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Article: Dissecting Neuronal Activation on a Brain-Wide Scale With Immediate Early Genes.

Franceschini, Alessandra / Costantini, Irene / Pavone, Francesco S / Silvestri, Ludovico

2020 Volume 14, Page(s) 569517

Abstract: Visualizing neuronal activation on a brain-wide scale yet with cellular resolution is a fundamental technical challenge for neuroscience. This would enable analyzing how different neuronal circuits are disrupted in pathology and how they could be rescued ...

Abstract	Visualizing neuronal activation on a brain-wide scale yet with cellular resolution is a fundamental technical challenge for neuroscience. This would enable analyzing how different neuronal circuits are disrupted in pathology and how they could be rescued by pharmacological treatments. Although this goal would have appeared visionary a decade ago, recent technological advances make it eventually feasible. Here, we review the latest developments in the fields of genetics, sample preparation, imaging, and image analysis that could be combined to afford whole-brain cell-resolution activation mapping. We show how the different biochemical and optical methods have been coupled to study neuronal circuits at different spatial and temporal scales, and with cell-type specificity. The inventory of techniques presented here could be useful to find the tools best suited for a specific experiment. We envision that in the next years, mapping of neuronal activation could become routine in many laboratories, allowing dissecting the neuronal counterpart of behavior.
Language	English
Publishing date	2020-10-23
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2411902-7
ISSN	1662-453X ; 1662-4548
ISSN (online)	1662-453X
ISSN	1662-4548
DOI	10.3389/fnins.2020.569517
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Article ; Online: KHz-rate volumetric voltage imaging of the whole Zebrafish heart.

Sacconi, Leonardo / Silvestri, Ludovico / Rodríguez, Esteban C / Armstrong, Gary A B / Pavone, Francesco S / Shrier, Alvin / Bub, Gil

Biophysical reports

2022 Volume 2, Issue 1, Page(s) 100046

Abstract: Fast volumetric imaging is essential for understanding the function of excitable tissues such as those found in the brain and heart. Measuring cardiac voltage transients in tissue volumes is challenging, especially at the high spatial and temporal ... ...

Abstract	Fast volumetric imaging is essential for understanding the function of excitable tissues such as those found in the brain and heart. Measuring cardiac voltage transients in tissue volumes is challenging, especially at the high spatial and temporal resolutions needed to give insight to cardiac function. We introduce a new imaging modality based on simultaneous illumination of multiple planes in the tissue and parallel detection with multiple cameras, avoiding compromises inherent in any scanning approach. The system enables imaging of voltage transients
Language	English
Publishing date	2022-02-03
Publishing country	United States
Document type	Journal Article
ISSN	2667-0747
ISSN (online)	2667-0747
DOI	10.1016/j.bpr.2022.100046
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Article ; Online: A Guide to Perform 3D Histology of Biological Tissues with Fluorescence Microscopy.

Laurino, Annunziatina / Franceschini, Alessandra / Pesce, Luca / Cinci, Lorenzo / Montalbano, Alberto / Mazzamuto, Giacomo / Sancataldo, Giuseppe / Nesi, Gabriella / Costantini, Irene / Silvestri, Ludovico / Pavone, Francesco Saverio

International journal of molecular sciences

2023 Volume 24, Issue 7

Abstract: The analysis of histological alterations in all types of tissue is of primary importance in pathology for highly accurate and robust diagnosis. Recent advances in tissue clearing and fluorescence microscopy made the study of the anatomy of biological ... ...

