Mapping Synaptic Input Fields of Neurons with Super-Resolution Imaging

Sigal, Yaron M.; Speer, Colenso M.; Babcock, Hazen P.; Zhuang, Xiaowei

doi:10.1016/j.cell.2015.08.033

Cited by 123 publications

(112 citation statements)

References 67 publications

(89 reference statements)

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“…6). A recent 4-color 3D STORM study of synaptic input fields in retinal interneurons confirmed the suitability of mouse retinal samples for such studies (Sigal et al, 2015). …”

Section: Evolution Of Methods For Studying Rod Cell Structurementioning

confidence: 79%

Structural and molecular bases of rod photoreceptor morphogenesis and disease

Wensel

Zhang

Anastassov

et al. 2016

Progress in Retinal and Eye Research

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The rod cell has an extraordinarily specialized structure that allows it to carry out its unique function of detecting individual photons of light. Both the structural features of the rod and the metabolic processes required for highly amplified light detection seem to have rendered the rod especially sensitive to structural and metabolic defects, so that a large number of gene defects are primarily associated with rod cell death and give rise to blinding retinal dystrophies. The structures of the rod, especially those of the sensory cilium known as the outer segment, have been the subject of structural, biochemical, and genetic analysis for many years, but the molecular bases for rod morphogenesis and for cell death in rod dystrophies are still poorly understood. Recent developments in imaging technology, such as cryo-electron tomography and super-resolution fluorescence microscopy, in gene sequencing technology, and in gene editing technology are rapidly leading to new breakthroughs in our understanding of these questions. A summary is presented of our current understanding of selected aspects of these questions, highlighting areas of uncertainty and contention as well as recent discoveries that provide new insights. Examples of structural data from emerging imaging technologies are presented.

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“…6). A recent 4-color 3D STORM study of synaptic input fields in retinal interneurons confirmed the suitability of mouse retinal samples for such studies (Sigal et al, 2015). …”

Section: Evolution Of Methods For Studying Rod Cell Structurementioning

confidence: 79%

Structural and molecular bases of rod photoreceptor morphogenesis and disease

Wensel

Zhang

Anastassov

et al. 2016

Progress in Retinal and Eye Research

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“…STORM imaging of Bassoon and the glutamate receptors GluR1 and GluR2 was also achieved in 4% paraformaldehyde‐fixed mouse brain tissue . A combination of immunohistochemistry for the synaptic scaffolding protein gephyrin, with transgenic expression of the fluorophores GFP and YFP, was performed in mouse retinal sections . This permitted detailed structural imaging of pre‐and postsynaptic domains in glycinergic and GABAergic synapses within the retina.…”

Section: Discussionmentioning

confidence: 99%

“…This permitted detailed structural imaging of pre‐and postsynaptic domains in glycinergic and GABAergic synapses within the retina. The tissue preparation protocol included refinements such as post‐fixing with glutaraldehyde and epoxy resin embedding followed by ultra‐thin sectioning (70 nm) . Similar protocols have been used for mitochondrial labelling in 10–20 μm sections of mouse brain, heart and kidney .…”

Section: Discussionmentioning

confidence: 99%

Super‐resolution imaging of subcortical white matter using stochastic optical reconstruction microscopy (STORM) and super‐resolution optical fluctuation imaging (SOFI)

