Cajal course on Biosensors and actuators for cellular and ... Gallery/CAJAL programme/2… ·...

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Cajalcourseon

Biosensorsandactuatorsforcellularandsystemsneuroscience

Projects

Project1:GettoknowyourtoolsWhen:Block1&Block2Who:2students/blockInstructor:JonasWietek(Humboldt-Universität,Berlin,Germany)Abstract:Sincethediscoveryofchannelrhodopsinsandtheirutilizationasoptogenetictools, thefieldvastlyexpandedover the recent years.Nowadays theoptogenetic toolboxhas grown to anoptogenetic“homedepot” inwhich it isquitedifficult to findyourwayaround.Moreandmoreactuatorsandsensorsarepublished.However,oftenthesetoolswon’tbehaveasnaivelyexpectedandsometimesshowundesiredsideeffects.Abetterunderstandingofthemolecularmachineryandthebiophysicalprinciplesunderlyingthespecificpropertiesofagivenoptogenetictoolmayhelptocircumventsuchexperimental limitations.Suchknowledgehelpstobettertailortheexperimentalconditionstothepropertiesofagiventoolandselectappropriateactuatorsandsensorsthatmeetthedemandsandlimitations of the experimental system. This project aims at characterizing both excitatory andinhibitory optogenetic actuators in a simple experimental system. A better understanding of howthese tools work will provide a solid background for appropriate experimental design andtroubleshooting.Wewillfocusonvariousbiophysicalpropertiessuchasspectralsensitivity,kineticsandthephotocycleofselectedtools,includingunpublishedones.Wewillfurtherexplorecombineduseofoptogenetic actuators fordual colormanipulationof cellular activity and combinationwithopticalsensorstoachieveall-opticalcontrolandread-outofcellularfunction.Methodology:

To characterize biophysical properties of optogenetic actuators, participants will get ahands-ontrainingonpatch-clampmeasurementsofopsinexpressingcellsin-vitro,includingall preparations necessary. Expression of optogenetic actuators will be studied byfluorescence imaging. The biophysical properties of various excitatory and inhibitoryrhodopsinswillbeassessedbywhole-cellpatch-clampmeasurements includingapplicationofvariousilluminationprotocols.Inaddition,dataanalysistrainingwillbeprovided.

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Project 2: Electrophysiological and optical detection of neurotransmitter interactions in thestriatumWhen:Block1&Block2Who:2students/block.Instructor:LiefFenno(Stanford,CA,USA)Abstract:The basal ganglia circuit involves complex interactions between neurons releasing “classical”neurotransmitterssuchasGABAandglutamate,neuronsreleasingmonoaminessuchasdopamine,and peptidergic neurons that release potent neuropeptides that can alter the excitability andsynapticpropertiesofotherneurons inthecircuit. It isnowpossibletodissectthecontributionofeach of these neuron types by combining optogenetics, calcium imaging and pharmacologicalinterventions.Inthisproject,studentswillrecordfromprimaryculturedneuronsandstriatalacuteslices to observe the effects of electrical and optogenetic stimulation through bothelectrophysiology and calcium imaging, and examine the impact of opioid receptors on theresponsesofstriatalneuronstodopaminergicinputfromtheventraltegmentalarea(VTA).Goals:

1. To familiarize students with the principles of optogenetics, including the theoreticalframework of the read-in (optogenetic) and read-out (genetically-encoded calciumindicators)toolboxes.2. To give students a hands-on, introductory experience in utilizing optogenetic tools andGECIinalab-basedsettingviaprimaryneuronelectrophysiology.3.Togive studentsahands-onexperience inutilizingoptogenetic toolsandGECI ina lab-basedsettingviasliceelectrophysiologyandepifluorescenceimaging.

