The Deubiquitinase USP2 Modulates Photic Entrainment of the Circadian Clock at the Level of the Suprachiasmatic Nucleus.

Ubiquitin-specific peptidase 2 (USP2) is a deubiquitinase (DUB) with a diversity of functions in physiology. One of these functions is the regulation of circadian rhythms, which are physiological rhythms with a period of ~24 h. Previous studies have indicated a role for USP2 in photic entrainment, the process by which circadian clocks synchronize to environmental light cues.

Surface-Enhanced Raman Scattering Nanoendoscope for Quantification of a Protein Released under Physiological Stimulation in Brain Tissue.

A surface-enhanced Raman scattering (SERS) biosensor with minimal invasiveness and high spatial resolution has been developed as a nanoendoscope to detect changes in protein concentrations at specific sites in biological tissues. While generally applicable to various tissues or proteins, the SERS nanoendoscope is demonstrated for the quantitative detection of S100β, an astrocytic protein whose plasmatic levels are known to vary in several neuropathologies such as Alzheimer's disease, schizophrenia, Down syndrome, Parkinson's disease and epilepsy, but for which intratissular levels have not been locally monitored, demonstrating key attributes of the SERS nanoendoscope. The SERS nanoendoscope is fabricated with densely and well-dispersed deposited gold nanoparticles modified with anti-S100β primary antibody on pulled optical fibers with a tip diameter of 700 nm, conducive to noninvasive and regiospecific detection of the S100β protein in different regions of mouse brain slices under different physiological stimuli with micrometer resolution.

Distal activity patterns shape the spatial specificity of neurovascular coupling.

Neurovascular coupling links brain activity to local changes in blood flow, forming the basis for non-invasive brain mapping. Using multiscale imaging, we investigated how vascular activity spatially relates to neuronal activity elicited by single whiskers across different columns and layers of mouse cortex. Here we show that mesoscopic hemodynamic signals quantitatively reflect neuronal activity across space but are composed of a highly heterogeneous pattern of responses across individual vessel segments that is poorly predicted by local neuronal activity.

Pannexin-1 opening in neuronal edema causes cell death but also leads to protection via increased microglia contacts.

Neuronal swelling during cytotoxic edema is triggered by Na and Cl entry and is Ca independent. However, the causes of neuronal death during swelling are unknown. Here, we investigate the role of large-conductance Pannexin-1 (Panx1) channels in neuronal death during cytotoxic edema.

Phase Aberration Correction for In Vivo Ultrasound Localization Microscopy Using a Spatiotemporal Complex-Valued Neural Network.

Ultrasound Localization Microscopy (ULM) can map microvessels at a resolution of a few micrometers ( [Formula: see text]). Transcranial ULM remains challenging in presence of aberrations caused by the skull, which lead to localization errors. Herein, we propose a deep learning approach based on recently introduced complex-valued convolutional neural networks (CV-CNNs) to retrieve the aberration function, which can then be used to form enhanced images using standard delay-and-sum beamforming.

Orai, RyR, and IPR channels cooperatively regulate calcium signaling in brain mid-capillary pericytes.

Pericytes are multifunctional cells of the vasculature that are vital to brain homeostasis, yet many of their fundamental physiological properties, such as Ca signaling pathways, remain unexplored. We performed pharmacological and ion substitution experiments to investigate the mechanisms underlying pericyte Ca signaling in acute cortical brain slices of PDGFRβ-Cre::GCaMP6f mice. We report that mid-capillary pericyte Ca signalling differs from ensheathing type pericytes in that it is largely independent of L- and T-type voltage-gated calcium channels.

Diversity of neurovascular coupling dynamics along vascular arbors in layer II/III somatosensory cortex.

The spatial-temporal sequence of cerebral blood flow (CBF), cerebral blood volume (CBV) and blood velocity changes triggered by neuronal activation is critical for understanding functional brain imaging. This sequence follows a stereotypic pattern of changes across different zones of the vasculature in the olfactory bulb, the first relay of olfaction. However, in the cerebral cortex, where most human brain mapping studies are performed, the timing of activity evoked vascular events remains controversial.

Transfer functions linking neural calcium to single voxel functional ultrasound signal.

Functional ultrasound imaging (fUS) is an emerging technique that detects changes of cerebral blood volume triggered by brain activation. Here, we investigate the extent to which fUS faithfully reports local neuronal activation by combining fUS and two-photon microscopy (2PM) in a co-registered single voxel brain volume. Using a machine-learning approach, we compute and validate transfer functions between dendritic calcium signals of specific neurons and vascular signals measured at both microscopic (2PM) and mesoscopic (fUS) levels.

