Biology subjects

Kuner, T.

Publications and source records attributed to Kuner, T..

At least 19 recordsLinked to original sources

bioRxiv2026

A Dual-Ensemble Model of Infralimbic Cortex Function in Behavioural Flexibility

The infralimbic cortex (IL) has been described as both facilitator and terminator of reward-seeking, yet both functions appear mutually exclusive. We reconcile this discrepancy by proposing a dual-ensemble model: one for action-outcome prediction and another for prediction correction. Using longitudinal miniscope imaging in rats, we show that during training, the action-outcome predictor ensemble sharpens its activity by suppressing the majority of the IL neuronal population. As contingencies stabilize, entropy decreases, reflecting a refined predictive model. During extinction, the prediction correction ensemble activates in response to reward omission. A computational simulation based on these two ensembles successfully recapitulated our experimental results. This framework reconciles conflicting models, where IL manipulation can either accelerate or abolish extinction by demonstrating that IL activity dynamically encodes predictive accuracy. Our findings indicate that IL functions as an action-outcome predictor that elegantly guides behavioural flexibility.

neuroscience↗

bioRxiv2026

Repetitive anatomical patterns for thalamocortical projections of higher-order thalamic nuclei

Cortico-thalamo-cortical circuits entail extensive trans-thalamic connectivity between cortical areas, yet their structural organization and function remain poorly understood. Here, the thalamocortical projections of several higher-order thalamic nuclei were characterized by retrograde tracing from two cortical areas, the primary somatosensory (S1) and motor (M1) cortices. Cholera toxin B conjugated with different fluorophores allowed for simultaneous detection of projection neurons targeting S1 and M1. A cell detection pipeline based on neural networks was developed to allow semi-automated analysis of large thalamic imaging volumes to quantitatively infer the spatial distribution of projection neurons in the posterior complex (PO) and the adjacent ethmoid nucleus (Eth), nucleus centrolateralis (CL), nucleus paracentralis (PCN), and the nucleus parafascicularis (PF). The arrangement of neurons projecting to both, primary somatosensory and motor cortices, occurs at different connection strengths and was topographically organized in all nuclei studied. Co-injections into both cortical areas revealed projection neurons with axons branching into both S1 and M1 cortices. Our work introduces a pipeline for semi-automated quantitative analysis of thalamic projection patterns that could be useful for connectivity analyses in general. This approach revealed repetitive anatomical patterns in different thalamic nuclei with regard to projection strength, spatial organization and fraction of projection neurons targeting two cortical areas simultaneously.

neuroscience↗

bioRxiv2025

Glucocorticoid receptors mediate reprogramming of astrocytes in depression.

Psychiatric disorders are among the most pressing problems of the modern society, with various forms of depression affecting more than 300 millions of people worldwide. Dysfunction of glial cells has consistently been reported in major depressive disorder (MDD); however, no comprehensive resource detailing glial dysfunction is available. To provide insight into neurobiological mechanisms behind severe psychiatric symptoms, we performed transcriptional analysis of post-mortem samples from a subpopulation of suicide completers with previously reported glial abnormalities. We focused on BA25, a subregion of the prefrontal cortex prioritized for targeted medical interventions, due to its metabolic aberrations in disease. We found that a significant portion of genes deregulated in MDD is enriched in glia, with astrocyte-specific genes representing the highest fraction. Then we employed a novel protocol for enriching astrocytic nuclei to provide a detailed molecular signature of astrocytes in MDD. The analysis of the gene set revealed the glucocorticoid receptor (GR) as a key regulatory transcription factor. We found that astrocyte-specific elimination of the GR in mice largely prevented transcriptional, metabolic and behavioral changes elicited by chronic stress. We also demonstrated that regional manipulation of glutamate turnover in astrocytes suffices to elicit discrete traits of depressive-like behavior. Our data points to astrocytes as a key cellular site of convergence of multiple traits of depression and provide a resource for exploring novel targets for glia-focused therapeutic approaches.

