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Neurosciences

In Vitro Wedge Slice Forberedelse til efterligne In Vivo Neuronal Circuit Connectivity

Published: August 18, 2020
doi:
10.3791/61664
,
1Section on Neuronal Circuitry, National Institute on Deafness and Other Communication Disorders,NIH

Summary

Integration af forskellige synaptiske input til neuroner måles bedst i et præparat, der bevarer alle præ-synaptiske kerner for naturlig timing og kredsløb plasticitet, men hjernen skiver typisk afskære mange forbindelser. Vi udviklede en modificeret hjerneskive til at efterligne in vivo kredsløbsaktivitet, samtidig med at vitro-eksperimenterne opretholdes.

Abstract

In vitro skive elektrofysiologi teknikker måle encellet aktivitet med præcis elektrisk og tidsmæssig opløsning. Hjerne skiver skal være relativt tynde til korrekt visualisere og få adgang til neuroner til patch-fastspænding eller billeddannelse, og in vitro undersøgelse af hjernen kredsløb er begrænset til kun, hvad der er fysisk til stede i den akutte skive. For at opretholde fordelene ved in vitro skive eksperimenter og samtidig bevare en større del af præsynaptiske kerner, udviklede vi en ny skive forberedelse. Denne “kile skive” var designet til patch-clamp elektrofysiologi optagelser til at karakterisere de forskellige monaural, lyd-drevne input til mediale olivocochlear (MOC) neuroner i hjernestammen. Disse neuroner får deres primære afferente excitatoriske og hæmmende input fra neuroner aktiveret af stimuli i det kontralaterale øre og tilsvarende cochlear kerne (CN). En asymmetrisk hjerne skive blev designet, som er tykkest i rostro-caudal domæne på sidekanten af en halvkugle og derefter tynder mod den laterale kant af den modsatte halvkugle. Denne skive indeholder, på den tykke side, den auditive nerve rod formidle oplysninger om auditive stimuli til hjernen, den iboende CN kredsløb, og både den disynaptiske excitatoriske og trisynaptiske hæmmende afferent veje, der konvergerer på kontralaterale MOC neuroner. Optagelse udføres fra MOC neuroner på den tynde side af skiven, hvor de visualiseres ved hjælp af DIC optik til typiske patch-clamp eksperimenter. Direkte stimulering af hørenerven udføres, da den kommer ind i den auditive hjernestamme, hvilket giver mulighed for iboende CN kredsløb aktivitet og synaptisk plasticitet at forekomme på synapser opstrøms for MOC neuroner. Med denne teknik, kan man efterligne in vivo kredsløb aktivering så tæt som muligt i skiven. Denne kile skive forberedelse gælder for andre hjerne kredsløb, hvor kredsløb analyser ville drage fordel af bevarelse af upstream tilslutningsmuligheder og langtrækkende input, i kombination med de tekniske fordele ved in vitro skive fysiologi.

Introduction

Observation af aktiviteten af neurale kredsløb er ideelt udført med indfødte sensoriske indgange og feedback, og intakt forbindelse mellem hjernen regioner, in vivo. Men, udfører eksperimenter, der giver encellet opløsning af neurale kredsløb funktion er stadig begrænset af tekniske udfordringer i den intakte hjerne. Mens in vivo ekstracellulære elektrofysiologi eller multiphoton billeddannelse metoder kan bruges til at undersøge aktivitet i intakte nervesystemer, fortolke, hvordan forskellige indgange integrere eller måle subthreshold synaptiske indgange er fortsat vanskeligt. In vivo helcelleoptagelser overvinder disse begrænsninger, men er udfordrende at udføre, selv i hjerneregioner, der er let tilgængelige. Tekniske udfordringer i forbindelse med enkeltcelleopløsningsforsøg forstærkes yderligere i visse neuronpopulationer, der er placeret dybt inde i hjernen, eller i rumligt diffuse populationer, der kræver enten genetiske værktøjer til at lokalisere celler in vivo (f.eks. genetisk ekspression af channelrhodopsin parret med optrodeoptagelse) eller post-hoc histoktisk identifikation efter registrering af mærkning af celler (f.eks. med neurotransmissionsspecifikke markører). At være placeret diffust nær den ventrale overflade af hjernestammen, mediale olivocochlear (MOC) neuroner lider af ovenståendebegrænsninger 1,hvilket gør dem yderst vanskelige at få adgang til in vivo eksperimenter.

