Serial Block Face Research Articles

Death in the taste bud: Morphological features of dying taste cells and engulfment by Type I cells.

Taste buds comprise 50-100 epithelial derived cells, which are renewed throughout the life of an organism. Immature cells enter the bud at its base, maturing into one of three distinct cell types. How taste cells die and/or exit the bud, however, remains unclear. Here we present morphological data obtained through Serial Blockface Scanning Electron Microscopy of murine circumvallate taste buds, revealing several taste cells at the end of their life (4-6 per bud). Cells we identify as dying share certain morphological features typical of apoptosis: swollen endoplasmic reticulum, large lysosomes, degrading organelles, distended outer nuclear membranes, heterochromatin reorganization, cell shrinkage, and cell and/or nuclear fragmentation. Based on these features, we divide the cells into "early" and "late" stage dying cells. Most early stage dying cells have Type II cell morphologies, while a few display Type III cell features. Many dying cells maintain contacts with nerve fibers, but those fibers often appear detached from the main trunk of an afferent nerve fiber. Dying cells, like mature Type II and Type III taste cells, are surrounded by Type I taste cells, the glial-like cells of the bud. In many instances Type I cells appear to be engulfing their dying neighbors, suggesting a novel, phagocytic role for Type I cells. Surprisingly, virtually no Type I cells, which have the shortest residence time in taste buds, display features of apoptosis. The ultimate fate of Type I cells therefore remains enigmatic.

Serial block-face scanning electron microscopy of adherent cells on thin plastic substrate

Abstract Serial block-face (SBF) scanning electron microscopy (SEM) is used for imaging the entire internal ultrastructure of cells, tissue samples or small organisms. Here, we present a workflow for SBF SEM of adherent cells, such as Giardia parasites and HeLa cells, attached to the surface of a plastic culture dish, which preserves the interface between cells and plastic substrate. Cells were embedded in situ on their substrate using silicone microwells and were mounted for cross-sectioning which allowed SBF imaging of large volumes and many cells. A standard sample preparation and embedding protocol for thin section electron microscopy provided already sufficient resolution and image quality to visualize larger structures. To improve resolution and image quality of SBF imaging, we stepwise tested modifications of the protocol, such as the moderate increase of the heavy metal content of the sample. Modifications of the embedding by either the reduction of the resin layer (minimal embedding) or the addition of silver colloid to the resin were evaluated at high and low vacuum imaging conditions. The optimized sample preparation protocol is very similar to the standard preparation protocol for thin section electron microscopy, so that the samples can also be used for this application. The protocol applies a higher concentration of osmium tetroxide, a higher temperature for heavy metal incubation and an additional lead en bloc staining. In summary, the presented workflow provides a generic and adaptable solution for studying adherent cells by SBF SEM.

HeLa cell segmentation using digital image processing

Computational cell segmentation is a vital area of research, particularly in the analysis of images of cancer cells. The use of cell lines, such as the widely utilized HeLa cell line, is crucial for studying cancer. While deep learning algorithms have been commonly employed for cell segmentation, their resource and data requirements can be impractical for many laboratories. In contrast, image processing algorithms provide a promising alternative due to their effectiveness and minimal resource demands. This article presents the development of an algorithm utilizing digital image processing to segment the nucleus and shape of HeLa cells. The research aims to segment the cell shape in the image center and accurately identify the nucleus. The study uses and processes 300 images obtained from Serial Block-Face Scanning Electron Microscopy (SBF-SEM). For cell segmentation, the morphological operation of erosion was used to separate the cells, and through distance calculation, the cell located at the center of the image was selected. Subsequently, the eroded shape was employed to restore the original cell shape. The nucleus segmentation uses parameters such as distances and sizes, along with the implementation of verification stages to ensure accurate detection. The accuracy of the algorithm is demonstrated by comparing it with another algorithm meeting the same conditions, using four segmentation similarity metrics. The evaluation results rank the proposed algorithm as the superior choice, highlighting significant outcomes. The algorithm developed represents a crucial initial step towards more accurate disease analysis. In addition, it enables the measurement of shapes and the identification of morphological alterations, damages, and changes in organelles within the cell, which can be vital for diagnostic purposes.

