Distributed and Localized Dynamics Emerge in the Mouse Neocortex during Reach-to-Grasp Behavior.

A long-standing question in systems neuroscience is to what extent task-relevant features of neocortical processing are localized or distributed. Coordinated activity across the neocortex has been recently shown to drive complex behavior in the mouse, while activity in selected areas is canonically associated with specific functions (e.g., movements in the case of the motor cortex). Reach-to-grasp (RtG) movements are known to be dependent on motor circuits of the neocortex; however, the global activity of the neocortex during these movements has been largely unexplored in the mouse. Here, we characterized, using wide-field calcium imaging, these neocortex-wide dynamics in mice of either sex engaging in an RtG task. We demonstrate that, beyond motor regions, several areas, such as the visual and the retrosplenial cortices, also increase their activity levels during successful RtGs, and homologous regions across the ipsilateral hemisphere are also involved. Functional connectivity among neocortical areas increases transiently around movement onset and decreases during movement. Despite this global phenomenon, neural activity levels correlate with kinematics measures of successful RtGs in sensorimotor areas only. Our findings establish that distributed and localized neocortical dynamics co-orchestrate efficient control of complex movements.SIGNIFICANCE STATEMENT Mammals rely on reaching and grasping movements for fine-scale interactions with the physical world. In the mouse, the motor cortex is critical for the execution of such behavior, yet little is known about the activity patterns across neocortical areas. Using the mesoscale-level networks as a model of cortical processing, we investigated the hypothesis that areas beyond the motor regions could participate in RtG planning and execution, and indeed a large network of areas is involved while performing RtGs. Movement kinematics correlates mostly with neural activity in sensorimotor areas. By demonstrating that distributed and localized neocortical dynamics for the execution of fine movements coexist in the mouse neocortex during RtG, we offer an unprecedented view on the neocortical correlates of mammalian motor control.

Optimization of highly inclined illumination for diffraction-limited and super-resolution microscopy.

In HILO microscopy, a highly inclined and laminated light sheet is used to illuminate the sample, thus drastically reducing background fluorescence in wide-field microscopy, but maintaining the simplicity of the use of a single objective for both illumination and detection. Although the technique has become widely popular, particularly in single molecule and super-resolution microscopy, a limited understanding of how to finely shape the illumination beam and of how this impacts on the image quality complicates the setting of HILO to fit the experimental needs. In this work, we build up a simple and comprehensive guide to optimize the beam shape and alignment in HILO and to predict its performance in conventional fluorescence and super-resolution microscopy. We model the beam propagation through Gaussian optics and validate the model through far- and near-field experiments, thus characterizing the main geometrical features of the beam. Further, we fully quantify the effects of a progressive reduction of the inclined beam thickness on the image quality of both diffraction-limited and super-resolution images and we show that the most relevant impact is obtained by reducing the beam thickness to sub-cellular dimensions (< 3 µm). Based on this, we present a simple optical solution that exploits a rectangular slit to reduce the inclined beam thickness down to 2.6 µm while keeping a field-of-view dimension suited for cell imaging and allowing an increase in the number of localizations in super-resolution imaging of up to 2.6 folds.

Imaging Approaches to Investigate Pathophysiological Mechanisms of Brain Disease in Zebrafish.

Zebrafish has become an essential model organism in modern biomedical research. Owing to its distinctive features and high grade of genomic homology with humans, it is increasingly employed to model diverse neurological disorders, both through genetic and pharmacological intervention. The use of this vertebrate model has recently enhanced research efforts, both in the optical technology and in the bioengineering fields, aiming at developing novel tools for high spatiotemporal resolution imaging. Indeed, the ever-increasing use of imaging methods, often combined with fluorescent reporters or tags, enable a unique chance for translational neuroscience research at different levels, ranging from behavior (whole-organism) to functional aspects (whole-brain) and down to structural features (cellular and subcellular). In this work, we present a review of the imaging approaches employed to investigate pathophysiological mechanisms underlying functional, structural, and behavioral alterations of human neurological diseases modeled in zebrafish.

A Guide to Perform 3D Histology of Biological Tissues with Fluorescence Microscopy.

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.

Cortical propagation tracks functional recovery after stroke.

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.

Design and Synthesis of Novel Raman Reporters for Bioorthogonal SERS Nanoprobes Engineering.

