Biology subjects

Thunemann, M.

Publications and source records attributed to Thunemann, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Trisomy 21 Perturbs Oligodendrocyte Developmental States and Oligo-Neuron Communication in Human Forebrain Assembloids

Intellectual disability is a hallmark of Down syndrome (DS), caused by the triplication of human chromosome 21. Abnormal neuronal connectivity and impaired myelination are consistently observed in DS, yet the underlying mechanisms remain poorly understood. Here, we used DS-derived isogenic induced pluripotent stem cell-derived forebrain assembloids (FAs) to investigate oligodendrocyte lineage cell (OLLC) development and neuron-glia interactions. Trisomic FAs exhibited an expansion of BCAS1+ pre-myelinating OLLCs, without a corresponding increase in myelin basic protein (MBP) expression. Transcriptomic analyses identified a euploid-enriched, trophic and maturation-supportive state (OLLC_2-TMS) and a trisomy-enriched synaptic interactions and differentiation state (OLLC_3-SID), lacking coordinated enrichment in myelination-promoting programs. Corresponding transcriptional signatures were identified in OLLCs from early postnatal human cortex. Increased excitatory neuronal contacts with pre-myelinating OLLCs accompanied ultrastructural myelin abnormalities in trisomic assembloids. Our findings suggest that trisomy redirects OLLC development toward a pre-myelinating state, uncoupling oligodendrocyte differentiation from productive myelination.

neuroscience↗

bioRxiv2026

Depth-Dependent Alterations in Cerebral Blood and Tissue Oxygenation Associated with Tau in Alzheimer's Disease Models

Vascular dysfunction is an increasingly recognized feature of Alzheimer's disease, and inadequate oxygen delivery may contribute to neuronal death. However, how cerebral oxygenation relates to neuronal pathology remains unclear. Here, we used two-photon phosphorescence lifetime microscopy with Oxyphor2P to measure intravascular and tissue partial pressure of oxygen (pO2) at 0, 100 and 200 um cortical depth in awake rTg4510 and THY-Tau22 mice. Relative to WT, capillary and venular pO2 in rTg4510 mice were elevated at 6 months but reduced at 9 months, mainly in deeper layers, with increased oxygen extraction fraction. Suppressing the tau transgene with doxycycline for three months did not restore vascular pO2. THY-Tau22 mice, despite limited tangle pathology, showed even greater reductions in capillary pO2 at every depth. In 6-month-old rTg4510 mice, tissue pO2 trended lower than in WT, and the fraction of low pO2 values (< 18 mmHg) increased with depth, reaching 33% at 200 um versus 4% in WT. Labeling neurofibrillary tangles with the tau-binding dye HS84 revealed significantly lower tissue pO2 in tangle-positive areas at 100 and 200 um. Longitudinal imaging of 2,282 neurons showed that disappearing neurons were not located in relatively hypoxic tissue within an animal. Finally, ex vivo immunostaining revealed neuronal loss and increased astrogliosis in rTg4510 mice, with unchanged vascular architecture. These results indicate that tau disrupts cerebrovascular and tissue oxygenation in a depth- and age-dependent manner, and that neuronal loss is not explained by a neuron's local oxygen microenvironment but may reflect a global reduction in cortical oxygenation.

neuroscience↗

bioRxiv2026

Flexible 3D multimodal organoid interface reveals functional subnetworks and coordinated circuit activity in cortical organoids

The balance between excitatory and inhibitory (E/I) neurons is fundamental to cortical function, and its disruption underlies a wide range of neurological disorders, including epilepsy, Alzheimers disease and autism spectrum disorder. Cortical organoids offer a promising model to study this balance, as they recapitulate key features of brain development, including the emergence of E/I circuitry. However, no existing technology can simultaneously resolve neuronal identity, spatial organization, and dynamic network activity within intact organoids, limiting precise interrogation of E/I circuit function. Here, we report INFORM, an INtegrated Flexible Organoid Recording Multimodal neural interface that conformally wraps organoids of any size and shape, enabling long-term recordings over 371 days with above 90% of channels detecting spikes. INFORM interface combines four distinct modalities; electrophysiology, two-photon calcium imaging, targeted electrical microstimulation, and chemical intervention to monitor and modulate neural activity across multiple spatial and temporal scales spanning from action potentials from single neurons to synchronized bursts from large-scale populations. Such high-resolution interrogation enables probing of emergence of functional connectivity in fusing assembloids and resolves spatially discrete functional subnetworks that can be selectively recruited and modulated by INFORM. Neurotransmission blockade confirms that stimulus-evoked suppression of neuronal spiking is mediated by GABAergic inputs providing direct functional evidence of the presence of inhibitory signaling in organoids. This work allows single-cell dissection of functional networks in brain organoids, providing a powerful platform for modelling and studying cortical development, neurological diseases, and the E/I imbalances underlying circuit dysfunction.