Energetics of the microsporidian polar tube invasion machinery

Microsporidia are eukaryotic, obligate intracellular parasites that infect a wide range of hosts, leading to health and economic burdens worldwide. Microsporidia use an unusual invasion organelle called the polar tube (PT), which is ejected from a dormant spore at ultra-fast speeds, to infect host cells. The mechanics of PT ejection are impressive. Anncaliia algerae microsporidia spores (3–4 μm in size) shoot out a 100-nm-wide PT at a speed of 300 μm/s, creating a shear rate of 3000 s-1. The infectious cargo, which contains two nuclei, is shot through this narrow tube for a distance of ∼60–140 μm (Jaroenlak et al, 2020) and into the host cell. Considering the large hydraulic resistance in an extremely thin tube and the low-Reynolds-number nature of the process, it is not known how microsporidia can achieve this ultrafast event. In this study, we use Serial Block-Face Scanning Electron Microscopy to capture 3-dimensional snapshots of A. algerae spores in different states of the PT ejection process. Grounded in these data, we propose a theoretical framework starting with a systematic exploration of possible topological connectivity amongst organelles, and assess the energy requirements of the resulting models. We perform PT firing experiments in media of varying viscosity, and use the results to rank our proposed hypotheses based on their predicted energy requirement. We also present a possible mechanism for cargo translocation, and quantitatively compare our predictions to experimental observations. Our study provides a comprehensive biophysical analysis of the energy dissipation of microsporidian infection process and demonstrates the extreme limits of cellular hydraulics.

Energetics of the microsporidian polar tube invasion machinery.

Microsporidia are eukaryotic, obligate intracellular parasites that infect a wide range of hosts, leading to health and economic burdens worldwide. Microsporidia use an unusual invasion organelle called the polar tube (PT), which is ejected from a dormant spore at ultra-fast speeds, to infect host cells. The mechanics of PT ejection are impressive. Anncaliia algerae microsporidia spores (3-4 μm in size) shoot out a 100-nm-wide PT at a speed of 300 μm/s, creating a shear rate of 3000 s-1. The infectious cargo, which contains two nuclei, is shot through this narrow tube for a distance of ∼60-140 μm (Jaroenlak et al, 2020) and into the host cell. Considering the large hydraulic resistance in an extremely thin tube and the low-Reynolds-number nature of the process, it is not known how microsporidia can achieve this ultrafast event. In this study, we use Serial Block-Face Scanning Electron Microscopy to capture 3-dimensional snapshots of A. algerae spores in different states of the PT ejection process. Grounded in these data, we propose a theoretical framework starting with a systematic exploration of possible topological connectivity amongst organelles, and assess the energy requirements of the resulting models. We perform PT firing experiments in media of varying viscosity, and use the results to rank our proposed hypotheses based on their predicted energy requirement. We also present a possible mechanism for cargo translocation, and quantitatively compare our predictions to experimental observations. Our study provides a comprehensive biophysical analysis of the energy dissipation of microsporidian infection process and demonstrates the extreme limits of cellular hydraulics.

Work smart, not hard: How array tomography can help increase the ultrastructure data output.

Transmission electron microscopy has been essential for understanding cell biology for over six decades. Volume electron microscopy tools, such as serial block face and focused ion beam scanning electron microscopy acquisition, brought a new era to ultrastructure analysis. 'Array Tomography' (AT) refers to sequential image acquisition of resin-embedded sample sections on a large support (coverslip, glass slide, silicon wafers) for immunolabelling with multiple fluorescent labels, occasionally combined with ultrastructure observation. Subsequently, the term was applied to generating and imaging a series of sections to acquire a 3D representation of a structure using scanning electron microscopy (SEM). Although this is a valuable application, the potential of AT is to facilitate many tasks that are difficult or even impossible to obtain by Transmission Electron Microscopy (TEM). Due to the straightforward nature and versatility of AT sample preparation and image acquisition, the technique can be applied practically to any biological sample for selected sections or volume electron microscopy analysis. Furthermore, in addition to the benefits described here, AT is compatible with morphological analysis, multiplex immunolabelling, immune-gold labelling, and correlative light and electron microscopy workflow applicable for single cells, tissue and small organisms. This versatility makes AT attractive not only for basic research but as a diagnostic tool with a simplified routine.

Biomechanics of the JCT and SC Inner Wall Endothelial Cells with Their Basement Membrane Using 3D Serial Block-Face Scanning Electron Microscopy.