Abstract	The analysis of histological alterations in all types of tissue is of primary importance in pathology for highly accurate and robust diagnosis. Recent advances in tissue clearing and fluorescence microscopy made the study of the anatomy of biological tissue possible in three dimensions. The combination of these techniques with classical hematoxylin and eosin (H&E) staining has led to the birth of three-dimensional (3D) histology. Here, we present an overview of the state-of-the-art methods, highlighting the optimal combinations of different clearing methods and advanced fluorescence microscopy techniques for the investigation of all types of biological tissues. We employed fluorescence nuclear and eosin Y staining that enabled us to obtain hematoxylin and eosin pseudo-coloring comparable with the gold standard H&E analysis. The computational reconstructions obtained with 3D optical imaging can be analyzed by a pathologist without any specific training in volumetric microscopy, paving the way for new biomedical applications in clinical pathology.
MeSH term(s)	Hematoxylin ; Eosine Yellowish-(YS) ; Microscopy, Fluorescence/methods ; Staining and Labeling ; Imaging, Three-Dimensional/methods ; Microscopy, Confocal
Chemical Substances	Hematoxylin (YKM8PY2Z55) ; Eosine Yellowish-(YS) (TDQ283MPCW)
Language	English
Publishing date	2023-04-04
Publishing country	Switzerland
Document type	Review ; Journal Article
ZDB-ID	2019364-6
ISSN	1422-0067 ; 1422-0067 ; 1661-6596
ISSN (online)	1422-0067
ISSN	1422-0067 ; 1661-6596
DOI	10.3390/ijms24076747
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Article ; Online: Brain-wide neuron quantification toolkit reveals strong sexual dimorphism in the evolution of fear memory.

Franceschini, Alessandra / Mazzamuto, Giacomo / Checcucci, Curzio / Chicchi, Lorenzo / Fanelli, Duccio / Costantini, Irene / Passani, Maria Beatrice / Silva, Bianca Ambrogina / Pavone, Francesco Saverio / Silvestri, Ludovico

Cell reports

2023 Volume 42, Issue 8, Page(s) 112908

Abstract: Fear responses are functionally adaptive behaviors that are strengthened as memories. Indeed, detailed knowledge of the neural circuitry modulating fear memory could be the turning point for the comprehension of this emotion and its pathological states. ... ...

Abstract	Fear responses are functionally adaptive behaviors that are strengthened as memories. Indeed, detailed knowledge of the neural circuitry modulating fear memory could be the turning point for the comprehension of this emotion and its pathological states. A comprehensive understanding of the circuits mediating memory encoding, consolidation, and retrieval presents the fundamental technological challenge of analyzing activity in the entire brain with single-neuron resolution. In this context, we develop the brain-wide neuron quantification toolkit (BRANT) for mapping whole-brain neuronal activation at micron-scale resolution, combining tissue clearing, high-resolution light-sheet microscopy, and automated image analysis. The robustness and scalability of this method allow us to quantify the evolution of activity patterns across multiple phases of memory in mice. This approach highlights a strong sexual dimorphism in recruited circuits, which has no counterpart in the behavior. The methodology presented here paves the way for a comprehensive characterization of the evolution of fear memory.
MeSH term(s)	Mice ; Animals ; Sex Characteristics ; Brain/physiology ; Fear/physiology ; Neurons/physiology
Language	English
Publishing date	2023-07-29
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2649101-1
ISSN	2211-1247 ; 2211-1247
ISSN (online)	2211-1247
ISSN	2211-1247
DOI	10.1016/j.celrep.2023.112908
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Article: Nogo-A antibody delivery through the olfactory mucosa mitigates experimental autoimmune encephalomyelitis in the mouse CNS.

Pernet, Vincent / Joly, Sandrine / Spiegel, Sebastian / Meli, Ivo / Idriss, Sherif / Maigler, Frank / Mdzomba, Julius Baya / Roenneke, Anna K / Franceschini, Alessandra / Silvestri, Ludovico / Pavone, Francesco S / Calamai, Martino / Schindowski, Katharina / Chan, Andrew

Cell death discovery

2023 Volume 9, Issue 1, Page(s) 290

Abstract: Systemic administration of Nogo-A-neutralizing antibody ameliorates experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, the blood-brain barrier (BBB) is a major obstacle limiting the passage of systemically ... ...