neuroscience↗

bioRxiv2026

Dual-Mode Bio-CM2: Multimodal Computational Miniature Mesoscope for Fluorescence and Label-Free Imaging

Understanding biological function often requires complementary information on molecular identity, morphology, behavior, and physiology acquired simultaneously from the same specimen. Achieving this capability in miniature microscopes remains challenging because integrating multiple imaging contrasts typically increases optical complexity while compromising field of view (FOV), spatial resolution, or form factor. Here we present dual-mode Bio-CM2, a multimodal computational miniature mesoscope that simultaneously captures fluorescence and label-free reflectance through a shared optical architecture based on distributed computational optics. The system enables frame-interleaved, co-registered dual-mode imaging over a [~]7.5 x 10 mm2 FOV with [~]6 m lateral resolution. We demonstrate the versatility of dual-mode Bio-CM2 across diverse biological systems. In freely moving Caenorhabditis elegans, reflectance captures body posture and locomotor behavior that contextualize fluorescently labeled protein aggregates. In freely swimming larval zebrafish, reflectance captures whole-body morphology and swimming behavior, while fluorescence visualizes cardiac activity. In head-fixed mice, simultaneous fluorescence and reflectance imaging across the bilateral dorsal cortex provides complementary measurements of neuronal calcium activity and intrinsic hemodynamic signals. By integrating molecularly specific fluorescence with complementary reflectance contrast, dual-mode Bio-CM2 extends distributed computational optics into a scalable platform for multimodal miniature imaging.

bioengineering↗

bioRxiv2026

Blood-Mediated Opto-Acoustic Stimulation of Brain Cortex at Sub-millimeter Precision

Direct modulation of neural activity with high spatiotemporal precision is a cornerstone in experimental neuroscience. Here, we present a blood-mediated optoacoustic stimulation (BOAS) approach that utilizes blood as an endogenous transducer for brain stimulation. By delivering 532-nm nanosecond pulsed laser to the cortex, we demonstrate that the absorption of hemoglobin generates sufficient acoustic pressure to trigger neuronal activity. By integrating BOAS with calcium imaging in GCaMP6f-expressing mice, localized neuronal responses were observed. Quantitative analysis reveals that BOAS produces responses comparable to natural visual stimulation and is significantly more efficient than the photothermal stimulation. Furthermore, we show that the response is dose-dependent. At high energy doses, BOAS induces cortical spreading depression. Histological evaluation confirmed that the brain maintains tissue integrity even under these stimulation parameters. Together, this work establishes a versatile method for precise brain stimulation as an alternative method for stimulating neuron at cortex.

bioengineering↗

bioRxiv2026

Bio-CM{superscript 2}: Distributed computational optics for cortex-widecellular imaging

Understanding distributed biological systems, particularly neural circuits, requires simultaneous cellular-resolution imaging across millimeter-scale fields of view (FOV). Existing miniature microscopes remain fundamentally constrained by trade-offs among FOV, spatial resolution, and optical complexity, limiting their ability to bridge cellular microscopy with cortex-scale imaging. Here we introduce distributed computational optics, a framework that distributes image formation across coordinated optical modules and computationally integrates their measurements into a unified image. We realize this framework in Bio-CM2, a computational miniature mesoscope that partitions the imaging field across four optical modules while converging their measurements onto a common image sensor. This architecture overcomes the aberration-scaling limitations of conventional miniature optics while avoiding the hardware complexity of multi-camera systems and the contrast degradation associated with optical multiplexing. Bio-CM2 achieves a 7.5 x 10 mm2 FOV while enabling cellular-resolution in vivo imaging at video rates. We demonstrate its utility through two complementary imaging modalities in head-fixed mice: cortex-wide functional vascular imaging, enabling simultaneous quantification of pial arteriole vasomotion and mesoscale hemodynamic functional connectivity, and cellular-resolution calcium imaging, resolving the activity of over 3,000 neurons together with mesoscale neuronal functional connectivity. We further demonstrate the versatility of the platform through cellular-resolution imaging of entire coronal mouse brain sections, population-scale imaging of freely behaving Caenorhabditis elegans, and odor-evoked calcium imaging of the main olfactory bulb in head-fixed mice, highlighting its broad applicability across diverse biological systems and imaging modalities. By overcoming the conventional trade-off between FOV and spatial resolution in a compact miniature platform, Bio-CM2 establishes distributed computational optics as a scalable framework for multiscale biological imaging.

