Imaging Of Brain Slices Research Articles

High-throughput high-dynamic range imaging by spatiotemporally structured illumination

Recent advances in biochemistry and optics have enabled observation of the faintest signals from even single molecules. However, although biological samples can have varying degrees of fluorescence expression ranging from a single to thousands of fluorescent molecules in an observation volume, the detection range is fundamentally limited by the dynamic range (DR) of current detectors. In other words, for many biological systems where faint and strong signal sources coexist, traditional imaging methods make a compromise and end up choosing a limited target signal range to be quantitatively measured while other signal levels are either lost beneath the background noise or saturated. The DR can be extended by taking multiple images with varying exposures, which, however, severely restricts data throughput. To overcome this limitation, we introduce structured illumination high dynamic range (SI-HDR) imaging, which enables real-time HDR imaging with a single measurement. We demonstrate the wide and easy applicability of the method by realizing various applications, such as high throughput gigapixel imaging of mouse brain slices, quantitative analysis of neuronal mitochondria structures, and fast 3D volumetric HDR imaging.

Breasi-CRISPR: an efficient genome-editing method to interrogate protein localization and protein-protein interactions in the embryonic mouse cortex.

In developing tissues, knowing the localization and interactors of proteins of interest is key to understanding their function. Here, we describe the Breasi-CRISPR approach (Brain Easi-CRISPR), combining Easi-CRISPR with in utero electroporation to tag endogenous proteins within embryonic mouse brains. Breasi-CRISPR enables knock-in of both short and long epitope tag sequences with high efficiency. We visualized epitope-tagged proteins with varied expression levels, such as ACTB, LMNB1, EMD, FMRP, NOTCH1 and RPL22. Detection was possible by immunohistochemistry as soon as 1 day after electroporation and we observed efficient gene editing in up to 50% of electroporated cells. Moreover, tagged proteins could be detected by immunoblotting in lysates from individual cortices. Next, we demonstrated that Breasi-CRISPR enables the tagging of proteins with fluorophores, allowing visualization of endogenous proteins by live imaging in organotypic brain slices. Finally, we used Breasi-CRISPR to perform co-immunoprecipitation mass-spectrometry analyses of the autism-related protein FMRP to discover its interactome in the embryonic cortex. Together, these data demonstrate that Breasi-CRISPR is a powerful tool with diverse applications that will propel the understanding of protein function in neurodevelopment.

Computational microscopy for fast widefield deep-tissue fluorescence imaging using a commercial dual-cannula probe.

A solid-glass cannula serves as a micro-endoscope that can deliver excitation light deep inside tissue while also collecting emitted fluorescence. Then, we utilize deep neural networks to reconstruct images from the collected intensity distributions. By using a commercially available dual-cannula probe, and training a separate deep neural network for each cannula, we effectively double the field of view compared to prior work. We demonstrated ex vivo imaging of fluorescent beads and brain slices and in vivo imaging from whole brains. We clearly resolved 4 μm beads, with FOV from each cannula of 0.2 mm (diameter), and produced images from a depth of ~1.2 mm in the whole brain, currently limited primarily by the labeling. Since no scanning is required, fast widefield fluorescence imaging limited primarily by the brightness of the fluorophores, collection efficiency of our system, and the frame rate of the camera becomes possible.

A new brain‐cutting device and ultraviolet resin‐mounted human brain slices as a teaching adjunct for neuroanatomy education