Abstract	Systemic administration of Nogo-A-neutralizing antibody ameliorates experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, the blood-brain barrier (BBB) is a major obstacle limiting the passage of systemically applied antibody to the CNS. To bypass the BBB, in the present study we tested the intranasal route of administration by targeting the olfactory mucosa with the Nogo-A-blocking antibody 11C7 mAb in myelin oligodendrocyte glycoprotein-induced EAE. Antibodies were specifically administered onto the olfactory mucosa using a microcatheter. Antibody distribution was examined in the CNS by ELISA and light-sheet microscopy. The effects of 11C7 mAb on Nogo-A signaling were assessed by Western blotting. EAE-induced deficits were monitored daily. Demyelination was observed on spinal cord histological sections. Gene expression changes were followed by trancriptomic analyses. A sensitive capture ELISA revealed a rapid and widespread distribution of 11C7 mAb in the CNS, including the olfactory bulb, the cerebellum and the lumbar spinal cord, but not in the CSF. Light-sheet microscopy allowed to observe antibody accumulation in the parenchyma, thus demonstrating nose-to-brain transfer of IgG. At the functional level, the widespread penetration of 11C7 mAb in the CNS, including the thoracolumbar spinal cord, resulted in the improvement of motor symptoms and in the preservation of myelin in the spinal cord of EAE mice. This was accompanied by Nogo-A signaling downregulation, as reflected by the decreased level of phosphorylated cofilin observed by Western blotting in the cerebellum. In the brain of EAE score-matched animals, 11C7 modified the expression of genes that can influence neurotransmission and cognitive functions, independently of the demyelination phenotype in the spinal cord. In conclusion, our data show the feasibility of olfactory mucosa-directed administration for the delivery of therapeutic antibodies targeting CNS antigens in EAE mice.
Language	English
Publishing date	2023-08-09
Publishing country	United States
Document type	Journal Article
ISSN	2058-7716
ISSN	2058-7716
DOI	10.1038/s41420-023-01588-7
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Article ; Online: Mesoscopic Optical Imaging of Whole Mouse Heart.

Giardini, Francesco / Lazzeri, Erica / Olianti, Camilla / Beconi, Giada / Costantini, Irene / Silvestri, Ludovico / Cerbai, Elisabetta / Pavone, Francesco S / Sacconi, Leonardo

Journal of visualized experiments : JoVE

2021 , Issue 176

Abstract: Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition (fibrosis) and cellular misalignment, can affect electrical conduction, introduce electromechanical ... ...

Abstract	Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition (fibrosis) and cellular misalignment, can affect electrical conduction, introduce electromechanical dysfunctions and, eventually, lead to arrhythmia. Current predictive models of these functional alterations are based on non-integrated and low-resolution structural information. Placing this framework on a different order of magnitude is challenging due to the inefficacy of standard imaging methods in performing high-resolution imaging in massive tissue. In this work, we describe a methodological framework that allows imaging of whole mouse hearts with micrometric resolution. The achievement of this goal has required a technological effort where advances in tissue transformation and imaging methods have been combined. First, we describe an optimized CLARITY protocol capable of transforming an intact heart into a nanoporous, hydrogel-hybridized, lipid-free form that allows high transparency and deep staining. Then, a fluorescence light-sheet microscope able to rapidly acquire images of a mesoscopic field of view (mm-scale) with the micron-scale resolution is described. Following the mesoSPIM project, the conceived microscope allows the reconstruction of the whole mouse heart with micrometric resolution in a single tomographic scan. We believe that this methodological framework will allow clarifying the involvement of the cytoarchitecture disarray in the electrical dysfunctions and pave the way for a comprehensive model that considers both the functional and structural data, thus enabling a unified investigation of the structural causes that lead to electrical and mechanical alterations after the tissue remodeling.
MeSH term(s)	Animals ; Heart/diagnostic imaging ; Mice ; Microscopy/methods ; Optical Imaging
Language	English
Publishing date	2021-10-14
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Video-Audio Media
ZDB-ID	2259946-0
ISSN	1940-087X ; 1940-087X
ISSN (online)	1940-087X
ISSN	1940-087X
DOI	10.3791/62795
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Article ; Online: Removing striping artifacts in light-sheet fluorescence microscopy: a review.

