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Secretagogin (SCGN) is a recently discovered calcium-binding protein belonging to the group of EF-hand calcium-binding proteins. SCGN immunostaining has been described in various regions of the human, rat and mouse brain. In these studies, it has been reported that, in general, the patterns of SCGN staining differ between rodents and human brains. These differences have been interpreted as uncovering phylogenetic differences in SCGN expression. Nevertheless, an important aspect that is not usually taken into account is that different methods are used for obtaining and processing brain tissue coming from humans and experimental animals. This is a critical issue since it has been shown that post-mortem time delay and the method of fixation (i.e., perfused vs. nonperfused brains) may influence the results of the immunostaining. Thus, it is not clear whether differences found in comparative studies with the human brain are simply due to technical factors or species-specific differences. In the present study, we analyzed the pattern of SCGN immunostaining in the adult human hippocampal formation (DG, CA1, CA2, CA3, subiculum, presubiculum, and parasubiculum) as well as in the entorhinal and perirhinal cortices. This pattern of immunostaining was compared with rat and mouse that were fixed either by perfusion or immersion and with different post-mortem time delays (up to 5 hr) to mimic the way the human brain tissue is usually processed. We found a number of clear similarities and differences in the pattern of labeling among the human, rat, and mouse in these brain regions as well as between the different brain regions examined within each species. These differences were not due to the fixation. ; This work was supported by grants from the following entities: Centro de Investigación en Red sobre Enfermedades Neurodegenerativas (CIBERNED, CB06/05/0066, Spain); the Spanish "Ministerio de Ciencia, Innovación y Universidades" (grant PGC2018-094307-BI00); and the European Union's Horizon 2020 Research and Innovation Programme under grant agreement No. 785907 (Human Brain Project, second specific grant agreement [SGA2]).

Most data are available in the main text or the Supplementary Materials. The datasets used and analyzed during the current study are published in the EBRAINS Knowledge Graph (DOI:10.25493/5F04-N97): Domínguez-Álvaro M, Montero M, Alonso-Nanclares L, Blazquez-Llorca L, Rodríguez J-R, DeFelipe J. (2020). Densities and 3D distributions of synapses using FIB/SEM imaging in the human neocortex (Temporal cortex, T2) [Data set]. Human Brain Project Neuroinformatics Platform. ; In the present study, we have used Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) to perform a study of the synaptic organization of layer III of Brodmann's area 21 in human tissue samples obtained from autopsies and biopsies. We analyzed the synaptic density, 3D spatial distribution, and type (asymmetric/symmetric), as well as the size and shape of each synaptic junction of 4945 synapses that were fully reconstructed in 3D. Significant differences in the mean synaptic density between autopsy and biopsy samples were found (0.49 and 0.66 synapses/μm3 , respectively). However, in both types of samples (autopsy and biopsy), the asymmetric:symmetric ratio was similar (93:7) and most asymmetric synapses were established on dendritic spines (75%), while most symmetric synapses were established on dendritic shafts (85%). We also compared several electron microscopy methods and analysis tools to estimate the synaptic density in the same brain tissue. We have shown that FIB/SEM is much more reliable and robust than the majority of the other commonly used EM techniques. The present work constitutes a detailed description of the synaptic organization of cortical layer III. Further studies on the rest of the cortical layers are necessary to better understand the functional organization of this temporal cortical region. ; This study was funded by grants from the following entities: Spanish "Ministerio de Ciencia e Innovación" grant PGC2018-094307-B-I00, and the Cajal Blue Brain Project made available under aCC-BY-NC 4.0 International license. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is bioRxiv preprint doi: https://doi.org/10.1101/2021.01.18.427131; this version posted April 8, 2021. The copyright holder for this preprint 59 (the Spanish partner of the Blue Brain Project initiative from EPFL [Switzerland]); Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, Spain, CB06/05/0066); and the European Union's Horizon 2020 Framework Programme for Research and Innovation under Specific Grant Agreement No. 945539 (Human Brain Project SGA3). N C-A was awarded a research fellowship from the Spanish "Ministerio de Ciencia e Innovación" (PRE2019-089228). ; Peer reviewed

