Frontiers in Neuroanatomy

Brain banks provide small tissue samples fixed in neutral-buffered-formalin (NBF), but human anatomy teaching laboratories could provide full brains fixed with solutions that are more appropriate for gross anatomy such as a saturated salt solution (SSS) or an alcohol-formaldehyde solution (AFS). Advanced aging and prolonged exposure to aldehydes are known to enhance brain tissue autofluorescence (AF), limiting the efficacy of immunofluorescence (IF) procedures. We have previously shown by IF staining the antigenicity preservation in mouse brains fixed with the three solutions. We now aimed to compare the quality of IF staining in human brains fixed with SSS, AFS and NBF. In addition, we compared the efficiency of AF quenching methods, namely the application of SudanBlackB (SBB) and the treatment of sections with sodium borohydride (NaBH4). Blocks of neocortex were extracted from 18 brains (NBF=6, SSS=6, AFS=6) and cut into 40{micro}m sections. Neurons (anti-NeuN, AlexaFluor-488) and astrocytes (anti-GFAP, AlexaFluor-555) were revealed with IF after an antigen retrieval protocol, while two treatments (SBB or NaBH4) were used to quench AF. We then assessed the degree of AF (criteria: background or cell AF) and the immunostaining quality with excitation wavelengths of 488nm, 555nm and 647nm. Brains fixed with all three solutions showed well-labeled astrocytes, whereas neurons werent always stained, but this was not associated to the fixative solution. The overall AF intensity was similar in sections from brains fixed with all three solutions. Finally, the SBB treatment was the most effective at reducing AF in all specimens. Given the similarity in AF and antigenicity assessment across the three solutions, we conclude that brains fixed with SSS and AFS could be good alternatives for NBF-fixed specimens in the context of IF experiments processed with a SBB protocol. Highlights- Immunofluorescence staining is feasible in brains fixed with anatomy labs solutions - GFAP is less affected by fixation than NeuN - Autofluorescence can be reduced by Sudan Black treatment

Postmortem human brains stored in brain banks are important research resources to study the mechanisms underlying normal brain functions as well as various neurodegenerative disorders. Immunohistochemical (IHC) and histochemical (HC) staining have been used to examine human brains post-fixed in neutral-buffered formalin (NBF) for months, years, and even decades. As such, it is essential to establish the effects of prolonged post-fixation in NBF on both IHC and HC stains. Previously, we found that prolonged NBF post-fixation resulted in differential effects on IHC and HC staining on postmortem brains. In this study, we further examined the effects of prolonged post-fixation on IHC stains targeting 6 antigens and 2 HC stains of known biomarkers of cerebrovascular diseases in prefrontal cortex of human brains post-fixed for 1, 5, 10, 15, and 20 years. The IHC targets included microvasculature markers of the blood brain barrier (Collagen-IV and Claudin-5), a type III intermediate filament marker (Vimentin), an activated microglia marker (CD68), a biomarker for oligodendrocytic myelin proteolipid protein (PLP) and a marker for iron accumulation (Ferritin). The HC included Massons Trichrome Stain (MTS) and Bielschowsky silver stain (BSS). We found that staining intensities of Ferritin, Vimentin, Collagen-IV and BSS decreased with prolonged post-fixation, while no significant differences were observed in the staining intensity of other markers. Hence, these differential alterations should be taken into consideration when interpreting the results from processed tissues with prolonged post-fixation. We recommend performing IHC and HC staining for human brains with the same post-fixation times to offset any impact on downstream neuropathological analyses, as well as adding the post-fixation duration as a covariate in the analysis.