Ricci, Pietro / Gavryusev, Vladislav / Müllenbroich, Caroline / Turrini, Lapo / de Vito, Giuseppe / Silvestri, Ludovico / Sancataldo, Giuseppe / Pavone, Francesco Saverio

Progress in biophysics and molecular biology

2021 Volume 168, Page(s) 52–65

Abstract: In recent years, light-sheet fluorescence microscopy (LSFM) has found a broad application for imaging of diverse biological samples, ranging from sub-cellular structures to whole animals, both in-vivo and ex-vivo, owing to its many advantages relative to ...

Abstract	In recent years, light-sheet fluorescence microscopy (LSFM) has found a broad application for imaging of diverse biological samples, ranging from sub-cellular structures to whole animals, both in-vivo and ex-vivo, owing to its many advantages relative to point-scanning methods. By providing the selective illumination of sample single planes, LSFM achieves an intrinsic optical sectioning and direct 2D image acquisition, with low out-of-focus fluorescence background, sample photo-damage and photo-bleaching. On the other hand, such an illumination scheme is prone to light absorption or scattering effects, which lead to uneven illumination and striping artifacts in the images, oriented along the light sheet propagation direction. Several methods have been developed to address this issue, ranging from fully optical solutions to entirely digital post-processing approaches. In this work, we present them, outlining their advantages, performance and limitations.
MeSH term(s)	Animals ; Artifacts ; Microscopy, Fluorescence
Language	English
Publishing date	2021-07-15
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	209302-9
ISSN	1873-1732 ; 0079-6107
ISSN (online)	1873-1732
ISSN	0079-6107
DOI	10.1016/j.pbiomolbio.2021.07.003
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Article ; Online: Optical clearing in cardiac imaging: A comparative study.

Olianti, Camilla / Giardini, Francesco / Lazzeri, Erica / Costantini, Irene / Silvestri, Ludovico / Coppini, Raffaele / Cerbai, Elisabetta / Pavone, Francesco S / Sacconi, Leonardo

Progress in biophysics and molecular biology

2021 Volume 168, Page(s) 10–17

Abstract: The optical clearing of the cardiac tissue has always been a challenging goal to obtain successful three-dimensional reconstructions of entire hearts. Typically, the developed protocols are targeted at the clearing of the brain; cardiac tissue requires ... ...

Abstract	The optical clearing of the cardiac tissue has always been a challenging goal to obtain successful three-dimensional reconstructions of entire hearts. Typically, the developed protocols are targeted at the clearing of the brain; cardiac tissue requires proper arrangements to the original protocols, which are usually tough and time-consuming to figure out. Here, we present the application of three different clearing methodologies on mouse hearts: uDISCO, CLARITY, and SHIELD. For each approach, we describe the required optimizations that we have developed to improve the outcome; in particular, we focus on comparing the features of the tissue after the application of each methodology, especially in terms of tissue preservation, transparency, and staining. We found that the uDISCO protocol induces strong fiber delamination of the cardiac tissue, thus reducing the reliability of structural analyses. The CLARITY protocol confers a high level of transparency to the heart and allows deep penetration of the fluorescent dyes; however, it requires long times for the clearing and the tissue loses its robustness. The SHIELD methodology, indeed, is very promising for tissue maintenance since it preserves its consistency and provides ideal transparency, but further approaches are needed to obtain homogeneous staining of the whole heart. Since the CLARITY procedure, despite the disadvantages in terms of tissue preservation and timings, is actually the most suitable approach to image labeled samples in depth, we optimized and performed the methodology also on human cardiac tissue from control hearts and hearts with hypertrophic cardiomyopathy.
MeSH term(s)	Animals ; Brain ; Heart/diagnostic imaging ; Imaging, Three-Dimensional ; Mice ; Optical Imaging ; Reproducibility of Results
Language	English
Publishing date	2021-08-04
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	209302-9
ISSN	1873-1732 ; 0079-6107
ISSN (online)	1873-1732
ISSN	0079-6107
DOI	10.1016/j.pbiomolbio.2021.07.012
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Article: In-vivo and ex-vivo optical clearing methods for biological tissues: review.

