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Neuromethods 187 Nicholas Graziane Yan Dong Editors Electrophysiological Analysis of Synaptic Transmission Second Edition N EUROMETHODS SeriesEditor Wolfgang Walz University ofSaskatchewan Saskatoon, SK,Canada Forfurther volumes: http://www.springer.com/series/7657 Neuromethodspublishescutting-edgemethodsandprotocolsinallareasofneuroscienceas wellastranslationalneurologicalandmentalresearch.Eachvolumeintheseriesofferstested laboratoryprotocols,step-by-stepmethodsforreproduciblelabexperimentsandaddresses methodologicalcontroversiesandpitfallsinordertoaidneuroscientistsinexperimentation. Neuromethodsfocusesontraditionalandemergingtopicswithwide-rangingimplicationsto brain function, such as electrophysiology, neuroimaging, behavioral analysis, genomics, neurodegeneration,translationalresearchandclinicaltrials.Neuromethodsprovidesinvesti- gators and trainees with highly useful compendiums of key strategies and approaches for successful research in animal and human brain function including translational “bench to bedside”approachestomentalandneurologicaldiseases. Electrophysiological Analysis of Synaptic Transmission Second Edition Edited by Nicholas Graziane Department of Anesthesiology, The Pennsylvania State University, Hershey, PA, USA Yan Dong Neuroscience Department, University of Pittsburgh, Pittsburgh, PA, USA Editors NicholasGraziane YanDong DepartmentofAnesthesiology NeuroscienceDepartment ThePennsylvaniaStateUniversity UniversityofPittsburgh Hershey,PA,USA Pittsburgh,PA,USA ISSN0893-2336 ISSN1940-6045 (electronic) Neuromethods ISBN978-1-0716-2588-0 ISBN978-1-0716-2589-7 (eBook) https://doi.org/10.1007/978-1-0716-2589-7 ©TheEditor(s)(ifapplicable)andTheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,part ofSpringerNature2022 Thisworkissubjecttocopyright.AllrightsaresolelyandexclusivelylicensedbythePublisher,whetherthewholeorpart of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting,reproductionon microfilmsorinanyotherphysicalway,andtransmissionorinformation storageand retrieval,electronicadaptation, computersoftware,orbysimilar ordissimilar methodologynow knownorhereafter developed. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations andthereforefreeforgeneraluse. Thepublisher,theauthorsandtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressedorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeen made.Thepublisherremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations. ThisHumanaimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLC,partofSpringer Nature. Theregisteredcompanyaddressis:1NewYorkPlaza,NewYork,NY10004,U.S.A. Preface to the Series Experimental life sciences have two basic foundations: concepts and tools. The Neuro- methods series focuses on the tools and techniques unique to the investigation of the nervous system and excitable cells. It will not, however, shortchange the concept side of thingsascarehasbeentakentointegratethesetoolswithinthecontextoftheconceptsand questions under investigation. In this way, the series is unique in that it not only collects protocols but also includes theoretical background information and critiques which led to themethodsandtheirdevelopment.Thus,itgivesthereaderabetterunderstandingofthe origin of the techniques and their potential future development. The Neuromethods publishing program strikes a balance between recent and exciting developments like those concerningnewanimalmodelsofdisease,imaging,invivomethods,andmore established techniques, including, for example, immunocytochemistry and electrophysiological tech- nologies.Newtraineesinneurosciencesstillneedasoundfootingintheseoldermethodsin order toapplyacriticalapproachtotheir results. Under theguidanceofitsfounders,AlanBoultonandGlenBaker,theNeuromethods serieshasbeenasuccesssinceitsfirstvolumepublishedthroughHumanaPressin1985.The seriescontinuestoflourishthroughmanychangesovertheyears.Itisnowpublishedunder theumbrellaofSpringerProtocols.Whilemethodsinvolvingbrainresearchhavechangeda lot since theseriesstarted, thepublishingenvironmentand technologyhavechanged even more radically. Neuromethods has the distinct layout and style of the Springer Protocols program,designedspecificallyfor readabilityandeaseofreferenceinalaboratorysetting. Thecarefulapplicationofmethodsispotentiallythemostimportantstepintheprocess of scientific inquiry. In the past, new methodologies led the way in developing new disciplines in the biological and medical sciences. For example, physiology emerged out of anatomyinthenineteenthcenturybyharnessingnewmethodsbasedonthenewlydiscov- eredphenomenonofelectricity.Nowadays,therelationshipsbetweendisciplinesandmeth- ods are more complex. Methods are now widely shared between disciplines and research areas. New developments in electronic publishing make