Studies of whole brain cryopreservation are rare but are potentially important for a variety of applications. It has been demonstrated that ultrastructure in whole rabbit and pig brains can be cryopreserved by vitrification (ice-free cryopreservation) after prior aldehyde fixation, but fixation limits the range of studies that can be done by neurobiologists, including studies that depend upon general molecular integrity, signal transduction, macromolecular synthesis, and other physiological processes. We now show that whole brain ultrastructure can be preserved by vitrification without prior aldehyde fixation. Rabbit brain perfusion with the M22 vitrification solution followed by vitrification, warming, and fixation showed an absence of visible ice damage and overall structural preservation, but osmotic brain shrinkage sufficient to distort and obscure neuroanatomical detail. Neuroanatomical preservation in the presence of M22 was also investigated in human cerebral cortical biopsies taken after whole brain perfusion with M22. These biopsies did not form ice upon cooling or warming, and high power electron microscopy showed dehydrated and electron-dense but predominantly intact cells, neuropil, and synapses with no signs of ice crystal damage, and partial dilution of these samples restored normal cortical pyramidal cell shapes. To further evaluate ultrastructural preservation within the severely dehydrated brain, rabbit brains were perfused with M22 and then partially washed free of M22 before fixation. Perfusion dilution of the brain to 3-5M M22 resulted in brain re-expansion and the re-appearance of well-defined neuroanatomical features, but rehydration of the brain to 1M M22 resulted in ultrastructural damage suggestive of preventable osmotic injury caused by incomplete removal of M22. We conclude that both animal and human brains can be cryopreserved by vitrification with predominant retention of ultrastructural integrity without the need for prior aldehyde fixation. This observation has direct relevance to the feasibility of human cryopreservation, for which direct evidence has been lacking until this report. It also provides a starting point for perfecting brain cryopreservation, which may be necessary for lengthy space travel and could allow future medical time travel.

Long-term storage of aldehyde-fixed brain tissue is commonly performed in the fluid state. This has the potential to maintain morphology for many decades, but has been found to cause progressive loss of antigenicity over time for some biomolecules. While cryoprotection and subzero storage has been successfully used for brain tissue sections or blocks, methods for preserving whole brains using this approach have not been widely characterized. Here we present a protocol for the preservation of fixed whole brains using graded immersion cryoprotection and subzero temperature storage, which is one type of a more general approach that we refer to as aldehyde-based cryopreservation (ABC). Our method uses a gradual ramp-up of the osmotic concentration of cryoprotectants, leading to a final solution containing 50% (v/v) ethylene glycol and 30% (w/v) sucrose. We used CT imaging to track cryoprotectant penetration, finding that with the use of our protocol, approximately 10 months is required to reach equilibration throughout whole human brains. In our initial histological validation, we found that insufficient equilibration time prior to freezing led to apparent ice crystal artifacts seen on ultrastructural imaging of the white matter. After refining the protocol to allow adequate diffusion time, histologic data at both the light and electron microscopic levels showed preserved cellular architecture and ultrastructure after the process of cryoprotectant loading, freezer storage, and unloading. This protocol can be implemented using laboratory freezers or freezer rooms and provides a degree of resilience against freezer failures because the morphology of the fixed tissue is expected to remain preserved long-term in the fluid state even if rewarmed. Our approach may be valuable for laboratories seeking to enhance the long-term preservation of antigenicity in large brain tissue specimens for future research applications.

Although commonly known as rapid and easy to use methodology, Golgi staining requires a range of staining solutions, impregnation periods, concentrations and slicing variables. The use of this methodology can help researchers identify and label individual neuronal components within the extended circuitry. The original Golgi stain technique, developed by Camillo Golgi in 1873, is a silver staining method that enabled scientists to visualize individual neurons in their entirety within nervous tissue for the first time. publications featuring the Golgi staining technique utilise cryostat or microtome slicing, with the combination of a readily purchased kit which comes with a cost and limited morphological detail. Here, we describe an optimised Golgi staining methodology that specifically targets the major drawbacks of traditional protocols; prolonged and inconsistent impregnation, slice fragility during sectioning, and variable visualization of fine dendritic structures. Through modest adjustments to impregnation duration and temperature, fixation, and vibratome sectioning conditions, this low-cost and simple protocol improves staining reliability, facilitates robust slicing without specialized embedding, and supports detailed analysis of neuronal morphology throughout the central nervous system. We validate our optimised protocol using tissue from on-going animal studies of pain and treatment. Representative images illustrate typical staining patterns, characterised by sparse background and high signal-to-noise ratio, facilitating unbiased neuronal tracing and analysis.

