Forbundne Helcelle optagelser i Organotypiske hippocampusskiver
Summary
Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.
Abstract
Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.
Introduction
Glutamatreceptorer medierer størstedelen af excitatorisk synaptisk transmission i centralnervesystemet synapser. De to store undertyper af ionotrope glutamatreceptorer lokaliseret ved ryggen leder af den postsynaptiske membran, er N-methyl-D-aspartat (NMDA) og α-amino-3-hydroxy-5-methylisoxazol-4-proprionic acid (AMPA)-receptorer. Ved hvilende membranpotentialer, AMPA-receptorer bære det meste af den postsynaptiske strøm under synaptisk transmission. I hippocampus, NMDA-receptoren spiller en central rolle i udløsningen ændringer i antallet af AMPA-receptorer i den postsynaptiske membran: ved at fungere som en "tilfældighed detektor" 1 at indlede ændringer i synaptisk styrke 1 NMDA-receptoren deltager i synaptiske mekanismer der menes at understøtte indlæring og hukommelse på subcellulært niveau. Som reaktion på depolarisering af den postsynaptiske neuron parallelt med præsynaptiske transmitterfrigivelse, calcium trænger ind via NMDAreceptoren indlede AMPA-receptoren indsættelse eller fjernelse 2. Disse receptor dynamik ligger til grund synapse plasticitet: en stigning i synaptisk styrke er langsigtet potensering 2,3 (LTP), mens en formindskelse i synaptisk styrke er langvarig depression 4 (LTD). Derfor AMPA-receptoren bevægelse menes at være ansvarlig for synaptisk plasticitet ekspression, mens NMDA-receptorer menes at regulere dens induktion.
Fastlæggelse af de præcise mekanismer, der ligger synaptisk transmission og plasticitet kræver studere små populationer af synapser, helst enkelt synapser. Mens nogle synapser er meget velegnet til undersøgelse på dette niveau, fx blomsterbægeret af Held 5, for de fleste synaptiske befolkninger dette er yderst vanskelig på grund af den lille og diffus karakter synaptiske forbindelser. To store elektrofysiologi teknikker er blevet udviklet til at undersøge enkelt synaptiske forbindelser: Den første er minimal stimulation, Where en præsynaptisk fibre antages stimuleret ekstracellulært. Den anden teknik er parret optagelser, hvor to samtidige helcelleproteiner optagelser fra synaptisk forbundne neuroner udføres. En stor fordel ved minimal stimulation er, at den er hurtig og relativt enkel at udføre, der involverer placering af en ekstracellulær stimulerende elektrode i axonal tarmkanalen samtidig optagelse fra en postsynaptisk neuron. Den primære bekymring, når du bruger denne teknik er, at pålidelige stimulering af en enkelt celle sjældent kan garanteres retssag efter retssagen.
I løbet af de sidste femten år har vi rutinemæssigt anvendes parret hel-celle optagelser fra to synaptisk forbundet pyramideneuroner 6-17. Den største fordel ved denne teknik er, at kun én præsynaptiske neuron konsekvent og pålideligt stimuleres. Det giver også mulighed for ikke kun elektrofysiologisk karakterisering, men også farmakologisk manipulation af den præsynaptiske neuron 6,18 </ Sup>. Sandsynligheden af synaptisk forbindelse mellem neuroner er lav, hvilket gør forbundne par vanskeligt at opnå 19. Anvendelsen af organotypiske hjerne skivekulturer omgår denne hindring som synaptisk forbindelse kan genoprette in vitro og desuden af arten af den resulterende forbindelse er den samme som i nativ hjernevæv 20. Desuden organotypiske kulturer udtrykker LTP, LTD 7-10,12-15,21 og andre former for kortvarig synaptisk plasticitet, herunder parret-puls lettelse (PPF) og depression (PPD) 6,22,23, så plasticitetsmodeller mekanismer til studeres i par af neuroner. Her beskriver vi den detaljerede metodologi, der er involveret i en vellykket nå parrede optagelser i denne in vitro-system. Denne information kan let tilpasses til andre eksperimentelle systemer, herunder akutte skiver og andre hjerneregioner.
Protocol
Representative Results
Discussion
Her har vi beskrevet kravene til oprettelse af vellykkede parrede helcelleproteiner optagelser i organotypiske hippocampus skivekulturer. Forbundne optagelser kan også udføres på flere præparater, herunder akutte skiver og dissocierede dyrkningssystemer 26,27. Mens fokus her har været på induktionen af længere former for synaptisk plasticitet (nemlig LTP og LTD), er det vigtigt at fremhæve, at parret helcelleproteiner optagelser i organotypisk, akut skive og dissocieret cellepræparater har give…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We would like to thank the members of the Montgomery and Madison labs for helpful discussion. We acknowledge the funding received from the following sources in this research: NFNZ, AMRF, Marsden Fund, HRC, and NIH.
Materials
| Organotypic cultures | Paired recordings | ||
| Minimum Essential Medium | Stable motorized micromanipulators | ||
| Penicillin-Streptomycin solution | Shallow tissue bath | ||
| HEPES buffer solution | DIC camera | ||
| 1M Tris stock solution | Amplifier | ||
| Hank’s Balanced Salt Solution | Computer | ||
| Horse Serum | Vibration isolation table | ||
| plastic-coated miniature spatulas | Upright microscope | ||
| soft paintbrush | Data acquistion and analysis software | ||
| manual tissue chopper | Electrode puller | ||
| #2 filter paper | Faraday cage | ||
| #5 forceps | |||
| Membrane inserts | |||
| CO2 incubator | |||
| Dissection hood | |||
| Class II hood |
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