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Article ; Online: In Vivo Imaging of the Structural Plasticity of Cortical Neurons After Stroke.

Conti, Emilia / Pavone, Francesco Saverio / Allegra Mascaro, Anna Letizia

Methods in molecular biology (Clifton, N.J.)

2023 Volume 2616, Page(s) 69–81

Abstract: The comprehension of the finest mechanisms underlying experience-dependent plasticity requires the investigation of neurons and synaptic terminals in the intact brain over prolonged periods of time. Longitudinal two-photon imaging together with the ... ...

Abstract	The comprehension of the finest mechanisms underlying experience-dependent plasticity requires the investigation of neurons and synaptic terminals in the intact brain over prolonged periods of time. Longitudinal two-photon imaging together with the expression of fluorescent proteins enables high-resolution imaging of dendritic spines and axonal varicosities of cortical neurons in vivo. Importantly, the study of the mechanisms of structural reorganization is relevant for a deeper understanding of the pathophysiological mechanisms of neurological diseases such as stroke and for the development of new therapeutic approaches. This protocol describes the principal steps for in vivo investigation of neuronal plasticity both in healthy conditions and after an ischemic lesion. First, we give a description of the surgery to perform a stable cranial window that allows optical access to the mouse brain cortex. Then we explain how to perform longitudinal two-photon imaging of dendrites, axonal branches, and synaptic terminals in the mouse brain cortex in vivo, in order to investigate the plasticity of synaptic terminals and orientation of neuronal processes. Finally, we describe how to induce an ischemic lesion in a target region of the mouse brain cortex through a cranial window by applying the photothrombotic stroke model.
MeSH term(s)	Mice ; Animals ; Neurons/metabolism ; Stroke/metabolism ; Axons/pathology ; Microscopy, Fluorescence, Multiphoton/methods ; Presynaptic Terminals ; Neuronal Plasticity/physiology ; Dendritic Spines/physiology
Language	English
Publishing date	2023-01-30
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-2926-0_8
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Article: Tracking the Effect of Therapy With Single-Trial Based Classification After Stroke.

Scaglione, Alessandro / Conti, Emilia / Allegra Mascaro, Anna Letizia / Pavone, Francesco Saverio

Frontiers in systems neuroscience

2022 Volume 16, Page(s) 840922

Abstract: Stroke is a debilitating disease that leads, in the 50% of cases, to permanent motor or cognitive impairments. The effectiveness of therapies that promote recovery after stroke depends on indicators of the disease state that can measure the degree of ... ...

Abstract	Stroke is a debilitating disease that leads, in the 50% of cases, to permanent motor or cognitive impairments. The effectiveness of therapies that promote recovery after stroke depends on indicators of the disease state that can measure the degree of recovery or predict treatment response or both. Here, we propose to use single-trial classification of task dependent neural activity to assess the disease state and track recovery after stroke. We tested this idea on calcium imaging data of the dorsal cortex of healthy, spontaneously recovered and rehabilitated mice while performing a forelimb retraction task. Results show that, at a single-trial level for the three experimental groups, neural activation during the reward pull can be detected with high accuracy with respect to the background activity in all cortical areas of the field of view and this activation is quite stable across trials and subjects of the same group. Moreover, single-trial responses during the reward pull can be used to discriminate between healthy and stroke subjects with areas closer to the injury site displaying higher discrimination capability than areas closer to this site. Finally, a classifier built to discriminate between controls and stroke at the single-trial level can be used to generate an index of the disease state, the therapeutic score, which is validated on the group of rehabilitated mice. In conclusion, task-related neural activity can be used as an indicator of disease state and track recovery without selecting a peculiar feature of the neural responses. This novel method can be used in both the development and assessment of different therapeutic strategies.
Language	English
Publishing date	2022-05-04
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2453005-0
ISSN	1662-5137
ISSN	1662-5137
DOI	10.3389/fnsys.2022.840922
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Article ; Online: Photothrombotic Middle Cerebral Artery Occlusion in Mice: A Novel Model of Ischemic Stroke.

