Frontiers in Neuroanatomy

Cortico-thalamo-cortical circuits entail extensive trans-thalamic connectivity between cortical areas, yet their structural organization and function remain poorly understood. Here, the thalamocortical projections of several higher-order thalamic nuclei were characterized by retrograde tracing from two cortical areas, the primary somatosensory (S1) and motor (M1) cortices. Cholera toxin B conjugated with different fluorophores allowed for simultaneous detection of projection neurons targeting S1 and M1. A cell detection pipeline based on neural networks was developed to allow semi-automated analysis of large thalamic imaging volumes to quantitatively infer the spatial distribution of projection neurons in the posterior complex (PO) and the adjacent ethmoid nucleus (Eth), nucleus centrolateralis (CL), nucleus paracentralis (PCN), and the nucleus parafascicularis (PF). The arrangement of neurons projecting to both, primary somatosensory and motor cortices, occurs at different connection strengths and was topographically organized in all nuclei studied. Co-injections into both cortical areas revealed projection neurons with axons branching into both S1 and M1 cortices. Our work introduces a pipeline for semi-automated quantitative analysis of thalamic projection patterns that could be useful for connectivity analyses in general. This approach revealed repetitive anatomical patterns in different thalamic nuclei with regard to projection strength, spatial organization and fraction of projection neurons targeting two cortical areas simultaneously.

This step-by-step protocol provides a cross-species, non-surgical approach that enables prenatal gene delivery to the developing nervous system in rats and marmosets. Under transabdominal ultrasound guidance, intracerebroventricular injection of recombinant adeno-associated virus vectors into the fetal brain achieves robust and long-term transduction from prenatal stages into adulthood. This approach can be adapted to other species and target sites outside nervous system, enabling safe and selective intrauterine manipulation and the generation of diverse experimental models for basic and preclinical research. For complete details on the use and execution of this protocol, please refer to Ribeiro Gomes et al (2026)1. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/737050v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@696364org.highwire.dtl.DTLVardef@fc3c7forg.highwire.dtl.DTLVardef@1e7c7caorg.highwire.dtl.DTLVardef@1edcef0_HPS_FORMAT_FIGEXP M_FIG C_FIG Before you beginExperimental procedures during gestation allow researchers to study developmental processes, including how manipulations of the fetus and its intrauterine environment influence biological outcomes. Ultrasound imaging guidance greatly facilitates such interventions by providing safe and targeted access to fetal compartments, including for prenatal gene delivery to developing neural cell populations. Critically, delivery of recombinant adeno-associated viruses (rAAVs) into the cerebrospinal fluid (CSF) of developing animals enables widespread gene transfer across the brain. The efficiency and distribution of transduction are strongly influenced by developmental stage, making the timing of delivery an important experimental variable. In altricial species such as mice, major developmental processes, including cortical lamination and the establishment of long-range connections, begin prenatally but continue throughout early postnatal life. In primates, however, development is more advanced at birth, and many equivalent developmental events are shifted to the prenatal period. Consequently, developmental stages that can be targeted postnatally in mice require prenatal access in primates. Here, we present a step-by-step protocol for ultrasound-guided fetal intracerebroventricular viral injection (FIVI) of rAAV in marmosets (Callithrix jacchus) and rats (Rattus norvegicus). The procedure was initially developed and optimized in rats before being translated to marmosets, small New World primates that share key developmental, anatomical, and functional characteristics with humans. Together, these models illustrate the cross-species applicability of the approach, while providing gene delivery strategies for both a genetically tractable rodent model and a translationally relevant nonhuman primate. FIVI enables broad gene transfer and stable, long-term transgene expression in wild type animals, facilitating the generation of complementary quasi-transgenic models for research and translational applications from prenatal development through adulthood.

