Two-photon microscopy

…Here, we used two-photon microscopy to image Ca2+ activity from hundreds of neurons and while delivering brief, focal excitatory input with near-cellular- resolution optogenetics. Specifically, we compared how the primary visual (V1) and the secondary motor (M2) cortices distinctly sustain this input while mice ran spontaneously. Single-neuron responses to focal stimulation were much more prevalent in M2 than V1. Moreover, evoked network-level dynamic

The navigational behavior of many animals relies on internal representations of space. These representations are often encoded in a world-centric (allocentric) reference frame. To use these representations to guide behavior, animals must transform them to a body-centric (egocentric) reference frame. We took advantage of the experimental tractability of Drosophila melanogaster to determine how neurons in its brain perform this computation. In the fly brain, directions are encoded as vectors in an allocentric reference frame by neurons in a region called the central complex. We investigated whet

Studying blood microcirculation is crucial for understanding vascular diseases, yet current imaging methods involving acute administration of fluorescent dyes are invasive and short-lived. To address this limitation, we present here two novel approaches for longitudinal vasculature study. Given that albumin constitutes ~60% of plasma protein and is​​​​ produced by liver hepatocytes, we developed adeno-associated viral vectors (AAVs) with the hepatocyte-specific P3 optimized transthyretin promoter to express the albumin-mNeonGreen or albumin-mScarlet fusion protein. These AAVs were packaged wit

Micro-coils were designed to selectively activate pyramidal neurons (PNs), avoiding axonal stimulation, and potentially overcoming the limitations of conventional electric stimulation[1][2]. Finite element simulations showed a significant impact of micro-coil design on neuron activation selectivity[3]. A V-shaped bend at the micro-coil tip-enhanced field gradient selectivity vertically, leading to spatially confined cortical activation. In previous in vivo experiments, extracellular electrodes were necessary for recording neuronal action potentials, potentially increasing brain tissue damage[4

The formation of new memories requires the hippocampus, a brain region which exhibits high levels of structural plasticity. Within the dorsal hippocampal CA1, specific interneuron subtypes synapse onto specific subcellular compartments of Pyramidal Neurons (PNs) such as dendrites, soma and axon initial segment (AIS) thus shaping the cellular computations underlying memory formation. Yet, very little is known about the long-term structural dynamics of these compartmentalized inhibitory synapses on PNs and their link to learning.To address these outstanding questions, we used longitudinal in viv

…5xFAD mice at 4-5 months of age with two-photon microscopy, ch

Alzheimer's Disease (AD) is the primary cause of dementia, characterized by irreversible neurodegeneration resulting in memory loss and cognitive decline. Growing evidence suggests that neuroinflammation plays a pivotal role in AD, with extracellular ATP (eATP) as one of the key players in this process. Our project aims at investigating whether eATP influences microglia activation, AD-associated neuroinflammation and neuronal Ca2+ excitability in an AD mouse model (B6.152H) expressing human PS2-N141I and APP Swedish mutations. We conducted experiments at various disease stages: pre- and post-A

Fluorescent drug conjugates enable the selective visualization of proteins in cell models, primary cultures, and tissue preparations. These compounds have become valuable tools in neuroscience in investigating the expression and function of ion channels, receptors, and transporters. In this project, we explored the utility of fluorescent drug conjugates in visualizing the serotonin transporter (SERT) in the brain. We made use of a recently developed class of fluorescent compounds called PyrAtes which offer interesting chemical properties, photostability, and large Stokes shifts. Herein, two co

Spontaneous neuronal activity (SNA) plays an important role in the maturation and refinement of neuronal connections. However, neurodegenerative diseases, like tauopathies, can significantly alter SNA. Changes in SNA after the expression of tau mutations have not yet been analysed at presymptomatic stages. Furthermore, the currently available models lack the necessary complexity of the human brain to fully understand the underlying mechanisms of tau pathology and its effects on SNA. To address this, we present a novel in vitro platform utilizing cortical organoids (COs), derived from hPSCs, to

…to examine changes in [Cl-]i using two-photon microscopy in brain slices. Depending on the binding of chloride, with this sensor, Fluorescence Resonance Energy Transfer (FRET) occurs

In this research, the primary focus was on the development and characterization of a drug delivery system, that integrates micro electrocorticography (ECoG) arrays with upconverting nanoparticles (UCNP) to create a surface-modified, light-triggerable nanosystem for nervous tissue application. Methodology involves using a soft ECoG with a silicone substrate and Pt/Ir measuring sites. First the electrode surface is exposed to oxygen plasma then azide groups bind to it. Simultaneously, the UCNP exterior is modified with an aminosilane layer then a BCN-NHS linker compound. This linker attaches the

…Using in vivo two-photon microscopy we found that ChABC treatment increased microglial branching and ECM phagocytic capacity and decreased spine elimination rate under basal conditions. Moreover, ECM attenuation largely prevented sy

Dendrites play a fundamental role in neuronal computation. It is thought that functionally defined regions of dendritic branches, termed ‘dendritic computational units’ are used to decode information transmitted by synaptic input. This information is encoded by the corresponding dendritic output, to perform complex neuronal computations. To investigate dendric input-output in vivo, it is necessary to record synaptic input and dendritic output simultaneously. To do this we perform simultaneous glutamate imaging and voltage imaging from the spiny dendrites of single Purkinje neurons in awake mic