Emotion and mood regulation critically depends on interactions between the anterior cingulate cortex (ACC) and the amygdala. However, the detailed architecture of ACC projections to their major targets, the basal (BA) and accessory (AcBA) basal nuclei of the amygdala, remains unclear. To address this issue, a combined retrograde and anterograde tracing with viral vectors were performed in macaques to map the projection patterns from pregenual (pgACC), subgenual (sgACC), and dorsal (dACC) subareas. Data revealed that ACC neurons projecting to the BA arose predominantly from the superficial layers (II/III) of all subareas and the deep layers (V/VI) of the sgACC, whereas ACC neurons projecting to the AcBA originated mainly in the deep layers of the sgACC and dACC. The present study defines the topographic and layer-specific organization of ACC-amygdala connectivity in primates and subserves to provide an anatomical basis for future causal and translational approaches, such as targeted interventions against ACC-related mood disorders. TeaserPrimate anterior cingulate cortex has topographic and layer-specific projections to amygdala that are involved in emotion and mood regulation.

IntroductionSupervised statistical learning for cell-level segmentation and morphometry in optical microscopy is limited less by algorithmic capacity than by the scarcity of reliable, expert-validated ground truth. In comparative neuroscience and quantitative histology, where classical stains such as Nissls method remain the primary means to study cellular morphology, this bottleneck is acute: manual annotation is expensive, subject to individual bias, and rarely performed at the scale or consistency that computational approaches demand. No existing platform integrates a stain-specific bioimage segmentation protocol, a structured multi-annotator workflow, and consensus-based quality control into a single pipeline from image ingestion to machine-readable training data. MethodsWe present Anatolution, an open-source, web-based platform designed to address the gap of quality annotations at https://anatolution.herokuapp.com/public-tool/. Anatolution organizes microscopy images, including 2D arrays or 3D volumes, into project workspaces where multiple annotators independently label cellular structures against a shared computer vision catalogue. This design enables systematic inter-rater and intra-rater reliability assessment, with consensus derived from agreement across annotators rather than from any single experts judgment. The platform enables the export of aggregated labels or annotation datasets for downstream statistical learning methods. We describe the systems architecture, its Nissl-specific segmentation pipeline, the consensus annotation workflow, and validation of inter-rater reliability. ConclusionAcross 20+ histological annotation containers annotated by up to 15 independent raters, consensus boundary agreement increased monotonically with annotator count, reaching a median Dice of 0.79 against the full-rater reference at seven annotators, with top-tier containers achieving leave-one-out ceiling values of 0.621-0.769 for cell-body segmentation. The segmentation pipeline provided effective spatial anchoring, with 88% of consensus-annotated polygons containing at least one algorithmically detected seed. Anatolution provides open-source infrastructure for producing consensus-validated training data from classical histological preparations, addressing the primary bottleneck limiting supervised learning for cell-level morphometry.

Left-right asymmetry of the brain is well recognized in various animals including C. elegans, drosophila and zebrafish. In primates, most of the brain studies describe side of the brain. However, in spite of huge amounts of accumulating rodent studies on neuroscience, most of rodent studies do not distinguish the brain side. The pig brain is considered to occupy an intermediate position between primates and rodents in terms of structural complexity and brain function. Moreover, the numbers of studies using genetic manipulation of pigs are drastically increasing. So, we investigated microminipig (MMP) brain mesoscopic anatomy focusing on left-right differences of its morphology. Here, we show the anterior cingulate cortex, perirhinal cortex, and cerebellum of male and female MMPs, are structurally asymmetrical. The cerebellar vermis, paravermis is tilted from the midline and the consequently the cerebellar cortex exhibits asymmetrical morphology. The anterior cingulate gurus exhibited protrusion and invagination toward the midline on the right and left side, respectively. The left perirhinal lobe exhibited distinct patterns of cortical gyration between left and right side. These data demonstrate that MMPs are one of the suitable model animals for investigating cerebral and cerebellar asymmetry.