bioengineering↗

bioRxiv2026

Arteriolar GMP signaling revealed by FRET-based real-time measurement in vivo

cGMP evokes arteriolar vasorelaxation and is generated in smooth muscle by the soluble guanylyl cyclase (sGC) stimulated by endothelial NO. Phosphodiesterases (PDEs) degrade cGMP and may contribute in diameter regulation. We examined arteriolar cGMP levels in real-time in vivo upon NO and acetylcholine (ACh) and effects of PDE inhibition. Changes in cGMP were measured by FRET using the indicator protein cGi500. Arterioles in the cremaster muscle of anesthetized mice expressing cGi500 were exposed for imaging. They were stimulated with various NO donors (DEA/NO, SNP, SNAP) and ACh with or without PDE inhibition (non-specific: IBMX; PDE-5 specific: sildenafil). Furthermore, dilations induced by ACh and SNP were studied in sGC-deficient mice. DEA/NO and SNAP led to a fast, concentration-dependent rise of fluorescence ratio of cGi500 (by 4.0% at 10{micro}M), that declined on removal slowly. These ratio changes indicated cGMP increases as verified by inhibition of the sGC using ODQ. PDE inhibition increased cGMP levels slowly despite NO synthase inhibition. However, it did not amplify NO-induced ratio increases, but the decline after drug removal was decelerated. Interestingly, ACh did not modify cGMP levels and dilations were not impaired in sGC-deficient mice. FRET-based imaging allows reliable real-time assessment of arteriolar cGMP levels in vivo. PDE activity does not limit the amplitude, but the duration of cGMP signaling after stimulation. Furthermore, we conclude that ACh does not release endothelial NO in murine arterioles. Preliminary experiments demonstrate that simultaneous measurement of cGMP and diameter is feasible using mice with expression of cGi500 in smooth muscle cells.

physiology↗

bioRxiv2026

A pipeline for cell migration analysis in live-cell imaging data from human iPSC-derived forebrain assembloids.

During forebrain development, inhibitory interneurons and oligodendrocyte progenitor cells migrate long distances into the developing dorsal cortex. Human induced pluripotent stem cell-derived forebrain assembloids (FAs) provide direct experimental access to this migratory process in vitro. Using viral labeling to express yellow fluorescent protein (EYFP) and tandem-dimer tomato (tdTomato) driven by EF1 or SOX10 promoters, respectively, we tracked cells in FAs over 15-17h using spinning disk confocal microscopy. We developed an end-to-end processing pipeline for 4D volumetric imaging data, consisting of background subtraction and drift correction, manual cell coordinate tracking, and an analysis workflow to describe migratory cell behavior. Image preprocessing significantly improved data quality for subsequent manual tracking in datasets with heterogeneous labeling density and brightness. Trajectory analysis of 336 EYFP- and 337 tdTomato-labeled cells from twelve FAs indicates that most cells show super-diffusive directed motility. Our pipeline represents a key resource for cell tracking in FAs and similar three-dimensional platforms. This pipeline represents the first open tracking resource for iPSC-derived FAs and can be used as a ground-truth resource for the development of automated cell detection and tracking algorithms.

neuroscience↗

bioRxiv2026

High-Field Multinuclear MRI Reveals Sodium Relaxation Heterogeneity in Cortical Organoids