Methodology:1. Primary neuron electrophysiology (whole-cell patch-clamp) of neurons expressing either theexcitatoryoptogenetictoolChR2orinhibitoryoptogenetictoolNpHR(introducedtoprimaryneuronculturesusingtransienttransfection)2. Primary neuron field stimulation of neurons expressing GECI (introduced to primary neuronculturesusingtransienttransfection)3. Primary neuron electrophysiology of neurons expressing “mystery” optogenetic tools tocharacterizetheiradsorptionwavelengthandionicselectivity4.Theoptiontopatchneuronsexpressingbothexcitatoryandinhibitoryoptogenetictools(e.g.ChR2ANDNpHR,orequivalent)5.SliceelectrophysiologyofmiceinjectedwithAdeno-associatedvirus(AAV)expressingexcitatoryoptogenetictools (ChR2orequivalent) intheVTAtoexaminethedirecteffectofoptogenetictoolexpressioninVTAslicesbypatchingneuronsidentifiedasexpressingtheoptogenetictoolbasedonfluorophoreexpression7.TheindirectnetworkeffectofoptogenetictoolexpressioninNAccslicesbypatchingneuronsthatarelikelytobedownstreamoftheVTA-expressingneurons8.Thecombinationofoptogeneticandchemicalmodulationthroughassayingchangesin

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optogenetically-modulated synaptic response in the presence of opioid receptor agonists andantagonists.Project3:Monitoringandmanipulatingintra-organellephysiologywithopticaltoolsWhen:Block1Who:2studentsInstructors:FabriceCordelieresandSandrinePouvreau(IINS,BIC,Bordeaux,France)Abstract:Intracellular calcium stores play central roles in neurons by acting as sources or sinks of calciumthereby allowing a fine modulation of spatiotemporal calcium dynamics. The precise tuning ofsubcellularcalciumsignalsunderliesdiverseneuronalfunctionsincludingbasalsynaptictransmissionpresynaptic short-term plasticity long-term plasticity and activity mediated gene expression.Impaired Ca2+ handling by intracellular calcium stores has been proposed to play an importantfunctioninthephysio-pathologicalmechanismsunderlyingseveralneurodegenerativedisease,suchas Alzheimer’s and Parkinson’s diseases. The regulation of Ca2+ dynamics by organelles has beenstudied for decades using electrophysiology and optical or electron microscopy combined withpharmacological and genetic alterations. More recently, organelles targeted genetically encodedcalciumsensorshavebeendesignedtorecordcalciummovementinlivesamples,athighspatialandtemporalresolution.Availabilityofnewprobesmightleadtotheconclusionthatmonitoringintracellularintraorganellescalcium is easy as pie. To the opposite lack of a proper probe characterizationmakesmonitoringcalciumstoresquitechallengingandpronetoerror.First, theseorganellescanconstituteextremeenvironmentswithunusualconditionsofpH,calciumconcentration,redoxsignaling,etc,potentiallyaffectingthesensor’sresponsetocalcium.Secondly,duetothesmallsizeofthesecompartments,calciummeasurementusingclassicalimagingtechniquescanbespatiallyinaccurateleadingtofalseinterpretationofdata.Super resolutionmicroscopymightbeanalternativebut itscombinationwithgeneticallyencodedsensorsisnotstraightforward.MethodologyInthisworkshop,wewillexploredifferentconstraintsunderlying intraorganellescalciumimaging.Using a panel of biological samples going from isolated cell cultures to organotypic slices thestudentswillexploreintraorganellescalciumsignalinginrelationtoneuronalactivity,usingconfocalandsuperresolutionmicroscopy.Topicssuchastargetingofthesensorsartefactskineticsaffinitiesandposthocanalysiswillbeaddressed.

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Project4:Mappingsynapticactivitywithtwo-photonmicroscopyWhen:Block2Who:2studentsInstructors:ChristineGeeandBrennaFearey(HamburgZMNH,Germany)Abstract:Two-photonmicroscopyisagreattechniquetomeasurethebriefcalciumsignalsgeneratedinsidesingleactivesynapse.Toscana largevolumeof tissuewithhundredsofsynapses,however, takesseveralminutes,whichmeans thatwewouldmissmost synaptic calcium transients. To solve thisproblem,wedevelopedageneticallyencodedcalciumsensorthatislocalizedtodendriticspinesandswitches color (from green to red) when illuminated with violet light. Importantly, thisphotoconversion happens only in spines with high internal [Ca 2+]. As photoconversion isirreversible,wehavenowplenty of time to find all active (= red) synapses in a large two-photonimagestack.Usingthisnewtool(SynTagMA),wewillinvestigatesynapticallydrivenactivityandtheback-propagationofactionpotentialsintothedendrite.Methodology:Wewillusesingle-cellelectroporationtotransfectindividualpyramidalneuronsinorganotypicslicecultures with SynTagMA. To acquire large 3D two-color stacks of labeled synapses, we use aFemtonicstwo-photonmicroscopeequippedwithatunableTi:Sapphirelaser(960nmand1040nmexcitation). We will stimulate either pharmacologically (block of inhibition), by somatic currentinjection(whole-cellpatchclamp)orviastimulationelectrodeplacedinstratumradiatum,whichwillresultindifferentactivitymaps.Achallengingpartoftheprojectistheanalysisofimagingdata.WewilluseImaris, ImageJandcustom-writtencode(Matlab)todeterminethechangeinred-to-greenfluorescence ratio inmany spinesbeforeandafter stimulation.PlottingΔR/Gvaluesback into3Dspace,wewillgeneratemapsofallsynapsesthatwereactivejustbeforethevioletlightpulse.