More than ~70% of the aqueous humor exits the eye through the conventional aqueous outflow pathway that is comprised of the trabecular meshwork (TM), juxtacanalicular tissue (JCT), the inner wall endothelium of Schlemm's canal (SC). The flow resistance in the JCT and SC inner wall basement membrane is thought to play an important role in the regulation of the intraocular pressure (IOP) in the eye, but current imaging techniques do not provide enough information about the mechanics of these tissues or the aqueous humor in this area. A normal human eye was perfusion-fixed and a radial wedge of the TM tissue from a high-flow region was dissected. The tissues were then sliced and imaged using serial block-face scanning electron microscopy. Slices from these images were selected and segmented to create a 3D finite element model of the JCT and SC cells with an inner wall basement membrane. The aqueous humor was used to replace the intertrabecular spaces, pores, and giant vacuoles, and fluid-structure interaction was employed to couple the motion of the tissues with the aqueous humor. Higher tensile stresses (0.8-kPa) and strains (25%) were observed in the basement membrane beneath giant vacuoles with open pores. The volumetric average wall shear stress was higher in SC than in JCT/SC. As the aqueous humor approached the inner wall basement membrane of SC, the velocity of the flow decreased, resulting in the formation of small eddies immediately after the flow left the inner wall. Improved modeling of SC and JCT can enhance our understanding of outflow resistance and funneling. Serial block-face scanning electron microscopy with fluid-structure interaction can achieve this, and the observed micro-segmental flow patterns in ex vivo perfused human eyes suggest a hypothetical mechanism.

Ultrastructure of Synaptic Connectivity within Subregions of the Suprachiasmatic Nucleus Revealed by a Genetically Encoded Tag and Serial Blockface Electron Microscopy.

The hypothalamic suprachiasmatic nucleus (SCN) is the central circadian pacemaker in vertebrates. The SCN receives photic information exclusively through melanopsin-expressing retinal ganglion cells (mRGCs) to synchronize circadian rhythms with the environmental light cycles. The SCN is composed of two major peptidergic neuron types in the core and shell regions of the SCN. Determining how mRGCs interact with the network of synaptic connections onto and between SCN neurons is key to understand how light regulates the circadian clock and to elucidate the relevant local circuits within the SCN. To map these connections, we used a newly developed Cre-dependent electron microscopy (EM) reporter, APEX2, to label the mitochondria of mRGC axons. Serial blockface scanning electron microscopy was then used to resolve the fine 3D structure of mRGC axons and synaptic boutons in the SCN of a male mouse. The resulting maps reveal patterns of connectomic organization in the core and shell of the SCN. We show that these regions are composed of different neuronal subtypes and differ with regard to the pattern of mRGC input, as the shell receives denser mRGC synaptic input compared with the core. This finding challenges the present view that photic information coming directly from the retina is received primarily by the core region of the SCN.

What Should I Do with My Serial Block-Face Data? Suggestions for Preparing, Analyzing, and Presenting Volume EM Datasets.

What Should I Do with My Serial Block-Face Data? Suggestions for Preparing, Analyzing, and Presenting Volume EM Datasets.

An Integrated Solution for the Complete Serial Block-Face Scanning Electron Microscopy Workflow: From Image Acquisition to Data Processing.

An Integrated Solution for the Complete Serial Block-Face Scanning Electron Microscopy Workflow: From Image Acquisition to Data Processing.

CRMP2 conditional knockout changes axonal function and ultrastructure of axons in mice corpus callosum