Sodium magnetic resonance imaging (23Na MRI) provides a unique opportunity to probe ionic microenvironments in neural tissue because sodium ions play central roles in membrane electrophysiology, ion transport, and cellular homeostasis. Unlike conventional proton ({superscript 1}H) MRI, which primarily reflects water distribution and tissue structure, {superscript 2}3Na MRI is sensitive to ionic compartmentation and quadrupolar interactions arising from the spin-3/2 nature of the sodium nucleus. However, sodium MRI remains technically challenging due to intrinsically low signal sensitivity and rapid biexponential relaxation, particularly when imaging small biological systems. Here, we establish a high-field multinuclear MRI platform for imaging human cerebral organoids at 14 Tesla. Cerebral organoids derived from human induced pluripotent stem cells provide a simplified three-dimensional neural tissue model that enables investigation of ionic microenvironments without vascular or systemic confounds. Using a dual-tuned {superscript 1}H/{superscript 2}3Na radiofrequency coil, we performed co-registered structural, diffusion, and sodium imaging of individual fixed organoids. High-resolution {superscript 1}H MRI (33-100 m) revealed pronounced microstructural heterogeneity, while multi-echo {superscript 2}3Na MRI (300-400 m) enabled voxel-wise characterization of quadrupolar relaxation behavior. Bi-exponential analysis of the sodium signal decay identified distinct relaxation components (T2*short {approx} 1 ms and T2*long {approx} 12 ms) and revealed spatial heterogeneity in sodium microenvironments across the organoid tissue. These results demonstrate the feasibility of quantitative sodium relaxometry in cortical organoids and establish a multinuclear imaging platform for investigating ionic microenvironment dynamics in three-dimensional neural tissue models.

bioengineering↗

bioRxiv2026

Seeing clearly with CLARI-O: a window into cellular architecture, interactions, and morphology of organoid models.

Cortical organoids (COs) represent a powerful in vitro model system that recapitulates key aspects of human brain development, enabling the study of neurodevelopmental processes, cellular diversity, and disease mechanisms in a physiologically relevant 3D environment. However, traditional histological analysis of COs relies on tissue sectioning, which limits the ability to capture the full spatial complexity of organoid architecture. In this study, we establish a framework for applying CLARI-O, an improved tissue-clearing technique, for intact COs and organoid-based systems, enabling comprehensive 3D visualization and analysis of 3D organizational features. Using CLARI-O in combination with high-resolution imaging, we demonstrate the utility of tissue clearing for studying glial populations, including oligodendrocytes and microglia, considered to be underrepresented in COs, and their interactions with neurons. Additionally, we apply this method to forebrain assembloids (FAs) to visualize cellular heterogeneity and the interface between ventral and dorsal regions. Finally, we use CLARI-O to study mouse brains containing xenotransplanted COs (MB-COs) to evaluate human cell integration, migration, vascularization, and structural connectivity. This is the first study to demonstrate how tissue clearing can be used after functional assays such as calcium imaging to correlate neural activity with post hoc structural analysis in MB-COs. Together, this work establishes CLARI-O as a powerful tool for advancing 3D structural and functional interrogation of human CO-derived systems, enhancing their value for disease modeling, drug screening, and translational neuroscience. MotivationCortical organoids have become an increasingly powerful tool in neuroscience. Their complexity has expanded substantially, now incorporating exogenous lineages, fusing organoids with distinct regional identities (assembloids), and enabling xenotransplantation into in-vivo environments. These advancements require more sophisticated technological approaches that are capable of capturing the intricate three-dimensional cyotarchitecture and organization of intact organoid systems both in vitro and after xenotransplantation in vivo. Tissue-clearing methodologies offer a unique opportunity to visualize these structural and cellular features with exceptional depth and resolution. Graphical abstract HighlightsO_LIWe optimized clearing protocols to develop an organoid specific clearing method (CLARI-O) that enables high-resolution visualization of diverse neuronal and glial populations without tissue sectioning, preserving long-range connections and cellular processes. C_LIO_LIForebrain assembloids used to study neuronal and oligodendrocyte migration can be effectively processed using CLARI-O, allowing detailed visualization of fusion interface. C_LIO_LIWe established a robust framework for CLARI-O-based clearing of mouse brain tissue containing xenotransplanted human cortical organoids, enabling comprehensive 3D analysis of graft development, integration, and vascularization in vivo. C_LI

neuroscience↗

bioRxiv2025

A neurorecording toolkit for longitudinal assessments of transplanted human cortical organoids in vivo