neuroscience↗

bioRxiv2025

Glyco-STORM: Nanoscale Mapping of the Cellular Glycosylation Landscape

Glycosylation is a crucial biochemical modification of proteins and other biomolecules in cells that generates an exceptional structural and functional diversity. Aberrant glycosylation is implicated in numerous diseases, including neurodegenerative disorders and cancer. Despite its significance, methodological constraints to date have limited the exploration of the nanometer scale spatial arrangement of glycans across entire cells. We developed Glyco-STORM, a super-resolution imaging approach that generates nano-structural maps of cellular glycosylation. Glyco-STORM employs fluorophore-labeled lectins and multiplexed single-molecule super-resolution microscopy, in combination with nanoscale spatial pattern analysis. For example, Glyco-STORM unraveled nanodomains within the endoplasmic reticulum, subdomains along the Golgi axes, and a polarized lysosomal clathrin coat. At synaptic contact sites, mature glycans delineate the synaptic cleft and subsynaptic tubules adjacent to the postsynaptic density. In summary, Glyco-STORM elucidates the spatial arrangement of glycosylation sites from subcellular to molecular levels, revealing the previously obscured glycosylation landscape at nanoscale and establishing a spatial glycosylation code that provides a unique perspective on cellular organization distinct from traditional protein-centric views.

cell biology↗

bioRxiv2024

Early-life adversity mediates a thalamo-amgydalar circuit dysfunction underlying chronic pain and anxiety

Childhood adversity increases the risk of developing a vicious cycle of chronic pain and comorbid anxious avoidance, yet the underlying biological mechanisms remain unclear. Here, we investigated the role of a brain circuit from the paraventricular thalamus (PVT) to the central amygdala (CeA) in mediating hyperalgesia and comorbid anxiety following early life stress. Using a vulnerability-stress model, we exposed both male and female mice to early social isolation (vulnerability) followed by nerve injury (stress) and showed increased hyperalgesia and anxious avoidance behavior in nerve-injured female mice following early adversity. Chemogenetic, electrophysiological, and optophysiological analyses revealed a causal contribution of a hyperexcitable PVT-CeA circuit dysfunction to chronic hyperalgesia and anxiety in nerve-injured female mice after early life stress. Our findings reveal a neural mechanism linking childhood adversity to chronic pain and anxiety, and suggest that reprogramming this pathway may reverse the impact of childhood adversity.

neuroscience↗

bioRxiv2024

A tactile discrimination task to study neuronal dynamics in freely-moving mice

Sensory discrimination tasks are valuable tools to study neuronal mechanisms of perception and learning. Most discrimination tasks for electrophysiological and imaging studies in rodents require the animals to be head-fixed. However, implementing neurophysiological recordings into more ethologically realistic settings with unrestrained animals has been challenging. Here, we introduce a whisker-dependent discrimination task for freely moving mice, integrating electrophysiology and calcium imaging with cellular resolution. In this go/no-go paradigm, male mice learned to discriminate aperture widths within days while foraging on a linear platform. The setups versatility enables exploration into diverse behavioral aspects, including tactile discrimination thresholds, valence-dependent behavior, and cognitive flexibility following repeated task rule reversals. Rule learning was highly stereotypical, fast and reproducible across individual mice. Electrophysiological recordings revealed distributed tactile processing across the thalamocortical system, with subsets of units encoding both movement- and stimulus-related features. Sensory encoding was strongly modulated by behavioral state, with neurons tuned to locomotion, whisking, whisker angle and phase, head angle, and spatial position. By enabling simultaneous extracellular recordings and calcium imaging within the same freely moving paradigm, this approach allows for precise synchronization of neural activity with multiple behavioral readouts. This paradigm provides a versatile tool to elucidate neural mechanisms of cognition and sensory processing in naturalistic conditions.

neuroscience↗

bioRxiv2024

Super-resolved protein imaging using bifunctional light-up aptamers

Efficient labeling methods for protein visualization with minimal tag size and appropriate photophysical properties are required for single-molecule localization microscopy (SMLM), providing insights into the organization and interactions of biomolecules in cells at the molecular level. Among the fluorescent light-up aptamers (FLAPs) originally developed for RNA imaging, RhoBAST stands out due to its remarkable brightness, photostability, fluorogenicity, and rapid exchange kinetics, enabling super-resolved imaging with high localization precision. Here, we expand the applicability of RhoBAST to protein imaging by fusing it to protein-binding aptamers. The versatility of such bifunctional aptamers is demonstrated by employing a variety of protein-binding DNA or RNA aptamers and different FLAPs. Moreover, fusing RhoBAST with the GFP-binding aptamer AP3 facilitates high- and super-resolution imaging of GFP-tagged proteins, which is particularly valuable in view of the widespread availability of plasmids and stable cell lines expressing proteins fused to GFP. The bifunctional aptamers compare favorably with standard antibody-based immunofluorescence protocols, as they are 7-fold smaller than antibody conjugates and exhibit higher bleaching-resistance. We demonstrate the effectiveness of our approach in super-resolution microscopy in secondary mammalian cell lines and primary neurons by RhoBAST-PAINT, an SMLM protein imaging technique that leverages the transient binding of the fluorogenic rhodamine dye SpyRho to RhoBAST.

