Biology subjects

Rose, C. R.

Publications and source records attributed to Rose, C. R..

12 recordsLinked to original sources

bioRxiv2025

Local differences in baseline sodium shape astrocytic potassium uptake by the NKA

Astrocytes are vital for the maintenance of ion and transmitter homeostasis in the extracellular space, with the inward Na gradient playing a pivotal role in these processes. Earlier studies not only reported a low baseline Na+ concentration ([Na+]) in astrocytes, but also suggested an equilibration of [Na+] within the gap-junction-coupled syncytium. This is consistent with the view that the basic homeostatic properties of astrocytes are largely identical due to their critical role in brain function. Here, we used multi-photon fluorescence lifetime imaging for a quantitative determination of astrocytic [Na+] in mouse forebrain tissue slices and in vivo. Contrary to the prevailing notion of a rather uniform Na distribution, we detected a previously unobserved subcellular and cellular heterogeneity in astrocytic [Na+], accompanied by differences in the capacity for Na+/K+-ATPase (NKA)-mediated uptake of extracellular K+. Biophysical modelling showed that this heterogeneity can be replicated by the reported differential expression of NKA isoforms in astrocytes together with a different strength of Na+ influx over the plasma membranes. Altogether, our results thus suggest the existence of functionally distinct astrocytes and astrocyte subdomains in which Na+ homeostasis is locally adapted to the specific requirements of surrounding neural networks.

neuroscience↗

bioRxiv2025

The role of the glutamate-glutamine cycle in synaptic transmission during ischemia and recovery

Cerebral ischemia impairs neuronal and glial function, ranging from synaptic failure to irreversible damage. The effects of ischemia on excitatory synaptic transmission remain incompletely understood. Here, we present a detailed biophysical model including the first full implementation of the glutamate-glutamine cycle (GG-cycle), which is essential for proper functioning of glutamatergic synapses. We simulate a presynaptic neuron and an astrocyte in a finite extracellular space (ECS), surrounded by an oxygen bath, as a proxy for energy supply. The model includes ionic currents with corresponding channels and transporters such as the sodium-potassium AT-Pase. To model synaptic transmission, we combine calcium-dependent glutamate release, its uptake by the sodium-dependent excitatory amino acid transporters (EAATs), and the GG-cycle, including glutamine synthesis. We simulate ischemia by blocking energy supply completely. The neuron enters a depolarization block, ion concentrations reach pathological values, and glutamate accumulates in the ECS while glutamate release is disrupted. Surprisingly, we found that synaptic transmission failure was not primarily caused by excessive glutamate release nor by failure of glutamine synthetase. Instead, it mainly resulted from EAAT dysfunction, driven by the collapse of the sodium gradient. Enhancing glutamate clearance alone was insufficient for recovery of synaptic transmission. However, inhibition of the voltage-gated Na+ channels restored ion gradients, recovered glutamate uptake, and re-enabled glutamate release. Taken together, our study highlights the critical role of ion homeostasis, in particular the sodium gradient, in maintaining synaptic function during metabolic stress. Moreover, the model provides a better understanding of synaptic transmission failure and potential recovery strategies during ischemia.

neuroscience↗

bioRxiv2025

Spatio-temporal dynamics of lateral Na+ diffusion in apical dendrites of mouse CA1 pyramidal neurons

Sodium ions (Na+) are major charge carriers mediating neuronal excitation and play a fundamental role in brain physiology. Glutamatergic synaptic activity is accompanied by large transient Na+ increases, but the spatio-temporal dynamics of Na+ signals and properties of Na+ diffusion within dendrites are largely unknown. To address these questions, we employed multi-photon Na+ imaging combined with whole-cell patch-clamp in dendrites of CA1 pyramidal neurons in tissue slices from mice of both sexes. Fluorescence lifetime microscopy revealed a dendritic baseline Na+ concentration of ~10 mM. Using intensity-based line-scan imaging we found that local, glutamate-evoked Na+ signals spread rapidly within dendrites, with peak amplitudes decreasing and latencies increasing with increasing distance from the site of stimulation. Spread of Na+ along dendrites was independent of dendrite diameter, order or overall spine density in the ranges measured. Our experiments also show that dendritic Na+ readily invades spines and suggest that spine necks may represent a partial diffusion barrier. Experimental data were well reproduced by mathematical simulations assuming normal diffusion with a diffusion coefficient of [Formula]. Modeling moreover revealed that lateral diffusion is key for the clearance of local Na+ increases at early time points, whereas when diffusional gradients are diminished, Na+/K+-ATPase becomes more relevant. Taken together, our study thus demonstrates that Na+ influx causes rapid lateral diffusion of Na+ within spiny dendrites. This results in an efficient redistribution and fast recovery from local Na+ transients which is mainly governed by concentration differences. Significance statementActivity of excitatory glutamatergic synapses generates large Na+ transients in postsynaptic cells. Na+ influx is a main driver of energy consumption and modulates cellular properties by modulating Na+-dependent transporters. Knowing the spatio-temporal dynamics of dendritic Na+ signals is thus critical for understanding neuronal function. To study propagation of Na+ signals within spiny dendrites, we performed fast Na+ imaging combined with mathematical simulations. Our data shows that normal diffusion, based on a diffusion coefficient of 600 {micro}m2/s, is crucial for fast clearance of local Na+ transients in dendrites, whereas Na+ export by the Na+/K+-ATPase becomes more relevant at later time points. This fast diffusive spread of Na+ will reduce the local metabolic burden imposed by synaptic Na+ influx.

