Characterization of two near-infrared genetically encoded voltage indicators.

Significance: Efforts starting more than 20 years ago led to increasingly well performing genetically encoded voltage indicators (GEVIs) for optical imaging at wavelengths . Although optical imaging in the wavelength range has many advantages over shorter wavelength approaches for mesoscopic monitoring of neuronal activity in the mammalian brain, the availability and evaluation of well performing near-infrared GEVIs are still limited.

Aim: Here, we characterized two recent near-infrared GEVIs, Archon1 and nirButterfly, to support interested tool users in selecting a suitable near-infrared GEVI for their specific research question requirements.

DIRECT, a low-cost system for high-speed, low-noise imaging of fluorescent bio-samples.

A targeted imaging system has been developed for applications requiring recording from stationary samples at high spatiotemporal resolutions. It works by illuminating regions of interest in rapid sequence, and recording the signal from the whole field of view onto a single photodetector. It can be implemented at low cost on an existing microscope without compromising existing functionality.

Cortical Correlates of Psychedelic-Induced Shaking Behavior Revealed by Voltage Imaging.

(1) From mouse to man, shaking behavior (head twitches and/or wet dog shakes) is a reliable readout of psychedelic drug action. Shaking behavior like psychedelia is thought to be mediated by serotonin 2A receptors on cortical pyramidal cells. The involvement of pyramidal cells in psychedelic-induced shaking behavior remains hypothetical, though, as experimental in vivo evidence is limited.

Complexity of cortical wave patterns of the wake mouse cortex.

Rich spatiotemporal dynamics of cortical activity, including complex and diverse wave patterns, have been identified during unconscious and conscious brain states. Yet, how these activity patterns emerge across different levels of wakefulness remain unclear. Here we study the evolution of wave patterns utilizing data from high spatiotemporal resolution optical voltage imaging of mice transitioning from barbiturate-induced anesthesia to wakefulness (N = 5) and awake mice (N = 4).

Inhibition is a prevalent mode of activity in the neocortex around awake hippocampal ripples in mice.

Coordinated peri-ripple activity in the hippocampal-neocortical network is essential for mnemonic information processing in the brain. Hippocampal ripples likely serve different functions in sleep and awake states. Thus, the corresponding neocortical activity patterns may differ in important ways.

Cortex-wide spontaneous activity non-linearly steers propagating sensory-evoked activity in awake mice.

The brain responds highly variably to identical sensory inputs, but there is no consensus on the nature of this variability. We explore this question using cortex-wide optical voltage imaging and whisker stimulation in awake mice. Clustering analysis reveals that the sensory-evoked activity propagates over the cortex via distinct pathways associated with distinct behavioral states.

Hippocampal gamma and sharp wave/ripples mediate bidirectional interactions with cortical networks during sleep.

Hippocampus-neocortex interactions during sleep are critical for memory processes: Hippocampally initiated replay contributes to memory consolidation in the neocortex and hippocampal sharp wave/ripples modulate cortical activity. Yet, the spatial and temporal patterns of this interaction are unknown. With voltage imaging, electrocorticography, and laminarly resolved hippocampal potentials, we characterized cortico-hippocampal signaling during anesthesia and nonrapid eye movement sleep.

Toward high-throughput oligomer detection and classification for early-stage aggregation of amyloidogenic protein.

Aggregation kinetics of proteins and peptides have been studied extensively due to their significance in many human diseases, including neurodegenerative disorders, and the roles they play in some key physiological processes. However, most of these studies have been performed as bulk measurements using Thioflavin T or other fluorescence turn-on reagents as indicators of fibrillization. Such techniques are highly successful in making inferences about the nucleation and growth mechanism of fibrils, yet cannot directly measure assembly reactions at low protein concentrations which is the case for amyloid-β (Aβ) peptide under physiological conditions.

DNA Methylation Profiles of in Human Cerebral Organoids of Autism Indicate Disrupted Epigenetic Regulation during Early Development.

DNA methylation profiling has become a promising approach towards identifying biomarkers of neuropsychiatric disorders including autism spectrum disorder (ASD). Epigenetic markers capture genetic risk factors and diverse exogenous and endogenous factors, including environmental risk factors and complex disease pathologies. We analysed the differential methylation profile of a regulatory region of the gene using cerebral organoids generated from induced pluripotent stem cells (iPSCs) from adults with a diagnosis of ASD and from age- and gender-matched healthy individuals.

