Tuning i Hippocampal Theta Band<em> In Vitro</em>: Metoder til optagelse fra den isolerede gnagere Septohippocampal Circuit
Summary
Her præsenterer vi en protokol til optagelse af rytmisk neuronalt netværk theta og gamma oscillationer fra et isoleret hele hippocampal præparat. Vi beskriver de eksperimentelle trin fra udvindingen af hippocampus til detaljer af felt-, enheds- og helcelle-patch-klemmeoptagelser samt optogenetisk pacing af theta-rytmen.
Abstract
Denne protokol beskriver fremgangsmåderne til fremstilling og registrering fra den isolerede hele hippocampus, af WT og transgene mus sammen med nylige forbedringer i metoder og anvendelser til undersøgelsen af theta-oscillationer. En simpel karakterisering af det isolerede hippocampale præparat præsenteres, hvorved forholdet mellem interne hippocampale theta-oscillatorer undersøges sammen med aktiviteten af pyramidale celler og GABAergiske interneuroner af cornu ammoni-1 (CA1) og subikulum (SUB) områder. Samlet viser vi, at den isolerede hippocampus er i stand til at generere indre theta-oscillationer in vitro, og at rytmicitet frembragt inden for hippocampus kan manipuleres nøjagtigt ved optogenetisk stimulering af parvalbumin-positive (PV) interneuroner. Det in vitro- isolerede hippocampale præparat giver en enestående mulighed for at anvende samtidige felt- og intracellulære patch-clamp-optagelser fra visuelt identificeret neuRons for bedre at forstå mekanismerne bagved theta rytmegenerering.
Introduction
Hippokampale theta-oscillationer (4-12 Hz) er blandt de mest fremtrædende former for rytmisk aktivitet i pattedyrs hjernen og antages at spille centrale roller i kognitive funktioner som behandling af spatiotemporale oplysninger og dannelse af episodiske minder 1 , 2 , 3 . Mens flere in vivo- undersøgelser, der fremhæver forholdet mellem theta-modulerede stedceller med rumlig navigation og læsionsundersøgelser samt kliniske beviser, understøtter synspunktet om, at hippocampale theta-oscillationer er involveret i hukommelsesdannelse 4 , 5 , 6 , de mekanismer, der er forbundet Med generering af hippocampale theta-oscillationer er stadig ikke fuldt ud forstået. Tidlige in vivo undersøgelser foreslog, at theta-aktivitet hovedsageligt afhænger af ekstrinsiske oscillatorer, især rytmisk inputFra afferente hjerne strukturer såsom septum og entorhinal cortex 7 , 8 , 9 , 10 . En rolle for indre faktorer – intern tilslutning af hippocampale neurale netværk sammen med egenskaberne af hippocampale neuroner – blev også postuleret på basis af in vitro observationer 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 . Men bortset fra nogle få landmærkeundersøgelser 19 , 20 , 21 er der vanskeligheder med at udvikle fremgangsmåder, som kunne replikere fysiologisk realistiske befolkningsaktiviteter i simpel in vitro- skiveforberedelseS har i lang tid forsinket mere detaljeret forsøgsundersøgelse af hippocampus 'indbyggede evner og relaterede områder til selvfrembringende theta-oscillationer.
En vigtig ulempe ved standard in vitro -tyndskiveeksperimentelle indstillinger er, at den 3D-cellulære og synaptiske organisation af hjernestrukturer sædvanligvis kompromitteres. Det betyder, at mange former for samordnede netværksaktiviteter baseret på rumligt fordelte celleanlæg, der spænder fra lokaliserede grupper (≤1 mm radius) til populationer af neuroner spredt over en eller flere hjerneområder (> 1 mm), ikke kan understøttes. I betragtning af disse overvejelser var der behov for en anden type tilgang til at studere, hvordan theta-svingninger dukker op i hippocampus og former for relaterede cortical og subcortical output strukturer.
I de senere år har den indledende udvikling af "komplet septo-hippocampal" -præparatet til at undersøge tovejsinteraCtioner af de to strukturer 22 og den efterfølgende udvikling af det "isolerede hippocampus" -præparat har vist, at intrinsiske theta-oscillationer forekommer spontant i hippocampus, der mangler ekstern rytmisk indgang 23 . Værdien af disse fremgangsmåder ligger på den indledende indsigt, at hele den funktionelle struktur af disse regioner skulle bevares for at fungere som en theta-rytmegenerator in vitro 22 .
Protocol
Representative Results
Discussion
Mens elektrofysiologiske optagelser fra akutte hippocampale skiver udgør en standard in vitro- teknik, adskiller de her fremlagte metoder sig væsentligt fra den klassiske tilgang. I modsætning til de tynde skivepræparater, hvor specifikke cellelag er synlige på overfladen og kan undersøges direkte, er de intakte hippocampale præparater mere beslægtede med in vivo konfigurationer, hvor elektroder sænkes til målrettede hjerneområder, mens de krydser gennem individuelle lag. Hippocampus integri…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af de canadiske institutter for sundhedsforskning og naturvidenskab.