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Project5:OptogeneticinterrogationofsynaptictransmissionathippocampalsynapsesWhen:Block1&Block2Who:2students/blockInstructors:SimonWiegertandMauroPulin(HamburgZMNH,Germany)AbstractIn recent years numerous different types of optogenetic and chemogenetic tools for neuronalsilencingwereevolved.Someof themarehighlysuitable tosuppressactionpotential firing,whileothers can be used to inhibit release of neurotransmitters at synaptic terminals or to shuntdepolarizationofpostsynapticdendriticcompartments.Theaimofthiscourseistoexplorevariousrecentlydevelopedsilencingtoolswithdifferentfunctionalpropertiesandtoassesstheirsuitabilityforhippocampalcircuitmanipulationondifferenttemporalandspatialscales.Methodology:Wewillexpressoptogeneticorchemogenetictoolsinorganotypichippocampalslicecultures,verifytheirexpressionwithtwo-photonmicroscopyandusewhole-cellpatch-clampelectrophysiologytoassesstheeffectofthesetoolsonmultiplebiophysicalneuronalparameters.Withrecordingsfrompairsof synaptically connectedCA3andCA1neuronswewill further testhow thesevarious toolsaffect synaptic transmission and plasticity. Moreover, we will use an in silico model neuron tosimulate optogenetic manipulations of neuronal function. Finally, we will run a closed-loopexperiment using the model neuron to drive optogenetic silencing of a hippocampal neuron inresponsetoitsspikingactivity.

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Project6:EffectofGABAonthalamocorticalprojectionsWhen:Block1&Block2Who:2students/block.Instructor:MathiasMahn(FMI,Basel,Switzerland)AbstractGABAisoftendescribedasthemaininhibitoryneurotransmitterinthemammaliannervoussystem.Itexertsitseffectonpostsynapticneuronsthroughionotropicandmetabotropicreceptors.Theeffectofpresynaptic ionotropicGABA(A)receptors isdeterminedbythereversalpotentialoftheconductedions:chlorideandbicarbonate.ThepredominantcurrenttheoryisthatGABA(A)typereceptorsexertaninhibitoryeffectinmatureneurons.However,evidenceforanexcitatoryeffectofGABA (A) type receptors in the axonal compartment is accumulating, for instance inhippocampalmossyfibers,cerebellargranulecells,pyramidalneurons inthebasalnucleusoftheamygdalaandbrainstemaxonterminals.Moreover,GABA(A)typereceptorsintheaxonalcompartmentseemtobemorecommonthanpreviouslythought.Werecentlyfound,thattheopeningoflight-gatedanionchannels leads to light onset triggered vesicle release in thalamocortical projections. However, ifchloride conductance plays a role in thalamocortical projections under physiological conditions isunknown.DuringtheprojectwewillperformapilotexperimenttoaddresstheopenquestionwhateffectGABAexertsonthalamocorticalprojections.GoalsCharacterizetheeffectofGABAon:1.axonalchloridelevels2.axonalcalciumlevels3.vesiclereleaseMethodology1.Viralinjectionsandopticalwindowimplantationinthemouse.2.Optogeneticexcitationofthalamocorticalprojectionneuronsandlocalinhibitoryneurons3. In vivo two-photon imaging of genetically encoded chloride, calcium and vesicle releaseindicators.4.Analysisoftheimagingdata