Surface-enhanced Raman spectroscopy (SERS) exploiting Raman reporter-labeled nanoparticles (RR@NPs) represents a powerful tool for the improvement of optical bio-assays due to RRs' narrow peaks, SERS high sensitivity, and potential for multiplexing. In the present work, starting from low-cost and highly available raw materials such as cysteamine and substituted benzoic acids, novel bioorthogonal RRs, characterized by strong signal (103 counts with FWHM < 15 cm−1) in the biological Raman-silent region (>2000 cm−1), RRs are synthesized by implementing a versatile, modular, and straightforward method with high yields and requiring three steps lasting 18 h, thus overcoming the limitations of current reported procedures. The resulting RRs' chemical structure has SH-pendant groups exploited for covalent conjugation to high anisotropic gold-NPs. RR@NPs constructs work as SERS nanoprobes demonstrating high colloidal stability while retaining NPs' physical and vibrational properties, with a limit of detection down to 60 pM. RR@NPs constructs expose carboxylic moieties for further self-assembling of biomolecules (such as antibodies), conferring tagging capabilities to the SERS nanoprobes even in heterogeneous samples, as demonstrated with in vitro experiments by transmembrane proteins tagging in cell cultures. Finally, thanks to their non-overlapping spectra, we envision and preliminary prove the possibility of exploiting RR@NPs constructs simultaneously, aiming at improving current SERS-based multiplexing bioassays.

Reconstruction scheme for excitatory and inhibitory dynamics with quenched disorder: application to zebrafish imaging.

An inverse procedure is developed and tested to recover functional and structural information from global signals of brains activity. The method assumes a leaky-integrate and fire model with excitatory and inhibitory neurons, coupled via a directed network. Neurons are endowed with a heterogenous current value, which sets their associated dynamical regime. By making use of a heterogenous mean-field approximation, the method seeks to reconstructing from global activity patterns the distribution of in-coming degrees, for both excitatory and inhibitory neurons, as well as the distribution of the assigned currents. The proposed inverse scheme is first validated against synthetic data. Then, time-lapse acquisitions of a zebrafish larva recorded with a two-photon light sheet microscope are used as an input to the reconstruction algorithm. A power law distribution of the in-coming connectivity of the excitatory neurons is found. Local degree distributions are also computed by segmenting the whole brain in sub-regions traced from annotated atlas.

Colored visual stimuli evoke spectrally tuned neuronal responses across the central nervous system of zebrafish larvae.

BACKGROUND: Visually guided behaviors such as optomotor and optokinetic responses, phototaxis, and prey capture are crucial for survival in zebrafish and become apparent after just a few days of development. Color vision, which in zebrafish is based on a spatially anisotropic tetrachromatic retina, provides an additional important component of world representation driving fundamental larval behaviors. However, little is known about the central nervous system (CNS) circuitry underlying color vision processing downstream of the retina, and its activity correlates with behavior. Here, we used the transparent larva of zebrafish to image CNS neurons and their activity in response to colored visual stimuli. RESULTS: To investigate the processing of chromatic information in the zebrafish larva brain, we mapped with cellular resolution, spectrally responsive neurons in the larva encephalon and spinal cord. We employed the genetically encoded calcium indicator GCaMP6s and two-photon microscopy to image the neuronal activity while performing visual stimulation with spectrally distinct stimuli at wavelengths matching the absorption peaks of the four zebrafish cone types. We observed the presence of a high number of wavelength-selective neurons not only in the optic tectum, but also in all other regions of the CNS, demonstrating that the circuitry involved in processing spectral information and producing color-selective responses extends to the whole CNS. CONCLUSIONS: Our measurements provide a map of neurons involved in color-driven responses, revealing that spectral information spreads in all regions of the CNS. This suggests the underlying complexity of the circuits involved and opens the way to their detailed future investigation.

Ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke.

We describe a dual-trap force-clamp configuration that applies constant loads between a binding protein and an intermittently interacting biological polymer. The method has a measurement delay of only ∼10 µs, allows detection of interactions as brief as ∼100 µs and probes sub-nanometer conformational changes with a time resolution of tens of microseconds. We tested our method on molecular motors and DNA-binding proteins. We could apply constant loads to a single motor domain of myosin before its working stroke was initiated (0.2-1 ms), thus directly measuring its load dependence. We found that, depending on the applied load, myosin weakly interacted (<1 ms) with actin without production of movement, fully developed its working stroke or prematurely detached (<5 ms), thus reducing the working stroke size with load. Our technique extends single-molecule force-clamp spectroscopy and opens new avenues for investigating the effects of forces on biological processes.