Materials
| Reagents | |||
| Sodium Chloride | Sigma Aldrich | S9625 | |
| Sucrose | Sigma Aldrich | S9378 | |
| Sodium Bicarbonate | Sigma Aldrich | S5761 | |
| NaH2PO4 – sodium phosphate monobasic | Sigma Aldrich | S8282 | |
| Magnesium sulfate | Sigma Aldrich | M7506 | |
| Potassium Chloride | Sigma Aldrich | P3911 | |
| D-(+)-Glucose | Sigma Aldrich | G7528 | |
| Calcium chloride dihydrate | Sigma Aldrich | C5080 | |
| Sodium Ascorbate | Sigma Aldrich | A7631-25G | |
| Name | Company | Catalog Number | Comments |
| Equipment | |||
| Standard Dissecting Scissors | Fisher Scientific | 08-951-25 | brain extraction |
| Scalpel Handle #4, 14cm | WPI | 500237 | brain extraction |
| Filter forceps, flat jaws, straight (11cm) | WPI | 500456 | brain extraction |
| Paragon Stainless Steel Scalpel Blades #20 | Ultident | 02-90010-20 | brain extraction |
| Fine Point Curved Dissecting Scissors | Thermo Fisher Scientific | 711999 | brain extraction |
| Teflon (PTFE) -coated thin spatula | VWR | 82027-534 | hippocampal preparation |
| Hayman Style Microspatula | Fisher Scientific | 21-401-25A | hippocampal preparation |
| Lab spoon | Fisher Scientific | 14-375-20 | hippocampal preparation |
| Borosilicate Glass Pasteur Pipets | Fisher Scientific | 13-678-20A | hippocampal preparation |
| Droper | Fisher Scientific | hippocampal preparation | |
| Razor blades Single edged | VWR | 55411-055 | hippocampal preparation |
| Lens paper (4X6 inch) | VWR | 52846-001 | hippocampal preparation |
| Glass petri dishes (100 x 20 mm) | VWR | 25354-080 | hippocampal preparation |
| Plastic tray for ice; size 30 x 20 x 5 cm | n.a. | n.a. | hippocampal preparation |
| Single Inline Solution Heater | Warner Instruments | SH-27B | perfusion system |
| Aquarium air stones for bubbling | n.a. | n.a. | perfusion system |
| Tygon E-3603 tubing (ID 1/16 OD 1/8) | Fisherbrand | 14-171-129 | perfusion system |
| Electric Skillet | Black & Decker | n.a. | perfusion system |
| 95% O2/5% CO2 gas mixture (carbogen) | Vitalaire | SG466204A | perfusion system |
| Glass bottles/flasks (4 x 1 L) | n.a. | n.a. | perfusion system |
| Submerged recording Chamber | custom design (FM) | n.a. | Commercial alternative may be used |
| Glass pipettes (1.5 / 0.84 OD/ID (mm) ) | WPI | 1B150F-4 | electrophysiology |
| Hum Bug 50/60 Hz Noise Eliminator | Quest Scientific | Q-Humbug | electrophysiology |
| Multiclamp 700B patch-clamp amplifier | Molecular devices | MULTICLAMP | electrophysiology |
| Multiclamp 700B Commander Program | Molecular devices | MULTICLAMP | electrophysiology |
| Digital/Analogue converter | Molecular devices | DDI440 | electrophysiology |
| PCLAMP10 | Molecular devices | PCLAMP10 | electrophysiology |
| Vibration isolation table | Newport | n.a. | electrophysiology |
| Micromanipulators (manually operated ) | Siskiyou | MX130 | electrophysiology (LFP) |
| Micromanipulators (automated) | Siskiyou | MC1000e | electrophysiology (patch) |
| Audio monitor | A-M Systems | Model 3300 | electrophysiology |
| Micropipette/Patch pipette puller | Sutter | P-97 | electrophysiology |
| Custom-built upright fluorescence microscope | Siskiyou | n.a. | Imaging |
| Analogue video camera | COHU | 4912-2000/0000 | Imaging |
| Digital frame grabber with imaging software | EPIX, Inc | PIXCI-SV7 | Imaging |
| Olympus 2.5x objective | Olympus | MPLFLN | Imaging |
| Olympus 40x water immersion objective | Olympus | UIS2 LUMPLFLN | Imaging |
| Custom-made light-emitting diode (LED) system | custom | n.a. | optogenetic stimulation (Amhilon et al., 2015) |
| Name | Company | Catalog Number | Comments |
| Animals | |||
| PV::Cre (KI) mice | Jackson Laboratory | stock number 008069 | Allow Cre-directed gene expression in PV interneurons |
| Constitutive-conditional Ai9 mice (R26-lox-stop-lox-tdTomato (KI)) | Jackson Laboratory | stock number 007905 | Express TdTomato following Cre-mediated recombination |
| Ai32 mice (R26-lox-stop-lox-ChR2(H134R)-EYFP | Jackson Laboratory | stock number 012569 |
Express the improved channelrhodopsin-2/EYFP fusion protein following exposure to Cre recombinase |
| PVChY mice | In house breeding | n.a. | Offspring obtained from cross-breeding the PV-Cre line with Ai32 mice (R26-lox-stop-lox-ChR2(H134R)-EYFP |
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