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Electrophysiological Methods for Investigating Inhibitory Synaptic Plasticity

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Part of the book series: Neuromethods ((NM,volume 81))

Abstract

This chapter provides a detailed electrophysiological protocol for investigating inhibitory synaptic plasticity using the gramicidin perforated patch clamp technique. Gramicidin is a polypeptide antibiotic that is used as a perforating agent because it forms pores in the neuronal membrane that are permeable to monovalent cations and small uncharged molecules, but not to Cl. Preserving the neuronal Cl gradient is essential for recording native inhibitory GABAA receptor (GABAAR) currents, which are largely carried by Cl ions.

Inhibitory synaptic plasticity is a change in the strength of GABA- or glycine-mediated synaptic transmission. The mechanisms underlying inhibitory synaptic plasticity include changes in synaptic conductance (pre- or postsynaptically), as well as changes in the strength and polarity of the neuronal Cl gradient. The gramicidin perforated patch clamp technique is preferable over the whole cell patch clamp technique, because it does not equilibrate the intracellular milieu with the artificial pipette solution, and thus permits the observation of changes in the native Cl gradient following the induction of inhibitory synaptic plasticity. This methods chapter describes how to electrophysiologically record GABAAR inhibitory synaptic plasticity between mono-synaptically connected pairs of cultured hippocampal neurons.

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References

  1. Bliss TV, Lomo T (1973) Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path. J Physiol 232:331–356

    PubMed  CAS  Google Scholar 

  2. Malinow R, Mainen ZF, Hayashi Y (2000) LTP mechanisms: from silence to four-lane traffic. Curr Opin Neurobiol 10:352–357

    Article  PubMed  CAS  Google Scholar 

  3. Malenka RC, Bear MF (2004) LTP and LTD: an embarrassment of riches. Neuron 44:5–21

    Article  PubMed  CAS  Google Scholar 

  4. Whitlock JR, Heynen AJ, Shuler MG, Bear MF (2006) Learning induces long-term potentiation in the hippocampus. Science 313:1093–1097

    Article  PubMed  CAS  Google Scholar 

  5. Lynch MA (2004) Long-term potentiation and memory. Physiol Rev 84:87–136

    Article  PubMed  CAS  Google Scholar 

  6. Lamsa K, Kullmann DM, Woodin MA (2010) Inhibitory circuit plasticity. Front Synaptic Neurosci 2. doi:10.3389

    Google Scholar 

  7. Kullmann DM, Lamsa KP (2011) LTP and LTD in cortical GABAergic interneurons: emerging rules and roles. Neuropharmacology 60:712–719

    Article  PubMed  CAS  Google Scholar 

  8. Pelletier JG, Lacaille JC (2008) Long-term synaptic plasticity in hippocampal feedback inhibitory networks. Prog Brain Res 169:241–250

    Article  PubMed  CAS  Google Scholar 

  9. Chevaleyre V, Castillo PE (2003) Heterosynaptic LTD of hippocampal GABAergic synapses: a novel role of endocannabinoids in regulating excitability. Neuron 38:461–472

    Article  PubMed  CAS  Google Scholar 

  10. Korn H, Oda Y, Faber DS (1992) Long-term potentiation of inhibitory circuits and synapses in the central nervous system. Proc Natl Acad Sci U S A 89:440–443

    Article  PubMed  CAS  Google Scholar 

  11. Kang J, Jiang L, Goldman SA, Nedergaard M (1998) Astrocyte-mediated potentiation of inhibitory synaptic transmission. Nat Neurosci 1:683–692

    Article  PubMed  CAS  Google Scholar 

  12. Lu YM, Mansuy IM, Kandel ER, Roder J (2000) Calcineurin-mediated LTD of GABAergic inhibition underlies the increased excitability of CA1 neurons associated with LTP. Neuron 26:197–205

    Article  PubMed  CAS  Google Scholar 

  13. McLean HA, Caillard O, Ben-Ari Y, Gaiarsa JL (1996) Bidirectional plasticity expressed by GABAergic synapses in the neonatal rat hippocampus. J Physiol 496(Pt 2):471–477

    PubMed  CAS  Google Scholar 

  14. Patenaude C, Chapman CA, Bertrand S, Congar P, Lacaille JC (2003) GABAB receptor- and metabotropic glutamate receptor-dependent cooperative long-term potentiation of rat hippocampal GABAA synaptic transmission. J Physiol 553:155–167

    Article  PubMed  CAS  Google Scholar 

  15. Stelzer A, Simon G, Kovacs G, Rai R (1994) Synaptic disinhibition during maintenance of long-term potentiation in the CA1 hippocampal subfield. Proc Natl Acad Sci U S A 91:3058–3062