Correlative two-photon and light sheet microscopy.

Information processing inside the central nervous system takes place on multiple scales in both space and time. A single imaging technique can reveal only a small part of this complex machinery. To obtain a more comprehensive view of brain functionality, complementary approaches should be combined into a correlative framework. Here, we describe a method to integrate data from in vivo two-photon fluorescence imaging and ex vivo light sheet microscopy, taking advantage of blood vessels as reference chart. We show how the apical dendritic arbor of a single cortical pyramidal neuron imaged in living thy1-GFP-M mice can be found in the large-scale brain reconstruction obtained with light sheet microscopy. Starting from the apical portion, the whole pyramidal neuron can then be segmented. The correlative approach presented here allows contextualizing within a three-dimensional anatomic framework the neurons whose dynamics have been observed with high detail in vivo.

Two-photon all-optical neurophysiology for the dissection of larval zebrafish brain functional and effective connectivity.

One of the most audacious goals of modern neuroscience is unraveling the complex web of causal relations underlying the activity of neuronal populations on a whole-brain scale. This endeavor, which was prohibitive only a couple of decades ago, has recently become within reach owing to the advancements in optical methods and the advent of genetically encoded indicators/actuators. These techniques, applied to the translucent larval zebrafish have enabled recording and manipulation of the activity of extensive neuronal populations spanning the entire vertebrate brain. Here, we present a custom two-photon optical system that couples light-sheet imaging and 3D excitation with acousto-optic deflectors for simultaneous high-speed volumetric recording and optogenetic stimulation. By employing a zebrafish line with pan-neuronal expression of both the calcium reporter GCaMP6s and the red-shifted opsin ReaChR, we implemented a crosstalk-free, noninvasive all-optical approach and applied it to reconstruct the functional and effective connectivity of the left habenula.

Microplastics reach the brain and interfere with honey bee cognition.

Scientific research on the impact of microplastics (MPs) in terrestrial systems is still emerging, but it has confirmed adverse health effects in organisms exposed to plastics. Although recent studies have shown the toxicological effects of individual MPs polymers on honey bees, the effects of different polymer combinations on cognitive and behavioural performance remain unknown. To fill this knowledge gap, we investigated the effects of oral exposure to spherical MPs on cognitive performance and brain accumulation in the honey bee Apis mellifera. We evaluated the acute toxicity, after a two-day exposure, of polystyrene (PS - 4.8-5.8 µm) and plexiglass (Poly(methyl methacrylate), or PMMA - 1-40 µm) MPs, and a combination of the two (MIX), at two environmentally relevant and one higher concentration (0.5, 5 and 50 mg L-1) and analysed their effects on sucrose responsiveness and appetitive olfactory learning and memory. We also used fluorescent thermoset amino formaldehyde MPs (1-5 µm) to explore whether microspheres of this diameter could penetrate the insect blood-brain barrier (BBB), using Two-Photon Fluorescence Microscopy (TPFM) in combination with an optimized version of the DISCO clearing technique. The results showed that PS reduced sucrose responsiveness, while PMMA had no significant effect; however, the combination had a marked negative effect on sucrose responsiveness. PMMA, PS, and MIX impaired bee learning and memory in bees, with PS showing the most severe effects. 3D brain imaging analysis using TFPM showed that 1-5 µm MPs penetrated and accumulated in the brain after only three days of oral exposure. These results raise concerns about the potential mechanical, cellular, and biochemical damage that MPs may cause to the central nervous system.

Hybrid lipid-AuNP clusters as highly efficient SERS substrates for biomedical applications.

