Marking synaptic activity in dendritic spines with a calpain substrate exhibiting fluorescence resonance energy transfer
Open accessAbstract
Excitatory synaptic activity can evoke transient and substantial elevations of postsynaptic calcium. Downstream effects of elevated calcium include the activation of the calcium-dependent protease calpain. We have developed a reagent that identifies dendritic spines in which calpain has been activated. A fusion protein was expressed that contained enhanced yellow and enhanced cyan fluorescent protein (EYFP and ECFP, respectively) linked by a peptide that included the μ-calpain cleavage site from α-spectrin. A PDZ-binding site fused to ECFP anchored this protein to postsynaptic densities. The fusion protein exhibited fluorescence resonance energy transfer (FRET), and diminution of FRET by proteolysis was used to localize calpain activity in situ by fluorescence microscopy. Incubation of the fusion protein with calpain in the presence of calcium resulted in the separation of EYFP and ECFP into monomeric fluorophores. In transiently transfected cell lines and dissociated hippocampal neurons, FRET was diminished by raising intracellular calcium levels with an ionophore or with glutamatergic agonists. Calpain inhibitors blocked these changes. Under control conditions, FRET levels in different dendritic spines of cultured neurons and in hippocampal slices were heterogeneous but showed robust decreases upon treatment with glutamatergic agonists. Immunostaining of cultured neurons with antibodies to a spectrin epitope produced by calpain-mediated digestion revealed an inverse correlation between the amount of FRET present at postsynaptic elements and the concentration of spectrin breakdown products. These results suggest that the FRET methodology identifies sites of synaptically induced calpain activity and that it may be useful in analyzing synapses undergoing changes in efficacy.
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TBS causes the breakdown of actin crosslinking proteins in the subsynaptic cytoskeleton.
“TBS, in addition to its actions on kinases, causes the breakdown of actin crosslinking proteins in the subsynaptic cytoskeleton; the relevant proteases also cleave transmembrane adhesion receptors that play a critical role in LTP-related actin polymerization (40–42).”
From Synaptic evidence for the efficacy of spaced learning (2012) · cites this paper as [40]
The relevant proteases cleave transmembrane adhesion receptors that play a critical role in LTP-related actin polymerization.
“TBS, in addition to its actions on kinases, causes the breakdown of actin crosslinking proteins in the subsynaptic cytoskeleton; the relevant proteases also cleave transmembrane adhesion receptors that play a critical role in LTP-related actin polymerization (40–42).”
From Synaptic evidence for the efficacy of spaced learning (2012) · cites this paper as [40]
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Cites
Fluorescent indicators for Ca2+based on green fluorescent proteins and calmodulin
Miyawaki, A., Llopis, J. L., Heim, R., McCaffery, J. M., Adams, J. A., Ikura, M., & Tsien, R. Y. (1997) · Nature · doi:10.1038/42264 · Cited by 3,250 · see citing works
Dendritic spine changes associated with hippocampal long-term synaptic plasticity
Engert, F., & Bonhoeffer, T. (1999) · Nature · doi:10.1038/19978 · Cited by 1,664 · see citing works
Evidence for silent synapses: Implications for the expression of LTP
Isaac, J. T. R., Nicoll, R. A., & Malenka, R. C. (1995) · Neuron · doi:10.1016/0896-6273(95)90046-2 · Cited by 1,233 · see citing works
Rapid Dendritic Morphogenesis in CA1 Hippocampal Dendrites Induced by Synaptic Activity
Maletić‐Savatić, M., Malinow, R., & Svoboda, K. (1999) · Science · doi:10.1126/science.283.5409.1923 · Cited by 1,189 · see citing works
Intracellular injections of EGTA block induction of hippocampal long-term potentiation
Lynch, G., Larson, J., Kelso, S., Barrionuevo, G., & Schottler, F. (1983) · Nature · doi:10.1038/305719a0 · Cited by 1,116 · see citing works
Larson, J., Wong, D., & Lynch, G. (1986) · Brain Research · doi:10.1016/0006-8993(86)90579-2 · Cited by 1,037 · see citing works
Kim, E., Niethammer, M., Rothschild, A., Jan, Y. N., & Sheng, M. (1995) · Nature · doi:10.1038/378085a0 · Cited by 999 · see citing works
LTP promotes formation of multiple spine synapses between a single axon terminal and a dendrite
Toni, N., Buchs, P.-A., Nikonenko, I., Bron, C. R., & Muller, D. (1999) · Nature · doi:10.1038/46574 · Cited by 949 · see citing works
Postsynaptic Calcium Is Sufficient for Potentiation of Hippocampal Synaptic Transmission
Malenka, R. C., Kauer, J. A., Zucker, R. S., & Nicoll, R. A. (1988) · Science · doi:10.1126/science.2845577 · Cited by 829 · see citing works
Steward, O., Wallace, C. S., Lyford, G. L., & Worley, P. F. (1998) · Neuron · doi:10.1016/s0896-6273(00)80591-7 · Cited by 811 · see citing works
Calcium-induced actin depolymerization reduces NMDA channel activity
Rosenmund, C., & Westbrook, G. L. (1993) · Neuron · doi:10.1016/0896-6273(93)90197-y · Cited by 503 · see citing works
Long-term potentiation in the amygdala: a mechanism for emotional learning and memory
Maren, S. (1999) · Trends in Neurosciences · doi:10.1016/s0166-2236(99)01465-4 · Cited by 456 · see citing works
Emptage, N. J., Bliss, T., & Fine, A. (1999) · Neuron · doi:10.1016/s0896-6273(00)80683-2 · Cited by 435 · see citing works
The Influence of Prior Synaptic Activity on the Induction of Long-Term Potentiation
Huang, Y.-Y., Colino, A., Selig, D. K., & Malenka, R. C. (1992) · Science · doi:10.1126/science.1346729 · Cited by 412 · see citing works
Roberts‐Lewis, J. M., Mj, S., Marcy, V. R., Pinsker, L. R., & Siman, R. (1994) · Journal of Neuroscience · doi:10.1523/jneurosci.14-06-03934.1994 · Cited by 316 · see citing works
Calcium‐dependent proteases: an enzyme system active at cellular membranes?
