Advertisement

Methods for the Study of Synaptic Receptor Functional Properties

  • Enrica Maria Petrini
  • Andrea BarberisEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1183)

Abstract

The generation of a synaptic current at the postsynaptic element (PSCs) is the result of a dynamic sequence of events including the release of the neurotransmitter, its diffusion in the synaptic cleft, and the activation of neurotransmitter receptors located at the postsynaptic side. It is widely accepted that the amplitude and the duration of PSCs are largely dictated by the gating properties of postsynaptic receptors. However, the knowledge of the properties of postsynaptic receptors is mostly derived from steady-state analysis, a condition that is substantially different from the non-equilibrium activation of synaptic receptors imposed by submillisecond neurotransmitter exposures. Given the technical limitations to reproduce the brief “synaptic-like” agonist pulse durations, the functioning of postsynaptic receptors during synaptic transmission is not fully elucidated and the “on-demand” postsynaptic activation of synapses cannot be easily achieved. In this chapter, we review the diverse approaches to study receptor gating at times relevant for synaptic transmission and novel optical/optogenetic techniques for controlling synaptic activity at the postsynaptic level. In addition, we emphasize the role of non-equilibrium in unmasking specific features of synaptic receptor gating and the recent advances in photonics for the light-control of neuronal activity at the single-receptor level.

Key words

Synaptic transmission Ligand-gated channels Receptor gating Neurotransmitter transient Ultrafast perfusion Concentration jumps Neurotransmitter uncaging Light-gated receptors Optogenetics Plasmonic devices 

Notes

Acknowledgments

This work was supported by the Telethon grant GGP11043 to A.B.