Although the level of neuroscience research is rapidly developing with the introduction of new technologies, the method of neuroanatomy education remains at the traditional level and requires improvement to meet the needs of educators and trainees. We developed a new three-dimensional (3D) printed device (human brain-cutting mold, HBCM) for creating human brain slices; moreover, we demonstrated a simple method for creating semi-permanent ultraviolet (UV) resin-mounted brain slice specimens for neuroanatomy education. We obtained brain slices of uniform thickness (3 mm) through the HBCM; the resultant brain slices were optimal for assessing morphological details of the human brain. Furthermore, we used an agar-embedding method for brain-slicing with the HBCM, which minimized geometrical distortions of the brain slices. Also, we prepared semi-permanent brain serial specimens using an acrylic brain slice frame and UV-curable resin, which was highly compatible with moist bio-specimens. During UV resin curing, neither air bubble formation nor color change occurred. The resultant UV resin-mounted brain slices produced definite coronal sections with high transparency and morphological accuracy. We also performed 3D modeling by stacking brain slice images that differentiated the cortical area and nine subcortical regions via manual segmentation. This method could be a reliable alternative for displaying high-quality human brain slices and would be helpful for students and trainee to understand anatomical orientation from 2D images to 3D structures. Also, this may present an innovative approach for preparing and preserving coronal sections of the normal or pathological human brain.

Ordered iterative colour channel selection enhances segmentation of brain slice image neurons of the sham fetal sheep model

• We propose a novel segmentation model to extract the densely populated overlapping cortical neurons from the background image, which is essential to rapidly qualify and quantify the brain cortical neurons from brain slice images of in utero fetal sheep model . • The ordered iterative colour channel selection (OICCS) segmentation model enhances the performance of the marker controlled watershed (MCW) K-means segmentation technique. • The OICCS model involves the iterative application of the MCW K-means segmentation method in the three colour channels (red, green, and blue) of an RGB image in a selective order. • The segmentation model is significant as it is the essential first step to accelerate the histological analysis process over the currently employed manual assessment. Hypoxic Ischemia (HI) accounts for 23% of annual worldwide neonatal death, brain damage and long-term disability. Histopathological studies of infants with HI are rare, so the assessment of neuronal survival in HI animal models compared to the neuronal survival of their sham control, such as the term in utero fetal sheep model, is pivotal to increasing our understanding of HI at the cellular level and for the development of novel therapeutics and treatments. Histopathological studies of the in utero fetal sheep model rely heavily on the manual identification of neurons in histological brain slice images. However, manual identification is prone to being highly subjective, costly and time-consuming. Thus, there is an urgent need to automate the segmentation process, which is complicated by the existence of densely populated overlapping cells with no prior information of shape or size, resulting in images of high complexity. In this article, we propose an ‘Ordered Iterative Colour Channel Selection (OICCS) method. Such that, when applied to the standard ‘Marker Controlled Watershed k-means segmentation’ (MCW k-means) method, serves to effectively segment cortical neurons from the background in brain slice images of the term equivalent sham control in utero fetal sheep model. Thus, tackling the over-segmentation issue that was encountered by MCW k-means alone. In the OICCS model, we determine that by iteratively applying MCW k-means segmentation: firstly, to the blue channel image; then again on the green channel image for cortical neurons that remained unsegmented from the blue channel segmentation; and finally again on the red channel image for cortical neurons that remained unsegmented from the green channel segmentation that optimal performance results could be achieved of 97.98% ± 0.97% over 90.65 ± 3.32% achieved by the standard MCW k-means applied to the R, G, B channel alone or other permutations of the colour channels. The new OICCS enhanced MCW k-means segmentation method presented here is a significant step in automating the segmentation process of cortical brain cells from brain slice images of the sham control fetal sheep model. This will serve to accelerate the histological segmentation process over the manually intensive identification process that is currently employed.

3D brain slice classification and feature extraction using Deformable Hierarchical Heuristic Model

Brain tumors are the most frequently occurring and severe type of cancer, with a life expectancy of only a few months in most advanced stages. As a result, planning the best course of therapy is critical to improve a patient's ability to fight cancer and their quality of life. Various imaging modalities, such as computed tomography (CT), magnetic resonance imaging (MRI) and ultrasound imaging, are commonly employed to assess a brain tumor. This research proposes a novel technique for extracting and classifying tumor features in 3D brain slice images. After input images are processed for noise removal, resizing, and smoothening, features of brain tumor are extracted using Volume of Interest (VOI). The extracted features are then classified using the Deformable Hierarchical Heuristic Model-Deep Deconvolutional Residual Network (DHHM-DDRN) based on surfaces, curves, and geometric patterns. Experimental results show that proposed approach obtained an accuracy of 95%, DSC of 83%, precision of 80%, recall of 85%, and F1 score of 55% for classifying brain cancer features.