Recombinant Adeno-associated viruses (AAVs) are widely used for gene delivery in the central nervous system and have become central tools in both gene therapy and basic neuroscience research. However, although AAV serotypes have been extensively characterized in rodent models, their performance in human neurons, particularly those derived from induced pluripotent stem cells (iPSCs), remains poorly characterized. While human iPSC-derived neurons are increasingly used for disease modeling and drug screening, their susceptibility to viral transduction varies and remains difficult to predict. In this study, we systematically evaluated the transduction efficiency and toxicity profiles of 18 wild-type and engineered AAV serotypes across three distinct types of iPSC-derived neurons, relevant to disease modeling and drug discovery: cortical projection neurons, NGN2- induced forebrain-like neurons, and dopaminergic neurons and four doses (1E3, 1E4, 1E5 and 2E5 genome copies per cell). Using automated high-throughput confocal imaging and quantification of reporter gene expression, we identified several serotypes with robust and efficient transduction across all neuronal subtypes. Among these, three serotypes AAV6, AAV6.2 and AAV2.7m8 showed consistently high performance. To assess safety, we quantified cell number and neurite morphology, finding that while high transduction and gene expression correlate with toxicity, sensitivity varied across neuronal subtypes, with NGN2 neurons being most vulnerable and dopaminergic neurons most resilient. Finally, we validated our findings in a more complex 3D model by testing one of the best-performing serotypes, AAV2.7m8, in both whole and dissociated human cerebellar organoids. Together, our results establish a benchmark dataset for AAV performance in human iPSC- derived neurons and provide practical guidance for AAV based gene delivery in human in vitro neural models. This resource will be valuable for both basic research and preclinical applications aiming to manipulate gene expression in human neurons and understanding AAV tropism in disease-relevant cell types.

Low take rates and inter-tumor variability in growth rates can limit the effectiveness of mouse xenograft models when comparing between groups. To address this problem we developed a simple method to compare multiple cell types within a single mixed xenograft. Individual cell lines or clones were transduced with a lentiviral vector that includes a unique PCR tag, allowing the use of qPCR to determine the proportion of each tagged cell type within a mixed xenograft tumor. We generated vectors with six distinct PCR tags, and two different selectable markers, and have optimized the approach for determining their relative proportions within a mix. An initial pre-amplification step is used to increase the amount of material for subsequent qPCR reactions. This also removes the bulk of the genomic DNA, increasing the specificity of the qPCR step. Samples are then used for qPCR with specific pairs of primers that distinguish between each of the individual PCR tags, and the relative proportion of each tag is determined relative to that in the starting mix. We have tested this approach for in vitro growth of mixed cell cultures and in an orthotopic cecal xenograft model using a human colon cancer cell line. Since each individual tumor is initiated with a mix of cells, multiple tumors within a single animal can be analyzed separately, and overall tumor size is not important. Similarly, multiple metastatic lesions from the same animal can be analyzed individually. Thus, each tumor provides a direct comparison between individually tagged cell lines or clones. This low throughput "bar-coding" approach is simple and cost effective and has the potential to reduce the number of animals needed for xenograft experiments.

Voltage-gated sodium channels (VGSCs) are conventionally described as heterotrimers composed of one alpha and two beta subunits. However, the patterns of co-expression of alpha- and beta-subunits in neurons remain unclear. In the present study, we report that alpha- (Nav1.1, Nav1.2, and Nav1.6) and beta- (beta-1 and beta-2) subunits are densely expressed in axon initial segments (AISs) of neurons in the neocortex, hippocampus and cerebellum at postnatal days 14-15 (P14-15) and 8-9 weeks (8-9W). These distributions are largely unique and partially overlapping among brain regions. Notably, in the neocortex and hippocampus, AISs of presumptive parvalbumin-positive inhibitory neurons are positive for Nav1.1 and beta-1, whereas those of excitatory ones are positive for Nav1.2 and beta-2. Similarly, AISs of cerebellar basket cells, which are inhibitory neurons, are positive for Nav1.1 and beta-1, whereas those of granule cells, which are excitatory neurons, are positive for Nav1.2 and beta-2. Nav1.6 is expressed in many of these neurons. Some subunits exhibited distinct distribution patterns at the two postnatal stages analyzed, possibly because of their developmental changes of subcellular localizations. Taken together, these results indicate that combinations of VGSC subunits are largely unique among different neuronal subpopulations. These findings provide a useful reference for understanding the distribution and interactions of VGSC subunits in the brain.

