Abstract
Current concepts of synaptic fine-structure are derived from electron microscopic studies of tissue fixed by chemical fixation using aldehydes. However, chemical fixation with glutaraldehyde and paraformaldehyde and subsequent dehydration in ethanol result in uncontrolled tissue shrinkage. While electron microscopy allows for the unequivocal identification of synaptic contacts, it cannot be used for real-time analysis of structural changes at synapses. For the latter purpose advanced fluorescence microscopy techniques are to be applied which, however, do not allow for the identification of synaptic contacts. Here, two approaches are described that may overcome, at least in part, some of these drawbacks in the study of synapses. By focusing on a characteristic, easily identifiable synapse, the mossy fiber synapse in the hippocampus, we first describe high-pressure freezing of fresh tissue as a method that may be applied to study subtle changes in synaptic ultrastructure associated with functional synaptic plasticity. Next, we propose to label presynaptic mossy fiber terminals and postsynaptic complex spines on CA3 pyramidal neurons by different fluorescent dyes to allow for the real-time monitoring of these synapses in living tissue over extended periods of time. We expect these approaches to lead to new insights into the structure and function of central synapses.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Acsády L, Kamondi A, Sik A, Freund T, Buzsáki G (1998) GABAergic cells are the major postsynaptic targets of mossy fibers in the rat hippocampus. J Neurosci 18:3386–3403
Andersen P, Bliss TVP, Skrede KK (1971) Lamellar organization of hippocampal excitatory pathways. Exp Brain Res 13:222–238
Bischofberger J, Engel D, Frotscher M, Jonas P (2006) Timing and efficacy of transmitter release at mossy fiber synapses in the hippocampal network. Pflügers Arch Eur J Physiol 453:361–372
Blackstad TW, Kjaerheim A (1961) Special axodendritic synapses in the hippocampal cortex: electron and light microscopic studies on the layer of mossy fibers. J Comp Neurol 117:113–159
Chicurel ME, Harris KM (1992) Three-dimensional analysis of the structure and composition of CA3 branched dendritic spines and their synaptic relationships with mossy fiber boutons in the rat hippocampus. J Comp Neurol 325:169–182
Engert E, Bonhoeffer T (1999) Dendritic spine changes associated with hippocampal long-term synaptic plasticity. Nature 339:66–70
Förster E, Zhao S, Frotscher M (2006) Laminating the hippocampus. Nat Rev Neurosci 7:259–267
Frotscher M (1985) Mossy fibres form synapses with identified pyramidal basket cells in the CA3 region of the guinea pig hippocampus: a combined Golgi-electron microscope study. J Neurocytol 14:245–259
Frotscher M (1989) Mossy fiber synapses on glutamate decarboxylase-immunoreactive neurons: evidence for feed-forward inhibition in the CA3 region of the hippocampus. Exp Brain Res 75:441–445
Frotscher M, Deller T (2005) Role of the spine apparatus in synaptic plasticity. In: Stanton PK, Bramham C, Scharfman HE (eds) Synaptic plasticity and transsynaptic signaling, Springer, Berlin pp 519–528
Frotscher M, Gähwiler BH (1988) Synaptic organization of intracellularly stained CA3 pyramidal neurons in slice cultures of rat hippocampus. Neuroscience 24:541–551
Frotscher M, Misgeld U, Nitsch C (1981) Ultrastructure of mossy fiber endings in in vitro hippocampal slices. Exp Brain Res 41:247–255
Frotscher M, Soriano E, Misgeld U (1994) Divergence of hippocampal mossy fibers. Synapse 16:148–160
Frotscher M, Zafirov S, Heimrich B (1995) Development of identified neuronal types and of specific synaptic connections in slice cultures of rat hippocampus. Prog Neurobiol 45:143–164