The M1 muscarinic receptor is present in situ as a ligand-regulated mixture of monomers and oligomeric complexes

The quaternary organization of rhodopsin-like G protein-coupled receptors in native tissues is unknown. To address this we generated mice in which the M1 muscarinic acetylcholine receptor was replaced with a C-terminally monomeric enhanced green fluorescent protein (mEGFP)-linked variant. Fluorescence imaging of brain slices demonstrated appropriate regional distribution, and using both anti-M1 and anti-green fluorescent protein antisera the expressed transgene was detected in both cortex and hippocampus only as the full-length polypeptide. M1-mEGFP was expressed at levels equal to the M1 receptor in wild-type mice and was expressed throughout cell bodies and projections in cultured neurons from these animals. Signaling and behavioral studies demonstrated M1-mEGFP was fully active. Application of fluorescence intensity fluctuation spectrometry to regions of interest within M1-mEGFP-expressing neurons quantified local levels of expression and showed the receptor was present as a mixture of monomers, dimers, and higher-order oligomeric complexes. Treatment with both an agonist and an antagonist ligand promoted monomerization of the M1-mEGFP receptor. The quaternary organization of a class A G protein-coupled receptor in situ was directly quantified in neurons in this study, which answers the much-debated question of the extent and potential ligand-induced regulation of basal quaternary organization of such a receptor in native tissue when present at endogenous expression levels.

Sex Differences in CGRP Regulation and Function in the Amygdala in a Rat Model of Neuropathic Pain

The amygdala has emerged as a key player in the emotional response to pain and pain modulation. The lateral and capsular regions of the central nucleus of the amygdala (CeA) represent the “nociceptive amygdala” due to their high content of neurons that process pain-related information. These CeA divisions are the targets of the spino-parabrachio-amygdaloid pain pathway, which is the predominant source of calcitonin gene-related peptide (CGRP) within the amygdala. Changes in lateral and capsular CeA neurons have previously been observed in pain models, and synaptic plasticity in these areas has been linked to pain-related behavior. CGRP has been demonstrated to play an important role in peripheral and spinal mechanisms, and in pain-related amygdala plasticity in male rats in an acute arthritis pain model. However, the role of CGRP in chronic neuropathic pain-related amygdala function and behaviors remains to be determined for both male and female rats. Here we tested the hypothesis that the CGRP1 receptor is involved in neuropathic pain-related amygdala activity, and that blockade of this receptor can inhibit neuropathic pain behaviors in both sexes. CGRP mRNA expression levels in the CeA of male rats were upregulated at the acute stage of the spinal nerve ligation (SNL) model of neuropathic pain, whereas female rats had significantly higher CGRP and CGRP receptor component expression at the chronic stage. A CGRP1 receptor antagonist (CGRP 8-37) administered into the CeA in chronic neuropathic rats reduced mechanical hypersensitivity (von Frey and paw compression tests) in both sexes but showed female-predominant effects on emotional-affective responses (ultrasonic vocalizations) and anxiety-like behaviors (open field test). CGRP 8-37 inhibited the activity of CeA output neurons assessed with calcium imaging in brain slices from chronic neuropathic pain rats. Together, these findings may suggest that CGRP1 receptors in the CeA are involved in neuropathic pain-related amygdala activity and contribute to sensory aspects in both sexes but to emotional-affective pain responses predominantly in females. The sexually dimorphic function of CGRP in the amygdala would make CGRP1 receptors a potential therapeutic target for neuropathic pain relief, particularly in females in chronic pain conditions.

High-Speed Ultraviolet Photoacoustic Microscopy for Histological Imaging with Virtual-Staining assisted by Deep Learning.

