34 research outputs found
Towards a comprehensive understanding of brain machinery by correlative microscopy.
Unraveling the complexity of brain structure and function is the biggest challenge of contemporary science. Due to their flexibility, optical techniques are the key to exploring this intricate network. However, a single imaging technique can reveal only a small part of this machinery due to its inherent multilevel organization. To obtain a more comprehensive view of brain functionality, complementary approaches have been combined. For instance, brain activity was monitored simultaneously on different spatiotemporal scales with functional magnetic resonance imaging and calcium imaging. On the other hand, dynamic information on the structural plasticity of neuronal networks has been contextualized in a wider framework combining two-photon and light-sheet microscopy. Finally, synaptic features have been revealed on previously in vivo imaged samples by correlative light-electron microscopy. Although these approaches have revealed important features of brain machinery, they provided small bridges between specific spatiotemporal scales, lacking an omni-comprehensive view. In this perspective, we briefly review the state of the art of correlative techniques and propose a wider methodological framework fusing multiple levels of brain investigation
Multi-Photon Nanosurgery in Live Brain
In the last few years two-photon microscopy has been used to perform in vivo high spatial resolution imaging of neurons, glial cells and vascular structures in the intact neocortex. Recently, in parallel to its applications in imaging, multi-photon absorption has been used as a tool for the selective disruption of neural processes and blood vessels in living animals. In this review we present some basic features of multi-photon nanosurgery and we illustrate the advantages offered by this novel methodology in neuroscience research. We show how the spatial localization of multi-photon excitation can be exploited to perform selective lesions on cortical neurons in living mice expressing fluorescent proteins. This methodology is applied to disrupt a single neuron without causing any visible collateral damage to the surrounding structures. The spatial precision of this method allows to dissect single processes as well as individual dendritic spines, preserving the structural integrity of the main neuronal arbor. The same approach can be used to breach the blood-brain barrier through a targeted photo-disruption of blood vessels walls. We show how the vascular system can be perturbed through laser ablation leading toward two different models of stroke: intravascular clot and extravasation. Following the temporal evolution of the injured system (either a neuron or a blood vessel) through time lapse in vivo imaging, the physiological response of the target structure and the rearrangement of the surrounding area can be characterized. Multi-photon nanosurgery in live brain represents a useful tool to produce different models of neurodegenerative disease
ADVANCED OPTICAL TECHNIQUES TO EXPLORE BRAIN STRUCTURE AND FUNCTION
Understanding brain structure and function, and the complex relationships between them, is one of the grand challenges of contemporary sciences. Thanks to their flexibility, optical techniques could be the key to explore this complex network. In this manuscript, we briefly review recent advancements in optical methods applied to three main issues: anatomy, plasticity and functionality. We describe novel implementations of light-sheet microscopy to resolve neuronal anatomy in whole fixed brains with cellular resolution. Moving to living samples, we show how real-time dynamics of brain rewiring can be visualized through two-photon microscopy with the spatial resolution of single synaptic contacts. The plasticity of the injured brain can also be dissected through cutting-edge optical methods that specifically ablate single neuronal processes. Finally, we report how nonlinear microscopy in combination with novel voltage sensitive dyes allow optical registrations of action potential across a population of neurons opening promising prospective in understanding brain functionality. The knowledge acquired from these complementary optical methods may provide a deeper comprehension of the brain and of its unique features
Whole-brain vasculature reconstruction at the single capillary level
The distinct organization of the brain’s vascular network ensures that it is adequately supplied with oxygen and nutrients. However, despite this fundamental role, a detailed reconstruction of the brain-wide vasculature at the capillary level remains elusive, due to insufficient image quality using the best available techniques. Here, we demonstrate a novel approach that improves vascular demarcation by combining CLARITY with a vascular staining approach that can fill the entire blood vessel lumen and imaging with light-sheet fluorescence microscopy. This method significantly improves image contrast, particularly in depth, thereby allowing reliable application of automatic segmentation algorithms, which play an increasingly important role in high-throughput imaging of the terabyte-sized datasets now routinely produced. Furthermore, our novel method is compatible with endogenous fluorescence, thus allowing simultaneous investigations of vasculature and genetically targeted neurons. We believe our new method will be valuable for future brain-wide investigations of the capillary network
Latency correction in sparse neuronal spike trains
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, 9 figure
Whole-brain vasculature reconstruction at the single capillary level
The distinct organization of the brain’s vascular network ensures that it is adequately supplied with oxygen and nutrients. However, despite this fundamental role, a detailed reconstruction of the brain-wide vasculature at the capillary level remains elusive, due to insufficient image quality using the best available techniques. Here, we demonstrate a novel approach that improves vascular demarcation by combining CLARITY with a vascular staining approach that can fill the entire blood vessel lumen and imaging with light-sheet fluorescence microscopy. This method significantly improves image contrast, particularly in depth, thereby allowing reliable application of automatic segmentation algorithms, which play an increasingly important role in high-throughput imaging of the terabyte-sized datasets now routinely produced. Furthermore, our novel method is compatible with endogenous fluorescence, thus allowing simultaneous investigations of vasculature and genetically targeted neurons. We believe our new method will be valuable for future brain-wide investigations of the capillary network
A versatile clearing agent for multi-modal brain imaging
Extensive mapping of neuronal connections in the central nervous system
requires high-throughput um-scale imaging of large volumes. In recent years,
different approaches have been developed to overcome the limitations due to
tissue light scattering. These methods are generally developed to improve the
performance of a specific imaging modality, thus limiting comprehensive
neuroanatomical exploration by multimodal optical techniques. Here, we
introduce a versatile brain clearing agent (2,2'-thiodiethanol; TDE) suitable
for various applications and imaging techniques. TDE is cost-efficient,
water-soluble and low-viscous and, more importantly, it preserves fluorescence,
is compatible with immunostaining and does not cause deformations at
sub-cellular level. We demonstrate the effectiveness of this method in
different applications: in fixed samples by imaging a whole mouse hippocampus
with serial two-photon tomography; in combination with CLARITY by
reconstructing an entire mouse brain with light sheet microscopy and in
translational research by imaging immunostained human dysplastic brain tissue.Comment: in Scientific Reports 201
Cobrawap: A pipeline for the analysis of wave activity at different brain states
Plenary talk at "WP2 Meeting: Networks underlying consciousness and cognition" held in Barcelona, Spain, from 19 to 21 June, 2023.Progetto EBRAINS-Italy IR00011, CUP B51E2200015006,Missione 4 - Istruzione e Ricerca, Componente 2, Azione 3.1.1
Funded by EU