Living Brain Tissue Research Articles

Patch-walking: Coordinated multi-pipette patch clamp for efficiently finding synaptic connections.

Significant technical challenges exist when measuring synaptic connections between neurons in living brain tissue. The patch clamping technique, when used to probe for synaptic connections, is manually laborious and time-consuming. To improve its efficiency, we pursued another approach: instead of retracting all patch clamping electrodes after each recording attempt, we cleaned just one of them and reused it to obtain another recording while maintaining the others. With one new patch clamp recording attempt, many new connections can be probed. By placing one pipette in front of the others in this way, one can "walk" across the tissue, termed "patch-walking." We performed 136 patch clamp attempts for two pipettes, achieving 71 successful whole cell recordings (52.2%). Of these, we probed 29 pairs (i.e., 58 bidirectional probed connections) averaging 91 μm intersomatic distance, finding 3 connections. Patch-walking yields 80-92% more probed connections, for experiments with 10-100 cells than the traditional synaptic connection searching method.

Single Molecule Imaging of Sp3 Functionalized Ultrashort Carbon Nanotubes for Deep Brain Tissue Investigation

Near-infrared (NIR) fluorescence microscopy has attracted significant attention for in vivo deep tissue imaging due to the combination of low light scattering and absorption by the tissues, resulting in good light penetration depth, especially in the NIR-II window (~1000–1350 nm). In this context, NIR photoluminescence (PL) (λem ~ 900–1400 nm) of single-walled carbon nanotubes overlapping with the so-called “tissue transparency window” laid the foundation for their use in bioimaging and sensing applications. Interestingly, although ultrashort carbon nanotubes (usCNTs) had long been sought for their advantages of mimicking bio-molecular dimensions, it is only when sp3 defects introduced bright fluorescent usCNTs could be generated [1]. In this work, we demonstrate the efficient preparation of bright fluorescent usCNTs specifically designed for biological applications, and we show how to create bright usCNTs wrapped by a biocompatible encapsulating agent (phospholipid-polyethylene glycol, PL-PEG). More precisely, we adapted a synthesis route where the sp3 functionalization is followed by chemical cutting [2]. We will present their full characterization and demonstrate their performance for deep tissue imaging in neuroscience. More precisely, our results performed on live brain tissue allow us to analyze their length specific behaviors in different compartments of mouse brains in comparison to the long sp3 functionalized CNTs [3].

Simultaneous Mapping of the Nanoscale Organization and Redox State of Extracellular Space in Living Brain Tissue.

The spatial organization characteristics and redox status of the extracellular space (ECS) are crucial in the development of brain diseases. However, it remains a challenge to simultaneously capture dynamic changes in microstructural features and redox states at the submicron level within the ECS. Here, we developed a reversible glutathione (GSH)-responsive nanoprobe (RGN) for mapping the spatial organization features and redox status of the ECS in brain tissues with nanoscale resolution. The RGN is composed of polymer nanoparticles modified with GSH-responsive molecules and amino-functionalized methoxypoly(ethylene glycol), which exhibit exceptional single-particle brightness and excellent free diffusion capability in the ECS of brain tissues. Tracking single RGNs in acute brain slices allowed us to dynamically map spatial organizational features and redox levels within the ECS of brain tissues in disease models. This provides a powerful super-resolution imaging method that offers a potential opportunity to study the dynamic changes in the ECS microenvironment and to understand the physiological and pathological roles of the ECS in vivo.

OpenSTED: open-source dynamic intensity minimum system for stimulated emission depletion microscopy.

Stimulated emission depletion (STED) is a powerful super-resolution microscopy technique that can be used for imaging live cells. However, the high STED laser powers can cause significant photobleaching and sample damage in sensitive biological samples. The dynamic intensity minimum (DyMIN) technique turns on the STED laser only in regions of the sample where there is fluorescence signal, thus saving significant sample photobleaching. The reduction in photobleaching allows higher resolution images to be obtained and longer time-lapse imaging of live samples. A stand-alone module to perform DyMIN is not available commercially. In this work, we developed an open-source design to implement three-step DyMIN on a STED microscope and demonstrated reduced photobleaching for timelapse imaging of beads, cells, and tissue. The DyMIN system uses a fast multiplexer circuit and inexpensive field-programmable gate array controlled by Labview software that operates as a stand-alone module for a STED microscope. All software and circuit diagrams are freely available. We compared time-lapse images of bead samples using our custom DyMIN system to conventional STED and recorded a higher signal when using DyMIN after a 50-image sequence. We further demonstrated the DyMIN system for time-lapse STED imaging of live cells and brain tissue slices. Our open-source DyMIN system is an inexpensive add-on to a conventional STED microscope that can reduce photobleaching. The system can significantly improve signal to noise for dynamic time-lapse STED imaging of live samples.

