Serotonin 1A receptor-mediated signaling through ERK and PKC? is essential for normal synaptogenesis in neonatal mouse hippocampus.

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by  Mogha, A

Citation:TranslPsychiatry(2012)2,e66, doi:10.1038/tp.2011.58
&2012MacmillanPublishersLimited Allrightsreserved2158-3188/12
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Serotonin 1A receptor-mediated signaling through
ERK and PKCa is essential for normal synaptogenesis
in neonatal mouse hippocampus
AMogha1,SRGuariglia2,PRDebata2,GYWen2,3andPBanerjee1,2,3,4
Aberrant expression ofthepresynapticserotonin1Areceptor (5-HT -R)becauseofapolymorphisminthe 5-HT -R geneis
1A 1A
associatedwithseveredepressioninhuman,whereasitsabsenceuptopostnatalday21(P21)intheforebrainofmiceresults
in heightened anxiety in adulthood. These observations collectively indicate that the 5-HT -R has a crucial role in brain
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development. To understand the mechanistic underpinnings of this phenomenon, we used organotypic slice cultures of
hippocampi from C57BL6 mice (C57) at P15, which coincides with the peak of neonatal synaptogenesis. Stimulation of the
hippocampal 5-HT -R caused a dramatic increase in PSD95 expression and dendritic spine and synapse formation through
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sequential activation of the mitogen-activated protein kinase isozymes Erk1/2 and protein kinase C (PKC). Intrahippocampal
infusionof5-HT -RagonistsandsignalinginhibitorsatP15revealedthatthesamepathwaythroughPKCaaugmentsPSD95
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expression and synaptogenesis in vivo in 24h in both C57 as well as Swiss Webster mice. Furthermore, intrahippocampal
infusionoftheantidepressantfluoxetine,aserotoninreuptakeinhibitor,alsoaugmentedPSD95expressionandsynaptogenesis
throughthesamepathway.Thisincreasedsynaptogenesiswasobservedeven5daysaftertreatment.Finally,comparedwiththe
wildtype,the5-HT -R((cid:2)/(cid:2))miceharborsignificantlylesssynapsesinthehippocampus,butinfusionofthePKC-stimulatorand
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Alzheimer drug bryostatin into the 5-HT -R((cid:2)/(cid:2)) mice to bypass the non-existent 5-HT -R boosted PSD95 expression and
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synaptogenesis.Theelucidatedsignalingcascadeexplainshow5-HT -Rregulateshippocampalsculptingandfunction,which
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maydeterminetheaffectivephenotypeofanadult.
TranslationalPsychiatry(2012)2,e66;doi:10.1038/tp.2011.58;publishedonline10January2012
Introduction autoreceptors) starting from early brain development
elicits increased behavioral despair, which is believed to be
Signalingactivitymediatedbythebrainserotonin1Areceptor the mouse equivalent of depression. Further emphasizing
(5–HT -R) has been implicated in a large number of the importance of brain 5-HT -R levels, an earlier report
1A 1A
behavioral abnormalities. Both decreased as well as in- showedthataC-1019Gpolymorphisminthepromoterofthe
creased5–HT -Rsignalinginthebrainhavebeenlinkedtoa 5-HT receptor gene causes de-repressed expression of
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numberofaffectivedisorders.Inpost-mortemhumanstudies, the5-HT autoreceptorselectivelyintheserotonergicraphe´
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increased5–HT -Ragonistbindinghasbeenobservedinthe neuronsandsuggestadownregulationofthesamereceptor
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frontal cortex of schizophrenia patients.1 Positron emission inthepostsynapticneurons.8Thisisassociatedwithsevere
tomographyhaveindicatedabove-normallevelsofserotonin depression and suicide.9 Similarly, attenuated 5-HT -R
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inthe brainofadolescentautisticchildren,2whichmayelicit expression has been reported in the cortex of suicide
elevated5–HT -Rsignaling.Similarly,inmousestudies,an victims.10Finally,post-mortemanalysisofhumanbrainsam-
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increase in brain serotonin because of monoamine oxidase plesrevealedthatreduced5-HT -Rbindinginthetemporal
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A-deficiencyduringthecriticalperiodofdevelopmentresulted cortex correlates with aggressive behavior in Alzheimer
in the lack of barrels in the somatosensory cortex.3 Con- patients.11
versely, other studies showed that a systemic absence of Thus,priorstudiesstronglyindicatethatanoptimallevelof
the 5-HT receptor was associated with elevated levels 5-HT -R signaling is required during brain development
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of anxiety.4–6 More recent experiments have shown that for normal affect in adulthood. However, the biochemical
eliminationofthepresynaptic5-HT autoreceptorsbutnot cascades, which are initiated by serotonin binding to the
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theheteroreceptorsfromP14issufficientforthedevelopment 5-HT -R and the identity of downstream signaling proteins
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ofanxietyphenotypeinadulthood.7Thesamereportsubmits that help in the formation of brain structures and circuitry
that elimination of the 5-HT heteroreceptors (but not the during neonatal brain development have remained unclear.
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1DoctoralPrograminBiology,CityUniversityofNewYork,NY,USA;2CSI/IBRCenterforDevelopmentalNeuroscience,TheCityUniversityofNewYorkattheCollegeof
StatenIsland(CUNY),StatenIsland,NY,USA;3InstituteforBasicResearchinDevelopmentalDisabilities(OPWDD),StatenIsland,NY,USAand4Departmentof
Chemistry,TheCollegeofStatenIsland(CUNY),StatenIsland,NY,USA
Correspondence:ProfessorPBanerjee,DepartmentofChemistryandtheCSI/IBRCenterforDevelopmentalNeuroscience,TheCityUniversityofNewYorkat
TheCollegeofStatenIsland,6S-326,2800VictoryBLVD,StatenIsland,NY10314,USA.