Combining Cortical Voltage Imaging and Hippocampal Electrophysiology for Investigating Global, Multi-Timescale Activity Interactions in the Brain.

A new generation of optogenetic tools for analyzing neural activity has been contributing to the elucidation of classical open questions in neuroscience. Specifically, voltage imaging technologies using enhanced genetically encoded voltage indicators have been increasingly used to observe the dynamics of large circuits at the mesoscale. Here, we describe how to combine cortical wide-field voltage imaging with hippocampal electrophysiology in awake, behaving mice.

Impaired phosphate transport in SLC34A2 variants in patients with pulmonary alveolar microlithiasis.

Background: Variants in SLC34A2 encoding the sodium-dependent phosphate transport protein 2b (NaPi-IIb) cause the rare lung disease pulmonary alveolar microlithiasis (PAM). PAM is characterised by the deposition of calcium-phosphate concretions in the alveoli usually progressing over time. No effective treatment is available.

Neurogenic and pericytic plasticity of conditionally immortalized cells derived from renal erythropoietin-producing cells.

In adult mammals, the kidney is the main source of circulating erythropoietin (Epo), the master regulator of erythropoiesis. In vivo data in mice demonstrated multiple subtypes of interstitial renal Epo-producing (REP) cells. To analyze the differentiation plasticity of fibroblastoid REP cells, we used a transgenic REP cell reporter mouse model to generate conditionally immortalized REP-derived (REPD) cell lines.

Fate-mapping of erythropoietin-producing cells in mouse models of hypoxaemia and renal tissue remodelling reveals repeated recruitment and persistent functionality.

Aim: Fibroblast-like renal erythropoietin (Epo) producing (REP) cells of the corticomedullary border region "sense" a decrease in blood oxygen content following anaemia or hypoxaemia. Burst-like transcription of Epo during tissue hypoxia is transient and is lost during fibrotic tissue remodelling, as observed in chronic kidney disease. The reason for this loss of Epo expression is under debate.

Cholinergic modulation of sensory processing in awake mouse cortex.

Cholinergic modulation of brain activity is fundamental for awareness and conscious sensorimotor behaviours, but deciphering the timing and significance of acetylcholine actions for these behaviours is challenging. The widespread nature of cholinergic projections to the cortex means that new insights require access to specific neuronal populations, and on a time-scale that matches behaviourally relevant cholinergic actions. Here, we use fast, voltage imaging of L2/3 cortical pyramidal neurons exclusively expressing the genetically-encoded voltage indicator Butterfly 1.

Reverse optogenetics of G protein signaling by zebrafish non-visual opsin Opn7b for synchronization of neuronal networks.

Opn7b is a non-visual G protein-coupled receptor expressed in zebrafish. Here we find that Opn7b expressed in HEK cells constitutively activates the G pathway and illumination with blue/green light inactivates G protein-coupled inwardly rectifying potassium channels. This suggests that light acts as an inverse agonist for Opn7b and can be used as an optogenetic tool to inhibit neuronal networks in the dark and interrupt constitutive inhibition in the light.

Cortex-Wide Dynamics of Intrinsic Electrical Activities: Propagating Waves and Their Interactions.

Cortical circuits generate patterned activities that reflect intrinsic brain dynamics that lay the foundation for any, including stimuli-evoked, cognition and behavior. However, the spatiotemporal organization properties and principles of this intrinsic activity have only been partially elucidated because of previous poor resolution of experimental data and limited analysis methods. Here we investigated continuous wave patterns in the 0.

Genetically Encoded Voltage Indicators.

Optogenetic approaches combine the power to allocate optogenetic tools (proteins) to specific cell populations (defined genetically or functionally) and the use of light-based interfaces between biological wetware (cells and tissues) and hardware (controllers and recorders). The optogenetic toolbox contains two main compartments: tools to interfere with cellular processes and tools to monitor cellular events. Among the latter are genetically encoded voltage indicators (GEVIs).

1,25(OH) vitamin D stimulates active phosphate transport but not paracellular phosphate absorption in mouse intestine.

Key Points: Intestinal absorption of phosphate proceeds via an active/transcellular route mostly mediated by NaPi-IIb/Slc34a2 and a poorly characterized passive/paracellular pathway. Intestinal phosphate absorption and expression of NaPi-IIb are stimulated by 1,25(OH) vitamin D but whether NaPi-IIb is the only target under hormonal control remains unknown. We report that administration of 1,25(OH) vitamin D to wild-type mice resulted in the expected increase in active transport of phosphate in jejunum, without changing paracellular fluxes.