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Project7:Mappingsynapticconnectivitywith2-photonholographicphotoactivationandvoltageimagingWhen:Block1&Block2Who:2-3students/block.Preferably,onestudentperblockshouldhaveexperiencewithwhole-cellpatchclamprecording.Instructors:DimitriiTanese(Emilianiteam,ParisDescartes,France),NicolòAccanto(Emilianiteam,ParisDescartes,France),SophieBouccara(Yizharteam,WeizmannInstituteofScience,Rehovot,Israel)AbstractDeficits in neuronal connectivity patterns have been associated with psychiatric diseases such asautism, schizophrenia anddepression. Theprefrontal cortex (PFC) is particularly involved in thesedisorders,butthereislittleknowledgeregardingtheconnectivitystructureofthisregion.Studyingfunctional synaptic connectivity of neurons requires the ability to both stimulate and recordneuronalactivity frommultiplecellswithsinglecell resolutionandhightemporalprecision. Inthisproject,wewillusetwo-photon(2P) imagingand2Pphotostimulationtocharacterizethesynapticinputsfrompyramidalcellstoparvalbumininterneurons(PVI)inthePFConmousebrainslices.MethodologyThe students will use a specialized microscope capable of performing 2P-scanning imaging, 2Ppatterned holographic illumination [1,2,3] and electrophysiological recording. They will performwholecellpatchclamprecordingonPVIsandopticalstimulationofopsinexpressingpyramidalcellsto identify synaptic connections. By varying illumination protocols and light patterns on single ormultiplepresynapticcells,connectionswillbemappedandcharacterized.Additionally, protocols for optical detection of photoinduced activity will also be investigated, byusingtherecentlydevelopedASAP3voltageindicator[4]and2Pimaging.Bibliography:[1]Chen,I.-W.,Papagiakoumou,E.,&Emiliani,V.(2018).Towardscircuitoptogenetics.CurrentOpinioninNeurobiology,50,179–189.[2]Ronzitti,E.,Emiliani,V.,&Papagiakoumou,E.(2018).MethodsforThree-DimensionalAll-OpticalManipulationofNeuralCircuits.FrontiersinCellularNeuroscience,12.[3]Shemesh,O.A.,Tanese,D.,Zampini,V.,Linghu,C.,Piatkevich,K.,Ronzitti,E.,…Emiliani,V.(2017).Temporallyprecisesingle-cell-resolutionoptogenetics.NatureNeuroscience,20(12),1796–1806.[4]Chavarna,M.,Villette,V.,DimovI,PradhanL,EvansS.,ShiD.,…Lin,M.(2017).Fasttwo-photonvolumetricimagingofanimprovedvoltageindicatorrevealselectricalactivityindeeplylocatedneuronsintheawakebrain.bioArxiv,10.1101/445064.

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Project8:All-opticaldynamicneuromodulationoftheE/IbalanceintheprefrontalcortexWhen:Block1&Block2Who:2students/blockInstructors:DamarisHolder&MatthiasPrigge(LIN,Magdeburg,Germany)Abstract:Neuromodulatorssuchasserotonin,dopamineornorepinephrinehavebeenlongrecognizedasanimmensely important factor in adaptive and maladaptive brain function. A majority of approvedpsychiatric interventions targeting theseneuromodulatory systemswithclassicalpharmacology. Insuch approach, specific agonist or antagonist for subset of receptors or transporters, are evenlydistributed through brain extracellular space. This is in vast contrast to the natural release ofneuromodulators in form of an uneven distributionwith local ‘hotspots’ and fluctuating levels ofneuromodulators. Inourproject,wewanttoexploredifferences inthedynamicsofneuralcircuitswhen expose to pharmacological application versus optogenetic-induced release ofneuromodulators. We will therefore use an all-optical approach in which cortical dynamic ismonitored either with red and blue-absorbing calcium sensors while optical-release ofneuromodulatorsistriggeredviablueandred-absorbingchannelrhodopsin,respectively.Furthermore, we will look carefully into cross-activation of spectrally separated sensors andactuators.Methodology:Practically,wewillprepareandimageacuteslicesfromanimalsexpressingcalciumindicatorsintheprefrontal cortex (AAV2/1-hSyn-GCaMP6f_dlx5/6-mScarlet-nls or AAV2/1-hSyn-jRCaMP1a_dlx5/6-mNeon-nls).Dlxpromoterwillenableustoidentifyinterneuronsinourpreparation.Light-sensitizeaxonal projections from neuromodulatory nuclei such as the Dorsal Raphe (serotonin), Ventraltegmental area (dopamine) or the Locus coeruleus (norepinephrine) will carry Chrimson (red-absorbingChR)orChronos (blue-absorbingChR).Wewilluseone-photonwidefieldmicroscopy toimagecalciumdynamicsfrominhibitoryandexcitatoryneuronsinalargefieldofview(~2x2mm).Inaddition,wearehappytotalktoyouaboutanyideasandscientificquestionsyouwanttostudytogetherwithus.