    Article  PubMed  CAS  Google Scholar 

  16. Ormond J, Woodin MA (2009) Disinhibition mediates a form of hippocampal long-term potentiation in area CA1. PLoS One 4:e7224

    Article  PubMed  Google Scholar 

  17. Balena T, Woodin MA (2008) Coincident pre- and postsynaptic activity downregulates NKCC1 to hyperpolarize E(Cl) during development. Eur J Neurosci 27:2402–2412

    Article  PubMed  Google Scholar 

  18. Woodin MA, Ganguly K, Poo MM (2003) Coincident pre- and postsynaptic activity modifies GABAergic synapses by postsynaptic changes in Cl- transporter activity. Neuron 39:807–820

    Article  PubMed  CAS  Google Scholar 

  19. Holmgren CD, Zilberter Y (2001) Coincident spiking activity induces long-term changes in inhibition of neocortical pyramidal cells. J Neurosci 21:8270–8277

    PubMed  CAS  Google Scholar 

  20. Kano M (1995) Plasticity of inhibitory synapses in the brain: a possible memory mechanism that has been overlooked. Neurosci Res 21:177–182

    Article  PubMed  CAS  Google Scholar 

  21. Komatsu Y (1996) GABAB receptors, monoamine receptors, and postsynaptic inositol trisphosphate-induced Ca2+ release are involved in the induction of long-term potentiation at visual cortical inhibitory synapses. J Neurosci 16:6342–6352

    PubMed  CAS  Google Scholar 

  22. Komatsu Y, Iwakiri M (1993) Long-term modification of inhibitory synaptic transmission in developing visual cortex. Neuroreport 4:907–910

    Article  PubMed  CAS  Google Scholar 

  23. Ouardouz M, Sastry BR (2000) Mechanisms underlying LTP of inhibitory synaptic transmission in the deep cerebellar nuclei. J Neurophysiol 84:1414–1421

    PubMed  CAS  Google Scholar 

  24. Carvalho TP, Buonomano DV (2009) Differential effects of excitatory and inhibitory plasticity on synaptically driven neuronal input–output functions. Neuron 61:774–785

    Article  PubMed  CAS  Google Scholar 

  25. Hartman KN, Pal SK, Burrone J, Murthy VN (2006) Activity-dependent regulation of inhibitory synaptic transmission in hippocampal neurons. Nat Neurosci 9:642–649

    Article  PubMed  CAS  Google Scholar 

  26. Fiumelli H, Cancedda L, Poo MM (2005) Modulation of GABAergic transmission by activity via postsynaptic Ca2+-dependent regulation of KCC2 function. Neuron 48:773–786

    Article  PubMed  CAS  Google Scholar 

  27. Woodin MA, Maffei A (eds) (2010) Inhibitory synaptic plasticity. Springer, New York

    Google Scholar 

  28. Gaiarsa JL, Ben-Ari Y (2006) Long-term plasticity at inhibitory synapses: a phenomenon that has been overlooked. In: The dynamic synapse. CRC Press, Boca Raton, FL

    Google Scholar 

  29. Gaiarsa JL, Caillard O, Ben-Ari Y (2002) Long-term plasticity at GABAergic and glycinergic synapses: mechanisms and functional significance. Trends Neurosci 25:564–570

    Article  PubMed  CAS  Google Scholar 

  30. Owens DF, Boyce LH, Davis MB, Kriegstein AR (1996) Excitatory GABA responses in embryonic and neonatal cortical slices demonstrated by gramicidin perforated-patch recordings and calcium imaging. J Neurosci 16:6414–6423

    PubMed  CAS  Google Scholar 

  31. Akaike N (1996) Gramicidin perforated patch recording and intracellular chloride activity in excitable cells. Prog Biophys Mol Biol 65:251–264

    Article  PubMed  CAS  Google Scholar 

  32. Kyrozis A, Reichling DB (1995) Perforated-patch recording with gramicidin avoids artifactual changes in intracellular chloride concentration. J Neurosci Methods 57:27–35

    Article  PubMed  CAS  Google Scholar 

  33. Balena T, Acton BA, Woodin MA (2010) GABAergic synaptic transmission regulates calcium influx during spike-timing dependent plasticity. Front Synaptic Neurosci 2. doi:10.2289