biophysics↗

bioRxiv2024

Direct excitatory synapses between neurons and tumor cells drive brain metastatic seeding of breast cancer and melanoma

Interactions between neurons and cancer cells are found in many malignancies, but their relevance for metastatic organ colonization remain largely unknown. It is also unclear whether any direct synaptic communication between neurons and cancer cells of non-neural tumor types exists, and if so, whether this can support metastasis and thus cancer progression. Here we show that excitatory synapses are formed between neurons and brain-metastatic melanoma and breast cancer cells. This starts at an early microscopic stage after extravasation into the brain parenchyma, during residence of cancer cells in the perivascular niche, a critical step for their survival. These neuron-cancer synapses showed a bona fide synaptic ultrastructure, and generated excitatory postsynaptic currents mediated by glutamate receptors of the AMPA subtype in cancer cells. In accordance, AMPA receptor signatures were consistently detected in preclinical and patient samples of melanoma and breast cancer brain metastases. Genetic perturbation and pharmacological inhibition of AMPA receptors with the approved antiepileptic drug perampanel in models of breast and melanoma cancer reduced the number of brain metastases and overall brain metastatic burden. These findings demonstrate for the first time that neurons can form biologically relevant direct synapses with non-neural cancer cells. In brain metastasis, a particularly challenging complication of many common malignancies, this non-canonical stimulatory synaptic interaction offers novel therapeutic opportunities.

cancer biology↗

bioRxiv2023

Deep intravital brain tumor imaging enabled by tailored three-photon microscopy and analysis

Intravital two-photon microscopy has emerged as a powerful technology to study brain tumor biology and its temporal dynamics, including invasion, proliferation and therapeutic resistance in the superficial layers of the mouse cortex. However, intravital microscopy of deeper cortical layers and especially the subcortical white matter, an important route of glioblastoma invasion and recurrence, has not yet been feasible due to low signal-to-noise ratios, missing spatiotemporal resolution and the inability to delineate myelinated axonal tracts. Here, we present a tailored intravital microscopy and artificial intelligence-based analysis methodology and workflow that enables routine deep imaging of glioblastoma over extended time periods, named Deep3P. We show that three-photon microscopy, adaptive optics, as well as customized deep learning-based denoising and machine learning segmentation together allow for deep brain intravital investigation of tumor biology up to 1.2 mm depth. Leveraging this approach, we find that perivascular invasion is a preferred invasion route into the corpus callosum as compared to intracortical glioblastoma invasion and uncover two vascular mechanisms of glioblastoma migration in the white matter. Furthermore, we can define an imaging biomarker of white matter disruption during early glioblastoma colonization. Taken together, Deep3P allows for an efficient and non-invasive investigation of brain tumor biology and its tumor microenvironment in unprecedented deep white and gray matter of the living mouse, opening up novel opportunities for studying the neuroscience of brain tumors and other model systems.

cancer biology↗

bioRxiv2023

Loss of secondary motor cortex neurons in chronic neuropathic pain

Chronic neuropathic pain is associated with structural plasticity of the brain on different spatial scales, yet, little is known on the mesoscopic scale of tissue composition. Here, we determined the cellular composition of cortical areas and structural variability of entire neurons during the development of chronic neuropathic pain using longitudinal in vivo two-photon microscopy and behavioral assessment. When monitoring cell type composition in response to spared-nerve injury in 84 cortical volumes containing {bsim}25000 cells each, we found neuronal loss in the secondary motor cortex region M2 immediately adjacent to the cingulate cortex already one week after surgery. Loss of mostly interneurons was also evident when monitoring individual M2 neurons over time. This neuronal loss was preceded by decreased spine density and loss of distal dendritic branches. In conclusion, our work delineates M2 as a novel site and neuronal loss as a so far underappreciated mechanism underlying chronic neuropathic pain states.

neuroscience↗

bioRxiv2022

3MDR, a microcomputer-controlled visual stimulation device for psychotherapy-like treatments of mice