BackgroundBiallelic loss-of-function mutations in PRKN gene (encoding Parkin protein) cause early-onset Parkinsons disease (EOPD). Parkin is a crucial component of PINK1-Parkin pathway, which marks damaged mitochondria for degradation via mitophagy. Without functional Parkin, damaged mitochondria accumulate, causing oxidative stress and neurodegeneration. ObjectiveInvestigate Parkin gene replacement via AAV gene therapy as a potential treatment for Parkin-dependent EOPD. MethodsWe initially validated phosphorylated ubiquitin Ser65 (pUbSer65) as an indicator of Parkin-mediated mitophagy initiation. We evaluated AAV-mediated PRKN replacement (hereafter, AAV-Parkin) in a Parkin knockout neuroblastoma cell line (SH-SY5Y cells) and feasibility of delivery in mouse and rat models. ResultsOur research showed pUbSer65 signal was reduced in Parkin-KO SH-SY5Y cells when compared to wild-type cells after mitochondrial stress, indicating deficiency in initiation of mitophagy. AAV-mediated human PRKN gene replacement successfully restored these pUbSer65 levels in knockout cells. We saw restoration in patient-derived fibroblasts following AAV-Parkin overexpression. We developed a translatable gene therapy approach using rodents. We demonstrated the feasibility of delivering AAV-Parkin directly into the substantia nigra (SN) of wild-type rats. Using an AAV1 capsid with Ef1a promoter, we achieved dose-dependent Parkin expression and identified a well-tolerated dose. We also evaluated multiple promoters in a proprietary Spark100 capsid, finding Ef1a and Synapsin1 (Syn1) were most effective for transducing dopaminergic neurons in the SN of mice without causing adverse effects. These findings established a well-tolerated vector dose and an optimal capsid-promoter combination. ConclusionsOur results support the potential of AAV-Parkin gene therapy as a disease-modifying approach for Parkin-deficient EOPD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/737487v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@16dd13corg.highwire.dtl.DTLVardef@c3dfcdorg.highwire.dtl.DTLVardef@19a310dorg.highwire.dtl.DTLVardef@a66f2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Birds evolved large, cognitively capable forebrains independently of mammals, yet comparative analyses of avian brain organization have been constrained by the lack of standardized resources capable of resolving internal parcellation and long-range connectivity across species. Here, we present a comparative MRI resource spanning 16 avian species representing major clades and diverse ecological niches. We analyzed high-resolution T2-weighted and diffusion-weighted datasets suitable for direct interspecific comparison. T2-weighted morphometry revealed pronounced region-specific variation in internal brain architecture, including lineage-dependent differences in the relative prominence of major brain divisions and commissural structures, supporting a pattern of mosaic diversification rather than uniform scaling. To validate MRI-derived anatomical boundaries, we compared MRI parcellations with complementary histological analyses in three representative taxa (the large-billed crow, gentoo penguin, and mandarin duck), demonstrating close correspondence between MRI-defined borders and cytoarchitectonic transitions identified by Nissl staining, as well as major myelinated compartments visualized by Luxol Fast Blue staining. Moreover, diffusion MRI tractography and fractional anisotropy (FA) mapping further revealed both conserved and species-specific features of large-scale brain organization. Seed-based tractography of the optic lobe, dorsal cortex, cerebellum, and anterior cortex in chick, gentoo penguin, and large-billed crow revealed conserved within-compartment trajectory patterns alongside marked region-specific interspecific differences, particularly in optic-lobe-associated long-range trajectories. Whole-brain FA maps revealed complementary variation in regional microstructural organization across taxa. Together, this comparative MRI framework provides a cross-validated foundation for linking internal brain anatomy and long-range connectivity to ecological and evolutionary diversification in birds, with broader applications to comparative neuroanatomy across amniotes.