Perivascular spaces in the brain: anatomy, physiology and pathology.

Perivascular spaces include a variety of passageways around arterioles, capillaries and venules in the brain, along which a range of substances can move. Although perivascular spaces were first identified over 150 years ago, they have come to prominence recently owing to advances in knowledge of their roles in clearance of interstitial fluid and waste from the brain, particularly during sleep, and in the pathogenesis of small vessel disease, Alzheimer disease and other neurodegenerative and inflammatory disorders. Experimental advances have facilitated in vivo studies of perivascular space function in intact rodent models during wakefulness and sleep, and MRI in humans has enabled perivascular space morphology to be related to cognitive function, vascular risk factors, vascular and neurodegenerative brain lesions, sleep patterns and cerebral haemodynamics.

In vivo imaging with a water immersion objective affects brain temperature, blood flow and oxygenation.

Previously, we reported the first oxygen partial pressure (Po2) measurements in the brain of awake mice, by performing two-photon phosphorescence lifetime microscopy at micrometer resolution (Lyons et al., 2016). However, this study disregarded that imaging through a cranial window lowers brain temperature, an effect capable of affecting cerebral blood flow, the properties of the oxygen sensors and thus Po2 measurements.

Mesoscopic and microscopic imaging of sensory responses in the same animal.

Imaging based on blood flow dynamics is widely used to study sensory processing. Here we investigated the extent to which local neuronal and capillary responses (two-photon microscopy) are correlated to mesoscopic responses detected with fast ultrasound (fUS) and BOLD-fMRI. Using a specialized chronic olfactory bulb preparation, we report that sequential imaging of the same mouse allows quantitative comparison of odour responses, imaged at both microscopic and mesoscopic scales.

Vascular Compartmentalization of Functional Hyperemia from the Synapse to the Pia.

Functional hyperemia, a regional increase of blood flow triggered by local neural activation, is used to map brain activity in health and disease. However, the spatial-temporal dynamics of functional hyperemia remain unclear. Two-photon imaging of the entire vascular arbor in NG2-creERT2;GCaMP6f mice shows that local synaptic activation, measured via oligodendrocyte precursor cell (OPC) Ca signaling, generates a synchronous Ca drop in pericytes and smooth muscle cells (SMCs) enwrapping all upstream vessels feeding the activated synapses.

Ca 3.2 drives sustained burst-firing, which is critical for absence seizure propagation in reticular thalamic neurons.

Objective: Genetic alterations have been identified in the CACNA1H gene, encoding the Ca 3.2 T-type calcium channel in patients with absence epilepsy, yet the precise mechanisms relating to seizure propagation and spike-wave-discharge (SWD) pacemaking remain unknown. Neurons of the thalamic reticular nucleus (TRN) express high levels of Ca 3.

Light controls cerebral blood flow in naive animals.

Optogenetics is increasingly used to map brain activation using techniques that rely on functional hyperaemia, such as opto-fMRI. Here we test whether light stimulation protocols similar to those commonly used in opto-fMRI or to study neurovascular coupling modulate blood flow in mice that do not express light sensitive proteins. Combining two-photon laser scanning microscopy and ultrafast functional ultrasound imaging, we report that in the naive mouse brain, light per se causes a calcium decrease in arteriolar smooth muscle cells, leading to pronounced vasodilation, without excitation of neurons and astrocytes.

Ca transients in astrocyte fine processes occur via Ca influx in the adult mouse hippocampus.

Astrocytes display complex morphologies with an array of fine extensions extending from the soma and the primary thick processes. Until the use of genetically encoded calcium indicators (GECIs) selectively expressed in astrocytes, Ca signaling was only examined in soma and thick primary processes of astrocytes where Ca -sensitive fluorescent dyes could be imaged. GECI imaging in astrocytes revealed a previously unsuspected pattern of spontaneous Ca transients in fine processes that has not been observed without chronic expression of GECIs, raising potential concerns about the effects of GECI expression.

The cellular mechanisms of neuronal swelling underlying cytotoxic edema.