Visual processing is the ability of the brain to analyze and interpret visual information received from the eyes, and it is extensively studied in rodent primary visual cortex (V1) by revealing the relationship between neural circuitry and its functional behaviour. Within the V1, binocular neurons are crucial for stereopsis establishment and for various innate behaviours including navigation and predation. The development of binocular neurons requires vision stimulation since opening of eyes to reconstruct and modify the neural circuit according to the receptive field tunings. However, this bi

In the realm of memory, environmental lighting exerts significant influence, impacting various cognitive processes such as fear and object memory. Within this context, our laboratory has previously conducted research demonstrating that brief exposure to light adversely affects social recognition memory (SRM) in mice. Specifically, we found that light exposure hampers the activity of oxytocin neurons located in the supraoptic nucleus (SON) through a pathway involving M1 SON-projecting intrinsically photosensitive retinal ganglion cells (ipRGCs) and GABAergic neurons situated in the perinuclear

The basolateral amygdala (BLA), traditionally known for its role in associative fear learning, may also play a role in self-initiated, incentive-motivated behaviors. However, it is unclear how the BLA contributes to learning to initiate neutral actions for a positive outcome. In particular, while the mouse secondary motor cortex (M2), a key region involved in spontaneous action initiation, is a major target of BLA glutamatergic output, it is unknown whether and how BLA-M2 communication is involved in the self-initiation of incentive-motivated actions. To address these questions, we trained hea

…Two-photon microscopy can provide exact localization and immediate identification of cell types. Our aim was to develop and test an imaging modality that is capable of deep-tissue long-term two-photon imaging while being light and compact for a mouse to carry while freely moving in its environment.Methods. All experimental procedures were approved by the French ethics committee (APAFiS #30959) and were con

Progress in brain research is widely based on experimental approaches having the potential to advance the knowledge on fundamental biology. In particular, the increasing availability of data obtained by experimental techniques has boosted the development of data-driven mathematical models reproducing neuronal circuits. While cellular and subcellular resolution data have been extensively collected in animal models, experimental constraints have limited the acquisition of information about human neuronal cytoarchitectures and morphologies. In this context, high resolution data can be efficiently

Proprioception is the sensation of the position and movement of the body in space. Forelimb proprioceptive afferents not only drive reflexes circuits in the spinal cord, but also ascend to the brain via projections to the external cuneate nucleus (Ecu) in the brainstem. Secondary neurons in the Ecu relay the incoming information via two major pathways: 1) the cuneo-cerebellar track which innervates the cerebellum and allows movement adaptation to unexpected perturbations and 2) the dorsal column-medial lemniscus pathway targeting the somatosensory cortex and allowing for the perception of prop

Neurons and glial cells are surrounded by interstitial fluid (ISF), which primarily originates from cerebrospinal fluid (CSF). The fluid dynamics of ISF and CSF have been examined by cisterna magna injection of fluid tracers such as fluorescent dextran or contrast agents for magnetic resonance imaging. While this approach has unveiled major CSF flow dynamics in the recent decade, cisterna magna injection introduces transient intracranial pressure changes that can lead to experimental variability. In this study, we attempted to label the CSF/ISF via endogenously expressed secretory fluorescent

Multi-electrode stimulation aims to induce sophisticated brain perception by eliciting specific spatial and temporal patterns of neural activity corresponding to sensory perceptions[1][2][3]. However, conventional implanted microelectrode stimulation often results in large-scale brain responses, from a few hundred microns to a few millimeters[2]. This spread of signals may lead to complex signal interactions and integration across adjacent electrodes.In our study, we positioned two monopolar micro-electrodes at various spacings ranging from 400 to 800µm between them, and simultaneously stimula

Recent studies have revealed an essential contribution of cortical astrocyte calcium activity in regulation of sleep depth and state transitions (Bojarskaite et al., 2020, Nat Comms; Vaidyanathan et al., 2021, Nat Comms). However, astrocytic activity is highly heterogeneous across brain regions, and it is unclear if astrocytes mediate sleep transitions in a brain region specific manner. A region of utmost interest to investigate astrocytic activity during sleep is the hippocampus, which not only display high astrocytic heterogeneity (Viana et al., 2023, Glia), but also shows sleep-specific pat

Liver-secreted albumin is the most abundant protein in blood plasma and cerebrospinal fluid. We have previously developed liver-targeting adeno-associated viral vectors (AAV8-P3-*) that express fluorescent protein-tagged albumin to visualize blood plasma in adult mice (DOI: 10.1016/j.crmeth.2022.100302). We have also established a virally induced CRISPR/Cas9-based knock-in of green fluorescent albumin in neonatal mice (DOI: 10.1101/2023.07.10.548084). Here, we have generated a new transgenic mouse model by CRISPR/Cas9 in which the bright red fluorescent protein mScarlet is knocked into the alb

Proprioception enables an organism to sense the position and motion of its limbs and body in space. Comparative evolutionary studies have proposed that the organization of the proprioceptive cortex does not emerge as a consequence of innervation density but rather as a result of the actual use of a limb. We tested this hypothesis by quantifying if and how the proprioceptive cortical representation reorganizes after skilled motor training in adult mice as well as enriched environmental exposure during development.We performed wide-filed calcium imaging of cortical activation in response to mult