Surgical margin analysis (SMA), an essential procedure to confirm the complete excision of cancerous tissue in tumor resection surgery, requires intraoperative diagnostic tools to avoid repeated surgeries due to a positive surgical margin. Recently, by taking the advantage of the high intrinsic optical absorption of DNA/RNA at 266 nm wavelength, ultraviolet photoacoustic microscopy (UV-PAM) has been developed to provide high-resolution histological images without labeling, showing great promise as an intraoperative tool for SMA. To enable the development of UV-PAM for SMA, here, a high-speed and open-top UV-PAM system is presented, which can be operated similarly to conventional optical microscopies. The UV-PAM system provides a high lateral resolution of 1.2 µm, and a high imaging speed of 55 kHz A-line rate with one-axis galvanometer mirror scanning. Moreover, to ensure UV-PAM images can be easily interpreted by pathologists without additional training, the original grayscale UV-PAM images are virtually stained by a deep-learning algorithm to mimic the standard hematoxylin- and eosin-stained images, enabling training-free histological analysis. Mouse brain slice imaging is performed to demonstrate the high performance of the open-top UV-PAM system, illustrating its great potential for SMA applications.

Real-time imaging of glutamate transients in the extracellular space of acute human brain slices using a single-wavelength glutamate fluorescence nanosensor

Glutamate is the most important excitatory neurotransmitter in the brain. The ability to assess glutamate release and re-uptake with high spatial and temporal resolution is crucial to understand the involvement of this primary excitatory neurotransmitter in both normal brain function and different neurological disorders. Real-time imaging of glutamate transients by fluorescent nanosensors has been accomplished in rat brain slices. We performed for the first time single-wavelength glutamate nanosensor imaging in human cortical brain slices obtained from patients who underwent epilepsy surgery. The glutamate fluorescence nanosensor signals of the electrically stimulated human cortical brain slices showed steep intensity increase followed by an exponential decrease. The spatial distribution and the time course of the signal were in good agreement with the position of the stimulation electrode and the dynamics of the electrical stimulation, respectively. Pharmacological manipulation of glutamate release and reuptake was associated with corresponding changes in the glutamate fluorescence nanosensor signals. We demonstrated that the recently developed fluorescent nanosensors for glutamate allow to detect neuronal activity in acute human cortical brain slices with high spatiotemporal precision. Future application to tissue samples from different pathologies may provide new insights into pathophysiology without the limitations of an animal model.

Imaging and analysis of neuronal mitochondria in murine acute brain slices

BackgroundMitochondrial alterations are common to many inflammatory, degenerative as well as metabolic diseases. However, due to the vulnerability of mitochondria in explanted tissue, there is a general lack of ex vivo models, especially of CNS tissue, that preserve mitochondria and allow investigation of mitochondrial dynamics. New methodsHere, we present a model of acute hippocampal slices to study neuronal mitochondria ex vivo. We used two-photon microscopy to image CFP fluorescent neuronal mitochondria in B6. Cg-Tg(Thy1-CFP/COX8A)S2Lich mice brain slices. To define the optimal processing and culturing conditions, we compared mitochondrial morphology and motility with three different sets of slicing and incubation solutions. The investigation of mitochondrial dynamics was performed on deconvoluted images. For morphological investigation, images were segmented into three different categories according to the shape of mitochondria, while motility was investigated using semi-automated tracking. ResultsThe imaging of acute brain slices by two-photon microscopy represented a suitable tool to monitor neuronal mitochondria ex vivo. We observed that mitochondrial dynamics were better preserved in slices incubated with HEPES aCSF, maintaining elongated rod-shaped morphology and the motility. Comparison with existing methodsWe showed for the first time a method that allows live imaging of mitochondria and its quantification, while the existing in vitro protocol are not suitable to investigate mitochondria in live tissue. ConclusionWe have established the best incubation conditions and microscopy tools to investigate living mitochondria in acute slices. We showed that preventing initial swelling with HEPES and addition of glucose, pyruvate, ascorbate and thiourea preserved mitochondria in adult brain slices, which could be monitored by two-photon microscopy.