Post-traumatic stress disorder and other mental disorders can be treated by an established psychotherapy called Eye Movement Desensitization and Reprocessing (EMDR). In EMDR, patients are confronted with traumatic memories while they are stimulated with alternating bilateral stimuli (ABS). How ABS affect the brain and whether ABS could be adapted to different patients or mental disorders is unknown. Interestingly, ABS reduced conditioned fear in mice. Yet, an approach to systematically test complex visual stimuli and compare respective differences in emotional processing based on (semi-)automated behavioral analysis is lacking. We developed 3MDR (Model for MultiModal visual stimulation to Desensitize Rodents) - a novel, open-source, low-cost, customizable device that can be integrated in and TTL-controlled by commercial rodent behavioral setups. 3MDR allows to design and precisely steer multimodal visual stimuli in the head direction of freely-moving mice. Optimized videography allows to semi-automatically analyze rodent behavior during visual stimulation. Detailed building, integration, and treatment instructions along with open-source software provide easy access for inexperienced users. Using 3MDR, we confirmed that EMDR-like ABS persistently improve fear extinction in mice and showed for the first time that ABS-mediated anxiolytic effects strongly depend on physical stimulus properties such as ABS brightness. 3MDR not only enables researchers to interfere with mouse behavior in an EMDR-like setting, but demonstrates that visual stimuli can be used as a noninvasive brain stimulation to differentially alter emotional processing in mice. SIGNIFICANCE STATEMENTAlternating bilateral stimuli (ABS) reduce fear in post-traumatic stress disorder patients and in mice. The mechanism of how classic ABS - typically used in Eye Movement Desensitization and Reprocessing (EMDR) - reduce fear is enigmatic. We provide detailed resources to build a cost-effective, computer-controlled device called 3MDR to perform and semi-automatically analyze EMDR-like treatments in freely-moving mice and to test behavioral effects of multiple ABS variants. Using the 3MDR device, this study confirmed that classic ABS strongly and persistently improve the extinction of conditioned fear in mice - an effect that depended on the brightness of ABS. This novel method may ultimately contribute to a deeper translational and neurobiological understanding of how visual stimuli affect emotional processing in mice.

neuroscience↗

bioRxiv2022

Comprehensive monitoring of tissue composition using in vivo imaging of cell nuclei and deep learning

Comprehensive analysis of tissue composition has so far been limited to ex-vivo approaches. Here, we introduce NuCLear (Nucleus-instructed tissue composition using deep learning), an approach combining in vivo two-photon imaging of histone 2B-eGFP-labeled cell nuclei with subsequent deep learning-based identification of cell types from structural features of the respective cell nuclei. Using NuCLear, we were able to classify almost all cells per imaging volume in the secondary motor cortex of the mouse brain (0.25 mm3 containing [~]25000 cells) and to identify their position in 3D space in a non-invasive manner using only a single label throughout multiple imaging sessions. Twelve weeks after baseline, cell numbers did not change yet astrocytic nuclei significantly decreased in size. NuCLear opens a window to study changes in relative abundance and location of different cell types in the brains of individual mice over extended time periods, enabling comprehensive studies of changes in cellular composition in physiological and pathophysiological conditions.

neuroscience↗

bioRxiv2022

Primary Somatosensory Cortex Bidirectionally Modulates Sensory Gain and Nociceptive Behavior in a Layer-Specific Manner

The primary somatosensory cortex (S1) is a hub for body sensation of both innocuous and noxious signals, yet its role in somatosensation versus pain is debated. Despite known contributions of S1 to sensory gain modulation, its causal involvement in subjective sensory experiences remains elusive. Here, in mouse S1, we reveal the involvement of cortical output neurons in layers 5 (L5) and 6 (L6) in the perception of innocuous and noxious somatosensory signals. We find that L6 activation can drive aversive hypersensitivity and spontaneous nocifensive behavior. Linking behavior to neuronal mechanisms, we find that L6 enhances thalamic somatosensory responses, and in parallel, strongly suppresses L5 neurons. Directly suppressing L5 reproduced the pronociceptive phenotype induced by L6 activation, suggesting an anti-nociceptive function for L5 output. Indeed, L5 activation reduced sensory sensitivity and reversed inflammatory allodynia. Together, these findings reveal a layer-specific and bidirectional role for S1 in modulating subjective sensory experiences.

neuroscience↗

bioRxiv2021

Visualizing multi-protein patterns at the synapse of neuronal tissue with DNA-assisted single-molecule localization microscopy

The development of super-resolution microscopy (SRM) has widened our understanding of biomolecular structure and function in biological materials. Imaging multiple targets within a single area would elucidate their spatial localization relative to the cell matrix and neighboring biomolecules, revealing multi-protein macromolecular structures and their functional co-dependencies. SRM methods are, however, limited to the number of suitable fluorophores that can be imaged during a single acquisition as well as the loss of antigens during antibody washing and restaining for organic dye multiplexing. We report the visualization of multiple protein targets within the pre- and postsynapse in 350-400 nm thick neuronal tissue sections using DNA-assisted single-molecule localization microscopy. Using antibodies labeled with short DNA oligonucleotides, multiple targets are visualized successively by sequential exchange of fluorophore-labeled complementary oligonucleotides present in the imaging buffer. The structural integrity of the tissue is maintained owing to only a single labelling step during sample preparation. Multiple targets are imaged using a single laser wavelength, minimizing chromatic aberration. This method proved robust for multi-target imaging in semi-thin tissue sections, paving the way towards structural cell biology with single-molecule super-resolution microscopy.