neuroscience↗

bioRxiv2025

Atypical plume-like events drive glutamate accumulation in metabolic stress conditions

Neural glutamate homeostasis plays a key role in health and disease. In ischemic conditions, such as stroke, this homeostasis is severely disrupted since energy depletion and ion imbalances lead to more glutamate release and less uptake. We here used the fluorescent glutamate sensor SF-iGluSnFR(A184V) to probe the effects of chemical ischemia on extracellular glutamate dynamics in situ, using organotypic slice cultures from mouse cortex. SF-iGluSnFR imaging reported spontaneous glutamate release events, which indicate synchronous network activity, similar to calcium signals detected with GCaMP6f. In addition, glutamate imaging revealed local, asynchronous release events, which were atypically large and long-lasting and showed plume-like characteristics. Under baseline conditions plumes occurred with low frequency, were independent of network activity, and persisted in the presence of TTX. Plume induction was strongly favored by blocking glutamate uptake with TFB-TBOA, whereas blocking ionotropic glutamate receptors (iGluRs) suppressed plumes. Upon inducing chemical ischemia plumes became more pronounced and overly abundant, which resulted in large-scale accumulation of extracellular glutamate. Similar plumes were recently also observed in models of cortical spreading depression and migraine. We therefore propose that plumes represent a more general phenomenon induced by glutamate uptake dysfunction, which may contribute to glutamate-related excitotoxicity in various neurodegenerative and neurological disorders.

neuroscience↗

bioRxiv2024

A 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 study 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 fluorophores employed exhibit low brightness and low Signal-to-Noise ratio (SNR). As a consequence, manual intervention may become a necessity, introducing variability in the analysis outcomes even for identical samples when analyzed by different users. This leads to the incorporation of blinded analysis which ensures that the outcome is free from user bias to a certain extent but is extremely time-consuming. To overcome these issues, we have developed a tool called DL-SCAN that automatically segments and analyzes fluorophore-stained regions of interest such as cell bodies in fluorescence microscopy images using a Deep Learning algorithm called Stardist. We demonstrate the programs ability to automate cell identification and study cellular ion dynamics using synthetic image stacks with varying SNR. This is followed by its application to experimental Na+ and Ca2+ imaging data from neurons and astrocytes in mouse brain tissue slices exposed to transient chemical ischemia. The results from DL-SCAN are consistent, reproducible, and free from user bias, allowing efficient and rapid analysis of experimental data in an objective manner. The open-source nature of the tool also provides room for modification and extension to analyze other forms of microscopy images specific to the dynamics of different ions in other cell types. Statement of SignificanceFluorescence microscopy is widely used to study the functional and morphological features of living cells. However, various factors, such as low SNR, background noise, drift in the signal, movement of the tissue, and the large size of the resulting imaging data, make the processing of fluorescence microscopy data prone to errors, user bias, and extremely time-consuming. These and other issues hinder the full utilization of these powerful experimental techniques. Our novel Deep Learning-based tool overcomes these issues by processing and analyzing fluorescence imaging data, e.g., enabling automated visualization of ion changes in living cells in brain slices. Yet the tool remains easy to use with a streamlined workflow.

neuroscience↗

bioRxiv2024

Discovery of orally active SARS-CoV-2 papain-like protease (PLpro) inhibitors

Vaccines and first-generation antiviral therapeutics have provided important protection against coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there remains a need for additional therapeutic options that provide enhanced efficacy and protection against potential viral resistance. The SARS-CoV-2 papain-like protease (PLpro) is one of two essential cysteine proteases involved in viral replication. While inhibitors of the SARS-CoV-2 main protease (Mpro) have demonstrated clinical efficacy, known PLpro inhibitors have to date lacked the inhibitory potency and requisite pharmacokinetics to demonstrate that targeting PLpro translates to in vivo efficacy in a preclinical setting. Herein, we report the machine learning-driven discovery of potent, selective, and orally available SARS-CoV-2 PLpro inhibitors, with lead compound PF-07957472 (4) providing robust efficacy in a mouse-adapted model of COVID-19 infection.