References

  1. 1.
    Branco T, Hausser M (2010) The single dendritic branch as a fundamental functional unit in the nervous system. Curr Opin Neurobiol 20(4):494–502. doi: 10.1016/j.conb.2010.07.009 PubMedCrossRefGoogle Scholar
  2. 2.
    Klausberger T, Somogyi P (2008) Neuronal diversity and temporal dynamics: the unity of hippocampal circuit operations. Science 321(5885):53–57. doi: 10.1126/science.1149381, 321/5885/53 [pii]PubMedCrossRefGoogle Scholar
  3. 3.
    Tang CM, Margulis M, Shi QY, Fielding A (1994) Saturation of postsynaptic glutamate receptors after quantal release of transmitter. Neuron 13(6):1385–1393PubMedCrossRefGoogle Scholar
  4. 4.
    Barberis A, Petrini EM, Cherubini E, Mozrzymas JW (2002) Allosteric interaction of zinc with recombinant alpha(1)beta(2)gamma(2) and alpha(1)beta(2) GABA(A) receptors. Neuropharmacology 43(4):607–618, S0028390802001090 [pii]PubMedCrossRefGoogle Scholar
  5. 5.
    Bowie D, Lange GD (2002) Functional stoichiometry of glutamate receptor desensitization. J Neurosci 22(9):3392–3403, 20026333PubMedGoogle Scholar
  6. 6.
    Dravid SM, Prakash A, Traynelis SF (2008) Activation of recombinant NR1/NR2C NMDA receptors. J Physiol 586(Pt 18):4425–4439. doi: 10.1113/jphysiol.2008.158634 PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Pitt SJ, Sivilotti LG, Beato M (2008) High intracellular chloride slows the decay of glycinergic currents. J Neurosci 28(45):11454–11467. doi: 10.1523/JNEUROSCI.3890-08.2008 PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Barberis A, Petrini EM, Mozrzymas JW (2011) Impact of synaptic neurotransmitter concentration time course on the kinetics and pharmacological modulation of inhibitory synaptic currents. Front Cell Neurosci 5:6. doi: 10.3389/fncel.2011.00006 PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Barberis A (2012) Fast perfusion methods for the study of ligand-gated ion channels. In: Fellin THM (ed) Neuronal network analysis, vol 67. Springer, New York, NY, pp 173–187. doi: 10.1007/7657_2011_20 CrossRefGoogle Scholar
  10. 10.
    Jonas P (1995) Fast application of agonist to isolated membrane patches. In: Sakmann B, Neher E (eds) Single channel recordings. Plenum Press, New York, NYGoogle Scholar
  11. 11.
    De Angelis F, Das G, Candeloro P et al (2010) Nanoscale chemical mapping using three-dimensional adiabatic compression of surface plasmon polaritons. Nat Nanotechnol 5(1):67–72. doi: 10.1038/nnano.2009.348 PubMedCrossRefGoogle Scholar
  12. 12.
    Franke C, Hatt H, Dudel J (1987) Liquid filament switch for ultra-fast exchanges of solutions at excised patches of synaptic membrane of crayfish muscle. Neurosci Lett 77(2):199–204PubMedCrossRefGoogle Scholar
  13. 13.
    Moffatt L, Hume RI (2007) Responses of rat P2X2 receptors to ultrashort pulses of ATP provide insights into ATP binding and channel gating. J Gen Physiol 130(2):183–201. doi: 10.1085/jgp.200709779 PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Clements JD, Lester RA, Tong G et al (1992) The time course of glutamate in the synaptic cleft. Science 258(5087):1498–1501PubMedCrossRefGoogle Scholar
  15. 15.
    Mozrzymas JW, Barberis A, Michalak K, Cherubini E (1999) Chlorpromazine inhibits miniature GABAergic currents by reducing the binding and by increasing the unbinding rate of GABAA receptors. J Neurosci 19(7):2474–2488PubMedGoogle Scholar
  16. 16.
    He L, Wu XS, Mohan R, Wu LG (2006) Two modes of fusion pore opening revealed by cell-attached recordings at a synapse. Nature 444(7115):102–105. doi: 10.1038/nature05250 PubMedCrossRefGoogle Scholar
  17. 17.
    Popescu G, Robert A, Howe JR, Auerbach A (2004) Reaction mechanism determines NMDA receptor response to repetitive stimulation. Nature 430(7001):790–793. doi: 10.1038/nature02775 PubMedCrossRefGoogle Scholar
  18. 18.
    Barberis A, Petrini EM, Cherubini E (2004) Presynaptic source of quantal size variability at GABAergic synapses in rat hippocampal neurons in culture. Eur J Neurosci 20(7):1803–1810. doi: 10.1111/j.1460-9568.2004.03624.x, EJN3624 [pii]PubMedCrossRefGoogle Scholar
  19. 19.
    Min MY, Rusakov DA, Kullmann DM (1998) Activation of AMPA, kainate, and metabotropic receptors at hippocampal mossy fiber synapses: role of glutamate diffusion. Neuron 21(3):561–570, S0896-6273(00)80566-8 [pii]PubMedCrossRefGoogle Scholar