E-mail:[email protected]
Keywords:hippocampus;5-HT receptor;neonatal;PKCalpha;synaptogenesis
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Received12July2011;revised18October2011;accepted1November2011
5-HT1Areceptor-linkedsynaptogenesis
AMoghaetal
2
This study focuses on the hippocampus, which harbors high hippocampiofthepups17attherateof1mlperminuteusinga
levels of the 5-HT heteroreceptors, to address four funda- stereotaxic set-up (KDS Model 310 plus infusion-withdrawal
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mental issues: (i) If increased 5-HT -R signaling affects syringepump).
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neonatal synaptogenesis; (ii) What signaling pathway is
involvedinthisprocess?(iii)Doestheabsenceofthiscascade Drug concentrations and infusion strategy. (See
inthe5-HT -R((cid:2)/(cid:2))miceaffectsynaptogenesis?(iv)Could Supplementary Methods for details). Drug concentrations
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we normalize synaptogenesis by stimulating a downstream were made for the observed hippocampal volume of 10ml
memberofthispathwayinthe5-HT -R((cid:2)/(cid:2))mice? at P15. Inhibitors were infused 30min before infusing
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Our earlier studies in mouse brain slices demonstrated a 8-OH-DPATor fluoxetine.
5-HT -R-evoked increase in field excitatory postsynaptic
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potential (fEPSP) in the Schaffer Collateral pathway of the Immunohistochemistry. (See Supplementary Methods
hippocampus at P15.12 This report also showed that a for details). Hippocampal sections were blocked in (0.1%
5-HT -R-linked mitogen-activated protein kinase pathway, Triton X-100, 10% serum from the animal used to raise the
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linkedtoproteinkinaseC(PKC),wasinvolvedintheboosted secondary antibody-in 1(cid:3)PBS) for overnight at 41C. This
synapticactivity.Thecurrentstudyaddressesthepossibility wasfollowedbytreatmentwithprimaryantibodyinantibody
thatthispathwayisoperantinvivointheP15hippocampus solution (0.1% Triton X-100, 2% serum-in 1(cid:3) PBS) for
andit regulatessynaptogenesis,whichis knowntobeatits 48h at 41C and then with fluorescent secondary antibody
peakinthecortexatP15.13 covalently linked to Alexa Fluor 488 (Green) or Alexa Fluor
Results presented here establish the importance of a 568(Red)for24h.
synaptogenicpathwayinvolving5-HT -R,ErkandPKCain
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the neonatal hippocampus. Hyper-normal levels of cortical Confocal microscopy of the immunostained slices,
serotoninisbelievedtooccurinautism2andalsowiththeuse fluorescencequantification,countingofspinenumbers
ofselectiveserotoninreuptakeinhibitorsduringadolescence. and statistical analysis. (See Supplementary Methods for
On the basis of our results, such a hyperserotonemic state details).Alltheimageswereacquiredwiththeframeaverage
couldelicitaberranthippocampaldevelopmentviaunwanted offourusingaNikonC1-LU3laser-scanningconfocalsystem
synaptogenesisthroughthesamesignalingpathway,thereby (NikonInstrumentsInc.,1300WaltWhitmanRoad,Melville,
causing local overconnectivity, which is believed to occur in NY, USA). The Nikon EZ-C1-system software was used to
autism.14 determine the total thickness of each slice after adjusting
channels to obtain pictures from each exciting wavelength
separately. In addition to images taken at (cid:3)4 and (cid:3)20,
Materialsandmethods
Z-stacks were also acquired at (cid:3)40 and such volume-
rendered representative images were used in the figures
Reagents. SeeSupplementary Methods.
shown in this report. All the values were then converted to
percent ‘Carrier-treated’. Statistical analysis was carried out
Animals. C57BL6 5-HT -R(þ/(cid:2)) mice were obtained as
1A usingANOVA withBonferroni posthoctest.
akindgiftfromDrLaurenceTecott4andthenbredtoobtain
5-HT -R((cid:2)/(cid:2)) mice. Genotyping was performed by PCR
1A Electronmicroscopicstudies. Animalswereanesthetized
using the following primers: Wild type: Fwd 50-ctgctcatgc
withtheketamineþxylazinemixtureaccordingtothesugges-
tggtcctctatg-30, Rvs 50-taggaggtagctcctgattcgc-30 (product:
ted dosage and were perfused intra-cardially with 2.5%
323bp);KO:Fwd50-caccttgctcctgccgagaaa-30,Rvs50-agaag
glutaraldehydeþ4% paraformaldehyde for 15min. Brains
gcgatagaaggcgatg-30 (product: 464bp). Swiss Webster
were removed and were post fixed in the same perfusion
(SW) 5-HT -R((cid:2)/(cid:2)) mice were obtained from Dr Toni
1A solution overnight. Hippocampi were removed and 1-mm
Shippenberg.15 Mice were housed in the College of Staten
thick sections were cut. After washing with 0.1M PBS for
IslandAnimalCareFacilityandhandledfollowingaprotocol
30min (3(cid:3)) brain sections were post-fixed in 1% osmium
approvedbytheCSIInstitutional AnimalCareCommittee.
