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High-throughput, detailed, cell-specific neuroanatomy of dendritic spines using microinjection and confocal microscopy

Nature Protocols volume 6, pages 1391–1411 (2011)Cite this article

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Abstract

Morphological features such as size, shape and density of dendritic spines have been shown to reflect important synaptic functional attributes and potential for plasticity. Here we describe in detail a protocol for obtaining detailed morphometric analysis of spines using microinjection of fluorescent dyes, high-resolution confocal microscopy, deconvolution and image analysis with NeuronStudio. Recent technical advancements include better preservation of tissue, resulting in prolonged ability to microinject, and algorithmic improvements that compensate for the residual z-smear inherent in all optical imaging. Confocal imaging parameters were probed systematically to identify both optimal resolution and the highest efficiency. When combined, our methods yield size and density measurements comparable to serial section transmission electron microscopy in a fraction of the time. An experiment containing three experimental groups with eight subjects each can take as little as 1 month if optimized for speed, or approximately 4–5 months if the highest resolution and morphometric detail is sought.

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Figure 1: The final high-resolution image obtained by our methods results from a two-step improvement of the PSF by deconvolution, followed by z-smear correction.
Figure 2: Microinjections can be performed immediately after or months to years following perfusion if tissue is stored in 0.
Figure 3: Empirical evaluation of the effect of spherical aberrations on 2D and 3D resolution.
Figure 4: Systematic analysis of the effect of imaging parameter choice on resolution and shot noise.
Figure 5: Bleaching rate as a function of laser power.
Figure 6: Multiple fluorophores can be used within an experiment, but the pinhole size should be kept at the same absolute diameter.
Figure 7: Detailed spine morphometric analysis.
Figure 8: Rat CA1 dendritic spine morphometry using this protocol is comparable to measurements obtained by ssTEM.

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Acknowledgements

This work was supported by NIA grants AG006647 (J.H.M.) and AG010606 to (J.H.M.) and NRSA training grant 1F30MH083402 (D.D.). We also acknowledge the late S. Wearne and P.R. Hof for the development of NeuronStudio, funded by US National Institutes of Health grants AG035071 and MH071818. We thank B. Janssen, Y. Grossman, R. Gonzaga and N. Dumitriu for technical assistance. We thank B. Janssen, E. Bloss and Q. Laplant for helpful suggestions on the manuscript. We are also deeply grateful to T. Hu and D. Chklovskii for their computational assistance with exponential decay curve fitting.

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Authors and Affiliations

  1. Fishberg Department of Neuroscience, Mount Sinai School of Medicine, New York, New York, USA

    Dani Dumitriu, Alfredo Rodriguez & John H Morrison

  2. Friedman Brain Institute, Mount Sinai School of Medicine, New York, New York, USA

    Dani Dumitriu, Alfredo Rodriguez & John H Morrison

  3. Computational Neurobiology and Imaging Center, Mount Sinai School of Medicine, New York, New York, USA

    Alfredo Rodriguez

  4. Department of Geriatrics and Palliative Care, Mount Sinai School of Medicine, New York, New York, USA

    John H Morrison

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Contributions

All authors contributed to the preparation of the manuscript. D.D. conducted all experiments, made the figures and wrote the initial draft of the manuscript. A.R. programmed the z-smear correction factor into NeuronStudio. J.H.M. provided guidance throughout the experimental phase and improved the manuscript.

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Correspondence to Dani Dumitriu.

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Supplementary information

Supplementary Fig. 1

Z-projections of the 23 dendrites used in the morphometric analysis of dendritic spines in the stratum radiatum of the male rat CA1. The 4-6 segments from each of the 5 animals included in the analysis are each boxed in a separate color. Note the diversity in spine density, spine size and dendritic diameter. Segments A2i and A2ii are the same as the examples shown in Figure 7A. Scale bar = 5µm. (TIFF 4122 kb)

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Dumitriu, D., Rodriguez, A. & Morrison, J. High-throughput, detailed, cell-specific neuroanatomy of dendritic spines using microinjection and confocal microscopy. Nat Protoc 6, 1391–1411 (2011). https://doi.org/10.1038/nprot.2011.389

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