Training induced changes in Npas4 (a) and c-Fos (b) expression Right B corresponds to the trained row of barrels right D tocontrol row of barrels in the same hemisphere and left B to control row B in the other hemisphere Npas4 and c-Fos have similar pattern ofexpression following the training procedure their expression is elevated in the trained row B in comparison with the control side mean plusmnSEM lowast119875
lt 005 lowastlowast119875
lt 001
To see if the observed changes in Npas4 expression arelimited to the stimulated row B barrels we evaluated the levelof its expression in row D barrels in the same hemisphereWe did not assess Npas4 mRNA level in row D of the left(control) hemisphere in the CS + UCS group but taking intoaccount results from the naıve group it can be presumedthat it is comparable to the control row B The level ofNpas4 expression in the right row D was an intermediatevalue between the levels of Npas4 mRNA expression in theright (trained) row B and left (control) row B it was notsignificantly different from any of the B rows (Figure 6(a))It can be interpreted that training affects row D so there areno differences between trained row B and row D in the samehemisphere but the impact of training upon rowD is weak sono differences can be observed when compared to the controlhemisphere
In the deprivation studyNpas4 turned out to have a differ-ent pattern of expression than other activity-regulated genesand we wondered if it is also the case in sensory conditioningWe evaluated c-Fos mRNA levels in samples obtained fromCS + UCS mice The pattern of c-Fos expression was thesame as for Npas4 (Figure 6(b)) The training procedureevoked differences among c-Fos expressions in examinedbarrel rows (ANOVA 119865(2 24) = 4407 119875 = 00299) c-Fos mRNA expression level was 484 higher in the trainedrow of barrels (row B in the right hemisphere) than in thecontralateral row B (in the left hemisphere 119875 lt 005)
4 Discussion
Knowledge on Npas4 in plasticity of adult cortex is limitedHerein we provide first description of Npas4 expression in
the barrel cortex undergoing plastic reorganization in twoparadigms deprivation and sensory conditioning We alsocompare changes in expression of Npas4 and c-Fos in bothexperimental models
We found that classical conditioning in which stimula-tion of a row of whiskers is paired with tail shock results inincreased expression of Npas4 mRNA in the cognate row ofcortical barrels As conditioning triggers inhibitory synapto-genesis in the barrels representing stimulated vibrissae [33]our results are in agreement with data demonstrating a roleof Npas4 in the formation of GABAergic synapses The workof Lin et al [3] found that Npas4 regulates expression ofa variety of genes including gene coding for BDNF Npas4binds to activity-dependent promoters I and IV of the BDNFgene [3] and expression of the BDNF gene from promoterIV contributes to the plasticity of inhibitory synapses [3839] Npas4 expression was demonstrated to drive inhibitorysynaptogenesis on excitatory neurons andNpas4 knockdownincreases interevent interval and decreases the amplitudeof mIPSCs [3] In the current experiment we observedan increase in Npas4 expression within the trained rowwhere previously we found increases of GABAergic synapsesdensity increased synaptic content of GABA and increasedspontaneous IPSCs [33 40] Our findings are also in linewith the recent results by Sim et al [4] who found thatincreased cell intrinsic activity results in via an Npas4dependent mechanism the addition of GABAergic inputsto the neuron In hippocampal pyramidal neurons behav-iorally induced expression of Npas4 drives redistribution ofinhibitory synapses increasing inhibitory synapse numberon the cell body while decreasing the number of inhibitorysynapses on the apical dendrites [5] In contrast in our
Neural Plasticity 7
previous experiments we observed an increase in density ofGABAergic synapses located on spines in barrels representingldquotrainedrdquo vibrissae [33] This discrepancy could be attributedto possibility that Npas4 impact on dendritic and somaticpool of GABAergic synapses depends on the type of neuronits location within the brain and the type of stimulation usedto evoke plasticity
Interestingly it was recently found that increased Npas4expression may also account for increased number of exci-tatory contacts made onto somatostatin interneurons [6] Inthe CS + UCS mice an increase in density of somatostatininterneurons within layer 4 of the barrel cortex is observed[41] which may be a result of elevated neuronal activity[42] It can be hypothesized that Npas4 expression drivesgeneration of additional excitatory input onto somatostatininterneurons which in turn express higher level of somato-statin
Herein we demonstrate that sensory conditioning pro-duces increase in Npas4 expression in the region whereinhibitory synaptogenesis was previously observed [33]Synapses formation and elimination accompanying learning-related behaviors may contribute to shift in excitation-inhibition balance Dysregulation of this balance has beenimplicated in number of human neuropsychiatric and neu-rodegenerative disorders and associated with impairmentof cognitive functions [43] Regarding Npas4 significancefor maintaining excitation-inhibition equilibrium it can bepresumed that Npas4 deficiency would result in cognitivedeficits Indeed such deficits were observed in Npas4 knock-out mice [44 45] So far Npas4 has not been directly linkedto human neuropsychiatric disorders However Bersten et al[46] identified human variants of Npas4 with reduced tran-scriptional activity so sequencing Npas4 in neuropsychiatricpatients might be helpful in detecting such a link if it exists
Previous experiments regarding fear conditioning andcontextual learning indicate that Npas4 is indispensable formemory formation [11 12]Therefore it is reasonable to thinkthat Npas4 deficiency should also impair sensory condition-ing Testing this hypothesis using Npas4 knockout animalsmight be misleading Npas4 knockout animals performedwell in amygdala-dependent fear conditioning [11] whileacute deletion of Npas4 in amygdala impaired fear memoryformation [12] Regionally selective depletion of Npas4mightbe more useful then but first structures required for learningassociation of sensory CS and UCS in paradigm used in thisstudy should be identified Basolateral amygdala is activatedduring the training procedure [37] and amygdala is requiredfor all forms of fear conditioning [47] therefore amygdalawould seem a first choice structure for local knockdown ofNpas4 and determining its impact on sensory conditioning
Npas4 expression in remote row D barrels did notdiffer from the results obtained for the control hemisphereand naıve group which is in agreement with our previousobservation that no changes typical for the trained row Bappear in remote row D (no AMPA and NMDA bindingincrease no GAD mRNA expression upregulation and noincrease in density of somatostatin containing inhibitoryinterneurons) [40 41 48 49] We did not do EM to evaluatethe synapse density of remote row D barrels but we found
no increase in spontaneous IPSC there which would indicateincreased GABA release in its excitatory neurons [40]
In the sensory conditioning paradigm Npas4 and c-Foslevels changed in the same wayThey were upregulated in therow of barrels that received the conditioned input
Depriving selected barrel rows of sensory input resultedin decreased Npas4 mRNA expression in the deprivedbarrel rows in comparison with spared rows Duration ofdeprivation (24 h versus 7 days) had no impact upon Npas4expression in spared rows and deprived rowsThis contrastedwith c-Fos the expression of which was increased in bothdeprived and spared regions after 7 days of deprivation incomparison with 24 hours of deprivation
Using immunohistochemical techniques we previouslydescribed the pattern of c-Fos protein expression followingwhisker deprivation of various durations [18] in the samedeprivation paradigm The density of c-Fos positive nucleiincreased in the barrel row deprived of whisker input for 7days and the effect was augmented as the deprivation periodwas prolonged We interpreted this result as illustrating theexpansion of the spared vibrissal input into neighboringfunctionally deafferented barrels The gradual increase in thenumber of immunoreactive nuclei could reflect the gradualrewiring of the barrel cortex in the course of prolongeddeprivation The present results confirm our previous data atthe mRNA level
Unlike c-Fos Npas4mRNA in the deprived B andD rowswas not upregulated by sensory stimulation when comparing24 h versus 7 days of deprivation We suppose that thestrength of the sensory signal coming from the spared row Cwhiskers was insufficient for changing the expression of thisIEG In the experimental paradigm used here we previouslyobserved pronounced changes in Zif268 and Arc expression[36] It is not surprising that differently regulated genes donot respond identically to an experimental situation (see[35] for comparison of several IEGs activations by enrichedenvironment) Perhaps the plastic rearrangement of connec-tions induced by deprivation although it already changesthe metabolic response [18] does not yet trigger activationin Npas4 mRNA expression Npas4 can regulate activity-dependent expression of Arc c-Fos and Zif268 [11] Takinginto consideration that Arc c-Fos and Zif268 expressionsincrease in deprived barrels after 7 days of deprivation it mayseem puzzling that expression of Npas4 which regulates thetranscription of these genes remains unaltered However itshould be noted that Npas4 itself is an immediate early geneand gets activated in response to stimulation Accordingly itprobably does not regulate the first phase of other immediateearly genes expression which is independent of de novoprotein synthesis It rather seems that Npas4 plays a rolein enhancing and sustaining other immediate early genes inlater phases [11]
To the best of our knowledge this is the first report onNpas4 expression in deprivation-induced plasticity Maya-Vetencourt et al [13] examined Npas4 expression in monoc-ularly deprived rats treated with fluoxetine but they concen-trated on influence of fluoxetine onNpas4 expression and noton the influence of deprivation
8 Neural Plasticity
In this paper the precise anatomical dissection of arow of barrels where a plastic change took place allowedfor a quantitative analysis of IEGs expression in two typesof experience dependent plasticity Activation of the barrelcortex undergoing reorganization triggered by removal ofselected rows of whiskers strongly affected c-Fos (but notNpas4) expression Activation of the cortex undergoing aplastic change due to being involved in sensory conditioningupregulated the expression of both Npas4 and c-Fos Takinginto consideration our observations that sensory condition-ing increases the number of inhibitory synapses withinthe trained barrels and studies of other groups showinginvolvement of Npas4 in synaptogenesis we presume thatNpas4 may be involved in reshaping of connectivity withinbarrel cortex after sensory conditioning
Conflict of Interests
The authors declare no conflict of interests
Acknowledgment
This project was supported by GrantMNiSWno 2486BP012010 to Malgorzata Kossut
References
[1] M Moser R Knoth C Bode and C Patterson ldquoLE-PAS anovel Arnt-dependent HLH-PAS protein is expressed in limbictissues and transactivates the CNS midline enhancer elementrdquoMolecular Brain Research vol 128 no 2 pp 141ndash149 2004
[2] N Ooe K Saito N Mikami I Nakatuka and H KanekoldquoIdentification of a novel basic helix-loop-helix-PAS factorNXF reveals a Sim2 competitive positive regulatory role indendritic-cytoskeleton modulator drebrin gene expressionrdquoMolecular and Cellular Biology vol 24 no 2 pp 608ndash616 2004
[3] Y Lin B L Bloodgood J L Hauser et al ldquoActivity-dependentregulation of inhibitory synapse development by Npas4rdquoNature vol 455 no 7217 pp 1198ndash1204 2008
[4] S Sim S Antolin C-W Lin Y-X Lin and C Lois ldquoIncreasedcell-intrinsic excitability induces synaptic changes in new neu-rons in the adult dentate gyrus that require Npas4rdquo Journal ofNeuroscience vol 33 no 18 pp 7928ndash7940 2013
[5] B L Bloodgood N Sharma H A Browne A Z Trepman andM E Greenberg ldquoThe activity-dependent transcription factorNPAS4 regulates domain-specific inhibitionrdquo Nature vol 503no 7474 pp 121ndash125 2013
[6] I Spiegel A R Mardinly H W Gabel et al ldquoNpas4 regulatesexcitatory-inhibitory balance within neural circuits throughcell-type-specific gene programsrdquo Cell vol 157 no 5 pp 1216ndash1229 2014
[7] J Yun T Nagai Y Furukawa-Hibi et al ldquoNeuronal per arnt sim(PAS) domain protein 4 (NPAS4) regulates neurite outgrowthand phosphorylation of synapsin Irdquo The Journal of BiologicalChemistry vol 288 no 4 pp 2655ndash2664 2013
[8] G Pouchelon F Gambino C Bellone et al ldquoModality-specificthalamocortical inputs instruct the identity of postsynaptic L4neuronsrdquo Nature vol 511 no 7510 pp 471ndash474 2014
[9] S Yoshihara H Takahashi N Nishimura et al ldquoNpas4 regu-lates Mdm2 and thus Dcx in experience-dependent dendritic
spine development of newborn olfactory bulb interneuronsrdquoCell Reports vol 8 no 3 pp 843ndash857 2014
[10] K Hayashi R Ishikawa L-H Ye et al ldquoModulatory role ofdrebrin on the cytoskeleton within dendritic spines in the ratcerebral cortexrdquo Journal of Neuroscience vol 16 no 22 pp 7161ndash7170 1996
[11] K Ramamoorthi R Fropf GM Belfort et al ldquoNpas4 regulatesa transcriptional program in CA3 required for contextualmemory formationrdquo Science vol 334 no 6063 pp 1669ndash16752011
[12] J E Ploski M S Monsey T Nguyen R J DiLeone and G ESchafe ldquoThe neuronal PAS domain protein 4 (Npas4) isrequired for new and reactivated fear memoriesrdquo PLoS ONEvol 6 no 8 Article ID e23760 2011
[13] JFMaya-Vetencourt E Tiraboschi DGreco et al ldquoExperience-dependent expression of NPAS4 regulates plasticity in adultvisual cortexrdquo Journal of Physiology vol 590 no 19 pp 4777ndash4787 2012
[14] M Kossut P J Hand J Greenberg and C L Hand ldquoSinglevibrissal cortical column in SI cortex of rat and its alterations inneonatal and adult vibrissa-deafferented animals a quantitative2DG studyrdquo Journal of Neurophysiology vol 60 no 2 pp 829ndash852 1988
[15] S Glazewski andK Fox ldquoTime course of experience-dependentsynaptic potentiation and depression in barrel cortex of adoles-cent ratsrdquo Journal of Neurophysiology vol 75 no 4 pp 1714ndash1729 1996
[16] H Wallace and K Fox ldquoLocal cortical interactions determinethe form of cortical plasticityrdquo Journal of Neurobiology vol 41no 1 pp 58ndash63 1999
[17] M A Lebedev G Mirabella I Erchova and M E DiamondldquoExperience-dependent plasticity of rat barrel cortex redistri-bution of activity across barrel-columnsrdquo Cerebral Cortex vol10 no 1 pp 23ndash31 2000
[18] A Kaliszewska M Bijata L Kaczmarek and M KossutldquoExperience-dependent plasticity of the barrel cortex in miceobserved with 2-DG brain mapping and c-Fos effects of MMP-9 KOrdquo Cerebral Cortex vol 22 no 9 pp 2160ndash2170 2012
[19] D J Margolis H Lutcke K Schulz et al ldquoReorganization ofcortical population activity imaged throughout long-term sen-sory deprivationrdquo Nature Neuroscience vol 15 no 11 pp 1539ndash1546 2012
[20] S AMarikH Yamahachi J N JMcManusG Szabo andCDGilbert ldquoAxonal dynamics of excitatory and inhibitory neuronsin somatosensory cortexrdquo PLoS Biology vol 8 no 6 Article IDe1000395 2010
[21] V C Wimmer P J Broser T Kuner and R M BrunoldquoExperience-induced plasticity of thalamocortical axons inboth juveniles and adultsrdquo Journal of Comparative Neurologyvol 518 no 22 pp 4629ndash4648 2010
[22] M Oberlaender A Ramirez and R M Bruno ldquoSensory expe-rience restructures thalamocortical axons during adulthoodrdquoNeuron vol 74 no 4 pp 648ndash655 2012
[23] D Katzel and G Miesenbock ldquoExperience-dependent rewiringof specific inhibitory connections in adult neocortexrdquo PLoSBiology vol 12 no 2 Article ID e1001798 2014
[24] C Tailby L LWright A BMetha andM B Calford ldquoActivity-dependent maintenance and growth of dendrites in adultcortexrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 102 no 12 pp 4631ndash4636 2005
Neural Plasticity 9
[25] C E J Cheetham M S L Hammond R McFarlane and G TFinnerty ldquoAltered sensory experience induces targeted rewiringof local excitatory connections in mature neocortexrdquo Journal ofNeuroscience vol 28 no 37 pp 9249ndash9260 2008
[26] K D Micheva and C Beaulieu ldquoAn anatomical substrate forexperience-dependent plasticity of the rat barrel field cortexrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 25 pp 11834ndash11838 1995
[27] Y Zuo G Yang E Kwon and W-B Gan ldquoLong-term sen-sory deprivation prevents dendritic spine loss in primarysomatosensory cortexrdquo Nature vol 436 no 7048 pp 261ndash2652005
[28] A Holtmaat L Wilbrecht G W Knott E Welker and KSvoboda ldquoExperience-dependent and cell-type-specific spinegrowth in the neocortexrdquo Nature vol 441 no 7096 pp 979ndash983 2006
[29] C E J Cheetham M S L Hammond C E J Edwards and GT Finnerty ldquoSensory experience alters cortical connectivity andsynaptic function site specificallyrdquo The Journal of Neurosciencevol 27 no 13 pp 3456ndash3465 2007
[30] L Wilbrecht A Holtmaat N Wright K Fox and K Svo-boda ldquoStructural plasticity underlies experience-dependentfunctional plasticity of cortical circuitsrdquo Journal of Neurosciencevol 30 no 14 pp 4927ndash4932 2010
[31] C E J Cheetham S J Barnes G Albieri G W Knott and GT Finnerty ldquoPansynaptic enlargement at adult cortical connec-tions strengthened by experiencerdquo Cerebral Cortex vol 24 no2 pp 521ndash531 2014
[32] E Siucinska and M Kossut ldquoShort-lasting classical condition-ing induces reversible changes of representational maps ofvibrissae inmouse SI cortex-A 2DG studyrdquoCerebral Cortex vol6 no 3 pp 506ndash513 1996
[33] M Jasinska E Siucinska A Cybulska-Klosowicz et al ldquoRapidlearning-induced inhibitory synaptogenesis in murine barrelfieldrdquo The Journal of Neuroscience vol 30 no 3 pp 1176ndash11842010
[34] R N Strominger and T AWoolsey ldquoTemplates for locating thewhisker area in fresh flattened mouse and rat cortexrdquo Journal ofNeuroscience Methods vol 22 no 2 pp 113ndash118 1987
[35] A Valles A J Boender S Gijsbers R A M Haast G J MMartens and P de Weerd ldquoGenomewide analysis of rat barrelcortex reveals time- and layer-specificmrna expression changesrelated to experience-dependent plasticityrdquo Journal of Neuro-science vol 31 no 16 pp 6140ndash6158 2011
[36] A Kaliszewska Expression of selected immediate early genesand involvement of matrix metalloproteinase 9 in functionalreorganization of mouse barrel cortex [Doctoral dissertation]Nencki Institute of Experimental Biology Polish Academy ofSciences Warsaw Poland 2012
[37] A Cybulska-Klosowicz R Zakrzewska and M Kossut ldquoBrainactivation patterns during classical conditioning with appetitiveor aversive UCSrdquo Behavioural Brain Research vol 204 no 1 pp102ndash111 2009
[38] B Lu K H Wang and A Nose ldquoMolecular mechanismsunderlying neural circuit formationrdquo Current Opinion in Neu-robiology vol 19 no 2 pp 162ndash167 2009
[39] Y Jiao Z Zhang C Zhang et al ldquoA key mechanism under-lying sensory experience-dependent maturation of neocorti-cal GABAergic circuits in vivordquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 108 no29 pp 12131ndash12136 2011
[40] K Tokarski J Urban-Ciecko M Kossut and G Hess ldquoSensorylearning-induced enhancement of inhibitory synaptic trans-mission in the barrel cortex of the mouserdquo European Journal ofNeuroscience vol 26 no 1 pp 134ndash141 2007
[41] A Cybulska-Klosowicz A Posluszny K Nowak E SiucinskaM Kossut and M Liguz-Lecznar ldquoInterneurons containingsomatostatin are affected by learning-induced cortical plastic-ityrdquo Neuroscience vol 254 pp 18ndash25 2013
[42] Z-H Hou and X Yu ldquoActivity-regulated somatostatin expres-sion reduces dendritic spine density and lowers excitatorysynaptic transmission via postsynaptic somatostatin receptor 4rdquoJournal of Biological Chemistry vol 288 no 4 pp 2501ndash25092013
[43] S A Eichler and J CMeier ldquoE-I balance and human diseasesmdashfrom molecules to networkingrdquo Frontiers in Molecular Neuro-science vol 1 article 2 2008
[44] L Coutellier S Beraki P M Ardestani N L Saw and MShamloo ldquoNpas4 a neuronal transcription factor with a keyrole in social and cognitive functions relevant to developmentaldisordersrdquo PLoS ONE vol 7 no 9 Article ID e46604 2012
[45] E J Jaehne T S Klaric S A Koblar B T Baune and MD Lewis ldquoEffects of Npas4 deficiency on anxiety depression-like cognition and sociability behaviourrdquo Behavioural BrainResearch vol 281 pp 276ndash282 2015
[46] D C Bersten J B Bruning D J Peet and M L WhitelawldquoHuman variants in the neuronal basic helix-loop-helixPer-Arnt-Sim (bHLHPAS) transcription factor complex NPAS4ARNT2 disrupt functionrdquo PLoS ONE vol 9 no 1 Article IDe85768 2014
[47] S Maren ldquoPavlovian fear conditioning as a behavioral assayfor hippocampus and amygdala function cautions and caveatsrdquoEuropean Journal of Neuroscience vol 28 no 8 pp 1661ndash16662008
[48] B Jabłonska M Kossut and J Skangiel-Kramska ldquoTransientincrease of AMPA and NMDA receptor binding in the barrelcortex of mice after tactile stimulationrdquo Neurobiology of Learn-ing and Memory vol 66 no 1 pp 36ndash43 1996
[49] M Gierdalski B Jablonska E SiucinskaM Lech A Skibinskaand M Kossut ldquoRapid regulation of GAD67 mRNA and pro-tein level in cortical neurons after sensory learningrdquo CerebralCortex vol 11 no 9 pp 806ndash815 2001
Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
2 Neural Plasticity
Local elimination of excitatory input to deprived corticalbarrels disrupts the equilibrium between excitation and inhi-bition which leads to modifications in functional activationand anatomical rewiring of cortical sensory areas Functionalrepresentation of spared inputs starts to expand to deprivedareas [14ndash19] This is accompanied by large-sale structuralchanges such as axonal retraction and sprouting [20ndash23]and dendritic reorganization [24 25] and also by moresubtle changes involving spines and synapses with decreaseddensity of inhibitory synapse in deprived barrels [26ndash31]Classical conditioning involving unilateral stimulation ofrow B vibrissae paired with tail shock results in behavioralchanges (increased immobility) expansion of functional cor-tical representation of the row of vibrissae stimulated duringconditioning and increased density of inhibitory synapseson spines in barrels that represent the stimulated vibrissae[32 33] As Npas4 is implicated in structural plasticity in thepresent study we aimed to determine how Npas4 expressionin the barrel cortex is influenced by sensory conditioning andsensory deprivation
2 Methods
21 Deprivation and Somatosensory Stimulation Six maleC57BL6 mice aged 8-9 weeks were used in the deprivationexperiment The mice were reared in a 12 12 lightdark cyclein standard cages and had ad libitum access to water andfood All experimental procedures were approved by the FirstEthical Commission in Warsaw Poland and were in accor-dance with the European Communities Council Directiveof 24 November 1986 (86609EEC) Mice were deprivedunder short (2-3 minutes) isoflurane anesthesia by pluckingout all whiskers except row C on one side of the snout(referred to as experimental side later in the text Figure 1(a))This experimental model of sensory deprivation leaves thecentrally situated row C of vibrissae intact with symmetricalspace for remodeling of the cortex in the medial and lateraldirection Regrowing vibrissae were removed every secondday On the 6th day (24 hours before the experiment) theother side of the snout (control side) was subjected to thesame deprivation procedure so thatmice were leftwith intactrow C whiskers on both sides Animals were deprived 24hours prior to exploration of the stimulatory cage to avoidincrease in Npas4 and c-Fos expression induced by whiskerplucking Mice were allowed to explore the stimulatory cageon the 7th day The walls and floor were made of bars andthe cage was equipped with objects of different shapes andtextures mouse wheels plastic toys maze-like constructionsand pieces of Styrofoam Animals were placed in the cageand left for 30min in a room without illumination to pro-mote sensory stimulation Next they were killed by cervicaldislocation The brains were dissected out and cortices wereflattened [34] and frozen in minus70∘C isopentane
22 Training Procedure 29 male Swiss albino mice aged 8-9weeks were used in training procedure Prior to behavioraltraining mice were habituated to a neck restraint for 10mina day 5 days a week for 2-3 weeks Animals were placed inseparate home cages a few days before onset of the training
Experimentalrow C
Controlrow C
Experimentalrow C
30min
6 days 24h
Enriched environment
