Ultrastructure of light-activated axons following optogenetic stimulation to produce late-phase long-term potentiation

Autoři: Masaaki Kuwajima aff001;  Olga I. Ostrovskaya aff001;  Guan Cao aff001;  Seth A. Weisberg aff002;  Kristen M. Harris aff001;  Boris V. Zemelman aff001
Působiště autorů: Center for Learning and Memory, The University of Texas at Austin, Austin, Texas, United States of America aff001;  Department of Neuroscience, The University of Texas at Austin, Austin, Texas, United States of America aff002
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: 10.1371/journal.pone.0226797


Analysis of neuronal compartments has revealed many state-dependent changes in geometry but establishing synapse-specific mechanisms at the nanoscale has proven elusive. We co-expressed channelrhodopsin2-GFP and mAPEX2 in a subset of hippocampal CA3 neurons and used trains of light to induce late-phase long-term potentiation (L-LTP) in area CA1. L-LTP was shown to be specific to the labeled axons by severing CA3 inputs, which prevented back-propagating recruitment of unlabeled axons. Membrane-associated mAPEX2 tolerated microwave-enhanced chemical fixation and drove tyramide signal amplification to deposit Alexa Fluor dyes in the light-activated axons. Subsequent post-embedding immunogold labeling resulted in outstanding ultrastructure and clear distinctions between labeled (activated), and unlabeled axons without obscuring subcellular organelles. The gold-labeled axons in potentiated slices were reconstructed through serial section electron microscopy; presynaptic vesicles and other constituents could be quantified unambiguously. The genetic specification, reliable physiology, and compatibility with established methods for ultrastructural preservation make this an ideal approach to link synapse ultrastructure and function in intact circuits.

Klíčová slova:

Axons – Electron microscopy – Functional electrical stimulation – Light pulses – Neurons – Synapses – Immunogold labeling – Glutaraldehyde fixation


1. Bourne JN, Harris KM. Coordination of size and number of excitatory and inhibitory synapses results in a balanced structural plasticity along mature hippocampal CA1 dendrites during LTP. Hippocampus. 2011;21: 354–373. doi: 10.1002/hipo.20768 20101601

2. Ostroff LE, Fiala JC, Allwardt B, Harris KM. Polyribosomes Redistribute from Dendritic Shafts into Spines with Enlarged Synapses during LTP in Developing Rat Hippocampal Slices. Neuron. 2002;35: 535–545. doi: 10.1016/s0896-6273(02)00785-7 12165474

3. Popov VI, Davies HA, Rogachevsky VV, Patrushev IV, Errington ML, Gabbott PLA, et al. Remodelling of synaptic morphology but unchanged synaptic density during late phase long-term potentiation(ltp): A serial section electron micrograph study in the dentate gyrus in the anaesthetised rat. Neuroscience. 2004;128: 251–262. doi: 10.1016/j.neuroscience.2004.06.029 15350638

4. Smith HL, Bourne JN, Cao G, Chirillo MA, Ostroff LE, Watson DJ, et al. Mitochondrial support of persistent presynaptic vesicle mobilization with age-dependent synaptic growth after LTP. eLife. 2016;5: e15275. doi: 10.7554/eLife.15275 27991850

5. Martell JD, Deerinck TJ, Sancak Y, Poulos TL, Mootha VK, Sosinsky GE, et al. Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat Biotechnol. 2012;30: 1143–1148. doi: 10.1038/nbt.2375 23086203

6. Lam SS, Martell JD, Kamer KJ, Deerinck TJ, Ellisman MH, Mootha VK, et al. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat Methods. 2015;12: 51–54. doi: 10.1038/nmeth.3179 25419960

7. Nabavi S, Fox R, Proulx CD, Lin JY, Tsien RY, Malinow R. Engineering a memory with LTD and LTP. Nature. 2014;511: 348–352. doi: 10.1038/nature13294 24896183

8. Oishi N, Nomoto M, Ohkawa N, Saitoh Y, Sano Y, Tsujimura S, et al. Artificial association of memory events by optogenetic stimulation of hippocampal CA3 cell ensembles. Mol Brain. 2019;12: 2. doi: 10.1186/s13041-018-0424-1 30621738

9. Chun S, Bayazitov IT, Blundon JA, Zakharenko SS. Thalamocortical Long-Term Potentiation Becomes Gated after the Early Critical Period in the Auditory Cortex. J Neurosci. 2013;33: 7345–7357. doi: 10.1523/JNEUROSCI.4500-12.2013 23616541