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Project9:Large-scaleelectrophysiologyandopto-taggingduringhead-fixedbehaviorWhen:Block1Who:2studentsInstructor:NikolasKaralis(FriedrichMiescherInstituteforBiomedicalResearch,Basel,Switzerland)AbstractThe coordinated activity of neuronal ensembles comprising hundreds or thousands of cells isbelieved to provide themechanism that enables the encoding and retrieval of information in thebrain.Theactivityofsuchensembles isorchestratedby inhibitory interneuronsthatmodulatetheinputoroutputofthecellstheytarget,effectivelycontrollingtheinformationflowinthenetwork.Recent advances in genetics andneurotechnologiesenableus to specifically label andmanipulatedistinct cell types. Using these approaches, paired with advanced electrophysiological recordingtechnologies, we can investigate the function of different interneuron classes in coordinatingneuronal ensembles and their role in controlling the learning and expression ofmemories duringbehavior.In this project, we will perform large-scale extracellular electrophysiological recordings frommultiplecorticalregionsduringbehavior,usinghigh-densitysiliconprobes,pairedwithoptogeneticmanipulationsinhead-fixedtransgenicmice.Theserecordingswillenableustoidentifythegeneticidentity of specific interneurons, while in parallel enabling us to characterize the effect of theseinterneurons on the formation and expression of neuronal ensembles during a discriminativelearningparadigm.Methodology1. Experiment planning: Participants will be involved in the process of designing an experiment,exploringthedifferentaspectsofthisprocess.2. Head-fixed electrophysiology setup building: Participants will get acquainted with the basiccomponents of building a functional setup for performing optogenetic and electrophysiologicalexperimentswithhead-fixedmice.2. Surgeries: Participantswill learn how to preparemice for head-fixed optogenetic experiments,including the head-bar implantation, stereotaxic intracerebral virus injection, and craniotomypreparationforlong-termrecordings.3. Head-fixed behavior: Participants will learn how to habituate mice to head-fixation and run adiscriminativelearningparadigminvolvingappetitiveandaversivecomponents.4. Physiological monitoring: Participants will learn how to establish and perform monitoring ofphysiological parameters from the behaving mice, including breathing, whisking, heart rate, andpupiltracking.5.Silicon-proberecordings:Participantswill learnhowtoperformacuteextracellularrecordingsofhundreds of cells frommultiple brain regions simultaneously, in head-fixedmice during behavior,usingmultishank,high-densitysiliconprobes(128-512channels).6.Opto-tagging:Participantswilllearnhowtouseoptogeneticstimulationsfromopticfiberspairedwith silicon-probe recordings, enabling the unambiguous identification of cells based on theirgenetic-andprojection-profile.

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7.Optogeneticmanipulations:Participantswill learnhowtoperformoptogeneticmanipulationsofspecificcell-typestoexplorethepotentialroleofthesecellsincontrollingneuronalensemblesandbehavior.8. Open-source hardware and software: Participants will learn to use a range of free and open-sourcetools fortheacquisitionandprocessingofthedata, includingOpenEphys,Bonsai,PulsePal,Cyclops,Arduino,Linux,KiloSort,andMountainSort.9. Data processing: Participants will learn how to handle the large data acquired during theexperiments, involving conversion, pre-processing, organization, cleaning and spike sorting,preparingthedataforlateranalyses.10. Data analysis: Participants will learn how to analyze local field potential (LFP) and single-unitrecordingsinrelationtobehavior,physiology,andoptogeneticstimulationsusingcustom-toolsinMatlab. They will learn basic approaches to characterize the spectral content of LFP signals andunderstandmethodsforcharacterizingsingle-unitandensembleactivity.