Pyramidal neurons are the most abundant and characteristic neuronal type in the cerebral cortex and their dendritic spines are the main postsynaptic elements of cortical excitatory synapses. Previous studies have shown that pyramidal cell structure differs across layers, cortical areas, and species. However, within the human cortex, the pyramidal dendritic morphology has been quantified in detail in relatively few cortical areas. In the present work, we performed intracellular injections of Lucifer Yellow at several distances from the temporal pole. We found regional differences in pyramidal cell morphology, which showed large inter-individual variability in most of the morphological variables measured. However, some values remained similar in all cases. The smallest and least complex cells in the most posterior temporal region showed the greatest dendritic spine density. Neurons in the temporal pole showed the greatest sizes with the highest number of spines. Layer V cells were larger, more complex, and had a greater number of dendritic spines than those in layer III. The present results suggest that, while some aspects of pyramidal structure are conserved, there are specific variations across cortical regions, and species. ; The Spanish "Ministerio de Ciencia e Innovación" (grant PGC2018-094307-B-I00); the Cajal Blue Brain Project (the Spanish partner of the Blue Brain Project initiative from EPFL, [Switzerland]); Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, Spain, CB06/05/0066); and the European Union's Horizon 2020 Framework Programme for Research and Innovation (under Specific grant agreement No. 785907, Human Brain Project SGA2, and Specific grant agreement No. 945539; Human Brain Project SGA3).

Multivesicular bodies (MVBs) are membrane-bound organelles that belong to the endosomal pathway. They participate in the transport, sorting, storage, recycling, degradation, and release of multiple substances. They interchange cargo with other organelles and participate in their renovation and degradation. We have used focused ion beam milling and scanning electron microscopy (FIB-SEM) to obtain stacks of serial sections from the neuropil of the somatosensory cortex of the juvenile rat. Using dedicated software, we have 3D-reconstructed 1618 MVBs. The mean density of MVBs was 0.21 per cubic micron. They were unequally distributed between dendrites (39.14%), axons (18.16%), and nonsynaptic cell processes (42.70%). About one out of five MVBs (18.16%) were docked on mitochondria, representing the process by which the endosomal pathway participates in mitochondrial maintenance. Other features of MVBs, such as the presence of tubular protrusions (6.66%) or clathrin coats (19.74%) can also be interpreted in functional terms, since both are typical of early endosomes. The sizes of MVBs follow a lognormal distribution, with differences across cortical layers and cellular compartments. The mean volume of dendritic MVBs is more than twice as large as the volume of axonic MVBs. In layer I, they are smaller, on average, than in the other layers. ; The Spanish "Ministerio de Ciencia, Innovación y Universidades" (grant PGC2018-094307-B-I00 and the Cajal Blue Brain Project [C080020-09; the Spanish partner of the Blue Brain Project initiative from EPFL, Switzerland]; the European Union's Horizon 2020 Research and Innovation Programme under grant agreement No. 785907 (Human Brain Project, SGA2) and; Centro de Investigacion en Red sobre Enfermedades Neurodegenerativas (CIBERNED, CB06/05/0066, Spain).

Background Analyzing images to accurately estimate the number of different cell types in the brain using automatic methods is a major objective in neuroscience. The automatic and selective detection and segmentation of neurons would be an important step in neuroanatomical studies. New method We present a method to improve the 3D reconstruction of neuronal nuclei that allows their segmentation, excluding the nuclei of non-neuronal cell types. Results We have tested the algorithm on stacks of images from rat neocortex, in a complex scenario (large stacks of images, uneven staining, and three different channels to visualize different cellular markers). It was able to provide a good identification ratio of neuronal nuclei and a 3D segmentation. Comparison with Existing Methods: Many automatic tools are in fact currently available, but different methods yield different cell count estimations, even in the same brain regions, due to differences in the labeling and imaging techniques, as well as in the algorithms used to detect cells. Moreover, some of the available automated software methods have provided estimations of cell numbers that have been reported to be inaccurate or inconsistent after evaluation by neuroanatomists. Conclusions It is critical to have a tool for automatic segmentation that allows discrimination between neurons, glial cells and perivascular cells. It would greatly speed up a task that is currently performed manually and would allow the cell counting to be systematic, avoiding human bias. Furthermore, the resulting 3D reconstructions of different cell types can be used to generate models of the spatial distribution of cells. ; This study was funded by grants from the following entities: Spanish "Ministerio de Ciencia e Innovación " grants PGC2018-094307-B-I00 and TIN2017-83132-C2-2-R; the Cajal Blue Brain Project (the Spanish partner of the Blue Brain Project initiative from EPFL, Switzerland); the European Union's Horizon 2020 Framework Programme for Research and Innovation under grant agreement No. 945,539 (Human Brain Project SGA3).