Knockout of the longevity gene Klotho perturbs aging and Alzheimer’s disease-linked brain microRNAs and tRNA fragments

Overexpression of the longevity gene Klotho prolongs lifespan, while its knockout shortens lifespan and impairs cognition via perturbation of myelination and synapse formation. However, comprehensive analysis of Klotho knockout effects on mammalian brain transcriptomics is lacking. Here, we report that Klotho knockout alters the levels of aging- and cognition related mRNAs, long non-coding RNAs, microRNAs and tRNA fragments. These include altered neuronal and glial regulators in murine models of aging and Alzheimer’s disease and in human Alzheimer’s disease post-mortem brains. We further demonstrate interaction of the knockout-elevated tRNA fragments with the spliceosome, possibly affecting RNA processing. Last, we present cell type-specific short RNA-seq datasets from FACS-sorted neurons and microglia of live human brain tissue demonstrating in-depth cell-type association of Klotho knockout-perturbed microRNAs. Together, our findings reveal multiple RNA transcripts in both neurons and glia from murine and human brain that are perturbed in Klotho deficiency and are aging- and neurodegeneration-related.

Monofluorophore-based Two-Photon Ratiometric Fluorescent Probe for the Quantitative Imaging of Fatty Acid Amide Hydrolase in Live Neurons and Mouse Brain Tissues

Monofluorophore-based Two-Photon Ratiometric Fluorescent Probe for the Quantitative Imaging of Fatty Acid Amide Hydrolase in Live Neurons and Mouse Brain Tissues

Effective cryopreservation of human brain tissue and neural organoids

Human brain tissue models and organoids are vital for studying and modeling human neurological disease. However, the high cost of long-term cultured organoids inhibits their wide-ranging application. It is therefore urgent to develop methods for the cryopreservation of brain tissue and organoids. Here, we establish a method using methylcellulose, ethylene glycol, DMSO, and Y27632 (termed MEDY) for the cryopreservation of cortical organoids without disrupting the neural cytoarchitecture or functional activity. MEDY can be applied to multiple brain-region-specific organoids, including the dorsal/ventral forebrain, spinal cord, optic vesicle brain, and epilepsy patient-derived brain organoids. Additionally, MEDY enables the cryopreservation of human brain tissue samples, and pathological features are retained after thawing. Transcriptomic analysis shows that MEDY can protect synaptic function and inhibit the endoplasmic reticulum-mediated apoptosis pathway. MEDY will enable the large-scale and reliable storage of diverse neural organoids and living brain tissue and will facilitate wide-ranging research, medical applications, and drug screening.

Structural, Functional, and Genetic Changes Surrounding Electrodes Implanted in the Brain.

Implantable neurotechnology enables monitoring and stimulating of the brain signals responsible for performing cognitive, motor, and sensory tasks. Electrode arrays implanted in the brain are increasingly used in the clinic to treat a variety of sources of neurological diseases and injuries. However, the implantation of a foreign body typically initiates a tissue response characterized by physical disruption of vasculature and the neuropil as well as the initiation of inflammation and the induction of reactive glial states. Likewise, electrical stimulation can induce damage to the surrounding tissue depending on the intensity and waveform parameters of the applied stimulus. These phenomena, in turn, are likely influenced by the surface chemistry and characteristics of the materials employed, but further information is needed to effectively link the biological responses observed to specific aspects of device design. In order to inform improved design of implantable neurotechnology, we are investigating the basic science principles governing device-tissue integration. We are employing multiple techniques to characterize the structural, functional, and genetic changes that occur in the cells surrounding implanted electrodes. First, we have developed a new "device-in-slice" technique to capture chronically implanted electrodes within thick slices of live rat brain tissue for interrogation with single-cell electrophysiology and two-photon imaging techniques. Our data revealed several new observations of tissue remodeling surrounding devices: (a) there was significant disruption of dendritic arbors in neurons near implants, where losses were driven asymmetrically on the implant-facing side. (b) There was a significant loss of dendritic spine densities in neurons near implants, with a shift toward more immature (nonfunctional) morphologies. (c) There was a reduction in excitatory neurotransmission surrounding implants, as evidenced by a reduction in the frequency of excitatory postsynaptic currents (EPSCs). Lastly, (d) there were changes in the electrophysiological underpinnings of neuronal spiking regularity. In parallel, we initiated new studies to explore changes in gene expression surrounding devices through spatial transcriptomics, which we applied to both recording and stimulating arrays. We found that (a) device implantation is associated with the induction of hundreds of genes associated with neuroinflammation, glial reactivity, oligodendrocyte function, and cellular metabolism and (b) electrical stimulation induces gene expression associated with damage or plasticity in a manner dependent upon the intensity of the applied stimulus. We are currently developing computational analysis tools to distill biomarkers of device-tissue interactions from large transcriptomics data sets. These results improve the current understanding of the biological response to electrodes implanted in the brain while producing new biomarkers for benchmarking the effects of novel electrode designs on responses. As the next generation of neurotechnology is developed, it will be increasingly important to understand the influence of novel materials, surface chemistries, and implant architectures on device performance as well as the relationship with the induction of specific cellular signaling pathways.