biophysics↗

bioRxiv2019

Automated highly multiplexed super-resolution imaging of protein nano-architecture in cells and tissues

Understanding the nano-architecture of protein machines in diverse sub-cellular compartments remains a challenge despite rapid progress in super-resolution microscopy. While singlemolecule localization microscopy techniques allow the visualization and identification of cellular structures with near-molecular resolution, multiplex-labeling of tens of target proteins within the same sample has not yet been achieved routinely. However, single sample multiplexing is essential to detect patterns that threaten to get lost in multi-sample averaging. Here, we report maS3TORM (multiplexed automated serial staining stochastic optical reconstruction microscopy), a microscopy approach capable of fully automated 3D dSTORM imaging and solution exchange employing a re-staining protocol to achieve highly multiplexed protein localization within individual biological samples. We demonstrate 3D super-resolution images of 15 target proteins in single cultured cells and 16 targets in individual neuronal tissue samples with <10 nm localization precision. This allowed us to define novel nano-architectural features of protein distribution within the presynaptic nerve terminal.

neuroscience↗

bioRxiv2019

Grey matter volume changes and corresponding cellular metrics identified in a longitudinal in vivo imaging approach

Magnetic resonance imaging (MRI) of the brain combined with voxel-based morphometry (VBM) has revealed structural changes of grey and white matter in a range of neurological and psychiatric disorders. However, the cellular basis of volume changes observed with VBM has remained unclear. We devised an approach to systematically correlate changes in grey matter volume (GMV) with cellular composition. Mice were alternately examined with structural MRI and two-photon in vivo microscopy at three time points, taking advantage of age-dependent changes in brain structure. We chose to image fluorescently labelled cell nuclei, because these can be readily imaged in large tissue volumes and allow inferences on several structural parameters: (1) the physical volume as determined from a subset of nuclei used to generate a geometrically defined space, (2) the number of cells, (3) the nearest neighbour distance measured between all nuclei as an indicator of cell clustering, and (4) the volume of the cell nuclei. Using this approach, we found that physical volume did not significantly correlate with GMV change, whereas mean nuclear volume was inversely correlated. When focusing on layers within the imaging volume, positive correlations of GMV were found with cell number near the cortical surface and nearest neighbour distance in deeper layers. Thus, the novel approach introduced here provided new insights into the factors underlying grey matter volume changes.

neuroscience↗

bioRxiv2019

retroLEAP: rAAV-based retrograde trans-synaptic labeling, expression and perturbation

Identifying and manipulating synaptically connected neurons across brain regions remains a core challenge in understanding complex nervous systems. RetroLEAP is a novel approach for retrograde trans-synaptic Labelling, Expression And Perturbation. GFP-dependent recombinase (Flp-DOG) detects trans-synaptic transfer of GFP-tetanus toxin heavy chain fusion protein (GFP-TTC) and activates expression of any gene of interest in synaptically connected cells. RetroLEAP overcomes existing limitations, is non-toxic, highly flexible, efficient, sensitive and easy to implement.

neuroscience↗

bioRxiv2019

Acute ethanol intoxication induces preferred loss of presynaptic boutons devoid of mitochondria in vivo

Acute alcohol intoxication is frequently observed in modern societies and carries a vast burden, ranging from traffic accidents to transient memory loss. Despite years of intense research, the effects of acute ethanol intoxication on brain function remain incompletely understood. Here, we studied the effect of acute ethanol intoxication on axonal organelle trafficking and presynaptic structure using in vivo two photon microscopy in anesthetized mice. After a single intraperitoneal injection of ethanol, inducing a blood alcohol concentration of roughly 250 mg/dl, the axonal mitochondrial mobility was doubled while dense core vesicle mobility remained unaffected. Simultaneously imaging mitochondria and presynaptic boutons revealed that unoccupied presynaptic boutons perished more frequently after ethanol exposure, while boutons stably occupied with mitochondria mostly persisted. Our results define a novel mechanism of ethanol action and may explain difficulties in permanently storing new memories after episodes of intense ethanol consumption with a loss of synapses.

neuroscience↗