Reliability of high-quantity human brain organoids for modeling microcephaly, glioma invasion and drug screening.

Brain organoids offer unprecedented insights into brain development and disease modeling and hold promise for drug screening. Significant hindrances, however, are morphological and cellular heterogeneity, inter-organoid size differences, cellular stress, and poor reproducibility. Here, we describe a method that reproducibly generates thousands of organoids across multiple hiPSC lines.

Deep-Learning-Based Segmentation of Cells and Analysis (DL-SCAN).

With the recent surge in the development of highly selective probes, fluorescence microscopy has become one of the most widely used approaches to studying cellular properties and signaling in living cells and tissues. Traditionally, microscopy image analysis heavily relies on manufacturer-supplied software, which often demands extensive training and lacks automation capabilities for handling diverse datasets. A critical challenge arises if the fluorophores employed exhibit low brightness and a low signal-to-noise ratio (SNR).

A dangerous liaison: Spreading depolarization and tissue acidification in cerebral ischemia.

Brain pH is precisely regulated, and pH transients associated with activity are rapidly restored under physiological conditions. During ischemia, the brain's ability to buffer pH changes is rapidly depleted. Tissue oxygen deprivation causes a shift from aerobic to anaerobic metabolism and the accumulation of lactic acid and protons.

Protocol for the generation of cultured cortical brain organoid slices.

Three-dimensional brain organoids from human pluripotent stem cells are a powerful tool for studying human neural networks. Here, we present a protocol for generating cortical brain organoid slices (cBOS) derived from regionalized cortical organoids and grown at the air-liquid interphase. We provide steps for slicing organoids and maintaining them in long-term culture.

pH regulating mechanisms of astrocytes: A critical component in physiology and disease of the brain.

Strict homeostatic control of pH in both intra- and extracellular compartments of the brain is fundamentally important, primarily due to the profound impact of free protons ([H]) on neuronal activity and overall brain function. Astrocytes, crucial players in the homeostasis of various ions in the brain, actively regulate their intracellular [H] (pH) through multiple membrane transporters and carbonic anhydrases. The activation of astroglial pH regulating mechanisms also leads to corresponding alterations in the acid-base status of the extracellular fluid.

Cortical brain organoid slices (cBOS) for the study of human neural cells in minimal networks.

Brain organoids derived from human pluripotent stem cells are a promising tool for studying human neurodevelopment and related disorders. Here, we generated long-term cultures of cortical brain organoid slices (cBOS) grown at the air-liquid interphase from regionalized cortical organoids. We show that cBOS host mature neurons and astrocytes organized in complex architecture.

Inward Operation of Sodium-Bicarbonate Cotransporter 1 Promotes Astrocytic Na Loading and Loss of ATP in Mouse Neocortex during Brief Chemical Ischemia.

Ischemic conditions cause an increase in the sodium concentration of astrocytes, driving the breakdown of ionic homeostasis and exacerbating cellular damage. Astrocytes express high levels of the electrogenic sodium-bicarbonate cotransporter1 (NBCe1), which couples intracellular Na homeostasis to regulation of pH and operates close to its reversal potential under physiological conditions. Here, we analyzed its mode of operation during transient energy deprivation via imaging astrocytic pH, Na, and ATP in organotypic slice cultures of the mouse neocortex, complemented with patch-clamp and ion-selective microelectrode recordings and computational modeling.

Sodium homeostasis and signalling: The core and the hub of astrocyte function.

Neuronal activity and neurochemical stimulation trigger spatio-temporal changes in the cytoplasmic concentration of Na ions in astrocytes. These changes constitute the substrate for Na signalling and are fundamental for astrocytic excitability. Astrocytic Na signals are generated by Na influx through neurotransmitter transporters, with primary contribution of glutamate transporters, and through cationic channels; whereas recovery from Na transients is mediated mainly by the plasmalemmal Na/K ATPase.

Ca-dependent rapid uncoupling of astrocytes upon brief metabolic stress.

Astrocytic gap junctional coupling is a major element in neuron-glia interaction. There is strong evidence that impaired coupling is involved in neurological disorders. Reduced coupling was, e.

Novel algorithms for improved detection and analysis of fluorescent signal fluctuations.

Fluorescent dyes and genetically encoded fluorescence indicators (GEFI) are common tools for visualizing concentration changes of specific ions and messenger molecules during intra- as well as intercellular communication. Using advanced imaging technologies, fluorescence indicators are a prerequisite for the analysis of physiological molecular signaling. Automated detection and analysis of fluorescence signals require to overcome several challenges, including correct estimation of fluorescence fluctuations at basal concentrations of messenger molecules, detection, and extraction of events themselves as well as proper segmentation of neighboring events.

Activation of TRPV4 channels promotes the loss of cellular ATP in organotypic slices of the mouse neocortex exposed to chemical ischemia.

The vertebrate brain has an exceptionally high energy need. During ischemia, intracellular ATP concentrations decline rapidly, resulting in the breakdown of ion gradients and cellular damage. Here, we employed the nanosensor ATeam1.

Modeling the heterogeneity of sodium and calcium homeostasis between cortical and hippocampal astrocytes and its impact on bioenergetics.

Emerging evidence indicates that neuronal activity-evoked changes in sodium concentration in astrocytes Na represent a special form of excitability, which is tightly linked to all other major ions in the astrocyte and extracellular space, as well as to bioenergetics, neurotransmitter uptake, and neurovascular coupling. Recently, one of us reported that Na transients in the neocortex have a significantly higher amplitude than those in the hippocampus. Based on the extensive data from that study, here we develop a detailed biophysical model to further understand the origin of this heterogeneity and how it affects bioenergetics in the astrocytes.