tetra-oxidefor90min.18Sectionswererinsedagainwith0.1M
PBS for 30min and dehydrated with graded ethanol (50%,
Hippocampal slice culture. Details of the procedure for
70%, 85%, 95%, 2(cid:3) 100%) for10mineachstep, infiltrated
hippocampal slice culture have been reported earlier and
with propylene oxide and plastic Spurr medium, and finally
alsoincludedinthe SupplementaryMethods.12 embeddedwithpureSpurrmedium19inBEEMcapsulesand
polymerized in oven at 701C overnight. Ultrathin sections
Westernblotting. Detailsofthewestern-blottingprocedure (70–100nm) were placed on uncoated 200 mesh copper
havebeenincludedinthe SupplementaryMethods. grids,stainedwithsaturateduranylacetatein50%ethanolfor
2min, rinsed with 0.22mM Millipore-filtered distilled water for
Intra-hippocampalinjections. (SeeSupplementaryMethods 2min to obtain clean sections without any trace of uranyl
for details). Injections were made at stereotaxic coordinates acetateresidue,stainedwithReynolds’sleadcitratefor90s,
correspondingtoBregma:anterioposterior¼(cid:2)1.8mm,medio- and rinsed with Millipore-filtered distilled water for 90s.20
lateral¼(cid:2)1.5mm, dorsoventral¼(cid:2)1.8mm. This corresponds Sections were examined and imaged with a Hitachi 7500
toasiteinthedorsalhippocampusintheapicaldendriticzones electron microscopy operated at 80kV. Quantification was
of the CA1 region near the hippocampal fissure.16 A 10-ml achieved using about 10 sections per mouse hippocampus
Hamilton syringe was used to deliver the drugs into the andthreemicewereusedforeachtreatmentset.
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Results or without the pharmacological inhibitors into the right
hippocampus of P15 C57BL6 muse pups (Figures 2c
Thepostsynapticdensity(PSD)proteinof95KDa,PSD95—
and d). After 24h, the pups were subjected to intracardiac
a postsynaptic membrane-associated protein with a guany-
perfusion of paraformaldehyde followed by isolation of the
late kinase-like domain without any kinase activity—is an
ipsilateral (right) andcontralateral hippocampifor sectioning
established postsynaptic marker of spine and synapse
maturation.21–24 In neuronal signaling, it is reported to into30-mMslices.Thesectionswereprocessedforimmuno-
histochemistry. The red staining for PSD95 was markedly
exert a major influence on synaptic strength and plasticity
higherintheipsilateralhippocampusfromtheDPAT-injected
by recruiting AMPA receptors to the postsynaptic mem-
brains than in the contralateral hippocampus or the hippo-
brane.22,23 On the basis of this information, we used
campifromcarrier-treatedbrains(Figure2d).Zoominginon
PSD95 expression and electron microscopy to demonstrate
a CA1 region (indicated by the square area), we found that
5-HT -R-mediatedsynaptogenesisintheP15hippocampus
1A this increase in the PSD95 staining was absent or sharply
and also elucidate the signaling cascade involved in this
diminishedinslicesthatreceivedWAY100635,U0126orRo
process.
31-8220 (a selective PKCa inhibitor p10nM; IC50¼5nM)
in addition to DPAT18 (Figures 2a and b). This strongly
Stimulation of the 5-HT1A-R in organotypic cultures of indicates that the same 5-HT1A-R mediated signaling path-
P15 hippocampal slices augments PSD95 expression, wayasshownaboveinFigure2alsobooststheexpression
spineformationandsynaptogenesis. Adramatic5-HT1A- ofPSD95 invivo.
R-evoked increase in PSD95 expression was evident from It should be noted that a P15 hippocampal slice cultured
immunohistochemistry (Figure 1a) and it was inhibited in 6 days in vitro (6DIV) maintains its overall structure but
presenceofWAY100635andthepharmacologicalinhibitors spreadsoutsignificantlyonthesubstratum(membrane).This
of MEK (U0126, 10mM) and PKC (bisindolylmaleimide I or allows the individual PSD95-immunopositive dendrites and
GFX,2mM),respectively(Figures1a–d).PSD95wasshown spines to be imaged clearly (Figure 1). In contrast, the
as punctuate staining in the spines. The number of puncta cryosectionsofthefreshlyisolatedhippocampifrominvivo-
per unit length of the dendritic spines and puncta intensity treatedbrainsharbordenselypackeddendrites,thusyielding
representing the number of PSD95molecules per spine strong PSD95 immunostaining in the entire hippocampus
showed a striking increase in presence of the 5-HT1A-R (Figure 2). High-magnification images acquired at (cid:3)100
agonist8-OH-DPAT(DPAT).ThisDPAT-evokedincreasein showthepresenceofpunctatePSD95immunostainingonthe
spineformation waseliminatedin presenceof WAY,U0126 dendrites and spines especially following 8-OH-DPAT treat-
and GFX, thereby confirming the involvement of MEK- ment(Figures2e–g).
