Annotation of multiple regions of interest across the whole mouse brain is an indispensable process for quantitative evaluation of a multitude of study endpoints in neuroscience digital pathology. Prior experience and domain expert knowledge are the key aspects for image annotation quality and consistency. At present, image annotation is often achieved manually by certified pathologists or trained technicians, limiting the total throughput of studies performed at neuroscience digital pathology labs. It may also mean that simpler and quicker methods of examining tissue samples are used by non-pathologists, especially in the early stages of research and preclinical studies. To address these limitations and to meet the growing demand for image analysis in a pharmaceutical setting, we developed AnNoBrainer, an open-source software tool that leverages deep learning, image registration, and standard cortical brain templates to automatically annotate individual brain regions on 2D pathology slides. Application of AnNoBrainer to a published set of pathology slides from transgenic mice models of synucleinopathy revealed comparable accuracy, increased reproducibility, and a significant reduction (~ 50%) in time spent on brain annotation, quality control and labelling compared to trained scientists in pathology. Taken together, AnNoBrainer offers a rapid, accurate, and reproducible automated annotation of mouse brain images that largely meets the experts' histopathological assessment standards (> 85% of cases) and enables high-throughput image analysis workflows in digital pathology labs.

• Klíčová slova
• Deep learning, Image registration, Mouse brain,
• MeSH
• deep learning * MeSH
• mozek * diagnostické zobrazování   patologie   MeSH
• myši transgenní MeSH
• myši MeSH
• počítačové zpracování obrazu * metody   MeSH
• reprodukovatelnost výsledků MeSH
• software normy   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

In the central nervous system (CNS), cannabinoid receptor 1 (CB1R) is preferentially expressed in axons where it has a unique property, namely resistance to agonist-driven endocytosis. This review aims to summarize what we know about molecular mechanisms of CB1R cell surface stability in axonal compartments, how these impact CB1R signaling, and to consider their physiological consequences. This review then focuses on a potential candidate for maintaining axonal CB1R at the cell surface, Src homology 3-domain growth factor receptor-bound 2-like endophilin interacting protein 1 (SGIP1). SGIP1 may contribute to the polarized distribution of CB1R and modify its signaling in axons. In addition, deletion of SGIP1 results in discrete behavioral changes in modalities controlled by the endocannabinoid system in vivo. Several drugs acting directly via CB1R have important therapeutic potential, however their adverse effects limit their clinical use. Future studies might reveal chemical approaches to target the SGIP1-CB1R interaction, with the aim to exploit the endocannabinoid system pharmaceutically in a discrete way, with minimized undesired consequences.

• Klíčová slova
• axon enrichment, cannabinoid receptor 1, clathrin-mediated endocytosis, internalization, synaptic transmission,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

The protease caspase-3 is a key mediator of apoptotic programmed cell death. But weak or transient caspase activity can contribute to neuronal differentiation, axonal pathfinding, and synaptic long-term depression. Despite the importance of sublethal, or nonapoptotic, caspase activity in neurodevelopment and neural plasticity, there has been no simple method for mapping and quantifying nonapoptotic caspase activity (NACA) in rodent brains. We therefore generated a transgenic mouse expressing a highly sensitive and specific fluorescent reporter of caspase activity, with peak signal localized to the nucleus. As a proof of concept, we first obtained evidence that NACA influences neurophysiology in an amygdalar circuit. Then focusing on the amygdala, we were able to quantify a sex-specific persistent elevation in caspase activity in females after restraint stress. This simple in vivo caspase activity reporter will facilitate systems-level studies of apoptotic and nonapoptotic phenomena in behavioral and pathologic models.

