Sammenkoblede hele cellen Recordings i organotypic hippocampus Slices
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
Glutamat-reseptorer medierer flertallet av eksitatorisk synaptisk transmisjon i sentralnervesystemet synapser. De to hoved subtyper av ionotrofiske glutamatreseptorer lokalisert på ryggraden hodet av postsynaptiske membranen er N-metyl-D-aspartat (NMDA) og α-amino-3-hydroksy-5-metylisoksazol-4-propionsyre (AMPA) reseptorer. Hos hvilende membranpotensialer, AMPA-reseptorer bære mesteparten av den postsynaptiske strøm under synaptisk transmisjon. I hippocampus, spiller NMDA-reseptor-en nøkkelrolle i å utløse endringer i antallet AMPA-reseptorer i den postsynaptiske membranen: ved å fungere som en "tilfeldighet detektor" 1 for å igangsette endringer i synaptisk styrke 1, er med NMDA-reseptoren i de synaptiske mekanismer som er tenkt å understøtte læring og hukommelse på et subcellulære nivå. I respons til depolarisering av den postsynaptiske nevroner parallelt med presynaptisk transmitterfrigjørelse, går kalsium via NMDAreseptor for å initiere AMPA reseptor innsetting eller fjerning to. Disse reseptor-dynamikken til grunn synapse plastisitet: en økning av synaptisk styrke er langvarig potense 2,3 (LTP), mens en reduksjon i synaptisk styrke er langvarig depresjon 4 (LTD). Derfor AMPA-reseptor-bevegelsen er antatt å være ansvarlig for synaptisk plastisitet ekspresjon, mens NMDA-reseptorer er antatt å kontrollere dens induksjon.
Bestemme de nøyaktige mekanismene bak synaptisk transmisjon og plastisitet krever studere små bestander av synapser, helst enkelt synapser. Mens noen synapser er godt egna for studier på dette nivået, for eksempel, den Calyx av Held 5, for de fleste synaptiske populasjoner dette er ekstremt vanskelig på grunn av den lille og diffuse natur synaptiske forbindelser. To store elektrofysiologiske teknikker har blitt utviklet for å undersøke enkelt synaptiske forbindelser: Den første er minimal stimulering, der hvore en presynaptisk fiber antas stimulert ekstracellulært. Den andre teknikken er paret innspillinger, der to samtidige hele celle opptak fra postsynaptisk koblet nevroner er utført. En stor fordel med minimal stimulering er at den er rask og relativt enkel å utføre, som omfatter plassering av et ekstracellulært stimulerende elektrode inn i den aksonal kanalen samtidig opptak fra en postsynaptiske nevroner. Den primære bekymring når du bruker denne teknikken er at pålitelig stimulering av en enkelt celle kan sjelden være garantert rettssak etter rettssak.
I løpet av de siste femten årene har vi rutinemessig brukt paret hel-celle opptak fra to postsynaptisk koblet pyramidale nevroner 6-17. Den store fordelen med denne teknikken er at bare én presynaptiske nevron er konsekvent og pålitelig stimulert. Den gjør det også ikke bare elektrofysiologisk karakterisering men også farmakologisk manipulasjon av den presynaptiske nervecellen 6,18 </ Sup>. Imidlertid er sannsynligheten for synaptisk forbindelse mellom nevroner lav, noe som gjør koblede par vanskelig å få 19. Bruken av organotypic hjernen skive kulturer omgår dette hinderet som synaptiske tilkobling kan reetablere in vitro og dessuten arten av den resulterende tilkobling er lik som i native hjernevev 20. I tillegg organotypic kulturer uttrykke LTP, LTD 7-10,12-15,21 og andre former for kortsiktig synaptisk plastisitet inkludert parvise puls tilrettelegging (PPF) og depresjon (PPD) 6,22,23, slik plastisitet mekanismer for å studeres i parene av neuroner. Her beskriver vi detaljert metodikk involvert i vellykket oppnå sammenkoblede opptak i denne in vitro system. Denne informasjonen kan lett tilpasses andre eksperimentelle systemer, inkludert akutte skiver og andre hjerneregioner.
Protocol
Representative Results
Discussion
Her har vi beskrevet kravene for å etablere vellykkede paret hele celleopptak i organotypic hippocampus skive kulturer. Sammenkoblede opptak kan også utføres i flere preparater, inkludert akutte skiver og dissosiert kultursystemer 26,27. Mens fokuset her har vært på induksjon av lengre former for synaptisk plastisitet (nemlig LTP og LTD), er det viktig å understreke at sammenkoblede hele celle opptak i organotypic, akutt skive og dissosiert celle forberedelser har gitt viktig innsikt i quantal størrels…
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 |
References
- Dingledine, R., Borges, K., Bowie, D., Traynelis, S. F. The glutamate receptor ion channels. Pharmacology Reviews. 51, 7-61 (1999).
- Malenka, R. C., Nicoll, R. A. Long-term potentiation – A decade of progress. Science. 285, 1870-1874 (1999).
- Bliss, T. V. P., Lomo, T. Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path. Journal of Physiology. 232, 331-356 (1973).
- Dudek, S. M., Bear, M. F. Homosynaptic long-term depression in area CA1 of hippocampus and effects of N-methyl-D-aspartate receptor blockade. Proceedings of the National Academy of Sciences USA. 89, 4363-4367 (1992).
- Borst, J. G., Soria van Hoeve, J. The calyx of held synapse: from model synapse to auditory relay. Annual Reviews in Physiology. 74, 199-224 (2012).