Human cortical organoids (hCOs) are three-dimensional neural cell aggregates that recapitulate certain structural and functional aspects of the developing human cortex. Xenotransplantation of hCOs into the rodent brain enables human-centric modeling of neurodevelopmental processes in a physiologically relevant environment. Here, we present a neurorecording toolkit for longitudinal structural and functional assessment of hCO xenografts as they mature in vivo. Single hCOs were implanted into the retrosplenial cortex of adult immunodeficient mice and monitored for up to 8 months. Optical coherence tomography was used for label-free imaging of xenograft vascularization and structure, enabling quantitative assessments of capillary density and graft volume. To probe neuronal activity, human neurons were labeled with a calcium sensor before implantation using either adeno-associated or lentivirus for sparse or dense neuronal labeling, respectively. Fluorescent imaging was conducted using two-photon, widefield, and swept confocally-aligned planar excitation microscopy for single cell, whole-graft, and volumetric calcium imaging, respectively. Results from these modalities indicate an increase in neuronal activity and synchronicity over time during in vivo graft maturation. Further, we chronically implanted surface graphene microelectrode arrays (gMEAs) and performed recordings of xenograft and host local field potential signals simultaneously with 2P calcium imaging, confirming the spatial localization and human origin of electrical signals recorded at the xenograft surface.

neuroscience↗

bioRxiv2024

Neurovascular Impulse Response Function (IRF) during spontaneous activity differentially reflects intrinsic neuromodulation across cortical regions

Ascending neuromodulatory projections from deep brain nuclei generate internal brain states that differentially engage specific neuronal cell types. Because neurovascular coupling is cell-type specific and neuromodulatory transmitters have vasoactive properties, we hypothesized that the impulse response function (IRF) linking spontaneous neuronal activity with hemodynamics would depend on neuromodulation. To test this hypothesis, we used optical imaging to measure (1) release of neuromodulatory transmitters norepinephrine (NE) or acetylcholine (ACh), (2) Ca2+ activity of local cortical neurons, and (3) changes in hemoglobin concentration and oxygenation across the dorsal surface of cerebral cortex during spontaneous neuronal activity in awake mice. A canonical convolution model with a global, stationary IRF (i.e., the convolution kernel) describing evolution of total hemoglobin (HbT, reflective of dilation dynamics) with respect to Ca2+, resulted in a poor fit to the data. However, the HbT time-course was well predicted, pixel-by-pixel, by a weighted sum of Ca2+ and NE time-courses. Consistent with this result, modeling HbT as a weighted sum of stationary Ca2+ - and NE-specific IRFs (IRFCa2+ and IRFNE) convolved with the respective time-courses dramatically improved the fit compared to the global IRF. IRFCa2+ and IRFNE, estimated from the data, were positive and negative, respectively. In contrast to NE, ACh was largely redundant with Ca2+ and therefore did not improve HbT estimation. Because NE covaried with arousal, we observed instances of the diminished hemodynamic coherence between cortical regions during high arousal despite coherent behavior of the underlying neuronal Ca2+ activity. We conclude that while neurovascular coupling with respect to neuronal Ca2+ is a dynamic and seemingly complex phenomenon, hemodynamic fluctuations can be captured by a simple linear model with stationary IRFs with respect to the underlying dilatory and constrictive forces. In the current study, these forces were captured by the positive IRFCa2+ (dilation) and negative IRFNE (constriction). Without accounting for NE neuromodulation and the associated vasoconstriction, diminished hemodynamic coherence, commonly referred to as "functional (dys)connectivity" in BOLD fMRI studies, can be falsely interpreted as neuronal desynchronizations.

neuroscience↗

bioRxiv2024

Visual stimulation drives retinotopic acetylcholine release in the mouse visual cortex

Cholinergic signaling is involved with a variety of brain functions including learning and memory, attention, and behavioral state modulation. The spatiotemporal characteristics of neocortical acetylcholine (ACh) release in response to sensory inputs are poorly understood, but a lack of intra-region topographic organization of cholinergic projections from the basal forebrain has suggested diffuse release patterns and volume transmission. Here, we use mesoscopic imaging of fluorescent ACh sensors to show that visual stimulation results in ACh release patterns that conform to a retinotopic map of visual space in the mouse primary visual cortex, suggesting new modes of functional cholinergic signaling in cortical circuits.x

neuroscience↗

bioRxiv2023

Widefield in vivo imaging system with two fluorescence and two reflectance channels, a single sCMOS detector, and shielded illumination