• Klíčová slova
• amygdala, caspases, in vivo reporter, mapping, nonapoptotic, sex differences, stress,
• MeSH
• apoptóza * fyziologie   MeSH
• kaspasa 9 MeSH
• mozek * MeSH
• myši transgenní MeSH
• myši MeSH
• neuroplasticita MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• kaspasa 9 MeSH

Multimode fibres show great potential for use as miniature endoscopes for imaging deep in tissue with minimal damage. When used for coherent anti-Stokes Raman scattering (CARS) microscopy with femtosecond excitation sources, a high band-width probe is required to efficiently focus the broadband laser pulses at the sample plane. Although graded-index (GRIN) fibres have a large bandwidth, it is accompanied by a strong background signal from four-wave mixing and other non-linear processes occurring inside the fibre. We demonstrate that using a composite probe consisting of a GRIN fibre with a spliced on step-index fibre reduces the intensity of the non-linear background by more than one order of magnitude without significantly decreasing the focusing performance of the probe. Using this composite probe we acquire CARS images of biologically relevant tissue such as myelinated axons in the brain with good contrast.

• Publikační typ
• časopisecké články MeSH

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay.

• MeSH
• buněčné linie MeSH
• fosforylace MeSH
• genetická transkripce MeSH
• genový knockdown MeSH
• lidé MeSH
• myši knockoutované MeSH
• myši MeSH
• neurony chemie   metabolismus   MeSH
• posttranskripční úpravy RNA MeSH
• proteinové domény MeSH
• regulace genové exprese MeSH
• RNA-polymerasa II chemie   genetika   metabolismus   MeSH
• RNA * chemie   genetika   metabolismus   MeSH
• stabilita RNA MeSH
• transkripční faktory genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• PHF3 protein, human MeSH Prohlížeč
• RNA-polymerasa II MeSH
• RNA * MeSH
• transkripční faktory MeSH

Information about features in the visual world is parsed by circuits in the retina and is then transmitted to the brain by distinct subtypes of retinal ganglion cells (RGCs). Axons from RGC subtypes are stratified in retinorecipient brain nuclei, such as the superior colliculus (SC), to provide a segregated relay of parallel and feature-specific visual streams. Here, we sought to identify the molecular mechanisms that direct the stereotyped laminar targeting of these axons. We focused on ipsilateral-projecting subtypes of RGCs (ipsiRGCs) whose axons target a deep SC sublamina. We identified an extracellular glycoprotein, Nephronectin (NPNT), whose expression is restricted to this ipsiRGC-targeted sublamina. SC-derived NPNT and integrin receptors expressed by ipsiRGCs are both required for the targeting of ipsiRGC axons to the deep sublamina of SC. Thus, a cell-extracellular matrix (ECM) recognition mechanism specifies precise laminar targeting of ipsiRGC axons and the assembly of eye-specific parallel visual pathways.

• Klíčová slova
• axon targeting, development, extracellular matrix, retina, superior colliculus,
• MeSH
• axony fyziologie   MeSH
• colliculus superior cytologie   metabolismus   fyziologie   MeSH
• extracelulární matrix fyziologie   MeSH
• integriny metabolismus   MeSH
• mozek fyziologie   MeSH
• myši MeSH
• retinální gangliové buňky fyziologie   MeSH
• signální transdukce MeSH
• zrakové dráhy * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• integriny MeSH

The choroid plexus (ChP) in each brain ventricle produces cerebrospinal fluid (CSF) and forms the blood-CSF barrier. Here, we construct a single-cell and spatial atlas of each ChP in the developing, adult, and aged mouse brain. We delineate diverse cell types, subtypes, cell states, and expression programs in epithelial and mesenchymal cells across ages and ventricles. In the developing ChP, we predict a common progenitor pool for epithelial and neuronal cells, validated by lineage tracing. Epithelial and fibroblast cells show regionalized expression by ventricle, starting at embryonic stages and persisting with age, with a dramatic transcriptional shift with maturation, and a smaller shift in each aged cell type. With aging, epithelial cells upregulate host-defense programs, and resident macrophages upregulate interleukin-1β (IL-1β) signaling genes. Our atlas reveals cellular diversity, architecture and signaling across ventricles during development, maturation, and aging of the ChP-brain barrier.