microbiology↗

bioRxiv2023

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 iPSC lines. These High Quantity brain organoids (Hi-Q brain organoids) exhibit reproducible cytoarchitecture, cell diversity, and functionality, are free from ectopically active cellular stress pathways, and allow cryopreservation and re-culturing. Using patient-derived Hi-Q brain organoids, we recapitulated distinct forms of microcephaly pathogenesis: primary microcephaly due to a mutation in centrosomal CDK5RAP2 and progeria-associated microcephaly in Cockayne syndrome. When modeling glioma invasion, hi-Q brain organoids displayed a similar invasion pattern for a given patient-derived glioma cell line. This enabled a medium-throughput screen to identify Selumetinib and Fulvestrant, which also perturbed glioma invasion in vivo. Thus, the Hi-Q approach can easily be adapted to reliably harness brain organoids utility for personalized disease modeling and drug discovery.

cell biology↗

bioRxiv2022

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 avnalysis of fluorescence signals requires 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. Moreover, event detection algorithms need to be sensitive enough to accurately capture localized and low amplitude events exhibiting a limited spatial extent. Here, we present two algorithms (PBasE and CoRoDe) for accurate baseline estimation of fluorescent detection of messenger molecules and automated detection of fluorescence fluctuations. Author summaryAnalyzing molecular signalling is crucial in understanding intra- and intercellular communication. These signals are visualized using fluorescent dyes or genetically encoded fluorescence indicators. In the brain, Ca2+ signals of glial cells are essential in deciphering complex regulatory functions in health and disease. Due to signal heterogeneity, detection and analysis are highly challenging. They can be stationary, with low amplitude and localized in cell processes, occur as prominent somatic signals or propagate as waves across cellular networks. We have developed two algorithms to analyze fluorescence transients, each tackling a specific problem. PBasE performs automatic and adaptive background correction, removing basal fluorescence fluctuations. CoRoDe automatically extracts regions of interest, explicitly including temporal information to obtain a precise segmentation, which is essential for accurate transient extraction. Combined, these algorithms are able to detect regions exhibiting low amplitude transients with small spatial extent as well as large, high amplitude signals. Extracted transients are categorized based on their peak amplitude, allowing detailed analyses by comparing changes of specific properties. In order to make these algorithms accessible, an interactive application, called Msparkles, has been designed.

neuroscience↗

bioRxiv2022

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

Elevated intracranial pressure (ICP) is observed in many neurological pathologies, e.g. hydrocephalus and stroke. This condition is routinely relieved with neurosurgical approaches, since effective and targeted pharmacological tools are still lacking. The carbonic anhydrase inhibitor, acetazolamide (AZE), may be employed to treat elevated ICP. However, its effectiveness is questioned, its location of action unresolved, and its tolerability low. Here, we employed in vivo and ex vivo approaches to reveal the efficacy and mode of action of AZE in the rat brain. The drug effectively reduced the ICP, irrespective of the mode of drug administration and level of anaesthesia. The effect occurred via a direct action on the choroid plexus and an associated decrease in cerebrospinal fluid secretion, and not indirectly via the systemic action of AZE on renal and vascular processes. Upon a single administration, the reduced ICP endured for approximately 10 h post-AZE delivery with no long-term changes of brain water content or choroidal transporter expression. However, a persistent reduction of ICP was secured with repeated AZE administrations throughout the day. Future specific targeting of choroidal carbonic anhydrases may limit the systemic side effects, and therefore enhance the treatment tolerability and effectiveness in select patient groups experiencing elevated ICP.

neuroscience↗

bioRxiv2021

Cerebrospinal fluid formation is controlled by membrane transporters to modulate intracranial pressure

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 molecular mechanisms governing cerebrospinal fluid (CSF) secretion are largely unresolved, thus preventing targeted and efficient pharmaceutical therapy of cerebral pathologies involving elevated ICP. Here, we employed experimental rats to demonstrate low osmotic water permeability of the choroid plexus, lack of an osmotic gradient across this tissue, and robust CSF secretion against osmotic gradients. Together, these results illustrate that CSF secretion occurs independently of conventional osmosis, which challenges the existing assumption that CSF production is driven entirely by bulk osmotic forces across the CSF-secreting choroid plexus. Instead, we reveal that the choroidal Na+/K+/Cl- cotransporter NKCC1, Na+/HCO3- cotransporter NBCe2, and Na+/K+-ATPase are actively involved in CSF production and propose a molecular mode of water transport supporting CSF production in this secretory tissue. Further, we demonstrate that inhibition of NKCC1 directly reduces the ICP, illustrating that altered CSF secretion may be employed as a strategy to modulate ICP. These insights identify new promising therapeutic targets against brain pathologies associated with elevated ICP.