  20. 20.
    Perrais D, Ropert N (2000) Altering the concentration of GABA in the synaptic cleft potentiates miniature IPSCs in rat occipital cortex. Eur J Neurosci 12(1):400–404, ejn957 [pii]PubMedCrossRefGoogle Scholar
  21. 21.
    Barberis A, Sachidhanandam S, Mulle C (2008) GluR6/KA2 kainate receptors mediate slow-deactivating currents. J Neurosci 28(25):6402–6406. doi:10.1523/JNEUROSCI.1204-08.2008, 28/25/6402 [pii]PubMedCrossRefGoogle Scholar
  22. 22.
    Jones MV, Westbrook GL (1995) Desensitized states prolong GABAA channel responses to brief agonist pulses. Neuron 15(1):181–191, 0896-6273(95)90075-6 [pii]PubMedCrossRefGoogle Scholar
  23. 23.
    Petrini EM, Nieus T, Ravasenga T et al (2011) Influence of GABAAR monoliganded states on GABAergic responses. J Neurosci 31(5):1752–1761. doi: 10.1523/JNEUROSCI.1453-10.2011 PubMedCrossRefGoogle Scholar
  24. 24.
    Mozrzymas JW, Barberis A, Mercik K, Zarnowska ED (2003) Binding sites, singly bound states, and conformation coupling shape GABA-evoked currents. J Neurophysiol 89(2):871–883. doi: 10.1152/jn.00951.2002 PubMedCrossRefGoogle Scholar
  25. 25.
    Lester RA, Jahr CE (1992) NMDA channel behavior depends on agonist affinity. J Neurosci 12(2):635–643PubMedGoogle Scholar
  26. 26.
    Barberis A, Mozrzymas JW, Ortinski PI, Vicini S (2007) Desensitization and binding properties determine distinct alpha1beta2gamma2 and alpha3beta2gamma2 GABA(A) receptor-channel kinetic behavior. Eur J Neurosci 25(9):2726–2740. doi: 10.1111/j.1460-9568.2007.05530.x, EJN5530 [pii]PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Bragina L, Marchionni I, Omrani A et al (2008) GAT-1 regulates both tonic and phasic GABA(A) receptor-mediated inhibition in the cerebral cortex. J Neurochem 105(5):1781–1793. doi: 10.1111/j.1471-4159.2008.05273.x PubMedCrossRefGoogle Scholar
  28. 28.
    Macdonald RL, Rogers CJ, Twyman RE (1989) Kinetic properties of the GABAA receptor main conductance state of mouse spinal cord neurones in culture. J Physiol 410:479–499PubMedCentralPubMedGoogle Scholar
  29. 29.
    Sachidhanandam S, Blanchet C, Jeantet Y et al (2009) Kainate receptors act as conditional amplifiers of spike transmission at hippocampal mossy fiber synapses. J Neurosci 29(15):5000–5008. doi: 10.1523/JNEUROSCI.5807-08.2009 PubMedCrossRefGoogle Scholar
  30. 30.
    Mott DD, Rojas A, Fisher JL, Dingledine RJ, Benveniste M (2010) Subunit-specific desensitization of heteromeric kainate receptors. J Physiol 588(Pt 4):683–700, jphysiol.2009.185207 [pii]PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Mozrzymas JW, Barberis A, Vicini S (2007) GABAergic currents in RT and VB thalamic nuclei follow kinetic pattern of alpha3- and alpha1-subunit-containing GABAA receptors. Eur J Neurosci 26(3):657–665. doi: 10.1111/j.1460-9568.2007.05693.x, EJN5693 [pii]PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Seeman P (1980) Brain dopamine receptors. Pharmacol Rev 32(3):229–313PubMedGoogle Scholar
  33. 33.
    Toone BK, Fenton GW (1977) Epileptic seizures induced by psychotropic drugs. Psychol Med 7(2):265–270PubMedCrossRefGoogle Scholar
  34. 34.
    Mozrzymas JW, Zarnowska ED, Pytel M, Mercik K (2003) Modulation of GABA(A) receptors by hydrogen ions reveals synaptic GABA transient and a crucial role of the desensitization process. J Neurosci 23(22):7981–7992, 23/22/7981 [pii]PubMedGoogle Scholar
  35. 35.
    DiGregorio DA, Rothman JS, Nielsen TA, Silver RA (2007) Desensitization properties of AMPA receptors at the cerebellar mossy fiber granule cell synapse. J Neurosci 27(31):8344–8357. doi: 10.1523/JNEUROSCI.2399-07.2007 PubMedCrossRefGoogle Scholar
  36. 36.
    Matsuzaki M, Ellis-Davies GC, Nemoto T et al (2001) Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons. Nat Neurosci 4(11):1086–1092. doi: 10.1038/nn736 PubMedCrossRefGoogle Scholar
  37. 37.
    Trigo FF, Papageorgiou G, Corrie JE, Ogden D (2009) Laser photolysis of DPNI-GABA, a tool for investigating the properties and distribution of GABA receptors and for silencing neurons in situ. J Neurosci Methods 181(2):159–169. doi: 10.1016/j.jneumeth.2009.04.022 PubMedCrossRefGoogle Scholar
  38. 38.
    Patneau DK, Mayer ML (1990) Structure-activity relationships for amino acid transmitter candidates acting at N-methyl-D-aspartate and quisqualate receptors. J Neurosci 10(7):2385–2399PubMedGoogle Scholar