• Klíčová slova
• aging, brain barrier, cerebrospinal fluid, choroid plexus, development, single-cell RNA sequencing, single-nucleus RNA sequencing,
• MeSH
• analýza jednotlivých buněk MeSH
• buněčná diferenciace genetika   MeSH
• buněčný rodokmen genetika   MeSH
• epitelové buňky metabolismus   MeSH
• hematoencefalická bariéra metabolismus   MeSH
• mozek metabolismus   fyziologie   MeSH
• myši inbrední C57BL MeSH
• myši embryologie   MeSH
• nemoci mozku genetika   patofyziologie   MeSH
• plexus chorioideus embryologie   metabolismus   fyziologie   MeSH
• signální transdukce MeSH
• stárnutí fyziologie   MeSH
• věkové faktory MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši embryologie   MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

The choroid plexus (ChP) produces cerebrospinal fluid and forms an essential brain barrier. ChP tissues form in each brain ventricle, each one adopting a distinct shape, but remarkably little is known about the mechanisms underlying ChP development. Here, we show that epithelial WNT5A is crucial for determining fourth ventricle (4V) ChP morphogenesis and size in mouse. Systemic Wnt5a knockout, or forced Wnt5a overexpression beginning at embryonic day 10.5, profoundly reduced ChP size and development. However, Wnt5a expression was enriched in Foxj1-positive epithelial cells of 4V ChP plexus, and its conditional deletion in these cells affected the branched, villous morphology of the 4V ChP. We found that WNT5A was enriched in epithelial cells localized to the distal tips of 4V ChP villi, where WNT5A acted locally to activate non-canonical WNT signaling via ROR1 and ROR2 receptors. During 4V ChP development, MEIS1 bound to the proximal Wnt5a promoter, and gain- and loss-of-function approaches demonstrated that MEIS1 regulated Wnt5a expression. Collectively, our findings demonstrate a dual function of WNT5A in ChP development and identify MEIS transcription factors as upstream regulators of Wnt5a in the 4V ChP epithelium.

• Klíčová slova
• Choroid plexus, Epithelium, Meis1, Meis2, Morphogenesis, WNT5a,
• MeSH
• buněčné linie MeSH
• CRISPR-Cas systémy genetika   MeSH
• čtvrtá mozková komora embryologie   MeSH
• epitel metabolismus   MeSH
• epitelové buňky metabolismus   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• mozek embryologie   MeSH
• myši knockoutované MeSH
• myši MeSH
• plexus chorioideus embryologie   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• protein Wnt 5a genetika   metabolismus   MeSH
• signální transdukce fyziologie   MeSH
• sirotčí receptory podobné receptoru tyrosinkinasy metabolismus   MeSH
• transkripční faktor Meis1 metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• Meis1 protein, mouse MeSH Prohlížeč
• protein Wnt 5a MeSH
• Ror1 protein, mouse MeSH Prohlížeč
• Ror2 protein, mouse MeSH Prohlížeč
• sirotčí receptory podobné receptoru tyrosinkinasy MeSH
• transkripční faktor Meis1 MeSH
• Wnt5a protein, mouse MeSH Prohlížeč