(a) Deprivation
05 s
Training session 40x CS + UCS 10min1 session daily on 3 consecutive days
UCSmdash05mA
CSmdashstroking row B
(b) Training
Figure 1 (a) Scheme of the deprivation procedure All whiskers onone side of the snout except for row C are removed After 6 days thesame deprivation procedure is applied to the other side of the snoutand the mouse is left with both C rows intact After 24 h the animalexplores a stimulatory cage for 30 minutes and is then immediatelykilled (b) Scheme of the training procedure Row B of whiskers onthe left side of the snout is stroked (CS) The CS lasts for 9 s Duringthe last second an electric shock (05mA for 05 s UCS) is deliveredand coterminates with the CS A single training session lasts for 10minutes and encompasses 40 CS and UCS pairings The animal issubjected to one session daily for three consecutive days Followingthe last training session the mouse is placed in its home cage for 20minutes to allow increase in Npas4 expression and is then killed
During training row B whiskers on the left side of the snoutwere stroked in the posterior to anterior direction (CS) witha fine handheld brush (Figure 1(b)) The CS lasted for 9 sDuring the last second an aversiveUCS (amild electric shockof 05mA for 05 s applied to the tail) was delivered andcoterminated with the CS This trial was repeated after a 6 sinterval and the routine was continued for 10min Trainingencompassed three training sessions on three consecutivedays Following the last training session animals were placedin their home cages for 20 minutes to allow for increase inNpas4 expression and then killed by cervical dislocationThebrainswere dissected out and corticeswere flattened [34] andfrozen in minus70∘C isopentane
Figure 2 (a) Single rows of barrels are microdissected from Nissl-stained sections RNA from dissected tissue is extracted and processed forRT-PCR (b) Nissl-stained section with row B dissected out Neighbouring row C is left intact ((c) (d)) Rows of barrels microdissected indeprivation (C) and sensory conditioning (D) experiment Microdissected barrels are colored in green and red Legend below the schemeexplains terminology used in the text for description of particular rows
(ii) PSEUDO (pseudoconditioned 119899 = 7) unpaired appli-cation of the same number of CS and UCS as duringconditioning
(iii) CS only (119899 = 8) sessions of whisker stroking(iv) naıve ndash (119899 = 5) unstimulated controls in neck restrain-
ing apparatus
23 Laser Microdissection Flattened cortices were cryosec-tioned (16 120583m) tangentially to the surface Slices from layerIV were mounted on membrane slides (MembraneSlide 10PEN Carl Zeiss MicroImaging GmbH) and stored at minus70∘Cuntil further usage Immediately prior to themicrodissectionslices were subjected to modified Nissl staining using Arc-turus HistoGene Staining Solution (KIT0401 Life Technolo-gies) which preserves nucleic acid integrity Sections fromboth hemispheres of the animal were processed togetherIsolation of tissue was performed using the ArcturusXTLCMSystem equipped with a Nikon Eclipse Ti-Emicroscope
(Figures 2(a) and 2(b)) Microdissected tissue was collectedon CapSureMacro LCMCaps (LCM0211 Life Technologies)In slices frommice used in the conditioning experiment rowsB and D from the right hemisphere were dissected and fromthe left hemisphere only row B was dissected (Figure 2(d))Row B from the right hemisphere in the CS + UCS groupis referred to as the trained row of whiskers In slices fromdeprived mice rows B C and D from both hemisphereswere microdissected (Figure 2(c)) Row C was collected ona separate membrane and rows B and Dwere collected on thesame membrane In further steps tissue from rows B and Dfrom the same hemisphere was pooled Samples from differ-ent animals were not pooled In summation three sampleswere collected from animals in the conditioning experiment(row B right hemisphere row D right hemisphere and rowB left hemisphere) and four samples were collected fromdeprived animals (rowC experimental hemisphere rows B +D experimental hemisphere row C control hemisphere androws B + D control hemisphere) RNA was recovered using
4 Neural Plasticity
Table 1 Sequences of primers used in real-time PCR
a PicoPure RNA Isolation Kit (KIT0204 Life Technologies)with concurrent genomic DNA elimination using DNase(RNase-Free DNase Set 79254 Qiagen)
24 Real-Time PCR Reversed transcription was performedusing aMaxima First Strand cDNASynthesisKit (K1641ThermoScientific-Fermentas) Real-time PCR was conducted usingPower SYBR Green PCR Master Mix (4368702 Life Tech-nologies) In real-time PCR experiment each sample wasrun in triplicate Amplification was carried out with a 7500real-time PCR System (Applied Biosystems) using PowerSYBR Green PCR Master Mix specific primers (Table 1)and cDNA for each sample The glyceraldehyde 3-phosphatedehydrogenase (GAPDH) gene was used as a housekeepinggene The amplification reaction was cycled 40 times witha 95∘C denaturation step for 15 s and a 60∘C annealing stepfor 1 minute A dissociation stage was performed to assessspecificity of primers Results were calculated using standardcurve method
25 Data Analysis and Statistics Ratios of a target gene andhousekeeping gene levels were used for analysis Statisticalanalysis was performed using GraphPad Prism 5 software(GraphPad Software Inc) Gene expression levels in differ-ent barrel rows were analyzed using ANOVA followed byNewman-Keuls post hoc tests Studentrsquos 119905-test was usedwhereapplicable
3 Results
31 Sensory Deprivation
311 Npas4 and c-Fos Mice were subjected to one week ofsensory deprivationmdashall vibrissae on one side of the snoutwere removed except for row C while vibrissae on the otherside were left intact On the day preceding stimulation theother side of the snout was subjected to the same procedureso that mice were left with two C rows intact Animalswere placed in the stimulatory cage for 30 minutesmdasha timeinterval demonstrated to be appropriate to observe increasein Npas4 expression in the barrel cortex after explorationof an enriched environment [35] Regions of interest weremicrodissected from slices of layer IV Real-timePCRmethodwas used to assess Npas4 mRNA level in individual barrel
rows Npas4 level was evaluated in spared rows C and indeprived rows B and D in the experimental and controlhemisphere
Elimination of sensory input to selected rows of vibrissaeevoked differences in Npas4 expression between spared anddeprived barrels (ANOVA 119865(318) = 1004 119875 = 00004Figure 3(a)) Post hoc analysis demonstrated that the levelof Npas4 transcript in the control hemisphere was 484lower in deprived rows B and D than in the spared row C(035 plusmn 005 versus 067 plusmn 006 119875 lt 001) There were nodifferences in Npas4 transcript levels between spared rowsC in both hemispheres (119875 gt 005) Also no differenceswere observed between deprived regions in both hemispheres(119875 gt 005) which shows that duration of deprivation (7days versus 24 hours) had no impact on Npas4 expressionin deprived barrel rows We have previously shown that thisdeprivation procedure followed by two hours of explorationof stimulatory cage resulted in an increased number of cellsexpressing c-Fos in deprived rows B and D after 7 daysof deprivation in comparison with 24 hours of deprivation[18] We also observed this phenomenon regarding the otheractivity-regulated genes Arc and Zif268 [36] As Npas4 isalso an activity-regulated gene we expected a similar patternof its expression As this was not the case we decided toassess c-Fos mRNA in the same samples to confirm thatthe Npas4 pattern of expression is atypical of other activity-regulated genes in this model of plasticity The deprivationprocedure produced differences in c-Fos expression amonganalyzed barrel rows (ANOVA 119865(316) = 8028 119875 = 00022Figure 3(b)) In the experimental hemisphere the level ofc-Fos mRNA was 742 higher in deprived rows B and D(138 plusmn 020 versus 079 plusmn 009 119875 lt 005) than in homotypicregions in the control hemisphere
32 Sensory Conditioning The training procedure encom-passed three training sessions on three consecutive dayseach lasting for 10 minutes and consisting of 40 CS (strokingrow B of vibrissae) and UCS (electric shock to the tail)pairings This paradigm evokes freezing-like behavior whichindicates that association of CS and UCS occurred [37] InCS + UCS animals Npas4 expression was on average 529higher in trained row B barrels than in the contralateral rowB (024 plusmn 003 versus 015 plusmn 003 119905-test 119875 lt 0001) We foundno differences in Npas4 expression between hemispheresin naıve animals and for further analysis we pooled datafrom both B rows Npas4 expression in the trained row Bfor the CS + UCS group was 481 higher than in naıvegroup B rows (119905-test 119875 lt 005) There were no differencesbetween B rows in the naıve group and left (control) rowB in the CS + UCS group (119905-test 119875 gt 005) Analysis ofratios of Npas4 expression in right row B (trained row inCS + UCS group) and left row B revealed significant effectof the training procedure (ANOVA 119865(325) = 6780 119875 =00017 Figure 4) Ratio of Npas4 expression in right row Band left row B was higher in CS + UCS group in comparisonwith other experimental groups (119875 lt 001) indicating thatinterhemispheric difference in Npas4 mRNA level is not justan effect of stimulation of the row B of vibrissae Npas4
Neural Plasticity 5
00
02
04
06
08
Npa
s4 re
lativ
e exp
ress
ion
leve
l
Con
trolC
Exp
C
Con
trolB
D
Exp
BD
lowastlowastlowastlowast
lowastlowast
(a)
0
1
2
3
c-Fo
s rela
tive e
xpre
ssio
n le
vel
Con
trolC
Exp
C
Con
trolB
D
Exp
BD
lowastlowastlowast
lowastlowast
lowastlowast
lowast
lowast
(b)
Figure 3 (a) Npas4 expression in spared C rows and in deprived regions (averaged rows B and D) Control C spared row C on controlside (deprived for 24 hours) Exp C spared row C on experimental side (deprived 7 days) Control BD deprived B and D rows on controlside Exp BD deprived B and D rows on experimental side mean plusmn SEM lowastlowast
119875
lt 001 Duration of deprivation had no impact on Npas4expression (b) In deprived B and D rows deprivation lasting 7 days induced increase in c-Fos expression mean plusmn SEM lowast
119875
lt 005 lowastlowast119875
lt 001lowastlowastlowast
119875
lt 0001
00
05
10
15
20
relat
ive e
xpre
ssio
n le
vel
Righ
t Ble
ft B
Npa
s4
Naıve CS + UCS PSEUDO CS
lowastlowastlowastlowast
lowastlowast
Figure 4 Changes in Npas4 expression induced by training Intrained (CS + UCS) animals expression of Npas4 in the right(trained) row B of barrels is elevated in comparison with left(control unstimulated) row B Sole stimulation of whiskers (CSonly) and application of unpaired CS and UCS (PSEUDO) do notproduce increase in Npas4 expression in comparison with controlside Data are presented as ratio of Npas4 expression level in rightand left row B of barrels mean plusmn SEM lowastlowast
119875
lt 001
expression was increased in the ldquotrainedrdquo row in every singleconditioned animal (Figure 5)
000
005
010
015
020
025
030
035
040
045
050
Npa
s4 re
lativ
e exp
ress
ion
leve
l
Trained 1
Trained 2
Trained 3
Trained 4
Trained 5
Trained 6
Trained 7
Trained 8
Trained 9
L R
Figure 5 Changes of Npas4 mRNA expression in individualconditionedmicemdashcomparison between trained rowB of barrels (Rright) and control row B (L left)
6 Neural Plasticity
00
01
02
03
Npa
s4 re
lativ
e exp
ress
ion
leve
l
Left B Right D Right B
lowastlowast
(a)
00
05
10
15
c-Fo
s rela
tive e
xpre
ssio
n le
vel
Left B Right D Right B
lowast
(b)
Figure 6 Training induced changes in Npas4 (a) and c-Fos (b) expression Right B corresponds to the trained row of barrels right D tocontrol row of barrels in the same hemisphere and left B to control row B in the other hemisphere Npas4 and c-Fos have similar pattern ofexpression following the training procedure their expression is elevated in the trained row B in comparison with the control side mean plusmnSEM lowast119875
lt 005 lowastlowast119875
lt 001
To see if the observed changes in Npas4 expression arelimited to the stimulated row B barrels we evaluated the levelof its expression in row D barrels in the same hemisphereWe did not assess Npas4 mRNA level in row D of the left(control) hemisphere in the CS + UCS group but taking intoaccount results from the naıve group it can be presumedthat it is comparable to the control row B The level ofNpas4 expression in the right row D was an intermediatevalue between the levels of Npas4 mRNA expression in theright (trained) row B and left (control) row B it was notsignificantly different from any of the B rows (Figure 6(a))It can be interpreted that training affects row D so there areno differences between trained row B and row D in the samehemisphere but the impact of training upon rowD is weak sono differences can be observed when compared to the controlhemisphere
In the deprivation studyNpas4 turned out to have a differ-ent pattern of expression than other activity-regulated genesand we wondered if it is also the case in sensory conditioningWe evaluated c-Fos mRNA levels in samples obtained fromCS + UCS mice The pattern of c-Fos expression was thesame as for Npas4 (Figure 6(b)) The training procedureevoked differences among c-Fos expressions in examinedbarrel rows (ANOVA 119865(2 24) = 4407 119875 = 00299) c-Fos mRNA expression level was 484 higher in the trainedrow of barrels (row B in the right hemisphere) than in thecontralateral row B (in the left hemisphere 119875 lt 005)
4 Discussion
Knowledge on Npas4 in plasticity of adult cortex is limitedHerein we provide first description of Npas4 expression in
the barrel cortex undergoing plastic reorganization in twoparadigms deprivation and sensory conditioning We alsocompare changes in expression of Npas4 and c-Fos in bothexperimental models
We found that classical conditioning in which stimula-tion of a row of whiskers is paired with tail shock results inincreased expression of Npas4 mRNA in the cognate row ofcortical barrels As conditioning triggers inhibitory synapto-genesis in the barrels representing stimulated vibrissae [33]our results are in agreement with data demonstrating a roleof Npas4 in the formation of GABAergic synapses The workof Lin et al [3] found that Npas4 regulates expression ofa variety of genes including gene coding for BDNF Npas4binds to activity-dependent promoters I and IV of the BDNFgene [3] and expression of the BDNF gene from promoterIV contributes to the plasticity of inhibitory synapses [3839] Npas4 expression was demonstrated to drive inhibitorysynaptogenesis on excitatory neurons andNpas4 knockdownincreases interevent interval and decreases the amplitudeof mIPSCs [3] In the current experiment we observedan increase in Npas4 expression within the trained rowwhere previously we found increases of GABAergic synapsesdensity increased synaptic content of GABA and increasedspontaneous IPSCs [33 40] Our findings are also in linewith the recent results by Sim et al [4] who found thatincreased cell intrinsic activity results in via an Npas4dependent mechanism the addition of GABAergic inputsto the neuron In hippocampal pyramidal neurons behav-iorally induced expression of Npas4 drives redistribution ofinhibitory synapses increasing inhibitory synapse numberon the cell body while decreasing the number of inhibitorysynapses on the apical dendrites [5] In contrast in our
Neural Plasticity 7
previous experiments we observed an increase in density ofGABAergic synapses located on spines in barrels representingldquotrainedrdquo vibrissae [33] This discrepancy could be attributedto possibility that Npas4 impact on dendritic and somaticpool of GABAergic synapses depends on the type of neuronits location within the brain and the type of stimulation usedto evoke plasticity
Interestingly it was recently found that increased Npas4expression may also account for increased number of exci-tatory contacts made onto somatostatin interneurons [6] Inthe CS + UCS mice an increase in density of somatostatininterneurons within layer 4 of the barrel cortex is observed[41] which may be a result of elevated neuronal activity[42] It can be hypothesized that Npas4 expression drivesgeneration of additional excitatory input onto somatostatininterneurons which in turn express higher level of somato-statin
Herein we demonstrate that sensory conditioning pro-duces increase in Npas4 expression in the region whereinhibitory synaptogenesis was previously observed [33]Synapses formation and elimination accompanying learning-related behaviors may contribute to shift in excitation-inhibition balance Dysregulation of this balance has beenimplicated in number of human neuropsychiatric and neu-rodegenerative disorders and associated with impairmentof cognitive functions [43] Regarding Npas4 significancefor maintaining excitation-inhibition equilibrium it can bepresumed that Npas4 deficiency would result in cognitivedeficits Indeed such deficits were observed in Npas4 knock-out mice [44 45] So far Npas4 has not been directly linkedto human neuropsychiatric disorders However Bersten et al[46] identified human variants of Npas4 with reduced tran-scriptional activity so sequencing Npas4 in neuropsychiatricpatients might be helpful in detecting such a link if it exists
Previous experiments regarding fear conditioning andcontextual learning indicate that Npas4 is indispensable formemory formation [11 12]Therefore it is reasonable to thinkthat Npas4 deficiency should also impair sensory condition-ing Testing this hypothesis using Npas4 knockout animalsmight be misleading Npas4 knockout animals performedwell in amygdala-dependent fear conditioning [11] whileacute deletion of Npas4 in amygdala impaired fear memoryformation [12] Regionally selective depletion of Npas4mightbe more useful then but first structures required for learningassociation of sensory CS and UCS in paradigm used in thisstudy should be identified Basolateral amygdala is activatedduring the training procedure [37] and amygdala is requiredfor all forms of fear conditioning [47] therefore amygdalawould seem a first choice structure for local knockdown ofNpas4 and determining its impact on sensory conditioning
Npas4 expression in remote row D barrels did notdiffer from the results obtained for the control hemisphereand naıve group which is in agreement with our previousobservation that no changes typical for the trained row Bappear in remote row D (no AMPA and NMDA bindingincrease no GAD mRNA expression upregulation and noincrease in density of somatostatin containing inhibitoryinterneurons) [40 41 48 49] We did not do EM to evaluatethe synapse density of remote row D barrels but we found
no increase in spontaneous IPSC there which would indicateincreased GABA release in its excitatory neurons [40]
In the sensory conditioning paradigm Npas4 and c-Foslevels changed in the same wayThey were upregulated in therow of barrels that received the conditioned input
Depriving selected barrel rows of sensory input resultedin decreased Npas4 mRNA expression in the deprivedbarrel rows in comparison with spared rows Duration ofdeprivation (24 h versus 7 days) had no impact upon Npas4expression in spared rows and deprived rowsThis contrastedwith c-Fos the expression of which was increased in bothdeprived and spared regions after 7 days of deprivation incomparison with 24 hours of deprivation
Using immunohistochemical techniques we previouslydescribed the pattern of c-Fos protein expression followingwhisker deprivation of various durations [18] in the samedeprivation paradigm The density of c-Fos positive nucleiincreased in the barrel row deprived of whisker input for 7days and the effect was augmented as the deprivation periodwas prolonged We interpreted this result as illustrating theexpansion of the spared vibrissal input into neighboringfunctionally deafferented barrels The gradual increase in thenumber of immunoreactive nuclei could reflect the gradualrewiring of the barrel cortex in the course of prolongeddeprivation The present results confirm our previous data atthe mRNA level
Unlike c-Fos Npas4mRNA in the deprived B andD rowswas not upregulated by sensory stimulation when comparing24 h versus 7 days of deprivation We suppose that thestrength of the sensory signal coming from the spared row Cwhiskers was insufficient for changing the expression of thisIEG In the experimental paradigm used here we previouslyobserved pronounced changes in Zif268 and Arc expression[36] It is not surprising that differently regulated genes donot respond identically to an experimental situation (see[35] for comparison of several IEGs activations by enrichedenvironment) Perhaps the plastic rearrangement of connec-tions induced by deprivation although it already changesthe metabolic response [18] does not yet trigger activationin Npas4 mRNA expression Npas4 can regulate activity-dependent expression of Arc c-Fos and Zif268 [11] Takinginto consideration that Arc c-Fos and Zif268 expressionsincrease in deprived barrels after 7 days of deprivation it mayseem puzzling that expression of Npas4 which regulates thetranscription of these genes remains unaltered However itshould be noted that Npas4 itself is an immediate early geneand gets activated in response to stimulation Accordingly itprobably does not regulate the first phase of other immediateearly genes expression which is independent of de novoprotein synthesis It rather seems that Npas4 plays a rolein enhancing and sustaining other immediate early genes inlater phases [11]
To the best of our knowledge this is the first report onNpas4 expression in deprivation-induced plasticity Maya-Vetencourt et al [13] examined Npas4 expression in monoc-ularly deprived rats treated with fluoxetine but they concen-trated on influence of fluoxetine onNpas4 expression and noton the influence of deprivation
8 Neural Plasticity
In this paper the precise anatomical dissection of arow of barrels where a plastic change took place allowedfor a quantitative analysis of IEGs expression in two typesof experience dependent plasticity Activation of the barrelcortex undergoing reorganization triggered by removal ofselected rows of whiskers strongly affected c-Fos (but notNpas4) expression Activation of the cortex undergoing aplastic change due to being involved in sensory conditioningupregulated the expression of both Npas4 and c-Fos Takinginto consideration our observations that sensory condition-ing increases the number of inhibitory synapses withinthe trained barrels and studies of other groups showinginvolvement of Npas4 in synaptogenesis we presume thatNpas4 may be involved in reshaping of connectivity withinbarrel cortex after sensory conditioning
Conflict of Interests
The authors declare no conflict of interests
Acknowledgment
This project was supported by GrantMNiSWno 2486BP012010 to Malgorzata Kossut
References
[1] M Moser R Knoth C Bode and C Patterson ldquoLE-PAS anovel Arnt-dependent HLH-PAS protein is expressed in limbictissues and transactivates the CNS midline enhancer elementrdquoMolecular Brain Research vol 128 no 2 pp 141ndash149 2004
[2] N Ooe K Saito N Mikami I Nakatuka and H KanekoldquoIdentification of a novel basic helix-loop-helix-PAS factorNXF reveals a Sim2 competitive positive regulatory role indendritic-cytoskeleton modulator drebrin gene expressionrdquoMolecular and Cellular Biology vol 24 no 2 pp 608ndash616 2004
[3] Y Lin B L Bloodgood J L Hauser et al ldquoActivity-dependentregulation of inhibitory synapse development by Npas4rdquoNature vol 455 no 7217 pp 1198ndash1204 2008
[4] S Sim S Antolin C-W Lin Y-X Lin and C Lois ldquoIncreasedcell-intrinsic excitability induces synaptic changes in new neu-rons in the adult dentate gyrus that require Npas4rdquo Journal ofNeuroscience vol 33 no 18 pp 7928ndash7940 2013
[5] B L Bloodgood N Sharma H A Browne A Z Trepman andM E Greenberg ldquoThe activity-dependent transcription factorNPAS4 regulates domain-specific inhibitionrdquo Nature vol 503no 7474 pp 121ndash125 2013
[6] I Spiegel A R Mardinly H W Gabel et al ldquoNpas4 regulatesexcitatory-inhibitory balance within neural circuits throughcell-type-specific gene programsrdquo Cell vol 157 no 5 pp 1216ndash1229 2014
[7] J Yun T Nagai Y Furukawa-Hibi et al ldquoNeuronal per arnt sim(PAS) domain protein 4 (NPAS4) regulates neurite outgrowthand phosphorylation of synapsin Irdquo The Journal of BiologicalChemistry vol 288 no 4 pp 2655ndash2664 2013
[8] G Pouchelon F Gambino C Bellone et al ldquoModality-specificthalamocortical inputs instruct the identity of postsynaptic L4neuronsrdquo Nature vol 511 no 7510 pp 471ndash474 2014
[9] S Yoshihara H Takahashi N Nishimura et al ldquoNpas4 regu-lates Mdm2 and thus Dcx in experience-dependent dendritic
spine development of newborn olfactory bulb interneuronsrdquoCell Reports vol 8 no 3 pp 843ndash857 2014
[10] K Hayashi R Ishikawa L-H Ye et al ldquoModulatory role ofdrebrin on the cytoskeleton within dendritic spines in the ratcerebral cortexrdquo Journal of Neuroscience vol 16 no 22 pp 7161ndash7170 1996