Src family kinases activity is required for transmitting purinergic P2X7 receptor signaling in cortical spreading depression and neuroinflammation

BackgroundPurinergic P2X7 receptor plays an important role in migraine pathophysiology. Yet precise molecular mechanism underlying P2X7R signaling in migraine remains unclear. This study explores the hypothesis that P2X7 receptor transmits signaling to Src family kinases (SFKs) during cortical spreading depression (CSD) and neuroinflammation after CSD.MethodsCSD was recorded using electrophysiology in rats and intrinsic optical imaging in mouse brain slices. Cortical IL-1β and TNFα mRNA levels were detected using qPCR. Glutamate release from mouse brain slices was detected using glutamate assay.ResultsThe data showed that deactivation of SFKs by systemic injection of PP2 reduced cortical susceptibility to CSD in rats and CSD-induced IL-1β and TNF-α gene expression in rat ipsilateral cortices. Consistently, in mouse brain slices, inhibition of SFKs activity by saracatinib and P2X7 receptor by A740003 similarly reduced cortical susceptibility to CSD. When the interaction of P2X7 receptor and SFKs was disrupted by TAT-P2X7, a marked reduction of cortical susceptibility to CSD, IL-1β gene expression and glutamate release after CSD induction were observed in mouse brain slices. The reduced cortical susceptibility to CSD by TAT-P2X7 was restored by NMDA, and disrupting the Fyn-NMDA interaction using TAT-Fyn (39-57) but not disrupting Src-NMDA receptor interaction using TAT-Src (40-49) reduced cortical susceptibility to CSD. Furthermore, activation of P2X7 receptor by BzATP restored the TAT-Fyn (39-57)-reduced cortical susceptibility to CSD.ConclusionThis study reveals that SFKs activity transmits P2X7 receptor signaling to facilitate CSD propagation via glutamatergic pathway and promote neuroinflammation, which is of particular relevance to migraine.

EVAP: A two-photon imaging tool to study conformational changes in endogenous Kv2 channels in live tissues.

A primary goal of molecular physiology is to understand how conformational changes of proteins affect the function of cells, tissues, and organisms. Here, we describe an imaging method for measuring the conformational changes of the voltage sensors of endogenous ion channel proteins within live tissue, without genetic modification. We synthesized GxTX-594, a variant of the peptidyl tarantula toxin guangxitoxin-1E, conjugated to a fluorophore optimal for two-photon excitation imaging through light-scattering tissue. We term this tool EVAP (Endogenous Voltage-sensor Activity Probe). GxTX-594 targets the voltage sensors of Kv2 proteins, which form potassium channels and plasma membrane-endoplasmic reticulum junctions. GxTX-594 dynamically labels Kv2 proteins on cell surfaces in response to voltage stimulation. To interpret dynamic changes in fluorescence intensity, we developed a statistical thermodynamic model that relates the conformational changes of Kv2 voltage sensors to degree of labeling. We used two-photon excitation imaging of rat brain slices to image Kv2 proteins in neurons. We found puncta of GxTX-594 on hippocampal CA1 neurons that responded to voltage stimulation and retain a voltage response roughly similar to heterologously expressed Kv2.1 protein. Our findings show that EVAP imaging methods enable the identification of conformational changes of endogenous Kv2 voltage sensors in tissue.

Blockade of TRPC Channels Limits Cholinergic-Driven Hyperexcitability and Seizure Susceptibility After Traumatic Brain Injury.