Live Mapping of the Brain Extracellular Matrix and Remodeling in Neurological Disorders.

Live imaging of the brain extracellular matrix (ECM) provides vital insights into changes that occur in neurological disorders. Current techniques such as second or third-harmonic generation offer limited contrast for live imaging of the brain ECM. Here, a new method, pan-ECM via chemical labeling of extracellular proteins, is introduced for live brain ECM imaging. pan-ECM labels all major ECM components in live tissue including the interstitial matrix, basement membrane, and perineuronal nets. pan-ECM enables in vivo observation of the ECM heterogeneity between the glioma core and margin, as well as the assessment of ECM deterioration under stroke condition, without ECM shrinkage from tissue fixation. These findings indicate that the pan-ECM approach is a novel way to image the entire brain ECM in live brain tissue with optical resolution. pan-ECM has the potential to advance the understanding of ECM in brain function and neurological diseases.

Shadow imaging for panoptical visualization of brain tissue in vivo

Progress in neuroscience research hinges on technical advances in visualizing living brain tissue with high fidelity and facility. Current neuroanatomical imaging approaches either require tissue fixation (electron microscopy), do not have cellular resolution (magnetic resonance imaging) or only give a fragmented view (fluorescence microscopy). Here, we show how regular light microscopy together with fluorescence labeling of the interstitial fluid in the extracellular space provide comprehensive optical access in real-time to the anatomical complexity and dynamics of living brain tissue at submicron scale. Using several common fluorescence microscopy modalities (confocal, light-sheet and 2-photon microscopy) in mouse organotypic and acute brain slices and the intact mouse brain in vivo, we demonstrate the value of this straightforward ‘shadow imaging’ approach by revealing neurons, microglia, tumor cells and blood capillaries together with their complete anatomical tissue contexts. In addition, we provide quantifications of perivascular spaces and the volume fraction of the extracellular space of brain tissue in vivo.

Microscopic-scale magnetic recording of brain neuronal electrical activity using a diamond quantum sensor

Quantum sensors using solid state qubits have demonstrated outstanding sensitivity, beyond that possible using classical devices. In particular, those based on colour centres in diamond have demonstrated high sensitivity to magnetic field through exploiting the field-dependent emission of fluorescence under coherent control using microwaves. Given the highly biocompatible nature of diamond, sensing from biological samples is a key interdisciplinary application. In particular, the microscopic-scale study of living systems can be possible through recording of temperature and biomagnetic field. In this work, we use such a quantum sensor to demonstrate such microscopic-scale recording of electrical activity from neurons in fragile living brain tissue. By recording weak magnetic field induced by ionic currents in mouse corpus callosum axons, we accurately recover signals from neuronal action potential propagation while demonstrating in situ pharmacology. Our sensor allows recording of the electrical activity in neural circuits, disruption of which can shed light on the mechanisms of disease emergence. Unlike existing techniques for recording activity, which can require potentially damaging direct interaction, our sensing is entirely passive and remote from the sample. Our results open a promising new avenue for the microscopic recording of neuronal signals, offering the eventual prospect of microscopic imaging of electrical activity in the living mammalian brain.

Dense 4D nanoscale reconstruction of living brain tissue.