Conti, Emilia / Carlini, Noemi / Piccardi, Benedetta / Allegra Mascaro, Anna Letizia / Pavone, Francesco Saverio

eNeuro

2023 Volume 10, Issue 2

Abstract: Stroke is one of the main causes of death and disability worldwide. Over the past decades, several animal models of focal cerebral ischemia have been developed allowing to investigate pathophysiological mechanisms underlying stroke progression. Despite ... ...

Abstract	Stroke is one of the main causes of death and disability worldwide. Over the past decades, several animal models of focal cerebral ischemia have been developed allowing to investigate pathophysiological mechanisms underlying stroke progression. Despite intense preclinical research efforts, the need for noninvasive mouse models of vascular occlusion targeting the middle cerebral artery yet avoiding mechanical intervention is still pressing. Here, by applying the photothrombotic stroke model to the distal branch of the middle cerebral artery, we developed a novel strategy to induce a targeted occlusion of a large blood vessel in mice. This approach induces unilateral damage encompassing most of the dorsal cortex from the motor up to the visual regions 1 week after stroke. Pronounced limb dystonia one day after the damage is partially recovered after one week. Furthermore, we observe the insurgence of blood vessel leakage and edema formation in the peri-infarct area. Finally, this model elicits a notable inflammatory response revealed as a strong increase in astrocyte density and morphologic complexity in the perilesional region of the cortex compared with both other regions of the ipsilesional and contralesional hemispheres, and in sham-operated mice. To conclude, the stroke model we developed induces in mice the light-mediated occlusion of one of the main targets of human ischemic stroke, the middle cerebral artery, free from the limitations of commonly used preclinical models.
MeSH term(s)	Mice ; Humans ; Animals ; Infarction, Middle Cerebral Artery/complications ; Ischemic Stroke/complications ; Stroke/complications ; Brain Ischemia/complications ; Middle Cerebral Artery/surgery ; Disease Models, Animal
Language	English
Publishing date	2023-02-08
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2800598-3
ISSN	2373-2822 ; 2373-2822
ISSN (online)	2373-2822
ISSN	2373-2822
DOI	10.1523/ENEURO.0244-22.2022
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Article ; Online: Large Scale Double-Path Illumination System with Split Field of View for the All-Optical Study of Inter-and Intra-Hemispheric Functional Connectivity on Mice.

Conti, Emilia / Allegra Mascaro, Anna Letizia / Pavone, Francesco Saverio

Methods and protocols

2019 Volume 2, Issue 1

Abstract: Recent improvements in optical tools that can perturb brain activity and simultaneously reveal the elicited alterations in the associated regions offer an exceptional means to understand and map the connectivity of the brain. In this work, we exploit a ... ...

Abstract	Recent improvements in optical tools that can perturb brain activity and simultaneously reveal the elicited alterations in the associated regions offer an exceptional means to understand and map the connectivity of the brain. In this work, we exploit a combination of recently developed optical tools to monitor neural population at the meso-scale level and to mould the cortical patterns of targeted neuronal population. Our goal was to investigate the propagation of neuronal activity over the mouse cortex that is triggered by optogenetic stimulation in the contralateral hemisphere. Towards this aim, we developed a wide-field fluorescence microscope that is characterized by a double illumination path allowing for the optogenetic stimulation of the transfected area in the left hemisphere and the simultaneous recording of cortical activity in the right hemisphere. The microscope was further implemented with a custom shutter in order to split the LED illumination path, resulting in a half-obscured field of view. By avoiding the spectral crosstalk between GCaMP6f and channelrhodopsin 2 (ChR2), this system offered the possibility of simultaneous "pumping and probing" of inter-hemispheric functional connectivity on Thy1-GCaMP6f mice.
Language	English
Publishing date	2019-01-29
Publishing country	Switzerland
Document type	Letter
ISSN	2409-9279
ISSN (online)	2409-9279
DOI	10.3390/mps2010011
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Article ; Online: Latency correction in sparse neuronal spike trains.

Kreuz, Thomas / Senocrate, Federico / Cecchini, Gloria / Checcucci, Curzio / Mascaro, Anna Letizia Allegra / Conti, Emilia / Scaglione, Alessandro / Pavone, Francesco Saverio

Journal of neuroscience methods

2022 Volume 381, Page(s) 109703

Abstract: Background: In neurophysiological data, latency refers to a global shift of spikes from one spike train to the next, either caused by response onset fluctuations or by finite propagation speed. Such systematic shifts in spike timing lead to a spurious ... ...

