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Deleterious Ca2+ accumulation is central to hypoxic cell death in the brain of most mammals. Conversely, hypoxia‐mediated increases in cytosolic Ca2+ are retarded in hypoxia‐tolerant naked mole‐rat brain. We hypothesized that naked mole‐rat brain mitochondria have an enhanced capacity to buffer exogenous Ca2+ and examined Ca2+ handling in naked mole‐rat cortical tissue. We report that naked mole‐rat brain mitochondria buffer >2‐fold more exogenous Ca2+ than mouse brain mitochondria, and that the half‐maximal inhibitory concentration (IC50) at which Ca2+ inhibits aerobic oxidative phosphorylation is >2‐fold higher in naked mole‐rat brain. The primary driving force of Ca2+ uptake is the mitochondrial membrane potential (Δψm), and the IC50 at which Ca2+ decreases Δψm is ∼4‐fold higher in naked mole‐rat than mouse brain. The ability of naked mole‐rat brain mitochondria to safely retain large volumes of Ca2+ may be due to ultrastructural differences that support the uptake and physical storage of Ca2+ in mitochondria. Specifically, and relative to mouse brain, naked mole‐rat brain mitochondria are larger and have higher crista density and increased physical interactions between adjacent mitochondrial membranes, all of which are associated with improved energetic homeostasis and Ca2+ management. We propose that excessive Ca2+ influx into naked mole‐rat brain is buffered by physical storage in large mitochondria, which would reduce deleterious Ca2+ overload and may thus contribute to the hypoxia and ischaemia‐tolerance of naked mole‐rat brain. Key points: Unregulated Ca2+ influx is a hallmark of hypoxic brain death; however, hypoxia‐mediated Ca2+ influx into naked mole‐rat brain is markedly reduced relative to mice.This is important because naked mole‐rat brain is robustly tolerant against in vitro hypoxia, and because Ca2+ is a key driver of hypoxic cell death in brain.We show that in hypoxic naked mole‐rat brain, oxidative capacity and mitochondrial membrane integrity are better preserved following exogenous Ca2+ stress.This is due to mitochondrial buffering of exogenous Ca2+ and is driven by a mitochondrial membrane potential‐dependant mechanism.The unique ultrastructure of naked mole‐rat brain mitochondria, as a large physical storage space, may support increased Ca2+ buffering and thus hypoxia‐tolerance. [ABSTRACT FROM AUTHOR]

Perineuronal nets (PNNs), a specialized form of extra cellular matrix (ECM), surround numerous neurons in the CNS and allow synaptic connectivity through holes in its structure. We hypothesize that PNNs serve as gatekeepers that guard and protect synaptic territory and thus may stabilize an engram circuit. We present high-resolution and 3D EM images of PNN-engulfed neurons in mice brains, showing that synapses occupy the PNN holes and that invasion of other cellular components is rare. PNN constituents in mice brains are long-lived and can be eroded faster in an enriched environment, while synaptic proteins have a high turnover rate. Preventing PNN erosion by using pharmacological inhibition of PNN-modifying proteases or matrix metalloproteases 9 (MMP9) knockout mice allowed normal fear memory acquisition but diminished long-term memory stabilization, supporting the above hypothesis. [ABSTRACT FROM AUTHOR]

Abstract Background While progress has been made to develop automatic segmentation techniques for mitochondria, there remains a need for more accurate and robust techniques to delineate mitochondria in serial blockface scanning electron microscopic data. Previously developed texture based methods are limited for solving this problem because texture alone is often not sufficient to identify mitochondria. This paper presents a new three-step method, the Cytoseg process, for automated segmentation of mitochondria contained in 3D electron microscopic volumes generated through serial block face scanning electron microscopic imaging. The method consists of three steps. The first is a random forest patch classification step operating directly on 2D image patches. The second step consists of contour-pair classification. At the final step, we introduce a method to automatically seed a level set operation with output from previous steps. Results We report accuracy of the Cytoseg process on three types of tissue and compare it to a previous method based on Radon-Like Features. At step 1, we show that the patch classifier identifies mitochondria texture but creates many false positive pixels. At step 2, our contour processing step produces contours and then filters them with a second classification step, helping to improve overall accuracy. We show that our final level set operation, which is automatically seeded with output from previous steps, helps to smooth the results. Overall, our results show that use of contour pair classification and level set operations improve segmentation accuracy beyond patch classification alone. We show that the Cytoseg process performs well compared to another modern technique based on Radon-Like Features. Conclusions We demonstrated that texture based methods for mitochondria segmentation can be enhanced with multiple steps that form an image processing pipeline. While we used a random-forest based patch classifier to recognize texture, it would be possible to replace this with other texture identifiers, and we plan to explore this in future work.

