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Title:d-LysergicaciddiethylamidehasmajorpotentialasacognitiveenhancerAuthors:Felipe Augusto Cini 1*, Isis Ornelas 2*, Encarni Marcos 3*, Livia Goto-Silva 2,4,Juliana Nascimento 2,5, Sergio Ruschi 1, José Salerno 2,4, Karina Karmirian 2,4,MarceloCosta2,4,EduardoSequerra1,DráuliodeAraújo1,LuisFernandoTófoli5,César Rennó-Costa 6, Daniel Martins-de-Souza 5, Amanda Feilding 7, StevensRehen2,4#,SidartaRibeiro1#*Equalcontribution#Correspondingauthors Affiliations:1- Brain Institute, Federal University of Rio Grande do Norte (UFRN), Natal,

Brazil2- D’OrInstitute,RiodeJaneiro,Brazil3- InstitutodeNeurocienciasdeAlicante,ConsejoSuperiordeInvestigaciones

Científicas-Universidad Miguel Hernández de Elche, San Juan de Alicante,Spain.

4- InstituteofBiomedicalSciences,FederalUniversityofRiodeJaneiro(UFRJ),RiodeJaneiro,Brazil.

5- UniversityofCampinas(UNICAMP),Campinas,Brazil6- Digital Metropolis Institute, Federal University of Rio Grande do Norte

(UFRN),Natal,Brazil7- TheBeckleyFoundation,Oxford,UK.

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Abstract:Psychedelic agonists of serotonin receptors induce neural plasticity and

synaptogenesis,buttheirpotentialtoenhancelearningremainsuncharted.Here

we show that a single dose of d-LSD, a potent serotonergic agonist, increased

novelobjectpreferenceinyoungandadultratsseveraldaysaftertreatment.d-

LSD alone did not increase preference in old animals, but could rescue it to

younglevelswhenfollowedbya6-dayexposuretoenrichedenvironment(EE).

Massspectrometry-basedproteomicsinhumanbrainorganoidstreatedwithd-

LSD showed upregulation of proteins from the presynaptic active zone. A

computationalmodelofsynapticconnectivityinthehippocampusandprefrontal

cortex suggests that d-LSD enhances novelty preference by combining local

synaptic changes inmnemonic and executive regions,with alterations of long-

rangesynapses.BetterpatternseparationwithinEEexplaineditssynergywith

d-LSD inrescuingnoveltypreference inoldanimals.Theseresultsadvance the

useofd-LSDincognitiveenhancement.

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Introduction

Normal aging is associated with a decline in cognitive abilities, such as

learning capacity, processing speed,workingmemory, and executive functions

(1,2).Cognitiveaging is thought toreflectdecreasedsynaptogenesis inelderly

individuals(3).Inthe1960sand1970s,psychedelicsubstancesthatareagonists

ofserotoninreceptorswereextensivelyusedinpsychotherapybecauseoftheir

potential to cause long-lasting psychological changes (4-7). Decades of

prohibitive regulation prevented research on the cognitive benefits of

serotonergicagonists,butinthepastyearspsychedelicresearchisundergoinga

major renaissance (8-11), particularly in psychiatric conditions. Agonists of

serotoninreceptorshavebeenshowntohaveforemostutilityforthetreatment

of depression (12, 13) and terminal anxiety (14-16). The use of serotonergic

psychedelics is associated with decreased psychological suffering and lower

suicide rates (17). These substances have also been shown to induce neural

plasticityandsynaptogenesisbothinvitroandinvivo(18-20),andaretherefore

likely to enhance learning capacity. Indeed, psychedelics have been shown to

enhancememoryconsolidationwhenadministeredafter fear learningornovel

object exploration (21, 22). In the former, a single dose immediately after

learning enhanced memory consolidation in adult mice, while treatment

immediatelybeforelearningfacilitatedtheextinctionoffearmemory(21).Inthe

latter,chronictreatmentofadultratswithd-LSDfor11daysimprovedlearning

inanimalsdeprivedofolfactionbybulbectomy,butnoeffectswereobservedin

shamcontrols(22).Thelesionincreaseshippocampal5-HT2Alevels,andd-LSD

down-regulated these to normal levels, with no effects in sham animals. The

main interpretation proposed for these findings was that the post-learning

activationof5-HT2Areceptors improvesmemoryconsolidation, andpromotes

positivemoodchanges.Neitherstudyinvestigatedtheeffectsofd-LSDtreatment

several days before the learning task. Thus, we hypothesized that d-LSD

treatmentbeforeagivenlearningtaskwouldincreasesynaptogenesis,creatinga

richsynapticlandscapethatshouldfavortheencodingofnewmemories.