it possible for scientists that encounter new methods to quickly find sources of information electronically. The design of individual volumes and chapters in this series takes this new access technology into account. Springer Protocols makes it possible to download single protocols separately. In addition, Springer makes its print-on-demand technology available globally. A print copy canthereforebeacquiredquicklyandforacompetitivepriceanywhereintheworld. Saskatoon,SK,Canada WolfgangWalz v Preface The central nervous system comprising the brain and spinal cord controls vertebrate responsestoexternalstimuli.Forexample,ourancestorsneededthecentralnervoussystem tovisuallylocatepredatorsandrespondbymakingquickmotormovementstoasafeplace. Simplyreadingthisbookrequiresinformationtobeprocessedinthebrainandstoredsothat it can be implemented at a future time in a research setting. This constant processing and responding to our external environment is mostly controlled by electrical signals, which formaquickandefficientmeansofcommunicationbetweendistinctregionsofthecentral nervoussystem. In modern neuroscience, electrophysiology often refers to the study or research approach implemented to investigate the electrical properties of brain cells and tissues. It issensitiveenoughtomeasurebasalvoltage/potentialorelectricalcurrentsfromasingleion channelorawholecell,whilealsopossessingtheabilitytostudyelectricalprocessingwithin anentirebrainregion. Thebrainfunctionsbyintegratingandcomputingelectricaleventsfromindividualbrain cells(includingnervouscellsandglialcells)andcommunicationbetweenthesecells.These electricaleventssometimeshappenslowly,butmostofthetimetheyoccur veryfast,within the submillisecond to millisecond range. Electrophysiology is one of the finest approaches capable of detecting and analyzing these electrical events during the endeavor of under- standingthefunctionofneurons,neuralsystems,andthebrain. Specifically, for studying synaptic transmission, electrophysiology offers great advan- tages that other techniques may not provide. First, and most important in our opinion, electrophysiology detects functional readouts of synaptic transmission. Under the physio- logical and pathophysiological conditions, the number of synapse-like structures is often much larger than the number of synapses that are actually functional. By definition, a synapse is the connection between cells, functioning to transfer signals from one cell to another. Thus, although abundant synapse-like structures are observed, it cannot be con- cludedthattransmissionbetweenthesetwocellsiseffective.Inatypicalelectrophysiological measurement,onlyfunctionalsynapses,namelythesynapsesthatmediatesynapticcurrents, are detected and recorded. To some extent, electrophysiology measures the electrical con- sequences of activating synapses. This property of the electrophysiological approach is significant for several reasons. First, it can detect synaptic efficacy. Under different physio- logicalconditionsoroverdifferentdevelopmentalphases,centralsynapsesexhibitdifferent efficacies, which are regulated by key mechanisms underlying synaptic plasticity and matu- ration.Manyoftheseregulationsoccuratthemolecularlevel,forexample,phosphorylation of synaptic proteins, without changing the shape/structure of synapses. Therefore, these changes can be readily and reliably detected electrophysiologically. An extreme example is thatsomesynapsesaregeneratedbutremaindormant;theydonotconductreliablesynaptic activity and thus are non- or semi-functional. During development or upon experience- dependent regulations, these synapses may evolve into fully functional synapses or, under other circumstances, completely lose their function and are primed for pruning/removal. Thesedynamicprocessescanalsobedetectedelectrophysiologically.Wewilldiscussthisand other relatedproceduresthoroughlyinthisbook. vii viii Preface Second,electrophysiologymeasurestherealtimingofsynaptictransmission.Timingis everything,andthisisalsotrueforsynaptictransmission.Fromstarttoend(neurotransmit- terreleasestimulatedbyapresynapticactionpotentialtopostsynapticreceptorresponseto neurotransmitterrelease),synaptictransmissioncanbeaccomplishedasfastaswithinafew milliseconds. In most central neurons, depolarization induced from a single excitatory synapse is usually not sufficient to trigger action potentials in the postsynaptic neuron. In order to trigger an action potential in a postsynaptic cell, a few synapses must be activated within the same time window in order to generate summed depolarization that is large enough to push the postsynaptic cell beyond the threshold of an action potential. With certain manipulations, electrophysiological measurements can distinguish the contribution of each synapse to the summed depolarization, and thus determine how effective/efficient