neuroscience↗

bioRxiv2021

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. The severity of ischemic damage depends on the age and the brain region in which a stroke occurs, but the reasons for this differential vulnerability are far from understood. In the present study, we address this question by developing a comprehensive biophysical model of a glutamatergic synapse to identify key determinants of synaptic failure during energy deprivation. Our model is based on fundamental biophysical principles, includes dynamics of the most relevant ions, i.e., Na+, K+, Ca2+, Cl- and glutamate, and is calibrated with experimental data. It confirms the critical role of the Na+/K+-ATPase in maintaining ion gradients, membrane potentials and cell volumes. Our simulations demonstrate that the system exhibits two stable states, one physiological and one pathological. During energy deprivation, the physiological state may disappear, forcing a transit to the pathological state, which can be reverted when blocking voltage-gated Na+ and K+ channels. Our model predicts that the transition to the pathological state is favoured if the extracellular space fraction is small. A reduction in the extracellular space volume fraction, as, e.g. observed with ageing, will thus promote the brains susceptibility to ischemic damage. Our work provides new insights into the brains ability to recover from energy deprivation, with translational relevance for diagnosis and treatment of ischemic strokes. Author summaryThe brain consumes energy to keep ion concentrations at normal working conditions. In the case of energy deprivation (ED), e.g., during a stroke, synaptic communication fails first. Inspired by our recent experimental work on ED, we formulated a novel computational model to explore initial events during ED. Our model reproduces time courses for several ions from different experimental data. In some cases, the system returns to baseline upon restoring energy supply. In others, we observe that neurons and astrocytes cannot recover accompanied by cell swelling. There is a threshold depending on the depth and duration of ATP depletion differentiating these cases. Also, smaller extracellular spaces hamper recovery more. This result may explain clinical observations of increased vulnerability to stroke as the size of the extracellular space shrinks with ageing.

biophysics↗

bioRxiv2020

On the Origin of Ultraslow Spontaneous Na+ Fluctuations in Neurons of the Neonatal Forebrain

Spontaneous neuronal and astrocytic activity in the neonate forebrain is believed to drive the maturation of individual cells and their integration into complex brain-region-specific networks. The previously reported forms include bursts of electrical activity and oscillations in intracellular Ca2+ concentration. Here, we use ratiometric Na+ imaging to demonstrate spontaneous fluctuations in the intracellular Na+ concentration of CA1 pyramidal neurons and astrocytes in tissue slices obtained from the hippocampus of mice at postnatal days 2-4 (P2-4). These occur at very low frequency ([~]2/h), can last minutes with amplitudes up to several mM, and mostly disappear after the first postnatal week. To further study the mechanisms that may generate such spontaneous fluctuations in neurons, we model a network consisting of pyramidal neurons and interneurons. Experimentally observed Na+ fluctuations are mimicked when GABAergic inhibition in the simulated network is inverted. Both our experiments and computational model show that the application of tetrodotoxin to block voltage-gated Na+ channels or of inhibitors targeting GABAergic signaling respectively, significantly diminish the neuronal Na+ fluctuations. On the other hand, blocking a variety of other ion channels, receptors, or transporters including glutamatergic pathways, does not have significant effects. In addition, our model shows that the amplitude and duration of Na+ fluctuations decrease as we increase the strength of glial K+ uptake. Furthermore, neurons with smaller somatic volumes exhibit fluctuations with higher frequency and amplitude. As opposed to this, the larger relative size of the extracellular with respect to intracellular space observed in neonatal brain exerts a dampening effect. Finally, our model also predicts that these periods of spontaneous Na+ influx leave neonatal neuronal networks more vulnerable to hyperactivity when compared to mature brain. Taken together, our model thus confirms the experimental observations, and offers additional insight into how the neonatal environment shapes early signaling in the brain. Author SummarySpontaneous neuronal and astrocytic activity during the early postnatal period is crucial to the development and physiology of the neonate forebrain. Elucidating the origin of this activity is key to our understanding of the cell maturation and formation of brain-region-specific networks. This study reports spontaneous, ultraslow, large-amplitude, long-lasting fluctuations in the intracellular Na+ concentration of neurons and astrocytes in the hippocampus of mice at postnatal days 2-4 that mostly disappear after the first postnatal week. We combine ratiometric Na+ imaging and pharmacological manipulations with a detailed computational model of neuronal networks in the neonatal and adult brain to provide key insights into the origin of these Na+ fluctuations. Furthermore, our model predicts that these periods of spontaneous Na+ influx leave neonatal neuronal networks more vulnerable to hyperactivity when compared to mature brain.

neuroscience↗