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Results&Discussion

Totestthishypothesiswefirstexploredhowapre-treatmentwithd-LSDat

variousconcentrationsandtimeintervals(Figure1A)wouldaffectsubsequent

novelobjectpreference(Figure1B)inyoung,adultandoldrats(Suppl.Table

S1).Thereferencedosechosenfortreatment(0.13mg/kg)wasdeterminedby

the current literature as required for the activation of 5-HT2A receptors, as

indicated by the occurrence of wet dog shakes (23). For comparison, we also

tested half and triple doses (0.065mg/kg and 0.39mg/kg, respectively). The

choice of serotonergic agonist – lysergic acid diethylamide (d-LSD) – was

determinedbyitsextremelylowtoxicityandeffectivedose,whichmakesd-LSD

standoutamongallothersimilarmolecules(24).

Using the reference dose, we found that d-LSD treatment significantly

increasednovelobjectpreference inadults (p=0.008), from44.16±7.49%in

saline controls to 62.65 ± 5.07% in d-LSD treated animals (median ± sd). In

younganimalstheeffectwasalsodetected(p=0.05),withanincreasefrom58.57

±9.06%insalinecontrolsto67.95%±13.13%ind-LSDtreatedanimals(median

± sd; Figure 1C; Suppl. Tables S2,S3). When different d-LSD doses were

comparedinadultanimals,amaximumofnovelobjectpreferencewasfoundfor

theintermediatedoseof0.13mg/kg(Figure1D).Dailydosesof0.13mg/kgfor

3 consecutive days were not as effective as the single dose; nor did a single

application of a triple dose (0.39 mg/kg) produce more gain with a longer

interval (Suppl. Figure S1A). Importantly, old animals did not display

differences in novel object exploration after a single d-LSD dose (Figure 1C).

Moreover, theefficiencyofd-LSD in increasingexplorativebehaviorcorrelated

negativelywithbothweightandage(Suppl.FiguresS1B,C;Suppl.TableS4).

Since exposure to an enriched environment (EE) enhances learning &

memory in aging rodents (25, 26), we hypothesized that d-LSD treatment

followedbyseveraldaysofEEexposurecouldpromoteacognitiverescueofthe

oldanimals.Totestthisideawetreatedoldratswith0.13mg/kgofd-LSDfor1

or 3 days, followed by 3h of EE exposure for 6 days (Figure 1E). Both

combinations of d-LSD treatment with EE led to a significant increase from

baseline levels (i.e. salinecontrolswithoutEE) (Figure 1F).Treatmentwithd-

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LSD for 1 day followed by EE also led to significantly more novel object

exploration than saline followed by EE; a non-significant trend in the same

directionwas detected for 3 days of d-LSD treatment followed by EE (Figure

1F). The different age groups did not show significant differences in the total

time exploring objects (Suppl. Figure S1D). Overall the data indicate that a

single dose of d-LSD increases cognitionwhenever plasticity is present, but is

insufficient to rescue cognitive losses caused by ageing. However, it also

indicates thatoncebrainplasticity isbyothermeans stimulated inold rats,d-

LSDcanboosttheeffect.

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Figure1:Treatmentwithd-LSDenhancednovelobjectpreferenceinadultsrats, andold rats showed similar effectswhend-LSDwas combinedwithenrichedenvironmentexposure.(A)Ratsofdifferentagesweretreatedwithd-LSD or saline and tested for novel object preference 6 days later. (B) Novelobjectpreferencewasassessedduringtestsessionswithonenewobjectandonefamiliarobject. (C)Inyoungandadultrats,butnotoldanimals,asingled-LSDdosesignificantlyincreasedthepreferencefornovelobjects6dayslater.(D)Inadult rats, the biggest effect was observed for the intermediary dose of 0.13mg/kg (medians marked by grey dashed lines). (E) Experimental designcombiningd-LSDwithEE inoldanimals.(F)Cognitive rescuewasobtained inoldratstreatedwithd-LSDtreatmentandthenexposedtoEE,asshownbytheincreased time exploring novelty in this group. # indicates a significantdifference from the performance of saline controls unexposed to EE (medianmarkedbygreydashedline).