Erk1/2 signaling and PKC in the 5-HT1A-R-mediated Two-dimensional cultures of hippocampal neurons form
inductionofPSD95. synapticconnectionsinthehorizontalplane,whichallowsfor
Wealsomonitoredtheinfluenceof5-HT1A-Rsignalingon doublestainingofpre-andpostsynapticterminalsforspecific
synaptogenesisinorganotypicculturesofhippocampalslices markers such as synaptophysin (pre-synaptic) and PSD95
byelectronmicroscopy.TheculturedP15hippocampalslices (postsynaptic) and confocal imaging of both in the same
at6DIVweretreatedwiththe5-HT1A-Ragonist8-OH-DPAT plane. In sharp contrast, hippocampal tissue in the 6DIV
(DPAT) or carrier to show that the DPAT-treated slices cultures as well as the cryosections from in vivo treatment
harboredasignificantlyhighernumberofexcitatorysynapses werethree-dimensionalunits,inwhichsimultaneousimaging
(Po0.0001), which were marked by the presence of post- ofpre-andpostsynapticterminalswasdifficulttoachieveby
synaptic densities (Supplementary Figures 1a and b). confocalmicroscopy.Therefore,inadditiontodemonstrating
Simultaneously, we used western blotting to monitor the the induction of the postsynaptic protein PSD95, we have
influence of 5-HT1A-R signaling on PSD-95 expression in performed electron microscopy to image and quantify the
cultured hippocampal slices fromP15 mice. We observed a hippocampalsynapses.
significantincreasein PSD95 expressionin thepresenceof
DPAT(100nM).ThePSD95levelsreachedapeakafter16h
ofDPATtreatment(unpublisheddata)andtheinductionwas Increased synaptogenesis via PKCa downstream of
abrogated in the presence of the 5-HT -R antagonist
1A 5-HT -R in C57BL6 mouse hippocampus. In order to
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WAY100635 (WAY) (4mM), thus confirming the involvement determine the role of 5-HT -R mediated pathway in
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of the 5-HT -R in the induction of PSD95 (Supplementary
1A synaptogenesis in vivo we performed electron microscopic
Figure1c).
analysisonhippocampiobtainedfrombrainsthatwereinfused
intra-hippocampally with DPAT (final intrahippocampal
Intra-hippocampal infusion of DPAT and inhibitors into concentration of 100nM) in the absence or presence of
P15 mice elicit PSD95 induction in vivo through the different pharmacological inhibitors. We observed that DPAT
5-HT -R,Erk1/2,PKCapathway. Ourearlierstudiesinthe treatment caused a significant increase in the number of
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hippocampal HN2-5 cells and cultured hippocampal slices excitatory synapses in the hippocampal CA1 region. This
indicatedthata5-HT -R--ERK-PKCapathwayhasan increaseinsynaptogenesiswasabolishedinpresenceofany
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important role in neuroprotection as well as increased of the inhibitors: U0126, Ro 31-822018 and WAY. These
synaptic activity.12,25 In order to confirm that the same resultsconfirmthat5-HT -R-mediatedsignalingviaErkand
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5-HT -R--ERK-PKCa pathway also functions in vivo, PKCa causes induced synaptogenesis in vivo in P15 mouse
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we performed intra-hippocampal injections26 of DPAT with hippocampus(Figure3).
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Intrahippocampal infusion of fluoxetine causes 5-HT - DownstreaminvolvementofPKCawasutilizedtoinduce
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R-, Erk1/2-, and PKCa-dependent induction of PSD95. PSD95 and synaptogenesis in C57BL6 5-HT -R((cid:2)/(cid:2))
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The optimum concentration of 5-HT agonist 8-OH-DPAT mice. To further confirm the role of 5-HT -R-mediated
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(100nM) for the activation of Erk1/2 had been determined signaling pathway in the expression of PSD95, we used
earlier from in vitro experiments using organotypic cultures 5-HT -R((cid:2)/(cid:2)) C57BL6 mice at P15. These mice were
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of P15 hippocampal slices.12 Although the same intra- injected with carrier, DPAT and bryostatin. Bryostatin is a
hippocampal concentration of 8-OH-DPAT allowed us to well-known memory-enhancing drug, which selectively
confirm that a 5-HT -R-mediated signaling through Erk1/2 activates PKC.18 We observed that PSD95 expression was
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and PKCa causes PSD95 induction and synaptogenesis, significantly higher (Po0.0001) in the bryostatin-treated
the physiological relevance of this phenomenon could be hippocampi than the carrier or DPAT-treated ones. This
elucidatedonlythroughanincreaseinintrasynapticserotonin enhancementwasobservedintheCA1regionofthehippo-
concentration above the basal levels (o1nM) in the P15 campus(Figures6aandb)aswellasCA3andDGregionsof
mouse hippocampus.15,27 This has been typically achieved the hippocampus (data not shown here). Simultaneous
byadministering fluoxetine (Prozac), which is an inhibitor of electron microscopy analysis revealed that the bryostatin-
theserotoninreuptakeproteinSERT.Albeitthesameeffect treated hippocampi harbored significantly more CA1
from systemic infusion in adult mice,15,27 direct intracortical excitatorysynapses(Figures6c–f).
infusion of a fluoxetine analog also causes a 3-5-fold
increase in extracellular serotonin within 60min.28 We
investigated whether such augmented serotonin levels at InvivoanalysisinSWmiceatP15showtheinvolvement
the hippocampal synapses would elicit the same 5-HT -R, of PKCa in the induction of PSD95. To address the
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ErkandPKCa-mediatedsynaptogenicsignalinginP15mice. question whether this 5-HT -R- and PKCa-mediated
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As shown in Figures 4 and 5, intrahippocampal infusion of signaling was strain-independent, we performed parallel
fluoxetine into P15 mice caused a significant increase in experiment with wild-type SW mice with intact 5-HT -Rs.