A recent study (Manjarrez et al., 2026) showed that the classification of cortical dendritic spines into stubby, thin, and mushroom subtypes is unstable under rotation. That result criticizes the categorical scheme but leaves an open question. What is the actual structure of spine morphology once the viewing angle is controlled? Here we answer it. We analyzed 228 spines from layer II pyramidal neurons in the H01 nanometer-resolution reconstruction of human temporal cortex. We first quantified the source of instability. We found that rotating dendritic segments by 90 degrees about their axes shifted the apparent spine height and head width in opposite directions across the population, thereby confirming orientation-dependent measurement error. Furthermore, to obtain measurements free of this artifact, we developed the Spine Morphometry Hub (SMH), a 12-point anatomical landmark framework that characterizes each spine in all three orthogonal planes and extracts geometric, voxel-based, and mesh-based metrics. All morphometric distributions were unimodal and right-skewed. Density-based clustering assigned most spines to noise, and a Monte-Carlo test against a discrete two-type null model confirmed that this pattern is incompatible with categorical subtypes. We also confirmed that apical and basal spines were statistically indistinguishable. Unlike previous reports of a spine continuum, all based on orientation-dependent measurements, our framework removes the viewing-angle confound itself, so the continuum we observe cannot be attributed to a projection artifact. Hence, our framework will be useful to quantify dendritic-spine remodeling in neurological disorders, in which spine shape has long been observed but never measured against an orientation-invariant morphometric standard. HighlightsO_LISpine Morphometry Hub (SMH) measures spines free of viewing-angle error C_LIO_LISMH was validated as an orientation-invariant morphometry framework C_LIO_LIRotating dendrites by 90{degrees} shifts spine height and head width oppositely C_LIO_LIAll morphometric distributions are unimodal and right-skewed, not categorical C_LIO_LISMH could be used to quantify dendritic-spine remodeling in neurological disorders C_LI

Bergmann glia-like progenitors (BGLPs) are transient astroglial progenitors in the postnatal cerebellum, but how their lineage potential changes during development remains incompletely understood. Our previous electroporation-based study suggested that P0 BGLPs possess broader lineage potential than P6 BGLPs. Here, we performed recombination-based lineage tracing by cerebellar surface application of tamoxifen to Ai9/+; GlastCreERT2/+ mice and temporally analyzed the progeny of BGLPs labeled at P0, P3, P6, and P8. We found that BGLPs undergo progressive lineage restriction during postnatal development. P0 BGLPs gave rise to Bergmann glial cells (BGs), inner granule cell layer astrocytes (IGL astrocytes), white matter astrocytes (WM astrocytes), and molecular layer inhibitory neurons (ML-INs), confirming our previous electroporation-based findings. In contrast, P3 BGLPs generated BGs, IGL astrocytes, and WM astrocytes, whereas P6 BGLPs generated BGs and IGL astrocytes, and P8 BGLPs generated predominantly BGs. Thus, BGLP lineage output was progressively restricted from four progeny categories at P0 to a predominantly BG-restricted output by P8, suggesting that BGLPs dynamically adjust their cellular output during postnatal cerebellar maturation. Additional temporal analyses suggested that ML-INs are unlikely to be generated directly from P0 BGLPs, but may arise indirectly through astrocyte-like progenitors (AsLPs) and inhibitory neuron progenitors (INPs). These findings identify postnatal BGLPs as a useful in vivo model for studying progressive lineage restriction and stage-specific cellular supply during cerebellar development.

Companion animal brain banking has been recognized as a valuable approach for translational aging and dementia research. However, realizing the full value of canine brain banks depends on optimizing the methods that are used to collect and preserve the tissue. Whole brain perfusion fixation is one promising approach, but it is not yet well described in dogs. Here we describe the development of methods for a canine brain bank (currently n = 55), including whole brain perfusion fixation via aortic cannulation and brain extraction. We assessed perfusion quality using gross examination, post-perfusion CT, and histological clearance of blood vessels. We found that body weight and average flow rate per body weight were each significantly correlated with perfusion quality in our cohort. To illustrate the kind of analysis the bank could facilitate, we next performed a preliminary study of brain aging, one of our primary planned research applications. Using a pixel classifier applied to whole slide images, we quantified lipofuscin burden, and in this preliminary cohort found that it increased strongly with age in both the thalamus and hippocampus. In the hippocampus, lipofuscin burden was also elevated in dogs with owner-reported cognitive dysfunction, although the current cohort is too small to determine to what extent this association is independent of age. Preliminary electron microscopy studies also confirmed that perfusion fixed tissue from the bank is amenable to ultrastructural analysis. This work describes one approach for canine brain perfusion fixation and introduces a brain tissue resource that may help support future neuroscience research.