Abstract	Background: In neurophysiological data, latency refers to a global shift of spikes from one spike train to the next, either caused by response onset fluctuations or by finite propagation speed. Such systematic shifts in spike timing lead to a spurious decrease in synchrony which needs to be corrected. New method: We propose a new algorithm of multivariate latency correction suitable for sparse data for which the relevant information is not primarily in the rate but in the timing of each individual spike. The algorithm is designed to correct systematic delays while maintaining all other kinds of noisy disturbances. It consists of two steps, spike matching and distance minimization between the matched spikes using simulated annealing. Results: We show its effectiveness on simulated and real data: cortical propagation patterns recorded via calcium imaging from mice before and after stroke. Using simulations of these data we also establish criteria that can be evaluated beforehand in order to anticipate whether our algorithm is likely to yield a considerable improvement for a given dataset. Comparison with existing method(s): Existing methods of latency correction rely on adjusting peaks in rate profiles, an approach that is not feasible for spike trains with low firing in which the timing of individual spikes contains essential information. Conclusions: For any given dataset the criterion for applicability of the algorithm can be evaluated quickly and in case of a positive outcome the latency correction can be applied easily since the source codes of the algorithm are publicly available.
MeSH term(s)	Action Potentials/physiology ; Algorithms ; Animals ; Calcium ; Mice ; Models, Neurological ; Neurons/physiology ; Noise
Chemical Substances	Calcium (SY7Q814VUP)
Language	English
Publishing date	2022-09-06
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	282721-9
ISSN	1872-678X ; 0165-0270
ISSN (online)	1872-678X
ISSN	0165-0270
DOI	10.1016/j.jneumeth.2022.109703
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Article ; Online: Optogenetic confirmation of transverse-tubular membrane excitability in intact cardiac myocytes.

Scardigli, Marina / Pásek, Michal / Santini, Lorenzo / Palandri, Chiara / Conti, Emilia / Crocini, Claudia / Campione, Marina / Loew, Leslie M / de Vries, Antoine A F / Pijnappels, Daniël A / Pavone, Francesco S / Poggesi, Corrado / Cerbai, Elisabetta / Coppini, Raffaele / Kohl, Peter / Ferrantini, Cecilia / Sacconi, Leonardo

The Journal of physiology

2024 Volume 602, Issue 5, Page(s) 791–808

Abstract: T-tubules (TT) form a complex network of sarcolemmal membrane invaginations, essential for well-co-ordinated excitation-contraction coupling (ECC) and thus homogeneous mechanical activation of cardiomyocytes. ECC is initiated by rapid depolarization of ... ...

Abstract	T-tubules (TT) form a complex network of sarcolemmal membrane invaginations, essential for well-co-ordinated excitation-contraction coupling (ECC) and thus homogeneous mechanical activation of cardiomyocytes. ECC is initiated by rapid depolarization of the sarcolemmal membrane. Whether TT membrane depolarization is active (local generation of action potentials; AP) or passive (following depolarization of the outer cell surface sarcolemma; SS) has not been experimentally validated in cardiomyocytes. Based on the assessment of ion flux pathways needed for AP generation, we hypothesize that TT are excitable. We therefore explored TT excitability experimentally, using an all-optical approach to stimulate and record trans-membrane potential changes in TT that were structurally disconnected, and hence electrically insulated, from the SS membrane by transient osmotic shock. Our results establish that cardiomyocyte TT can generate AP. These AP show electrical features that differ substantially from those observed in SS, consistent with differences in the density of ion channels and transporters in the two different membrane domains. We propose that TT-generated AP represent a safety mechanism for TT AP propagation and ECC, which may be particularly relevant in pathophysiological settings where morpho-functional changes reduce the electrical connectivity between SS and TT membranes. KEY POINTS: Cardiomyocytes are characterized by a complex network of membrane invaginations (the T-tubular system) that propagate action potentials to the core of the cell, causing uniform excitation-contraction coupling across the cell. In the present study, we investigated whether the T-tubular system is able to generate action potentials autonomously, rather than following depolarization of the outer cell surface sarcolemma. For this purpose, we developed a fully optical platform to probe and manipulate the electrical dynamics of subcellular membrane domains. Our findings demonstrate that T-tubules are intrinsically excitable, revealing distinct characteristics of self-generated T-tubular action potentials. This active electrical capability would protect cells from voltage drops potentially occurring within the T-tubular network.
MeSH term(s)	Myocytes, Cardiac/metabolism ; Optogenetics ; Sarcolemma/metabolism ; Cell Membrane ; Membrane Potentials ; Action Potentials/physiology
Language	English
Publishing date	2024-02-13
Publishing country	England
Document type	Journal Article
ZDB-ID	3115-x
ISSN	1469-7793 ; 0022-3751
ISSN (online)	1469-7793
ISSN	0022-3751
DOI	10.1113/JP285202
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Book ; Online: Latency correction in sparse neuronal spike trains