Hainsworth

Lee

Foot

et al. 2017

Neuropathology Appl Neurobio

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Aims The spatial resolution of light microscopy is limited by the wavelength of visible light (the ‘diffraction limit’, approximately 250 nm). Resolution of sub-cellular structures, smaller than this limit, is possible with super resolution methods such as stochastic optical reconstruction microscopy (STORM) and super-resolution optical fluctuation imaging (SOFI). We aimed to resolve subcellular structures (axons, myelin sheaths and astrocytic processes) within intact white matter, using STORM and SOFI. Methods Standard cryostat-cut sections of subcortical white matter from donated human brain tissue and from adult rat and mouse brain were labelled, using standard immunohistochemical markers (neurofilament-H, myelin-associated glycoprotein, glial fibrillary acidic protein, GFAP). Image sequences were processed for STORM (effective pixel size 8–32 nm) and for SOFI (effective pixel size 80 nm). Results In human, rat and mouse, subcortical white matter high-quality images for axonal neurofilaments, myelin sheaths and filamentous astrocytic processes were obtained. In quantitative measurements, STORM consistently underestimated width of axons and astrocyte processes (compared with electron microscopy measurements). SOFI provided more accurate width measurements, though with somewhat lower spatial resolution than STORM. Conclusions Super resolution imaging of intact cryo-cut human brain tissue is feasible. For quantitation, STORM can under-estimate diameters of thin fluorescent objects. SOFI is more robust. The greatest limitation for super-resolution imaging in brain sections is imposed by sample preparation. We anticipate that improved strategies to reduce autofluorescence and to enhance fluorophore performance will enable rapid expansion of this approach.

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“…Recently, improved automated or semi-automated techniques have been developed for in vitro and in vivo quantification of synapses using fluorescence microscopy (Ippolito and Eroglu 2010; Schätzle et al 2012; Dumitriu et al 2012; Busse and Smith 2013; Fogarty et al 2013; Danielson and Lee 2014; Sanders et al 2015; Klenowski et al 2015; Sigal et al 2015). The combination of fluorescence microscopy, immunolabeling of key pre- and postsynaptic markers of excitatory/inhibitory synapses and automated software analysis, has afforded new high-throughput methodology allowing for rapid quantification of putative neurochemical synapses.…”

Section: Introductionmentioning

confidence: 99%

Mapping the connectivity of serotonin transporter immunoreactive axons to excitatory and inhibitory neurochemical synapses in the mouse limbic brain

et al. 2016

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Serotonin neurons arise from the brainstem raphe nuclei and send their projections throughout the brain to release 5-HT which acts as a modulator of several neuronal populations. Previous electron microscopy studies in rats have morphologically determined the distribution of 5-HT release sites (boutons) in certain brain regions and have shown that 5-HT containing boutons form synaptic contacts that are either symmetric or asymmetric. In addition, 5-HT boutons can form synaptic triads with the pre- and postsynaptic specializations of either symmetrical or asymmetrical synapses. However, due to the labor intensive processing of serial sections required by electron microscopy, little is known about the neurochemical properties or the quantitative distribution of 5-HT triads within whole brain or discrete subregions. Therefore, we used a semi-automated approach that combines immunohistochemistry and high-resolution confocal microscopy to label serotonin transporter (SERT) immunoreactive axons and reconstruct in 3D their distribution within limbic brain regions. We also used antibodies against key pre- (synaptophysin) and postsynaptic components of excitatory (PSD95) or inhibitory (gephyrin) synapses to (1) identify putative 5-HTergic boutons within SERT immunoreactive axons and, (2) quantify their close apposition to neurochemical excitatory or inhibitory synapses. We provide a 5-HTergic axon density map and have determined the ratio of synaptic triads consisting of a 5-HT bouton in close proximity to either neurochemical excitatory or inhibitory synapses within different limbic brain areas. The ability to model and map changes in 5-HTergic axonal density and the formation of triadic connectivity within whole brain regions using this rapid and quantitative approach offers new possibilities for studying neuroplastic changes in the 5-HTergic pathway.Electronic supplementary materialThe online version of this article (doi:10.1007/s00429-016-1278-x) contains supplementary material, which is available to authorized users.

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Mapping Synaptic Input Fields of Neurons with Super-Resolution Imaging

Cited by 123 publications

References 67 publications

Structural and molecular bases of rod photoreceptor morphogenesis and disease

Structural and molecular bases of rod photoreceptor morphogenesis and disease

Super‐resolution imaging of subcortical white matter using stochastic optical reconstruction microscopy (STORM) and super‐resolution optical fluctuation imaging (SOFI)

Mapping the connectivity of serotonin transporter immunoreactive axons to excitatory and inhibitory neurochemical synapses in the mouse limbic brain

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