SignificanceDigital Holographic Microscopy (DHM) provides label-free quantitative phase images (QPIs) of living cells and has become a powerful tool for studying cellular morphology and dynamics. While most DHM studies have focused on cell-level analysis, the quantitative characterization of neuronal network organization and maturation from DHM images remains largely unexplored, highlighting the need for dedicated computational approaches. AimWe aimed to develop an automated framework combining deep-learning-based image analysis and graph theory to quantitatively characterize the organization, connectivity, and maturation of neuronal networks in primary rat cortical cultures imaged by DHM. ApproachTwo U-Net convolutional neural networks were trained on manually annotated DHM phase images to segment neuronal cell bodies and neurites. The resulting segmentation maps were used to infer putative morphological connections between neurons and generate graph representations of neuronal networks, referred to as graph fingerprints. A panel of 18 connectomics-inspired graph features was then computed to characterize local and global properties of network organization across four stages of culture maturation. ResultsThe mean area under the receiver operating characteristic curves was 0.98 for cell-body and 0.91 for neurite segmentation, indicating near-perfect identification. Graph-theoretical analysis revealed reproducible topological changes during network maturation in vitro, including increased density, reduced modularity, and progressive network integration. Correlation analysis showed that the 18 graph features grouped into two highly correlated families. A Random Forest classifier identified density and modularity as the most informative descriptors, achieving an accuracy of 87% in classifying maturation stages of neuronal cultures. ConclusionsOur results demonstrate that combining DHM, deep-learning-based segmentation, and graphtheoretical analysis enables quantitative characterization of neuronal network organization and maturation from label-free phase images. This framework provides a foundation for future studies of pharmacological experiments, neuronal network phenotyping, and human induced pluripotent stem cell (hiPSC)-derived neuronal cultures, where quantitative assessment of network organization remains a major challenge.

The corpus callosum is a major white matter bundle responsible for connecting both hemispheres. In mammals, due to a variety of causes, the development of the corpus callosum can be impaired - this brain malformation is known as corpus callosum dysgenesis (CCD). The clinical presentation of CCD varies, with patients exhibiting three morphological phenotypes: agenesis, partial dysgenesis, and hypoplasia. Although the first two presentations are easily detectable on MRI scans, the latter is more challenging, as the structure is fully formed but has a reduced area. In this study, we develop (1) a pipeline to generate synthetic MRI scans with apparent anatomical variation and (2) train a U-Net-based tool to automatically segment the corpus callosum of marmosets in both healthy and disease contexts. Methodologically, a custom script was devised to apply rotation and translation to T1-weighted MRI scans at the volume level. Because the slicing grid remains unchanged, these rigid transformations translate into apparent anatomical variations at the slice level. We compared corpus callosum measurements obtained from automatically segmented masks with those from manually delineated masks. The average Dice score was above 0.90, and the Hausdorff distance was below 0.4 mm. We also stratified our cohort according to phenotype (healthy controls and hypoplastic animals). The magnitude of the effect and the significance level observed between the voxel counts of healthy and hypoplastic animals using manually delineated masks were comparable to those obtained via automatic segmentations. These results show that our pipeline can generate a sufficiently varied training pool to build an accurate U-Net segmentation model with high diagnostic capability.

The thalamic reticular nucleus (TRN) is a thin, inhibitory shell surrounding the thalamus. It regulates the thalamocortical information flow, and thereby plays a central role in attention, task switching, and the sleep-wake cycle. Despite its importance, the TRN remains poorly studied in the human brain. This is largely because its small size and deep anatomical location limit its visibility with conventional non-invasive neuroimaging techniques. Here, we assessed whether the human TRN can be reliably visualised and segmented in vivo using ultra-high field (UHF) magnetic resonance imaging (MRI) at 7 Tesla. High resolution (0.35 mm isotropic) partial-brain T2* and T1 scans were acquired from healthy individuals, followed by manual delineation of the TRN. These in vivo segmentations were compared with TRN estimates obtained from two high-quality postmortem datasets serving as an anatomical reference. In vivo segmentations of TRN volume and thickness closely matched measurements derived from the postmortem reference datasets, and quantitative comparisons showed high consistency in TRN shape and location across individuals while also capturing meaningful inter-individual variability. Using these segmentations, we constructed a publicly available probabilistic atlas of the human TRN. This atlas provides a new resource for incorporating TRN anatomy into functional, structural, and clinical neuroimaging studies. Our findings demonstrate that the human TRN can be robustly mapped in vivo at 7T and establish a foundation for future investigations into its structure and function.