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PSD95 expression and synaptogenesis and this effect was InhibitionofthedownstreameffectorPKCawithRo31-8220
eliminated upon inhibition of the Erk activator MEK with againcausedeliminationofthe5-HT -R-mediatedinduction
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U0126andPKCawithRo31-8220. of PSD95 (Supplementary Figures 3a and b). In order to
confirm the involvement of PKCa in the increased
The hierarchy of signaling molecules in the 5-HT - hippocampal expression of PSD95 at P15 in this mouse
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R--Erk1/2-PKCa pathway in C57BL6 mouse strain,weusedSW5-HT -R((cid:2)/(cid:2))miceandstimulatedthe
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hippocampus. The widespread induction of PSD95 over downstream molecule, PKCa, by injecting the suggested
the entire hippocampus (Figure 3d) enabled us to perform dose of bryostatin18 intra-hippocampally. As expected,
western-blotanalysisonwholehippocampallysatestostudy DPAT treatment elicited no increase in PSD95 staining in
thepossiblelinkamong5-HT -R,ErkandPKCa.Weused these 5-HT -R-deficient mice, but bryostatin-treatment
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T638 phosphorylation of PKCa and dual phosphorylation resulted in a significantly higher level of PSD95 expression
of T202 and Y204 on Erk1/2 as a measure of stimulation of (Supplementary Figures 3c and d). This re-confirms that
thesekinases.AsshowninSupplementaryFigures2aandb, the 5-HT -R-mediated signaling pathway leads to higher
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short-term stimulation of the hippocampal 5-HT -R by expression ofPSD95 inthehippocampusofP15SWmice.
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intrahippocampal infusion of 8-OH-DPAT (final concen-
tration of 100nM) in the absence and presence of inhibitors
followedbykillingofthetreatedmiceafter60min,removalof Absence of the 5-HT -R results in a decreased density
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hippocampi,andlysisinproteaseinhibitor-containingbuffers of excitatory synapses in the hippocampus of P15
yielded protein samples that could be analyzed by western mice. On the basis of our observation that 5-HT -R
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blotting for P-PKCa and P-Erk. We observed a significant signaling in the hippocampus was critical in synapto-
inductionofP-PKCaintheDPAT-infusedhippocampi,which genesis, an obvious question remains, ‘Is the hippocampal
was eliminated in the presence of WAY, U0126 and Ro 31- synaptic density significantly lower in the 5-HT -R((cid:2)/(cid:2))
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8220 (Supplementary Figure 2a). In contrast, 5-HT -R- mice?’AsshowninSupplementaryFigures4a,b,andc,the
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mediatedinduction of P-Erk was eliminated in the presence 5-HT -R((cid:2)/(cid:2)) mice harbor a significantly lower number
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of WAY and U0126 but not Ro 31-8220 (Supplementary of excitatory synapses in the CA1 region (Po0.0001). As
Figure 2b). These two data sets place Erk above PKCa explained in the discussion, this could have a detrimental
in a 5-HT -R signaling pathway the long-term effect of effect on hippocampal maturation, which in turn could
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which would be increased synaptogenesis (Supplementary account for the behavioral abnormalities observed by
Figure2c). othersinthesemice.4–7
Figure1 IncreasedPSD95indendriticspinesupon5-HT -Ractivationinculturedhippocampalslices:involvementofErk1/2andPKC.OrganotypicculturesofP15
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hippocampalslicesat6DIVweretreatedwithDPAT(100nM)(abbreviatedasDinthegraphs)intheabsenceandpresenceofWAY100635(4mM)(WAY),U0126(10mM)(U),
orGFX(2mM)for16h(Scalebar:50mM).ThesliceswerefixedandimmunostainedtodetectPSD95expressionindendriticspines.DPATtreatmentcausedasignificant
increaseinbothPSD95expression(punctaintensity,c,e,andg)aswellasspinenumberperunitlength(b,d,andf).ThisincreaseinPSD95andspinedensitywas
eliminatedinthepresenceofWAY100635,U0126andGFX(a-g).Quantificationfromthreeexperimentseachwithtriplicateslicesrevealedsignificance(b,d,andf:
*Po0.001DPATversusallothergroups);(c,e,andg:*Po0.001DPATversusallothergroups;n¼3).
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N
u
e
N
5
9
D
S
P
d
e
g
r
e
M
Carrier DPAT WAY WAY+DPAT U+DPAT Ro+DPAT BAY3702
200 Ipsilateral Contralateral
*
Expression in15 (% Carrier) 115000 ** Carrier
PSD95 CA1 at P 50
0
CARRIER DPAT (D) WAY WAY+D U01026+D Ro31-8220+D BAY DPAT
Carrier DPAT WAY + D
Figure2 A5-HT1A-R-mediatedsignalingpathwayinvolvingErk1/2andPKCacausesheightenedexpressionofPSD95inP15mousehippocampus.(a)ADPAT(100nM)-
evokedinductioninPSD95expressionin24hiseliminatedinthepresenceofWAY100635(4mM)(WAY),U0126(10mM)(U),andtheselectivePKCainhibitorRo31-8220at
10nM(Ro).Asecond5-HT1A-RagonistBAY3702(100nM)alsocausesinducedPSD95expression(Scalebar:50mM).(b)Densitometricquantificationfromthreeinfusions
(threemiceandquadruplicatesectionsfromeachmouse)showsasignificantinductionofPSD95followinginfusionofDPATorBAY3702(BAY)(*Po0.001DPATversusall
othergroupsexceptBAY3702;**P¼0.1345DPATversusBAY3702)(n¼3).(c)Theinjectiontrackandthesiteofinjecteddrugsinthedorsalhippocampusmarkedby
CoommassieBlue.(d)TheipsilateralhippocampusshowsDPAT-evokedinductionofPSD95(red),whereasthecontralateralhippocampusdoesnot.Theseimageswere
acquiredat (cid:3)4magnification(Scale:500mM).ThesquareareaintheCA1regionoftheipsilateralhippocampuswasimagedat (cid:3)40andthenusedforthecomparisonof
histochemicalstainingpresentedinpanela.(e–g)High-magnificationimagesacquiredat (cid:3)100showstrong,punctate,PSD95immunoreactivityinnumerousdendritesand
spinesintheCA1regionofaDPAT-infusedhippocampus.