Pyramidal neurons are the most common cell type in the cerebral cortex. Understanding how they differ between species is a key challenge in neuroscience. A recent study provided a unique set of human and mouse pyramidal neurons of the CA1 region of the hippocampus, and used it to compare the morphology of apical and basal dendritic branches of the two species. The study found inter-species differences in the magnitude of the morphometrics and similarities regarding their variation with respect to morphological determinants such as branch type and branch order. We use the same data set to perform additional comparisons of basal dendrites. In order to isolate the heterogeneity due to intrinsic differences between species from the heterogeneity due to differences in morphological determinants, we fit multivariate models over the morphometrics and the determinants. In particular, we use conditional linear Gaussian Bayesian networks, which provide a concise graphical representation of the independencies and correlations among the variables. We also extend the previous study by considering additional morphometrics and by formally testing whether a morphometric increases or decreases with the distance from the soma. This study introduces a multivariate methodology for inter-species comparison of morphology. ; Tis work has been partially supported by the Spanish Ministry of Economy and Competitiveness through the TIN2016-79684-P project. Tis project has received funding from the European Union's Horizon 2020 Framework Programme for Research and Innovation under the Specifc Grant Agreement No. 785907 (Human Brain Project SGA2).

There is currently no unique catalog of cortical GABAergic interneuron types. In 2013, we asked 48 leading neuroscientists to classify 320 interneurons by inspecting images of their morphology. That study was the first to quantify the degree of agreement among neuroscientists in morphology-based interneuron classification, showing high agreement for the chandelier and Martinotti types, yet low agreement for most of the remaining types considered. Here we present the dataset containing the classification choices by the neuroscientists according to interneuron type as well as to five prominent morphological features. These data can be used as crisp or soft training labels for learning supervised machine learning interneuron classifiers, while further analyses can try to pinpoint anatomical characteristics that make an interneuron especially difficult or especially easy to classify. ; This work has been partially supported by the Spanish Ministry of Economy and Competitiveness through the TIN2016-79684-P project. This project has received funding from the European Union's Horizon 2020 Framework Programme for Research and Innovation under the Specific Grant Agreement No. 785907 (Human Brain Project SGA2).

[Background] The challenge of classifying cortical interneurons is yet to be solved. Data-driven classification into established morphological types may provide insight and practical values. [Results] We trained models using 217 high-quality morphologies of rat somatosensory neocortex interneurons reconstructed by a single laboratory and pre-classified into eight types. We quantified 103 axonal and dendritic morphometrics, including novel ones that capture features such as arbor orientation, extent in layer one, and dendritic polarity. We trained a one-versus-rest classifier for each type, combining well-known supervised classification algorithms with feature selection and over- and under-sampling. We accurately classified the nest basket, Martinotti, and basket cell types with the Martinotti model outperforming 39 out of 42 leading neuroscientists. We had moderate accuracy for the double bouquet, small and large basket types, and limited accuracy for the chandelier and bitufted types. We characterized the types with interpretable models or with up to ten morphometrics. [Conclusion] Except for large basket, 50 high-quality reconstructions sufficed to learn an accurate model of a type. Improving these models may require quantifying complex arborization patterns and finding correlates of bouton-related features. Our study brings attention to practical aspects important for neuron classification and is readily reproducible, with all code and data available online. ; This project has received funding from the European Union's Horizon 2020 Framework Programme for Research and Innovation under Specific Grant Agreement No. 785907 (HBP SGA2), the Spanish Ministry of Economy and Competitiveness through the Cajal Blue Brain (C080020-09; the Spanish partner of the EPFL Blue Brain initiative) and TIN2016-79684-P projects, from the Regional Government of Madrid through theS2013/ICE-2845-CASI-CAM-CM project, and from Fundación BBVA grants to Scientific Research Teams in Big Data 2016. ; Peer reviewed