Traumatic brain injury produces widespread axonal damage can be assessed histologically using amyloid precursor protein (APP) immunohistochemistry, which labels injured axonal profiles at cellular resolution [1, 2]. However, quantification of APP pathology remains a major bottleneck: annotation is manual, time-consuming, spatially localized, and variable across raters, limiting scalability and reproducibility. This limitation is particularly important in studies that use histology as a reference for neuroimaging or other tissue-level measurements, where cellular APP pathology must be quantified in a spatial form that can be aligned with imaging abnormalities. Here, we introduce PIGMENT, an annotation-efficient deep-learning framework for automated segmentation and quantification of APP-positive pathology in porcine white matter histology. PIGMENT uses a compact SegFormer-B0 architecture trained on 525 expert-annotated 512 x 512-pixel tiles from four APP-stained sections across three pigs. Because APP-positive profiles are sparse, fragmented, stain-variable, and morphologically diverse, PIGMENT combines limited expert labels with APP-specific augmentation designed to model variation in APP-positive intensity, size, continuity, fragmentation, and local tissue context. We evaluated PIGMENT using an instance-level detection rate that measures whether discrete APP-positive components are localized. Across held-out APP-stained data, PIGMENT achieved a mean instance-level detection rate of 0.86. Across the configurations tested, the highest mean detection rate was achieved by a training set that included sections from different animals, suggesting that annotation diversity may be an important factor under limited-label conditions. By extending limited high-confidence expert annotations into whole-section APP burden maps, PIGMENT provides a scalable framework for characterizing the extent and spatial distribution of traumatic axonal injury. These maps may support future studies that align histological injury burden with imaging-derived measures.

Synaptojanin 1 is a brain enriched phosphoinositide phosphatase implicated in endocytosis at the synapse. A mutation (R258Q) that selectively impairs its Sac1 phosphatase domain causes early onset familial Parkinsonism. Neurons of mice with this mutation display synaptic vesicle traffic defects across the brain, but selective dystrophic changes in a subset of dopaminergic axons in the dorsolateral striatum. Using correlative light microscopy-FIB-SEM of mutant mouse striata to visualize in 3D these abnormal structures we show that they represent clusters of focal axonal dilations harboring massive, onion-like DAT enriched plasma membrane infoldings, generally localized next to cell bodies of neighboring cells, often engulfing evaginations of such cells. This dysmorphia was associated with a deficit in dopamine release in the same striatal region. Given the involvement of Synj1 in endocytic mechanisms, these structures may reflect an imbalance between exocytosis and endocytosis. Their occurrence only in a subset of axons suggest a vulnerability threshold of these axons beyond which the expansion of the plasma membrane is not counteracted by compensatory mechanisms.