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Toinvestigatethemolecularmechanismsunderlyingthecognitivebenefits

ofd-LSDtreatment,wetestedwhetherd-LSDisabletoalterexpressionofgenes

associatedwithbrainplasticityinvitro.Whilethishasbeendemonstratedinrats

both in vitro and in vivo (20), similar effects remain to be shown in human

neurons.Oneofthegreatestcaveats forstudyingcellularandmoleculareffects

of any psychedelic compound is the relative difficulty of obtaining brain cells

from human donors. To overcome this limitation, we used human induced

pluripotentstemcells(iPSCs)togeneratebrainorganoidsthatrecapitulatesome

aspectsofpatterning,organization,andconnectivityobservedintheembryonic

humanbrain(27).

BrainorganoidsgeneratedfromhumaniPSCsweregrowninagitationand

used for the experiments at day 45, when they already express most of the

transcription factors and a protein profile consistentwith differentiated brain

regions(28).Organoidsweretreatedfor24hwith10nMd-LSDandprocessed

bymassspectrometry-basedproteomicsinordertosystematicallyapproachthe

lysergic effects in human neural cells. Liquid chromatography–mass

spectrometry (LC-MS) based proteomics of control and d-LSD identified 3,448

proteinsin3biologicalreplicates(Suppl.TableS5).WeusedaStringnetwork

toanalyzeatotalof234proteinswithexpressionsignificantlymodifiedbyd-LSD

treatment (p<0.05). This tool allows searching for experimental and

correlational data connecting proteins in an interaction network (Figure 2A).

Fromthisnetworkweidentifiedenrichedcategoriesofcellcomponents,suchas

theterm‘neuronpart’(redcircles,14%ofhits,q=0.01),andtheterm‘synapse’

(bluecircles,9%ofhits,q=0.02).

In addition, gene ontology (GO) enrichment analysis, using the DAVID

bioinformaticstoolshowsother‘cellcompartment’termsenriched(Figure2B).

Seehighlightedtheenrichmentfortheterm‘presynapticactivezone’(p=0,005;

q<0.2). The proteins synaptophysin (SYP = 3.63 ± 0.92), PTPRF interacting

protein alpha 3 (PPFIA3 = 1.28 ± 0.38), glutamate metabotropic receptor 7

(GRM7=-2.4±0.55),andsynapticvesicleglycoprotein2A(SV2A=1.17±0.29)

aresomeoftheproteinspresentinthisenrichedGOcategory(valuesexpressed

as normalized fold change ± SD). Typical neuronal markers like class III β-

tubulin,MAP2andTbr1didnotshowasignificantdifferenceintheirexpression

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(Suppl.TableS4),suggestingthattheincreaseinsynapticproteinswasnotdue

toanincreaseinthenumberofneurons.

SYPisamajorintegralmembraneproteinthatbindstosynaptobrevinand

is ubiquitously expressed in synapses throughout the mammalian brain (29).

Synapseformation isoneof themainneuroplasticchangesandcorrelateswith

cognition. Interestingly, SYP stands out as a highly upregulated proteinwithin

the categories of the GO analysis. In order to confirm whether d-LSD was

affecting synaptic proteins, and SYP in particular, we performed an

immunofluorescencestainingforSYPinbrainorganoidskeptinthepresenceor

absence of d-LSD for 4 days. SYP was mainly expressed in the part of the

organoidthatresemblesthestructuralorganizationofthecorticalplate(27),the

preferentiallocationoftheneurons(Figure2C-F).d-LSDinducedamorethan2-

fold increase in the levelsof SYP in the corticalplate-like regionof thehuman

brain organoids (Figure 2H). These data suggest that d-LSD stimulates young

neurons to increase theexpressionofSYP, thusbecomingmoreprone to form

additionalnewsynapses.