Although Surface Enhanced Raman Scattering (SERS) is widely applied for ultrasensitive diagnostics and imaging, its potential is largely limited by the difficult preparation of SERS tags, typically metallic nanoparticles (NPs) functionalized with Raman-active molecules (RRs), whose production often involves complex synthetic approaches, low colloidal stability and poor reproducibility. Here, we introduce LipoGold Tags, a simple platform where gold NPs (AuNPs) clusters form via self-assembly on lipid vesicle. RRs embedded in the lipid bilayer experience enhanced electromagnetic field, significantly increasing their Raman signals. We modulate RRs and lipid vesicle concentrations to achieve optimal SERS enhancement and we provide robust structural characterization. We further demonstrate the versatility of LipoGold Tags by functionalizing them with biomolecular probes, including antibodies. As proof of concept, we successfully detect intracellular GM1 alterations, distinguishing healthy donors from patients with infantile GM1 gangliosidosis, showcasing LipoGold Tags as advancement in SERS probes production.

Mapping brain state-dependent sensory responses across the mouse cortex.

Sensory information must be integrated across a distributed brain network for stimulus processing and perception. Recent studies have revealed specific spatiotemporal patterns of cortical activation for the early and late components of sensory-evoked responses, which are associated with stimulus features and perception, respectively. Here, we investigated how the brain state influences the sensory-evoked activation across the mouse cortex. We utilized isoflurane to modulate the brain state and conducted wide-field calcium imaging of Thy1-GCaMP6f mice to monitor distributed activation evoked by multi-whisker stimulation. Our findings reveal that the level of anesthesia strongly shapes the spatiotemporal features and the functional connectivity of the sensory-activated network. As anesthesia levels decrease, we observe increasingly complex responses, accompanied by the emergence of the late component within the sensory-evoked response. The persistence of the late component under anesthesia raises new questions regarding the potential existence of perception during unconscious states.

A modular and adaptable analysis pipeline to compare slow cerebral rhythms across heterogeneous datasets.

Neuroscience is moving toward a more integrative discipline where understanding brain function requires consolidating the accumulated evidence seen across experiments, species, and measurement techniques. A remaining challenge on that path is integrating such heterogeneous data into analysis workflows such that consistent and comparable conclusions can be distilled as an experimental basis for models and theories. Here, we propose a solution in the context of slow-wave activity (<1 Hz), which occurs during unconscious brain states like sleep and general anesthesia and is observed across diverse experimental approaches. We address the issue of integrating and comparing heterogeneous data by conceptualizing a general pipeline design that is adaptable to a variety of inputs and applications. Furthermore, we present the Collaborative Brain Wave Analysis Pipeline (Cobrawap) as a concrete, reusable software implementation to perform broad, detailed, and rigorous comparisons of slow-wave characteristics across multiple, openly available electrocorticography (ECoG) and calcium imaging datasets.

Acute ischemic STROKE - from laboratory to the Patient's BED (STROKELABED): A translational approach to reperfusion injury. Study Protocol.

Cerebral edema (CE) and hemorrhagic transformation (HT) are frequent and unpredictable events in patients with acute ischemic stroke (AIS), even when an effective vessel recanalization has been achieved. These complications, related to blood-brain barrier (BBB) disruption, remain difficult to prevent or treat and may offset the beneficial effect of recanalization, and lead to poor outcomes. The aim of this translational study is to evaluate the association of circulating and imaging biomarkers with subsequent CE and HT in stroke patients with the dual purpose of investigating possible predictors as well as molecular dynamics underpinning those events and functional outcomes. Concurrently, the preclinical study will develop a new mouse model of middle cerebral artery (MCA) occlusion and recanalization to explore BBB alterations and their potentially harmful effects on tissue. The clinical section of the study is based on a single-center observational design enrolling consecutive patients with AIS in the anterior circulation territory, treated with recanalization therapies from October 1, 2015 to May 31, 2020. The study will employ an innovative evaluation of routine CT scans: in fact, we will assess and quantify the presence of CE and HT after stroke in CT scans at 24 h, through the quantification of anatomical distortion (AD), a measure of CE and HT. We will investigate the relationship of AD and several blood biomarkers of inflammation and extracellular matrix, with functional outcomes at 3 months. In parallel, we will employ a newly developed mouse model of stroke and recanalization, to investigate the emergence of BBB changes 24 h after the stroke onset. The close interaction between clinical and preclinical research can enhance our understanding of findings from each branch of research, enabling a deeper interpretation of the underlying mechanisms of reperfusion injury following recanalization treatment for AIS.