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Carrier DPAT WAY+DPAT
200 *
mber of synapse per2µ (% carruer)00m11505000
u1
N
0
CARRIER DPAT (D) WAY+D U0126+D Ro1-8220+D
3
U0126+DPAT Ro+DPAT
Figure3 The5-HT -R,Erk,PKCasignalingcascadeinducessynaptogenesisintheP15hippocampus.(a)Electronmicroscopyrevealedasignificantincreasein
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synapsenumberinthehippocampalCA1regionoftheDPAT-infusedmice.ThisDPAT-stimulationofsynaptogenesiswaseliminatedinthepresenceofWAY100635(4mM),
U0126(10mM)andRo31-8820(Ro)(Scalebar:500nm;post-synapticterminalshighlightedinyellow).(b)Quantificationfromthreemicewitheightsectionspermouse
revealedasignificanteffectof5-HT1A-Rsignaling,whichwaseliminatedinthepresenceofU0126andRo31-8820(*Po0.001forDPATversusallothergroups;n¼3).
Theinducedsynaptogenesiswaspersistenteven5days enhancing drug bryostatin (Figure 6 and Supplementary
after infusion of 8-OH-DPAT or Fluoxetine. A pertinent Figure3).18Thesame5-HT -R-mediatedPSD95induction
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argument is that the observed increase in synaptogenesis and synaptogenesis is observed in the presence of prozac
could be only transient and therefore of insignificant (Flx) (Figures 4 and 5), which causes an increase in
consequence. To address this important point, we per- extracellularserotonin.Thisraisesthepossibilitythatalocal
formedelectronmicroscopy5daysaftercarrier,8-OH-DPAT increaseinserotoninduringinappropriatestagesmayresultin
orfluoxetinetreatment.AsshowninSupplementaryFigure5, unwanted overconnectivity in cortical centers. By applying
the boosted density of excitatory synapses observed in intra-hippocampal infusion of drugs, we have avoided all
theCA1regionafter24hpersistedeven5daysafterDPAT confounds ensuing from systemic application of agonists or
or Flx treatment at P15. Intriguingly, systemic infusion Flx, which could cause differential activation of somato-
(intraperitoneal) of fluoxetine from P15, both once as well dendritic and hetero 5-HT -R molecules. Furthermore, our
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as chronic (daily) for 5 days, caused an inhibition of the experiments involved infusion of pharmacological agents
number of CA1 synapses compared with the carrier-infused into the right hippocampus, which left the contralateral
controls(SupplementaryFigure6). hippocampusessentiallyunaffected,therebyconfirmingthat
the 5-HT -R agonists produce a specific effect in the
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ipsilateralhippocampus(Figure2d).
Discussion
Confirming our concern about confounds ensuing from
Mice open their eyes between P12 and P14 and closely systemingadministrationofFlx,intraperitonealinfusionofFlx
thereafter (P15) is a stage of heightened synaptogenesis.29 into P15 mice caused an inhibition in the number of CA1
Our report shows that 5-HT -R signaling has a crucial synapsescomparedwiththatobservedincarrier-infusedmice
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role at this stage by triggering PSD95 expression and (SupplementaryFigure6).Itshouldbenotedthattheraphe´
formation of excitatory synapses in the hippocampus at neurons,whichactuallysynthesize5-HTandexpress5-HT
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P15.30 A deficiency in 5-HT -R expression is associated receptors and SERT, develop much earlier than the hippo-
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withasignificantdecreaseinthesesynapses(Supplementary campalneurons.Therefore,itisquitelikelythatperipherally
Figure 4) and the 5-HT -R-linked increase in excitatory administered fluoxetine first reaches brain-stem raphe´ neu-
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synapsesinthewild-typehippocampalCA1regionpersistsfor rons, blocks the local SERT molecules, causing a local
at least 5 days (Supplementary Figure 5). By elucidating a increaseinextracellular5-HT,whichinturnbindstotheample
5-HT signalingpathwaylinkedtosynaptogenesis,wealso somatodendritic 5-HT receptors to cause an inhibition of
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submitapossiblestrategyofcombating5-HT -Rdeficiency 5-HT firing at the hippocampal terminals. In contrast, when
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via downstream stimulation of PKCa with the memory- systemic fluoxetine molecules eventually diffuse to the
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Figure4 InfusionoffluoxetineintoP15mousehippocampuscausesinductionofPSD95via5-HT -R,Erk1/2,andPKCa.(a)Intrahippocampalinfusionoffluoxetine(Flx)
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causedasignificantincreaseinPSD95expressioninallregionsofthehippocampus,butconfocalimagesfromCA1areshown(Scalebar:50mM).(b–d)Quantificationfrom
threemiceperformedwithquadruplicatesectionspermouseshowedasignificantincreaseinPSD95expressioninthepresenceofFlx,whichwaseliminatedinthepresence
ofWAY100635,U0126andRo31-8820(Po0.0001forFlxversusallothergroupsforeachhippocampalregion;n¼3).