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Project10:Optogeneticidentificationofcorticalparvalbumin-expressingaxo-axonicinterneuronsWhen:Block2Who:2studentsInstructor:CyrilHerry(MagendieInstitute,Bordeaux,France)AbstractOver the past decades, tremendous progresses have been made in the identification of thededicatedcircuitrymediatingspecificfearbehaviors.Inparticularitiswellknownthatprefrontalparvalbumin-expressinginterneurons(PVIN)controlfearexpression.CorticalPVINarecomposedofseveralclassesofcelltargetingdifferentcellularcompartment.Inparticularthewell-knownbasketcells (BC) target the soma and proximal dendrites whereas axo-axonic cells (AAC) preferentiallycontacttheaxon initiationsegment.Until recentlytherewasnogeneticmousemodelavailabletoseparateandidentifyspecificallyAACfromBC.NewtransgenicmicemodelsallowingidentifyingandmanipulatingAAChavebeenrecentlydeveloped(Heetal2016). Inthetimecourseoftheprojectwe will perform single unit recordings coupled to optogenetic manipulation of prefrontal AACneuronsduring fear-related tasks to causally relate changes inAACneuronal activitywith specificfearbehaviors.Goals1.Constructionofsingleelectrode/tetrodesforinvivorecordings2.Viralinjection3.Optogeneticidentification,manipulationsandrecordingsduringfearbehavior4.BehavioralanalysesMethodology1.Electrodeconstruction:Participantwilllearnhowtobuildsingleelectrodesandtetrodes2. Viral injection: Participantwill learn how to do intracerebral injections to deliver opsins in thetargetareaofinterest.3.Behavior:Participantwill learnhowtoruntwowayactiveavoidancebehaviorandclassical fearconditioning4. Optogenetic identification and stimulations during behavior: Here will use photo-taggingapproaches tounambiguously identifyAACandperformoptogenetic stimulations to change firingactivityofprefrontalAACduringfearbehavior.5.Behavioralanalyses:Participantwilllearntoanalysebehavioralandoptogeneticdata

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Project11:In-vivoimaginginbehavingmicewithopen-sourcehardwareWhen:Block1Who:2-4studentsInstructors:DeniseCaiandTristanSchuman(IcahnSchoolofMedicine,MountSinai,NY,USA)AbstractRecentdevelopmentshavemadein-vivocalciumimagingpossibleinfreelymovingmice.Pioneeringstudieshavedemonstrated the feasibilityof thisapproach ina rangeofneural circuits [REFS]andtheminiscope technology has beenwidely disseminated through thework of companies such asInscopixandDoricLenses.Recently,ateamattheUniversityofCaliforniaLosAngeles(UCLA)ledbyProf.PeymanGolshaniandDr. Daniel Aharoni has developed an open-source version of this technology, which is widelyapplicable at a small fraction of the cost of the commercially available systems. Denise Cai andTristanSchumanwereamongthefirstdevelopersandusersofthistechnology,andtheywillprovideacompleteworkshoponbuildingtheUCLAminiscope,implantingitinmiceandperformingcalciumimagingfromthemousehippocampus.MethodologyInthisproject,studentswill:1.Understandthebasicsofminiscopeimaging2.Buildafunctionalminiscopesystem3.PerformimplantationsurgeriesinmiceexpressingGCaMP6sinthedorsalhippocampus4.Performcalciumimaginginfreelymovingmice5.Analyzetherecordeddatausingopen-sourcesoftware.