[11] K Ramamoorthi R Fropf GM Belfort et al ldquoNpas4 regulatesa transcriptional program in CA3 required for contextualmemory formationrdquo Science vol 334 no 6063 pp 1669ndash16752011
[12] J E Ploski M S Monsey T Nguyen R J DiLeone and G ESchafe ldquoThe neuronal PAS domain protein 4 (Npas4) isrequired for new and reactivated fear memoriesrdquo PLoS ONEvol 6 no 8 Article ID e23760 2011
[13] JFMaya-Vetencourt E Tiraboschi DGreco et al ldquoExperience-dependent expression of NPAS4 regulates plasticity in adultvisual cortexrdquo Journal of Physiology vol 590 no 19 pp 4777ndash4787 2012
[14] M Kossut P J Hand J Greenberg and C L Hand ldquoSinglevibrissal cortical column in SI cortex of rat and its alterations inneonatal and adult vibrissa-deafferented animals a quantitative2DG studyrdquo Journal of Neurophysiology vol 60 no 2 pp 829ndash852 1988
[15] S Glazewski andK Fox ldquoTime course of experience-dependentsynaptic potentiation and depression in barrel cortex of adoles-cent ratsrdquo Journal of Neurophysiology vol 75 no 4 pp 1714ndash1729 1996
[16] H Wallace and K Fox ldquoLocal cortical interactions determinethe form of cortical plasticityrdquo Journal of Neurobiology vol 41no 1 pp 58ndash63 1999
[17] M A Lebedev G Mirabella I Erchova and M E DiamondldquoExperience-dependent plasticity of rat barrel cortex redistri-bution of activity across barrel-columnsrdquo Cerebral Cortex vol10 no 1 pp 23ndash31 2000
[18] A Kaliszewska M Bijata L Kaczmarek and M KossutldquoExperience-dependent plasticity of the barrel cortex in miceobserved with 2-DG brain mapping and c-Fos effects of MMP-9 KOrdquo Cerebral Cortex vol 22 no 9 pp 2160ndash2170 2012
[19] D J Margolis H Lutcke K Schulz et al ldquoReorganization ofcortical population activity imaged throughout long-term sen-sory deprivationrdquo Nature Neuroscience vol 15 no 11 pp 1539ndash1546 2012
[20] S AMarikH Yamahachi J N JMcManusG Szabo andCDGilbert ldquoAxonal dynamics of excitatory and inhibitory neuronsin somatosensory cortexrdquo PLoS Biology vol 8 no 6 Article IDe1000395 2010
[21] V C Wimmer P J Broser T Kuner and R M BrunoldquoExperience-induced plasticity of thalamocortical axons inboth juveniles and adultsrdquo Journal of Comparative Neurologyvol 518 no 22 pp 4629ndash4648 2010
[22] M Oberlaender A Ramirez and R M Bruno ldquoSensory expe-rience restructures thalamocortical axons during adulthoodrdquoNeuron vol 74 no 4 pp 648ndash655 2012
[23] D Katzel and G Miesenbock ldquoExperience-dependent rewiringof specific inhibitory connections in adult neocortexrdquo PLoSBiology vol 12 no 2 Article ID e1001798 2014
[24] C Tailby L LWright A BMetha andM B Calford ldquoActivity-dependent maintenance and growth of dendrites in adultcortexrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 102 no 12 pp 4631ndash4636 2005
Neural Plasticity 9
[25] C E J Cheetham M S L Hammond R McFarlane and G TFinnerty ldquoAltered sensory experience induces targeted rewiringof local excitatory connections in mature neocortexrdquo Journal ofNeuroscience vol 28 no 37 pp 9249ndash9260 2008
[26] K D Micheva and C Beaulieu ldquoAn anatomical substrate forexperience-dependent plasticity of the rat barrel field cortexrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 25 pp 11834ndash11838 1995
[27] Y Zuo G Yang E Kwon and W-B Gan ldquoLong-term sen-sory deprivation prevents dendritic spine loss in primarysomatosensory cortexrdquo Nature vol 436 no 7048 pp 261ndash2652005
[28] A Holtmaat L Wilbrecht G W Knott E Welker and KSvoboda ldquoExperience-dependent and cell-type-specific spinegrowth in the neocortexrdquo Nature vol 441 no 7096 pp 979ndash983 2006
[29] C E J Cheetham M S L Hammond C E J Edwards and GT Finnerty ldquoSensory experience alters cortical connectivity andsynaptic function site specificallyrdquo The Journal of Neurosciencevol 27 no 13 pp 3456ndash3465 2007
[30] L Wilbrecht A Holtmaat N Wright K Fox and K Svo-boda ldquoStructural plasticity underlies experience-dependentfunctional plasticity of cortical circuitsrdquo Journal of Neurosciencevol 30 no 14 pp 4927ndash4932 2010
[31] C E J Cheetham S J Barnes G Albieri G W Knott and GT Finnerty ldquoPansynaptic enlargement at adult cortical connec-tions strengthened by experiencerdquo Cerebral Cortex vol 24 no2 pp 521ndash531 2014
[32] E Siucinska and M Kossut ldquoShort-lasting classical condition-ing induces reversible changes of representational maps ofvibrissae inmouse SI cortex-A 2DG studyrdquoCerebral Cortex vol6 no 3 pp 506ndash513 1996
[33] M Jasinska E Siucinska A Cybulska-Klosowicz et al ldquoRapidlearning-induced inhibitory synaptogenesis in murine barrelfieldrdquo The Journal of Neuroscience vol 30 no 3 pp 1176ndash11842010
[34] R N Strominger and T AWoolsey ldquoTemplates for locating thewhisker area in fresh flattened mouse and rat cortexrdquo Journal ofNeuroscience Methods vol 22 no 2 pp 113ndash118 1987
[35] A Valles A J Boender S Gijsbers R A M Haast G J MMartens and P de Weerd ldquoGenomewide analysis of rat barrelcortex reveals time- and layer-specificmrna expression changesrelated to experience-dependent plasticityrdquo Journal of Neuro-science vol 31 no 16 pp 6140ndash6158 2011
[36] A Kaliszewska Expression of selected immediate early genesand involvement of matrix metalloproteinase 9 in functionalreorganization of mouse barrel cortex [Doctoral dissertation]Nencki Institute of Experimental Biology Polish Academy ofSciences Warsaw Poland 2012
[37] A Cybulska-Klosowicz R Zakrzewska and M Kossut ldquoBrainactivation patterns during classical conditioning with appetitiveor aversive UCSrdquo Behavioural Brain Research vol 204 no 1 pp102ndash111 2009
[38] B Lu K H Wang and A Nose ldquoMolecular mechanismsunderlying neural circuit formationrdquo Current Opinion in Neu-robiology vol 19 no 2 pp 162ndash167 2009
[39] Y Jiao Z Zhang C Zhang et al ldquoA key mechanism under-lying sensory experience-dependent maturation of neocorti-cal GABAergic circuits in vivordquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 108 no29 pp 12131ndash12136 2011
[40] K Tokarski J Urban-Ciecko M Kossut and G Hess ldquoSensorylearning-induced enhancement of inhibitory synaptic trans-mission in the barrel cortex of the mouserdquo European Journal ofNeuroscience vol 26 no 1 pp 134ndash141 2007
[41] A Cybulska-Klosowicz A Posluszny K Nowak E SiucinskaM Kossut and M Liguz-Lecznar ldquoInterneurons containingsomatostatin are affected by learning-induced cortical plastic-ityrdquo Neuroscience vol 254 pp 18ndash25 2013
[42] Z-H Hou and X Yu ldquoActivity-regulated somatostatin expres-sion reduces dendritic spine density and lowers excitatorysynaptic transmission via postsynaptic somatostatin receptor 4rdquoJournal of Biological Chemistry vol 288 no 4 pp 2501ndash25092013
[43] S A Eichler and J CMeier ldquoE-I balance and human diseasesmdashfrom molecules to networkingrdquo Frontiers in Molecular Neuro-science vol 1 article 2 2008
[44] L Coutellier S Beraki P M Ardestani N L Saw and MShamloo ldquoNpas4 a neuronal transcription factor with a keyrole in social and cognitive functions relevant to developmentaldisordersrdquo PLoS ONE vol 7 no 9 Article ID e46604 2012
[45] E J Jaehne T S Klaric S A Koblar B T Baune and MD Lewis ldquoEffects of Npas4 deficiency on anxiety depression-like cognition and sociability behaviourrdquo Behavioural BrainResearch vol 281 pp 276ndash282 2015
[46] D C Bersten J B Bruning D J Peet and M L WhitelawldquoHuman variants in the neuronal basic helix-loop-helixPer-Arnt-Sim (bHLHPAS) transcription factor complex NPAS4ARNT2 disrupt functionrdquo PLoS ONE vol 9 no 1 Article IDe85768 2014
[47] S Maren ldquoPavlovian fear conditioning as a behavioral assayfor hippocampus and amygdala function cautions and caveatsrdquoEuropean Journal of Neuroscience vol 28 no 8 pp 1661ndash16662008
[48] B Jabłonska M Kossut and J Skangiel-Kramska ldquoTransientincrease of AMPA and NMDA receptor binding in the barrelcortex of mice after tactile stimulationrdquo Neurobiology of Learn-ing and Memory vol 66 no 1 pp 36ndash43 1996
[49] M Gierdalski B Jablonska E SiucinskaM Lech A Skibinskaand M Kossut ldquoRapid regulation of GAD67 mRNA and pro-tein level in cortical neurons after sensory learningrdquo CerebralCortex vol 11 no 9 pp 806ndash815 2001
Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Neural Plasticity 3
LCM
RT-PCR
(a)
100120583m
(b)
7 days of deprivation
Experimental C (spared 7 days)
Experimental B and D (deprived 7 days)
A B CDE
Deprivation
Control C (spared 24h)
Control B and control D (spared 24h)
24h of deprivation
(c)
Right B (trained B in CS + UCS)
Left B right D
Learning
(d)
Figure 2 (a) Single rows of barrels are microdissected from Nissl-stained sections RNA from dissected tissue is extracted and processed forRT-PCR (b) Nissl-stained section with row B dissected out Neighbouring row C is left intact ((c) (d)) Rows of barrels microdissected indeprivation (C) and sensory conditioning (D) experiment Microdissected barrels are colored in green and red Legend below the schemeexplains terminology used in the text for description of particular rows
(ii) PSEUDO (pseudoconditioned 119899 = 7) unpaired appli-cation of the same number of CS and UCS as duringconditioning
(iii) CS only (119899 = 8) sessions of whisker stroking(iv) naıve ndash (119899 = 5) unstimulated controls in neck restrain-
ing apparatus
23 Laser Microdissection Flattened cortices were cryosec-tioned (16 120583m) tangentially to the surface Slices from layerIV were mounted on membrane slides (MembraneSlide 10PEN Carl Zeiss MicroImaging GmbH) and stored at minus70∘Cuntil further usage Immediately prior to themicrodissectionslices were subjected to modified Nissl staining using Arc-turus HistoGene Staining Solution (KIT0401 Life Technolo-gies) which preserves nucleic acid integrity Sections fromboth hemispheres of the animal were processed togetherIsolation of tissue was performed using the ArcturusXTLCMSystem equipped with a Nikon Eclipse Ti-Emicroscope
(Figures 2(a) and 2(b)) Microdissected tissue was collectedon CapSureMacro LCMCaps (LCM0211 Life Technologies)In slices frommice used in the conditioning experiment rowsB and D from the right hemisphere were dissected and fromthe left hemisphere only row B was dissected (Figure 2(d))Row B from the right hemisphere in the CS + UCS groupis referred to as the trained row of whiskers In slices fromdeprived mice rows B C and D from both hemisphereswere microdissected (Figure 2(c)) Row C was collected ona separate membrane and rows B and Dwere collected on thesame membrane In further steps tissue from rows B and Dfrom the same hemisphere was pooled Samples from differ-ent animals were not pooled In summation three sampleswere collected from animals in the conditioning experiment(row B right hemisphere row D right hemisphere and rowB left hemisphere) and four samples were collected fromdeprived animals (rowC experimental hemisphere rows B +D experimental hemisphere row C control hemisphere androws B + D control hemisphere) RNA was recovered using
4 Neural Plasticity
Table 1 Sequences of primers used in real-time PCR
a PicoPure RNA Isolation Kit (KIT0204 Life Technologies)with concurrent genomic DNA elimination using DNase(RNase-Free DNase Set 79254 Qiagen)
24 Real-Time PCR Reversed transcription was performedusing aMaxima First Strand cDNASynthesisKit (K1641ThermoScientific-Fermentas) Real-time PCR was conducted usingPower SYBR Green PCR Master Mix (4368702 Life Tech-nologies) In real-time PCR experiment each sample wasrun in triplicate Amplification was carried out with a 7500real-time PCR System (Applied Biosystems) using PowerSYBR Green PCR Master Mix specific primers (Table 1)and cDNA for each sample The glyceraldehyde 3-phosphatedehydrogenase (GAPDH) gene was used as a housekeepinggene The amplification reaction was cycled 40 times witha 95∘C denaturation step for 15 s and a 60∘C annealing stepfor 1 minute A dissociation stage was performed to assessspecificity of primers Results were calculated using standardcurve method
25 Data Analysis and Statistics Ratios of a target gene andhousekeeping gene levels were used for analysis Statisticalanalysis was performed using GraphPad Prism 5 software(GraphPad Software Inc) Gene expression levels in differ-ent barrel rows were analyzed using ANOVA followed byNewman-Keuls post hoc tests Studentrsquos 119905-test was usedwhereapplicable
3 Results
31 Sensory Deprivation
311 Npas4 and c-Fos Mice were subjected to one week ofsensory deprivationmdashall vibrissae on one side of the snoutwere removed except for row C while vibrissae on the otherside were left intact On the day preceding stimulation theother side of the snout was subjected to the same procedureso that mice were left with two C rows intact Animalswere placed in the stimulatory cage for 30 minutesmdasha timeinterval demonstrated to be appropriate to observe increasein Npas4 expression in the barrel cortex after explorationof an enriched environment [35] Regions of interest weremicrodissected from slices of layer IV Real-timePCRmethodwas used to assess Npas4 mRNA level in individual barrel
rows Npas4 level was evaluated in spared rows C and indeprived rows B and D in the experimental and controlhemisphere
Elimination of sensory input to selected rows of vibrissaeevoked differences in Npas4 expression between spared anddeprived barrels (ANOVA 119865(318) = 1004 119875 = 00004Figure 3(a)) Post hoc analysis demonstrated that the levelof Npas4 transcript in the control hemisphere was 484lower in deprived rows B and D than in the spared row C(035 plusmn 005 versus 067 plusmn 006 119875 lt 001) There were nodifferences in Npas4 transcript levels between spared rowsC in both hemispheres (119875 gt 005) Also no differenceswere observed between deprived regions in both hemispheres(119875 gt 005) which shows that duration of deprivation (7days versus 24 hours) had no impact on Npas4 expressionin deprived barrel rows We have previously shown that thisdeprivation procedure followed by two hours of explorationof stimulatory cage resulted in an increased number of cellsexpressing c-Fos in deprived rows B and D after 7 daysof deprivation in comparison with 24 hours of deprivation[18] We also observed this phenomenon regarding the otheractivity-regulated genes Arc and Zif268 [36] As Npas4 isalso an activity-regulated gene we expected a similar patternof its expression As this was not the case we decided toassess c-Fos mRNA in the same samples to confirm thatthe Npas4 pattern of expression is atypical of other activity-regulated genes in this model of plasticity The deprivationprocedure produced differences in c-Fos expression amonganalyzed barrel rows (ANOVA 119865(316) = 8028 119875 = 00022Figure 3(b)) In the experimental hemisphere the level ofc-Fos mRNA was 742 higher in deprived rows B and D(138 plusmn 020 versus 079 plusmn 009 119875 lt 005) than in homotypicregions in the control hemisphere
32 Sensory Conditioning The training procedure encom-passed three training sessions on three consecutive dayseach lasting for 10 minutes and consisting of 40 CS (strokingrow B of vibrissae) and UCS (electric shock to the tail)pairings This paradigm evokes freezing-like behavior whichindicates that association of CS and UCS occurred [37] InCS + UCS animals Npas4 expression was on average 529higher in trained row B barrels than in the contralateral rowB (024 plusmn 003 versus 015 plusmn 003 119905-test 119875 lt 0001) We foundno differences in Npas4 expression between hemispheresin naıve animals and for further analysis we pooled datafrom both B rows Npas4 expression in the trained row Bfor the CS + UCS group was 481 higher than in naıvegroup B rows (119905-test 119875 lt 005) There were no differencesbetween B rows in the naıve group and left (control) rowB in the CS + UCS group (119905-test 119875 gt 005) Analysis ofratios of Npas4 expression in right row B (trained row inCS + UCS group) and left row B revealed significant effectof the training procedure (ANOVA 119865(325) = 6780 119875 =00017 Figure 4) Ratio of Npas4 expression in right row Band left row B was higher in CS + UCS group in comparisonwith other experimental groups (119875 lt 001) indicating thatinterhemispheric difference in Npas4 mRNA level is not justan effect of stimulation of the row B of vibrissae Npas4
Neural Plasticity 5
00
02
04
06
08
Npa
s4 re
lativ
e exp
ress
ion
leve
l
Con
trolC
Exp
C
Con
trolB
D
Exp
BD
lowastlowastlowastlowast
lowastlowast
(a)
0
1
2
3
c-Fo
s rela
tive e
xpre
ssio
n le
vel
Con
trolC
Exp
C
Con
trolB
D
Exp
BD
lowastlowastlowast
lowastlowast
lowastlowast
lowast
lowast
(b)
Figure 3 (a) Npas4 expression in spared C rows and in deprived regions (averaged rows B and D) Control C spared row C on controlside (deprived for 24 hours) Exp C spared row C on experimental side (deprived 7 days) Control BD deprived B and D rows on controlside Exp BD deprived B and D rows on experimental side mean plusmn SEM lowastlowast
119875
lt 001 Duration of deprivation had no impact on Npas4expression (b) In deprived B and D rows deprivation lasting 7 days induced increase in c-Fos expression mean plusmn SEM lowast
119875
lt 005 lowastlowast119875
lt 001lowastlowastlowast
119875
lt 0001
00
05
10
15
20
relat
ive e
xpre
ssio
n le
vel
Righ
t Ble
ft B
Npa
s4
Naıve CS + UCS PSEUDO CS
lowastlowastlowastlowast
lowastlowast
Figure 4 Changes in Npas4 expression induced by training Intrained (CS + UCS) animals expression of Npas4 in the right(trained) row B of barrels is elevated in comparison with left(control unstimulated) row B Sole stimulation of whiskers (CSonly) and application of unpaired CS and UCS (PSEUDO) do notproduce increase in Npas4 expression in comparison with controlside Data are presented as ratio of Npas4 expression level in rightand left row B of barrels mean plusmn SEM lowastlowast
119875
lt 001
expression was increased in the ldquotrainedrdquo row in every singleconditioned animal (Figure 5)
000
005
010
015
020
025
030
035
040
045
050
Npa
s4 re
lativ
e exp
ress
ion
leve
l
Trained 1
Trained 2
Trained 3
Trained 4
Trained 5
Trained 6
Trained 7
Trained 8
Trained 9
L R
Figure 5 Changes of Npas4 mRNA expression in individualconditionedmicemdashcomparison between trained rowB of barrels (Rright) and control row B (L left)
6 Neural Plasticity
00
01
02
03
Npa
s4 re
lativ
e exp
ress
ion
leve
l
Left B Right D Right B
lowastlowast
(a)
00
05
10
15
c-Fo
s rela
tive e
xpre
ssio
n le
vel
Left B Right D Right B
lowast
(b)
Figure 6 Training induced changes in Npas4 (a) and c-Fos (b) expression Right B corresponds to the trained row of barrels right D tocontrol row of barrels in the same hemisphere and left B to control row B in the other hemisphere Npas4 and c-Fos have similar pattern ofexpression following the training procedure their expression is elevated in the trained row B in comparison with the control side mean plusmnSEM lowast119875
lt 005 lowastlowast119875
lt 001
To see if the observed changes in Npas4 expression arelimited to the stimulated row B barrels we evaluated the levelof its expression in row D barrels in the same hemisphereWe did not assess Npas4 mRNA level in row D of the left(control) hemisphere in the CS + UCS group but taking intoaccount results from the naıve group it can be presumedthat it is comparable to the control row B The level ofNpas4 expression in the right row D was an intermediatevalue between the levels of Npas4 mRNA expression in theright (trained) row B and left (control) row B it was notsignificantly different from any of the B rows (Figure 6(a))It can be interpreted that training affects row D so there areno differences between trained row B and row D in the samehemisphere but the impact of training upon rowD is weak sono differences can be observed when compared to the controlhemisphere
In the deprivation studyNpas4 turned out to have a differ-ent pattern of expression than other activity-regulated genesand we wondered if it is also the case in sensory conditioningWe evaluated c-Fos mRNA levels in samples obtained fromCS + UCS mice The pattern of c-Fos expression was thesame as for Npas4 (Figure 6(b)) The training procedureevoked differences among c-Fos expressions in examinedbarrel rows (ANOVA 119865(2 24) = 4407 119875 = 00299) c-Fos mRNA expression level was 484 higher in the trainedrow of barrels (row B in the right hemisphere) than in thecontralateral row B (in the left hemisphere 119875 lt 005)
4 Discussion
Knowledge on Npas4 in plasticity of adult cortex is limitedHerein we provide first description of Npas4 expression in
the barrel cortex undergoing plastic reorganization in twoparadigms deprivation and sensory conditioning We alsocompare changes in expression of Npas4 and c-Fos in bothexperimental models
We found that classical conditioning in which stimula-tion of a row of whiskers is paired with tail shock results inincreased expression of Npas4 mRNA in the cognate row ofcortical barrels As conditioning triggers inhibitory synapto-genesis in the barrels representing stimulated vibrissae [33]our results are in agreement with data demonstrating a roleof Npas4 in the formation of GABAergic synapses The workof Lin et al [3] found that Npas4 regulates expression ofa variety of genes including gene coding for BDNF Npas4binds to activity-dependent promoters I and IV of the BDNFgene [3] and expression of the BDNF gene from promoterIV contributes to the plasticity of inhibitory synapses [3839] Npas4 expression was demonstrated to drive inhibitorysynaptogenesis on excitatory neurons andNpas4 knockdownincreases interevent interval and decreases the amplitudeof mIPSCs [3] In the current experiment we observedan increase in Npas4 expression within the trained rowwhere previously we found increases of GABAergic synapsesdensity increased synaptic content of GABA and increasedspontaneous IPSCs [33 40] Our findings are also in linewith the recent results by Sim et al [4] who found thatincreased cell intrinsic activity results in via an Npas4dependent mechanism the addition of GABAergic inputsto the neuron In hippocampal pyramidal neurons behav-iorally induced expression of Npas4 drives redistribution ofinhibitory synapses increasing inhibitory synapse numberon the cell body while decreasing the number of inhibitorysynapses on the apical dendrites [5] In contrast in our
Neural Plasticity 7
previous experiments we observed an increase in density ofGABAergic synapses located on spines in barrels representingldquotrainedrdquo vibrissae [33] This discrepancy could be attributedto possibility that Npas4 impact on dendritic and somaticpool of GABAergic synapses depends on the type of neuronits location within the brain and the type of stimulation usedto evoke plasticity
Interestingly it was recently found that increased Npas4expression may also account for increased number of exci-tatory contacts made onto somatostatin interneurons [6] Inthe CS + UCS mice an increase in density of somatostatininterneurons within layer 4 of the barrel cortex is observed[41] which may be a result of elevated neuronal activity[42] It can be hypothesized that Npas4 expression drivesgeneration of additional excitatory input onto somatostatininterneurons which in turn express higher level of somato-statin
Herein we demonstrate that sensory conditioning pro-duces increase in Npas4 expression in the region whereinhibitory synaptogenesis was previously observed [33]Synapses formation and elimination accompanying learning-related behaviors may contribute to shift in excitation-inhibition balance Dysregulation of this balance has beenimplicated in number of human neuropsychiatric and neu-rodegenerative disorders and associated with impairmentof cognitive functions [43] Regarding Npas4 significancefor maintaining excitation-inhibition equilibrium it can bepresumed that Npas4 deficiency would result in cognitivedeficits Indeed such deficits were observed in Npas4 knock-out mice [44 45] So far Npas4 has not been directly linkedto human neuropsychiatric disorders However Bersten et al[46] identified human variants of Npas4 with reduced tran-scriptional activity so sequencing Npas4 in neuropsychiatricpatients might be helpful in detecting such a link if it exists
Previous experiments regarding fear conditioning andcontextual learning indicate that Npas4 is indispensable formemory formation [11 12]Therefore it is reasonable to thinkthat Npas4 deficiency should also impair sensory condition-ing Testing this hypothesis using Npas4 knockout animalsmight be misleading Npas4 knockout animals performedwell in amygdala-dependent fear conditioning [11] whileacute deletion of Npas4 in amygdala impaired fear memoryformation [12] Regionally selective depletion of Npas4mightbe more useful then but first structures required for learningassociation of sensory CS and UCS in paradigm used in thisstudy should be identified Basolateral amygdala is activatedduring the training procedure [37] and amygdala is requiredfor all forms of fear conditioning [47] therefore amygdalawould seem a first choice structure for local knockdown ofNpas4 and determining its impact on sensory conditioning