Mellgren, R. L. (1987) · The FASEB Journal · doi:10.1096/fasebj.1.2.2886390 · Cited by 307 · see citing works
Calpain Regulates Actin Remodeling during Cell Spreading
Potter, D. A., Tirnauer, J. S., Janssen, R. A. J., Croall, D. E., Hughes, C. N., Fiacco, K. A., Mier, J. W., Maki, M., & Herman, I. M. (1998) · The Journal of Cell Biology · doi:10.1083/jcb.141.3.647 · Cited by 274 · see citing works
Spatial resolution of fodrin proteolysis in postischemic brain.
Saido, T. C., Yokota, M., Nagao, S., Yamaura, I., Tani, E., Tsuchiya, T., Suzuki, K., & Kawashima, S. (1993) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(19)74593-1 · Cited by 270 · see citing works
Detection of programmed cell death using fluorescence energy transfer
Xu, X. M. (1998) · Nucleic Acids Research · doi:10.1093/nar/26.8.2034 · Cited by 207 · see citing works
The calmodulin-binding site in alpha-fodrin is near the calcium-dependent protease-I cleavage site.
Harris, A. S., Croall, D. E., & Morrow, J. S. (1988) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(19)37652-5 · Cited by 152 · see citing works
Mahajan, N. P., Harrison-Shostak, D. C., Michaux, J., et al. (1999) · Chemistry & Biology · doi:10.1016/s1074-5521(99)80051-9 · Cited by 144 · see citing works
Postsynaptically Silent Synapses in Single Neuron Cultures
Gomperts, S. N., Rao, A., Craig, A. M., Malenka, R. C., & Nicoll, R. A. (1998) · Neuron · doi:10.1016/s0896-6273(00)80662-5 · Cited by 142 · see citing works
Micromolar Ca2+ transients in dendritic spines of hippocampal pyramidal neurons in brain slice
Petrozzino, J. J., Pozzo‐Miller, L. D., & Connor, J. A. (1995) · Neuron · doi:10.1016/0896-6273(95)90269-4 · Cited by 137 · see citing works
Memory and the Brain: Unexpected Chemistries and a New Pharmacology
LYNCH, G. S. (1998) · Neurobiology of Learning and Memory · doi:10.1006/nlme.1998.3840 · Cited by 125 · see citing works
Vanderklish, P. W., Saido, T. C., Gal, C., et al. (1995) · Molecular Brain Research · doi:10.1016/0169-328x(95)00057-y · Cited by 112 · see citing works
Self-citationWickens, J. R. (1988) · Progress in Neurobiology · doi:10.1016/0301-0082(88)90013-5 · Cited by 112 · see citing works
del Cerro, S., Larson, J., Oliver, M. W., & LYNCH, G. S. (1990) · Brain Research · doi:10.1016/0006-8993(90)90660-4 · Cited by 104 · see citing works
Activity‐dependent formation of perforated synapses in cultured hippocampal neurons
Neuhoff, H., Roeper, J., & Schweizer, M. (1999) · European Journal of Neuroscience · doi:10.1046/j.1460-9568.1999.00856.x · Cited by 99 · see citing works
McBain, C. J., Boden, P., & Hill, R. G. (1989) · Journal of Neuroscience Methods · doi:10.1016/0165-0270(89)90051-4 · Cited by 93 · see citing works
Variations in Synaptic Plasticity and Types of Memory in Corticohippocampal Networks
Lynch, G., & Granger, R. (1992) · Journal of Cognitive Neuroscience · doi:10.1162/jocn.1992.4.3.189 · Cited by 83 · see citing works
Postsynaptic actin and neuronal plasticity
Matus, A. I. (1999) · Current Opinion in Neurobiology · doi:10.1016/s0959-4388(99)00018-5 · Cited by 82 · see citing works
Elimination of autofluorescence in immunofluorescence microscopy with digital image processing.
