Biology subjects

Lohse, M.

Publications and source records attributed to Lohse, M..

12 recordsLinked to original sources

bioRxiv2024

Zapit: Open Source Random-Access Photostimulation For Neuroscience

Optogenetic tools are indispensable for understanding the causal neural mechanisms underlying animal behavior,providing millisecond-precision control over genetically defined neural populations. Random-access laser-scanning optogenetics is a powerful and underutilized method for transiently manipulating cortical activity in mice. Despite the utility of this technique, no general-purpose open-source implementation currently exists, and potential users need expertise across optics, real-time hardware control, and programming. This represents a major barrier to adoption, particularly for newly established research groups. Here we present 'Zapit', the first open-source general-purpose platform for random-access laser-scanning optogenetic experiments in head-fixed mice. Zapit is fully documented, has a user-friendly GUI, works in stereotaxic coordinates, and comes with easy to build hardware options that extend functionality beyond any published system. We validate Zapit's performance through electrophysiological recordings and cortical photoinhibition in behaving mice. Zapit is a novel and innovative tool with the potential to democratize laser-scanning optogenetics and significantly increase uptake throughout the scientific community.

neuroscience↗

bioRxiv2024

Subcortical origin of nonlinear sound encoding in auditory cortex

A major challenge in neuroscience is to understand how neural representations of sensory information are transformed by the network of ascending and descending connections in each sensory system. By recording from neurons at several levels of the auditory pathway, we show that much of the nonlinear encoding of complex sounds in auditory cortex can be explained by transformations in the midbrain and thalamus. Modeling cortical neurons in terms of their inputs across these subcortical populations enables their responses to be predicted with unprecedented accuracy. In contrast, subcortical responses cannot be predicted from descending cortical inputs, indicating that ascending transformations are irreversible, resulting in increasingly lossy, higher-order representations across the auditory pathway. Rather, auditory cortex selectively modulates the nonlinear aspects of thalamic auditory responses and the functional connectivity between subcortical neurons, without affecting the linear encoding of sound. These findings reveal the fundamental role of subcortical transformations in shaping cortical responses.

neuroscience↗

bioRxiv2023

Nanobodies against SARS-CoV-2 non-structural protein Nsp9 inhibit viral replication by targeting innate immunity

Nanobodies are emerging as critical tools for drug design. Several have been recently created to serve as inhibitors of SARS-Cov-2 entry in the host cell by targeting surface-exposed Spike protein. However, due to the high frequency of mutations that affect Spike, these nanobodies may not target it to their full potential and as a consequence, inhibition of viral entry may not be efficient. Here we have established a pipeline that instead targets highly conserved viral proteins that are made only after viral entry into the host cell when the SARS-Cov-2 RNA-based genome is translated. As proof of principle, we designed nanobodies against the SARS-CoV-2 non-structural protein Nsp9, required for viral genome replication. To find out if this strategy efficiently blocks viral replication, one of these anti-Nsp9 nanobodies, 2NSP23, previously characterized using immunoassays and NMR spectroscopy for epitope mapping, was encapsulated into lipid nanoparticles (LNP) as mRNA. We show that this nanobody, hereby referred to as LNP-mRNA- 2NSP23, is internalized and translated in HEK293 cells. We next infected HEK293-ACE2 cells with multiple SARS-CoV-2 variants and subjected them to LNP-mRNA-2NSP23 treatment. Analysis of total RNA isolated from infected cells treated or untreated with LNP-mRNA-2NSP23 using qPCR and RNA deep sequencing shows that the LNP-mRNA-2NSP23 nanobody protects HEK293-ACE2 cells and suppresses replication of several SARS-CoV-2 variants. These observations indicate that following translation, the nanobody 2NSP23 inhibits viral replication by targeting Nsp9 in living cells. We speculate that LNP-mRNA-2NSP23 may be translated into an innovative technology to generate novel antiviral drugs highly efficient across coronaviruses.