Hjerne skiver (~ 100-500 μm tykkelse) har længe været brugt til at studere hjernen kredsløb, herunder auditive hjernestamme kredsløb, på grund af den fysiske adskillelse af tilsluttede neuroner, der er indeholdt i samme skive2,3,4,5,6,7,8,9. Der er i andre laboratorier anvendt forsøg med meget tykkere skiver (>1 mm) for atforstå,hvordan bilaterale input integreres i områder af det overlegne olivary-kompleks (SOC), herunder den mediale overlegneoliven 10,11. Disse skiver blev udarbejdet således, at axoner af hørenerven (AN) forblev intakt i skiven og blev elektrisk stimuleret til at indlede synaptisk neurotransmitter frigivelse i CN, efterligne aktivitet første orden auditive neuroner, som de ville reagere på lyd. En væsentlig ulempe ved disse tykke skiver er synligheden af neuroner til patch-clamp elektrofysiologiske optagelser (“patching”). Patching bliver stadig vanskeligere som de mange axoner i dette område bliver myelinated medalderen 12,13,14,15, hvilket gør vævet optisk tæt og tilsløre neuroner selv i en typisk, tynd hjerne skive. Vores mål er at skabe in vitro præparater, der mere ligner kredsløbet tilslutning smuligheder in vivo optagelser, men med den høje kapacitet og høj opløsning optagelse evner visuelt guidet patch-clamp elektrofysiologi i hjernen skiver.

Vores laboratorium undersøger fysiologien af neuroner i det auditive efferent-system, herunder MOC-neuroner. Disse kolinerge neuroner giver efferent feedback til cochlea ved at modulere aktiviteten af ydre hårceller (OHCs)16,17,18,19,20. Tidligere undersøgelser harvist,at denne graduering spiller en rolle i at opnå kontrol i cochlea21,22,23,24,25,26 ogbeskyttelse mod akustisktraume27,28,29,30,31,32,33. I mus, MOC neuroner er diffust placeret i ventrale kernen i trapez kroppen (VNTB) i auditivehjernestammen 1. Vores gruppe har udnyttet ChAT-IRES-Cre muselinjen krydset med tdTomato reporter muselinjen til at målrette MOC neuroner i hjernestamme skiver under epifluorescerende belysning. Vi viste, at MOC neuroner modtager afferent hæmmende input fra ipsilateral mediale kerne af trapez kroppen (MNTB), som er ophidset, igen, af axoner fra kugleformede buskede celler (GBC) i den kontralaterale cochlear kerne (CN)34,35,36,37,38. Derudover, MOC neuroner sandsynligvis modtage deres excitatoriske input fra T-stellate celler i den kontralaterale CN39,40,41. Tilsammen viser disse undersøgelser, at MOC-neuroner får både excitatoriske og hæmmende input fra det samme (kontralaterale) øre. Men, de præsynaptiske neuroner, og deres axoner konvergerende på MOC neuroner, er ikke helt tæt nok til hinanden til at være helt intakt i en typisk koronal skive forberedelse. For at undersøge, hvordan integration af synaptiske input til MOC neuroner påvirker deres indsats potentielle fyring mønstre, med fokus på nyligt beskrevne hæmning, vi udviklet et præparat, hvor vi kunne stimulere de forskellige afferenter til MOC neuroner fra det ene øre i den mest fysiologisk realistiske måde muligt, men med de tekniske fordele ved in vitro hjerne skive eksperimenter.

Kilen skive er en modificeret tyk skive forberedelse designet til undersøgelse af kredsløb integration i MOC neuroner (schematized i figur 1A). På den tykke side af skiven indeholder kilen de afhuggede axoner af hørenerven (benævnt “auditiv nerverod” herefter), da de kommer ind i hjernestammen fra periferien og synapser i CN. Den auditive nerverod kan stimuleres elektrisk til at fremkalde neurotransmitter frigivelse og synaptisk aktivering af celler i den fuldt intakte KN42,43,44,45,46. Dette stimuleringsformat har flere fordele for kredsløbsanalyse. For det første, i stedet for direkte at stimulere T-stellate og GBC axoner, der giver afferent input til MOC neuroner, vi stimulerer AN at tillade aktivering af iboende kredsløb rigelige i CN. Disse kredsløb modulere produktionen af CN neuroner til deres mål i hele hjernen, herunder MOC neuroner46,47,48,49,50,51. For det andet giver den polysynaptiske aktivering af afferentkredsløb fra AN gennem CN opstrøms for MOC-neuroner mulighed for mere naturlig aktiveringstidspunktet og for plasticitet ved disse synapser, som de ville in vivo under auditiv stimulation. For det tredje kan vi variere vores stimulationsmønstre for at efterligne EN aktivitet. Endelig er både excitatoriske og hæmmende monaural projektioner til MOC neuroner intakt i kilen skive, og deres integration kan måles ved en MOC neuron med præcisionen af patch-clamp elektrofysiologi. Som helhed, denne aktivering ordning giver en mere intakt kredsløb til MOC neuroner i forhold til en typisk hjerne skive forberedelse. Denne hjernestamme kile skive kan også bruges til at undersøge andre auditiveområder,der modtager hæmmende input fra ipsilateral MNTB herunder lateral overlegen oliven, overlegen olivary kerne og mediale overlegne oliven10,11,52,53,54,55,56. Ud over vores specifikke præparat kan denne udskæringsmetode bruges eller ændres til at evaluere andre systemer med fordelene ved at opretholde tilslutning af langtrækkende indgange og forbedre visualiseringen af neuroner til en række elektrofysiologi eller billeddannelsesteknikker med en enkelt celleopløsning.