Cytotoxic brain edema triggered by neuronal swelling is the chief cause of mortality following brain trauma and cerebral infarct. Using fluorescence lifetime imaging to analyze contributions of intracellular ionic changes in brain slices, we find that intense Na(+) entry triggers a secondary increase in intracellular Cl(-) that is required for neuronal swelling and death. Pharmacological and siRNA-mediated knockdown screening identified the ion exchanger SLC26A11 unexpectedly acting as a voltage-gated Cl(-) channel that is activated upon neuronal depolarization to membrane potentials lower than -20 mV.

Activation of neuronal NMDA receptors triggers transient ATP-mediated microglial process outgrowth.

Microglia are morphologically dynamic cells that rapidly extend their processes in response to various stimuli including extracellular ATP. In this study, we tested the hypothesis that stimulation of neuronal NMDARs trigger ATP release leading to communication with microglia. We used acute mouse hippocampal brain slices and two-photon laser scanning microscopy to study microglial dynamics and developed a novel protocol for fixation and immunolabeling of microglia processes.

Lipid Nanoparticle Delivery of siRNA to Silence Neuronal Gene Expression in the Brain.

Manipulation of gene expression in the brain is fundamental for understanding the function of proteins involved in neuronal processes. In this article, we show a method for using small interfering RNA (siRNA) in lipid nanoparticles (LNPs) to efficiently silence neuronal gene expression in cell culture and in the brain in vivo through intracranial injection. We show that neurons accumulate these LNPs in an apolipoprotein E-dependent fashion, resulting in very efficient uptake in cell culture (100%) with little apparent toxicity.

Regenerative glutamate release by presynaptic NMDA receptors contributes to spreading depression.

Spreading depression (SD) is a slowly propagating neuronal depolarization that underlies certain neurologic conditions. The wave-like pattern of its propagation suggests that SD arises from an unusual form of neuronal communication. We used enzyme-based glutamate electrodes to show that during SD induced by transiently raising extracellular K(+) concentrations ([K(+)]o) in rat brain slices, there was a rapid increase in the extracellular glutamate concentration that required vesicular exocytosis but unlike fast synaptic transmission, still occurred when voltage-gated sodium and calcium channels (VGSC and VGCC) were blocked.

Metabolic communication between astrocytes and neurons via bicarbonate-responsive soluble adenylyl cyclase.

Astrocytes are proposed to participate in brain energy metabolism by supplying substrates to neurons from their glycogen stores and from glycolysis. However, the molecules involved in metabolic sensing and the molecular pathways responsible for metabolic coupling between different cell types in the brain are not fully understood. Here we show that a recently cloned bicarbonate (HCO₃⁻) sensor, soluble adenylyl cyclase (sAC), is highly expressed in astrocytes and becomes activated in response to HCO₃⁻ entry via the electrogenic NaHCO₃ cotransporter (NBC).

Contribution of calcium-dependent facilitation to synaptic plasticity revealed by migraine mutations in the P/Q-type calcium channel.

The dynamics, computational power, and strength of neural circuits are essential for encoding and processing information in the CNS and rely on short and long forms of synaptic plasticity. In a model system, residual calcium (Ca(2+)) in presynaptic terminals can act through neuronal Ca(2+) sensor proteins to cause Ca(2+)-dependent facilitation (CDF) of P/Q-type channels and induce short-term synaptic facilitation. However, whether this is a general mechanism of plasticity at intact central synapses and whether mutations associated with human disease affect this process have not been described to our knowledge.

Activation of pannexin-1 hemichannels augments aberrant bursting in the hippocampus.

Pannexin-1 (Px1) is expressed at postsynaptic sites in pyramidal neurons, suggesting that these hemichannels contribute to dendritic signals associated with synaptic function. We found that, in pyramidal neurons, N-methyl-d-aspartate receptor (NMDAR) activation induced a secondary prolonged current and dye flux that were blocked with a specific inhibitory peptide against Px1 hemichannels; knockdown of Px1 by RNA interference blocked the current in cultured neurons. Enhancing endogenous NMDAR activation in brain slices by removing external magnesium ions (Mg2+) triggered epileptiform activity, which had decreased spike amplitude and prolonged interburst interval during application of the Px1 hemichannel blocking peptide.

Brain metabolism dictates the polarity of astrocyte control over arterioles.

Calcium signalling in astrocytes couples changes in neural activity to alterations in cerebral blood flow by eliciting vasoconstriction or vasodilation of arterioles. However, the mechanism for how these opposite astrocyte influences provide appropriate changes in vessel tone within an environment that has dynamic metabolic requirements remains unclear. Here we show that the ability of astrocytes to induce vasodilations over vasoconstrictions relies on the metabolic state of the rat brain tissue.