Transportable hyperspectral imaging setup based on fast, high-density spectral scanning for in situ quantitative biochemical mapping of fresh tissue biopsies.

Significance: Histopathological examination of surgical biopsies, such as in glioma and glioblastoma resection, is hindered in current clinical practice by the long time required for the laboratory analysis and pathological screening, typically taking several days or even weeks to be completed. Aim: We propose here a transportable, high-density, spectral scanning-based hyperspectral imaging (HSI) setup, named HyperProbe1, that can provide in situ, fast biochemical analysis, and mapping of fresh surgical tissue samples, right after excision, and without the need for fixing, staining nor compromising the integrity of the tissue properties. Approach: HyperProbe1 is based on spectral scanning via supercontinuum laser illumination filtered with acousto-optic tunable filters. Such methodology allows the user to select any number and type of wavelength bands in the visible and near-infrared range between 510 and 900 nm (up to a maximum of 79) and to reconstruct 3D hypercubes composed of high-resolution (4 to 5 µ m ), widefield images ( 0.9 × 0.9 mm 2 ) of the surgical samples, where each pixel is associated with a complete spectrum. Results: The HyperProbe1 setup is here presented and characterized. The system is applied to 11 fresh surgical biopsies of glioma from routine patients, including different grades of tumor classification. Quantitative analysis of the composition of the tissue is performed via fast spectral unmixing to reconstruct the mapping of major biomarkers, such as oxy-( HbO 2 ) and deoxyhemoglobin (HHb), as well as cytochrome-c-oxidase (CCO). We also provided a preliminary attempt to infer tumor classification based on differences in composition in the samples, suggesting the possibility of using lipid content and differential CCO concentrations to distinguish between lower and higher-grade gliomas. Conclusions: A proof of concept of the performances of HyperProbe1 for quantitative, biochemical mapping of surgical biopsies is demonstrated, paving the way for improving current post-surgical, histopathological practice via non-destructive, in situ streamlined screening of fresh tissue samples in a matter of minutes after excision.

In Vivo Imaging of the Structural Plasticity of Cortical Neurons After Stroke.

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.

Dysplasia and tumor discrimination in brain tissues by combined fluorescence, Raman, and diffuse reflectance spectroscopies.

Identification of neoplastic and dysplastic brain tissues is of paramount importance for improving the outcomes of neurosurgical procedures. This study explores the combined application of fluorescence, Raman and diffuse reflectance spectroscopies for the detection and classification of brain tumor and cortical dysplasia with a label-free modality. Multivariate analysis was performed to evaluate classification accuracies of these techniques-employed both in individual and multimodal configuration-obtaining high sensitivity and specificity. In particular, the proposed multimodal approach allowed discriminating tumor/dysplastic tissues against control tissue with 91%/86% sensitivity and 100%/100% specificity, respectively, whereas tumor from dysplastic tissues were discriminated with 89% sensitivity and 86% specificity. Hence, multimodal optical spectroscopy allows reliably differentiating these pathologies using a non-invasive, label-free approach that is faster than the gold standard technique and does not require any tissue processing, offering the potential for the clinical translation of the technology.

Fiber enhancement and 3D orientation analysis in label-free two-photon fluorescence microscopy.

Fluorescence microscopy can be exploited for evaluating the brain's fiber architecture with unsurpassed spatial resolution in combination with different tissue preparation and staining protocols. Differently from state-of-the-art polarimetry-based neuroimaging modalities, the quantification of fiber tract orientations from fluorescence microscopy volume images entails the application of specific image processing techniques, such as Fourier or structure tensor analysis. These, however, may lead to unreliable outcomes as they do not isolate myelinated fibers from the surrounding tissue. In this work, we describe a novel image processing pipeline that enables the computation of accurate 3D fiber orientation maps from both grey and white matter regions, exploiting the selective multiscale enhancement of tubular structures of varying diameters provided by a 3D implementation of the Frangi filter. The developed software tool can efficiently generate orientation distribution function maps at arbitrary spatial scales which may support the histological validation of modern diffusion-weighted magnetic resonance imaging tractography. Despite being tested here on two-photon scanning fluorescence microscopy images, acquired from tissue samples treated with a label-free technique enhancing the autofluorescence of myelinated fibers, the presented pipeline was developed to be employed on all types of 3D fluorescence images and fiber staining.