cell biology↗

bioRxiv2023

Direct and indirect responses of the Arabidopsis transcriptome to an induced increase in trehalose 6-phosphate

Trehalose 6-phosphate (Tre6P) is an essential signal metabolite that reports and regulates the level of sucrose, linking growth and development to the metabolic status. We hypothesized that Tre6P plays a role in mediating the regulation of gene expression by sucrose. To test this, we performed transcriptomic profiling on Arabidopsis plants that expressed a bacterial trehalose-6-phosphate synthase (TPS) under the control of an ethanol-inducible promoter. Induction led to a 4-fold rise in Tre6P levels, a concomitant decrease in sucrose, and significant changes of over 13,000 transcripts and two-fold or larger changes of over 5000 transcripts. Comparison with nine published responses to sugar availability allowed some of these changes to be linked to the rise in Tre6P, while others were probably due to lower sucrose or other indirect effects. Changes linked to Tre6P included repression of photosynthesis and induction of many growth-related processes including ribosome biogenesis. About 500 starvation-related genes are known to be induced by SUCROSE-NON-FERMENTING-1-RELATED KINASE 1 (SnRK1). They were largely repressed by Tre6P in a manner consistent with Tre6P acting to inhibit SnRK1. SnRK1 also represses many genes that are involved in biosynthesis and growth. These responded to Tre6P in a more complex manner, pointing to Tre6P also interacting with further C-signaling pathways. In addition, elevated Tre6P modified expression of genes encoding regulatory subunits of the SnRK1 complex and TPS class II and FLZ proteins that are thought to modulate SnRK1 function, and genes involved in the circadian clock and in TOR, light, abscisic acid and other hormone signaling. One sentence summaryAn induced increase in trehalose 6-phosphate levels has direct effects on gene expression via inhibition of SUCROSE-NON-FERMENTING-1-RELATED KINASE 1 and interactions with light, circadian clock and phytohormone signaling, and widespread indirect effects on gene expression from reciprocal changes in sucrose levels.

plant biology↗

bioRxiv2023

Cell swelling enhances GPCR ternary complex formation, underpinning the potentiation of beta2 adrenergic receptor-mediated cAMP response

G protein-coupled receptors conformational landscape can be affected by their local, microscopic interactions within the cell plasma membrane. We employ here a pleiotropic stimulus, namely osmotic swelling, to alter the cortical environment within intact cells and monitor the response in terms of receptor function and downstream signaling. We observe that in osmotically swollen cells the {beta}2-adrenergic receptor, a prototypical GPCR, favors an active conformation, resulting in cAMP transient responses to adrenergic stimulation that have increased amplitude. The results are validated in primary cell types such as adult cardiomyocytes, a model system where swelling occurs upon ischemia-reperfusion injury. Our results suggest that receptors function is finely modulated by their biophysical context, and specifically that osmotic swelling acts as a potentiator of downstream signaling, not only for the {beta}2-adrenergic receptor, but also for other receptors, hinting at a more general regulatory mechanism.

cell biology↗

bioRxiv2022

Why the brown ghost chirps at night

Since the pioneering work by Moeller, Szabo, and Bullock, weakly electric fish have served as a valuable model for investigating spatial and social cognitive abilities in a vertebrate taxon usually less accessible than mammals or other terrestrial vertebrates. These fish, through their electric organ, generate low-intensity electric fields to navigate and interact with conspecifics, even in complete darkness. The brown ghost knifefish is appealing as a study subject due to a rich electric "vocabulary", made by individually variable and sex-specific electric signals. These are mainly characterized by brief frequency modulations of the oscillating dipole moment continuously generated by their electric organ, and are known as chirps. Different types of chirps are believed to convey specific and behaviorally salient information, serving as behavioral readouts for different internal states during behavioral observations. Despite the success of this model in neuroethology over the past seven decades, the code to decipher their electric communication remains unknown. To this aim, in this study we re-evaluate the correlations between signals and behavior offering an alternative, and possibly complementary, explanation for why these freshwater bottom dwellers emit electric chirps. By uncovering correlations among chirping, electric field geometry, and detectability in enriched environments, we present evidence for a previously unexplored role of chirps as specialized self-directed signals, enhancing conspecific electrolocation during social encounters.