SignificanceWidefield microscopy of the entire dorsal part of mouse cerebral cortex enables large-scale ("mesoscopic") imaging of different aspects of neuronal activity with spectrally compatible fluorescent indicators as well as hemodynamics via oxy- and deoxyhemoglobin absorption. Versatile and cost-effective imaging systems are needed for large-scale, color-multiplexed imaging of multiple fluorescent and intrinsic contrasts. AimDevelop a system for mesoscopic imaging of two fluorescent and two reflectance channels. ApproachExcitation of red and green fluorescence is achieved through epi-illumination. Hemoglobin absorption imaging is achieved using 525- and 625-nm LEDs positioned around the objective lens. An aluminum hemisphere placed between objective and cranial window provides diffuse illumination of the brain. Signals are recorded sequentially by a single sCMOS detector. ResultsWe demonstrate performance of our imaging system by recording large-scale spontaneous and stimulus-evoked neuronal, cholinergic, and hemodynamic activity in awake head-fixed mice with a curved "crystal skull" window expressing the red calcium indicator jRGECO1a and the green acetylcholine sensor GRABACh3.0. Shielding of illumination light through the aluminum hemisphere enables concurrent recording of pupil diameter changes. ConclusionsOur widefield microscope design with single camera can be used to acquire multiple aspects of brain physiology and is compatible with behavioral readouts of pupil diameter.

neuroscience↗

bioRxiv2023

Guide to the construction and use of an adaptive optics two-photon microscope with direct wavefront sensing

Two-photon microscopy, combined with appropriate optical labeling, has enabled the study of structure and function throughout nervous systems. This methodology enables, for example, the measurement and tracking of sub-micrometer structures within brain cells, the spatio-temporal mapping of spikes in individual neurons, and the spatio-temporal mapping of transmitter release in individual synapses. Yet the spatial resolution of two-photon microscopy rapidly degrades as imaging is attempted at depths more than a few scattering lengths into tissue, i.e., below the superficial layers that constitute the top 300 to 400 {micro}m of neocortex. To obviate this limitation, we measure the wavefront at the focus of the excitation beam and utilize adaptive optics that alters the incident wavefront to achieve an improved focal volume. We describe the constructions, calibration, and operation of a two-photon microscopy that incorporates adaptive optics to restore diffraction-limited resolution throughout the nearly 900 {micro}m depth of mouse cortex. Our realization utilizes a guide star formed by excitation of red-shifted dye within the blood serum to directly measure the wavefront. We incorporate predominantly commercial optical, optomechanical, mechanical, and electronic components; computer aided design models of the exceptional custom components are supplied. The design is modular and allows for expanded imaging and optical excitation capabilities. We demonstrate our methodology in mouse neocortex by imaging the morphology of somatostatin-expressing neurons at 700 {micro}m beneath the pia, calcium dynamics of layer 5b projection neurons, and glutamate transmission to L4 neurons.

neuroscience↗

bioRxiv2022

Imaging through Windansee electrode arrays reveals a small fraction of local neurons following surface MUA