Collapsin response mediator protein 2 (CRMP2) is a member of a protein family, which is highly involved in neurodevelopment, but most of its members become heavily downregulated in adulthood. CRMP2 is an important factor in neuronal polarization, axonal formation and growth cone collapse. The protein remains expressed in adulthood, but is more region specific. CRMP2 is present in adult corpus callosum (CC) and in plastic areas like prefrontal cortex and hippocampus. CRMP2 has been implicated as one of the risk-genes for Schizophrenia (SZ). Here, a CRMP2 conditional knockout (CRMP2-cKO) mouse was used as a model of SZ to investigate how it could affect the white matter and therefore brain connectivity.Multielectrode electrophysiology (MEA) was used to study the function of corpus callosum showing an increase in conduction velocity (CV) measured as Compound Action Potentials (CAPs) in acute brain slices. Light- and electron-microscopy, specifically Serial Block-face Scanning Electron Microscopy (SBF-SEM), methods were used to study the structure of CC in CRMP2-cKO mice. A decrease in CC volume of CRMP2-cKO mice as compared to controls was observed. No differences were found in numbers nor in the size of CC oligodendrocytes (OLs). Similarly, no differences were found in myelin thickness or in node of Ranvier (NR) structure. In contrast, abnormally smaller axons were measured in the CRMP2-cKO mice.Using these state-of-the-art methods it was possible to shed light on specific parts of the dysconnectivity aspect of deletion of CRMP2 related to SZ and add details to previous findings helping further understanding the disease. This paper substantiates the white matter changes in the absence of CRMP2 and ties it to the role it plays in this complex disorder.

Serial Block Face-Scanning Electron Microscopy as a Burgeoning Technology.

Serial block face scanning electron microscopy (SBF-SEM), also referred to as serial block-face electron microscopy, is an advanced ultrastructural imaging technique that enables three-dimensional visualization that provides largerx- and y-axis ranges than other volumetric EM techniques. While SEM is first introduced in the 1930s, SBF-SEM is developed as a novel method to resolve the 3D architecture of neuronal networks across large volumes with nanometer resolution by Denk and Horstmann in 2004.Here, the authors provide an accessible overview of the advantages and challenges associated with SBF-SEM. Beyond this, the applications of SBF-SEM in biochemical domains as well as potential future clinical applications are briefly reviewed. Finally, the alternative forms of artificial intelligence-based segmentation which may contribute to devising a feasible workflow involving SBF-SEM, are also considered.

Femtosecond laser preparation of resin embedded samples for correlative microscopy workflows in life sciences.

Correlative multimodal imaging is a useful approach to investigate complex structural relations in life sciences across multiple scales. For these experiments, sample preparation workflows that are compatible with multiple imaging techniques must be established. In one such implementation, a fluorescently labeled region of interest in a biological soft tissue sample can be imaged with light microscopy before staining the specimen with heavy metals, enabling follow-up higher resolution structural imaging at the targeted location, bringing context where it is required. Alternatively, or in addition to fluorescence imaging, other microscopy methods, such as synchrotron x-ray computed tomography with propagation-based phase contrast or serial blockface scanning electron microscopy, might also be applied. When combining imaging techniques across scales, it is common that a volumetric region of interest (ROI) needs to be carved from the total sample volume before high resolution imaging with a subsequent technique can be performed. In these situations, the overall success of the correlative workflow depends on the precise targeting of the ROI and the trimming of the sample down to a suitable dimension and geometry for downstream imaging. Here, we showcase the utility of a femtosecond laser (fs laser) device to prepare microscopic samples (1) of an optimized geometry for synchrotron x-ray tomography as well as (2) for volume electron microscopy applications and compatible with correlative multimodal imaging workflows that link both imaging modalities.