    Google Scholar 

  34. Caporale N, Dan Y (2008) Spike timing-dependent plasticity: a Hebbian learning rule. Annu Rev Neurosci 31:25–46

    Article  PubMed  CAS  Google Scholar 

  35. Kaila K (1994) Ionic basis of GABAA receptor channel function in the nervous system. Prog Neurobiol 42:489–537

    Article  PubMed  CAS  Google Scholar 

  36. Farrant M, Kaila K (2007) The cellular, molecular and ionic basis of GABA(A) receptor signalling. Prog Brain Res 160:59–87

    Article  PubMed  CAS  Google Scholar 

  37. Wafford KA (2005) GABAA receptor subtypes: any clues to the mechanism of benzodiazepine dependence? Curr Opin Pharmacol 5:47–52

    Article  PubMed  CAS  Google Scholar 

  38. Hebert SC, Mount DB, Gamba G (2004) Molecular physiology of cation-coupled Cl cotransport: the SLC12 family. Pflugers Arch 447:580–593

    Article  PubMed  CAS  Google Scholar 

  39. Rivera C, Voipio J, Payne JA, Ruusuvuori E, Lahtinen H, Lamsa K, Pirvola U, Saarma M, Kaila K (1999) The K+/Cl co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation. Nature 397:251–255

    Article  PubMed  CAS  Google Scholar 

  40. Blaesse P, Airaksinen MS, Rivera C, Kaila K (2009) Cation-chloride cotransporters and neuronal function. Neuron 61:820–838

    Article  PubMed  CAS  Google Scholar 

  41. Payne JA, Rivera C, Voipio J, Kaila K (2003) Cation-chloride co-transporters in neuronal communication, development and trauma. Trends Neurosci 26:199–206

    Article  PubMed  CAS  Google Scholar 

  42. Song L, Mercado A, Vazquez N, Xie Q, Desai R, George AL Jr, Gamba G, Mount DB (2002) Molecular, functional, and genomic characterization of human KCC2, the neuronal K–Cl cotransporter. Brain Res Mol Brain Res 103:91–105

    Article  PubMed  CAS  Google Scholar 

  43. Wardle RA, Poo MM (2003) Brain-derived neurotrophic factor modulation of GABAergic synapses by postsynaptic regulation of chloride transport. J Neurosci 23:8722–8732

    PubMed  CAS  Google Scholar 

  44. van den Pol AN, Obrietan K, Chen G (1996) Excitatory actions of GABA after neuronal trauma. J Neurosci 16:4283–4292

    PubMed  Google Scholar 

  45. Nabekura J, Ueno T, Okabe A, Furuta A, Iwaki T, Shimizu-Okabe C, Fukuda A, Akaike N (2002) Reduction of KCC2 expression and GABAA receptor-mediated excitation after in vivo axonal injury. J Neurosci 22:4412–4417

    PubMed  CAS  Google Scholar 

  46. Coull JA, Beggs S, Boudreau D, Boivin D, Tsuda M, Inoue K, Gravel C, Salter MW, De Koninck Y (2005) BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain. Nature 438:1017–1021

    Article  PubMed  CAS  Google Scholar 

  47. Toyoda H, Ohno K, Yamada J, Ikeda M, Okabe A, Sato K, Hashimoto K, Fukuda A (2003) Induction of NMDA and GABAA receptor-mediated Ca2+ oscillations with KCC2 mRNA downregulation in injured facial motoneurons. J Neurophysiol 89:1353–1362

    Article  PubMed  CAS  Google Scholar 

  48. Kahle KT, Staley KJ, Nahed BV, Gamba G, Hebert SC, Lifton RP, Mount DB (2008) Roles of the cation-chloride cotransporters in neurological disease. Nat Clin Pract Neurol 4:490–503

    Article  PubMed  CAS  Google Scholar 

  49. Pond BB, Galeffi F, Ahrens R, Schwartz-Bloom RD (2004) Chloride transport inhibitors influence recovery from oxygen-glucose deprivation-induced cellular injury in adult hippocampus. Neuropharmacology 47:253–262

    Article  PubMed  CAS  Google Scholar 

  50. Jaenisch N, Witte OW, Frahm C (2010) Downregulation of potassium chloride cotransporter KCC2 after transient focal cerebral ischemia. Stroke 41:e151–e159

    Article  PubMed  CAS  Google Scholar 

  51. Rivera C, Li H, Thomas-Crusells J, Lahtinen H, Viitanen T, Nanobashvili A, Kokaia Z, Airaksinen MS, Voipio J, Kaila K et al (2002) BDNF-induced TrkB activation down-regulates the K+-Cl cotransporter KCC2 and impairs neuronal Cl extrusion. J Cell Biol 159:747–752