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Figure 2: d-LSD regulated synaptic protein expression in human brainorganoids.BrainorganoidsderivedfromhumaniPSCsweregrownfor45daysand treated with 10 nM d-LSD for proteomic analysis. (A) String networkanalysisshowingneuronpart(red,q=0.01)andsynapse(blueq=0.02)enrichedproteinsinthedataset.(B)Cellcompartmentgeneontologycategoriesenrichedinproteomichitsfromthedataset(p<0.05).Presynapticactivezoneproteinsarelisted.–logp-valueofenrichedcategoriesandfoldincreaseenrichment(bubblesize) are plotted. (C-F) Representative images of SYP (red) and DAPI (blue,nuclear) stainingof control andd-LSD-treatedorganoids. The toppanels (C,D)show a section of the whole organoid and the bottom panels (E,F) representmagnified pictures. (G) Phase contrast images of control and d-LSD-treatedorganoidsdisplayingsimilarmorphologyandappearance.(H)QuantificationofSYP intensity expressed as fold change of control. Plot represents themean±SEM,*p<0.05.Organoidswerecollectedfrom4independentexperimentsandatleast4organoidspercondition.Scalebar=250µminC,D,Gand50µminE,F.

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To understand the association of SYP up-regulation with cognitive

enhancement, we manipulated the proprieties of synaptic connectivity in a

composite model of the hippocampus (30) and the prefrontal cortex (31, 32)

used to emulate the novel object preference task (Figure 3A). Altering the

balanceofpatternseparationandcompletioninthehippocampus(Figure3B),

the internal connectivity of prefrontal cortex (Figure 3C), and the strength of

coupling between these areas (Figure 3D) impacted the estimated preference

fornoveltyindependentlyandallowedforaninterpretationofthedifferencesin

thebehavioral resultsofadultandold rats (Figure 3E).Theuseof long-range

connectivity as a proxy for age (33, 34) provided an explanation for age

differences in baseline performance and cognitive enhancement (Figure 3D):

Young rats start at a high baseline level, but stillwith room for improvement;

adult andold rats startatneutralperformance level,butwith theconnectivity

levels foradult ratssittingnext toan inflectionand foroldratsataplateau.A

small increase in connectivitywithin the local circuits of the prefrontal cortex

further impedes the cognitive enhancement in old rats (Figure 3C). Superior

pattern separation in an enriched environment (35) does not affect baseline

performancelevelforoldratsbutamplifiesthegainaddedforextraconnectivity

(Figure3B).

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Figure 3: Composite mechanism for d-LSD associated behavioralalterations.(A)Cortico-hippocampalmodel.Themnemoniccircuitoutputsthelevel of novelty of an input pattern. Familiar patterns serve as a reference intrainingcyclesbeforetesting.Thenoveltysignalfeedsthedownstreamdecisioncircuitthroughamagnifiedlong-rangesynapse.Thedecisioncircuitpromotesaconvergent competitiveprocessbetween twoneuronal pools, one excitedby aconstantsignalthatbiasesthenetworktowardsexplorationandtheotherbythenovelty signal. The pools are, respectively, associated with the decision toexplore or not. The simulations include multiple trials with novel or familiarinputs, allowing an estimation of the time exploring novelty. Time exploringnoveltyreportedasa functionof(B)hippocampalpatternseparation;(C) localcorticalconnectivity;and(D)long-rangeconnectivitybetweenhippocampusandcortex. (E) The behavioral results were replicated by considering long-rangeconnectivityasaproxyforage(baselineof+0.2foryoung,-0.1foradultand-0.4forold)andSYPlevel(increaseof+0.1forlowdoseand+0.3forregulardose);and increaseofpatternseparationasasurrogateofenvironmentalenrichment(10%fornormaland20%forenriched).

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The results indicate that d-LSD can substantially increase synaptic

plasticity in human neurons in vitro, and novelty preference in behaving rats.

Altogether, they support the use of d-LSD to enhance learning, chart the

molecularpathwaysunderlyingthiseffect,andshowforthefirsttimethatd-LSD

modulates synaptic proteins in human neurons. Of note, the computational

modeling of synaptic connectivity in mnemonic and executive brain regions

demonstratedthatsuperiorpatternseparationintheEEissufficienttoexplain

itssynergywithd-LSDinthecognitiverescueofoldanimals.