Denne protokol kræver brug af en vibratome fase eller platform, som kan vippes ca 15°. Her bruger vi en kommercielt tilgængelig 2-delt magnetisk fase, hvor “scenen” er en metalskive med en buet bund placeret i en konkav magnetisk “scenebase.” Scenen kan derefter flyttes for at justere udsnitsvinklen. Koncentriske cirkler på scenebasen bruges til at estimere vinklen reproducerbart. Scene- og scenebasen er placeret i udskæringskammeret, hvor den magnetiske scenebase også kan drejes.

Protocol

Alle eksperimentelle procedurer blev godkendt af National Institute of Neurological Disorders and Stroke /National Institute on Døvhed og andre kommunikationsforstyrrelser Animal Care and Use Committee. 1. Forsøgspræparater BEMÆRK: Nærmere oplysninger om skiveforberedelse, herunder udskæringsopløsning, udskæringstemperatur, skiveinkubationstemperatur og apparater (osv.), er specifikke for det præparat for hjernestammer, der udføres i dette eksperiment. Slice…

Representative Results

Histologisk undersøgelse af kile skiveTil vores undersøgelse af auditive hjernestammen neuron funktion, kilen skive forberedelse var designet til at indeholde auditive nerve rod og CN kontralaterale til MOC neuroner målrettet til optagelser (eksempel skive vist i figur 1B). Indledende histologisk undersøgelse af præparatet er vigtigt for at bekræfte, at skiven indeholder de kerner, der er nødvendige for kredsløbsaktivering, og at axonale projektioner er intakte. …

Discussion

Udskæringsproceduren beskrevet her kaldes en kile skive er kraftfuld til at opretholde intakt præsynaptiske neuronal kredsløb, men med tilgængeligheden af hjernen skive eksperimenter til analyse af neuronal funktion. Der skal være stor omhu i flere indledende skridt for at maksimere nytten af forberedelsen til kredsløbsanalyse. Kilens dimensioner skal bekræftes ved hjælp af histologisk undersøgelse, som er integreret for bekræftelse af, at både præsynaptiske kerner og deres aksonale projektioner er indeholdt …

Divulgations

The authors have nothing to disclose.

Acknowledgements

Denne forskning blev støttet af Intramural Research Program af NIH, NIDCD, Z01 DC000091 (CJCW).