Three-dimensional (3D) reconstruction of living brain tissue down to an individual synapse level would create opportunities for decoding the dynamics and structure-function relationships of the brain's complex and dense information processing network; however, this has been hindered by insufficient 3D resolution, inadequate signal-to-noise ratio and prohibitive light burden in optical imaging, whereas electron microscopy is inherently static. Here we solved these challenges by developing an integrated optical/machine-learning technology, LIONESS (live information-optimized nanoscopy enabling saturated segmentation). This leverages optical modifications to stimulated emission depletion microscopy in comprehensively, extracellularly labeled tissue and previous information on sample structure via machine learning to simultaneously achieve isotropic super-resolution, high signal-to-noise ratio and compatibility with living tissue. This allows dense deep-learning-based instance segmentation and 3D reconstruction at a synapse level, incorporating molecular, activity and morphodynamic information. LIONESS opens up avenues for studying the dynamic functional (nano-)architecture of living brain tissue.

LIONESS enables 4D nanoscale reconstruction of living brain tissue.

LIONESS enables 4D nanoscale reconstruction of living brain tissue.

Stimulated Raman scattering microscopy reveals a unique and steady nature of brain water dynamics

The biological activities of substances in the brain are shaped by their spatiotemporal dynamics in brain tissues, all of which are regulated by water dynamics. In contrast to solute dynamics, water dynamics have been poorly characterized, owing to the lack of appropriate analytical tools. To overcome this limitation, we apply stimulated Raman scattering multimodal multiphoton microscopy to live brain tissues. The microscopy system allows for the visualization of deuterated water, fluorescence-labeled solutes, and cellular structures at high spatiotemporal resolution, revealing that water moves faster than fluorescent molecules in brain tissues. Detailed analyses demonstrate that water, unlike solutes, diffuses homogeneously in brain tissues without differences between the intra- and the extracellular routes. Furthermore, we find that the water dynamics are steady during development and ischemia, when diffusions of solutes are severely affected. Thus, our approach reveals routes and uniquely robust properties of water diffusion in brain tissues.

Spatiotemporal Imaging of Zinc Ions in Zebrafish Live Brain Tissue Enabled by Fluorescent Bionanoprobes.

The zebrafish is a powerful model organism to study the mechanisms governing transition metal ions within whole brain tissue. Zinc is one of the most abundant metal ions in the brain, playing a critical pathophysiological role in neurodegenerative diseases. The homeostasis of free, ionic zinc (Zn2+) is a key intersection point in many of these diseases, including Alzheimer's disease and Parkinson's disease. A Zn2+ imbalance can eventuate several disturbances that may lead to the development of neurodegenerative changes. Therefore, compact, reliable approaches that allow the optical detection of Zn2+ across the whole brain would contribute to our current understanding of the mechanisms that underlie neurological disease pathology. We developed an engineered fluorescence protein-based nanoprobe that can spatially and temporally resolve Zn2+ in living zebrafish brain tissue. The self-assembled engineered fluorescence protein on gold nanoparticles was shown to be confined to defined locations within the brain tissue, enabling site specific studies, compared to fluorescent protein-based molecular tools, which diffuse throughout the brain tissue. Two-photon excitation microscopy confirmed the physical and photometrical stability of these nanoprobes in living zebrafish (Danio rerio) brain tissue, while the addition of Zn2+ quenched the nanoprobe fluorescence. Combining orthogonal sensing methods with our engineered nanoprobes will enable the study of imbalances in homeostatic Zn2+ regulation. The proposed bionanoprobe system offers a versatile platform to couple metal ion specific linkers and contribute to the understanding of neurological diseases.

Cell-Surface Targeting of Fluorophores in Drosophila for Rapid Neuroanatomy Visualization.

Visualizing neuronal anatomy often requires labor-intensive immunohistochemistry on fixed and dissected brains. To facilitate rapid anatomical staining in live brains, we used genetically targeted membrane tethers that covalently link fluorescent dyes for in vivo neuronal labeling. We generated a series of extracellularly trafficked small-molecule tethering proteins, HaloTag-CD4 (Kirk et al. Front. Neurosci. 2021, 15, 754027) and SNAPf-CD4, which directly label transgene-expressing cells with commercially available ligand-substituted fluorescent dyes. We created stable transgenic Drosophila reporter lines, which express extracellular HaloTag-CD4 and SNAPf-CD4 with LexA and Gal4 drivers. Expressing these enzymes in live Drosophila brains, we labeled the expression patterns of various Gal4 driver lines recapitulating histological staining in live-brain tissues. Pan-neural expression of SNAPf-CD4 enabled the registration of live brains to an existing template for anatomical comparisons. We predict that these extracellular platforms will not only become a valuable complement to existing anatomical methods but will also prove useful for future genetic targeting of other small-molecule probes, drugs, and actuators.