The results also suggest that the potent anti-depressant effects of

serotonergic psychedelics (12-16, 36-38) may stem in part from a boost in

synaptogenesis that increases learning capacity and drives curiosity outwards.

The same mechanisms make this class of drugs one of the greatest promises

amongcognitiveenhancers(39,40),sidebysidewithΔ-9-tetrahydrocannabinol,

whichalso involvesSYP(41),andlikelyoverlapwiththemechanismsofsleep-

induced synaptogenesis (42). Future studiesmust determine how to optimize

the action of serotonergic psychedelics so as to rescue the learning deficits

broughtbynaturalorpathologicalaging(1-3).

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Support: Thisprojectwas supportedby theBeckleyFoundation; Fundaçãode

Amparo à Pesquisa do Estado do Rio de Janeiro, Coordenação de

Aperfeiçoamento de Pessoal de Nível Superior, Conselho Nacional de

Desenvolvimento Científico e Tecnológico grants 308775/2015-5 and

408145/2016-1, Sao Paulo Research Foundation grants 2013/07699-0

(Neuromathematics), 2014/10068-4, 2017/25588-1 and 2019/00098-7,

intramural grants fromD’Or Institute andFederalUniversityofRioGrandedo

Norte,andaJuandelaCierva-IncorporaciónScholarship(IJCI-2016-27864from

theSpanishMinistryofScience,InnovationandUniversities).

Acknowledgments:

WethanktheInstituteofChemistryofUFRNforNMRanalysis,F.G.Menezesand

R.S.Fernandesforanalyticalcharacterizationsupport,A.C.Souzaforbehavioral

advice,M.Medeirosand J.Pontes foranimalcare,D.Costaand I.S.Pereira for

documentation support, and A.E.A. Oliveira, G. Santana and K. Rocha for

miscellaneoussupport.

Authorcontributions:

SR, SR, AF, IO, FAC, DM-S, CR-C, LFT and DA contributed to the design of the

work;FAC, IO,LG-S, JN,SR, JS,KK,MC,ES,SR,SR,AF,DM-S,CR-C,LFTandDA

contributed to theacquisition,analysis,or interpretationofdata;EMandCR-C

contributedtothecreationofnewsoftwareusedinthework;SR,SR,AF,IO,FAC,

DM-S,ES,CR-C,LFTandDAhavedraftedtheworkorsubstantivelyrevisedit.

Theauthorsdeclarenocompetinginterests.

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Material&Methods

Research permits: ANVISAAEP no. 003/2018, ANVISAAEP no. 019/2019 and

CEUAUFRN#011.2015.

Drug: The study used high-purity d-LSD dissolved in ultrapure water (18.2

MΩ.cm).Thesolutionwasmanipulatedatroomtemperatureandlightexposure

was avoided. The analytical characterization is described on Suppl.

Information.

Animals: Wistar rats (n=96) on different age groups comprising young (2

months),adult(9months)andold(12-18months)animalsreceived1or3daily

intra-peritoneal injections of saline or d-LSD doses of 0.065mg/kg, 0.13mg/kg

and0.39mg/kg(Suppl.TableS1).Beforetheinjectiontheanimalswerehoused

withotherindividuals,aftertheapplicationofsalineord-LSDthe animalswere

housed individually.For tenhours following the injection,animalswerevideo-

recordedandstayedaloneduringtheacuteeffectofthedrug/saline.Afterthat,

eachratwashousedincageswith4moreindividuals.

Novel object preference task: The square arena where the novel object

preferencetasktookplace(44x44x41cm)hadtwo3Dprintedbasesfixedon

the floor 11 cm apart, for the firm placement of the objects, which were

assembled with the same number and types of LEGO™ pieces. On the 3 dayspreceding the task,animalswerehabituated for10min to the taskarena.Two

identical objects were used for the training session, with 12 edges and 12

verticeseach.Forthetestsession,anewobject,with14edgesand14vertices,

replaced one of the previous objects. Novel and familiar objects had identical

heightandbaseareadimensions.Attheonsetofeachsession,theanimalswere

always placed facing the samewall of the arena,with their backs towards the

objects. The test session was performed 30 min after the end of the training

session.NoveltypreferencewascalculatedasB/(A+B)*100,whereBisthetime

exploring the new object, and A the time exploring the familiar object. New

objectpreferencewasanalyzedusingtwo-way(AgeandDose)ANOVA,followed

byTukeyHSD(post-hocanalysissuitedtocomparegroupwithdifferentsample

sizes).