Materials

Experimental Preparations
Agar, powder Fisher Scientific BP1423500 4% agar block used to stabilize brain tissue during vibratome sectioning
AlexaFluor Hydrazide 488 Invitrogen A10436 Fluorophore used in internal solution to confirm successful MOC neuron patch
Analytical Balance Geneses Scientific (Intramalls) AV114 Weighing chemicals
Double edged razor blade Ted Pella 121-6 Vibratome cutting blade
Kynurenic acid (5g) Sigma Aldrich K3375-5G Slicing ACSF additive used to reduce neuron activity during dissection and slicing in order to improve tissue health for patch clamping
pH Meter Fisher Scientific (Intramalls) 13-620-451 Solution pH tester
Plastic petri dishes 100mm dia X 20mm Fisher Scientific (Intramalls) 12-556-002 4% Agar Prep
Stirring Hotplate Fisher Scientific (Intramalls) 11-500-150 Heating for 4% Agar preparation
Dissection and Slicing
Biocytin Sigma Aldrich B4261-250MG Chemical used for axonal tracing (conjugated to Streptavidin 488)
Dissecting Microscope Amscope SM-1BN For precision dissection during brain removal
Dumont #5 Forceps Fine Science Tools 11252-20 Fine forceps dissection tool
Economy tweezers #3 WPI 501976 Forceps dissection tool
Glass Petri Dish 150mm dia x 15mm H Fisher Scientific (Intramalls) 08-747E Dissection dish
Interface paper (203 X 254mm PCTE Membrane 10um) Thomas Scientific 1220823 Slice incubation/biocytin application
Leica VT1200S Vibratome Leica 1491200S001 Vibratome for wedge slice sectioning
Mayo scissors Roboz RS-6872 Dissection tool
Single-edged carbon steel blades Fisher Scientific (Intramalls) 12-640 Razor blade for dissection
Specimen disc, orienting Leica 14048142068 Specialized vibratome stage for reproducible tilting
Spoonula FisherSci 14-375-10 Dissection tool
Super Glue Newegg 15187 Used for glueing tissue to vibratome stage
Vannas Spring Scissors Fine Science Tools 91500-09 Dissection tool
Electrophysiology
A1R Upright Confocal Microscope Nikon Instruments Electrophysiology and imaging microscope, can be any microscope compatible with electrophysiology
Electrode Borosilicate glass w/ Filament OD 1.5mm, ID 1.1mm, 10 cm long Sutter Instrument BF150-110-10 Patch clamping pipette glass
Electrode Filler MicroFil WPI CMF20G Patch electrode pipette filler
In-line solution heater Warner Instruments (GSAdvantage) SH-27B Slice perfusion system heater
Multi-Micromanipulator Systems Sutter Intruments MPC-200 with MP285 Micromanipulators for patch clamp and stimulation electrode placement
P-1000 horizontal pipette puller for glass micropipettes Sutter instruments FG-P1000 Patch clamp pipetter puller
Patch-clamp amplifier and Software HEKA EPC-10 / Patchmaster Next Can be any amplifier/software
Recording Chamber Warner Instruments RC26G Slice "bath" during recording
Recording Chamber Harp Warner Instruments 640253 Stablizes slice during electrophysiology recording
Slice Incubation Chamber Custom Build Heated, oxygenated holding chamber for slices during recovery after slicing
Stimulus isolation unit A.M.P.I. Iso-Flex Stimulus isolation unit for electrophysiology
Syringe 60CC Fischer Scientific (Intramalls) 14-820-11 Electrophysiology perfusion fluid handling
Temperature controller Warner Instruments (GSAdvantage) TC-324C Slice perfusion system temperature controller
Tubing 1/8 OD 1/16 ID Fischer Scientific (Intramalls) 14-171-129 Electrophysiology perfusion fluid handling
Tugsten concentric bipolar microelectrode WPI TM33CCINS Stimulating electrode for electrophysiology
Histology
24 well Plate Fisher Scientific (Intramalls) 12-556006 Histology slice collection and immunostaining
Alexa Fluor 488 Streptavidin Jackson Immuno labs 016-540-084 Secondary antibody for biocytin visualization
Corning Orbital Shaker Sigma CLS6780FP Shaker for immunohistochemistry agitation
Cresyl Violet Acetate Sigma Aldrich (Intramalls) C5042-10G Cellular stain for histology
Disposable Microtome Blades Fisher Scientific 22-210-052 Sliding microtome blade
Filter-syringe Nalgene 4mm Cellulose Acetate 0.2um Fisher Scientific (Intramalls) 09-740-34A Syringe filter for filling recording pipettes with internal solution
Fluoromount-G Slide Mounting Medium Fisher Scientific OB100-01 Immunohistochemistry fluorescence mounting medium
glass slide staining dish with rack Fisher Scientific (Intramalls) 08-812 Cresyl Violet staining chamber
Microm HM450 Sliding Microtome ThermoFisher 910020 Freezing microtome for histology
Microscope Cover Glasses: Rectangles 50mm X 24mm Fisher Scientific (Intramalls) 12-543D Histochemistry slide cover glass
Permount mounting medium Fisher Scientific SP15-100 Cresyl violet section mounting medium
Superfrost Slides Fisher Scientific 22-034980 Histology slides
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In VitroWedge SliceNeuronal Circuit ConnectivityBrain SlicePresynaptic CircuitryPatch Clamp RecordingsPharmacologyActivity ImagingHistologySkullBrain DissectionCranial NervesSpinal CordBrain Fixation
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Fischl, M. J., Weisz, C. J. C. In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity. J. Vis. Exp. (162), e61664, doi:10.3791/61664 (2020).
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