10. Hashimotodani Y, Nasrallah K, Jensen KR, Chávez AE, Carrera D, Castillo PE. LTP at Hilar Mossy Cell-Dentate Granule Cell Synapses Modulates Dentate Gyrus Output by Increasing Excitation/Inhibition Balance. Neuron. 2017;95: 928–943.e3. doi: 10.1016/j.neuron.2017.07.028 28817805

11. Kohl MM, Shipton OA, Deacon RM, Rawlins JNP, Deisseroth K, Paulsen O. Hemisphere-specific optogenetic stimulation reveals left-right asymmetry of hippocampal plasticity. Nat Neurosci. 2011;14: 1413–1415. doi: 10.1038/nn.2915 21946328

12. Zhang Y-P, Oertner TG. Optical induction of synaptic plasticity using a light-sensitive channel. Nat Methods. 2007;4: 139–141. doi: 10.1038/nmeth988 17195846

13. O’Riordan KJ, Hu N-W, Rowan MJ. Aß Facilitates LTD at Schaffer Collateral Synapses Preferentially in the Left Hippocampus. Cell Rep. 2018;22: 2053–2065. doi: 10.1016/j.celrep.2018.01.085 29466733

14. Berndt A, Schoenenberger P, Mattis J, Tye KM, Deisseroth K, Hegemann P, et al. High-efficiency channelrhodopsins for fast neuronal stimulation at low light levels. Proc Natl Acad Sci. 2011;108: 7595–7600. doi: 10.1073/pnas.1017210108 21504945

15. Pédelacq J-D, Cabantous S, Tran T, Terwilliger TC, Waldo GS. Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol. 2006;24: 79–88. doi: 10.1038/nbt1172 16369541

16. Stockklausner C, Ludwig J, Ruppersberg JP, Klöcker N. A sequence motif responsible for ER export and surface expression of Kir2.0 inward rectifier K+ channels. FEBS Lett. 2001;493: 129–133. doi: 10.1016/s0014-5793(01)02286-4 11287009

17. Mittler R, Zilinskas BA. Molecular cloning and nucleotide sequence analysis of a cDNA encoding pea cytosolic ascorbate peroxidase. FEBS Lett. 1991;289: 257–259. doi: 10.1016/0014-5793(91)81083-k 1915856

18. Moriyoshi K, Richards LJ, Akazawa C, O’Leary DDM, Nakanishi S. Labeling Neural Cells Using Adenoviral Gene Transfer of Membrane-Targeted GFP. Neuron. 1996;16: 255–260. doi: 10.1016/s0896-6273(00)80044-6 8789941

19. Kim JH, Lee S-R, Li L-H, Park H-J, Park J-H, Lee KY, et al. High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines, Zebrafish and Mice. PLOS ONE. 2011;6: e18556. doi: 10.1371/journal.pone.0018556 21602908

20. Borghuis BG, Tian L, Xu Y, Nikonov SS, Vardi N, Zemelman BV, et al. Imaging Light Responses of Targeted Neuron Populations in the Rodent Retina. J Neurosci. 2011;31: 2855–2867. doi: 10.1523/JNEUROSCI.6064-10.2011 21414907

21. Grieger JC, Choi VW, Samulski RJ. Production and characterization of adeno-associated viral vectors. Nat Protoc. 2006;1: 1412–1428. doi: 10.1038/nprot.2006.207 17406430

22. Aurnhammer C, Haase M, Muether N, Hausl M, Rauschhuber C, Huber I, et al. Universal Real-Time PCR for the Detection and Quantification of Adeno-Associated Virus Serotype 2-Derived Inverted Terminal Repeat Sequences. Hum Gene Ther Methods. 2012;23: 18–28. doi: 10.1089/hgtb.2011.034 22428977

23. Yuste R, Miller RB, Holthoff K, Zhang S, Miesenbo¨ck G. Synapto-pHluorins: Chimeras between pH-sensitive mutants of green fluorescent protein and synaptic vesicle membrane proteins as reporters of neurotransmitter release. In: Thorner J, Emr SD, Abelson JN, editors. Methods in Enzymology. Academic Press; 2000. pp. 522–546. doi: 10.1016/S0076-6879(00)27300-X