Npas4 expression in remote row D barrels did notdiffer from the results obtained for the control hemisphereand naıve group which is in agreement with our previousobservation that no changes typical for the trained row Bappear in remote row D (no AMPA and NMDA bindingincrease no GAD mRNA expression upregulation and noincrease in density of somatostatin containing inhibitoryinterneurons) [40 41 48 49] We did not do EM to evaluatethe synapse density of remote row D barrels but we found
no increase in spontaneous IPSC there which would indicateincreased GABA release in its excitatory neurons [40]
In the sensory conditioning paradigm Npas4 and c-Foslevels changed in the same wayThey were upregulated in therow of barrels that received the conditioned input
Depriving selected barrel rows of sensory input resultedin decreased Npas4 mRNA expression in the deprivedbarrel rows in comparison with spared rows Duration ofdeprivation (24 h versus 7 days) had no impact upon Npas4expression in spared rows and deprived rowsThis contrastedwith c-Fos the expression of which was increased in bothdeprived and spared regions after 7 days of deprivation incomparison with 24 hours of deprivation
Using immunohistochemical techniques we previouslydescribed the pattern of c-Fos protein expression followingwhisker deprivation of various durations [18] in the samedeprivation paradigm The density of c-Fos positive nucleiincreased in the barrel row deprived of whisker input for 7days and the effect was augmented as the deprivation periodwas prolonged We interpreted this result as illustrating theexpansion of the spared vibrissal input into neighboringfunctionally deafferented barrels The gradual increase in thenumber of immunoreactive nuclei could reflect the gradualrewiring of the barrel cortex in the course of prolongeddeprivation The present results confirm our previous data atthe mRNA level
Unlike c-Fos Npas4mRNA in the deprived B andD rowswas not upregulated by sensory stimulation when comparing24 h versus 7 days of deprivation We suppose that thestrength of the sensory signal coming from the spared row Cwhiskers was insufficient for changing the expression of thisIEG In the experimental paradigm used here we previouslyobserved pronounced changes in Zif268 and Arc expression[36] It is not surprising that differently regulated genes donot respond identically to an experimental situation (see[35] for comparison of several IEGs activations by enrichedenvironment) Perhaps the plastic rearrangement of connec-tions induced by deprivation although it already changesthe metabolic response [18] does not yet trigger activationin Npas4 mRNA expression Npas4 can regulate activity-dependent expression of Arc c-Fos and Zif268 [11] Takinginto consideration that Arc c-Fos and Zif268 expressionsincrease in deprived barrels after 7 days of deprivation it mayseem puzzling that expression of Npas4 which regulates thetranscription of these genes remains unaltered However itshould be noted that Npas4 itself is an immediate early geneand gets activated in response to stimulation Accordingly itprobably does not regulate the first phase of other immediateearly genes expression which is independent of de novoprotein synthesis It rather seems that Npas4 plays a rolein enhancing and sustaining other immediate early genes inlater phases [11]
To the best of our knowledge this is the first report onNpas4 expression in deprivation-induced plasticity Maya-Vetencourt et al [13] examined Npas4 expression in monoc-ularly deprived rats treated with fluoxetine but they concen-trated on influence of fluoxetine onNpas4 expression and noton the influence of deprivation
8 Neural Plasticity
In this paper the precise anatomical dissection of arow of barrels where a plastic change took place allowedfor a quantitative analysis of IEGs expression in two typesof experience dependent plasticity Activation of the barrelcortex undergoing reorganization triggered by removal ofselected rows of whiskers strongly affected c-Fos (but notNpas4) expression Activation of the cortex undergoing aplastic change due to being involved in sensory conditioningupregulated the expression of both Npas4 and c-Fos Takinginto consideration our observations that sensory condition-ing increases the number of inhibitory synapses withinthe trained barrels and studies of other groups showinginvolvement of Npas4 in synaptogenesis we presume thatNpas4 may be involved in reshaping of connectivity withinbarrel cortex after sensory conditioning
Conflict of Interests
The authors declare no conflict of interests
Acknowledgment
This project was supported by GrantMNiSWno 2486BP012010 to Malgorzata Kossut
References
[1] M Moser R Knoth C Bode and C Patterson ldquoLE-PAS anovel Arnt-dependent HLH-PAS protein is expressed in limbictissues and transactivates the CNS midline enhancer elementrdquoMolecular Brain Research vol 128 no 2 pp 141ndash149 2004
[2] N Ooe K Saito N Mikami I Nakatuka and H KanekoldquoIdentification of a novel basic helix-loop-helix-PAS factorNXF reveals a Sim2 competitive positive regulatory role indendritic-cytoskeleton modulator drebrin gene expressionrdquoMolecular and Cellular Biology vol 24 no 2 pp 608ndash616 2004
[3] Y Lin B L Bloodgood J L Hauser et al ldquoActivity-dependentregulation of inhibitory synapse development by Npas4rdquoNature vol 455 no 7217 pp 1198ndash1204 2008
[4] S Sim S Antolin C-W Lin Y-X Lin and C Lois ldquoIncreasedcell-intrinsic excitability induces synaptic changes in new neu-rons in the adult dentate gyrus that require Npas4rdquo Journal ofNeuroscience vol 33 no 18 pp 7928ndash7940 2013
[5] B L Bloodgood N Sharma H A Browne A Z Trepman andM E Greenberg ldquoThe activity-dependent transcription factorNPAS4 regulates domain-specific inhibitionrdquo Nature vol 503no 7474 pp 121ndash125 2013
[6] I Spiegel A R Mardinly H W Gabel et al ldquoNpas4 regulatesexcitatory-inhibitory balance within neural circuits throughcell-type-specific gene programsrdquo Cell vol 157 no 5 pp 1216ndash1229 2014
[7] J Yun T Nagai Y Furukawa-Hibi et al ldquoNeuronal per arnt sim(PAS) domain protein 4 (NPAS4) regulates neurite outgrowthand phosphorylation of synapsin Irdquo The Journal of BiologicalChemistry vol 288 no 4 pp 2655ndash2664 2013
[8] G Pouchelon F Gambino C Bellone et al ldquoModality-specificthalamocortical inputs instruct the identity of postsynaptic L4neuronsrdquo Nature vol 511 no 7510 pp 471ndash474 2014
[9] S Yoshihara H Takahashi N Nishimura et al ldquoNpas4 regu-lates Mdm2 and thus Dcx in experience-dependent dendritic
spine development of newborn olfactory bulb interneuronsrdquoCell Reports vol 8 no 3 pp 843ndash857 2014
[10] K Hayashi R Ishikawa L-H Ye et al ldquoModulatory role ofdrebrin on the cytoskeleton within dendritic spines in the ratcerebral cortexrdquo Journal of Neuroscience vol 16 no 22 pp 7161ndash7170 1996
[11] K Ramamoorthi R Fropf GM Belfort et al ldquoNpas4 regulatesa transcriptional program in CA3 required for contextualmemory formationrdquo Science vol 334 no 6063 pp 1669ndash16752011
[12] J E Ploski M S Monsey T Nguyen R J DiLeone and G ESchafe ldquoThe neuronal PAS domain protein 4 (Npas4) isrequired for new and reactivated fear memoriesrdquo PLoS ONEvol 6 no 8 Article ID e23760 2011
[13] JFMaya-Vetencourt E Tiraboschi DGreco et al ldquoExperience-dependent expression of NPAS4 regulates plasticity in adultvisual cortexrdquo Journal of Physiology vol 590 no 19 pp 4777ndash4787 2012
[14] M Kossut P J Hand J Greenberg and C L Hand ldquoSinglevibrissal cortical column in SI cortex of rat and its alterations inneonatal and adult vibrissa-deafferented animals a quantitative2DG studyrdquo Journal of Neurophysiology vol 60 no 2 pp 829ndash852 1988
[15] S Glazewski andK Fox ldquoTime course of experience-dependentsynaptic potentiation and depression in barrel cortex of adoles-cent ratsrdquo Journal of Neurophysiology vol 75 no 4 pp 1714ndash1729 1996
[16] H Wallace and K Fox ldquoLocal cortical interactions determinethe form of cortical plasticityrdquo Journal of Neurobiology vol 41no 1 pp 58ndash63 1999
[17] M A Lebedev G Mirabella I Erchova and M E DiamondldquoExperience-dependent plasticity of rat barrel cortex redistri-bution of activity across barrel-columnsrdquo Cerebral Cortex vol10 no 1 pp 23ndash31 2000
[18] A Kaliszewska M Bijata L Kaczmarek and M KossutldquoExperience-dependent plasticity of the barrel cortex in miceobserved with 2-DG brain mapping and c-Fos effects of MMP-9 KOrdquo Cerebral Cortex vol 22 no 9 pp 2160ndash2170 2012
[19] D J Margolis H Lutcke K Schulz et al ldquoReorganization ofcortical population activity imaged throughout long-term sen-sory deprivationrdquo Nature Neuroscience vol 15 no 11 pp 1539ndash1546 2012
[20] S AMarikH Yamahachi J N JMcManusG Szabo andCDGilbert ldquoAxonal dynamics of excitatory and inhibitory neuronsin somatosensory cortexrdquo PLoS Biology vol 8 no 6 Article IDe1000395 2010
[21] V C Wimmer P J Broser T Kuner and R M BrunoldquoExperience-induced plasticity of thalamocortical axons inboth juveniles and adultsrdquo Journal of Comparative Neurologyvol 518 no 22 pp 4629ndash4648 2010
[22] M Oberlaender A Ramirez and R M Bruno ldquoSensory expe-rience restructures thalamocortical axons during adulthoodrdquoNeuron vol 74 no 4 pp 648ndash655 2012
[23] D Katzel and G Miesenbock ldquoExperience-dependent rewiringof specific inhibitory connections in adult neocortexrdquo PLoSBiology vol 12 no 2 Article ID e1001798 2014
[24] C Tailby L LWright A BMetha andM B Calford ldquoActivity-dependent maintenance and growth of dendrites in adultcortexrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 102 no 12 pp 4631ndash4636 2005
Neural Plasticity 9
[25] C E J Cheetham M S L Hammond R McFarlane and G TFinnerty ldquoAltered sensory experience induces targeted rewiringof local excitatory connections in mature neocortexrdquo Journal ofNeuroscience vol 28 no 37 pp 9249ndash9260 2008
[26] K D Micheva and C Beaulieu ldquoAn anatomical substrate forexperience-dependent plasticity of the rat barrel field cortexrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 25 pp 11834ndash11838 1995
[27] Y Zuo G Yang E Kwon and W-B Gan ldquoLong-term sen-sory deprivation prevents dendritic spine loss in primarysomatosensory cortexrdquo Nature vol 436 no 7048 pp 261ndash2652005
[28] A Holtmaat L Wilbrecht G W Knott E Welker and KSvoboda ldquoExperience-dependent and cell-type-specific spinegrowth in the neocortexrdquo Nature vol 441 no 7096 pp 979ndash983 2006
[29] C E J Cheetham M S L Hammond C E J Edwards and GT Finnerty ldquoSensory experience alters cortical connectivity andsynaptic function site specificallyrdquo The Journal of Neurosciencevol 27 no 13 pp 3456ndash3465 2007
[30] L Wilbrecht A Holtmaat N Wright K Fox and K Svo-boda ldquoStructural plasticity underlies experience-dependentfunctional plasticity of cortical circuitsrdquo Journal of Neurosciencevol 30 no 14 pp 4927ndash4932 2010
[31] C E J Cheetham S J Barnes G Albieri G W Knott and GT Finnerty ldquoPansynaptic enlargement at adult cortical connec-tions strengthened by experiencerdquo Cerebral Cortex vol 24 no2 pp 521ndash531 2014
[32] E Siucinska and M Kossut ldquoShort-lasting classical condition-ing induces reversible changes of representational maps ofvibrissae inmouse SI cortex-A 2DG studyrdquoCerebral Cortex vol6 no 3 pp 506ndash513 1996
[33] M Jasinska E Siucinska A Cybulska-Klosowicz et al ldquoRapidlearning-induced inhibitory synaptogenesis in murine barrelfieldrdquo The Journal of Neuroscience vol 30 no 3 pp 1176ndash11842010
[34] R N Strominger and T AWoolsey ldquoTemplates for locating thewhisker area in fresh flattened mouse and rat cortexrdquo Journal ofNeuroscience Methods vol 22 no 2 pp 113ndash118 1987
[35] A Valles A J Boender S Gijsbers R A M Haast G J MMartens and P de Weerd ldquoGenomewide analysis of rat barrelcortex reveals time- and layer-specificmrna expression changesrelated to experience-dependent plasticityrdquo Journal of Neuro-science vol 31 no 16 pp 6140ndash6158 2011
[36] A Kaliszewska Expression of selected immediate early genesand involvement of matrix metalloproteinase 9 in functionalreorganization of mouse barrel cortex [Doctoral dissertation]Nencki Institute of Experimental Biology Polish Academy ofSciences Warsaw Poland 2012
[37] A Cybulska-Klosowicz R Zakrzewska and M Kossut ldquoBrainactivation patterns during classical conditioning with appetitiveor aversive UCSrdquo Behavioural Brain Research vol 204 no 1 pp102ndash111 2009
[38] B Lu K H Wang and A Nose ldquoMolecular mechanismsunderlying neural circuit formationrdquo Current Opinion in Neu-robiology vol 19 no 2 pp 162ndash167 2009
[39] Y Jiao Z Zhang C Zhang et al ldquoA key mechanism under-lying sensory experience-dependent maturation of neocorti-cal GABAergic circuits in vivordquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 108 no29 pp 12131ndash12136 2011
[40] K Tokarski J Urban-Ciecko M Kossut and G Hess ldquoSensorylearning-induced enhancement of inhibitory synaptic trans-mission in the barrel cortex of the mouserdquo European Journal ofNeuroscience vol 26 no 1 pp 134ndash141 2007
[41] A Cybulska-Klosowicz A Posluszny K Nowak E SiucinskaM Kossut and M Liguz-Lecznar ldquoInterneurons containingsomatostatin are affected by learning-induced cortical plastic-ityrdquo Neuroscience vol 254 pp 18ndash25 2013
[42] Z-H Hou and X Yu ldquoActivity-regulated somatostatin expres-sion reduces dendritic spine density and lowers excitatorysynaptic transmission via postsynaptic somatostatin receptor 4rdquoJournal of Biological Chemistry vol 288 no 4 pp 2501ndash25092013
[43] S A Eichler and J CMeier ldquoE-I balance and human diseasesmdashfrom molecules to networkingrdquo Frontiers in Molecular Neuro-science vol 1 article 2 2008
[44] L Coutellier S Beraki P M Ardestani N L Saw and MShamloo ldquoNpas4 a neuronal transcription factor with a keyrole in social and cognitive functions relevant to developmentaldisordersrdquo PLoS ONE vol 7 no 9 Article ID e46604 2012
[45] E J Jaehne T S Klaric S A Koblar B T Baune and MD Lewis ldquoEffects of Npas4 deficiency on anxiety depression-like cognition and sociability behaviourrdquo Behavioural BrainResearch vol 281 pp 276ndash282 2015
[46] D C Bersten J B Bruning D J Peet and M L WhitelawldquoHuman variants in the neuronal basic helix-loop-helixPer-Arnt-Sim (bHLHPAS) transcription factor complex NPAS4ARNT2 disrupt functionrdquo PLoS ONE vol 9 no 1 Article IDe85768 2014
[47] S Maren ldquoPavlovian fear conditioning as a behavioral assayfor hippocampus and amygdala function cautions and caveatsrdquoEuropean Journal of Neuroscience vol 28 no 8 pp 1661ndash16662008
[48] B Jabłonska M Kossut and J Skangiel-Kramska ldquoTransientincrease of AMPA and NMDA receptor binding in the barrelcortex of mice after tactile stimulationrdquo Neurobiology of Learn-ing and Memory vol 66 no 1 pp 36ndash43 1996
[49] M Gierdalski B Jablonska E SiucinskaM Lech A Skibinskaand M Kossut ldquoRapid regulation of GAD67 mRNA and pro-tein level in cortical neurons after sensory learningrdquo CerebralCortex vol 11 no 9 pp 806ndash815 2001
a PicoPure RNA Isolation Kit (KIT0204 Life Technologies)with concurrent genomic DNA elimination using DNase(RNase-Free DNase Set 79254 Qiagen)
24 Real-Time PCR Reversed transcription was performedusing aMaxima First Strand cDNASynthesisKit (K1641ThermoScientific-Fermentas) Real-time PCR was conducted usingPower SYBR Green PCR Master Mix (4368702 Life Tech-nologies) In real-time PCR experiment each sample wasrun in triplicate Amplification was carried out with a 7500real-time PCR System (Applied Biosystems) using PowerSYBR Green PCR Master Mix specific primers (Table 1)and cDNA for each sample The glyceraldehyde 3-phosphatedehydrogenase (GAPDH) gene was used as a housekeepinggene The amplification reaction was cycled 40 times witha 95∘C denaturation step for 15 s and a 60∘C annealing stepfor 1 minute A dissociation stage was performed to assessspecificity of primers Results were calculated using standardcurve method
25 Data Analysis and Statistics Ratios of a target gene andhousekeeping gene levels were used for analysis Statisticalanalysis was performed using GraphPad Prism 5 software(GraphPad Software Inc) Gene expression levels in differ-ent barrel rows were analyzed using ANOVA followed byNewman-Keuls post hoc tests Studentrsquos 119905-test was usedwhereapplicable
3 Results
31 Sensory Deprivation
311 Npas4 and c-Fos Mice were subjected to one week ofsensory deprivationmdashall vibrissae on one side of the snoutwere removed except for row C while vibrissae on the otherside were left intact On the day preceding stimulation theother side of the snout was subjected to the same procedureso that mice were left with two C rows intact Animalswere placed in the stimulatory cage for 30 minutesmdasha timeinterval demonstrated to be appropriate to observe increasein Npas4 expression in the barrel cortex after explorationof an enriched environment [35] Regions of interest weremicrodissected from slices of layer IV Real-timePCRmethodwas used to assess Npas4 mRNA level in individual barrel
rows Npas4 level was evaluated in spared rows C and indeprived rows B and D in the experimental and controlhemisphere
Elimination of sensory input to selected rows of vibrissaeevoked differences in Npas4 expression between spared anddeprived barrels (ANOVA 119865(318) = 1004 119875 = 00004Figure 3(a)) Post hoc analysis demonstrated that the levelof Npas4 transcript in the control hemisphere was 484lower in deprived rows B and D than in the spared row C(035 plusmn 005 versus 067 plusmn 006 119875 lt 001) There were nodifferences in Npas4 transcript levels between spared rowsC in both hemispheres (119875 gt 005) Also no differenceswere observed between deprived regions in both hemispheres(119875 gt 005) which shows that duration of deprivation (7days versus 24 hours) had no impact on Npas4 expressionin deprived barrel rows We have previously shown that thisdeprivation procedure followed by two hours of explorationof stimulatory cage resulted in an increased number of cellsexpressing c-Fos in deprived rows B and D after 7 daysof deprivation in comparison with 24 hours of deprivation[18] We also observed this phenomenon regarding the otheractivity-regulated genes Arc and Zif268 [36] As Npas4 isalso an activity-regulated gene we expected a similar patternof its expression As this was not the case we decided toassess c-Fos mRNA in the same samples to confirm thatthe Npas4 pattern of expression is atypical of other activity-regulated genes in this model of plasticity The deprivationprocedure produced differences in c-Fos expression amonganalyzed barrel rows (ANOVA 119865(316) = 8028 119875 = 00022Figure 3(b)) In the experimental hemisphere the level ofc-Fos mRNA was 742 higher in deprived rows B and D(138 plusmn 020 versus 079 plusmn 009 119875 lt 005) than in homotypicregions in the control hemisphere
32 Sensory Conditioning The training procedure encom-passed three training sessions on three consecutive dayseach lasting for 10 minutes and consisting of 40 CS (strokingrow B of vibrissae) and UCS (electric shock to the tail)pairings This paradigm evokes freezing-like behavior whichindicates that association of CS and UCS occurred [37] InCS + UCS animals Npas4 expression was on average 529higher in trained row B barrels than in the contralateral rowB (024 plusmn 003 versus 015 plusmn 003 119905-test 119875 lt 0001) We foundno differences in Npas4 expression between hemispheresin naıve animals and for further analysis we pooled datafrom both B rows Npas4 expression in the trained row Bfor the CS + UCS group was 481 higher than in naıvegroup B rows (119905-test 119875 lt 005) There were no differencesbetween B rows in the naıve group and left (control) rowB in the CS + UCS group (119905-test 119875 gt 005) Analysis ofratios of Npas4 expression in right row B (trained row inCS + UCS group) and left row B revealed significant effectof the training procedure (ANOVA 119865(325) = 6780 119875 =00017 Figure 4) Ratio of Npas4 expression in right row Band left row B was higher in CS + UCS group in comparisonwith other experimental groups (119875 lt 001) indicating thatinterhemispheric difference in Npas4 mRNA level is not justan effect of stimulation of the row B of vibrissae Npas4
Neural Plasticity 5
00
02
04
06
08
Npa
s4 re
lativ
e exp
ress
ion
leve
l
Con
trolC
Exp
C
Con
trolB
D
Exp
BD
lowastlowastlowastlowast
lowastlowast
(a)
0
1
2
3
c-Fo
s rela
tive e
xpre
ssio
n le
vel
Con
trolC
Exp
C
Con
trolB
D
Exp
BD
lowastlowastlowast
lowastlowast
lowastlowast
lowast
lowast
(b)
Figure 3 (a) Npas4 expression in spared C rows and in deprived regions (averaged rows B and D) Control C spared row C on controlside (deprived for 24 hours) Exp C spared row C on experimental side (deprived 7 days) Control BD deprived B and D rows on controlside Exp BD deprived B and D rows on experimental side mean plusmn SEM lowastlowast
119875
lt 001 Duration of deprivation had no impact on Npas4expression (b) In deprived B and D rows deprivation lasting 7 days induced increase in c-Fos expression mean plusmn SEM lowast
119875
lt 005 lowastlowast119875
lt 001lowastlowastlowast
119875
lt 0001
00
05
10
15
20
relat
ive e
xpre
ssio
n le
vel
Righ
t Ble
ft B
Npa
s4
Naıve CS + UCS PSEUDO CS
lowastlowastlowastlowast
lowastlowast
Figure 4 Changes in Npas4 expression induced by training Intrained (CS + UCS) animals expression of Npas4 in the right(trained) row B of barrels is elevated in comparison with left(control unstimulated) row B Sole stimulation of whiskers (CSonly) and application of unpaired CS and UCS (PSEUDO) do notproduce increase in Npas4 expression in comparison with controlside Data are presented as ratio of Npas4 expression level in rightand left row B of barrels mean plusmn SEM lowastlowast
119875
lt 001
expression was increased in the ldquotrainedrdquo row in every singleconditioned animal (Figure 5)
000
005
010
015
020
025
030
035
040
045
050
Npa
s4 re
lativ
e exp
ress
ion
leve
l
Trained 1
Trained 2
Trained 3
Trained 4
Trained 5
Trained 6
Trained 7
Trained 8
Trained 9
L R
Figure 5 Changes of Npas4 mRNA expression in individualconditionedmicemdashcomparison between trained rowB of barrels (Rright) and control row B (L left)
6 Neural Plasticity
00
01
02
03
Npa
s4 re
lativ
e exp
ress
ion
leve
l
Left B Right D Right B
lowastlowast
(a)
00
05
10
15
c-Fo
s rela
tive e
xpre
ssio
n le
vel
Left B Right D Right B
lowast
(b)
Figure 6 Training induced changes in Npas4 (a) and c-Fos (b) expression Right B corresponds to the trained row of barrels right D tocontrol row of barrels in the same hemisphere and left B to control row B in the other hemisphere Npas4 and c-Fos have similar pattern ofexpression following the training procedure their expression is elevated in the trained row B in comparison with the control side mean plusmnSEM lowast119875
lt 005 lowastlowast119875
lt 001
To see if the observed changes in Npas4 expression arelimited to the stimulated row B barrels we evaluated the levelof its expression in row D barrels in the same hemisphereWe did not assess Npas4 mRNA level in row D of the left(control) hemisphere in the CS + UCS group but taking intoaccount results from the naıve group it can be presumedthat it is comparable to the control row B The level ofNpas4 expression in the right row D was an intermediatevalue between the levels of Npas4 mRNA expression in theright (trained) row B and left (control) row B it was notsignificantly different from any of the B rows (Figure 6(a))It can be interpreted that training affects row D so there areno differences between trained row B and row D in the samehemisphere but the impact of training upon rowD is weak sono differences can be observed when compared to the controlhemisphere
In the deprivation studyNpas4 turned out to have a differ-ent pattern of expression than other activity-regulated genesand we wondered if it is also the case in sensory conditioningWe evaluated c-Fos mRNA levels in samples obtained fromCS + UCS mice The pattern of c-Fos expression was thesame as for Npas4 (Figure 6(b)) The training procedureevoked differences among c-Fos expressions in examinedbarrel rows (ANOVA 119865(2 24) = 4407 119875 = 00299) c-Fos mRNA expression level was 484 higher in the trainedrow of barrels (row B in the right hemisphere) than in thecontralateral row B (in the left hemisphere 119875 lt 005)
4 Discussion
Knowledge on Npas4 in plasticity of adult cortex is limitedHerein we provide first description of Npas4 expression in
the barrel cortex undergoing plastic reorganization in twoparadigms deprivation and sensory conditioning We alsocompare changes in expression of Npas4 and c-Fos in bothexperimental models