Costantini, Irene / Cicchi, Riccardo / Silvestri, Ludovico / Vanzi, Francesco / Pavone, Francesco Saverio

Biomedical optics express

2019 Volume 10, Issue 10, Page(s) 5251–5267

Abstract: Every optical imaging technique is limited in its penetration depth by scattering occurring in biological tissues. Possible solutions to overcome this problem consist of limiting the detrimental effects of scattering by reducing optical inhomogeneities ... ...

Abstract	Every optical imaging technique is limited in its penetration depth by scattering occurring in biological tissues. Possible solutions to overcome this problem consist of limiting the detrimental effects of scattering by reducing optical inhomogeneities within the sample. This can be achieved either by using physical methods (such as refractive index matching solutions) or by chemical methods (such as the removal of scatterers), based on tissue transformation protocols. This review provides an overview of the current state-of-the-art methods used for both ex-vivo and in-vivo optical clearing of biological tissues. We start with a brief history of the development of the most widespread clearing methods across the new millennium, then we describe the working principles of both physical and chemical methods. Clearing methods are then reviewed, pointing the attention of the reader on both physical and chemical methods, classified based on the tissue size and type for each specific application. A small section is reserved for methods that have already found in-vivo applications at the research level. Finally, a detailed discussion highlighting both the most relevant results achieved and the new ongoing developments in this field is reported in the last part, together with future perspectives for the clearing methodology.
Language	English
Publishing date	2019-09-19
Publishing country	United States
Document type	Journal Article
ZDB-ID	2572216-5
ISSN	2156-7085
ISSN	2156-7085
DOI	10.1364/BOE.10.005251
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Article: Fast whole-brain imaging of seizures in zebrafish larvae by two-photon light-sheet microscopy.

de Vito, Giuseppe / Turrini, Lapo / Müllenbroich, Caroline / Ricci, Pietro / Sancataldo, Giuseppe / Mazzamuto, Giacomo / Tiso, Natascia / Sacconi, Leonardo / Fanelli, Duccio / Silvestri, Ludovico / Vanzi, Francesco / Pavone, Francesco Saverio

Biomedical optics express

2022 Volume 13, Issue 3, Page(s) 1516–1536

Abstract: Light-sheet fluorescence microscopy (LSFM) enables real-time whole-brain functional imaging in zebrafish larvae. Conventional one-photon LSFM can however induce undesirable visual stimulation due to the use of visible excitation light. The use of two- ... ...

Abstract	Light-sheet fluorescence microscopy (LSFM) enables real-time whole-brain functional imaging in zebrafish larvae. Conventional one-photon LSFM can however induce undesirable visual stimulation due to the use of visible excitation light. The use of two-photon (2P) excitation, employing near-infrared invisible light, provides unbiased investigation of neuronal circuit dynamics. However, due to the low efficiency of the 2P absorption process, the imaging speed of this technique is typically limited by the signal-to-noise-ratio. Here, we describe a 2P LSFM setup designed for non-invasive imaging that enables quintuplicating state-of-the-art volumetric acquisition rate of the larval zebrafish brain (5 Hz) while keeping low the laser intensity on the specimen. We applied our system to the study of pharmacologically-induced acute seizures, characterizing the spatial-temporal dynamics of pathological activity and describing for the first time the appearance of caudo-rostral ictal waves (CRIWs).
Language	English
Publishing date	2022-02-16
Publishing country	United States
Document type	Journal Article
ZDB-ID	2572216-5
ISSN	2156-7085
ISSN	2156-7085
DOI	10.1364/BOE.434146
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