24. Bourne JN, Kirov SA, Sorra KE, Harris KM. Warmer preparation of hippocampal slices prevents synapse proliferation that might obscure LTP-related structural plasticity. Neuropharmacology. 2007;52: 55–59. doi: 10.1016/j.neuropharm.2006.06.020 16895730

25. Bourne JN, Harris KM. Nanoscale analysis of structural synaptic plasticity. Curr Opin Neurobiol. 2012;22: 372–382. doi: 10.1016/j.conb.2011.10.019 22088391

26. Harris KM, Teyler TJ. Developmental onset of long-term potentiation in area CA1 of the rat hippocampus. J Physiol. 1984;346: 27–48. doi: 10.1113/jphysiol.1984.sp015005 6699775

27. Jensen FE, Harris KM. Preservation of neuronal ultrastructure in hippocampal slices using rapid microwave-enhanced fixation. J Neurosci Methods. 1989;29: 217–230. doi: 10.1016/0165-0270(89)90146-5 2507828

28. Harris KM, Perry E, Bourne J, Feinberg M, Ostroff L, Hurlburt J. Uniform Serial Sectioning for Transmission Electron Microscopy. J Neurosci. 2006;26: 12101–12103. doi: 10.1523/JNEUROSCI.3994-06.2006 17122034

29. Kuwajima M, Mendenhall JM, Harris KM. Large-Volume Reconstruction of Brain Tissue from High-Resolution Serial Section Images Acquired by SEM-Based Scanning Transmission Electron Microscopy. In: Sousa AA, Kruhlak MJ, editors. Nanoimaging. Humana Press; 2013. pp. 253–273. doi: 10.1007/978-1-62703-137-0_15

30. Reynolds ES. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol. 1963;17: 208–212. doi: 10.1083/jcb.17.1.208 13986422

31. Kuwajima M, Mendenhall JM, Lindsey LF, Harris KM. Automated Transmission-Mode Scanning Electron Microscopy (tSEM) for Large Volume Analysis at Nanoscale Resolution. PLoS ONE. 2013;8: e59573. doi: 10.1371/journal.pone.0059573 23555711

32. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9: 676–682. doi: 10.1038/nmeth.2019 22743772

33. Cardona A, Saalfeld S, Schindelin J, Arganda-Carreras I, Preibisch S, Longair M, et al. TrakEM2 Software for Neural Circuit Reconstruction. PLoS ONE. 2012;7: e38011. doi: 10.1371/journal.pone.0038011 22723842

34. Saalfeld S, Fetter R, Cardona A, Tomancak P. Elastic volume reconstruction from series of ultra-thin microscopy sections. Nat Methods. 2012;9: 717–720. doi: 10.1038/nmeth.2072 22688414

35. Fiala JC. Reconstruct: a free editor for serial section microscopy. J Microsc. 2005;218: 52–61. doi: 10.1111/j.1365-2818.2005.01466.x 15817063

36. Fiala JC, Harris KM. Cylindrical diameters method for calibrating section thickness in serial electron microscopy. J Microsc. 2001;202: 468–472. doi: 10.1046/j.1365-2818.2001.00926.x 11422668

37. Fiala JC, Harris KM. Extending Unbiased Stereology of Brain Ultrastructure to Three-dimensional Volumes. J Am Med Inform Assoc. 2001;8: 1–16. doi: 10.1136/jamia.2001.0080001 11141509

38. Cao G, Harris KM. Developmental regulation of the late phase of long-term potentiation (L-LTP) and metaplasticity in hippocampal area CA1 of the rat. J Neurophysiol. 2012;107: 902–912. doi: 10.1152/jn.00780.2011 22114158

39. Isomura Y, Fujiwara-Tsukamoto Y, Imanishi M, Nambu A, Takada M. Distance-Dependent Ni2+-Sensitivity of Synaptic Plasticity in Apical Dendrites of Hippocampal CA1 Pyramidal Cells. J Neurophysiol. 2002;87: 1169–1174. doi: 10.1152/jn.00536.2001 11826086

40. Kloosterman F, Peloquin P, Leung LS. Apical and Basal Orthodromic Population Spikes in Hippocampal CA1 In Vivo Show Different Origins and Patterns of Propagation. J Neurophysiol. 2001;86: 2435–2444. doi: 10.1152/jn.2001.86.5.2435 11698533

41. Leung LW. Potentials evoked by alvear tract in hippocampal CA1 region of rats. I. Topographical projection, component analysis, and correlation with unit activities. J Neurophysiol. 1979;42: 1557–1570. doi: 10.1152/jn.1979.42.6.1557 501389