van de Lest, C. H. A., Versteeg, E. M. M., Veerkamp, J. H., et al. (1995) · Journal of Histochemistry & Cytochemistry · doi:10.1177/43.7.7608528 · Cited by 78 · see citing works
Long-term potentiation in rat piriform cortex following discrimination learning
Roman, F. S., Chaillan, F. A., & Soumireu-Mourat, B. (1993) · Brain Research · doi:10.1016/0006-8993(93)91719-9 · Cited by 69 · see citing works
Stimulation of NMDA receptors activates calpain in cultured hippocampal slices
del Cerro, S., Arai, A., Kessler, M., et al. (1994) · Neuroscience Letters · doi:10.1016/0304-3940(94)91049-9 · Cited by 66 · see citing works
Bednarski, E., Vanderklish, P. W., Gall, C. M., Saido, T. C., Bahr, B. A., & LYNCH, G. S. (1995) · Brain Research · doi:10.1016/0006-8993(95)00851-g · Cited by 59 · see citing works
Self-citationNerve growth factor-induced decrease in the calpain activity of PC12 cells
Oshima, M., Koizumi, S., Fujita, K., et al. (1989) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(19)47134-2 · Cited by 53 · see citing works
Spontaneous activity and recurrent inhibition in cultured hippocampal networks
Siebler, M., Köller, H., Stichel, C. C., Müller, H. W., & Freund, H. (1993) · Synapse · doi:10.1002/syn.890140304 · Cited by 46 · see citing works
Long-lasting physiological effects of bath applied N-methyl-d-aspartate
Thibault, O., Joly, M., Müller, D., Schottler, F., Dudek, S. M., & LYNCH, G. S. (1989) · Brain Research · doi:10.1016/0006-8993(89)91553-9 · Cited by 45 · see citing works
Sporns, O., & Jenkinson, S. (1997) · Neuroscience · doi:10.1016/s0306-4522(97)00152-8 · Cited by 30 · see citing works
LYNCH, G. S., & Seubert, P. A. (1989) · Annals of the New York Academy of Sciences · doi:10.1111/j.1749-6632.1989.tb12505.x · Cited by 25 · see citing works
Sagot, I., Bonneu, M., Balguerie, A., et al. (1999) · FEBS Letters · doi:10.1016/s0014-5793(99)00258-6 · Cited by 21 · see citing works
Najm, I., Vanderklish, P. W., LYNCH, G. S., et al. (1991) · Developmental Brain Research · doi:10.1016/0165-3806(91)90088-z · Cited by 16 · see citing works
Self-citationCompartmentation and glycoprotein substrates of calpain in the developing rat brain
Sheppard, A. M., Wu, J. C., Bahr, B. A., et al. (1991) · Synapse · doi:10.1002/syn.890090310 · Cited by 12 · see citing works
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Shors, T. J., Chua, C., & Falduto, J. (2001) · The Journal of Neuroscience · doi:10.1523/jneurosci.21-16-06292.2001 · Cited by 496 · see citing works
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- Authors
- Vanderklish, P. W., Krushel, L. A., Holst, B. H., Gally, J. A., Crossin, K. L., & Edelman, G. M.
- Affiliations
- Scripps Research Institute (US)
- Department of Neurobiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037; and The Neurosciences Institute, 10640 John Jay Hopkins Drive, San Diego, CA 92121
- Venue
- Proceedings of the National Academy of Sciences
- Publisher
- National Academy of Sciences
- Published
- 2000-02-25
- Volume
- 97
- Issue
- 5
- Pages
- 2253-2258
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- PMC
- PMC15787
- OpenAlex
- W2022054103
- Cited by
- 163
- References
- 43
- Citations by year
- 2026: 2 · 2025: 1 · 2024: 1 · 2023: 3 · 2021: 1 · 2020: 3 · 2019: 4 · 2018: 2 · 2017: 6 · 2016: 6 · 2015: 7 · 2014: 6 · 2013: 10 · 2012: 9
- FWCI
- 4.37
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Proceedings of the National Academy of Sciences (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Calpain Protease Function and Regulation
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
- Keywords
- calpain, dendritic spines, synaptic activity, fluorescence resonance energy transfer, postsynaptic calcium, calpain substrates, hippocampal neurons, postsynaptic density, spectrin breakdown products, synaptic plasticity
- MeSH terms
- Amino Acid Sequence, Animals, Calcium, Calpain, Dendrites, Energy Transfer, Fluorescence, Hippocampus, Intracellular Fluid, Luminescent Proteins, Microscopy, Fluorescence, Molecular Sequence Data, Synaptic Transmission, Recombinant Fusion Proteins, Spectrin, Spectrometry, Fluorescence, Substrate Specificity, Biomarkers, N-Methylaspartate, Receptors, Glutamate, Long-Term Potentiation, Glutamic Acid, COS Cells, Culture Techniques, Green Fluorescent Proteins, Rats
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