animal behavior and cognition↗

bioRxiv2021

Dual-color Fluorescence Cross-Correlation Spectroscopy to study Protein-Protein Interaction and Protein Dynamics in Live Cells

We present a protocol and workflow to perform live cell dual-color fluorescence crosscorrelation spectroscopy (FCCS) combined with Forster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques. In dual-color FCCS, where the fluctuations in fluorescence intensity represents the dynamical "fingerprint" of the respective fluorescent biomolecule, we can probe co-diffusion or binding of the receptors. FRET, with its high sensitivity to molecular distances, serves as a well-known "nanoruler" to monitor intramolecular changes. Taken together, conformational changes and key parameters such as local receptor concentrations, and mobility constants become accessible in cellular settings. Quantitative fluorescence approaches are challenging in cells due to high noise levels and the vulnerable sample itself. We will show how to perform the experiments including the calibration steps. We use dual-color labeled {beta}2-adrenergic receptor ({beta}2AR) labeled (eGFP and SNAPtag-TAMRA). We will guide you step-by-step through the data analysis procedure using open-source software and provide templates that are easy to customize. Our guideline enables researchers to unravel molecular interactions of biomolecules in live cells in situ with high reliability despite the limited signal-to-noise levels in live cell experiments. The operational window of FRET and particularly FCCS at low concentrations allows quantitative analysis near-physiological conditions. Link to accompanying video: https://tr240.uni-wuerzburg.de/vippclass/index.php/s/TL8aWmwE9RjGfLE

biophysics↗

bioRxiv2021

Differences in interactions between transmembrane domains tune the activation of metabotropic glutamate receptors

The metabotropic glutamate receptors (mGluRs) form a family of neuromodulatory G protein-coupled receptors that contain both a seven-helix transmembrane domain (TMD) and a large extracellular ligand-binding domain (LBD) which enables stable dimerization. While numerous studies have revealed variability across subtypes in the initial activation steps at the level of LBD dimers, an understanding of inter-TMD interaction and rearrangement remains limited. Here we use a combination of single molecule fluorescence, molecular dynamics, functional assays, and conformational sensors to reveal that distinct TMD assembly properties drive differences between mGluR subtypes. We uncover a variable region within transmembrane helix 4 (TM4) that contributes to homo- and heterodimerization in a subtype-specific manner and tunes orthosteric, allosteric and basal activation. We also confirm a critical role for a conserved inter-TM6 interface in stabilizing the active state during orthosteric or allosteric activation. Together this study informs a working model of inter-TMD rearrangement that drives mGluR function.

neuroscience↗

bioRxiv2020

Structural and functional characterization of allatostatin receptor type-C of Thaumetopoea pityocampa revealed the importance of Q2716.55 residue in G protein-dependent activation pathway

