Summary
Development, ageing, and a variety of neurological disorders are characterized by selective alterations in specific populations of nerve cells which are, in turn, associated with changes in the numbers of synapses in the target fields of these neurons. To begin to delineate the significance of changes in synapses in development, ageing, and disease, it is first essential to quantify the number of synapses in defined regions of the CNS. In the past, investigators have used EM methods to assess synapse numbers or density, but these approaches are costly, labour intensive, and technically difficult, particularly in autopsy material. To begin to define reliable strategies useful for studies of both animals and humans, we used three techniques to measure synaptophysin-immunoreactivity in rat brain. The levels of synaptophysin protein were determined by Western blots of five hippocampal subregions; the intensity of synaptophysin-immunoreactivity in dentate gyrus and stratum oriens was determined by optical densitometry of immunocytochemically stained sections; and the total number of synaptophysin-immunoreactivity presynaptic boutons in dentate gyrus and stratum oriens was assessed by unbiased stereology. Each approach has advantages and disadvantages. Western blotting is the least time-consuming of the three methods and allows simultaneous processing of multiple samples. In systematically sampled histological sections, both densitometry and stereology allow precise definition of the region of interest, and the sterological optical disector method allows quantitation of the numbers of synaptophysin-immunoreactive boutons. Stereology was the only method that clearly demonstrated greater synaptophysin-immunoreactivity in the dentate gyrus as compared to stratum oriens. The use of systematic sampling and the disector technique offer a high degree of anatomical resolution (lacking in Western blot methods) and has quantitative advantage over the greyscale-based density approach. Thus, at present, stereology is the most useful method for estimating synaptic numbers in defined regions of the brain.
Similar content being viewed by others
References
Chen, K. S., Masliah, E., Mallory M. &Gage, F. H. (1995) Synaptic loss in cognitively impaired aged rats is ameliorated by chronic human nerve growth factor infusion.Neuroscience 68, 19–27.
Colonnier, M. &Beaulieu, C. (1985) An empirical assessment of stereological formulae applied to the counting of synaptic disks in the cerebral cortex.Journal of Comparative Neurology,231, 175–9.
Cragg, B. G. (1975) The development of synapses in the visual system of the cat.Journal of Comparative Neurology 160, 147–66.
De Groot, D. M. G. &Bierman, E. P. B. (1986) A critical evaluation of methods for estimating the numerical density of synapses.Journal of Neuroscience Methods 18, 79–101.
Dekosky, S. T. &Scheff, S. W. (1990) Synapse loss in frontal cortex biopsies in Alzheimer's disease: correlation with cognitive severity.Annals of Neurology 27, 457–63.
Eastwood, S. L. &Harrison, P. J. (1995) Decreased synaptophysin in the medial temporal lobe in schizophrenia demonstrated using immunoautoradiography.Neuroscience 69, 339–43.
Gibson, P. H. (1983) EM study of the numbers of cortical synapses in the brains of ageing people and people with Alzheimer-type dementia.Acta Neuropathologica 62, 127–33.
Goto, S. &Hirano, A. (1990) Synaptophysin expression in the striatum in Huntington's disease.Acta Neuropathologica 80, 88–91.
Greengard, P., Valtorta, F., Czernik, A. J. &Benfenati, F. (1993) Synaptic vesicle phosphoproteins and regulation of synaptic function.Science 259, 780–5.
Gundersen, H. J. G. &Jensen, E. B. (1987) The efficiency of systematic sampling in stereology and its prediction.Journal of Microscience 147, 229–63.
Hamos, J. E., Degennaro, L. J. &Drachman, D. A. (1989) Synaptic loss in Alzheimer's disease and other dementias.Neurology 39, 355–61.
Honer, W. G., Dickson, D. W., Gleeson, J. &Davies, P. (1992) Regional synaptic pathology in Alzheimer's disease.Neurobiology of Aging 13, 375–82.
Hoog, A., Gould, V. E., Grimelius, L., Franke, W. W., Falkmer, S. &Chejfec, G. (1988) Tissue fixation methods alter the immunohistochemical demonstrability of synaptophysin.Ultrastructural Pathology 12, 673–8.
Ince, P. G., Slade, J., Chinnery, R. M., McKenzie, J., Royston, C., Roberts, G. W. &Shaw, P. J. (1995) Quantitative study of synaptophysin immunoreactivity of cerebral cortex and spinal cord in motor neuron disease.Journal of Neuropathology and Experimental Neurology 54, 673–9.