42. Wheal HV, Lancaster B, Bliss TVP. Long-term potentiation in Schaffer collateral and commissural systems of the hippocampus: In vitro study in rats pretreated with kainic acid. Brain Res. 1983;272: 247–253. doi: 10.1016/0006-8993(83)90570-x 6311340

43. Jackman SL, Beneduce BM, Drew IR, Regehr WG. Achieving High-Frequency Optical Control of Synaptic Transmission. J Neurosci. 2014;34: 7704–7714. doi: 10.1523/JNEUROSCI.4694-13.2014 24872574

44. Zakharenko SS, Zablow L, Siegelbaum SA. Visualization of changes in presynaptic function during long-term synaptic plasticity. Nat Neurosci. 2001;4: 711. doi: 10.1038/89498 11426227

45. Hainfeld JF, Powell RD. New Frontiers in Gold Labeling. J Histochem Cytochem. 2000;48: 471–480. doi: 10.1177/002215540004800404 10727288

46. Bourne JN, Chirillo MA, Harris KM. Presynaptic Ultrastructural Plasticity Along CA3→CA1 Axons During Long-Term Potentiation in Mature Hippocampus. J Comp Neurol. 2013;521: 3898–3912. doi: 10.1002/cne.23384 23784793

47. Shinohara Y, Hirase H, Watanabe M, Itakura M, Takahashi M, Shigemoto R. Left-right asymmetry of the hippocampal synapses with differential subunit allocation of glutamate receptors. Proc Natl Acad Sci. 2008;105: 19498–19503. doi: 10.1073/pnas.0807461105 19052236

48. Bliss TV, Lancaster B, Wheal HV. Long-term potentiation in commissural and Schaffer projections to hippocampal CA1 cells: an in vivo study in the rat. J Physiol. 1983;341: 617–626. doi: 10.1113/jphysiol.1983.sp014828 6620191

49. Martin SJ, Shires KL, da Silva BM. Hippocampal Lateralization and Synaptic Plasticity in the Intact Rat: No Left–Right Asymmetry in Electrically Induced CA3-CA1 Long-Term Potentiation. Neuroscience. 2019;397: 147–158. doi: 10.1016/j.neuroscience.2018.11.044 30513373

50. Habib D, Dringenberg HC. Low-frequency-induced synaptic potentiation: A paradigm shift in the field of memory-related plasticity mechanisms? Hippocampus. 2010;20: 29–35. doi: 10.1002/hipo.20611 19405136

51. Magee JC, Johnston D. A Synaptically Controlled, Associative Signal for Hebbian Plasticity in Hippocampal Neurons. Science. 1997;275: 209–213. doi: 10.1126/science.275.5297.209 8985013

52. Markram H, Lübke J, Frotscher M, Sakmann B. Regulation of Synaptic Efficacy by Coincidence of Postsynaptic APs and EPSPs. Science. 1997;275: 213–215. doi: 10.1126/science.275.5297.213 8985014

53. Bortolotto ZA, Collingridge GL. Characterisation of LTP induced by the activation of glutamate metabotropic receptors in area CA1 of the hippocampus. Neuropharmacology. 1993;32: 1–9. doi: 10.1016/0028-3908(93)90123-k 8381524

54. Kang H, Schuman EM. Long-lasting neurotrophin-induced enhancement of synaptic transmission in the adult hippocampus. Science. 1995;267: 1658–1662. doi: 10.1126/science.7886457 7886457

55. Lee J, Song EK, Bae Y, Min J, Rhee H-W, Park TJ, et al. An enhanced ascorbate peroxidase 2/antibody-binding domain fusion protein (APEX2–ABD) as a recombinant target-specific signal amplifier. Chem Commun. 2015;51: 10945–10948. doi: 10.1039/C5CC02409A 26063640

56. Coleman RA, Liu J, Wade JB. Use of anti-fluorophore antibody to achieve high-sensitivity immunolocalizations of transporters and ion channels. J Histochem Cytochem Off J Histochem Soc. 2006;54: 817–827. doi: 10.1369/jhc.6A6929.2006 16549505

57. Oberti D, Kirschmann MA, Hahnloser RHR. Correlative microscopy of densely labeled projection neurons using neural tracers. Front Neuroanat. 2010;4: 24. doi: 10.3389/fnana.2010.00024 20676237

Článek vyšel v časopise


2020 Číslo 1