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EnrichedEnvironment(EE):OldanimalswereplacedinanEEfor3heveryday,

during6days.On the last3days, theywerealsohabituated for10min to the

arenausedinthenovelobjectpreferencetask.TheEEwasmadeofcardboxes

with8differentrooms, inwhich itwaspossible to findPVCtubes, toiletpaper

cardboard tubes, wood-made objects and hidden fruit loops to stimulate

exploratorybehavior.Animalsexploredtheenvironmentfreelyingroupsof3-5

animals.Theeffectofd-LSDfollowedbyEEwasanalyzedusingone-wayANOVA,

followed by Tukey HSD. The statistical analyses were performed using open

sourceprogramminglanguageR(Version3.6.0).TheMann-WhitneyUtestwas

used to compare the performance of old animals exposed to EE to that of old

animalstreatedwithsalineandunexposedtoEE.

Brain organoid preparation: GM23279A, an induced pluripotent cell (iPSC)

linefromtheNIGMSHumanGeneticCellRepositoryobtainedandcertifiedby

theCoriellcellrepositorywasusedinthisstudy.iPSCsweremaintainedwith

mTeSR1mediumanduponconfluencepassagedmanuallyorwithEDTA.iPSC

cultures with no differentiated cells were used to prepare brain organoids

following apreviouslydescribedprotocol (28). Shortly, iPSCsweredetached

using Accutase for 5 min at 37°C, detached cells were then added to

phosphate-buffered saline (PBS; LGC Biotechnology, USA) containing 10µM

ROCKinhibitor(ROCKi,Y27632;MerckMillipore,USA)toafinalconcentration

of10μManddissociatedtosinglecells.Cellswerecentrifugedat300rpmfor

5 min and pellet was resuspended in hESC medium (20% knockout serum

replacement;LifeTechnologies),3%ESC-quality fetalbovineserum(Thermo

Fisher Scientific, USA), 1% GlutaMAX (Life Technologies, Canada), 1%

minimum essential medium non-essential amino acids (MEM-NEAAs; Life

Technologies), 0.7% 2-mercapto-ethanol, and 1% penicillin-streptomycin

(P/S; Life Technologies). 9,000 cells were plated per well of an ultra-low

attachment 96 well plate in hESC medium containing 50 µM ROCKi and 4

ng/ml b-FGF. After plating the plate was spinned at 300 rpm for 1min. On

days3and5mediumwaschanged.Atthisstage,embryoidbodies(EBs)were

formed and grew larger. On day 7 EBs were transferred to ultra-low

attachment 24well plates andmediawas changed to neuroinductionmedia

[1% N2 supplement (Gibco), 1% GlutaMAX (Life Technologies), 1% MEM-

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NEAAs,1%P/S,and1μg/mlheparininDMEM/F12(LifeTechnologies)].The

neuroinduction media was changed at day 9 and at day 11 the tissue was

submitted to 1 h Matrigel bath. After that, media was changed to

differentiationmediaminusvitaminA(50%neurobasalmedium,0.5%N2,1%

B27 supplement without vitamin A, 1:100 2-mercapto-ethanol, 0.5% MEM-

NEAA,1%GlutaMAX,and1:100P/SinDMEM/F12)andonday13themedia

was changed. On day 15 organoids were transferred to agitation in 6 well

platesat90rpm,mediawaschanged todifferentiationmediaplusvitaminA

(50% neurobasal medium, 0.5% N2, 1% B27 supplement with vitamin A,

1:1002-mercapto-ethanol,0.5%MEM-NEAA,1%GlutaMAX,and1:100P/Sin

DMEM/F12),andreplacedevery4daysuntilday45.

