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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-
5
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
17
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
18
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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