- Pavlidis, P., Madison, D. V. Synaptic transmission in pair recordings from CA3 pyramidal cells in organotypic culture. Journal of Neurophysiology. 81, 2787-2797 (1999).
- Pavlidis, P., Montgomery, J. M., Madison, D. V. Presynaptic protein kinase activity supports long-term potentiation at synapses between individual hippocampal neurons. Journal of Neuroscience. 20 (12), 4497-4505 (2000).
- Montgomery, J. M., Pavlidis, P., Madison, D. V. Pair recordings reveal all-silent synaptic connections and the postsynaptic expression of long-term potentiation. Neuron. 29, 691-701 (2001).
- Montgomery, J. M., Madison, D. V. State-dependent heterogeneity in synaptic depression between pyramidal cell pairs. Neuron. 33, 765-777 (2002).
- Montgomery, J. M., Selcher, J. C., Hansen, J. E., Madison, D. V. Dynamin-dependent NMDAR endocytosis during LTD and its dependence on synaptic state. BMC Neuroscience. 6, 48 (2005).
- Waites, C. L., et al. Synaptic SAP97 isoforms regulate AMPA receptor dynamics and access to presynaptic glutamate. Journal of Neuroscience. 29 (14), 4332-4345 (2009).
- Emond, M., et al. AMPA receptor subunits define properties of state-dependent synaptic plasticity. Journal of Physiology. 588, 1929-1946 (2010).
- Li, D., et al. SAP97 directs NMDA receptor spine targeting and synaptic plasticity. Journal of Physiology. 589, 4491-4510 (2011).
- Genoux, D., Bezerra, P., Montgomery, J. M. Intra-spaced stimulation and protein phosphatase 1 dictate the direction of synaptic plasticity. European Journal of Neuroscience. 33 (10), 1761-1770 (2011).
- Selcher, J. C., Xu, W., Hanson, J. E., Malenka, R. C., Madison, D. V. Glutamate receptor subunit GluA1 is necessary for long-term potentiation and synapse unsilencing, but not long-term depression in mouse hippocampus. Brain Research. 1435, 8-14 (2012).
- Arons, M. H., et al. Autism-associated mutations in ProSAP2/Shank3 impair synaptic transmission and neurexin-neuroligin-mediated transsynaptic signaling. Journal of Neuroscience. 32 (43), 14966-14978 (2012).
- Montgomery, J. M., Madison, D. V. Discrete synaptic states define a major mechanism of synapse plasticity. Trends in Neuroscience. 27 (12), 744-750 (2004).
- Miles, R., Poncer, J. C. Pair recordings from neurones. Current Opinion in Neurobiology. 6 (3), 387-394 (1996).
- Malinow, R. Transmission between pairs of hippocampal slice neurons: quantal levels, oscillations and LTP. Science. 252 (5006), 722-724 (1991).
- Gähwiler, B. H., Capogna, M., Debanne, D., McKinney, R. A., Thompson, S. M. Organotypic slice cultures: a technique has come of age. Trends in Neuroscience. 20 (10), 471-477 (1997).
- Stoppini, L., Buchs, P. A., Muller, D. A simple method for organotypic cultures of nervous tissue. Journal of Neuroscience Methods. 37 (2), 173-182 (1991).
- Debanne, D., Guérineau, N. C., Gähwiler, B. H., Thompson, S. M. Paired-pulse facilitation and depression at unitary synapses in rat hippocampus: quantal fluctuation affects subsequent release. Journal of Physiology. 491, 163-176 (1996).
- Debanne, D., Gähwiler, B. H., Thompson, S. M. Cooperative interactions in the induction of long-term potentiation and depression of synaptic excitation between hippocampal CA3-CA1 cell pairs in vitro. Proceedings of the National Academy of Sciences U S A. 93 (20), 11225-11230 (1996).
- Malinow, R., Tsien, R. W. Presynaptic enhancement shown by whole cell recordings of long-term potentiation in hippocampal slices. Nature. 346 (6280), 177-180 (1990).
- DeBello, W. M., et al. SNAP-mediated protein-protein interactions essential for neurotransmitter release. Nature. 373 (6515), 626-630 (1995).
- Bekkers, J. M., Stevens, C. F. Origin of variability in quantal size in cultured hippocampal neurons and hippocampal slices. Proceedings of the National Academy of Sciences U S A. 87, 5359-5362 (1990).
- Malinow, R. Transmission between pairs of hippocampal slice neurons: quantal levels, oscillations, and LTP. Science. 252, 722-724 (1991).
- Debanne, D., Guérineau, N. C., Gähwiler, B. H., Thompson, S. M. Physiology and pharmacology of unitary synaptic connections between pairs of cells in areas CA3 and CA1 of rat hippocampal slice cultures. Journal of Neurophysiology. 73 (3), 1282-1294 (1995).
- Mitra, A., Blank, M., Madison, D. V. Developmentally altered inhibition in Ts65Dn, a mouse model of Down syndrome. Brain Research. 1440, 1-8 (2012).
- Hanson, J. E., Madison, D. V. Presynaptic FMR1 genotype influences the degree of synaptic connectivity in a mosaic mouse model of fragile X syndrome. Journal of Neuroscience. 27 (15), 4014-4018 (2007).
- Hanson, J. E., Blank, M., Valenzuela, R. A., Garner, C. C., Madison, D. V. The functional nature of synaptic circuitry is altered in area CA3 of the hippocampus in a mouse model of Down’s syndrome. Journal of Physiology. 579, 53-67 (2007).