theisolatedsetofsynapsescontributestothetemporalsummation. Third, electrophysiology sensitively measures the functional changes at synapses. Most synapseneuroscientistsholdthehypothesisthatexternalexperienceschangethefunctionof synapses, thus reshaping future behaviors. Much of this hypothesis has been formulated based on electrophysiological studies of long-term potentiation (LTP) and long-term depression (LTD), two forms of long-lasting synaptic modifications critically contributing tolearningandmemory.ElectrophysiologyispowerfulincellularmodelsofLTPandLTD in which the efficacy or strength of synaptic transmission is continuously monitored for several tens of minutes and sometimes hours. As such, an increase or decrease of synaptic efficacy can be detected during and after experimental manipulations mimicking external stimulation. Fourth, combined with other techniques, electrophysiology can address very sophisti- catedmolecularandcellularquestionsofsynaptictransmission.Forexample,usingcurrent technology, neurotransmitter receptors can be easily labeled fluorescently such that their locationandmovementcanbemonitoredinrealtimeusingimagingmethods.Thecorrela- tive synaptic recruitment/internalization of these receptors and increases/decreases in synaptic strength have been used as strong evidence showing the postsynaptic mechanism ofsynapticplasticity. Likeallotherexperimentalapproaches,electrophysiologyhasitslimitationsinexamin- ing synaptic transmission. Probably the most glaring one is that it cannot unequivocally distinguishpre-vs.postsynapticalterationsuponchangesinsynapticefficacy.Electrophysi- ologicaldataalonecanoftenbeinterpretedeitherway,althoughsometimesonewayappears tobemoreparsimoniousthantheother.Thislimitationisindeedoneofthemajorreasons for thedecade-longdebateoftheexpressionmechanismsunderlyingLTP. Another limitation is that spatial and temporal effects are often involved in synaptic electrophysiological experiments. If these factors are not sufficiently considered, false con- clusions can be drawn. To help the new electrophysiologist avoid such mistakes, we make surethatthepotentialexperimentalandinterpretationalcaveatsarethoroughlydiscussedin eachelectrophysiologicalapproachweintroduce. Preface ix Therearemanyoutstandingbooksandpublishedmanuscripts,whicharecitedthrough- outthisbook,explainingthemanyfacetsofelectrophysiologyusedtobetterassistscholars lackingaccessibilitytoanexperiencedelectrophysiologist.Ourgoalinwritingthisbookisto create a guide, which introduces and highlights important topics in the field while at the same time attempting to extend these topics to practical electrophysiological approaches throughtheperspectiveofthetwoauthors.Weexpectthatthisbookholdsenoughtechnical information for graduate students or junior postdoctoral fellows to get started in their journeyofsynapseneuroscience. Hershey,PA,USA NicholasGraziane Pittsburgh,PA,USA YanDong Contents PrefacetotheSeries ........................................................... v Preface ..................................................................... vii Contributors................................................................. xiii AbouttheEditors............................................................. xv PART I BASIC CONCEPTS 1 ExtracellularandIntracellularRecordings.................................. 3 NicholasGrazianeandYanDong 2 ElectricalTheory........................................................ 17 NicholasGrazianeandYanDong 3 Amplifiers.............................................................. 33 NicholasGrazianeandYanDong 4 SaltEnvironment ....................................................... 55 NicholasGrazianeandYanDong 5 PatchPipettes(Micropipettes)............................................ 69 NicholasGrazianeandYanDong 6 SpatiotemporalEffectsofSynapticCurrent ................................ 79 NicholasGrazianeandYanDong 7 PerforatedPatch........................................................ 91 NicholasGrazianeandYanDong PART II RECORDING OF SYNAPTIC CURRENT 8 IsolationofSynapticCurrent............................................. 101 NicholasGrazianeandYanDong 9 FastandSlowSynapticCurrents.......................................... 111 NicholasGrazianeandYanDong 10 MeasuringKineticsofSynapticCurrent ................................... 123 NicholasGrazianeandYanDong 11 MeasuringPresynapticReleaseProbability................................. 135 NicholasGrazianeandYanDong 12 Long-TermMeasurements............................................... 147 NicholasGrazianeandYanDong 13 MeasuringReversalPotentials............................................ 159 NicholasGrazianeandYanDong PART III BASIC EXPERIMENTATIONS OF SYNAPTIC TRANSMISSION 14 Amplitude ............................................................. 167 NicholasGrazianeandYanDong xi