Prior studies have shown that neuronal spikes can be recorded with microelectrode arrays placed on the cortical surface. However, the etiology of these spikes remains unclear. Because the top cortical layer (layer 1) contains very few neuronal cell bodies, it has been proposed that these spikes originate from neurons with cell bodies in layer 2. To address this question, we combined two-photon calcium imaging with electrophysiological recordings from the cortical surface in awake mice using chronically implanted PEDOT:PSS electrode arrays on transparent parylene C substrate. Our electrode arrays (termed Windansee) were integrated with cortical windows offering see-through optical access while also providing measurements of local field potentials (LFP) and multiunit activity (MUA) from the cortical surface. To enable longitudinal data acquisition, we have developed a mechanical solution for installation, connectorization, and protection of Windansee devices aiming for an unhindered access for high numerical aperture microscope objectives and a lifetime of several months while worn by a mouse. Contrary to the common notion, our measurements revealed that only a small fraction of layer 2 neurons from the sampled pool (~13%) faithfully followed MUA recorded from the surface above the imaging field-of-view. Surprised by this result, we turned to computational modeling for an alternative explanation of the MUA signal. Using realistic modeling of neurons with back-propagating dendritic properties, we computed the extracellular action potential at the cortical surface due to firing of local cortical neurons and compared the result to that due to axonal inputs to layer 1. Assuming the literature values for the cell/axon density and firing rates, our modeling results show that surface MUA due to axonal inputs is over an order of magnitude larger than that due to firing of layer 2 pyramidal neurons. Thus, a combination of surface MUA recordings with two-photon calcium imaging can provide complementary information about the input to a cortical column and the local circuit response. Cortical layer I plays an important role in integration of a broad range of cortico-cortical, thalamocortical and neuromodulatory inputs. Therefore, detecting their activity as MUA while combining electrode recording with two-photon imaging using optically transparent surface electrode arrays would facilitate studies of the input/output relationship in cortical circuits, inform computational circuit models, and improve the accuracy of the next generation brain-machine interfaces.

neuroscience↗

bioRxiv2022

Multimodal monitoring of human cortical organoids implanted in mice using transparent graphene microelectrodes reveal functional connection between organoid and mouse visual cortex

Human cortical organoids, three-dimensional neuronal cell cultures derived from human induced pluripotent stem cells, have recently emerged as promising models of human brain development and dysfunction. Transplantation of human brain organoids into the mouse brain has been shown to be a successful in vivo model providing vascularization for long term chronic experiments. However, chronic functional connectivity and responses evoked by external sensory stimuli has yet to be demonstrated, due to limitations of chronic recording technologies. Here, we develop an experimental paradigm based on transparent graphene microelectrode arrays and two-photon imaging for longitudinal, multimodal monitoring of human organoids transplanted in the mouse cortex. The transparency of graphene microelectrodes permits visual and optical inspection of the transplanted organoid and the surrounding cortex throughout the chronic experiments where local field potentials and multi-unit activity (MUA) are recorded during spontaneous activity and visual stimuli. These experiments reveal that visual stimuli evoke electrophysiological responses in the organoid, matching the responses from the surrounding cortex. Increases in the power of the gamma and MUA bands as well as phase locking of MUA events to slow oscillations evoked by visual stimuli suggest functional connectivity established between the human and mouse tissue. Optical imaging through the transparent microelectrodes shows vascularization of the organoids. Postmortem histological analysis exhibits morphological integration and synaptic connectivity with surrounding mouse cortex as well as migration of organoid cells into the surrounding cortex. This novel combination of stem cell and neural recording technologies could serve as a unique platform for comprehensive evaluation of organoids as models of brain development and dysfunction and as personalized neural prosthetics to restore lost, degenerated, or damaged brain regions.

neuroscience↗

bioRxiv2021

Microscopic Quantification of Oxygen Consumption across Cortical Layers

The cerebral cortex is organized in cortical layers that differ in their cellular density, composition, and wiring. Cortical laminar architecture is also readily revealed by staining for cytochrome oxidase - the last enzyme in the respiratory electron transport chain located in the inner mitochondrial membrane. It has been hypothesized that a high-density band of cytochrome oxidase in cortical layer IV reflects higher oxygen consumption under baseline (unstimulated) conditions. Here, we tested the above hypothesis using direct measurements of the partial pressure of O2 (pO2) in cortical tissue by means of 2-photon phosphorescence lifetime microscopy (2PLM). We revisited our previously developed method for extraction of the cerebral metabolic rate of O2 (CMRO2) based on 2-photon pO2 measurements around diving arterioles and applied this method to estimate baseline CMRO2 in awake mice across cortical layers. To our surprise, our results revealed a decrease in baseline CMRO2 from layer I to layer IV. This decrease of CMRO2 with cortical depth was paralleled by an increase in tissue oxygenation. Higher baseline oxygenation and cytochrome density in layer IV may serve as an O2 reserve during surges of neuronal activity or certain metabolically active brain states rather than baseline energy needs. Our study provides the first quantification of microscopically resolved CMRO2 across cortical layers as a step towards informed interpretation and modeling of cortical-layer-specific Blood Oxygen Level Dependent (BOLD) functional Magnetic Resonance Imaging (fMRI) signals.

neuroscience↗