Ex vivo diffusion weighted imaging (DWI) enables high-resolution characterization of brain connectivity and is increasingly applied in comparative and evolutionary neuroscience. However, variability in tissue preparation and contrast agent exposure can substantially affect relaxation properties and compromise reproducibility, particularly in non-mammalian species. Here, we systematically assess the impact of different gadolinium-based contrast agent exposure protocols on relaxation stability and DWI compatibility in fixed pigeon brains. Brains were perfusion-fixed with 2% paraformaldehyde and assigned to four preparation protocols: (i) contrast agent exposure during perfusion, post-fixation, and rehydration; (ii) post-fixation and rehydration only; (iii) rehydration only; (iv) no contrast agent. Quantitative T1, T2, T2*, and DWI data were acquired at five time points over 70 days using a 7T MRI system. Protocols involving contrast agent during perfusion or post-fixation produced comparable relaxation trajectories, with T1, T2, and T2* stabilizing by Day 13. On day 13 the T1 values of tissue that was exposed to contrast agent, regardless of the application protocol were between 230.86 ms and 266.89 ms, while the T1 values of the control group were over 1100 ms at this point in time. T2 values of the experimental groups were between 39.97 ms and 56.17 ms while T2 values of the control group were between 58.68 ms and 77.82 ms. T2* values of the experimental groups were between 27.27 ms and 43.33 ms while T2* values of the control group were between 46.16 ms and 65.93 ms. Importantly, contrast agent exposure during rehydration alone resulted in equivalent stabilization after two weeks, reflecting gradual contrast agent diffusion into the tissue. In contrast, control samples without contrast agent exhibited significantly elevated T2 and T2* at later time points. These results demonstrate that post-fixation contrast agent exposure during rehydration is sufficient to achieve stable relaxation parameters and DWI compatibility, assessed via fractional anisotropy (FA) and mean diffusivity (MD) in ex vivo avian brain tissue. This minimal preparation protocol enhances reproducibility, reduces handling complexity, and supports standardized cross-species neuroimaging of brain connectivity.

Traumatic brain injury (TBI) is a characteristic feature of neurodegenerative diseases such as Alzheimers disease and chronic traumatic encephalopathy. Small animal models have been used to establish clinically relevant biomarkers of neuropathology, however, they show significant anatomical differences from humans and are affected by artificial experimental manipulations, making them often unsuitable for longitudinal study of repetitive mild TBI. Building on a previous study of neuropathology in headbutting bovids in the wild, this pilot study investigated whether freely headbutting domestic goats, which naturally engage in low-intensity, high-frequency head impacts, accumulate measurable biomarkers of neurodegeneration in cerebrospinal fluid (CSF) and brain tissue. Over a six-month period, three male goats (Capra hircus) were allowed to freely headbutt under continuous video surveillance. Monthly CSF samples were collected, and concentrations of key neurodegeneration biomarkers were measured via multiplex immunoassays, including amyloid {beta} ; peptides (A {beta} 40, A {beta} 42), total and phosphorylated tau (tTau and pTau), glial fibrillary acidic protein (GFAP), S100 calcium-binding protein B (S100B), and neurofilament M (NF-M). Postmortem immunohistochemistry was conducted on prefrontal cortical tissues using antibodies targeting pTau, GFAP, and S100B. Head impact kinematics were quantified using horn-mounted accelerometer and inclinometer sensors that recorded linear acceleration, rotational velocity, and head orientation during naturally occurring headbutting events. Several notable trends were observed. Phosphorylated tau as well as reactive astrocytes were detected in the brain tissue, mirrored by elevated GFAP detected in the CSF. PET TSPO was unsuccessful, however, FDG PET revealed frontal-dominant activity in all goats, and one with asymmetrical activation. Overall, the goats sustained 5,000-7,000 head impacts each over six months, with forces up to 388 N and peak acceleration up to 16.5 g. This multi-modal observational study is the first to characterize neurodegeneration biomarkers and kinematics in headbutting goats. Even at one year old, the combination of pTau and gliosis in both the brain tissue and CSF indicates that the goat s repetitive head impacts begin to show neurodegenerative consequences early in life. Likely, the severity of these consequences increases with headbutts and age, eventually resulting in chronic neurodegeneration. This system shows promise as a large-animal model for the longitudinal study of the onset and progression of neurodegenerative disease.

A critical step in visual cortical maturation is refinement of receptive field (RF) size, producing higher acuity vision. This was previously studied using species with well-developed vision (e.g., carnivores, primates), in which visual experience was necessary for refinement but not maintenance of RFs in visual cortex. In contrast, in Syrian hamsters, a crepuscular species with low visual acuity, dark rearing had no effect on RF refinement in juveniles, but RFs re-enlarged in adulthood, resulting in reduced acuity. These inter-species differences raise the question of whether the need for visual experience is primarily related to the phylogenetic position of the species or to its ecological niche. Here we report that dark rearing had no effect on development or maintenance of RF properties of visual cortical neurons in nocturnal mice. Mice with lifelong visual deprivation refined and maintained their RF size over time. Furthermore, the development of stimulus direction tuning was unaffected by dark rearing. In contrast, surround suppression, orientation tuning and the sharpness of direction tuning were abnormal in dark reared mice. These and our previous results from hamsters show that species living in an ecological niche with minimal daylight exposure require little to no visual experience to develop and maintain refined RFs. This study is an important step in developing a better understanding of the role of visual experience in the development of visual processing circuitry and suggests that diurnal mammals may be a better model for human visual cortical development than mice.