    Article  PubMed  CAS  Google Scholar 

  52. Woo NS, Lu J, England R, McClellan R, Dufour S, Mount DB, Deutch AY, Lovinger DM, Delpire E (2002) Hyperexcitability and epilepsy associated with disruption of the mouse neuronal-specific K-Cl cotransporter gene. Hippocampus 12:258–268

    Article  PubMed  CAS  Google Scholar 

  53. Galeffi F, Sah R, Pond BB, George A, Schwartz-Bloom RD (2004) Changes in intracellular chloride after oxygen-glucose deprivation of the adult hippocampal slice: effect of diazepam. J Neurosci 24:4478–4488

    Article  PubMed  CAS  Google Scholar 

  54. Brumback AC, Staley KJ (2008) Thermodynamic regulation of NKCC1-mediated Cl cotransport underlies plasticity of GABA(A) signaling in neonatal neurons. J Neurosci 28:1301–1312

    Article  PubMed  CAS  Google Scholar 

  55. Yamada J, Okabe A, Toyoda H, Kilb W, Luhmann HJ, Fukuda A (2004) Cl uptake promoting depolarizing GABA actions in immature rat neocortical neurones is mediated by NKCC1. J Physiol 557:829–841

    Article  PubMed  CAS  Google Scholar 

  56. Ben-Ari Y, Gaiarsa JL, Tyzio R, Khazipov R (2007) GABA: a pioneer transmitter that excites immature neurons and generates primitive oscillations. Physiol Rev 87:1215–1284

    Article  PubMed  CAS  Google Scholar 

  57. Ben-Ari Y (2002) Excitatory actions of gaba during development: the nature of the nurture. Nat Rev Neurosci 3:728–739

    Article  PubMed  CAS  Google Scholar 

  58. Fiumelli H, Woodin MA (2007) Role of activity-dependent regulation of neuronal chloride homeostasis in development. Curr Opin Neurobiol 17:81–86

    Article  PubMed  CAS  Google Scholar 

  59. Sivakumaran S, Mohajerani MH, Cherubini E (2009) At immature mossy-fiber-CA3 synapses, correlated presynaptic and postsynaptic activity persistently enhances GABA release and network excitability via BDNF and cAMP-dependent PKA. J Neurosci 29:2637–2647

    Article  PubMed  CAS  Google Scholar 

  60. Kaech S, Banker G (2006) Culturing hippocampal neurons. Nat Protoc 1:2406–2415

    Article  PubMed  CAS  Google Scholar 

  61. Vicario-Abejon C (2004) Long-term culture of hippocampal neurons. Curr Protoc Neurosci 3:Unit 3.2

    Google Scholar 

  62. Nunez J (2008) Primary culture of hippocampal neurons from P0 newborn rats. J Vis Exp 29:895

    Google Scholar 

  63. Barry PH (1994) JPCalc, a software package for calculating liquid junction potential corrections in patch-clamp, intracellular, epithelial and bilayer measurements and for correcting junction potential measurements. J Neurosci Methods 51:107–116

    Article  PubMed  CAS  Google Scholar 

  64. Neher E (1992) Correction for liquid junction potentials in patch clamp experiments. Methods Enzymol 207:123–131

    Article  PubMed  CAS  Google Scholar 

  65. Saraga F, Balena T, Wolansky T, Dickson CT, Woodin MA (2008) Inhibitory synaptic plasticity regulates pyramidal neuron spiking in the rodent hippocampus. Neuroscience 155:64–75

    Article  PubMed  CAS  Google Scholar 

  66. Williams JR, Payne JA (2004) Cation transport by the neuronal K(+)-Cl(−) cotransporter KCC2: thermodynamics and kinetics of alternate transport modes. Am J Physiol Cell Physiol 287:C919–C931

    Article  PubMed  CAS  Google Scholar 

  67. Huberfeld G, Wittner L, Clemenceau S, Baulac M, Kaila K, Miles R, Rivera C (2007) Perturbed chloride homeostasis and GABAergic signaling in human temporal lobe epilepsy. J Neurosci 27:9866–9873

    Article  PubMed  CAS  Google Scholar 

  68. Akaike N (2009) Gramicidin perforated patch. In: Alvarez-Leefmans FJ, Delpire E (eds) Physiology and pathology of chloride transporters and channels in the nervous system. Elsevier, Amsterdam, pp 141–148

    Google Scholar 

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Woodin, M.A. (2013). Electrophysiological Methods for Investigating Inhibitory Synaptic Plasticity. In: Nguyen, P. (eds) Multidisciplinary Tools for Investigating Synaptic Plasticity. Neuromethods, vol 81. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-517-0_10

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  • DOI: https://doi.org/10.1007/978-1-62703-517-0_10

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