In adult superficial dorsal horn, 90% of Reelin (Reln+) and 70% of Disabled-1 (Dab1+) neurons co-express the transcription factor LIM-homeobox 1-beta (Lmx1b+) and therefore are glutamatergic neurons. Here we asked if embryonic Reln+Lmx1b+ and Dab1+Lmx1b+ dorsal horn neurons are derived from Lmx1b-expressing early-born dI5 or late-born dILB dorsal neurons. On Embryonic day (E)11.5, Reln+ and Dab1+ neurons appear to be part of the migration of early-born dI5 Lmx1b-expressing neurons. Between E12.5-E15.5, the lateral Reln+Lmx1b+ and Dab1+Lmx1b+ neurons migrate circumferentially along the rim of what will become the superficial dorsal horn, whereas medial Reln+Lmx1b+ and Dab1+Lmx1b+ neurons move into the dorsal midline and then migrate into lamina V. The small, late-born dILB Reln+Lmx1b+ and Dab1-Lmx1b+ neurons fill the superficial dorsal horn. In Reln mutants, large Dab1+Lmx1b+ neurons were mispositioned in lamina I and at the border between the superficial and deep dorsal horn. To confirm the identity of the circumferential and midline Reln+Lmx1b+ and Dab1+Lmx1b+ neurons, we asked if they expressed the transcription factor Zfhx3, a marker of dI5 projection neurons. We detected examples of Reln+Lmx1b+Zfhx3+ and Dab1+Lmx1b+Zfhx3+ projection neurons that migrated along the outer rim of the superficial dorsal horn and others that migrated from the midline into lamina V. Taken together, our study demonstrates that the larger Reln+Lmx1b+Zfhx3+ and Dab+Lmx1b+Zfhx3+ neurons represent two subsets of dI5 projections neurons, whereas smaller Reln+Lmx1b+ and Dab1+Lmx1b+ neurons concentrated in lamina II are likely dILB interneurons.

Serotonin is one of the main neuromodulators in the brain, involved in regulating mood, complex behaviors and sensory input. Serotonin reaches primary somatosensory cortex (S1) via axons of neurons located in the dorsal raphe nucleus (DRN). DRN neurons can be modulated, amongst others, by reward, sensory stimulation, or movement but the activity pattern of serotonergic neurons targeting S1 is not known. Therefore, it is unclear under which circumstances serotonin is released in S1. Here, we expressed GCaMP8 in serotonergic neurons of the DRN to analyze the activity of their axons in S1 using two-photon Ca2+-imaging. Cluster analysis of axonal activities suggests that one to four functional groups of serotonergic axon segments project to a 0.3 mm2 horizontal plane of S1. We show that activity in serotonergic axons is strongly driven by reward and weakly by sensory stimulation of the whiskers. Movement, however, is preceded by a modulation, up and down, of the serotonergic signal seconds before the running onset. In summary, rewards and sensory stimulation lead to activity in serotonergic axons which is likely to adjust signal processing in S1 upon these events. The serotonergic signal changes seconds before movement onset probably preparing the neural network in S1 for the state change that accompanies running.

1.Functional neuronal circuits in the vertebrate retina emerge through coordinated developmental events, yet the timeline by which bipolar cells acquire visual feature selectivity remains unclear. A major barrier is the limited genetic access to bipolar subtypes during early postnatal stages. Recent comprehensive transcriptomic study points to Igfn1 as a molecular marker for type-7 bipolar cells (BC 7), a subtype that exhibits direction-selective glutamate releases in adult. Here, we generated an Igfn1iCre knock-in mouse line and characterized Igfn1-positive cell morphology from postnatal day 4(P4) to adult using Cre-dependent tdTomato reporter mice. We found Igfn1-positive cells in the inner retina by P12-P15, predominantly labelling bipolar cells and some amacrine populations. At P15, about 71% of labelled bipolar cells stratified their axons in the S4 sublamina of the inner plexiform layer, consistent with BC 7 morphology. In adult retina, the widespread Igfn1-labelling appears slightly dominated in amacrine cells. To validate these observations, we analysed Igfn1 expression in the Mouse Retina Cell Atlas and confirmed strong Igfn1 enrichment in BC 7 and expression in additional retinal cell types, mirroring experimental results. Overall, these results reveal Igfn1iCre as a potential developmental tool for BC 7 access in the retina.