We found that classical conditioning in which stimula-tion of a row of whiskers is paired with tail shock results inincreased expression of Npas4 mRNA in the cognate row ofcortical barrels As conditioning triggers inhibitory synapto-genesis in the barrels representing stimulated vibrissae [33]our results are in agreement with data demonstrating a roleof Npas4 in the formation of GABAergic synapses The workof Lin et al [3] found that Npas4 regulates expression ofa variety of genes including gene coding for BDNF Npas4binds to activity-dependent promoters I and IV of the BDNFgene [3] and expression of the BDNF gene from promoterIV contributes to the plasticity of inhibitory synapses [3839] Npas4 expression was demonstrated to drive inhibitorysynaptogenesis on excitatory neurons andNpas4 knockdownincreases interevent interval and decreases the amplitudeof mIPSCs [3] In the current experiment we observedan increase in Npas4 expression within the trained rowwhere previously we found increases of GABAergic synapsesdensity increased synaptic content of GABA and increasedspontaneous IPSCs [33 40] Our findings are also in linewith the recent results by Sim et al [4] who found thatincreased cell intrinsic activity results in via an Npas4dependent mechanism the addition of GABAergic inputsto the neuron In hippocampal pyramidal neurons behav-iorally induced expression of Npas4 drives redistribution ofinhibitory synapses increasing inhibitory synapse numberon the cell body while decreasing the number of inhibitorysynapses on the apical dendrites [5] In contrast in our
Neural Plasticity 7
previous experiments we observed an increase in density ofGABAergic synapses located on spines in barrels representingldquotrainedrdquo vibrissae [33] This discrepancy could be attributedto possibility that Npas4 impact on dendritic and somaticpool of GABAergic synapses depends on the type of neuronits location within the brain and the type of stimulation usedto evoke plasticity
Interestingly it was recently found that increased Npas4expression may also account for increased number of exci-tatory contacts made onto somatostatin interneurons [6] Inthe CS + UCS mice an increase in density of somatostatininterneurons within layer 4 of the barrel cortex is observed[41] which may be a result of elevated neuronal activity[42] It can be hypothesized that Npas4 expression drivesgeneration of additional excitatory input onto somatostatininterneurons which in turn express higher level of somato-statin
Herein we demonstrate that sensory conditioning pro-duces increase in Npas4 expression in the region whereinhibitory synaptogenesis was previously observed [33]Synapses formation and elimination accompanying learning-related behaviors may contribute to shift in excitation-inhibition balance Dysregulation of this balance has beenimplicated in number of human neuropsychiatric and neu-rodegenerative disorders and associated with impairmentof cognitive functions [43] Regarding Npas4 significancefor maintaining excitation-inhibition equilibrium it can bepresumed that Npas4 deficiency would result in cognitivedeficits Indeed such deficits were observed in Npas4 knock-out mice [44 45] So far Npas4 has not been directly linkedto human neuropsychiatric disorders However Bersten et al[46] identified human variants of Npas4 with reduced tran-scriptional activity so sequencing Npas4 in neuropsychiatricpatients might be helpful in detecting such a link if it exists
Previous experiments regarding fear conditioning andcontextual learning indicate that Npas4 is indispensable formemory formation [11 12]Therefore it is reasonable to thinkthat Npas4 deficiency should also impair sensory condition-ing Testing this hypothesis using Npas4 knockout animalsmight be misleading Npas4 knockout animals performedwell in amygdala-dependent fear conditioning [11] whileacute deletion of Npas4 in amygdala impaired fear memoryformation [12] Regionally selective depletion of Npas4mightbe more useful then but first structures required for learningassociation of sensory CS and UCS in paradigm used in thisstudy should be identified Basolateral amygdala is activatedduring the training procedure [37] and amygdala is requiredfor all forms of fear conditioning [47] therefore amygdalawould seem a first choice structure for local knockdown ofNpas4 and determining its impact on sensory conditioning
Npas4 expression in remote row D barrels did notdiffer from the results obtained for the control hemisphereand naıve group which is in agreement with our previousobservation that no changes typical for the trained row Bappear in remote row D (no AMPA and NMDA bindingincrease no GAD mRNA expression upregulation and noincrease in density of somatostatin containing inhibitoryinterneurons) [40 41 48 49] We did not do EM to evaluatethe synapse density of remote row D barrels but we found
no increase in spontaneous IPSC there which would indicateincreased GABA release in its excitatory neurons [40]
In the sensory conditioning paradigm Npas4 and c-Foslevels changed in the same wayThey were upregulated in therow of barrels that received the conditioned input
Depriving selected barrel rows of sensory input resultedin decreased Npas4 mRNA expression in the deprivedbarrel rows in comparison with spared rows Duration ofdeprivation (24 h versus 7 days) had no impact upon Npas4expression in spared rows and deprived rowsThis contrastedwith c-Fos the expression of which was increased in bothdeprived and spared regions after 7 days of deprivation incomparison with 24 hours of deprivation
Using immunohistochemical techniques we previouslydescribed the pattern of c-Fos protein expression followingwhisker deprivation of various durations [18] in the samedeprivation paradigm The density of c-Fos positive nucleiincreased in the barrel row deprived of whisker input for 7days and the effect was augmented as the deprivation periodwas prolonged We interpreted this result as illustrating theexpansion of the spared vibrissal input into neighboringfunctionally deafferented barrels The gradual increase in thenumber of immunoreactive nuclei could reflect the gradualrewiring of the barrel cortex in the course of prolongeddeprivation The present results confirm our previous data atthe mRNA level
Unlike c-Fos Npas4mRNA in the deprived B andD rowswas not upregulated by sensory stimulation when comparing24 h versus 7 days of deprivation We suppose that thestrength of the sensory signal coming from the spared row Cwhiskers was insufficient for changing the expression of thisIEG In the experimental paradigm used here we previouslyobserved pronounced changes in Zif268 and Arc expression[36] It is not surprising that differently regulated genes donot respond identically to an experimental situation (see[35] for comparison of several IEGs activations by enrichedenvironment) Perhaps the plastic rearrangement of connec-tions induced by deprivation although it already changesthe metabolic response [18] does not yet trigger activationin Npas4 mRNA expression Npas4 can regulate activity-dependent expression of Arc c-Fos and Zif268 [11] Takinginto consideration that Arc c-Fos and Zif268 expressionsincrease in deprived barrels after 7 days of deprivation it mayseem puzzling that expression of Npas4 which regulates thetranscription of these genes remains unaltered However itshould be noted that Npas4 itself is an immediate early geneand gets activated in response to stimulation Accordingly itprobably does not regulate the first phase of other immediateearly genes expression which is independent of de novoprotein synthesis It rather seems that Npas4 plays a rolein enhancing and sustaining other immediate early genes inlater phases [11]
To the best of our knowledge this is the first report onNpas4 expression in deprivation-induced plasticity Maya-Vetencourt et al [13] examined Npas4 expression in monoc-ularly deprived rats treated with fluoxetine but they concen-trated on influence of fluoxetine onNpas4 expression and noton the influence of deprivation
8 Neural Plasticity
In this paper the precise anatomical dissection of arow of barrels where a plastic change took place allowedfor a quantitative analysis of IEGs expression in two typesof experience dependent plasticity Activation of the barrelcortex undergoing reorganization triggered by removal ofselected rows of whiskers strongly affected c-Fos (but notNpas4) expression Activation of the cortex undergoing aplastic change due to being involved in sensory conditioningupregulated the expression of both Npas4 and c-Fos Takinginto consideration our observations that sensory condition-ing increases the number of inhibitory synapses withinthe trained barrels and studies of other groups showinginvolvement of Npas4 in synaptogenesis we presume thatNpas4 may be involved in reshaping of connectivity withinbarrel cortex after sensory conditioning
Conflict of Interests
The authors declare no conflict of interests
Acknowledgment
This project was supported by GrantMNiSWno 2486BP012010 to Malgorzata Kossut
References
[1] M Moser R Knoth C Bode and C Patterson ldquoLE-PAS anovel Arnt-dependent HLH-PAS protein is expressed in limbictissues and transactivates the CNS midline enhancer elementrdquoMolecular Brain Research vol 128 no 2 pp 141ndash149 2004
[2] N Ooe K Saito N Mikami I Nakatuka and H KanekoldquoIdentification of a novel basic helix-loop-helix-PAS factorNXF reveals a Sim2 competitive positive regulatory role indendritic-cytoskeleton modulator drebrin gene expressionrdquoMolecular and Cellular Biology vol 24 no 2 pp 608ndash616 2004
[3] Y Lin B L Bloodgood J L Hauser et al ldquoActivity-dependentregulation of inhibitory synapse development by Npas4rdquoNature vol 455 no 7217 pp 1198ndash1204 2008
[4] S Sim S Antolin C-W Lin Y-X Lin and C Lois ldquoIncreasedcell-intrinsic excitability induces synaptic changes in new neu-rons in the adult dentate gyrus that require Npas4rdquo Journal ofNeuroscience vol 33 no 18 pp 7928ndash7940 2013
[5] B L Bloodgood N Sharma H A Browne A Z Trepman andM E Greenberg ldquoThe activity-dependent transcription factorNPAS4 regulates domain-specific inhibitionrdquo Nature vol 503no 7474 pp 121ndash125 2013
[6] I Spiegel A R Mardinly H W Gabel et al ldquoNpas4 regulatesexcitatory-inhibitory balance within neural circuits throughcell-type-specific gene programsrdquo Cell vol 157 no 5 pp 1216ndash1229 2014
[7] J Yun T Nagai Y Furukawa-Hibi et al ldquoNeuronal per arnt sim(PAS) domain protein 4 (NPAS4) regulates neurite outgrowthand phosphorylation of synapsin Irdquo The Journal of BiologicalChemistry vol 288 no 4 pp 2655ndash2664 2013
[8] G Pouchelon F Gambino C Bellone et al ldquoModality-specificthalamocortical inputs instruct the identity of postsynaptic L4neuronsrdquo Nature vol 511 no 7510 pp 471ndash474 2014
[9] S Yoshihara H Takahashi N Nishimura et al ldquoNpas4 regu-lates Mdm2 and thus Dcx in experience-dependent dendritic
spine development of newborn olfactory bulb interneuronsrdquoCell Reports vol 8 no 3 pp 843ndash857 2014
[10] K Hayashi R Ishikawa L-H Ye et al ldquoModulatory role ofdrebrin on the cytoskeleton within dendritic spines in the ratcerebral cortexrdquo Journal of Neuroscience vol 16 no 22 pp 7161ndash7170 1996
[11] K Ramamoorthi R Fropf GM Belfort et al ldquoNpas4 regulatesa transcriptional program in CA3 required for contextualmemory formationrdquo Science vol 334 no 6063 pp 1669ndash16752011
[12] J E Ploski M S Monsey T Nguyen R J DiLeone and G ESchafe ldquoThe neuronal PAS domain protein 4 (Npas4) isrequired for new and reactivated fear memoriesrdquo PLoS ONEvol 6 no 8 Article ID e23760 2011
[13] JFMaya-Vetencourt E Tiraboschi DGreco et al ldquoExperience-dependent expression of NPAS4 regulates plasticity in adultvisual cortexrdquo Journal of Physiology vol 590 no 19 pp 4777ndash4787 2012
[14] M Kossut P J Hand J Greenberg and C L Hand ldquoSinglevibrissal cortical column in SI cortex of rat and its alterations inneonatal and adult vibrissa-deafferented animals a quantitative2DG studyrdquo Journal of Neurophysiology vol 60 no 2 pp 829ndash852 1988
[15] S Glazewski andK Fox ldquoTime course of experience-dependentsynaptic potentiation and depression in barrel cortex of adoles-cent ratsrdquo Journal of Neurophysiology vol 75 no 4 pp 1714ndash1729 1996
[16] H Wallace and K Fox ldquoLocal cortical interactions determinethe form of cortical plasticityrdquo Journal of Neurobiology vol 41no 1 pp 58ndash63 1999
[17] M A Lebedev G Mirabella I Erchova and M E DiamondldquoExperience-dependent plasticity of rat barrel cortex redistri-bution of activity across barrel-columnsrdquo Cerebral Cortex vol10 no 1 pp 23ndash31 2000
[18] A Kaliszewska M Bijata L Kaczmarek and M KossutldquoExperience-dependent plasticity of the barrel cortex in miceobserved with 2-DG brain mapping and c-Fos effects of MMP-9 KOrdquo Cerebral Cortex vol 22 no 9 pp 2160ndash2170 2012
[19] D J Margolis H Lutcke K Schulz et al ldquoReorganization ofcortical population activity imaged throughout long-term sen-sory deprivationrdquo Nature Neuroscience vol 15 no 11 pp 1539ndash1546 2012
[20] S AMarikH Yamahachi J N JMcManusG Szabo andCDGilbert ldquoAxonal dynamics of excitatory and inhibitory neuronsin somatosensory cortexrdquo PLoS Biology vol 8 no 6 Article IDe1000395 2010
[21] V C Wimmer P J Broser T Kuner and R M BrunoldquoExperience-induced plasticity of thalamocortical axons inboth juveniles and adultsrdquo Journal of Comparative Neurologyvol 518 no 22 pp 4629ndash4648 2010
[22] M Oberlaender A Ramirez and R M Bruno ldquoSensory expe-rience restructures thalamocortical axons during adulthoodrdquoNeuron vol 74 no 4 pp 648ndash655 2012
[23] D Katzel and G Miesenbock ldquoExperience-dependent rewiringof specific inhibitory connections in adult neocortexrdquo PLoSBiology vol 12 no 2 Article ID e1001798 2014
[24] C Tailby L LWright A BMetha andM B Calford ldquoActivity-dependent maintenance and growth of dendrites in adultcortexrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 102 no 12 pp 4631ndash4636 2005
Neural Plasticity 9
[25] C E J Cheetham M S L Hammond R McFarlane and G TFinnerty ldquoAltered sensory experience induces targeted rewiringof local excitatory connections in mature neocortexrdquo Journal ofNeuroscience vol 28 no 37 pp 9249ndash9260 2008
[26] K D Micheva and C Beaulieu ldquoAn anatomical substrate forexperience-dependent plasticity of the rat barrel field cortexrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 25 pp 11834ndash11838 1995
[27] Y Zuo G Yang E Kwon and W-B Gan ldquoLong-term sen-sory deprivation prevents dendritic spine loss in primarysomatosensory cortexrdquo Nature vol 436 no 7048 pp 261ndash2652005
[28] A Holtmaat L Wilbrecht G W Knott E Welker and KSvoboda ldquoExperience-dependent and cell-type-specific spinegrowth in the neocortexrdquo Nature vol 441 no 7096 pp 979ndash983 2006
[29] C E J Cheetham M S L Hammond C E J Edwards and GT Finnerty ldquoSensory experience alters cortical connectivity andsynaptic function site specificallyrdquo The Journal of Neurosciencevol 27 no 13 pp 3456ndash3465 2007
[30] L Wilbrecht A Holtmaat N Wright K Fox and K Svo-boda ldquoStructural plasticity underlies experience-dependentfunctional plasticity of cortical circuitsrdquo Journal of Neurosciencevol 30 no 14 pp 4927ndash4932 2010
[31] C E J Cheetham S J Barnes G Albieri G W Knott and GT Finnerty ldquoPansynaptic enlargement at adult cortical connec-tions strengthened by experiencerdquo Cerebral Cortex vol 24 no2 pp 521ndash531 2014
[32] E Siucinska and M Kossut ldquoShort-lasting classical condition-ing induces reversible changes of representational maps ofvibrissae inmouse SI cortex-A 2DG studyrdquoCerebral Cortex vol6 no 3 pp 506ndash513 1996
[33] M Jasinska E Siucinska A Cybulska-Klosowicz et al ldquoRapidlearning-induced inhibitory synaptogenesis in murine barrelfieldrdquo The Journal of Neuroscience vol 30 no 3 pp 1176ndash11842010
[34] R N Strominger and T AWoolsey ldquoTemplates for locating thewhisker area in fresh flattened mouse and rat cortexrdquo Journal ofNeuroscience Methods vol 22 no 2 pp 113ndash118 1987
[35] A Valles A J Boender S Gijsbers R A M Haast G J MMartens and P de Weerd ldquoGenomewide analysis of rat barrelcortex reveals time- and layer-specificmrna expression changesrelated to experience-dependent plasticityrdquo Journal of Neuro-science vol 31 no 16 pp 6140ndash6158 2011
[36] A Kaliszewska Expression of selected immediate early genesand involvement of matrix metalloproteinase 9 in functionalreorganization of mouse barrel cortex [Doctoral dissertation]Nencki Institute of Experimental Biology Polish Academy ofSciences Warsaw Poland 2012
[37] A Cybulska-Klosowicz R Zakrzewska and M Kossut ldquoBrainactivation patterns during classical conditioning with appetitiveor aversive UCSrdquo Behavioural Brain Research vol 204 no 1 pp102ndash111 2009
[38] B Lu K H Wang and A Nose ldquoMolecular mechanismsunderlying neural circuit formationrdquo Current Opinion in Neu-robiology vol 19 no 2 pp 162ndash167 2009
[39] Y Jiao Z Zhang C Zhang et al ldquoA key mechanism under-lying sensory experience-dependent maturation of neocorti-cal GABAergic circuits in vivordquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 108 no29 pp 12131ndash12136 2011
[40] K Tokarski J Urban-Ciecko M Kossut and G Hess ldquoSensorylearning-induced enhancement of inhibitory synaptic trans-mission in the barrel cortex of the mouserdquo European Journal ofNeuroscience vol 26 no 1 pp 134ndash141 2007
[41] A Cybulska-Klosowicz A Posluszny K Nowak E SiucinskaM Kossut and M Liguz-Lecznar ldquoInterneurons containingsomatostatin are affected by learning-induced cortical plastic-ityrdquo Neuroscience vol 254 pp 18ndash25 2013
[42] Z-H Hou and X Yu ldquoActivity-regulated somatostatin expres-sion reduces dendritic spine density and lowers excitatorysynaptic transmission via postsynaptic somatostatin receptor 4rdquoJournal of Biological Chemistry vol 288 no 4 pp 2501ndash25092013
[43] S A Eichler and J CMeier ldquoE-I balance and human diseasesmdashfrom molecules to networkingrdquo Frontiers in Molecular Neuro-science vol 1 article 2 2008
[44] L Coutellier S Beraki P M Ardestani N L Saw and MShamloo ldquoNpas4 a neuronal transcription factor with a keyrole in social and cognitive functions relevant to developmentaldisordersrdquo PLoS ONE vol 7 no 9 Article ID e46604 2012
[45] E J Jaehne T S Klaric S A Koblar B T Baune and MD Lewis ldquoEffects of Npas4 deficiency on anxiety depression-like cognition and sociability behaviourrdquo Behavioural BrainResearch vol 281 pp 276ndash282 2015
[46] D C Bersten J B Bruning D J Peet and M L WhitelawldquoHuman variants in the neuronal basic helix-loop-helixPer-Arnt-Sim (bHLHPAS) transcription factor complex NPAS4ARNT2 disrupt functionrdquo PLoS ONE vol 9 no 1 Article IDe85768 2014
[47] S Maren ldquoPavlovian fear conditioning as a behavioral assayfor hippocampus and amygdala function cautions and caveatsrdquoEuropean Journal of Neuroscience vol 28 no 8 pp 1661ndash16662008
[48] B Jabłonska M Kossut and J Skangiel-Kramska ldquoTransientincrease of AMPA and NMDA receptor binding in the barrelcortex of mice after tactile stimulationrdquo Neurobiology of Learn-ing and Memory vol 66 no 1 pp 36ndash43 1996
[49] M Gierdalski B Jablonska E SiucinskaM Lech A Skibinskaand M Kossut ldquoRapid regulation of GAD67 mRNA and pro-tein level in cortical neurons after sensory learningrdquo CerebralCortex vol 11 no 9 pp 806ndash815 2001
Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Neural Plasticity 5
00
02
04
06
08
Npa
s4 re
lativ
e exp
ress
ion
leve
l
Con
trolC
Exp
C
Con
trolB
D
Exp
BD
lowastlowastlowastlowast
lowastlowast
(a)
0
1
2
3
c-Fo
s rela
tive e
xpre
ssio
n le
vel
Con
trolC
Exp
C
Con
trolB
D
Exp
BD
lowastlowastlowast
lowastlowast
lowastlowast
lowast
lowast
(b)
Figure 3 (a) Npas4 expression in spared C rows and in deprived regions (averaged rows B and D) Control C spared row C on controlside (deprived for 24 hours) Exp C spared row C on experimental side (deprived 7 days) Control BD deprived B and D rows on controlside Exp BD deprived B and D rows on experimental side mean plusmn SEM lowastlowast
119875
lt 001 Duration of deprivation had no impact on Npas4expression (b) In deprived B and D rows deprivation lasting 7 days induced increase in c-Fos expression mean plusmn SEM lowast
119875
lt 005 lowastlowast119875
lt 001lowastlowastlowast
119875
lt 0001
00
05
10
15
20
relat
ive e
xpre
ssio
n le
vel
Righ
t Ble
ft B
Npa
s4
Naıve CS + UCS PSEUDO CS
lowastlowastlowastlowast
lowastlowast
Figure 4 Changes in Npas4 expression induced by training Intrained (CS + UCS) animals expression of Npas4 in the right(trained) row B of barrels is elevated in comparison with left(control unstimulated) row B Sole stimulation of whiskers (CSonly) and application of unpaired CS and UCS (PSEUDO) do notproduce increase in Npas4 expression in comparison with controlside Data are presented as ratio of Npas4 expression level in rightand left row B of barrels mean plusmn SEM lowastlowast
119875
lt 001
expression was increased in the ldquotrainedrdquo row in every singleconditioned animal (Figure 5)
000
005
010
015
020
025
030
035
040
045
050
Npa
s4 re
lativ
e exp
ress
ion
leve
l
Trained 1
Trained 2
Trained 3
Trained 4
Trained 5
Trained 6
Trained 7
Trained 8
Trained 9
L R
Figure 5 Changes of Npas4 mRNA expression in individualconditionedmicemdashcomparison between trained rowB of barrels (Rright) and control row B (L left)
6 Neural Plasticity
00
01
02
03
Npa
s4 re
lativ
e exp
ress
ion
leve
l
Left B Right D Right B
lowastlowast
(a)
00
05
10
15
c-Fo
s rela
tive e
xpre
ssio
n le
vel
Left B Right D Right B
lowast
(b)
Figure 6 Training induced changes in Npas4 (a) and c-Fos (b) expression Right B corresponds to the trained row of barrels right D tocontrol row of barrels in the same hemisphere and left B to control row B in the other hemisphere Npas4 and c-Fos have similar pattern ofexpression following the training procedure their expression is elevated in the trained row B in comparison with the control side mean plusmnSEM lowast119875
lt 005 lowastlowast119875
lt 001
To see if the observed changes in Npas4 expression arelimited to the stimulated row B barrels we evaluated the levelof its expression in row D barrels in the same hemisphereWe did not assess Npas4 mRNA level in row D of the left(control) hemisphere in the CS + UCS group but taking intoaccount results from the naıve group it can be presumedthat it is comparable to the control row B The level ofNpas4 expression in the right row D was an intermediatevalue between the levels of Npas4 mRNA expression in theright (trained) row B and left (control) row B it was notsignificantly different from any of the B rows (Figure 6(a))It can be interpreted that training affects row D so there areno differences between trained row B and row D in the samehemisphere but the impact of training upon rowD is weak sono differences can be observed when compared to the controlhemisphere
In the deprivation studyNpas4 turned out to have a differ-ent pattern of expression than other activity-regulated genesand we wondered if it is also the case in sensory conditioningWe evaluated c-Fos mRNA levels in samples obtained fromCS + UCS mice The pattern of c-Fos expression was thesame as for Npas4 (Figure 6(b)) The training procedureevoked differences among c-Fos expressions in examinedbarrel rows (ANOVA 119865(2 24) = 4407 119875 = 00299) c-Fos mRNA expression level was 484 higher in the trainedrow of barrels (row B in the right hemisphere) than in thecontralateral row B (in the left hemisphere 119875 lt 005)
4 Discussion
Knowledge on Npas4 in plasticity of adult cortex is limitedHerein we provide first description of Npas4 expression in
the barrel cortex undergoing plastic reorganization in twoparadigms deprivation and sensory conditioning We alsocompare changes in expression of Npas4 and c-Fos in bothexperimental models
We found that classical conditioning in which stimula-tion of a row of whiskers is paired with tail shock results inincreased expression of Npas4 mRNA in the cognate row ofcortical barrels As conditioning triggers inhibitory synapto-genesis in the barrels representing stimulated vibrissae [33]our results are in agreement with data demonstrating a roleof Npas4 in the formation of GABAergic synapses The workof Lin et al [3] found that Npas4 regulates expression ofa variety of genes including gene coding for BDNF Npas4binds to activity-dependent promoters I and IV of the BDNFgene [3] and expression of the BDNF gene from promoterIV contributes to the plasticity of inhibitory synapses [3839] Npas4 expression was demonstrated to drive inhibitorysynaptogenesis on excitatory neurons andNpas4 knockdownincreases interevent interval and decreases the amplitudeof mIPSCs [3] In the current experiment we observedan increase in Npas4 expression within the trained rowwhere previously we found increases of GABAergic synapsesdensity increased synaptic content of GABA and increasedspontaneous IPSCs [33 40] Our findings are also in linewith the recent results by Sim et al [4] who found thatincreased cell intrinsic activity results in via an Npas4dependent mechanism the addition of GABAergic inputsto the neuron In hippocampal pyramidal neurons behav-iorally induced expression of Npas4 drives redistribution ofinhibitory synapses increasing inhibitory synapse numberon the cell body while decreasing the number of inhibitorysynapses on the apical dendrites [5] In contrast in our
