Biology subjects

Knabbe, J.

Publications and source records attributed to Knabbe, J..

8 recordsLinked to original sources

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

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↗

bioRxiv2021

Genetic engineering for in vivo optical interrogation of neuronal responses to cell type-specific silencing

Genetic engineering of quintuple transgenic brain tissue was used to establish a low background, Cre-dependent version of the inducible Tet-On system for fast, cell type-specific transgene expression in vivo. Co-expression of a constitutive, Cre-dependent fluorescent marker selectively allowed single cell analyses before and after inducible, tet-dependent transgene expression. Here, we used this method for acute, high-resolution manipulation of neuronal activity in the living brain. Single induction of the potassium channel Kir2.1 produced cell type-specific silencing within hours that lasted for at least three days. Longitudinal in vivo imaging of spontaneous calcium transients and neuronal morphology demonstrated that prolonged silencing did not alter spine densities or synaptic input strength. Furthermore, selective induction of Kir2.1 in parvalbumin interneurons increased the activity of surrounding neurons in a distance-dependent manner. This high-resolution, inducible interference and interval imaging of individual cells (high I5, HighFive) method thus allows visualizing temporally precise, genetic perturbations of defined cells.

neuroscience↗

bioRxiv2020

Single-dose ethanol intoxication causes acute and lasting neuronal changes in the brain

Alcohol intoxication at early ages is a risk factor for development of addictive behavior. To uncover neuronal molecular correlates of acute ethanol intoxication, we used stable-isotope labeled mice combined with quantitative mass spectrometry to screen over 2000 hippocampal proteins of which 72 changed synaptic abundance up to two-fold after ethanol exposure. Among those were mitochondrial proteins and proteins important for neuronal morphology, including MAP6 and Ankyrin-G. Based on these candidate proteins, we found acute and lasting molecular, cellular, and behavioral changes following a single intoxication in alcohol-naive mice. Immunofluorescence analysis revealed a shortening of axon initial segments. Longitudinal two-photon in vivo imaging showed increased synaptic dynamics and mitochondrial trafficking in axons. Knockdown of mitochondrial trafficking in dopaminergic neurons abolished conditioned alcohol preference in Drosophila. This introduces mitochondrial trafficking as a process implicated in reward learning, and highlights the potential of high-resolution proteomics to identify cellular mechanisms relevant for addictive behavior.

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

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↗

bioRxiv2018

Secretory vesicle trafficking in awake and anesthetized mice: differential speeds in axons versus synapses

Neuronal dense core vesicles (DCVs) transport many cargo molecules like neuropeptides and neurotrophins to their release sites in dendrites or axons. The transport properties of DCVs in axons of the intact mammalian brain are unknown. We used viral expression of a DCV cargo reporter (NPY-Venus/Cherry) in the thalamus and two-photon in vivo imaging to visualize axonal DCV trafficking in thalamo-cortical projections of anesthetized and awake mice. We found an average speed of 1 m/s, maximal speeds of up to 5 m/s and a pausing fraction of ~11%. Directionality of transport differed between anesthetized and awake mice. In vivo microtubule +-end extension imaging using Macf18-GFP revealed microtubular growth at 0.12 m/s and provided positive identification of antero- and retrograde axonal transport. Consistent with previous reports, anterograde transport was faster (~2.1 m/s) than retrograde transport (~1.4 m/s). In summary, DCVs are transported with faster maximal speeds and lower pausing fraction in vivo compared to previous results obtained in vitro. Finally, we found that DCVs slowed down upon presynaptic bouton approach. We propose that this mechanism promotes synaptic localization and cargo release.\n\nKey pointsO_LIDespite their immense physiological and pathophysiological importance, we know very little about the biology of dense core vesicle (DCV) trafficking in the intact mammalian brain.\nC_LIO_LIDCVs are transported at similar average speeds in the anesthetized and awake mouse brain compared to neurons in culture, yet maximal speed and pausing fraction of transport were higher.\nC_LIO_LIMicrotubule +-end extension imaging visualized microtubular growth at 0.12 m/s and revealed that DCVs were transported faster in the anterograde direction.\nC_LIO_LIDCV transport slowed down upon presynaptic bouton approach, possibly promoting synaptic localization and cargo release.\nC_LIO_LIOur work provides a basis to extrapolate DCV transport properties determined in cultured neurons to the intact mouse brain and reveal novel features such as slowing upon bouton approach and brain state-dependent trafficking directionality.\nC_LI

neuroscience↗

bioRxiv2017

KCC2-dependent Steady-state Intracellular Chloride Concentration and pH in Cortical Layer 2/3 Neurons of Anesthetized and Awake Mice

Neuronal intracellular Cl- concentration ([Cl-]i) influences a wide range of processes such as neuronal inhibition, membrane potential dynamics, intracellular pH (pHi) or cell volume. Up to date, neuronal [Cl-]i has predominantly been studied in model systems of reduced complexity. Here, we implemented the genetically encoded ratiometric Cl- indicator Superclomeleon (SCLM) to estimate the steady-state [Cl-]i in cortical neurons from anesthetized and awake mice using 2-photon microscopy. Additionally, we implemented superecliptic pHluorin as a ratiometric sensor to estimate the intracellular steady-state pH (pHi) of mouse cortical neurons in vivo. We estimated an average resting [Cl-]i of 6 {+/-} 2 mM with no evidence of subcellular gradients in the proximal somato-dendritic domain and an average somatic pHi of 7.1 {+/-} 0.1. Neither [Cl-]i nor pHi were affected by isoflurane anesthesia. We deleted the cation-Cl- co-transporter KCC2 in single identified neurons of adult mice and found an increase of [Cl-]i to approximately 26 {+/-} 8 mM, demonstrating that under in vivo conditions KCC2 produces low [Cl-]i in adult mouse neurons. In summary, neurons of the brain of awake adult mice exhibit a low and evenly distributed [Cl-]i in the proximal somato-dendritic compartment that is independent of anesthesia and requires KCC2 expression for its maintenance.

neuroscience↗