BACKGROUND AND PURPOSE: Src homology 3-domain growth factor receptor-bound 2-like endophilin interacting protein 1 (SGIP1) interacts with cannabinoid CB1 receptors. SGIP1 is abundantly and principally expressed within the nervous system. SGIP1 and CB1 receptors co-localize in axons and presynaptic boutons. SGIP1 interferes with the internalization of activated CB1 receptors in transfected heterologous cells. Consequently, the transient association of CB1 receptors with β-arrestin2 is enhanced and prolonged, and CB1 receptor-mediated ERK1/2 signalling is decreased. Because of these actions, SGIP1 may modulate affect, anxiety, pain processing, and other physiological processes controlled by the endocannabinoid system (ECS). EXPERIMENTAL APPROACH: Using a battery of behavioural tests, we investigated the consequences of SGIP1 deletion in tasks regulated by the ECS in SGIP1 constitutive knockout (SGIP1-/- ) mice. KEY RESULTS: In SGIP1-/- mice, sensorimotor gating, exploratory levels, and working memory are unaltered. SGIP1-/- mice have decreased anxiety-like behaviours. Fear extinction to tone is facilitated in SGIP1-/- females. Several cannabinoid tetrad behaviours are altered in the absence of SGIP1. SGIP1-/- males exhibit abnormal behaviours on Δ9 -tetrahydrocannabinol withdrawal. SGIP1 deletion also reduces acute nociception, and SGIP1-/- mice are more sensitive to analgesics. CONCLUSION AND IMPLICATIONS: SGIP1 was detected as a novel protein associated with CB1 receptors, and profoundly modified CB1 receptor signalling. Genetic deletion of SGIP1 particularly affected behavioural tests of mood-related assessment and the cannabinoid tetrad. SGIP1-/- mice exhibit decreased nociception and augmented responses to CB1 receptor agonists and morphine. These in vivo findings suggest that SGIP1 is a novel modulator of CB1 receptor-mediated behaviour.

• Klíčová slova
• GPCR, anxiety, cannabinoid receptor 1, endocannabinoid system, pain, tolerance,
• MeSH
• adaptorové proteiny signální transdukční fyziologie   MeSH
• afekt MeSH
• emoce MeSH
• extinkce (psychologie) MeSH
• kanabinoidy MeSH
• myši knockoutované MeSH
• myši MeSH
• nocicepce * MeSH
• receptor kanabinoidní CB1 * genetika   MeSH
• receptory kanabinoidní MeSH
• strach MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• kanabinoidy MeSH
• receptor kanabinoidní CB1 * MeSH
• receptory kanabinoidní MeSH
• SGIP1 protein, mouse MeSH Prohlížeč

The deletion of M4 muscarinic receptors (MRs) changes biological rhythm parameters in females. Here, we searched for the mechanisms responsible for these changes. We performed biological rhythm analysis in two experiments: in experiment 1, the mice [C57Bl/6NTac (WT) and M4 MR -/- mice (KO)] were first exposed to a standard LD regime (12/12-h light/dark cycle) for 8 days and then subsequently exposed to constant darkness (for 24 h/day, DD regime) for another 16 days. In experiment 2, the mice (after the standard LD regime) were exposed to the DD regime and to one light pulse (zeitgeber time 14) on day 9. We also detected M1 MRs in brain areas implicated in locomotor biological rhythm regulation. In experiment 1, the biological rhythm activity curves differed: the period (τ, duration of diurnal cycle) was shorter in the DD regime. Moreover, the day mean, mesor (midline value), night mean and their difference were higher in KO animals. The time in which the maximal slope occurred was lower in the DD regime than in the LD regime in both WT and KO but was lower in KO than in WT mice. In experiment 2, there were no differences in biological rhythm parameters between WT and KO mice. The densities of M1 MRs in the majority of areas implicated in locomotor biological rhythm were low. A significant amount of M1 MR was found in the striatum. These results suggest that although core clock output is changed by M4 MR deletion, the structures involved in biological rhythm regulation in WT and KO animals are likely the same, and the most important areas are the striatum, thalamus and intergeniculate leaflet.

• Klíčová slova
• Biorhythm, Intergeniculate leaflet, Locomotor activity, M1 muscarinic receptors, M4 muscarinic receptors,
• MeSH
• aktigrafie MeSH
• lokomoce fyziologie   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• neostriatum fyziologie   MeSH
• periodicita * MeSH
• receptor muskarinový M4 genetika   fyziologie   MeSH
• thalamus fyziologie   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• receptor muskarinový M4 MeSH