Neural Plasticity 7
previous experiments we observed an increase in density ofGABAergic synapses located on spines in barrels representingldquotrainedrdquo vibrissae [33] This discrepancy could be attributedto possibility that Npas4 impact on dendritic and somaticpool of GABAergic synapses depends on the type of neuronits location within the brain and the type of stimulation usedto evoke plasticity
Interestingly it was recently found that increased Npas4expression may also account for increased number of exci-tatory contacts made onto somatostatin interneurons [6] Inthe CS + UCS mice an increase in density of somatostatininterneurons within layer 4 of the barrel cortex is observed[41] which may be a result of elevated neuronal activity[42] It can be hypothesized that Npas4 expression drivesgeneration of additional excitatory input onto somatostatininterneurons which in turn express higher level of somato-statin
Herein we demonstrate that sensory conditioning pro-duces increase in Npas4 expression in the region whereinhibitory synaptogenesis was previously observed [33]Synapses formation and elimination accompanying learning-related behaviors may contribute to shift in excitation-inhibition balance Dysregulation of this balance has beenimplicated in number of human neuropsychiatric and neu-rodegenerative disorders and associated with impairmentof cognitive functions [43] Regarding Npas4 significancefor maintaining excitation-inhibition equilibrium it can bepresumed that Npas4 deficiency would result in cognitivedeficits Indeed such deficits were observed in Npas4 knock-out mice [44 45] So far Npas4 has not been directly linkedto human neuropsychiatric disorders However Bersten et al[46] identified human variants of Npas4 with reduced tran-scriptional activity so sequencing Npas4 in neuropsychiatricpatients might be helpful in detecting such a link if it exists
Previous experiments regarding fear conditioning andcontextual learning indicate that Npas4 is indispensable formemory formation [11 12]Therefore it is reasonable to thinkthat Npas4 deficiency should also impair sensory condition-ing Testing this hypothesis using Npas4 knockout animalsmight be misleading Npas4 knockout animals performedwell in amygdala-dependent fear conditioning [11] whileacute deletion of Npas4 in amygdala impaired fear memoryformation [12] Regionally selective depletion of Npas4mightbe more useful then but first structures required for learningassociation of sensory CS and UCS in paradigm used in thisstudy should be identified Basolateral amygdala is activatedduring the training procedure [37] and amygdala is requiredfor all forms of fear conditioning [47] therefore amygdalawould seem a first choice structure for local knockdown ofNpas4 and determining its impact on sensory conditioning
Npas4 expression in remote row D barrels did notdiffer from the results obtained for the control hemisphereand naıve group which is in agreement with our previousobservation that no changes typical for the trained row Bappear in remote row D (no AMPA and NMDA bindingincrease no GAD mRNA expression upregulation and noincrease in density of somatostatin containing inhibitoryinterneurons) [40 41 48 49] We did not do EM to evaluatethe synapse density of remote row D barrels but we found
no increase in spontaneous IPSC there which would indicateincreased GABA release in its excitatory neurons [40]
In the sensory conditioning paradigm Npas4 and c-Foslevels changed in the same wayThey were upregulated in therow of barrels that received the conditioned input
Depriving selected barrel rows of sensory input resultedin decreased Npas4 mRNA expression in the deprivedbarrel rows in comparison with spared rows Duration ofdeprivation (24 h versus 7 days) had no impact upon Npas4expression in spared rows and deprived rowsThis contrastedwith c-Fos the expression of which was increased in bothdeprived and spared regions after 7 days of deprivation incomparison with 24 hours of deprivation
Using immunohistochemical techniques we previouslydescribed the pattern of c-Fos protein expression followingwhisker deprivation of various durations [18] in the samedeprivation paradigm The density of c-Fos positive nucleiincreased in the barrel row deprived of whisker input for 7days and the effect was augmented as the deprivation periodwas prolonged We interpreted this result as illustrating theexpansion of the spared vibrissal input into neighboringfunctionally deafferented barrels The gradual increase in thenumber of immunoreactive nuclei could reflect the gradualrewiring of the barrel cortex in the course of prolongeddeprivation The present results confirm our previous data atthe mRNA level
Unlike c-Fos Npas4mRNA in the deprived B andD rowswas not upregulated by sensory stimulation when comparing24 h versus 7 days of deprivation We suppose that thestrength of the sensory signal coming from the spared row Cwhiskers was insufficient for changing the expression of thisIEG In the experimental paradigm used here we previouslyobserved pronounced changes in Zif268 and Arc expression[36] It is not surprising that differently regulated genes donot respond identically to an experimental situation (see[35] for comparison of several IEGs activations by enrichedenvironment) Perhaps the plastic rearrangement of connec-tions induced by deprivation although it already changesthe metabolic response [18] does not yet trigger activationin Npas4 mRNA expression Npas4 can regulate activity-dependent expression of Arc c-Fos and Zif268 [11] Takinginto consideration that Arc c-Fos and Zif268 expressionsincrease in deprived barrels after 7 days of deprivation it mayseem puzzling that expression of Npas4 which regulates thetranscription of these genes remains unaltered However itshould be noted that Npas4 itself is an immediate early geneand gets activated in response to stimulation Accordingly itprobably does not regulate the first phase of other immediateearly genes expression which is independent of de novoprotein synthesis It rather seems that Npas4 plays a rolein enhancing and sustaining other immediate early genes inlater phases [11]
To the best of our knowledge this is the first report onNpas4 expression in deprivation-induced plasticity Maya-Vetencourt et al [13] examined Npas4 expression in monoc-ularly deprived rats treated with fluoxetine but they concen-trated on influence of fluoxetine onNpas4 expression and noton the influence of deprivation
8 Neural Plasticity
In this paper the precise anatomical dissection of arow of barrels where a plastic change took place allowedfor a quantitative analysis of IEGs expression in two typesof experience dependent plasticity Activation of the barrelcortex undergoing reorganization triggered by removal ofselected rows of whiskers strongly affected c-Fos (but notNpas4) expression Activation of the cortex undergoing aplastic change due to being involved in sensory conditioningupregulated the expression of both Npas4 and c-Fos Takinginto consideration our observations that sensory condition-ing increases the number of inhibitory synapses withinthe trained barrels and studies of other groups showinginvolvement of Npas4 in synaptogenesis we presume thatNpas4 may be involved in reshaping of connectivity withinbarrel cortex after sensory conditioning
Conflict of Interests
The authors declare no conflict of interests
Acknowledgment
This project was supported by GrantMNiSWno 2486BP012010 to Malgorzata Kossut
References
[1] M Moser R Knoth C Bode and C Patterson ldquoLE-PAS anovel Arnt-dependent HLH-PAS protein is expressed in limbictissues and transactivates the CNS midline enhancer elementrdquoMolecular Brain Research vol 128 no 2 pp 141ndash149 2004
[2] N Ooe K Saito N Mikami I Nakatuka and H KanekoldquoIdentification of a novel basic helix-loop-helix-PAS factorNXF reveals a Sim2 competitive positive regulatory role indendritic-cytoskeleton modulator drebrin gene expressionrdquoMolecular and Cellular Biology vol 24 no 2 pp 608ndash616 2004
[3] Y Lin B L Bloodgood J L Hauser et al ldquoActivity-dependentregulation of inhibitory synapse development by Npas4rdquoNature vol 455 no 7217 pp 1198ndash1204 2008
[4] S Sim S Antolin C-W Lin Y-X Lin and C Lois ldquoIncreasedcell-intrinsic excitability induces synaptic changes in new neu-rons in the adult dentate gyrus that require Npas4rdquo Journal ofNeuroscience vol 33 no 18 pp 7928ndash7940 2013
[5] B L Bloodgood N Sharma H A Browne A Z Trepman andM E Greenberg ldquoThe activity-dependent transcription factorNPAS4 regulates domain-specific inhibitionrdquo Nature vol 503no 7474 pp 121ndash125 2013
[6] I Spiegel A R Mardinly H W Gabel et al ldquoNpas4 regulatesexcitatory-inhibitory balance within neural circuits throughcell-type-specific gene programsrdquo Cell vol 157 no 5 pp 1216ndash1229 2014
[7] J Yun T Nagai Y Furukawa-Hibi et al ldquoNeuronal per arnt sim(PAS) domain protein 4 (NPAS4) regulates neurite outgrowthand phosphorylation of synapsin Irdquo The Journal of BiologicalChemistry vol 288 no 4 pp 2655ndash2664 2013
[8] G Pouchelon F Gambino C Bellone et al ldquoModality-specificthalamocortical inputs instruct the identity of postsynaptic L4neuronsrdquo Nature vol 511 no 7510 pp 471ndash474 2014
[9] S Yoshihara H Takahashi N Nishimura et al ldquoNpas4 regu-lates Mdm2 and thus Dcx in experience-dependent dendritic
spine development of newborn olfactory bulb interneuronsrdquoCell Reports vol 8 no 3 pp 843ndash857 2014
[10] K Hayashi R Ishikawa L-H Ye et al ldquoModulatory role ofdrebrin on the cytoskeleton within dendritic spines in the ratcerebral cortexrdquo Journal of Neuroscience vol 16 no 22 pp 7161ndash7170 1996
[11] K Ramamoorthi R Fropf GM Belfort et al ldquoNpas4 regulatesa transcriptional program in CA3 required for contextualmemory formationrdquo Science vol 334 no 6063 pp 1669ndash16752011
[12] J E Ploski M S Monsey T Nguyen R J DiLeone and G ESchafe ldquoThe neuronal PAS domain protein 4 (Npas4) isrequired for new and reactivated fear memoriesrdquo PLoS ONEvol 6 no 8 Article ID e23760 2011
[13] JFMaya-Vetencourt E Tiraboschi DGreco et al ldquoExperience-dependent expression of NPAS4 regulates plasticity in adultvisual cortexrdquo Journal of Physiology vol 590 no 19 pp 4777ndash4787 2012
[14] M Kossut P J Hand J Greenberg and C L Hand ldquoSinglevibrissal cortical column in SI cortex of rat and its alterations inneonatal and adult vibrissa-deafferented animals a quantitative2DG studyrdquo Journal of Neurophysiology vol 60 no 2 pp 829ndash852 1988
[15] S Glazewski andK Fox ldquoTime course of experience-dependentsynaptic potentiation and depression in barrel cortex of adoles-cent ratsrdquo Journal of Neurophysiology vol 75 no 4 pp 1714ndash1729 1996
[16] H Wallace and K Fox ldquoLocal cortical interactions determinethe form of cortical plasticityrdquo Journal of Neurobiology vol 41no 1 pp 58ndash63 1999
[17] M A Lebedev G Mirabella I Erchova and M E DiamondldquoExperience-dependent plasticity of rat barrel cortex redistri-bution of activity across barrel-columnsrdquo Cerebral Cortex vol10 no 1 pp 23ndash31 2000
[18] A Kaliszewska M Bijata L Kaczmarek and M KossutldquoExperience-dependent plasticity of the barrel cortex in miceobserved with 2-DG brain mapping and c-Fos effects of MMP-9 KOrdquo Cerebral Cortex vol 22 no 9 pp 2160ndash2170 2012
[19] D J Margolis H Lutcke K Schulz et al ldquoReorganization ofcortical population activity imaged throughout long-term sen-sory deprivationrdquo Nature Neuroscience vol 15 no 11 pp 1539ndash1546 2012
[20] S AMarikH Yamahachi J N JMcManusG Szabo andCDGilbert ldquoAxonal dynamics of excitatory and inhibitory neuronsin somatosensory cortexrdquo PLoS Biology vol 8 no 6 Article IDe1000395 2010
[21] V C Wimmer P J Broser T Kuner and R M BrunoldquoExperience-induced plasticity of thalamocortical axons inboth juveniles and adultsrdquo Journal of Comparative Neurologyvol 518 no 22 pp 4629ndash4648 2010
[22] M Oberlaender A Ramirez and R M Bruno ldquoSensory expe-rience restructures thalamocortical axons during adulthoodrdquoNeuron vol 74 no 4 pp 648ndash655 2012
[23] D Katzel and G Miesenbock ldquoExperience-dependent rewiringof specific inhibitory connections in adult neocortexrdquo PLoSBiology vol 12 no 2 Article ID e1001798 2014
[24] C Tailby L LWright A BMetha andM B Calford ldquoActivity-dependent maintenance and growth of dendrites in adultcortexrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 102 no 12 pp 4631ndash4636 2005
Neural Plasticity 9
[25] C E J Cheetham M S L Hammond R McFarlane and G TFinnerty ldquoAltered sensory experience induces targeted rewiringof local excitatory connections in mature neocortexrdquo Journal ofNeuroscience vol 28 no 37 pp 9249ndash9260 2008
[26] K D Micheva and C Beaulieu ldquoAn anatomical substrate forexperience-dependent plasticity of the rat barrel field cortexrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 25 pp 11834ndash11838 1995
[27] Y Zuo G Yang E Kwon and W-B Gan ldquoLong-term sen-sory deprivation prevents dendritic spine loss in primarysomatosensory cortexrdquo Nature vol 436 no 7048 pp 261ndash2652005
[28] A Holtmaat L Wilbrecht G W Knott E Welker and KSvoboda ldquoExperience-dependent and cell-type-specific spinegrowth in the neocortexrdquo Nature vol 441 no 7096 pp 979ndash983 2006
[29] C E J Cheetham M S L Hammond C E J Edwards and GT Finnerty ldquoSensory experience alters cortical connectivity andsynaptic function site specificallyrdquo The Journal of Neurosciencevol 27 no 13 pp 3456ndash3465 2007
[30] L Wilbrecht A Holtmaat N Wright K Fox and K Svo-boda ldquoStructural plasticity underlies experience-dependentfunctional plasticity of cortical circuitsrdquo Journal of Neurosciencevol 30 no 14 pp 4927ndash4932 2010
[31] C E J Cheetham S J Barnes G Albieri G W Knott and GT Finnerty ldquoPansynaptic enlargement at adult cortical connec-tions strengthened by experiencerdquo Cerebral Cortex vol 24 no2 pp 521ndash531 2014
[32] E Siucinska and M Kossut ldquoShort-lasting classical condition-ing induces reversible changes of representational maps ofvibrissae inmouse SI cortex-A 2DG studyrdquoCerebral Cortex vol6 no 3 pp 506ndash513 1996
[33] M Jasinska E Siucinska A Cybulska-Klosowicz et al ldquoRapidlearning-induced inhibitory synaptogenesis in murine barrelfieldrdquo The Journal of Neuroscience vol 30 no 3 pp 1176ndash11842010
[34] R N Strominger and T AWoolsey ldquoTemplates for locating thewhisker area in fresh flattened mouse and rat cortexrdquo Journal ofNeuroscience Methods vol 22 no 2 pp 113ndash118 1987
[35] A Valles A J Boender S Gijsbers R A M Haast G J MMartens and P de Weerd ldquoGenomewide analysis of rat barrelcortex reveals time- and layer-specificmrna expression changesrelated to experience-dependent plasticityrdquo Journal of Neuro-science vol 31 no 16 pp 6140ndash6158 2011
[36] A Kaliszewska Expression of selected immediate early genesand involvement of matrix metalloproteinase 9 in functionalreorganization of mouse barrel cortex [Doctoral dissertation]Nencki Institute of Experimental Biology Polish Academy ofSciences Warsaw Poland 2012
[37] A Cybulska-Klosowicz R Zakrzewska and M Kossut ldquoBrainactivation patterns during classical conditioning with appetitiveor aversive UCSrdquo Behavioural Brain Research vol 204 no 1 pp102ndash111 2009
[38] B Lu K H Wang and A Nose ldquoMolecular mechanismsunderlying neural circuit formationrdquo Current Opinion in Neu-robiology vol 19 no 2 pp 162ndash167 2009
[39] Y Jiao Z Zhang C Zhang et al ldquoA key mechanism under-lying sensory experience-dependent maturation of neocorti-cal GABAergic circuits in vivordquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 108 no29 pp 12131ndash12136 2011
[40] K Tokarski J Urban-Ciecko M Kossut and G Hess ldquoSensorylearning-induced enhancement of inhibitory synaptic trans-mission in the barrel cortex of the mouserdquo European Journal ofNeuroscience vol 26 no 1 pp 134ndash141 2007
[41] A Cybulska-Klosowicz A Posluszny K Nowak E SiucinskaM Kossut and M Liguz-Lecznar ldquoInterneurons containingsomatostatin are affected by learning-induced cortical plastic-ityrdquo Neuroscience vol 254 pp 18ndash25 2013
[42] Z-H Hou and X Yu ldquoActivity-regulated somatostatin expres-sion reduces dendritic spine density and lowers excitatorysynaptic transmission via postsynaptic somatostatin receptor 4rdquoJournal of Biological Chemistry vol 288 no 4 pp 2501ndash25092013
[43] S A Eichler and J CMeier ldquoE-I balance and human diseasesmdashfrom molecules to networkingrdquo Frontiers in Molecular Neuro-science vol 1 article 2 2008
[44] L Coutellier S Beraki P M Ardestani N L Saw and MShamloo ldquoNpas4 a neuronal transcription factor with a keyrole in social and cognitive functions relevant to developmentaldisordersrdquo PLoS ONE vol 7 no 9 Article ID e46604 2012
[45] E J Jaehne T S Klaric S A Koblar B T Baune and MD Lewis ldquoEffects of Npas4 deficiency on anxiety depression-like cognition and sociability behaviourrdquo Behavioural BrainResearch vol 281 pp 276ndash282 2015
[46] D C Bersten J B Bruning D J Peet and M L WhitelawldquoHuman variants in the neuronal basic helix-loop-helixPer-Arnt-Sim (bHLHPAS) transcription factor complex NPAS4ARNT2 disrupt functionrdquo PLoS ONE vol 9 no 1 Article IDe85768 2014
[47] S Maren ldquoPavlovian fear conditioning as a behavioral assayfor hippocampus and amygdala function cautions and caveatsrdquoEuropean Journal of Neuroscience vol 28 no 8 pp 1661ndash16662008
[48] B Jabłonska M Kossut and J Skangiel-Kramska ldquoTransientincrease of AMPA and NMDA receptor binding in the barrelcortex of mice after tactile stimulationrdquo Neurobiology of Learn-ing and Memory vol 66 no 1 pp 36ndash43 1996
[49] M Gierdalski B Jablonska E SiucinskaM Lech A Skibinskaand M Kossut ldquoRapid regulation of GAD67 mRNA and pro-tein level in cortical neurons after sensory learningrdquo CerebralCortex vol 11 no 9 pp 806ndash815 2001
Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
6 Neural Plasticity
00
01
02
03
Npa
s4 re
lativ
e exp
ress
ion
leve
l
Left B Right D Right B
lowastlowast
(a)
00
05
10
15
c-Fo
s rela
tive e
xpre
ssio
n le
vel
Left B Right D Right B
lowast
(b)
Figure 6 Training induced changes in Npas4 (a) and c-Fos (b) expression Right B corresponds to the trained row of barrels right D tocontrol row of barrels in the same hemisphere and left B to control row B in the other hemisphere Npas4 and c-Fos have similar pattern ofexpression following the training procedure their expression is elevated in the trained row B in comparison with the control side mean plusmnSEM lowast119875
lt 005 lowastlowast119875
lt 001
To see if the observed changes in Npas4 expression arelimited to the stimulated row B barrels we evaluated the levelof its expression in row D barrels in the same hemisphereWe did not assess Npas4 mRNA level in row D of the left(control) hemisphere in the CS + UCS group but taking intoaccount results from the naıve group it can be presumedthat it is comparable to the control row B The level ofNpas4 expression in the right row D was an intermediatevalue between the levels of Npas4 mRNA expression in theright (trained) row B and left (control) row B it was notsignificantly different from any of the B rows (Figure 6(a))It can be interpreted that training affects row D so there areno differences between trained row B and row D in the samehemisphere but the impact of training upon rowD is weak sono differences can be observed when compared to the controlhemisphere
In the deprivation studyNpas4 turned out to have a differ-ent pattern of expression than other activity-regulated genesand we wondered if it is also the case in sensory conditioningWe evaluated c-Fos mRNA levels in samples obtained fromCS + UCS mice The pattern of c-Fos expression was thesame as for Npas4 (Figure 6(b)) The training procedureevoked differences among c-Fos expressions in examinedbarrel rows (ANOVA 119865(2 24) = 4407 119875 = 00299) c-Fos mRNA expression level was 484 higher in the trainedrow of barrels (row B in the right hemisphere) than in thecontralateral row B (in the left hemisphere 119875 lt 005)
4 Discussion
Knowledge on Npas4 in plasticity of adult cortex is limitedHerein we provide first description of Npas4 expression in
the barrel cortex undergoing plastic reorganization in twoparadigms deprivation and sensory conditioning We alsocompare changes in expression of Npas4 and c-Fos in bothexperimental models
We found that classical conditioning in which stimula-tion of a row of whiskers is paired with tail shock results inincreased expression of Npas4 mRNA in the cognate row ofcortical barrels As conditioning triggers inhibitory synapto-genesis in the barrels representing stimulated vibrissae [33]our results are in agreement with data demonstrating a roleof Npas4 in the formation of GABAergic synapses The workof Lin et al [3] found that Npas4 regulates expression ofa variety of genes including gene coding for BDNF Npas4binds to activity-dependent promoters I and IV of the BDNFgene [3] and expression of the BDNF gene from promoterIV contributes to the plasticity of inhibitory synapses [3839] Npas4 expression was demonstrated to drive inhibitorysynaptogenesis on excitatory neurons andNpas4 knockdownincreases interevent interval and decreases the amplitudeof mIPSCs [3] In the current experiment we observedan increase in Npas4 expression within the trained rowwhere previously we found increases of GABAergic synapsesdensity increased synaptic content of GABA and increasedspontaneous IPSCs [33 40] Our findings are also in linewith the recent results by Sim et al [4] who found thatincreased cell intrinsic activity results in via an Npas4dependent mechanism the addition of GABAergic inputsto the neuron In hippocampal pyramidal neurons behav-iorally induced expression of Npas4 drives redistribution ofinhibitory synapses increasing inhibitory synapse numberon the cell body while decreasing the number of inhibitorysynapses on the apical dendrites [5] In contrast in our
Neural Plasticity 7
previous experiments we observed an increase in density ofGABAergic synapses located on spines in barrels representingldquotrainedrdquo vibrissae [33] This discrepancy could be attributedto possibility that Npas4 impact on dendritic and somaticpool of GABAergic synapses depends on the type of neuronits location within the brain and the type of stimulation usedto evoke plasticity
Interestingly it was recently found that increased Npas4expression may also account for increased number of exci-tatory contacts made onto somatostatin interneurons [6] Inthe CS + UCS mice an increase in density of somatostatininterneurons within layer 4 of the barrel cortex is observed[41] which may be a result of elevated neuronal activity[42] It can be hypothesized that Npas4 expression drivesgeneration of additional excitatory input onto somatostatininterneurons which in turn express higher level of somato-statin
Herein we demonstrate that sensory conditioning pro-duces increase in Npas4 expression in the region whereinhibitory synaptogenesis was previously observed [33]Synapses formation and elimination accompanying learning-related behaviors may contribute to shift in excitation-inhibition balance Dysregulation of this balance has beenimplicated in number of human neuropsychiatric and neu-rodegenerative disorders and associated with impairmentof cognitive functions [43] Regarding Npas4 significancefor maintaining excitation-inhibition equilibrium it can bepresumed that Npas4 deficiency would result in cognitivedeficits Indeed such deficits were observed in Npas4 knock-out mice [44 45] So far Npas4 has not been directly linkedto human neuropsychiatric disorders However Bersten et al[46] identified human variants of Npas4 with reduced tran-scriptional activity so sequencing Npas4 in neuropsychiatricpatients might be helpful in detecting such a link if it exists
Previous experiments regarding fear conditioning andcontextual learning indicate that Npas4 is indispensable formemory formation [11 12]Therefore it is reasonable to thinkthat Npas4 deficiency should also impair sensory condition-ing Testing this hypothesis using Npas4 knockout animalsmight be misleading Npas4 knockout animals performedwell in amygdala-dependent fear conditioning [11] whileacute deletion of Npas4 in amygdala impaired fear memoryformation [12] Regionally selective depletion of Npas4mightbe more useful then but first structures required for learningassociation of sensory CS and UCS in paradigm used in thisstudy should be identified Basolateral amygdala is activatedduring the training procedure [37] and amygdala is requiredfor all forms of fear conditioning [47] therefore amygdalawould seem a first choice structure for local knockdown ofNpas4 and determining its impact on sensory conditioning