Ito, H., Goto, S., Hirano, A. &Yen, S. H. (1991) Immunohistochemical study of the hippocampus in parkinsonism-dementia complex on Guam.Journal of Geriatric Psychiatry and Neurology 4, 134–42.
Jahn, R., Schiebler, W., Ouimet, C. &Greengard, P. (1985) A 38000-dalton membrane protein (p38) present in synaptic vesicles.Proceedings of the National Academy of Sciences (USA) 82, 4137–41.
Jones, D. G. &Calverley, R. K. S. (1991) Frequency of occurrence of perforated synapses in developing rat neocortex.Neuroscience Letters 129, 189–92.
Lahtinen, H., Miettinen, R., Ylinen, A., Halonen, T. &Riekkinen, P. J. (1993) Biochemical and morphological changes in the rat hippocampus following transection of the fimbria-fornix.Brain Research Bulletin 31, 311–18.
Leclerc, N., Beesley, P. W., Brown, I., Colonnier, M., Gurd, J. W., Paladino, T. &Hawkes, R. (1989) Synaptophysin expression during synaptogenesis in the rat cerebellar cortex.Journal of Comparative Neurology 280, 197–202.
Mahata, S. K., Marksteiner, J., Sperk, G., Mahata, M., Gruber, B., Fischer-Colbrie, R. &Winkler, H. (1992) Temporal lobe epilepsy of the rat: differential expression of mRNAs of chromogranin B, secretogranin II, synaptin/synaptophysin and p65 in subfields of the hippocampus.Molecular Brain Research 16, 1–12.
Masliah, E., Hansen, L., Albright, T., Mallory, M. &Terry R. D. (1991) Immunoelectron microscopic study of synaptic pathology in Alzheimer's disease.Acta Neuropathologica 81, 428–33.
Navone, F., Jahn, R., Digioia, G., Stukenbrok, H., Greengard, P. &Decamilli, P. (1986) Protein p38: an integral membrane protein specific for small vesicles of neurons and neuroendocrine cells.Journal of Cell Biology 103, 2511–27.
Perdahl, E., Adolfsson, R., Alafuzoff, I., Albert, K. A., Nestler, E. J., Greengard, P. &Winblad, B. (1984) Synapsin I (Protein I) in different brain regions in senile dementia of Alzheimer type and in multiinfarct dementia.Journal of Neural Transmission 60, 133–41.
Peters, A., Palay, S. L. &Webster, H. (1991) Synapses. InThe Fine Structure of the Nervous System: Neurons and their Supporting Cells, pp. 138–211. New York: Oxford University Press.
Scheff, S. W., Scott, S. A. &Dekosky, S. T. (1991) Quantitation of synaptic density in the septal nuclei of young and aged Fisher 344 rats.Neurobiology of Aging 12, 3–12.
Sterio, D. C. (1984) The unbiased estimation of number and sizes of arbitrary particles using the disector.Journal of Microscopy 134, 127–36.
Südhof, T. C. (1995) The synaptic vesicle cycle: a cascade of protein-protein interactions.Nature 375, 645–53.
Terry, R. D., Masliah, E., Salmon, D. P., Butters, N., Deteresa, R., Hill, R., Hansen, L. A. &Katzman, R. (1991) Physical basis of cognitive alterations in Alzheimer's disease: synapse loss is the major correlate of cognitive impairment.Annals of Neurology 30, 572–80.
Volknandt, W. (1995) The synaptic vesicle and its targets.Neuroscience 64, 277–300.
Wakabayashi, K., Honer, W. G. &Masliah, E. (1994) Synapse alterations in the hippocampal-entorhinal formation in Alzheimer's disease with and without Lewy body disease.Brain Research 667, 24–32.
West, M. J. (1993) New stereological methods for counting neurons.Neurobiology of Aging 14, 275–85.
West, M. J., Slomianka, L. &Gundersen, H. J. G. (1991) Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator.Anatomical Record 231, 482–97.
Wiedenmann, B. &Franke, W. W. (1985) Identification and localization of synaptophysin, an integral membrane glycoprotein of Mr 38000 characteristic of presynaptic vesciles.Cell 41, 1017–28.
Author information
Authors and Affiliations
Additional information
25th Anniversary Issue
Rights and permissions
About this article
Cite this article
Calhoun, M.E., Jucker, M., Martin, L.J. et al. Comparative evaluation of synaptophysin-based methods for quantification of synapses. J Neurocytol 25, 821–828 (1996). https://doi.org/10.1007/BF02284844
Issue Date:
DOI: https://doi.org/10.1007/BF02284844