  39. 39.
    Glykys J, Mody I (2007) Activation of GABAA receptors: views from outside the synaptic cleft. Neuron 56(5):763–770. doi: 10.1016/j.neuron.2007.11.002 PubMedCrossRefGoogle Scholar
  40. 40.
    Levi S, Triller A (2006) Neurotransmitter dynamics. In: Kittler JT, Moss SJ (eds) The dynamic synapse: molecular methods in ionotropic receptor biology, Frontiers in neuroscience. CRC Press, Boca Raton, FLGoogle Scholar
  41. 41.
    Fino E, Araya R, Peterka DS et al (2009) RuBi-glutamate: two-photon and visible-light photoactivation of neurons and dendritic spines. Front Neural Circ 3:2. doi: 10.3389/neuro.04.002.2009 Google Scholar
  42. 42.
    Fino E, Yuste R (2011) Dense inhibitory connectivity in neocortex. Neuron 69(6):1188–1203. doi: 10.1016/j.neuron.2011.02.025 PubMedCentralPubMedCrossRefGoogle Scholar
  43. 43.
    Hayama T, Noguchi J, Watanabe S et al (2013) GABA promotes the competitive selection of dendritic spines by controlling local Ca2+ signaling. Nat Neurosci 16(10):1409–1416. doi: 10.1038/nn.3496 PubMedCrossRefGoogle Scholar
  44. 44.
    Gorostiza P, Isacoff EY (2008) Optical switches for remote and noninvasive control of cell signaling. Science 322(5900):395–399. doi: 10.1126/science.1166022 PubMedCrossRefGoogle Scholar
  45. 45.
    Szobota S, Gorostiza P, Del Bene F et al (2007) Remote control of neuronal activity with a light-gated glutamate receptor. Neuron 54(4):535–545. doi: 10.1016/j.neuron.2007.05.010 PubMedCrossRefGoogle Scholar
  46. 46.
    Fenno L, Yizhar O, Deisseroth K (2011) The development and application of optogenetics. Annu Rev Neurosci 34:389–412. doi: 10.1146/annurev-neuro-061010-113817 PubMedCrossRefGoogle Scholar
  47. 47.
    Bartels E, Wassermann NH, Erlanger BF (1971) Photochromic activators of the acetylcholine receptor. Proc Natl Acad Sci U S A 68(8):1820–1823PubMedCentralPubMedCrossRefGoogle Scholar
  48. 48.
    Banghart M, Borges K, Isacoff E et al (2004) Light-activated ion channels for remote control of neuronal firing. Nat Neurosci 7(12):1381–1386. doi: 10.1038/nn1356 PubMedCentralPubMedCrossRefGoogle Scholar
  49. 49.
    Chambers JJ, Banghart MR, Trauner D, Kramer RH (2006) Light-induced depolarization of neurons using a modified Shaker K(+) channel and a molecular photoswitch. J Neurophysiol 96(5):2792–2796. doi: 10.1152/jn.00318.2006 PubMedCrossRefGoogle Scholar
  50. 50.
    Mathur BN, Tanahira C, Tamamaki N, Lovinger DM (2013) Voltage drives diverse endocannabinoid signals to mediate striatal microcircuit-specific plasticity. Nat Neurosci 16(9):1275–1283. doi: 10.1038/nn.3478 PubMedCentralPubMedCrossRefGoogle Scholar
  51. 51.
    Tye KM, Deisseroth K (2012) Optogenetic investigation of neural circuits underlying brain disease in animal models. Nat Rev Neurosci 13(4):251–266. doi: 10.1038/nrn3171 PubMedCrossRefGoogle Scholar
  52. 52.
    Specht CG, Izeddin I, Rodriguez PC et al (2013) Quantitative nanoscopy of inhibitory synapses: counting gephyrin molecules and receptor binding sites. Neuron 79(2):308–321. doi: 10.1016/j.neuron.2013.05.013 PubMedCrossRefGoogle Scholar
  53. 53.
    MacGillavry HD, Song Y, Raghavachari S, Blanpied TA (2013) Nanoscale scaffolding domains within the postsynaptic density concentrate synaptic AMPA receptors. Neuron 78(4):615–622. doi: 10.1016/j.neuron.2013.03.009 PubMedCentralPubMedCrossRefGoogle Scholar
  54. 54.
    Nair D, Hosy E, Petersen JD et al (2013) Super-resolution imaging reveals that AMPA receptors inside synapses are dynamically organized in nanodomains regulated by PSD95. J Neurosci 33(32):13204–13224. doi: 10.1523/JNEUROSCI.2381-12.2013 PubMedCrossRefGoogle Scholar
  55. 55.
    Deshpande VV, Bockrath M, Glazman LI, Yacoby A (2010) Electron liquids and solids in one dimension. Nature 464(7286):209–216. doi: 10.1038/nature08918 PubMedCrossRefGoogle Scholar
  56. 56.
    De Angelis F, Patrini M, Das G et al (2008) A hybrid plasmonic-photonic nanodevice for label-free detection of a few molecules. Nano Lett 8(8):2321–2327. doi: 10.1021/nl801112e PubMedCrossRefGoogle Scholar
  57. 57.
    Raether H (1988) Surface plasmons. Springer, BerlinGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Neuroscience and Brain TechnologiesIstituto Italiano di TecnologiaGenoaItaly

Personalised recommendations