P192 Investigating the link between pleomorphism and virulence in Cryptococcus

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMObjectivesFungal pathogens Cryptococcus neoformans and C. gattii are responsible for hundreds of thousands of annual deaths in immunocompromised individuals. Considerable phenotypic variation is exhibited by strains in response to stresses encountered during host infection, including increased capsule and cell size, the release of shed capsule, and the production of giant (>15 μm), micro (<1 μm), and irregular cells. We aimed to investigate whether the production of these morphological variants is associated with virulence using two sets of strains. The first is a collection of diverse clinical isolates obtained from HIV/AIDS patients in Botswana with accompanying clinical data. The second is a collection of lineages derived from the C. neoformans type strain H99 with high genetic similarity but differing levels of virulence. Some lineages in this set possess a mutation in SGF29, which encodes a component of the SAGA histone acetylation complex that has previously been implicated in their hypervirulence.MethodsIsolates were cultured under conditions that simulate stresses encountered in vivo (DMEM, 5% CO2, 37°C) as these are known to enhance capsule production and induce cell size changes. Cells were counterstained with India Ink, visualized by light microscopy, and phenotypes were scored. For clinical isolates, MLST analysis was performed to determine their relatedness. For H99 strains, Galleria mellonella larval infection assays, growth curves, and antifungal susceptibility testing was performed to confirm their relative virulence and growth profiles. Serial block face and regular scanning electron microscopy were used to investigate the internal morphology of the giant, micro, and irregular cells to confirm that they possess attributes of functional cells.ResultsSubstantial pleomorphism was seen across both collections. In the clinical strain set, phenotypic variables fell into two groups associated with differing symptoms. The production of ‘large’ phenotypes was associated with a higher CD4 count and was negatively correlated with intracranial pressure indicators, suggesting that these are induced in early-stage infection. ‘Small’ phenotypes were associated with lower CD4 counts, negatively correlated with meningeal inflammation indicators, and positively correlated with intracranial pressure indicators, suggesting that they are produced later during infection and may promote proliferation and dissemination. Isolates possessing giant cells, microcells, and shed capsule were rare, but strikingly, they were associated with patient death.In the H99 set, strains from hypervirulent lineages had larger average capsule size, greater variation in cell size, and increased production of microcells and shed capsule. Deletion of SGF29 in an intermediate virulence lineage substantially increased its production of microcells and released capsule, consistent with a switch to hypervirulence. SGF29 loss-of-function mutations were subsequently identified in clinical isolates and were found to be significantly correlated with patient death. Expansion of a TA repeat in the second intron of SGF29 in clinical isolates was positively correlated with cell and capsule size, suggesting it also affects SGF29 function.ConclusionOur results extend the evidence for a link between pleomorphism and virulence, with a likely role for epigenetic mechanisms mediated by SAGA-induced histone acetylation.

S5.3c Investigating the link between pleomorphism and virulence in Cryptococcus

S5.3 Cellular pleomorphism and fungal virulence, September 22, 2022, 3:00 PM - 4:30 PMObjectivesFungal pathogens Cryptococcus neoformans and Cryptococcus gattii are responsible for hundreds of thousands of annual deaths in immunocompromised individuals. Considerable phenotypic variation is exhibited by strains in response to stresses encountered during host infection, including increased capsule and cell size, the release of shed capsule, and the production of giant (> 15 μm), micro (< 1 μm), and irregular cells. We aimed to investigate whether the production of these morphological variants is associated with virulence using two sets of strains. The first is a collection of diverse clinical isolates obtained from HIV/AIDS patients in Botswana with accompanying clinical data. The second is a collection of lineages derived from the C. neoformans type strain H99 with high genetic similarity but differing levels of virulence. Some lineages in this set possess a mutation in SGF29, which encodes a component of the SAGA histone acetylation complex that has previously been implicated in their hypervirulence.MethodsIsolates were cultured under conditions that simulate stresses encountered in vivo (DMEM, 5% CO2, 37°C) as these are known to enhance capsule production and induce cell size changes. Cells were counterstained with india ink, visualized by light microscopy, and phenotypes were scored. For clinical isolates, MLST analysis was performed to determine their relatedness. For H99 strains, Galleria mellonella larval infection assays, growth curves, and antifungal susceptibility testing was performed to confirm their relative virulence and growth profiles. Serial block face and regular scanning electron microscopy were used to investigate the internal morphology of the giant, micro, and irregular cells to confirm that they possess attributes of functional cells.ResultsSubstantial pleomorphism was seen across both collections. In the clinical strain set, phenotypic variables fell into two groups associated with differing symptoms. The production of ‘large’ phenotypes was associated with a higher CD4 count and was negatively correlated with intracranial pressure indicators, suggesting that these are induced in early-stage infection. ‘Small’ phenotypes were associated with lower CD4 counts, negatively correlated with meningeal inflammation indicators, and positively correlated with intracranial pressure indicators, suggesting that they are produced later during infection and may promote proliferation and dissemination. Isolates possessing giant cells, microcells, and shed capsules were rare, but strikingly, they were associated with patient death.In the H99 set, strains from hypervirulent lineages had larger average capsule size, greater variation in cell size, and increased production of microcells and shed capsules. Deletion of SGF29 in an intermediate virulence lineage substantially increased its production of microcells and released capsule, consistent with a switch to hypervirulence. SGF29 loss-of-function mutations were subsequently identified in clinical isolates and were found to be significantly correlated with patient death. Expansion of a TA repeat in the second intron of SGF29 in clinical isolates was positively correlated with cell and capsule size, suggesting it also affects Sgf29 function.ConclusionOur results extend the evidence for a link between pleomorphism and virulence, with a likely role for epigenetic mechanisms mediated by SAGA-induced histone acetylation.