Vesicular glutamate transporter 3 (VGluT3) is expressed in a large subset of serotonin (5-HT) neurons of the dorsal raphe nucleus (DRN), suggesting a potential for glutamate co-transmission. Although VGluT3 has been implicated in the physiology of several non-glutamatergic neuronal populations, its specific role in the organization and function of 5-HT axons remains unclear. Here, we used CRISPR-Cas9 mediated knockdown and viral overexpression of VGluT3 in DRN 5-HT neurons of adult mice to assess its contribution to synaptic architecture in the lateral hypothalamus (LHA) and to 5-HT-related behaviors. VGluT3 depletion did not significantly alter synaptic incidence or organization of 5-HT DRN terminals in the LHA. In contrast, VGluT3 overexpression increased the proportion of asymmetric synapses without changing the overall synaptic incidence. In behavioral assays, VGluT3 depletion impaired motor coordination and increased anxiety-like, repetitive, and social behavior, whereas VGluT3 overexpression selectively reduced repetitive behavior. Basal locomotion and depressive-like behaviors were unchanged by either manipulation. Together, these findings indicate that VGluT3 modulates both the structural organization and behavioral output of DRN 5-HT neurons, supporting a modulatory role for VGluT3-dependent signaling within 5-HT circuits.

Although a differential vulnerability of dopaminergic neurons to degeneration based on their specific location within the dorsal and ventral tiers of the substantia nigra pars compacta (SNcD and SNcV, respectively) has long been postulated, the underlying mechanisms sustaining these tier-specific differences remain poorly understood. Here, upon inducing a viral-mediated enhancement of neuromelanin (NMel) accumulation within dopaminergic neurons in non-human primates, the distribution of Lewy body-like inclusions (LBs) was analyzed within identified SNcD and SNcV neurons, together with their intracellular NMel levels. Results showed that the vast majority of intracytoplasmic inclusions were found in SNcV neurons, and indeed correlated to higher pigmentation levels. By contrast, only very few LBs were found in calbindin-positive neurons of the SNcD, which in parallel exhibited very low levels of NMel accumulation. These results postulate an additive effect made of a tier-specific location of LB burden together with high pigmentation levels as synergistic drivers sustaining the preferential vulnerability of SNcV dopaminergic neurons. Moreover, the evidence obtained here supported that NMel accumulation beyond a given threshold triggers the aggregation of endogenous -Syn in the form of LBs; therefore, approaches intended to reduce pigmentation levels in SNcV neurons would likely induce a neuroprotective effect by preventing the subsequent aggregation of -Syn.

The medial subdivision of the inferior pulvinar (PIm) has been implicated in motion processing, visuomotor integration, and residual visual function, yet a comprehensive account of its cortical inputs remains unresolved. Previous studies often relied on indirect cortical injections or tracer deposits spanning multiple pulvinar subdivisions, limiting anatomical specificity. Here, we used MRI-guided, cytoarchitectonically restricted retrograde tracer injections to selectively target PI in the common marmoset (Callithrix jacchus) and systematically map its cortical afferents. Across four cases, retrogradely labeled neurons were widely distributed throughout occipital, temporal, parietal, and cingulate cortices, with a strong predominance in layer V, consistent with driver-like corticothalamic projections. Early and middle-tier visual areas (V1, V2, V3, V3A, V4, V6/DM) contributed substantial input, with labeling patterns corresponding to peripheral visual field representations. The middle temporal complex (MT, MTc, MST, FST) represented one of the densest sources of cortical projections. Prominent inputs also arose from posterior parietal regions, including LIP, MIP, VIP, AIP, and inferior parietal areas (e.g., PFG, OPt), linking PIm to visuospatial and action-related networks. Semi-quantitative analyses indicated that occipital cortex and the MT complex together accounted for approximately 60% of total cortical input, while parietal cortex contributed roughly 20%. Additional projections from retrosplenial and posterior cingulate cortices were observed. These findings identify PIm as a central integrative node embedded within distributed visual and visuomotor networks. Rather than functioning as a restricted visual relay, PIm appears positioned to coordinate motion, spatial, and action-relevant signals within cortico-thalamocortical circuits supporting adaptive visually-guided behavior.