Screening and Cellular Characterization of Genetically Encoded Voltage Indicators Based on Near-Infrared Fluorescent Proteins.

We developed genetically encoded voltage indicators using a transmembrane voltage-sensing domain and bright near-infrared fluorescent proteins derived from bacterial phytochromes. These new voltage indicators are excited by 640 nm light and emission is measured at 670 nm, allowing imaging in the near-infrared tissue transparency window. The spectral properties of our new indicators permit seamless voltage imaging with simultaneous blue-green light optogenetic actuator activation as well as simultaneous voltage-calcium imaging when paired with green calcium indicators.

Subcellular resolution three-dimensional light-field imaging with genetically encoded voltage indicators.

Light-field microscopy (LFM) enables high signal-to-noise ratio (SNR) and light efficient volume imaging at fast frame rates. Voltage imaging with genetically encoded voltage indicators (GEVIs) stands to particularly benefit from LFM's volumetric imaging capability due to high required sampling rates and limited probe brightness and functional sensitivity. We demonstrate subcellular resolution GEVI light-field imaging in acute mouse brain slices resolving dendritic voltage signals in three spatial dimensions.

Intestinal epithelial ablation of Pit-2/Slc20a2 in mice leads to sustained elevation of vitamin D upon dietary restriction of phosphate.

Aim: Several Na -dependent phosphate cotransporters, namely NaPi-IIb/SLC34A2, Pit-1/SLC20A1 and Pit-2/SLC20A2, are expressed at the apical membrane of enterocytes but their contribution to active absorption of phosphate is unclear. The aim of this study was to compare their pattern of mRNA expression along the small and large intestine and to analyse the effect of intestinal depletion of Pit-2 on phosphate homeostasis.

Methods: Intestinal epithelial Pit-2-deficient mice were generated by crossing floxed Pit-2 with villin-Cre mice.

A Novel Aβ Assembly at Physiological Concentration.

Aggregates of amyloid-β (Aβ) are characteristic of Alzheimer's disease, but there is no consensus as to either the nature of the toxic molecular complex or the mechanism by which toxic aggregates are produced. We report on a novel feature of amyloid-lipid interactions where discontinuities in the lipid continuum can serve as catalytic centers for a previously unseen microscale aggregation phenomenon. We show that specific lipid membrane conditions rapidly produce long contours of lipid-bound peptide, even at sub-physiological concentrations of Aβ.

Cre-mediated, loxP independent sequential recombination of a tripartite transcriptional stop cassette allows for partial read-through transcription.

One of the widely used applications of the popular Cre-loxP method for targeted recombination is the permanent activation of marker genes, such as reporter genes or antibiotic resistance genes, by excision of a preceding transcriptional stop signal. The STOP cassette consists of three identical SV40-derived poly(A) signal repeats and is flanked by two loxP sites. We found that in addition to complete loxP-mediated recombination, limiting levels of the Cre recombinase also cause incomplete recombination of the STOP cassette.

The proton-activated ovarian cancer G protein-coupled receptor 1 (OGR1) is responsible for renal calcium loss during acidosis.

Hypercalciuria is a common feature during metabolic acidosis and associates to nephrolithiasis and nephrocalcinosis. The mechanisms sensing acidosis and inducing increased urinary calcium excretion are still unknown. Here we tested whether mice deficient for proton-activated Ovarian cancer G-protein coupled receptor 1 (OGR1 or Gpr68) have reduced urinary excretion of calcium during chronic metabolic acidosis.

The serotonin 2A receptor agonist 25CN-NBOH increases murine heart rate and neck-arterial blood flow in a temperature-dependent manner.

Background: Serotonin 2A receptors, the molecular target of psychedelics, are expressed by neuronal and vascular cells, both of which might contribute to brain haemodynamic characteristics for the psychedelic state.

Aim: Aiming for a systemic understanding of psychedelic vasoactivity, here we investigated the effect of N-(2-hydroxybenzyl)-2,5-dimethoxy-4-cyanophenylethylamine - a new-generation agonist with superior serotonin 2A receptor selectivity - on brain-supplying neck-arterial blood flow.

Methods: We recorded core body temperature and employed non-invasive, collar-sensor based pulse oximetry in anesthetised mice to extract parameters of local blood perfusion, oxygen saturation, heart and respiration rate.