Npas4 expression in remote row D barrels did notdiffer from the results obtained for the control hemisphereand naıve group which is in agreement with our previousobservation that no changes typical for the trained row Bappear in remote row D (no AMPA and NMDA bindingincrease no GAD mRNA expression upregulation and noincrease in density of somatostatin containing inhibitoryinterneurons) [40 41 48 49] We did not do EM to evaluatethe synapse density of remote row D barrels but we found
no increase in spontaneous IPSC there which would indicateincreased GABA release in its excitatory neurons [40]
In the sensory conditioning paradigm Npas4 and c-Foslevels changed in the same wayThey were upregulated in therow of barrels that received the conditioned input
Depriving selected barrel rows of sensory input resultedin decreased Npas4 mRNA expression in the deprivedbarrel rows in comparison with spared rows Duration ofdeprivation (24 h versus 7 days) had no impact upon Npas4expression in spared rows and deprived rowsThis contrastedwith c-Fos the expression of which was increased in bothdeprived and spared regions after 7 days of deprivation incomparison with 24 hours of deprivation
Using immunohistochemical techniques we previouslydescribed the pattern of c-Fos protein expression followingwhisker deprivation of various durations [18] in the samedeprivation paradigm The density of c-Fos positive nucleiincreased in the barrel row deprived of whisker input for 7days and the effect was augmented as the deprivation periodwas prolonged We interpreted this result as illustrating theexpansion of the spared vibrissal input into neighboringfunctionally deafferented barrels The gradual increase in thenumber of immunoreactive nuclei could reflect the gradualrewiring of the barrel cortex in the course of prolongeddeprivation The present results confirm our previous data atthe mRNA level
Unlike c-Fos Npas4mRNA in the deprived B andD rowswas not upregulated by sensory stimulation when comparing24 h versus 7 days of deprivation We suppose that thestrength of the sensory signal coming from the spared row Cwhiskers was insufficient for changing the expression of thisIEG In the experimental paradigm used here we previouslyobserved pronounced changes in Zif268 and Arc expression[36] It is not surprising that differently regulated genes donot respond identically to an experimental situation (see[35] for comparison of several IEGs activations by enrichedenvironment) Perhaps the plastic rearrangement of connec-tions induced by deprivation although it already changesthe metabolic response [18] does not yet trigger activationin Npas4 mRNA expression Npas4 can regulate activity-dependent expression of Arc c-Fos and Zif268 [11] Takinginto consideration that Arc c-Fos and Zif268 expressionsincrease in deprived barrels after 7 days of deprivation it mayseem puzzling that expression of Npas4 which regulates thetranscription of these genes remains unaltered However itshould be noted that Npas4 itself is an immediate early geneand gets activated in response to stimulation Accordingly itprobably does not regulate the first phase of other immediateearly genes expression which is independent of de novoprotein synthesis It rather seems that Npas4 plays a rolein enhancing and sustaining other immediate early genes inlater phases [11]
To the best of our knowledge this is the first report onNpas4 expression in deprivation-induced plasticity Maya-Vetencourt et al [13] examined Npas4 expression in monoc-ularly deprived rats treated with fluoxetine but they concen-trated on influence of fluoxetine onNpas4 expression and noton the influence of deprivation
8 Neural Plasticity
In this paper the precise anatomical dissection of arow of barrels where a plastic change took place allowedfor a quantitative analysis of IEGs expression in two typesof experience dependent plasticity Activation of the barrelcortex undergoing reorganization triggered by removal ofselected rows of whiskers strongly affected c-Fos (but notNpas4) expression Activation of the cortex undergoing aplastic change due to being involved in sensory conditioningupregulated the expression of both Npas4 and c-Fos Takinginto consideration our observations that sensory condition-ing increases the number of inhibitory synapses withinthe trained barrels and studies of other groups showinginvolvement of Npas4 in synaptogenesis we presume thatNpas4 may be involved in reshaping of connectivity withinbarrel cortex after sensory conditioning
Conflict of Interests
The authors declare no conflict of interests
Acknowledgment
This project was supported by GrantMNiSWno 2486BP012010 to Malgorzata Kossut
References
[1] M Moser R Knoth C Bode and C Patterson ldquoLE-PAS anovel Arnt-dependent HLH-PAS protein is expressed in limbictissues and transactivates the CNS midline enhancer elementrdquoMolecular Brain Research vol 128 no 2 pp 141ndash149 2004
[2] N Ooe K Saito N Mikami I Nakatuka and H KanekoldquoIdentification of a novel basic helix-loop-helix-PAS factorNXF reveals a Sim2 competitive positive regulatory role indendritic-cytoskeleton modulator drebrin gene expressionrdquoMolecular and Cellular Biology vol 24 no 2 pp 608ndash616 2004
[3] Y Lin B L Bloodgood J L Hauser et al ldquoActivity-dependentregulation of inhibitory synapse development by Npas4rdquoNature vol 455 no 7217 pp 1198ndash1204 2008
[4] S Sim S Antolin C-W Lin Y-X Lin and C Lois ldquoIncreasedcell-intrinsic excitability induces synaptic changes in new neu-rons in the adult dentate gyrus that require Npas4rdquo Journal ofNeuroscience vol 33 no 18 pp 7928ndash7940 2013
[5] B L Bloodgood N Sharma H A Browne A Z Trepman andM E Greenberg ldquoThe activity-dependent transcription factorNPAS4 regulates domain-specific inhibitionrdquo Nature vol 503no 7474 pp 121ndash125 2013
[6] I Spiegel A R Mardinly H W Gabel et al ldquoNpas4 regulatesexcitatory-inhibitory balance within neural circuits throughcell-type-specific gene programsrdquo Cell vol 157 no 5 pp 1216ndash1229 2014
[7] J Yun T Nagai Y Furukawa-Hibi et al ldquoNeuronal per arnt sim(PAS) domain protein 4 (NPAS4) regulates neurite outgrowthand phosphorylation of synapsin Irdquo The Journal of BiologicalChemistry vol 288 no 4 pp 2655ndash2664 2013
[8] G Pouchelon F Gambino C Bellone et al ldquoModality-specificthalamocortical inputs instruct the identity of postsynaptic L4neuronsrdquo Nature vol 511 no 7510 pp 471ndash474 2014
[9] S Yoshihara H Takahashi N Nishimura et al ldquoNpas4 regu-lates Mdm2 and thus Dcx in experience-dependent dendritic
spine development of newborn olfactory bulb interneuronsrdquoCell Reports vol 8 no 3 pp 843ndash857 2014
[10] K Hayashi R Ishikawa L-H Ye et al ldquoModulatory role ofdrebrin on the cytoskeleton within dendritic spines in the ratcerebral cortexrdquo Journal of Neuroscience vol 16 no 22 pp 7161ndash7170 1996
[11] K Ramamoorthi R Fropf GM Belfort et al ldquoNpas4 regulatesa transcriptional program in CA3 required for contextualmemory formationrdquo Science vol 334 no 6063 pp 1669ndash16752011
[12] J E Ploski M S Monsey T Nguyen R J DiLeone and G ESchafe ldquoThe neuronal PAS domain protein 4 (Npas4) isrequired for new and reactivated fear memoriesrdquo PLoS ONEvol 6 no 8 Article ID e23760 2011
[13] JFMaya-Vetencourt E Tiraboschi DGreco et al ldquoExperience-dependent expression of NPAS4 regulates plasticity in adultvisual cortexrdquo Journal of Physiology vol 590 no 19 pp 4777ndash4787 2012
[14] M Kossut P J Hand J Greenberg and C L Hand ldquoSinglevibrissal cortical column in SI cortex of rat and its alterations inneonatal and adult vibrissa-deafferented animals a quantitative2DG studyrdquo Journal of Neurophysiology vol 60 no 2 pp 829ndash852 1988
[15] S Glazewski andK Fox ldquoTime course of experience-dependentsynaptic potentiation and depression in barrel cortex of adoles-cent ratsrdquo Journal of Neurophysiology vol 75 no 4 pp 1714ndash1729 1996
[16] H Wallace and K Fox ldquoLocal cortical interactions determinethe form of cortical plasticityrdquo Journal of Neurobiology vol 41no 1 pp 58ndash63 1999
[17] M A Lebedev G Mirabella I Erchova and M E DiamondldquoExperience-dependent plasticity of rat barrel cortex redistri-bution of activity across barrel-columnsrdquo Cerebral Cortex vol10 no 1 pp 23ndash31 2000
[18] A Kaliszewska M Bijata L Kaczmarek and M KossutldquoExperience-dependent plasticity of the barrel cortex in miceobserved with 2-DG brain mapping and c-Fos effects of MMP-9 KOrdquo Cerebral Cortex vol 22 no 9 pp 2160ndash2170 2012
[19] D J Margolis H Lutcke K Schulz et al ldquoReorganization ofcortical population activity imaged throughout long-term sen-sory deprivationrdquo Nature Neuroscience vol 15 no 11 pp 1539ndash1546 2012
[20] S AMarikH Yamahachi J N JMcManusG Szabo andCDGilbert ldquoAxonal dynamics of excitatory and inhibitory neuronsin somatosensory cortexrdquo PLoS Biology vol 8 no 6 Article IDe1000395 2010
[21] V C Wimmer P J Broser T Kuner and R M BrunoldquoExperience-induced plasticity of thalamocortical axons inboth juveniles and adultsrdquo Journal of Comparative Neurologyvol 518 no 22 pp 4629ndash4648 2010
[22] M Oberlaender A Ramirez and R M Bruno ldquoSensory expe-rience restructures thalamocortical axons during adulthoodrdquoNeuron vol 74 no 4 pp 648ndash655 2012
[23] D Katzel and G Miesenbock ldquoExperience-dependent rewiringof specific inhibitory connections in adult neocortexrdquo PLoSBiology vol 12 no 2 Article ID e1001798 2014
[24] C Tailby L LWright A BMetha andM B Calford ldquoActivity-dependent maintenance and growth of dendrites in adultcortexrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 102 no 12 pp 4631ndash4636 2005
Neural Plasticity 9
[25] C E J Cheetham M S L Hammond R McFarlane and G TFinnerty ldquoAltered sensory experience induces targeted rewiringof local excitatory connections in mature neocortexrdquo Journal ofNeuroscience vol 28 no 37 pp 9249ndash9260 2008
[26] K D Micheva and C Beaulieu ldquoAn anatomical substrate forexperience-dependent plasticity of the rat barrel field cortexrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 25 pp 11834ndash11838 1995
[27] Y Zuo G Yang E Kwon and W-B Gan ldquoLong-term sen-sory deprivation prevents dendritic spine loss in primarysomatosensory cortexrdquo Nature vol 436 no 7048 pp 261ndash2652005
[28] A Holtmaat L Wilbrecht G W Knott E Welker and KSvoboda ldquoExperience-dependent and cell-type-specific spinegrowth in the neocortexrdquo Nature vol 441 no 7096 pp 979ndash983 2006
[29] C E J Cheetham M S L Hammond C E J Edwards and GT Finnerty ldquoSensory experience alters cortical connectivity andsynaptic function site specificallyrdquo The Journal of Neurosciencevol 27 no 13 pp 3456ndash3465 2007
[30] L Wilbrecht A Holtmaat N Wright K Fox and K Svo-boda ldquoStructural plasticity underlies experience-dependentfunctional plasticity of cortical circuitsrdquo Journal of Neurosciencevol 30 no 14 pp 4927ndash4932 2010
[31] C E J Cheetham S J Barnes G Albieri G W Knott and GT Finnerty ldquoPansynaptic enlargement at adult cortical connec-tions strengthened by experiencerdquo Cerebral Cortex vol 24 no2 pp 521ndash531 2014
[32] E Siucinska and M Kossut ldquoShort-lasting classical condition-ing induces reversible changes of representational maps ofvibrissae inmouse SI cortex-A 2DG studyrdquoCerebral Cortex vol6 no 3 pp 506ndash513 1996
[33] M Jasinska E Siucinska A Cybulska-Klosowicz et al ldquoRapidlearning-induced inhibitory synaptogenesis in murine barrelfieldrdquo The Journal of Neuroscience vol 30 no 3 pp 1176ndash11842010
[34] R N Strominger and T AWoolsey ldquoTemplates for locating thewhisker area in fresh flattened mouse and rat cortexrdquo Journal ofNeuroscience Methods vol 22 no 2 pp 113ndash118 1987
[35] A Valles A J Boender S Gijsbers R A M Haast G J MMartens and P de Weerd ldquoGenomewide analysis of rat barrelcortex reveals time- and layer-specificmrna expression changesrelated to experience-dependent plasticityrdquo Journal of Neuro-science vol 31 no 16 pp 6140ndash6158 2011
[36] A Kaliszewska Expression of selected immediate early genesand involvement of matrix metalloproteinase 9 in functionalreorganization of mouse barrel cortex [Doctoral dissertation]Nencki Institute of Experimental Biology Polish Academy ofSciences Warsaw Poland 2012
[37] A Cybulska-Klosowicz R Zakrzewska and M Kossut ldquoBrainactivation patterns during classical conditioning with appetitiveor aversive UCSrdquo Behavioural Brain Research vol 204 no 1 pp102ndash111 2009
[38] B Lu K H Wang and A Nose ldquoMolecular mechanismsunderlying neural circuit formationrdquo Current Opinion in Neu-robiology vol 19 no 2 pp 162ndash167 2009
[39] Y Jiao Z Zhang C Zhang et al ldquoA key mechanism under-lying sensory experience-dependent maturation of neocorti-cal GABAergic circuits in vivordquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 108 no29 pp 12131ndash12136 2011
[40] K Tokarski J Urban-Ciecko M Kossut and G Hess ldquoSensorylearning-induced enhancement of inhibitory synaptic trans-mission in the barrel cortex of the mouserdquo European Journal ofNeuroscience vol 26 no 1 pp 134ndash141 2007
[41] A Cybulska-Klosowicz A Posluszny K Nowak E SiucinskaM Kossut and M Liguz-Lecznar ldquoInterneurons containingsomatostatin are affected by learning-induced cortical plastic-ityrdquo Neuroscience vol 254 pp 18ndash25 2013
[42] Z-H Hou and X Yu ldquoActivity-regulated somatostatin expres-sion reduces dendritic spine density and lowers excitatorysynaptic transmission via postsynaptic somatostatin receptor 4rdquoJournal of Biological Chemistry vol 288 no 4 pp 2501ndash25092013
[43] S A Eichler and J CMeier ldquoE-I balance and human diseasesmdashfrom molecules to networkingrdquo Frontiers in Molecular Neuro-science vol 1 article 2 2008
[44] L Coutellier S Beraki P M Ardestani N L Saw and MShamloo ldquoNpas4 a neuronal transcription factor with a keyrole in social and cognitive functions relevant to developmentaldisordersrdquo PLoS ONE vol 7 no 9 Article ID e46604 2012
[45] E J Jaehne T S Klaric S A Koblar B T Baune and MD Lewis ldquoEffects of Npas4 deficiency on anxiety depression-like cognition and sociability behaviourrdquo Behavioural BrainResearch vol 281 pp 276ndash282 2015
[46] D C Bersten J B Bruning D J Peet and M L WhitelawldquoHuman variants in the neuronal basic helix-loop-helixPer-Arnt-Sim (bHLHPAS) transcription factor complex NPAS4ARNT2 disrupt functionrdquo PLoS ONE vol 9 no 1 Article IDe85768 2014
[47] S Maren ldquoPavlovian fear conditioning as a behavioral assayfor hippocampus and amygdala function cautions and caveatsrdquoEuropean Journal of Neuroscience vol 28 no 8 pp 1661ndash16662008
[48] B Jabłonska M Kossut and J Skangiel-Kramska ldquoTransientincrease of AMPA and NMDA receptor binding in the barrelcortex of mice after tactile stimulationrdquo Neurobiology of Learn-ing and Memory vol 66 no 1 pp 36ndash43 1996
[49] M Gierdalski B Jablonska E SiucinskaM Lech A Skibinskaand M Kossut ldquoRapid regulation of GAD67 mRNA and pro-tein level in cortical neurons after sensory learningrdquo CerebralCortex vol 11 no 9 pp 806ndash815 2001
Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Neural Plasticity 7
previous experiments we observed an increase in density ofGABAergic synapses located on spines in barrels representingldquotrainedrdquo vibrissae [33] This discrepancy could be attributedto possibility that Npas4 impact on dendritic and somaticpool of GABAergic synapses depends on the type of neuronits location within the brain and the type of stimulation usedto evoke plasticity
Interestingly it was recently found that increased Npas4expression may also account for increased number of exci-tatory contacts made onto somatostatin interneurons [6] Inthe CS + UCS mice an increase in density of somatostatininterneurons within layer 4 of the barrel cortex is observed[41] which may be a result of elevated neuronal activity[42] It can be hypothesized that Npas4 expression drivesgeneration of additional excitatory input onto somatostatininterneurons which in turn express higher level of somato-statin
Herein we demonstrate that sensory conditioning pro-duces increase in Npas4 expression in the region whereinhibitory synaptogenesis was previously observed [33]Synapses formation and elimination accompanying learning-related behaviors may contribute to shift in excitation-inhibition balance Dysregulation of this balance has beenimplicated in number of human neuropsychiatric and neu-rodegenerative disorders and associated with impairmentof cognitive functions [43] Regarding Npas4 significancefor maintaining excitation-inhibition equilibrium it can bepresumed that Npas4 deficiency would result in cognitivedeficits Indeed such deficits were observed in Npas4 knock-out mice [44 45] So far Npas4 has not been directly linkedto human neuropsychiatric disorders However Bersten et al[46] identified human variants of Npas4 with reduced tran-scriptional activity so sequencing Npas4 in neuropsychiatricpatients might be helpful in detecting such a link if it exists
Previous experiments regarding fear conditioning andcontextual learning indicate that Npas4 is indispensable formemory formation [11 12]Therefore it is reasonable to thinkthat Npas4 deficiency should also impair sensory condition-ing Testing this hypothesis using Npas4 knockout animalsmight be misleading Npas4 knockout animals performedwell in amygdala-dependent fear conditioning [11] whileacute deletion of Npas4 in amygdala impaired fear memoryformation [12] Regionally selective depletion of Npas4mightbe more useful then but first structures required for learningassociation of sensory CS and UCS in paradigm used in thisstudy should be identified Basolateral amygdala is activatedduring the training procedure [37] and amygdala is requiredfor all forms of fear conditioning [47] therefore amygdalawould seem a first choice structure for local knockdown ofNpas4 and determining its impact on sensory conditioning
Npas4 expression in remote row D barrels did notdiffer from the results obtained for the control hemisphereand naıve group which is in agreement with our previousobservation that no changes typical for the trained row Bappear in remote row D (no AMPA and NMDA bindingincrease no GAD mRNA expression upregulation and noincrease in density of somatostatin containing inhibitoryinterneurons) [40 41 48 49] We did not do EM to evaluatethe synapse density of remote row D barrels but we found
no increase in spontaneous IPSC there which would indicateincreased GABA release in its excitatory neurons [40]
In the sensory conditioning paradigm Npas4 and c-Foslevels changed in the same wayThey were upregulated in therow of barrels that received the conditioned input
Depriving selected barrel rows of sensory input resultedin decreased Npas4 mRNA expression in the deprivedbarrel rows in comparison with spared rows Duration ofdeprivation (24 h versus 7 days) had no impact upon Npas4expression in spared rows and deprived rowsThis contrastedwith c-Fos the expression of which was increased in bothdeprived and spared regions after 7 days of deprivation incomparison with 24 hours of deprivation
Using immunohistochemical techniques we previouslydescribed the pattern of c-Fos protein expression followingwhisker deprivation of various durations [18] in the samedeprivation paradigm The density of c-Fos positive nucleiincreased in the barrel row deprived of whisker input for 7days and the effect was augmented as the deprivation periodwas prolonged We interpreted this result as illustrating theexpansion of the spared vibrissal input into neighboringfunctionally deafferented barrels The gradual increase in thenumber of immunoreactive nuclei could reflect the gradualrewiring of the barrel cortex in the course of prolongeddeprivation The present results confirm our previous data atthe mRNA level
Unlike c-Fos Npas4mRNA in the deprived B andD rowswas not upregulated by sensory stimulation when comparing24 h versus 7 days of deprivation We suppose that thestrength of the sensory signal coming from the spared row Cwhiskers was insufficient for changing the expression of thisIEG In the experimental paradigm used here we previouslyobserved pronounced changes in Zif268 and Arc expression[36] It is not surprising that differently regulated genes donot respond identically to an experimental situation (see[35] for comparison of several IEGs activations by enrichedenvironment) Perhaps the plastic rearrangement of connec-tions induced by deprivation although it already changesthe metabolic response [18] does not yet trigger activationin Npas4 mRNA expression Npas4 can regulate activity-dependent expression of Arc c-Fos and Zif268 [11] Takinginto consideration that Arc c-Fos and Zif268 expressionsincrease in deprived barrels after 7 days of deprivation it mayseem puzzling that expression of Npas4 which regulates thetranscription of these genes remains unaltered However itshould be noted that Npas4 itself is an immediate early geneand gets activated in response to stimulation Accordingly itprobably does not regulate the first phase of other immediateearly genes expression which is independent of de novoprotein synthesis It rather seems that Npas4 plays a rolein enhancing and sustaining other immediate early genes inlater phases [11]
To the best of our knowledge this is the first report onNpas4 expression in deprivation-induced plasticity Maya-Vetencourt et al [13] examined Npas4 expression in monoc-ularly deprived rats treated with fluoxetine but they concen-trated on influence of fluoxetine onNpas4 expression and noton the influence of deprivation
8 Neural Plasticity
In this paper the precise anatomical dissection of arow of barrels where a plastic change took place allowedfor a quantitative analysis of IEGs expression in two typesof experience dependent plasticity Activation of the barrelcortex undergoing reorganization triggered by removal ofselected rows of whiskers strongly affected c-Fos (but notNpas4) expression Activation of the cortex undergoing aplastic change due to being involved in sensory conditioningupregulated the expression of both Npas4 and c-Fos Takinginto consideration our observations that sensory condition-ing increases the number of inhibitory synapses withinthe trained barrels and studies of other groups showinginvolvement of Npas4 in synaptogenesis we presume thatNpas4 may be involved in reshaping of connectivity withinbarrel cortex after sensory conditioning
Conflict of Interests
The authors declare no conflict of interests
Acknowledgment
This project was supported by GrantMNiSWno 2486BP012010 to Malgorzata Kossut
References
[1] M Moser R Knoth C Bode and C Patterson ldquoLE-PAS anovel Arnt-dependent HLH-PAS protein is expressed in limbictissues and transactivates the CNS midline enhancer elementrdquoMolecular Brain Research vol 128 no 2 pp 141ndash149 2004
[2] N Ooe K Saito N Mikami I Nakatuka and H KanekoldquoIdentification of a novel basic helix-loop-helix-PAS factorNXF reveals a Sim2 competitive positive regulatory role indendritic-cytoskeleton modulator drebrin gene expressionrdquoMolecular and Cellular Biology vol 24 no 2 pp 608ndash616 2004
[3] Y Lin B L Bloodgood J L Hauser et al ldquoActivity-dependentregulation of inhibitory synapse development by Npas4rdquoNature vol 455 no 7217 pp 1198ndash1204 2008
[4] S Sim S Antolin C-W Lin Y-X Lin and C Lois ldquoIncreasedcell-intrinsic excitability induces synaptic changes in new neu-rons in the adult dentate gyrus that require Npas4rdquo Journal ofNeuroscience vol 33 no 18 pp 7928ndash7940 2013
[5] B L Bloodgood N Sharma H A Browne A Z Trepman andM E Greenberg ldquoThe activity-dependent transcription factorNPAS4 regulates domain-specific inhibitionrdquo Nature vol 503no 7474 pp 121ndash125 2013
[6] I Spiegel A R Mardinly H W Gabel et al ldquoNpas4 regulatesexcitatory-inhibitory balance within neural circuits throughcell-type-specific gene programsrdquo Cell vol 157 no 5 pp 1216ndash1229 2014
[7] J Yun T Nagai Y Furukawa-Hibi et al ldquoNeuronal per arnt sim(PAS) domain protein 4 (NPAS4) regulates neurite outgrowthand phosphorylation of synapsin Irdquo The Journal of BiologicalChemistry vol 288 no 4 pp 2655ndash2664 2013
[8] G Pouchelon F Gambino C Bellone et al ldquoModality-specificthalamocortical inputs instruct the identity of postsynaptic L4neuronsrdquo Nature vol 511 no 7510 pp 471ndash474 2014
[9] S Yoshihara H Takahashi N Nishimura et al ldquoNpas4 regu-lates Mdm2 and thus Dcx in experience-dependent dendritic
spine development of newborn olfactory bulb interneuronsrdquoCell Reports vol 8 no 3 pp 843ndash857 2014
[10] K Hayashi R Ishikawa L-H Ye et al ldquoModulatory role ofdrebrin on the cytoskeleton within dendritic spines in the ratcerebral cortexrdquo Journal of Neuroscience vol 16 no 22 pp 7161ndash7170 1996
[11] K Ramamoorthi R Fropf GM Belfort et al ldquoNpas4 regulatesa transcriptional program in CA3 required for contextualmemory formationrdquo Science vol 334 no 6063 pp 1669ndash16752011