We investigated the contribution of excitatory transient receptor potential canonical (TRPC) cation channels to posttraumatic hyperexcitability in the brain 7 days following controlled cortical impact model of traumatic brain injury (TBI) to the parietal cortex in male adult mice. We investigated if TRPC1/TRPC4/TRPC5 channel expression is upregulated in excitatory neurons after TBI in contribution to epileptogenic hyperexcitability in key hippocampal and cortical circuits that have substantial cholinergic innervation. This was tested by measuring TRPC1/TRPC4/TRPC5 protein and messenger RNA (mRNA) expression, assays of cholinergic function, neuronal Ca2+ imaging in brain slices, and seizure susceptibility after TBI. We found region-specific increases in expression of TRPC1, TRPC4, and TRPC5 subunits in the hippocampus and cortex following TBI. The dentate gyrus, CA3 region, and cortex all exhibited robust upregulation of TRPC4 mRNA and protein. TBI increased cFos activity in dentate gyrus granule cells (DGGCs) and layer 5 pyramidal neurons both at the time of TBI and 7 days post-TBI. DGGCs displayed greater magnitude and duration of acetylcholine-induced rises in intracellular Ca2+ in brain slices from mice subjected to TBI. The TBI mice also exhibited greater seizure susceptibility in response to pentylenetetrazol-induced kindling. Blockade of TRPC4/TRPC5 channels with M084 reduced neuronal hyperexcitation and impeded epileptogenic progression of kindling. We observed that the time-dependent upregulation of TRPC4/TRPC5-containing channels alters cholinergic responses and activity of principal neurons acting to increase proexcitatory sensitivity. The underlying mechanism includes acutely decreased acetylcholinesterase function, resulting in greater Gq/11-coupled muscarinic receptor activation of TRPC channels. Overall, our evidence suggests that TBI-induced plasticity of TRPC channels strongly contributes to overt hyperexcitability and primes the hippocampus and cortex for seizures.

A novel dynamic network imaging analysis method reveals aging-related fragmentation of cortical networks in mouse.

Network analysis of large-scale neuroimaging data is a particularly challenging computational problem. Here, we adapt a novel analytical tool, the community dynamic inference method (CommDy), for brain imaging data from young and aged mice. CommDy, which was inspired by social network theory, has been successfully used in other domains in biology; this report represents its first use in neuroscience. We used CommDy to investigate aging-related changes in network metrics in the auditory and motor cortices by using flavoprotein autofluorescence imaging in brain slices and in vivo. We observed that auditory cortical networks in slices taken from aged brains were highly fragmented compared to networks observed in young animals. CommDy network metrics were then used to build a random-forests classifier based on NMDA receptor blockade data, which successfully reproduced the aging findings, suggesting that the excitatory cortical connections may be altered during aging. A similar aging-related decline in network connectivity was also observed in spontaneous activity in the awake motor cortex, suggesting that the findings in the auditory cortex reflect general mechanisms during aging. These data suggest that CommDy provides a new dynamic network analytical tool to study the brain and that aging is associated with fragmentation of intracortical networks.

Subcellular Distribution of Persistent Sodium Conductance in Cortical Pyramidal Neurons.

Cortical pyramidal neurons possess a persistent Na+ current (I NaP), which, in contrast to the larger transient current, does not undergo rapid inactivation. Although relatively quite small, I NaP is active at subthreshold voltages and therefore plays an important role in neuronal input-output processing. The subcellular distribution of channels responsible for I NaP and the mechanisms that render them persistent are not known. Using high-speed fluorescence Na+ imaging and whole-cell recordings in brain slices obtained from mice of either sex, we reconstructed the I NaP elicited by slow voltage ramps in soma and processes of cortical pyramidal neurons. We found that in all neuronal compartments, the relationship between persistent Na+ conductance and membrane voltage has the shape of a Boltzmann function. Although the density of channels underlying I NaP was about twofold lower in the axon initial segment (AIS) than in the soma, the axonal channels were activated by ∼10 mV less depolarization than were somatic channels. This difference in voltage dependence explains why, at functionally critical subthreshold voltages, most I NaP originates in the AIS. Finally, we show that endogenous polyamines constrain I NaP availability in both somatodendritic and axonal compartments of nondialyzed cortical neurons.SIGNIFICANCE STATEMENT The most salient characteristic of neuronal sodium channels is fast inactivation. However, a fraction of the sodium current does not inactivate. In cortical neurons, persistent current (I NaP) plays a prominent role in many important functions. Its subcellular distribution and generation mechanisms are, however, elusive. Using high-speed fluorescence Na+ imaging and electrical recordings, we reconstructed the I NaP in soma and processes of cortical pyramidal neurons. We found that at near-threshold voltages I NaP originates predominately from the axon, because of the distinctive voltage dependence of the underlying channels and not because of their high density. Finally, we show that the presence of endogenous polyamines significantly constrains I NaP availability in all compartments of nondialyzed cortical neurons.