Sporadic Creutzfeldt–Jakob disease infected human cerebral organoids retain the original human brain subtype features following transmission to humanized transgenic mice

Human cerebral organoids (COs) are three-dimensional self-organizing cultures of cerebral brain tissue differentiated from induced pluripotent stem cells. We have recently shown that COs are susceptible to infection with different subtypes of Creutzfeldt–Jakob disease (CJD) prions, which in humans cause different manifestations of the disease. The ability to study live human brain tissue infected with different CJD subtypes opens a wide array of possibilities from differentiating mechanisms of cell death and identifying neuronal selective vulnerabilities to testing therapeutics. However, the question remained as to whether the prions generated in the CO model truly represent those in the infecting inoculum. Mouse models expressing human prion protein are commonly used to characterize human prion disease as they reproduce many of the molecular and clinical phenotypes associated with CJD subtypes. We therefore inoculated these mice with COs that had been infected with two CJD subtypes (MV1 and MV2) to see if the original subtype characteristics (referred to as strains once transmitted into a model organism) of the infecting prions were maintained in the COs when compared with the original human brain inocula. We found that disease characteristics caused by the molecular subtype of the disease associated prion protein were similar in mice inoculated with either CO derived material or human brain material, demonstrating that the disease associated prions generated in COs shared strain characteristics with those in humans. As the first and only in vitro model of human neurodegenerative disease that can faithfully reproduce different subtypes of prion disease, these findings support the use of the CO model for investigating human prion diseases and their subtypes.

Getting sharper: the brain under the spotlight of super-resolution microscopy.

Brain cells such as neurons and astrocytes exhibit an extremely elaborate morphology, and their functional specializations like synapses and glial processes often fall below the resolution limit of conventional light microscopy. This is a huge obstacle for neurobiologists because the nanoarchitecture critically shapes fundamental functions like synaptic transmission and Ca2+ signaling. Super-resolution microscopy can overcome this problem, offering the chance to visualize the structural and molecular organization of brain cells in a living and dynamic tissue context, unlike traditional methods like electron microscopy or atomic force microscopy. This review covers the basic principles of the main super-resolution microscopy techniques in use today and explains how their specific strengths can illuminate the nanoscale mechanisms that govern brain physiology.

‘wildDISCO’ whole-mouse mapping; high-resolution imaging of living brain tissue

‘wildDISCO’ whole-mouse mapping; high-resolution imaging of living brain tissue

Susceptibility of aged pericytes to Aβ1‐42 oligomers

AbstractBackgroundAging is associated with increased incidence of Alzheimer’s disease, blood brain barrier breakdown, and increased cellular senescence (Saez‐Atienzar et. al, 2020). Senescence is the permanent arrest of the proliferative state of the cell and is initiated as a result of aging and cellular stress (Martínez‐Cué et.al, 2020). Senescent cells induce production of proinflammatory cytokines. In the brain, chronic inflammation leads to synapse damage and cognitive decline (Saez‐Atienzar et. al, 2020). The objective of this study is to examine the impact of cellular senescence on brain vascular pericytes, which contribute to the regulation of cerebral blood flow.MethodFor this study, both in vitro as well as in vivo experiments were performed. To induce senescence by oxidative stress in vitro in a culture of mouse brain vascular pericytes, 100 uM of hydrogen peroxide was added to culture flasks. Senescence was confirmed in the treated cells via staining for β‐galactosidase, a known biomarker of senescent cells. The next steps for the in vitro aspects of this study include performing western blotting on senescent pericytes to determine if there is a differential expression of pericyte p75 neurotrophin receptor (p75NTR), which has a potential role in the impaired hemodynamic response and cell death in pericytes. Finally, cranial window procedures were performed on 18‐month‐old C57BL/6 mice followed by imaging the live brain tissue with a multiphoton microscope. While imaging, artificial cerebral spinal fluid (control animals) or 72 nM amyloid‐beta (Aβ)1‐42 oligomers were topically delivered to the brain surface to determine if Aβ1‐42 oligomers cause a significant reduction in erythrocyte flow through pericyte covered capillaries.ResultPreliminary data from the β‐galactosidase staining suggest that the hydrogen peroxide is an effective method for inducing senescence in mouse brain vascular pericytes. We have observed a decreased growth rate in cells treated with the hydrogen peroxide, further suggesting senescence in these cells.ConclusionThese findings characterize the impact of Aβ on pericyte contractility and could illuminate the role of senescence and aging on impaired hemodynamic responses because of dysfunctional pericytes.