Microcomputed tomography (micro-CT) is a useful tool that can be utilised for 3D structural characterisation and volumetric quantification of small biological specimens. Its potential application is particularly valuable within the field of cardiac development, where phenotypic profiling at the whole organ, cell, and molecular level is often most informative within the same sample. Consequently, this study sought to develop a multimodal imaging protocol to enable 3D phenotypic characterisation of embryonic avian hearts (iodine-based contrast X-ray imaging) prior to immunohistochemistry-based cell and molecular analysis. Micro-CT parameters were tested to establish an optimal protocol for 3D analysis of embryonic cardiac specimens across multiple developmental timepoints. Optimised parameters provided reliable and reproducible 3D analysis of cardiac macrostructures. Sodium thiosulphate treatment of X-ray imaged hearts effectively reversed the iodine-based contrast stain whilst maintaining antigen availability of nuclear, membranous, and cytoplasmic targets in traditional downstream imaging studies. Together, this study demonstrates a robust and highly efficient multimodal imaging strategy to comprehensively characterise cardiac morphology in avian embryos and may serve as a versatile foundation for a broad range of bioimaging applications within the wider scientific community.

STRUCTURED ABSTRACT Purpose of Research: The generation of iPSC lines expressing 21, 56 and 79 glutamine repeats within the HTT protein and homozygous KO of HTT in the KOLF2.1J background as an additional disease series within the iPSC Neurodegenerative Disease Initiative (iNDI) collection. Major Findings: All iPSCs, even those expressing long repeats of 79Q or HTT KO, were capable of differentiating to striatal and cortical neurons, astrocytes and microglia using established protocols. General quality control stains and morphological analyses are described for each differentiation. A selected set of assays were carried out on differentiated cells; expanded repeat expressing astrocytes showed altered expression of astrocyte protein markers and morphological characteristics, and striatal neurons showed altered DARPP-32/CTIP2 colocalization. mRNAseq carried out for striatal neurons showed high similarities in gene expression changes between 79Q and KO lines compared to the unexpanded repeat. Conclusions: The KOLF2.1J isogenic CAG repeat series serves as a community resource to study HD mechanisms with the potential for direct comparison across other neurodegenerative diseases through the iNDI collection.

Optical recording from large numbers of neurons is an indispensable technique for studying neuronal ensembles. We use optical sectioning through pinhole illumination to reduce the background fluorescence (F0) and increase the optical signal ({Delta}F/F0) in ex vivo brain slices densely labeled with GCaMP6f, allowing an ordinary fluorescence microscope to capture calcium transients from over 300 individual CA1 neurons - a marked increase compared to ordinary wide field fluorescence illumination. Multiple layers of overlapping neurons can be identified by their locations and the shape in space of their {Delta}F/F0 images. A single pinhole mask was placed at the field stop of a wide field illuminator, and the image of the pinhole was projected onto the tissue by a 20X NA 0.95 water immersion objective (Olympus). This created an illuminated disk with a diameter of [~]200 m and optical sections of hippocampal CA1 pyramidal layer tissue [~]100 m thick. This illumination blocked a large fraction of the F0, which in turn increased the {Delta}F/F0 5-10-fold compared to that of wide field illumination. When putative pyramidal neurons fire sparsely in the brain slice, up to 300 partially superimposed neurons can be identified by their shape and spatial location in the thick ([~]480 m) ex vivo slice in the CA1 area surrounding the pinhole image. The signal-to-noise ratio was adequate even at a low excitation light level of [~]20k photoelectrons per pixel well on the camera, allowing for 3,000 seconds of total recording time without significant bleaching. This pinhole "half confocal" method has created a useful way to sample calcium transient signals in thick tissue with a large population of neurons densely labeled with GCaMP-6f.