Liquid-chromatography mass spectrometry (LC-MS): 45-day old human brain

organoids(fourtofivepercondition)weretreatedfor24hourswith10nMd-

LSDinwater,oronlymedium(control).Theorganoidswerecollectedfromthree

independentexperimentalbatches.Organoidswerelysedinbuffercontaining7

MUrea,2Mthiourea,1%CHAPS,70mMDTT,andCompleteProteaseInhibitor

Cocktail(Roche).Supernatantproteinextracts(50µg)weredigestedingelwith

trypsin (1:100, w/w) overnight. Peptides were injected into a reverse-phase

liquid chromatographer [Acquity UPLC M-Class System (Waters Corporation,

USA)],coupledtoaSynaptG2-Simassspectrometer(WatersCorporation,USA).

DatawasacquiredwithData-IndependentAcquisitions(DIA),withionmobility

separation(high-definitiondata-independentmassspectrometry;HDMSE)(43).

Peptideswere loaded in first-dimensionchromatographyontoanM-ClassBEH

C18Column(130Å,5µm,300µmX50mm,WatersCorporation,Milford,MA).

Threediscontinuous fractionation stepswereperformed (13%,18%, and50%

acetonitrile).Peptide loadsweredirected,aftereachstep, tosecond-dimension

chromatographyonananoACQUITYUPLCHSST3Column(1.8µm,75µmX150

mm; Waters Corporation, USA). Peptides were then eluted using a 7 to 40%

(v/v)acetonitrilegradientfor54minataflowrateof0.4µL/mindirectlyintoa

SynaptG2-Si.MS/MSanalyseswereperformedbynano-electrosprayionization

in positive ionmode nanoESI (+) and aNanoLock Spray (Waters Corporation,

Manchester,UK)ionizationsource.Thelockmasschannelwassampledevery30

sec.[Glu1]-FibrinopeptideBhuman(Glu-Fib)solutionwasusedtocalibratethe

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mass spectrometer with an MS/MS spectrum reference, using the NanoLock

Spray source. Samples were all run in technical duplicates of biological

triplicates.

Database search and quantification: All HDMSE raw files were processed for

identification and quantification using Progenesis® QI for proteomics version

4.0, including software package with Apex3D, peptide 3D, and ion accounting

informatics (Waters). Search algorithms and cross-matched with the Uniprot

humanproteomedatabase, version2018/09 (reviewedandunreviewed),with

thedefaultparametersforionaccountingandquantitation.Toassessthefalse-

positive identification rate, reversed database queries were appended to the

originaldatabase.Proteinandpeptidelevelfalsediscoveryrate(FDR)wereset

at 1%. Digestion by trypsin allowed a limit of one missed cleavage, and

methionine oxidation was considered a variable modification and

carbamidomethylation (C), a fixed modification. Identifications that did not

satisfythesecriteriawererejected.ThesoftwarestartswithLC-MSdataloading,

thenperformsalignmentandpeakdetection,whichcreatesa listof interesting

peptide ions (peptides) that are explored within Peptide Ion Stats by

multivariate statistical methods; the final step is protein identity. Relative

quantitation of proteins used the Hi-N (3) method of comparison of peptides

(44).

Dataanalysis:Furtherstatisticalanalysesofthelistwithidentifiedproteinsand

normalized intensity datawas performedusing the Perseus software (45, 46).

Fold change of the intensities assigned to each protein was calculated within

each experimental batch. Fold change values were normalized using the vsn

package in RStudio (47, 48). Statistical analyses were performed with one

sample t-test (p<0.05) against 0 value (no change). Significant hits were

subjected to gene enrichment using String (49) and DAVID (50) online

bioinformaticstools.