Quantitative assessment of optic nerve health requires metrics beyond axon counts alone. Axon density and glial coverage fraction correlate with clinical measures of visual function, yet no existing automated tool extracts optic nerve cross-sectional boundaries to enable their calculation. We developed MONICA (Morphometrics from Optic Nerve Imaging Contour Analysis), a web application that integrates AxonDeepSeg deep learning segmentation with a novel morphology-based contour extraction algorithm to automatically derive whole nerve boundaries alongside axon and myelin masks. The contour extraction algorithm was validated against manual ground truth annotations using 15 optic nerve cross-sections spanning two taxonomic orders (mouse, rabbit), two mouse strains (BXD29, BXD51), and varying preparation quality levels (modern and archival samples). Automated contour extraction demonstrated excellent agreement with manual annotations, achieving an overall Dice similarity coefficient (a measure of segmentation overlap) of 0.987 {+/-} 0.009. Balanced precision (0.985) and recall (0.989) values indicated that the algorithm neither systematically over-segments nor under-segments nerve boundaries. MONICA requires no local software installation and runs entirely in-browser, providing batch processing for high-throughput phenotyping alongside a full suite of per-axon morphometrics. MONICA provides researchers with an accessible tool for complete nerve cross-section morphometry.

BackgroundRecent studies revealed key immunological mechanisms within the central nervous system (CNS) that contribute to Alzheimers disease pathology. Additionally, analyses of human AD datasets have also associated viral encephalitis exposure (i.e., viral-induced neuroinflammation) with the development of AD and dementia, highlighting the need to better understand how viral encephalitis and neuroimmune mechanisms within the brain may impact AD pathologies such as A{beta} plaque deposition. Intracranial infection of susceptible mice with the neurotropic JHM strain of murine coronavirus (JHMV) results in acute encephalomyelitis characterized by viral infection of glia and a robust inflammatory response comprised of monocytes/macrophages and T cells that aid in controlling viral replication. MethodsTo determine how coronavirus-induced encephalitis may impact established A{beta} plaque deposition, we intracranially inoculated JHMV into aged 5xFAD model of amyloidosis. We utilize immunohistochemical and biochemical analysis to assess the impact on existing A{beta} pathology. We also utilize spatial transcriptomic imaging to explore how viral encephalitis affects cellular responses to plaque pathology with single-cell resolution. ResultsIn aged 5xFAD mice, JHMV-induced encephalitis at 12 days p.i. resulted in minimal changes to overall A{beta} protein within the brain. However, viral encephalitis induces CD4+ and CD8+ T cell infiltration and more Lgals3/MAC2-expressing macrophages surrounding dense-core A{beta} plaques, which appear more compacted in JHMV-infected 5xFAD brains compared to uninfected 5xFAD controls. We compared gene expression within JHMV-infected 5xFAD mice and uninfected controls to identify distinct cellular responses to A{beta} plaques that differed. Utilizing differential gene expression and pathway analysis, we found that viral encephalitis increased the proportion of myeloid cells in the 5xFAD brain, which also showed down-regulated disease-associated (DAM) pathways involving A{beta} clearance, response to lipids, and macrophage activation within the post-encephalitis 5xFAD brains. ConclusionsTogether, these findings suggest an attenuated myeloid cell response to A{beta} plaque burden in 5xFAD mice following acute viral encephalitis. Future experiments aim to further dissect inflammatory mechanisms between infiltrating myeloid cells, T cells, and the progression of A{beta} and tau pathology. Data derived from these experiments will further elucidate the viral-induced neuroimmune mechanisms that affect AD pathology and offer an opportunity to determine how these neuropathologic changes, such as subsequent neuronal damage, occur.

The clitoris is one of the least studied organs of the human body. The detailed anatomy of the clitoris is challenging to address through a gross dissection, as most of its parts are embedded internally, surrounded by pubic bone and several pelvic organs. While clinical imaging methods such as magnetic resonance imaging can capture the gross 3D morphology, they lack the spatial resolution required to resolve the detailed structures. In this study, we generated micron-scale computed tomography images of the female pelvises, leveraging a synchrotron radiation X-ray source. This unique data revealed the complex trajectory of the dorsal nerve of the clitoris, the main sensory nerve of the clitoris. Notably, the nerve trunks within the clitoral glans were revealed, with the maximum diameter ranging from 0.2 to 0.7 mm. They showed a tree-like branching pattern projecting towards the surface of the glans. We also revealed that some branches of the dorsal nerve of the clitoris ramify to innervate the clitoral hood and mons pubis. Finally, the posterior labial nerve, a branch of the perineal nerves, was shown to innervate the surroundings of the clitoris and the labial structures. These findings have an immediate impact on operations performed around the vulva area, such as gender-affirmation surgery and reconstruction surgery after genital mutilation.