Frotscher M, Jonas P, Sloviter RS (2006) Synapses formed by normal and abnormal hippocampal mossy fibers. Cell Tissue Res 326:361–367
Gähwiler MH, Capogna M, Debanne D, McKinney RA, Thompson SM (1997) Organotypic slice cultures: a technique has come of age. TINS 20:471–477
Gray EG (1959) Axo-somatic and axo-dendritic synapses of the cerebral cortex: an electron microscopic study. J Anat 83:420–433
Hallermann S, Pawlu C, Jonas P, Heckmann M (2003) A large pool of releasable vesicles in a cortical glutamatergic synapse. Proc Natl Acad Sci USA 100:8975–8980
Hamlyn LH (1962) The fine structure of the mossy fibre endings in the hippocampus of the rabbit. J Anat 97:112–120
Matus A (2000) Actin-based plasticity in dendritic spines. Science 290:754–758
Matus A, Frotscher M (2005) Synapse. In: Nadel L (ed) Encyclopedia of cognitive science, vol 4. Wiley, Chichester, pp 301–307
Nadarajah B, Parnavelas JG (2002) Modes of neuronal migration in the developing cerebral cortex. Nat Rev Neurosci 3:423–432
Peters A, Palay SL, Webster HD (1991) The fine structure of the nervous system. Neurons and their supporting cells. Oxford University Press, Oxford
Rizzoli SO, Betz WJ (2005) Synaptic vesicle pools. Nat Rev Neurosci 6:57–69
Rostaing P, Real E, Siksou L, Lechaire J-P, Boudier T, Boeckers TM, Gertler F, Gundelfinger ED, Triller A, Marty S (2006) Analysis of synaptic ultrastructure without fixative using high-pressure freezing and tomography. Eur J Neurosci 24:3463–3473
Schikorski T, Stevens CF (2001) Morphological correlates of functionally defined synaptic vesicle populations. Nat Neurosci 4:391–395
Spacek J (1985) Three-dimensional analysis of dendritic spines. II. Spine apparatus and other cytoplasmic components. Anat Embryol 171:235–243
Spacek J, Harris KM (1997) Three-dimensional organization of smooth endoplasmic reticulum in hippocampal CA1 dendrites and dendritic spines of the immature and mature rat. J Neurosci 17:190–203
Squire LR, Stark CE, Clark RE (2004) The medial temporal lobe. Annu Rev Neurosci 27:279–306
Steward O, Levy WB (1982) Preferential localization of polyribosomes under the base of dendritic spines in granule cells of the dentate gyrus. J Neurosci 2:284–291
Studer D, Michel M, Müller M (1989) High pressure freezing comes of age. Scanning Microsc Suppl 3:253–269
Studer D, Michel M, Wohlwend M, Hunziker EB, Buschmann MD (1995) Vitrification of articular cartilage by high-pressure freezing. J Microsc 179:321–332
Studer D, Graber W, Al-Amoudi A, Eggli P (2001) A new approach for cryofixation by high-pressure freezing. J Microsc 203:285–294
Toni N, Buchs P-A, Nikonenko I, Bron CR, Muller D (1999) LTP induces synaptogenesis by duplication of synaptic spines contacting a single axon terminal. Nature 402:421–425
Willig KI, Rizzoli SO, Westphal V, Jahn R, Hell SW (2006) STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440:935–939
Acknowledgements
These studies were supported by the Deutsche Forschungsgemeinschaft (SFB 505, project A3) to MF and AD and the Swiss National Foundation (Grant No.: 105822) to DS. We thank Sigrun Nestel and Cornelia Hofmann for excellent technical assistance.
Author information
Authors and Affiliations
Corresponding author
Additional information
Robert Feulgen Lecture presented at the 49th Symposium of the Society for Histochemistry in Freiburg i.Br., Germany, 26-29 September 2007.
Rights and permissions
About this article
Cite this article
Frotscher, M., Zhao, S., Graber, W. et al. New ways of looking at synapses. Histochem Cell Biol 128, 91–96 (2007). https://doi.org/10.1007/s00418-007-0305-7
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00418-007-0305-7