relativelyless-developedhippocampalregion,theireffecton taryFigures2bandc).Thisinferencewasconsistentwithour
thestill-developingsynapseswiththematuringpostsynaptic earlierobservation that in hippocampal neuron-derived HN2
hippocampal neurons is likely to be far less than that in the cells, Erk physically bound to and phosphorylated PKCa at
raphe´ region. This out-of-balance high Flx-evoked 5-HT T638, which was crucial for the activation of PKCa.31 Earlier
activityintheraphe´ regionisexpectedtocauseadecrease studies have also shown that 5-HT -R-mediated activation
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innet5-HTfiringatthehippocampalsynapses,whichcould of PLC(cid:2) occurs in a hippocampal neuron-derived cell line
explain the apparent decrease in the number of CA1 HN2-525,32 and many other neuronal cell lines through
synapsesaftersystemicFlxtreatmentofP15mice(Supple- Gi(cid:2)g.33,34ThispathwaycouldberesponsibleforIP3-mediated
mentary Figure 6). This should not be compared with calcium release from the endoplasmic reticulum, thereby
the effect of systemic administration of Flx in adult animals causingstimulationoftheRas-Raf-1-MEK-Erk1/2path-
wherein both the raphe´ as well as the hippocampal regions way via Ca2þ-sensitive signaling proteins like Pyk2 and
arefullydevelopedwiththefullrepertoireofSERTmolecules Src.35–38
and5-HTreceptors. The 5-HT -R agonist 8-OH-DPAT also stimulates the
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TheeffectsoftheinhibitorsoftheErkpathway(U0126)and adenylylcyclase-coupled5-HT receptorcausinganincrease
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PKCa(Ro31-8220)on5-HT -R-linkedPSD95inductionand inintracellularcAMPlevels.Thiswouldcausestimulationof
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synaptogenesisweremutuallyocclusive,whichindicatedthat proteinkinaseA,whichisknowntostimulatetheRashomolog
these two signaling molecules functioned in the same path- Rap-1 through phosphorylation. Stimulated Rap-1 binds to
way.Short-termDPATtreatment(60min)followedbywestern andactivatesB-RafthatphosphorylatesMEKtostimulatethe
blottingrevealedthattheactivationofPKCawassensitiveto Erkpathway.39Theconcentrationofthe5-HT -Rintheadult
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U0126,whichplacedPKCadownstreamofErk(Supplemen- cortexismuchlowerthanthatofthe5-HT -R.Nonetheless,
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Figure5 Intra-hippocampalinfusionoffluoxetineintoP15hippocampuscausesinductionofsynaptogenesisthrough5-HT -R,Erk1/2andPKCa.(a)Asignificant
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increaseinsynapseswasobservedintheCA1regionoftheFlx-infusedhippocampi.(b)Quantificationfromthreemiceperformedwitheightsectionspermouserevealeda
significantincreasethenumberofsynapsesinthepresenceofFlx,whichwaseliminatedinthepresenceofWAY,U0126andRo31-8820(Po0.0001forFlxversusallother
groups;n¼3).
based on 5-HT -R-mediated membrane depolarization, the in the pyramidal neurons of the CA1 area (fEPSP), which
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corticalexpressionofthetworeceptorsatP15appearstobe couldcauseincreasedsynapticstrength,inductioninPSD95,
comparable.40Although8-OH-DPATbindsmuchmoreavidly and augmented synaptogenesis as observed in the hippo-
to5-HT1A-R(KDB1nM)thanto5-HT7-R(KD¼50nM),41,42the campusafter24-hofinvivo8-OH-DPATtreatment.
relativeabundanceofthetwo8-OH-DPAT-bindingreceptors Alternative Erk and PKC-dependent mechanisms could
atP15evokestheprospectthatinthe5-HT -R((cid:2)/(cid:2))miceat alsooperatebeyondtheacutephaseoffEPSP(1h),causing
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P15, 8-OH-DPAT may cause 5-HT -R-mediated activation an induction in PSD95. Prior studies by Elkobi et al. have
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of Erk, thereby stimulating PKCa, which would then elicit demonstratedErk-dependentPSD95 inductioninthe gusta-
induced expression of PSD95. Contrary to this possibility, torycortexasanessentialstepintastelearning;48however,
we observed no 8-OH-DPAT-evoked increase in PSD95 the mechanistic link between Erk activity and PSD95
expression in 5-HT -R((cid:2)/(cid:2)) mice of either C57BL6 or SW induction has not been elucidated. Our results strongly
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background. Although volume-renderings of some z-stack indicate that Erk-dependent induction of PSD95 in the
images showed a slight increase (Supplementary Figure 3; hippocampus involves PKCa. Among molecules that may
SW5-HT -R(cid:2)/(cid:2))oraslightdecrease(Figure6)inPSD95 linkPKCtotheinductionofPSD95,neuroligin-1(Nrg),which
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immunostaininginthe8-OH-DPAT-treatedhippocampi,these elicits induced expression of PSD95,49 is a likely candidate.
changeswerenotsignificantamongthethreesetsofmiceand TheintracellulardomainofNrgisshedinaPKC-dependent
largenumberhippocampalslicesusedforstatisticalanalysis. manner,50 following which the released Nrg fragment com-
Insharpcontrast,inbothstrainsofmice,bryostatincauseda plexeswiththetranscriptionfactorEostobindtoanenhancer
dramaticandsignificantincreaseinPSD95expressioninthe elementinthePSD95promotertoelicitinducedexpressionof
CA1 region. Thus, 5-HT -R-mediated signaling is unlikely PSD95.49Furtherinvestigationwilltestthispossibility.