Abstract K+ channel interacting protein 1 (KChIP1) is a neuronal calcium sensor (NCS) protein that interacts with multiple intracellular molecules. Its physiological function, however, remains largely unknown. We report that KChIP1 is predominantly expressed at GABAergic synapses of a subset of parvalbumin-positive neurons in the brain. Forced expression of KChIP1 in cultured hippocampal neurons increased the frequency of miniature inhibitory postsynaptic currents (mIPSCs), reduced paired pulse facilitation of autaptic IPSCs, and decreases potassium current density. Furthermore, genetic ablation of KChIP1 potentiated potassium current density in neurons and caused a robust enhancement of anxiety-like behavior in mice. Our study suggests that KChIP1 is a synaptic protein that regulates behavioral anxiety by modulating inhibitory synaptic transmission, and drugs that act on KChIP1 may help to treat patients with mood disorders including anxiety.

Early postnatal brain development involves complex interactions among maturing neurons and glial cells that drive tissue organization. We previously analyzed gene expression in tissue from the mouse medial nucleus of the trapezoid body (MNTB) during the first postnatal week to study changes that surround rapid growth of the large calyx of Held (CH) nerve terminal. Here, we present genes that show significant changes in gene expression level during the second postnatal week, a developmental timeframe that brackets the onset of airborne sound stimulation and the early stages of myelination. Gene Ontology analysis revealed that many of these genes are related to the myelination process. Further investigation of these genes using a previously published cell type‐specific bulk RNA‐Seq data set in cortex and our own single‐cell RNA‐Seq data set in the MNTB revealed enrichment of these genes in the oligodendrocyte lineage (OL) cells. Combining the postnatal day (P)6–P14 microarray gene expression data with the previously published P0–P6 data provided fine temporal resolution to investigate the initiation and subsequent waves of gene expression related to OL cell maturation and the process of myelination. Many genes showed increasing expression levels between P2 and P6 in patterns that reflect OL cell maturation. Correspondingly, the first myelin proteins were detected by P4. Using a complementary, developmental series of electron microscopy 3D image volumes, we analyzed the temporal progression of axon wrapping and myelination in the MNTB. By employing a combination of established ultrastructural criteria to classify reconstructed early postnatal glial cells in the 3D volumes, we demonstrated for the first time that astrocytes within the mouse MNTB extensively wrap the axons of the growing CH terminal prior to OL cell wrapping and compaction of myelin. Our data revealed significant expression of several myelin genes and enrichment of multiple genes associated with lipid metabolism in astrocytes, which may subserve axon wrapping in addition to myelin formation. The transition from axon wrapping by astrocytes to OL cells occurs rapidly between P4 and P9 and identifies a potential new role of astrocytes in priming calyceal axons for subsequent myelination. [ABSTRACT FROM AUTHOR]

Cristae are high‐curvature structures in the inner mitochondrial membrane (IMM) that are crucial for ATP production. While cristae‐shaping proteins have been defined, analogous lipid‐based mechanisms have yet to be elucidated. Here, we combine experimental lipidome dissection with multi‐scale modeling to investigate how lipid interactions dictate IMM morphology and ATP generation. When modulating phospholipid (PL) saturation in engineered yeast strains, we observed a surprisingly abrupt breakpoint in IMM topology driven by a continuous loss of ATP synthase organization at cristae ridges. We found that cardiolipin (CL) specifically buffers the inner mitochondrial membrane against curvature loss, an effect that is independent of ATP synthase dimerization. To explain this interaction, we developed a continuum model for cristae tubule formation that integrates both lipid and protein‐mediated curvatures. This model highlighted a snapthrough instability, which drives IMM collapse upon small changes in membrane properties. We also showed that cardiolipin is essential in low‐oxygen conditions that promote PL saturation. These results demonstrate that the mechanical function of cardiolipin is dependent on the surrounding lipid and protein components of the IMM. Synopsis: Dedicated proteins are known to control the architecture of the inner mitochondrial membrane; however, the role of specific lipids is less well defined. Here, a combination of methods including tomography and lipidomics is used to characterise the role of lipid composition in defining cristae architecture. As lipid ratios change, a critical breakpoint phenocopies the loss of cristae‐shaping proteins in the IMM of yeast mitochondria.Phospholipid saturation controls membrane mechanical properties and modulates ATP synthase oligomerization.The mitochondria‐specific lipid cardiolipin functionally compensates for increased phospholipid saturation and is required for cristae formation in low‐oxygen environments.A mathematical model for cristae membrane tubules predicts a snap‐through instability mediated by both protein and lipid‐encoded curvatures. [ABSTRACT FROM AUTHOR]