Benzopyrylium conjugated quinolone constructing 705 nm long wavelength emission for evaluation of sulfur dioxide in brain slices

Although sulfur dioxide shows a variety of physiological and pathological functions in the body, the lack of detection tools still limits its in situ analysis. Fluorescent probes have the advantages of visualization, non-destructive and multi-level imaging, and have potential application prospects for in-situ detection of organisms. However, fluorescent probes capable of in-situ resolution of substances need to have specific and rapid response to targets, as well as near-red and long-wavelength emission. In our study, benzopyrylium moiety as a versatile fluorophore with a built-in site for SO2, good water solubility and the ability to target mitochondria was employed to construct probe Mito-NQ together with quinoline moiety for extension of conjugate. The probe Mito-NQ exhibited a near infrared emission at 705 nm based on the designed d-π-A-π-D structure. Moreover, we have successfully applied Mito-NQ to the detection of SO2 targeting mitochondria in cells, zebrafish and nude mice. Brain slice imaging showed that long wavelength emission has deep tissue penetration compared to short wavelength emission.

Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing.

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the morphology of processes and substructures within these neurons. As such, much of what is known about brain function has arisen alongside developments in imaging technologies that allow further insight into how neurons are organized and connected in the brain. Improvements in tissue clearing have allowed for high-resolution imaging of thick brain slices, facilitating morphological reconstruction and analyses of neuronal substructures, such as dendritic arbors and spines. In tandem, advances in image processing software provide methods of quickly analyzing large imaging datasets. This work presents a relatively rapid method of processing, visualizing, and analyzing thick slices of labeled neural tissue at high-resolution using CLARITY tissue clearing, confocal microscopy, and image analysis. This protocol will facilitate efforts toward understanding the connectivity patterns and neuronal morphologies that characterize healthy brains, and the changes in these characteristics that arise in diseased brain states.

Norepinephrine-Induced Calcium Signaling and Store-Operated Calcium Entry in Olfactory Bulb Astrocytes.

It is well-established that astrocytes respond to norepinephrine with cytosolic calcium rises in various brain areas, such as hippocampus or neocortex. However, less is known about the effect of norepinephrine on olfactory bulb astrocytes. In the present study, we used confocal calcium imaging and immunohistochemistry in mouse brain slices of the olfactory bulb, a brain region with a dense innervation of noradrenergic fibers, to investigate the calcium signaling evoked by norepinephrine in astrocytes. Our results show that application of norepinephrine leads to a cytosolic calcium rise in astrocytes which is independent of neuronal activity and mainly mediated by PLC/IP3-dependent internal calcium release. In addition, store-operated calcium entry (SOCE) contributes to the late phase of the response. Antagonists of both α1- and α2-adrenergic receptors, but not β-receptors, largely reduce the adrenergic calcium response, indicating that both α-receptor subtypes mediate norepinephrine-induced calcium transients in olfactory bulb astrocytes, whereas β-receptors do not contribute to the calcium transients.

Sensitive label-free imaging of brain samples using FxClear-based tissue clearing technique

SummaryOptical clearing has emerged as a powerful tool for volume imaging. Although volume imaging with immunostaining have been successful in many protocols, yet obtaining homogeneously stained thick samples remains challenging. Here, we propose a method for label-free imaging of brain slices by enhancing the regional heterogeneity of the optical properties using the tissue clearing principle. We used FxClear, a method for delipidation of brain tissue, to retain a larger proportion of lipids at the white matter (WM). Furthermore, the embedding media affected the contrasts for the lipid-rich or extracellular matrix-rich areas, depending on their chemical properties. Thus, we tailored clearing conditions for the enhancement of the refractive indices (RIs) differences between gray and WM, or several pathological features. RI differences can be imaged using conventional light microscopy or optical coherence tomography. We propose that our protocol is simple, reliable, and flexible for label-free imaging, easily implementable to routine histology laboratory.