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to have any significant effect on PSD95 expression in the Performedinmiceof129S6/SvEvTac;C57BL/6;CBAmixed
P15hippocampus. background, pharmacological inhibition of 5-HT -R from
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Priorstudies indicate that multiple subunitsof the GABA P13-34hasbeenshowntophenocopytheeffectofneonatal
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receptor are phosphorylated by Ca2þ-dependent conven- absenceofthisreceptor,therebylinking5-HT -R-mediated
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tional PKCs,43–46 thereby causing inhibition of GABA CaMIIa stimulation to the maintenance of normal anxiety
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signaling and internalization of the receptor.47 Thus, the levels in adulthood.51 Our analyses, performed in mice of
activationofPKCa(aconventionalCa2þ-dependentPKC)via differentbackgrounds(C57BL6andSW),submitErk1/2and
5-HT -R-mediatedsignalingisexpectedtocauseaninhibi- PKCa as possible substrates for affective disorders, linked
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tionofGABA receptorfunctioninthetargetpyramidalCA1 to aberrant 5-HT -R expression/signaling. Intriguingly, in
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neurons of the SC pathway. Such reduction of inhibitory dissociated cell cultures, Ferreira et al. report the opposite
signalswouldleadtoanincreaseinthenetexcitatorysignals findingthat5-HT -Rsignalingcausessuppressionofgrowth
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Figure6 BryostatintreatmentofP155-HT -R((cid:2)/(cid:2))C57BL6miceleadstoheightenedexpressionofPSD95andboostedsynaptogenesis.(a)Inthe5-HT -R-deficient
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mice,PSD95stainingisincreasedintheCA1regioninbryostatin-,butnotDPAT-infusedmice(Scalebar50mM).(b)Quantificationofvolume-renderedimagesfromthree
experimentswithtriplicatesectionspertreatmentrevealedasignificantincreaseinPSD95inthepresenceofbryostatin(Po0.0001bryostatinversusallothergroups;n¼3).
(c–f)Electronmicroscopyrevealedasimultaneousincreaseinthenumberofsynapsesinthebryostatin-treatedanimals(Scalebar:500nm)(f,Po0.0001bryostatinversus
allothergroups;n¼3).
and sprouting of oblique dendrites.52 However, at 3–5 Ourexperimentshaveusedketamineforanesthesiaanda
postnatal weeks, the mice used in these studies were older recent study has demonstrated ketamine regulates hippo-
than the P15 mice used here. More consistent with our campal synapse formation.55 As both control and 5-HT
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observation, Chen et al. have demonstrated 5-HT -R- agonist-treatedmiceusedinourexperimentswereanesthe-
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mediatedmobilizationofmitochondria,53whichmaystimulate tizedusingthesameconcentrationofketamine,itisunlikely
synaptogenesis.54However,thespecificstageofP15hasnot that a different anesthetic would alter our conclusion. How-
beenthefocusofthesestudies,whichmaycontributetothe ever,itwillbeimportanttoanalyzehowanestheticsregulate
mechanistic difference (involving Akt-GSK3b) observed by synaptogenesisatP15.
Chenetal.53 Normal brain development requires that early postnatal
Inmouseandratpups,theeventofeyeopeningatP12-14 synaptogenesisbefollowedbyaphaseofsynapticpruningfor
is marked by a dramatic increase in PSD95 expression and therefinementofneuronalconnections.Basedonourresults
redistribution into synapases.29 Thus, as mentioned in the frominra-hippocampalinfusionofFlx,weinitiallyconjectured
introduction,P15isattheepicenterofamajorsynaptogenic that the use of prozac during the period of synaptic pruning
event,13andasignificantpartofitisvisualactivity-driven.By could impair brain development by triggering unwanted
elucidating 5-HT -R, Erk, and PKCa-mediated PSD95 synaptogenesisthroughthesame5-HT -Rpathway.Intrigu-
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induction and synaptogenesis and also demonstrating that ingly, systemic administration of Flx in P15 mice actually
the absence of the 5-HT -R in the 5-HT -R ((cid:2)/(cid:2)) mice is caused a significant decrease in the number of excitatory
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associated with a decrease in synapse density in the CA1 synapses in the CA1 region (Supplementary Figure 6).
region at P15, we have strengthened the possibility that the Therefore,administrationofprozacmayimpairneonatalbrain
hippocampal5-HT -Rhastheessentialroleoforchestrating developmentbyinhibitingsynaptogenesis.
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synaptogenesis. Our study also suggests that sluggish Our strategy of increasing local extracellular 5-HT by
hippocampal development because of impaired 5-HT -R, intrahippocampaladministrationofFlxmayreplicatethestate
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Erk,PKCasignalingcouldberemediedbytheuseofthePKC- of increased cortical serotonin, which has been reported in
activatingdrugbryostatin. juvenile autistic subjects.2 The resulting synaptogenesis
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