Role of PKC and AMPK in hypertonicity-stimulated water reabsorption in rat inner medullary collecting ducts.

Hypertonicity increases water permeability, independently of vasopressin, in the inner medullary collecting duct (IMCD) by increasing aquaporin-2 (AQP2) membrane accumulation. We investigated whether protein kinase C (PKC) and adenosine monophosphate kinase (AMPK) are involved in hypertonicity-regulated water permeability. Increasing perfusate osmolality from 150 to 290 mosmol/kgH2O and bath osmolality from 290 to 430 mosmol/kgH2O significantly stimulated osmotic water permeability. The PKC inhibitors chelerythrine (10 µM) and rottlerin (50 µM) significantly reversed the increase in osmotic water permeability stimulated by hypertonicity in perfused rat terminal IMCDs. Chelerythrine significantly increased phosphorylation of AQP2 at S261 but not at S256. Previous studies show that AMPK is stimulated by osmotic stress. We tested AMPK phosphorylation under hypertonic conditions. Hypertonicity significantly increased AMPK phosphorylation in inner medullary tissues. Blockade of AMPK with Compound C decreased hypertonicity-stimulated water permeability but did not alter phosphorylation of AQP2 at S256 and S261. AICAR, an AMPK stimulator, caused a transient increase in osmotic water permeability and increased phosphorylation of AQP2 at S256. When inner medullary tissue was treated with the PKC activator phorbol dibutyrate (PDBu), the AMPK activator metformin, or both, AQP2 phosphorylation at S261 was decreased with PDBu or metformin alone, but there was no additive effect on phosphorylation with PDBu and metformin together. In conclusion, hypertonicity regulates water reabsorption by activating PKC. Hypertonicity-stimulated water reabsorption by PKC may be related to the decrease in endocytosis of AQP2. AMPK activation promotes water reabsorption, but the mechanism remains to be determined. PKC and AMPK do not appear to act synergistically to regulate water reabsorption.

Protocol for using serial two-photon tomography to map cell types and cerebrovasculature at single-cell resolution in the whole adult mouse brain.

Here, we present a protocol using serial two-photon tomography (STPT) to quantitatively map genetically defined cell types and cerebrovasculature at single-cell resolution across the entire adult mouse brain. We describe the preparation of brain tissue and sample embedding for cell type and vascular STPT imaging and image processing using MATLAB codes. We detail the computational analyses for cell signal detection, vascular tracing, and three-dimensional image registration to anatomical atlases, which can be implemented for brain-wide mapping of different cell types. For complete details on the use and execution of this protocol, please refer to Wu et al. (2022),1 Son et al. (2022),2 Newmaster et al. (2020),3 Kim et al. (2017),4 and Ragan et al. (2012).5.

epDevAtlas: Mapping GABAergic cells and microglia in postnatal mouse brains.

During development, brain regions follow encoded growth trajectories. Compared to classical brain growth charts, high-definition growth charts could quantify regional volumetric growth and constituent cell types, improving our understanding of typical and pathological brain development. Here, we create high-resolution 3D atlases of the early postnatal mouse brain, using Allen CCFv3 anatomical labels, at postnatal days (P) 4, 6, 8, 10, 12, and 14, and determine the volumetric growth of different brain regions. We utilize 11 different cell type-specific transgenic animals to validate and refine anatomical labels. Moreover, we reveal region-specific density changes in γ-aminobutyric acid-producing (GABAergic), cortical layer-specific cell types, and microglia as key players in shaping early postnatal brain development. We find contrasting changes in GABAergic neuronal densities between cortical and striatal areas, stabilizing at P12. Moreover, somatostatin-expressing cortical interneurons undergo regionally distinct density reductions, while vasoactive intestinal peptide-expressing interneurons show no significant changes. Remarkably, microglia transition from high density in white matter tracks to gray matter at P10, and show selective density increases in sensory processing areas that correlate with the emergence of individual sensory modalities. Lastly, we create an open-access web-visualization (https://kimlab.io/brain-map/epDevAtlas) for cell-type growth charts and developmental atlases for all postnatal time points.

Developmental Mouse Brain Common Coordinate Framework.

3D standard reference brains serve as key resources to understand the spatial organization of the brain and promote interoperability across different studies. However, unlike the adult mouse brain, the lack of standard 3D reference atlases for developing mouse brains has hindered advancement of our understanding of brain development. Here, we present a multimodal 3D developmental common coordinate framework (DevCCF) spanning mouse embryonic day (E) 11.5, E13.5, E15.5, E18.5, and postnatal day (P) 4, P14, and P56 with anatomical segmentations defined by a developmental ontology. At each age, the DevCCF features undistorted morphologically averaged atlas templates created from Magnetic Resonance Imaging and co-registered high-resolution templates from light sheet fluorescence microscopy. Expert-curated 3D anatomical segmentations at each age adhere to an updated prosomeric model and can be explored via an interactive 3D web-visualizer. As a use case, we employed the DevCCF to unveil the emergence of GABAergic neurons in embryonic brains. Moreover, we integrated the Allen CCFv3 into the P56 template with stereotaxic coordinates and mapped spatial transcriptome cell-type data with the developmental ontology. In summary, the DevCCF is an openly accessible resource that can be used for large-scale data integration to gain a comprehensive understanding of brain development.

Dissecting the fatigue experience: A scoping review of fatigue definitions, dimensions, and measures in non-oncologic medical conditions.

INTRODUCTION: Fatigue is a prevalent and potentially debilitating symptom that impacts the health-related quality-of-life of individuals diagnosed with acute and chronic medical conditions. Yet, its etiologic mechanism is not fully understood. Additionally, the assessment and determination of the clinical meaning of fatigue and its multidimensionality may vary by medical condition. METHODS: A scoping literature review was conducted to investigate how fatigue is defined and measured, including its dimensions, in non-oncologic medical conditions. The PubMed database was searched using keywords. RESULTS: Overall, 8376 articles were screened at the title/abstract levels, where 293 articles were chosen for full-text review that mentioned fatigue or included fatigue measures. The review of the full text excluded 246 articles that did not assess at least one fatigue dimension using validated questionnaires and clinical tests. The final set included 47 articles. Physical fatigue was the most assessed fatigue dimension and the Multidimensional Fatigue Inventory was the most widely used questionnaire to assess fatigue in this review. LIMITATIONS: This review was limited by including only English-language publications and using PubMed as the sole database for the search. CONCLUSIONS: This review affirms that fatigue is a multidimensional construct, agnostic of medical condition, and that individual fatigue dimensions can be measured by validated clinical measures. Future research should focus on expanding the repertoire of clinical measures to assess specific fatigue dimensions.