Kreuz, Thomas / Senocrate, Federico / Cecchini, Gloria / Checcucci, Curzio / Mascaro, Anna Letizia Allegra / Conti, Emilia / Scaglione, Alessandro / Pavone, Francesco Saverio

2022

Abstract	Background: In neurophysiological data, latency refers to a global shift of spikes from one spike train to the next, either caused by response onset fluctuations or by finite propagation speed. Such systematic shifts in spike timing lead to a spurious decrease in synchrony which needs to be corrected. New Method: We propose a new algorithm of multivariate latency correction suitable for sparse data for which the relevant information is not primarily in the rate but in the timing of each individual spike. The algorithm is designed to correct systematic delays while maintaining all other kinds of noisy disturbances. It consists of two steps, spike matching and distance minimization between the matched spikes using simulated annealing. Results: We show its effectiveness on simulated and real data: cortical propagation patterns recorded via calcium imaging from mice before and after stroke. Using simulations of these data we also establish criteria that can be evaluated beforehand in order to anticipate whether our algorithm is likely to yield a considerable improvement for a given dataset. Comparison with Existing Method(s): Existing methods of latency correction rely on adjusting peaks in rate profiles, an approach that is not feasible for spike trains with low firing in which the timing of individual spikes contains essential information. Conclusions: For any given dataset the criterion for applicability of the algorithm can be evaluated quickly and in case of a positive outcome the latency correction can be applied easily since the source codes of the algorithm are publicly available. Comment: 15 pages, 10 figures
Keywords	Physics - Data Analysis ; Statistics and Probability ; Mathematics - Dynamical Systems ; Physics - Biological Physics ; Statistics - Methodology
Subject code	612
Publishing date	2022-05-19
Publishing country	us
Document type	Book ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Cortical propagation tracks functional recovery after stroke.

Cecchini, Gloria / Scaglione, Alessandro / Allegra Mascaro, Anna Letizia / Checcucci, Curzio / Conti, Emilia / Adam, Ihusan / Fanelli, Duccio / Livi, Roberto / Pavone, Francesco Saverio / Kreuz, Thomas

PLoS computational biology

2021 Volume 17, Issue 5, Page(s) e1008963

Abstract: Stroke is a debilitating condition affecting millions of people worldwide. The development of improved rehabilitation therapies rests on finding biomarkers suitable for tracking functional damage and recovery. To achieve this goal, we perform a ... ...

Abstract	Stroke is a debilitating condition affecting millions of people worldwide. The development of improved rehabilitation therapies rests on finding biomarkers suitable for tracking functional damage and recovery. To achieve this goal, we perform a spatiotemporal analysis of cortical activity obtained by wide-field calcium images in mice before and after stroke. We compare spontaneous recovery with three different post-stroke rehabilitation paradigms, motor training alone, pharmacological contralesional inactivation and both combined. We identify three novel indicators that are able to track how movement-evoked global activation patterns are impaired by stroke and evolve during rehabilitation: the duration, the smoothness, and the angle of individual propagation events. Results show that, compared to pre-stroke conditions, propagation of cortical activity in the subacute phase right after stroke is slowed down and more irregular. When comparing rehabilitation paradigms, we find that mice treated with both motor training and pharmacological intervention, the only group associated with generalized recovery, manifest new propagation patterns, that are even faster and smoother than before the stroke. In conclusion, our new spatiotemporal propagation indicators could represent promising biomarkers that are able to uncover neural correlates not only of motor deficits caused by stroke but also of functional recovery during rehabilitation. In turn, these insights could pave the way towards more targeted post-stroke therapies.
MeSH term(s)	Animals ; Cerebral Cortex/physiopathology ; Disease Models, Animal ; Humans ; Mice ; Recovery of Function/physiology ; Stroke/physiopathology ; Stroke Rehabilitation/methods
Language	English
Publishing date	2021-05-17
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2193340-6
ISSN	1553-7358 ; 1553-734X
ISSN (online)	1553-7358
ISSN	1553-734X
DOI	10.1371/journal.pcbi.1008963
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Article ; Online: Combining Optogenetic Stimulation and Motor Training Improves Functional Recovery and Perilesional Cortical Activity.