Integrating single cell transcriptomics and volume electron microscopy confirms the presence of pancreatic acinar-like cells in sea urchins.

The identity and function of a given cell type relies on the differential expression of gene batteries that promote diverse phenotypes and functional specificities. Therefore, the identification of the molecular and morphological fingerprints of cell types across taxa is essential for untangling their evolution. Here we use a multidisciplinary approach to identify the molecular and morphological features of an exocrine, pancreas-like cell type harbored within the sea urchin larval gut. Using single cell transcriptomics, we identify various cell populations with a pancreatic-like molecular fingerprint that are enriched within the S. purpuratus larva digestive tract. Among these, in the region where they reside, the midgut/stomach domain, we find that populations of exocrine pancreas-like cells have a unique regulatory wiring distinct from the rest the of the cell types of the same region. Furthermore, Serial Block-face scanning Electron Microscopy (SBEM) of the exocrine cells shows that this reported molecular diversity is associated to distinct morphological features that reflect the physiological and functional properties of this cell type. Therefore, we propose that these sea urchin exocrine cells are homologous to the well-known mammalian pancreatic acinar cells and thus we trace the origin of this particular cell type to the time of deuterostome diversification. Overall, our approach allows a thorough characterization of a complex cell type and shows how both the transcriptomic and morphological information contribute to disentangling the evolution of cell types and organs such as the pancreatic cells and pancreas.

Optimization Of Scanning Electron Microscopy and Serial Block Face – Scanning Electron Microscopy for Investigating Bacteria and Whipworm Egg Interactions

Optimization Of Scanning Electron Microscopy and Serial Block Face – Scanning Electron Microscopy for Investigating Bacteria and Whipworm Egg Interactions

Three-dimensional Structural Analysis of Ant Hair Sensors by X-ray and Serial Block-Face Scanning Electron Microscope Imaging

Three-dimensional Structural Analysis of Ant Hair Sensors by X-ray and Serial Block-Face Scanning Electron Microscope Imaging

How innovations in methodology offer new prospects for volume electron microscopy

Detailed knowledge of biological structure has been key in understanding biology at several levels of organisation, from organs to cells and proteins. Volume electron microscopy (volume EM) provides high resolution 3D structural information about tissues on the nanometre scale. However, the throughput rate of conventional electron microscopes has limited the volume size and number of samples that can be imaged. Recent improvements in methodology are currently driving a revolution in volume EM, making possible the structural imaging of whole organs and small organisms. In turn, these recent developments in image acquisition have created or stressed bottlenecks in other parts of the pipeline, like sample preparation, image analysis and data management. While the progress in image analysis is stunning due to the advent of automatic segmentation and server‐based annotation tools, several challenges remain. Here we discuss recent trends in volume EM, emerging methods for increasing throughput and implications for sample preparation, image analysis and data management.

Identifying long-range synaptic inputs using genetically encoded labels and volume electron microscopy

Enzymes that facilitate the local deposition of electron dense reaction products have been widely used as labels in electron microscopy (EM) for the identification of synaptic contacts in neural tissue. Peroxidases, in particular, can efficiently metabolize 3,3′-diaminobenzidine tetrahydrochloride hydrate (DAB) to produce precipitates with high contrast under EM following heavy metal staining, and can be genetically encoded to facilitate the labeling of specific cell-types or organelles. Nevertheless, the peroxidase/DAB method has so far not been reported to work in a multiplexed manner in combination with 3D volume EM techniques (e.g. Serial blockface electron microscopy, SBEM; Focused ion beam electron microscopy, FIBSEM) that are favored for the large-scale ultrastructural assessment of synaptic architecture However, a recently described peroxidase with enhanced enzymatic activity (dAPEX2) can efficienty deposit EM-visible DAB products in thick tissue without detergent treatment opening the possibility for the multiplex labeling of genetically defined cell-types in combination with volume EM methods. Here we demonstrate that multiplexed dAPEX2/DAB tagging is compatible with both FIBSEM and SBEM volume EM approaches and use them to map long-range genetically identified synaptic inputs from the anterior cingulate cortex to the periaqueductal gray in the mouse brain.