Immunostaining:Brainorganoidsweretreatedwith10nMd-LSDfor4days,and

fixedovernight in4%paraformaldehyde(Sigma-Aldrich,USA).Organoidswere

then incubated in 30% sucrose for dehydration, frozen in optimal cutting

temperature compound on dry ice, and stored at -80°C. The organoids were

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sectioned (20 μm thickness)with a cryostat (Leica Biosystems, Germany). For

immunofluorescence slides were thawed for 30 min, washed in PBS and

permeabilizedin0.3%TritonX-100inPBSfor15min.Sectionswereblockedin

a solution containing 3% BSA in PSB for 1 h. Primary antibody anti-

synaptophysin(1:200,Millipore,#MAB368)wasdilutedinblockingsolutionand

incubatedat4°Covernight.Afterprimaryincubation,slideswerewashedinPBS

3 times for 5 min. Sections were then incubated in AlexaFluor secondary

antibody goat anti-mouse (1:400, Invitrogen, #A-11003) for 1 hour at room

temperatureandwashed3timesfor5mininPBS.Fornuclearstaining,sections

wereincubatedwithDAPIfor5min.Slideswerewashedagain3timesfor5min

inPBSandthencover-slippedwithAqua-Poly/Mount(Polysciences,Inc).Images

were acquired using a Leica TCS SP8 confocal microscope. For quantification,

images were taken from 4-6 different fields of the borders of each brain

organoid.Withinanexperiment4-5organoidspercondition(controlandd-LSD)

were evaluated. Organoids were collected from 4 independent experiments.

ImageJsoftwarewasusedfortheanalysis.Foreachsectiontheareaofthetissue

was delineated and integrated density used as parameter to estimate SYP

expression. Statistical testing was performed using two-tailed t-test with

GraphPadPrism6software.Statisticalsignificancewasdefinedasp< 0.05.

Computational model: The architecture of the neural network included two

modules:amnemoniccircuit,inspiredbythehippocampustocomputethelevel

ofnoveltyofaninputpattern,andadecisioncircuit,modeledwiththeprefrontal

cortex as a reference to decide to explore or not to explore. The mnemonic

circuit projects the spiking activity of 800 neurons to the decision circuit.

Independent Poisson processes determine the activity of the neurons. On

familiartrials,thebasalfiringrateis50Hz.Noveltyisencodedasareductionof

thebasalfiringratewithastandardvalueof45Hz(-10%).Insomeexperiments,

thefiringratefornoveltytrialswaschangedtoemulatevariationsinthelevelof

patternseparation.Inemulationoftheenrichedenvironments,wereducedthe

firing rate to 40Hz (-20%). The projection of the mnemonic circuit to the

decisioncircuitissubjecttoamodulationfactor(standardvalueof1.0),analog

tothestrengthoflong-rangesynapsesconnectingthetwoareas.Thestrengthof

this connection can be modified. The decision circuit comprises 2000 leaky

19

integrate-and-fireneurons(80%excitatoryand20%inhibitory)andrepresents

pools of neurons in the prefrontal cortex that implement decision-making

mechanisms.Allparametersareidenticaltothosepresentedinapreviouswork

(31) except for the weight of the connections, which are changed during

simulations.Thenetworkhasall-to-allconnectivityviathreetypesofreceptors:

AMPA,NMDA,andGABAA.Allneuronsinthenetworkreceiveanexternalinput

via excitatory connections (AMPA mediated) that simulates the background

activity with a mean rate of νext = 2.4kHz, following an independent Poisson

process for each neuron. Of the excitatory neurons, 240 (15%) form a non-

exploratory pool that receives input from the mnemonic circuit. Other 240

excitatoryneuronsformanexploratorypoolthatreceivesanadditionalinputof

constantfiringratecalculatedastheaveragehippocampalinputduringtraining

with familiar patterns. Neurons sharing same external input have recurrent

connections of w+= 1.7 and connect to other excitatory neurons with w-= 1 -

f(w+-1)/(1-f),withf=0.15(31).Inhibitoryneuronshaverecurrentconnections

andconnecttoexcitatoryneuronswithunitarystrength.Thetwopoolscompete

with each other through shared recurrent inhibitory connectionsmediated by

theinterneurons.Allconnectionswithinthedecisioncircuitcouldalsobealtered

to allow the manipulation of decision dynamics. In the model, a decision

occurredwhenthedifferenceinmeanactivitybetweentheexploratoryandnon-

exploratorypoolwasabove12Hz(32).Toestimatethetimeexploringnovelty,

we computed the proportion of novel trials in which a decision was made

towards exploration compared to all cases, i.e., novel and familiar pattern

simulations.We simulated100blocksof 100 trialsper condition (connectivity

variationandnovel/familiarpatternpresented).

20

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