Many nonhuman primate species recapitulate the neuropathological features of sporadic Alzheimer's disease (AD) to varying degrees. As with humans, the assessment of AD-related pathology in nonhuman primates has historically relied on the use of postmortem brain tissues. In vivo alternatives, such as PET imaging tracers and fluid biomarkers, have been developed for use in humans but require further validation in nonhuman primates before replacing postmortem analyses. Here we employed the Nucleic Acid-Linked Immuno-Sandwich Assay (NULISATM) CNS Disease panel to compare age-related changes in plasma biomarkers in two nonhuman primate species (rhesus monkeys and baboons). In addition, we examined whether amyloid and tau biomarkers were associated with brain atrophy, as measured by gray matter volume. We found significant associations between age and multiple biomarkers of neurodegeneration for both species, as well as significant differences in the patterns of these associations between the two species. For the phosphorylated tau measures, though rhesus monkeys had higher values, baboons showed significant and stronger associations with age. By contrast, rhesus monkeys exhibited an earlier age-related decline in A{beta}42/A{beta}40 ratio than baboons. Finally, in both species, lower A{beta}42/A{beta}40 ratios were associated with lower gray matter volumes. This is the first systematic comparative study of age-related changes in neurodegeneration biomarkers in two closely related nonhuman primates using comparable age ranges and sample sizes, and the same multiplex assay. Future studies should examine longitudinal changes in these biomarkers as well as validate the plasma findings using cerebral spinal fluid.

Purpose: To investigate sex differences in cerebral blood flow through densely parcellated cortical and subcortical regions using explainable artificial intelligence methods and identify neurobiologically interpretable perfusion biomarkers. Methods: High-resolution pseudo-continuous arterial spin labelling (1.875 mm x 1.875 mm x 3 mm) and structural MRI data were curated from 215 healthy young adults (150 females, 95 males; age 18-30 years) from the publicly available I See your Brains (ISYB) dataset. Cerebral blood flow was quantified using atlas-based regional analysis with the Brainnetome Atlas (246 regions) and optimized registration procedures. Sex classification employed diverse machine learning paradigms including linear classifiers, ensemble methods, and kernel-based approaches for regional CBF features, with deep convolutional neural networks (CNN) applied to whole-brain 3D imaging data. Model interpretability was achieved using SHapley Additive exPlanations (SHAP), computed over an ensemble of 500 logistic regression models (100 iterations x 5-fold cross-validation). Regions appearing among the top 20% of discriminative features more than 289 times were considered statistically significant using binomial testing. GradCAM was used to obtain class-specific attribution maps from the CNN model. Results: Perfusion-based features demonstrated superior sex classification performance compared to structural morphometry. Regional CBF analysis using logistic regression achieved 91 +/- 2% balanced accuracy and 0.95 +/- 0.05 ROC-AUC, substantially outperforming morphometric features (85 +/- 8% balanced accuracy, 0.88 +/- 0.06 ROC-AUC). Deep learning classification of 3D CBF maps achieved a performance of 92 +/- 5% balanced accuracy, 0.92 +/- 0.05 ROC-AUC. SHAP analysis identified 30 statistically significant aggregation-agnostic CBF-based biomarker regions using regional CBF, predominantly involving frontoparietal control networks (27%) and default mode networks (17%). Grad-CAM revealed that the 3D CNN model primarily focused on regions within the frontal lobe. Morphometry-based analysis identified 28 discriminative regions with markedly different anatomical distribution (r = 0.21) emphasizing visual (32%) and default mode (14%) networks. Conclusion: Cerebral blood flow patterns provide highly sensitive and biologically interpretable markers of sex differences in young adult brain. The identification of robust perfusion biomarkers through explainable AI demonstrates the clinical potential of ASL imaging for precision medicine applications in neuroscience. We establish a methodological framework for investigating sex-specific brain physiology using non-invasive neuroimaging.