Insect neuropeptide receptors are among the potential targets for designing next-generation pesticides. Activation of allatostatin receptor type C (AstR-C), a G Protein-coupled receptor (GPCR), upon stimulation with its endogenous ligand, allatostatin C (AST-C), leads to the inhibition of juvenile hormone (JH) secretion that consequently regulates physiology of insects. Here we conducted in silico and in vitro approaches to characterize the structure and function of AstR-C of Thaumetopoea pityocampa (T.pit), a well-known pest in Mediterranean countries. The sequence of AstR-C and AST-C were derived from whole genome sequencing (WGS) data. Resonance energy transfer (RET) methods were used to investigate the downstream effectors of the receptor and the temporal kinetics of G protein activation. Three-dimensional (3D) structure of AstR-C constructed via homology modeling methods was subjected to molecular dynamics (MD) simulations and docking studies to identify the orthosteric pocket. Our results showed that T.pit AstR-C couples to Gi/o subtype of G proteins at sub-nanomolar ranges of the the ligand with the G protein recruitment and activation kinetics of [~]4 and 6 seconds, respectively, when 1 nM AST-C is administered. At the increasing concentration of native ligand, {beta}arrestin was shown to be recruited at nanomolar ranges the ligand. Docking and MD simulation studies revealed the importance of extracellular loop 2 (ECL2) in T.pit AstRC/AST-C interaction, and combination of in silico and in vitro methods supported the accuracy of the built model and the predicted orthosteric pocket. Q2716.55 (Ballesteros-Weinstein generic numbering) was found to have a substantial role in G protein dependent activation of AstR-C possibly via contributing to the flexibility of the structure.

bioinformatics↗

bioRxiv2020

Neural circuits for somatosensory control of auditory thalamocortical processing

Integration of information across the senses is critical for perception and is a common property of neurons in the cerebral cortex, where it is thought to arise primarily from corticocortical connections. Much less is known about the role of subcortical circuits in shaping the multisensory properties of cortical neurons. We show that stimulation of the whiskers causes widespread suppression of sound-evoked activity in mouse primary auditory cortex (A1). This suppression depends on the primary somatosensory cortex (S1), and is implemented through a descending circuit that links S1, via the auditory midbrain, with thalamic neurons that project to A1. Furthermore, a direct pathway from S1 has a facilitatory effect on auditory responses in higher-order thalamic nuclei that project to other brain areas. Crossmodal corticofugal projections to the auditory midbrain and thalamus therefore play a pivotal role in integrating multisensory signals and in enabling communication between different sensory cortical areas.

neuroscience↗

bioRxiv2019

Auditory contrast gain control predicts perceptual performance and is not dependent on cortical activity

Neural adaptation enables sensory information to be represented optimally in the brain despite large fluctuations over time in the statistics of the environment. Auditory contrast gain control represents an important example, which is thought to arise primarily from cortical processing. We find, however, that neurons in both the auditory thalamus and midbrain of mice show robust contrast gain control, and that this is implemented independently of cortical activity. Although neurons at each level exhibit contrast gain control to similar degrees, adaptation time constants become longer at later stages of the processing hierarchy, resulting in progressively more stable representations. We also show that auditory discrimination thresholds in human listeners compensate for changes in contrast, and that the strength of this perceptual adaptation can be predicted from physiological measurements. Contrast adaptation is therefore a robust property of both the subcortical and cortical auditory system and accounts for the short-term adaptability of perceptual judgments.

neuroscience↗

bioRxiv2018

Motor cortex can modulate somatosensory processing via cortico-thalamo-cortical pathway

The somatosensory and motor systems are intricately linked, providing several routes for the sensorimotor interactions necessary for haptic processing. Here, we used electrical and optogenetic stimulation to study the circuits that enable primary motor cortex (M1) to exert top-down modulation of whisker-evoked responses, at the levels of brain stem, thalamus and somatosensory cortex (S1). We find that activation of M1 drives somatosensory responsive cells at all levels, and that this excitation is followed by a period of tactile suppression, which gradually increases in strength along the ascending somatosensory pathway. Using optogenetic stimulation in the layer-specific Cre driver lines, we find that activation of layer VI cortico-thalamic neurons is sufficient to drive spiking in higher order thalamus, and that this is reliably followed by excitation of S1, suggesting a cross-modal cortico-thalamo-cortical pathway. Cortico-thalamic excitation predicts the degree of subsequent tactile suppression, consistent with a strong role for thalamic circuits in the expression of inhibitory sensorimotor interactions. These results provide evidence of a role for M1 in dynamic modulation of S1, largely under cortico-thalamic control.

neuroscience↗