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Project12:MonitoringmitochondrialcalciummovementsinastrocytesusinggeneticallyencodedprobesWhen:Block2Who:2studentsInstructor:RomanSerrat(MagendieInstitute,Bordeaux,France)AbstractIn the recentyearsmitochondriahaveevolvedasone important keyelement in the regulationofbrainfunction.Inparticular,mitochondrialcalciumbufferingcapacitymakestheseorganellescrucialfor brain cells, as neurons and astrocytes are cells highly dependent to calcium signaling. In ourlaboratorywefocusonthestudyofmitochondrialcalciumactivityusingthemitochondrial-targetedgenetically encoded calcium sensor Gcamp6s that allows highly sensitive detection of calciumactivity.Methodology:Inthisproject,primaryculturesofboth,neuronsandastrocytesfromneonatalmicewillbepreparedand then transfected with the indicated mitochondrial sensor using a low damaging calcium-phosphate method. In parallel, a cytosolic sensor (Rcamp2) will be transfected, allowing us tomeasure also cytosolic activity for correlation purposes. After allowing gene expression,mitochondrialandcytosoliccalciumlevelswillbemodifiedusingdifferentdrugsandrecordedusinglive-imagingrecordings inaspinningdiskmicroscope.Finally,using Image Jsoftwarethecollecteddatawillbeanalyzedandquantified.

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Project 13: The impact of Taupathologyonhippocampal CA3networkdynamics in organotypichippocampalslicesWhen:Block2Who:2studentsInstructors:AniaGoncalvesandRuthBetterton(IINS,Bordeaux,France)AbstractThe hippocampus is an area of the brain responsible for the rapid encoding of memory anddegeneration of the hippocampus is associated with memory dysfunction. In conditions such asAlzheimer’s disease, neurodegeneration and cognitive decline are correlated with a pathologicalaccumulation of the microtubule-associated protein Tau. In healthy neurons, Tau is enriched inaxons and involved in microtubule stabilisation and axonal transport. However, in pathologicalconditionsTaudetachesfromthemicrotubulesandformsaggregatesaffectingsynapticfunction.Subregions of the hippocampus have specific roles in the encoding of memory. Mossy fibres ofdentategranulecells synapseontoCA3pyramidal cellswithanunusualpresynaptic compartmentknown as the giant bouton. This unusual synapse has several properties which allow rapid pre-synapticpotentiationleadingtoproposalsthatitactsasaconditionaldetonatordrivingfiringintheCA3network.Goals-MethodologyThisprojectwillinvestigatewhetherneuronalactivityinCA3isalteredinatransgenicmousemodelof Taupathology.Usinggenetically encoded calcium indicators in combinationwith2-photonandconfocal imaging, we will investigate both spontaneous activity and the impact of mossy fibrestimulationaninvitrohippocampalmodel:theorganotypicsliceculture.Project14:Recordingsub-thresholdsynapticpotentialswithgeneticallyencodedvoltagesensors

• When:Block2• Who:2/3students.• TA:SophieBouccara(Yizharteam)

AbstractRecentdevelopmentsinvoltagesensortechnologyhasyieldedgenetically-encodedvoltagesensors(GEVIs) that can report sub-threshold activitywith high precision. These sensors could potentiallyrevolutionizethestudyofsynapticconnectivityastheycouldallowall-opticaldetectionofsynapticconnections using high-throughput systems. In this project, we will focus on two specific GEVIs:ArcLight-MT [1] and ASAP2s [2].Wewill use a combination of patch-clamp recordings with two-photonsingle-cellimagingandcharacterizetheopticalresponsestosingle-cellstimulation(throughthe patch pipette) and to electrical full field stimulations triggering subthreshold events throughsynapticconnections.MethodologyThestudentswill learnhowtousea2-photonmicroscopecapableofperformingsingle-neuron2Pimaging and electrophysiology recordings. We will perform whole-cell patch clamp recording ofGEVI-expressing cells andwill simultaneously image the optical response induced by an electricalfieldstimulation.Byvaryingtheelectricalstimulationamplitudewewilldeterminetherangevaluesneeded to induce subthreshold responses and then correlate theoptical response (∆𝐹 𝐹) to thevoltage depolarization. Synaptic blockers and TTX will eventully be also added for a deepercharacterization. Upon successful completion of this simple experiment, students will be able torecordopticalsynapticresponsetostimulationofsingleneuronsintheneuronalculture,aimingto

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identify synaptically connected partners by optically screening multiple putative post-synapticpartners foreachstimulatedneuron.Bytheendof theproject, studentsshouldbeable torecordelectrically and optically from single neurons, characterize the signal-to-noise ratio of the twovoltage sensors and demonstrate optical detection of synaptic connections between identifiedneurons.