Membrane transporters control cerebrospinal fluid formation independently of conventional osmosis to modulate intracranial pressure.

Background: Disturbances in the brain fluid balance can lead to life-threatening elevation in the intracranial pressure (ICP), which represents a vast clinical challenge. Nevertheless, the details underlying the molecular mechanisms governing cerebrospinal fluid (CSF) secretion are largely unresolved, thus preventing targeted and efficient pharmaceutical therapy of cerebral pathologies involving elevated ICP.

Methods: Experimental rats were employed for in vivo determinations of CSF secretion rates, ICP, blood pressure and ex vivo excised choroid plexus for morphological analysis and quantification of expression and activity of various transport proteins.

Cell-Type Dependent Regulation of the Electrogenic Na/HCO Cotransporter 1 (NBCe1) by Hypoxia and Acidosis in Glioblastoma.

Glioblastoma multiforme (GBM) is the most common and malignant brain tumour. It is characterised by transcriptionally distinct cell populations. In tumour cells, physiological pH gradients between the intracellular and extracellular compartments are reversed, compared to non-cancer cells.

Acetazolamide modulates intracranial pressure directly by its action on the cerebrospinal fluid secretion apparatus.

Background: Elevated intracranial pressure (ICP) is observed in many neurological pathologies, e.g. hydrocephalus and stroke.

Changes in Astroglial K upon Brief Periods of Energy Deprivation in the Mouse Neocortex.

Malfunction of astrocytic K regulation contributes to the breakdown of extracellular K homeostasis during ischemia and spreading depolarization events. Studying astroglial K changes is, however, hampered by a lack of suitable techniques. Here, we combined results from fluorescence imaging, ion-selective microelectrodes, and patch-clamp recordings in murine neocortical slices with the calculation of astrocytic [K].

Rapid Fluorescence Lifetime Imaging Reveals That TRPV4 Channels Promote Dysregulation of Neuronal Na in Ischemia.

Fluorescence imaging is an indispensable method for analysis of diverse cellular and molecular processes, enabling, for example, detection of ions, second messengers, or metabolites. Intensity-based approaches, however, are prone to artifacts introduced by changes in fluorophore concentrations. This drawback can be overcome by fluorescence lifetime imaging (FLIM) based on time-correlated single-photon counting.

Glial Chloride Homeostasis Under Transient Ischemic Stress.

High water permeabilities permit rapid adjustments of glial volume upon changes in external and internal osmolarity, and pathologically altered intracellular chloride concentrations ([Cl]) and glial cell swelling are often assumed to represent early events in ischemia, infections, or traumatic brain injury. Experimental data for glial [Cl] are lacking for most brain regions, under normal as well as under pathological conditions. We measured [Cl] in hippocampal and neocortical astrocytes and in hippocampal radial glia-like (RGL) cells in acute murine brain slices using fluorescence lifetime imaging microscopy with the chloride-sensitive dye MQAE at room temperature.

Generation of Human Brain Organoids for Mitochondrial Disease Modeling.

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental defects whose underlying mechanisms remain to be elucidated. A major roadblock is the lack of effective models recapitulating the early-onset neuronal impairment seen in the patients.

Human Induced Pluripotent Stem Cell-Derived Neural Progenitor Cells Produce Distinct Neural 3D In Vitro Models Depending on Alginate/Gellan Gum/Laminin Hydrogel Blend Properties.

Stable and predictive neural cell culture models are a necessary premise for many research fields. However, conventional 2D models lack 3D cell-material/-cell interactions and hence do not reflect the complexity of the in vivo situation properly. Here two alginate/gellan gum/laminin (ALG/GG/LAM) hydrogel blends are presented for the fabrication of human induced pluripotent stem cell (hiPSC)-based 3D neural models.

Ion dynamics at the energy-deprived tripartite synapse.

The anatomical and functional organization of neurons and astrocytes at 'tripartite synapses' is essential for reliable neurotransmission, which critically depends on ATP. In low energy conditions, synaptic transmission fails, accompanied by a breakdown of ion gradients, changes in membrane potentials and cell swelling. The resulting cellular damage and cell death are causal to the often devastating consequences of an ischemic stroke.

Dysregulation of Astrocyte Ion Homeostasis and Its Relevance for Stroke-Induced Brain Damage.

Ischemic stroke is a leading cause of mortality and chronic disability. Either recovery or progression towards irreversible failure of neurons and astrocytes occurs within minutes to days, depending on remaining perfusion levels. Initial damage arises from energy depletion resulting in a failure to maintain homeostasis and ion gradients between extra- and intracellular spaces.

Disruption of Glutamate Transport and Homeostasis by Acute Metabolic Stress.

High-affinity, Na-dependent glutamate transporters are the primary means by which synaptically released glutamate is removed from the extracellular space. They restrict the spread of glutamate from the synaptic cleft into the perisynaptic space and reduce its spillover to neighboring synapses. Thereby, glutamate uptake increases the spatial precision of synaptic communication.

Reactive astrocyte nomenclature, definitions, and future directions.

Reactive astrocytes are astrocytes undergoing morphological, molecular, and functional remodeling in response to injury, disease, or infection of the CNS. Although this remodeling was first described over a century ago, uncertainties and controversies remain regarding the contribution of reactive astrocytes to CNS diseases, repair, and aging. It is also unclear whether fixed categories of reactive astrocytes exist and, if so, how to identify them.