Conti, Emilia / Scaglione, Alessandro / de Vito, Giuseppe / Calugi, Francesco / Pasquini, Maria / Pizzorusso, Tommaso / Micera, Silvestro / Allegra Mascaro, Anna Letizia / Pavone, Francesco Saverio

Neurorehabilitation and neural repair

2021 Volume 36, Issue 2, Page(s) 107–118

Abstract: ... ...

Abstract	Background
MeSH term(s)	Animals ; Behavior, Animal/physiology ; Channelrhodopsins ; Disease Models, Animal ; Exercise Therapy/instrumentation ; Exercise Therapy/methods ; Female ; Ischemic Stroke/rehabilitation ; Ischemic Stroke/therapy ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Motor Activity/physiology ; Motor Cortex/metabolism ; Motor Cortex/physiopathology ; Neuronal Plasticity/physiology ; Optogenetics/methods ; Physical Conditioning, Animal/physiology ; Recovery of Function/physiology ; Robotics ; Stroke Rehabilitation/instrumentation ; Stroke Rehabilitation/methods
Chemical Substances	Channelrhodopsins
Language	English
Publishing date	2021-11-11
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1491637-x
ISSN	1552-6844 ; 1545-9683 ; 0888-4390
ISSN (online)	1552-6844
ISSN	1545-9683 ; 0888-4390
DOI	10.1177/15459683211056656
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Article ; Online: Translational Stroke Research Review: Using the Mouse to Model Human Futile Recanalization and Reperfusion Injury in Ischemic Brain Tissue.

Conti, Emilia / Piccardi, Benedetta / Sodero, Alessandro / Tudisco, Laura / Lombardo, Ivano / Fainardi, Enrico / Nencini, Patrizia / Sarti, Cristina / Allegra Mascaro, Anna Letizia / Baldereschi, Marzia

Cells

2021 Volume 10, Issue 12

Abstract: The approach to reperfusion therapies in stroke patients is rapidly evolving, but there is still no explanation why a substantial proportion of patients have a poor clinical prognosis despite successful flow restoration. This issue of futile ... ...

Abstract	The approach to reperfusion therapies in stroke patients is rapidly evolving, but there is still no explanation why a substantial proportion of patients have a poor clinical prognosis despite successful flow restoration. This issue of futile recanalization is explained here by three clinical cases, which, despite complete recanalization, have very different outcomes. Preclinical research is particularly suited to characterize the highly dynamic changes in acute ischemic stroke and identify potential treatment targets useful for clinical translation. This review surveys the efforts taken so far to achieve mouse models capable of investigating the neurovascular underpinnings of futile recanalization. We highlight the translational potential of targeting tissue reperfusion in fully recanalized mouse models and of investigating the underlying pathophysiological mechanisms from subcellular to tissue scale. We suggest that stroke preclinical research should increasingly drive forward a continuous and circular dialogue with clinical research. When the preclinical and the clinical stroke research are consistent, translational success will follow.
MeSH term(s)	Animals ; Brain Ischemia/complications ; Brain Ischemia/diagnostic imaging ; Disease Models, Animal ; Humans ; Mice ; Optical Imaging ; Reperfusion Injury/complications ; Reperfusion Injury/diagnostic imaging ; Stroke/diagnostic imaging ; Stroke/therapy ; Translational Research, Biomedical
Language	English
Publishing date	2021-11-25
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	2661518-6
ISSN	2073-4409 ; 2073-4409
ISSN (online)	2073-4409
ISSN	2073-4409
DOI	10.3390/cells10123308
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