[12] J E Ploski M S Monsey T Nguyen R J DiLeone and G ESchafe ldquoThe neuronal PAS domain protein 4 (Npas4) isrequired for new and reactivated fear memoriesrdquo PLoS ONEvol 6 no 8 Article ID e23760 2011
[13] JFMaya-Vetencourt E Tiraboschi DGreco et al ldquoExperience-dependent expression of NPAS4 regulates plasticity in adultvisual cortexrdquo Journal of Physiology vol 590 no 19 pp 4777ndash4787 2012
[14] M Kossut P J Hand J Greenberg and C L Hand ldquoSinglevibrissal cortical column in SI cortex of rat and its alterations inneonatal and adult vibrissa-deafferented animals a quantitative2DG studyrdquo Journal of Neurophysiology vol 60 no 2 pp 829ndash852 1988
[15] S Glazewski andK Fox ldquoTime course of experience-dependentsynaptic potentiation and depression in barrel cortex of adoles-cent ratsrdquo Journal of Neurophysiology vol 75 no 4 pp 1714ndash1729 1996
[16] H Wallace and K Fox ldquoLocal cortical interactions determinethe form of cortical plasticityrdquo Journal of Neurobiology vol 41no 1 pp 58ndash63 1999
[17] M A Lebedev G Mirabella I Erchova and M E DiamondldquoExperience-dependent plasticity of rat barrel cortex redistri-bution of activity across barrel-columnsrdquo Cerebral Cortex vol10 no 1 pp 23ndash31 2000
[18] A Kaliszewska M Bijata L Kaczmarek and M KossutldquoExperience-dependent plasticity of the barrel cortex in miceobserved with 2-DG brain mapping and c-Fos effects of MMP-9 KOrdquo Cerebral Cortex vol 22 no 9 pp 2160ndash2170 2012
[19] D J Margolis H Lutcke K Schulz et al ldquoReorganization ofcortical population activity imaged throughout long-term sen-sory deprivationrdquo Nature Neuroscience vol 15 no 11 pp 1539ndash1546 2012
[20] S AMarikH Yamahachi J N JMcManusG Szabo andCDGilbert ldquoAxonal dynamics of excitatory and inhibitory neuronsin somatosensory cortexrdquo PLoS Biology vol 8 no 6 Article IDe1000395 2010
[21] V C Wimmer P J Broser T Kuner and R M BrunoldquoExperience-induced plasticity of thalamocortical axons inboth juveniles and adultsrdquo Journal of Comparative Neurologyvol 518 no 22 pp 4629ndash4648 2010
[22] M Oberlaender A Ramirez and R M Bruno ldquoSensory expe-rience restructures thalamocortical axons during adulthoodrdquoNeuron vol 74 no 4 pp 648ndash655 2012
[23] D Katzel and G Miesenbock ldquoExperience-dependent rewiringof specific inhibitory connections in adult neocortexrdquo PLoSBiology vol 12 no 2 Article ID e1001798 2014
[24] C Tailby L LWright A BMetha andM B Calford ldquoActivity-dependent maintenance and growth of dendrites in adultcortexrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 102 no 12 pp 4631ndash4636 2005
Neural Plasticity 9
[25] C E J Cheetham M S L Hammond R McFarlane and G TFinnerty ldquoAltered sensory experience induces targeted rewiringof local excitatory connections in mature neocortexrdquo Journal ofNeuroscience vol 28 no 37 pp 9249ndash9260 2008
[26] K D Micheva and C Beaulieu ldquoAn anatomical substrate forexperience-dependent plasticity of the rat barrel field cortexrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 25 pp 11834ndash11838 1995
[27] Y Zuo G Yang E Kwon and W-B Gan ldquoLong-term sen-sory deprivation prevents dendritic spine loss in primarysomatosensory cortexrdquo Nature vol 436 no 7048 pp 261ndash2652005
[28] A Holtmaat L Wilbrecht G W Knott E Welker and KSvoboda ldquoExperience-dependent and cell-type-specific spinegrowth in the neocortexrdquo Nature vol 441 no 7096 pp 979ndash983 2006
[29] C E J Cheetham M S L Hammond C E J Edwards and GT Finnerty ldquoSensory experience alters cortical connectivity andsynaptic function site specificallyrdquo The Journal of Neurosciencevol 27 no 13 pp 3456ndash3465 2007
[30] L Wilbrecht A Holtmaat N Wright K Fox and K Svo-boda ldquoStructural plasticity underlies experience-dependentfunctional plasticity of cortical circuitsrdquo Journal of Neurosciencevol 30 no 14 pp 4927ndash4932 2010
[31] C E J Cheetham S J Barnes G Albieri G W Knott and GT Finnerty ldquoPansynaptic enlargement at adult cortical connec-tions strengthened by experiencerdquo Cerebral Cortex vol 24 no2 pp 521ndash531 2014
[32] E Siucinska and M Kossut ldquoShort-lasting classical condition-ing induces reversible changes of representational maps ofvibrissae inmouse SI cortex-A 2DG studyrdquoCerebral Cortex vol6 no 3 pp 506ndash513 1996
[33] M Jasinska E Siucinska A Cybulska-Klosowicz et al ldquoRapidlearning-induced inhibitory synaptogenesis in murine barrelfieldrdquo The Journal of Neuroscience vol 30 no 3 pp 1176ndash11842010
[34] R N Strominger and T AWoolsey ldquoTemplates for locating thewhisker area in fresh flattened mouse and rat cortexrdquo Journal ofNeuroscience Methods vol 22 no 2 pp 113ndash118 1987
[35] A Valles A J Boender S Gijsbers R A M Haast G J MMartens and P de Weerd ldquoGenomewide analysis of rat barrelcortex reveals time- and layer-specificmrna expression changesrelated to experience-dependent plasticityrdquo Journal of Neuro-science vol 31 no 16 pp 6140ndash6158 2011
[36] A Kaliszewska Expression of selected immediate early genesand involvement of matrix metalloproteinase 9 in functionalreorganization of mouse barrel cortex [Doctoral dissertation]Nencki Institute of Experimental Biology Polish Academy ofSciences Warsaw Poland 2012
[37] A Cybulska-Klosowicz R Zakrzewska and M Kossut ldquoBrainactivation patterns during classical conditioning with appetitiveor aversive UCSrdquo Behavioural Brain Research vol 204 no 1 pp102ndash111 2009
[38] B Lu K H Wang and A Nose ldquoMolecular mechanismsunderlying neural circuit formationrdquo Current Opinion in Neu-robiology vol 19 no 2 pp 162ndash167 2009
[39] Y Jiao Z Zhang C Zhang et al ldquoA key mechanism under-lying sensory experience-dependent maturation of neocorti-cal GABAergic circuits in vivordquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 108 no29 pp 12131ndash12136 2011
[40] K Tokarski J Urban-Ciecko M Kossut and G Hess ldquoSensorylearning-induced enhancement of inhibitory synaptic trans-mission in the barrel cortex of the mouserdquo European Journal ofNeuroscience vol 26 no 1 pp 134ndash141 2007
[41] A Cybulska-Klosowicz A Posluszny K Nowak E SiucinskaM Kossut and M Liguz-Lecznar ldquoInterneurons containingsomatostatin are affected by learning-induced cortical plastic-ityrdquo Neuroscience vol 254 pp 18ndash25 2013
[42] Z-H Hou and X Yu ldquoActivity-regulated somatostatin expres-sion reduces dendritic spine density and lowers excitatorysynaptic transmission via postsynaptic somatostatin receptor 4rdquoJournal of Biological Chemistry vol 288 no 4 pp 2501ndash25092013
[43] S A Eichler and J CMeier ldquoE-I balance and human diseasesmdashfrom molecules to networkingrdquo Frontiers in Molecular Neuro-science vol 1 article 2 2008
[44] L Coutellier S Beraki P M Ardestani N L Saw and MShamloo ldquoNpas4 a neuronal transcription factor with a keyrole in social and cognitive functions relevant to developmentaldisordersrdquo PLoS ONE vol 7 no 9 Article ID e46604 2012
[45] E J Jaehne T S Klaric S A Koblar B T Baune and MD Lewis ldquoEffects of Npas4 deficiency on anxiety depression-like cognition and sociability behaviourrdquo Behavioural BrainResearch vol 281 pp 276ndash282 2015
[46] D C Bersten J B Bruning D J Peet and M L WhitelawldquoHuman variants in the neuronal basic helix-loop-helixPer-Arnt-Sim (bHLHPAS) transcription factor complex NPAS4ARNT2 disrupt functionrdquo PLoS ONE vol 9 no 1 Article IDe85768 2014
[47] S Maren ldquoPavlovian fear conditioning as a behavioral assayfor hippocampus and amygdala function cautions and caveatsrdquoEuropean Journal of Neuroscience vol 28 no 8 pp 1661ndash16662008
[48] B Jabłonska M Kossut and J Skangiel-Kramska ldquoTransientincrease of AMPA and NMDA receptor binding in the barrelcortex of mice after tactile stimulationrdquo Neurobiology of Learn-ing and Memory vol 66 no 1 pp 36ndash43 1996
[49] M Gierdalski B Jablonska E SiucinskaM Lech A Skibinskaand M Kossut ldquoRapid regulation of GAD67 mRNA and pro-tein level in cortical neurons after sensory learningrdquo CerebralCortex vol 11 no 9 pp 806ndash815 2001
Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
8 Neural Plasticity
In this paper the precise anatomical dissection of arow of barrels where a plastic change took place allowedfor a quantitative analysis of IEGs expression in two typesof experience dependent plasticity Activation of the barrelcortex undergoing reorganization triggered by removal ofselected rows of whiskers strongly affected c-Fos (but notNpas4) expression Activation of the cortex undergoing aplastic change due to being involved in sensory conditioningupregulated the expression of both Npas4 and c-Fos Takinginto consideration our observations that sensory condition-ing increases the number of inhibitory synapses withinthe trained barrels and studies of other groups showinginvolvement of Npas4 in synaptogenesis we presume thatNpas4 may be involved in reshaping of connectivity withinbarrel cortex after sensory conditioning
Conflict of Interests
The authors declare no conflict of interests
Acknowledgment
This project was supported by GrantMNiSWno 2486BP012010 to Malgorzata Kossut
References
[1] M Moser R Knoth C Bode and C Patterson ldquoLE-PAS anovel Arnt-dependent HLH-PAS protein is expressed in limbictissues and transactivates the CNS midline enhancer elementrdquoMolecular Brain Research vol 128 no 2 pp 141ndash149 2004
[2] N Ooe K Saito N Mikami I Nakatuka and H KanekoldquoIdentification of a novel basic helix-loop-helix-PAS factorNXF reveals a Sim2 competitive positive regulatory role indendritic-cytoskeleton modulator drebrin gene expressionrdquoMolecular and Cellular Biology vol 24 no 2 pp 608ndash616 2004
[3] Y Lin B L Bloodgood J L Hauser et al ldquoActivity-dependentregulation of inhibitory synapse development by Npas4rdquoNature vol 455 no 7217 pp 1198ndash1204 2008
[4] S Sim S Antolin C-W Lin Y-X Lin and C Lois ldquoIncreasedcell-intrinsic excitability induces synaptic changes in new neu-rons in the adult dentate gyrus that require Npas4rdquo Journal ofNeuroscience vol 33 no 18 pp 7928ndash7940 2013
[5] B L Bloodgood N Sharma H A Browne A Z Trepman andM E Greenberg ldquoThe activity-dependent transcription factorNPAS4 regulates domain-specific inhibitionrdquo Nature vol 503no 7474 pp 121ndash125 2013
[6] I Spiegel A R Mardinly H W Gabel et al ldquoNpas4 regulatesexcitatory-inhibitory balance within neural circuits throughcell-type-specific gene programsrdquo Cell vol 157 no 5 pp 1216ndash1229 2014
[7] J Yun T Nagai Y Furukawa-Hibi et al ldquoNeuronal per arnt sim(PAS) domain protein 4 (NPAS4) regulates neurite outgrowthand phosphorylation of synapsin Irdquo The Journal of BiologicalChemistry vol 288 no 4 pp 2655ndash2664 2013
[8] G Pouchelon F Gambino C Bellone et al ldquoModality-specificthalamocortical inputs instruct the identity of postsynaptic L4neuronsrdquo Nature vol 511 no 7510 pp 471ndash474 2014
[9] S Yoshihara H Takahashi N Nishimura et al ldquoNpas4 regu-lates Mdm2 and thus Dcx in experience-dependent dendritic
spine development of newborn olfactory bulb interneuronsrdquoCell Reports vol 8 no 3 pp 843ndash857 2014
[10] K Hayashi R Ishikawa L-H Ye et al ldquoModulatory role ofdrebrin on the cytoskeleton within dendritic spines in the ratcerebral cortexrdquo Journal of Neuroscience vol 16 no 22 pp 7161ndash7170 1996
[11] K Ramamoorthi R Fropf GM Belfort et al ldquoNpas4 regulatesa transcriptional program in CA3 required for contextualmemory formationrdquo Science vol 334 no 6063 pp 1669ndash16752011
[12] J E Ploski M S Monsey T Nguyen R J DiLeone and G ESchafe ldquoThe neuronal PAS domain protein 4 (Npas4) isrequired for new and reactivated fear memoriesrdquo PLoS ONEvol 6 no 8 Article ID e23760 2011
[13] JFMaya-Vetencourt E Tiraboschi DGreco et al ldquoExperience-dependent expression of NPAS4 regulates plasticity in adultvisual cortexrdquo Journal of Physiology vol 590 no 19 pp 4777ndash4787 2012
[14] M Kossut P J Hand J Greenberg and C L Hand ldquoSinglevibrissal cortical column in SI cortex of rat and its alterations inneonatal and adult vibrissa-deafferented animals a quantitative2DG studyrdquo Journal of Neurophysiology vol 60 no 2 pp 829ndash852 1988
[15] S Glazewski andK Fox ldquoTime course of experience-dependentsynaptic potentiation and depression in barrel cortex of adoles-cent ratsrdquo Journal of Neurophysiology vol 75 no 4 pp 1714ndash1729 1996
[16] H Wallace and K Fox ldquoLocal cortical interactions determinethe form of cortical plasticityrdquo Journal of Neurobiology vol 41no 1 pp 58ndash63 1999
[17] M A Lebedev G Mirabella I Erchova and M E DiamondldquoExperience-dependent plasticity of rat barrel cortex redistri-bution of activity across barrel-columnsrdquo Cerebral Cortex vol10 no 1 pp 23ndash31 2000
[18] A Kaliszewska M Bijata L Kaczmarek and M KossutldquoExperience-dependent plasticity of the barrel cortex in miceobserved with 2-DG brain mapping and c-Fos effects of MMP-9 KOrdquo Cerebral Cortex vol 22 no 9 pp 2160ndash2170 2012
[19] D J Margolis H Lutcke K Schulz et al ldquoReorganization ofcortical population activity imaged throughout long-term sen-sory deprivationrdquo Nature Neuroscience vol 15 no 11 pp 1539ndash1546 2012
[20] S AMarikH Yamahachi J N JMcManusG Szabo andCDGilbert ldquoAxonal dynamics of excitatory and inhibitory neuronsin somatosensory cortexrdquo PLoS Biology vol 8 no 6 Article IDe1000395 2010
[21] V C Wimmer P J Broser T Kuner and R M BrunoldquoExperience-induced plasticity of thalamocortical axons inboth juveniles and adultsrdquo Journal of Comparative Neurologyvol 518 no 22 pp 4629ndash4648 2010
[22] M Oberlaender A Ramirez and R M Bruno ldquoSensory expe-rience restructures thalamocortical axons during adulthoodrdquoNeuron vol 74 no 4 pp 648ndash655 2012
[23] D Katzel and G Miesenbock ldquoExperience-dependent rewiringof specific inhibitory connections in adult neocortexrdquo PLoSBiology vol 12 no 2 Article ID e1001798 2014
[24] C Tailby L LWright A BMetha andM B Calford ldquoActivity-dependent maintenance and growth of dendrites in adultcortexrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 102 no 12 pp 4631ndash4636 2005
Neural Plasticity 9
[25] C E J Cheetham M S L Hammond R McFarlane and G TFinnerty ldquoAltered sensory experience induces targeted rewiringof local excitatory connections in mature neocortexrdquo Journal ofNeuroscience vol 28 no 37 pp 9249ndash9260 2008
[26] K D Micheva and C Beaulieu ldquoAn anatomical substrate forexperience-dependent plasticity of the rat barrel field cortexrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 25 pp 11834ndash11838 1995
[27] Y Zuo G Yang E Kwon and W-B Gan ldquoLong-term sen-sory deprivation prevents dendritic spine loss in primarysomatosensory cortexrdquo Nature vol 436 no 7048 pp 261ndash2652005
[28] A Holtmaat L Wilbrecht G W Knott E Welker and KSvoboda ldquoExperience-dependent and cell-type-specific spinegrowth in the neocortexrdquo Nature vol 441 no 7096 pp 979ndash983 2006
[29] C E J Cheetham M S L Hammond C E J Edwards and GT Finnerty ldquoSensory experience alters cortical connectivity andsynaptic function site specificallyrdquo The Journal of Neurosciencevol 27 no 13 pp 3456ndash3465 2007
[30] L Wilbrecht A Holtmaat N Wright K Fox and K Svo-boda ldquoStructural plasticity underlies experience-dependentfunctional plasticity of cortical circuitsrdquo Journal of Neurosciencevol 30 no 14 pp 4927ndash4932 2010
[31] C E J Cheetham S J Barnes G Albieri G W Knott and GT Finnerty ldquoPansynaptic enlargement at adult cortical connec-tions strengthened by experiencerdquo Cerebral Cortex vol 24 no2 pp 521ndash531 2014
[32] E Siucinska and M Kossut ldquoShort-lasting classical condition-ing induces reversible changes of representational maps ofvibrissae inmouse SI cortex-A 2DG studyrdquoCerebral Cortex vol6 no 3 pp 506ndash513 1996
[33] M Jasinska E Siucinska A Cybulska-Klosowicz et al ldquoRapidlearning-induced inhibitory synaptogenesis in murine barrelfieldrdquo The Journal of Neuroscience vol 30 no 3 pp 1176ndash11842010
[34] R N Strominger and T AWoolsey ldquoTemplates for locating thewhisker area in fresh flattened mouse and rat cortexrdquo Journal ofNeuroscience Methods vol 22 no 2 pp 113ndash118 1987
[35] A Valles A J Boender S Gijsbers R A M Haast G J MMartens and P de Weerd ldquoGenomewide analysis of rat barrelcortex reveals time- and layer-specificmrna expression changesrelated to experience-dependent plasticityrdquo Journal of Neuro-science vol 31 no 16 pp 6140ndash6158 2011
[36] A Kaliszewska Expression of selected immediate early genesand involvement of matrix metalloproteinase 9 in functionalreorganization of mouse barrel cortex [Doctoral dissertation]Nencki Institute of Experimental Biology Polish Academy ofSciences Warsaw Poland 2012
[37] A Cybulska-Klosowicz R Zakrzewska and M Kossut ldquoBrainactivation patterns during classical conditioning with appetitiveor aversive UCSrdquo Behavioural Brain Research vol 204 no 1 pp102ndash111 2009
[38] B Lu K H Wang and A Nose ldquoMolecular mechanismsunderlying neural circuit formationrdquo Current Opinion in Neu-robiology vol 19 no 2 pp 162ndash167 2009
[39] Y Jiao Z Zhang C Zhang et al ldquoA key mechanism under-lying sensory experience-dependent maturation of neocorti-cal GABAergic circuits in vivordquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 108 no29 pp 12131ndash12136 2011
[40] K Tokarski J Urban-Ciecko M Kossut and G Hess ldquoSensorylearning-induced enhancement of inhibitory synaptic trans-mission in the barrel cortex of the mouserdquo European Journal ofNeuroscience vol 26 no 1 pp 134ndash141 2007
[41] A Cybulska-Klosowicz A Posluszny K Nowak E SiucinskaM Kossut and M Liguz-Lecznar ldquoInterneurons containingsomatostatin are affected by learning-induced cortical plastic-ityrdquo Neuroscience vol 254 pp 18ndash25 2013
[42] Z-H Hou and X Yu ldquoActivity-regulated somatostatin expres-sion reduces dendritic spine density and lowers excitatorysynaptic transmission via postsynaptic somatostatin receptor 4rdquoJournal of Biological Chemistry vol 288 no 4 pp 2501ndash25092013
[43] S A Eichler and J CMeier ldquoE-I balance and human diseasesmdashfrom molecules to networkingrdquo Frontiers in Molecular Neuro-science vol 1 article 2 2008
[44] L Coutellier S Beraki P M Ardestani N L Saw and MShamloo ldquoNpas4 a neuronal transcription factor with a keyrole in social and cognitive functions relevant to developmentaldisordersrdquo PLoS ONE vol 7 no 9 Article ID e46604 2012
[45] E J Jaehne T S Klaric S A Koblar B T Baune and MD Lewis ldquoEffects of Npas4 deficiency on anxiety depression-like cognition and sociability behaviourrdquo Behavioural BrainResearch vol 281 pp 276ndash282 2015
[46] D C Bersten J B Bruning D J Peet and M L WhitelawldquoHuman variants in the neuronal basic helix-loop-helixPer-Arnt-Sim (bHLHPAS) transcription factor complex NPAS4ARNT2 disrupt functionrdquo PLoS ONE vol 9 no 1 Article IDe85768 2014
[47] S Maren ldquoPavlovian fear conditioning as a behavioral assayfor hippocampus and amygdala function cautions and caveatsrdquoEuropean Journal of Neuroscience vol 28 no 8 pp 1661ndash16662008
[48] B Jabłonska M Kossut and J Skangiel-Kramska ldquoTransientincrease of AMPA and NMDA receptor binding in the barrelcortex of mice after tactile stimulationrdquo Neurobiology of Learn-ing and Memory vol 66 no 1 pp 36ndash43 1996
[49] M Gierdalski B Jablonska E SiucinskaM Lech A Skibinskaand M Kossut ldquoRapid regulation of GAD67 mRNA and pro-tein level in cortical neurons after sensory learningrdquo CerebralCortex vol 11 no 9 pp 806ndash815 2001
Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Neural Plasticity 9
[25] C E J Cheetham M S L Hammond R McFarlane and G TFinnerty ldquoAltered sensory experience induces targeted rewiringof local excitatory connections in mature neocortexrdquo Journal ofNeuroscience vol 28 no 37 pp 9249ndash9260 2008
[26] K D Micheva and C Beaulieu ldquoAn anatomical substrate forexperience-dependent plasticity of the rat barrel field cortexrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 25 pp 11834ndash11838 1995
[27] Y Zuo G Yang E Kwon and W-B Gan ldquoLong-term sen-sory deprivation prevents dendritic spine loss in primarysomatosensory cortexrdquo Nature vol 436 no 7048 pp 261ndash2652005
[28] A Holtmaat L Wilbrecht G W Knott E Welker and KSvoboda ldquoExperience-dependent and cell-type-specific spinegrowth in the neocortexrdquo Nature vol 441 no 7096 pp 979ndash983 2006
[29] C E J Cheetham M S L Hammond C E J Edwards and GT Finnerty ldquoSensory experience alters cortical connectivity andsynaptic function site specificallyrdquo The Journal of Neurosciencevol 27 no 13 pp 3456ndash3465 2007
[30] L Wilbrecht A Holtmaat N Wright K Fox and K Svo-boda ldquoStructural plasticity underlies experience-dependentfunctional plasticity of cortical circuitsrdquo Journal of Neurosciencevol 30 no 14 pp 4927ndash4932 2010
[31] C E J Cheetham S J Barnes G Albieri G W Knott and GT Finnerty ldquoPansynaptic enlargement at adult cortical connec-tions strengthened by experiencerdquo Cerebral Cortex vol 24 no2 pp 521ndash531 2014
[32] E Siucinska and M Kossut ldquoShort-lasting classical condition-ing induces reversible changes of representational maps ofvibrissae inmouse SI cortex-A 2DG studyrdquoCerebral Cortex vol6 no 3 pp 506ndash513 1996
[33] M Jasinska E Siucinska A Cybulska-Klosowicz et al ldquoRapidlearning-induced inhibitory synaptogenesis in murine barrelfieldrdquo The Journal of Neuroscience vol 30 no 3 pp 1176ndash11842010
[34] R N Strominger and T AWoolsey ldquoTemplates for locating thewhisker area in fresh flattened mouse and rat cortexrdquo Journal ofNeuroscience Methods vol 22 no 2 pp 113ndash118 1987
[35] A Valles A J Boender S Gijsbers R A M Haast G J MMartens and P de Weerd ldquoGenomewide analysis of rat barrelcortex reveals time- and layer-specificmrna expression changesrelated to experience-dependent plasticityrdquo Journal of Neuro-science vol 31 no 16 pp 6140ndash6158 2011
[36] A Kaliszewska Expression of selected immediate early genesand involvement of matrix metalloproteinase 9 in functionalreorganization of mouse barrel cortex [Doctoral dissertation]Nencki Institute of Experimental Biology Polish Academy ofSciences Warsaw Poland 2012
[37] A Cybulska-Klosowicz R Zakrzewska and M Kossut ldquoBrainactivation patterns during classical conditioning with appetitiveor aversive UCSrdquo Behavioural Brain Research vol 204 no 1 pp102ndash111 2009
[38] B Lu K H Wang and A Nose ldquoMolecular mechanismsunderlying neural circuit formationrdquo Current Opinion in Neu-robiology vol 19 no 2 pp 162ndash167 2009
[39] Y Jiao Z Zhang C Zhang et al ldquoA key mechanism under-lying sensory experience-dependent maturation of neocorti-cal GABAergic circuits in vivordquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 108 no29 pp 12131ndash12136 2011
[40] K Tokarski J Urban-Ciecko M Kossut and G Hess ldquoSensorylearning-induced enhancement of inhibitory synaptic trans-mission in the barrel cortex of the mouserdquo European Journal ofNeuroscience vol 26 no 1 pp 134ndash141 2007
[41] A Cybulska-Klosowicz A Posluszny K Nowak E SiucinskaM Kossut and M Liguz-Lecznar ldquoInterneurons containingsomatostatin are affected by learning-induced cortical plastic-ityrdquo Neuroscience vol 254 pp 18ndash25 2013
[42] Z-H Hou and X Yu ldquoActivity-regulated somatostatin expres-sion reduces dendritic spine density and lowers excitatorysynaptic transmission via postsynaptic somatostatin receptor 4rdquoJournal of Biological Chemistry vol 288 no 4 pp 2501ndash25092013
[43] S A Eichler and J CMeier ldquoE-I balance and human diseasesmdashfrom molecules to networkingrdquo Frontiers in Molecular Neuro-science vol 1 article 2 2008
[44] L Coutellier S Beraki P M Ardestani N L Saw and MShamloo ldquoNpas4 a neuronal transcription factor with a keyrole in social and cognitive functions relevant to developmentaldisordersrdquo PLoS ONE vol 7 no 9 Article ID e46604 2012
[45] E J Jaehne T S Klaric S A Koblar B T Baune and MD Lewis ldquoEffects of Npas4 deficiency on anxiety depression-like cognition and sociability behaviourrdquo Behavioural BrainResearch vol 281 pp 276ndash282 2015
[46] D C Bersten J B Bruning D J Peet and M L WhitelawldquoHuman variants in the neuronal basic helix-loop-helixPer-Arnt-Sim (bHLHPAS) transcription factor complex NPAS4ARNT2 disrupt functionrdquo PLoS ONE vol 9 no 1 Article IDe85768 2014
[47] S Maren ldquoPavlovian fear conditioning as a behavioral assayfor hippocampus and amygdala function cautions and caveatsrdquoEuropean Journal of Neuroscience vol 28 no 8 pp 1661ndash16662008
[48] B Jabłonska M Kossut and J Skangiel-Kramska ldquoTransientincrease of AMPA and NMDA receptor binding in the barrelcortex of mice after tactile stimulationrdquo Neurobiology of Learn-ing and Memory vol 66 no 1 pp 36ndash43 1996
[49] M Gierdalski B Jablonska E SiucinskaM Lech A Skibinskaand M Kossut ldquoRapid regulation of GAD67 mRNA and pro-tein level in cortical neurons after sensory learningrdquo CerebralCortex vol 11 no 9 pp 806ndash815 2001