Abstract
The dendritic spines of pyramidal cells are the main postsynaptic target of excitatory glutamatergic synapses. Morphological alterations have been described in hippocampal dendritic spines during hibernation—a state of inactivity and metabolic depression that occurs via a transient neuronal tau hyperphosphorylation. Here, we have used the hibernating Syrian hamster to investigate the effect of hyperphosphorylated tau regarding neocortical neuronal structure. In particular, we examined layer Va pyramidal neurons. Our results indicate that hibernation does not promote significant changes in dendritic spine density. However, tau hyperphosphorylated neurons show a decrease in complexity, an increase in the tortuosity of the apical dendrites, and an increase in the diameter of the basal dendrites. Tau protein hyperphosphorylation and aggregation have been associated with loss or alterations of dendritic spines in neurodegenerative diseases, such as Alzheimer's disease (AD). Our results may shed light on the correlation between tau hyperphosphorylation and the neuropathological processes in AD. Moreover, we observed changes in the length and area of the apical and basal dendritic spines during hibernation regardless of tau hyperphosphorylation. The morphological changes observed here also suggest region specificity, opening up debate about a possible relationship with the differential brain activity registered in these regions in previous studies.

The entorhinal cortex (EC) is a brain region that has been shown to be essential for memory functions and spatial navigation. However, detailed three-dimensional (3D) synaptic morphology analysis and identification of postsynaptic targets at the ultrastructural level have not been performed before in the human EC. In the present study, we used Focused Ion Beam/Scanning Electron Microscopy to perform a 3D analysis of the synapses in the neuropil of medial EC in layers II and III from human brain autopsies. Specifically, we studied synaptic structural parameters of 3561 synapses, which were fully reconstructed in 3D. We analyzed the synaptic density, 3D spatial distribution, and type (excitatory and inhibitory), as well as the shape and size of each synaptic junction. Moreover, the postsynaptic targets of synapses could be clearly determined. The present work constitutes a detailed description of the synaptic organization of the human EC, which is a necessary step to better understand the functional organization of this region in both health and disease ; Spanish "Ministerio de Ciencia e Innovación" (grant PGC2018-094307-B-I00); the Cajal Blue Brain Project (the Spanish partner of the Blue Brain Project initiative from EPFL, [Switzerland]); Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Spain (CB06/05/0066); the Alzheimer's Association (ZEN-15-321663); the European Union's Horizon 2020 Framework Programme for Research and Innovation (grant agreement No. 945539) to Human Brain Project SGA3; the Spanish "Ministerio de Educación y Formación Profesional" (FPU14/02245 to M.M.-C.); UNED (Plan de Promoción de la Investigación, 2014-040-UNED-POST to L.B.-L.) We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

© The Author(s) 2016 ; In this article, we analyze branching angles of the basal dendrites of pyramidal neurons of layers III and V of the human temporal cortex. For this, we use a novel probability directional statistical distribution called truncated von Mises distribution that is able to describe more accurately the dendritic-branching angles than the previous proposals. Then, we perform comparative studies using this statistical method to determine similarities and/or differences between branches and branching angles that belong to different cortical layers and regions. Using this methodology, we found that common design principles exist and govern the patterns found in the different branches that compose the basal dendrites of human pyramidal cells of the temporal cortex. However, particular differences were found between supra and infragranular cells. Furthermore, we compared the branching angles of human layer III pyramidal neurons with data obtained in the previous studies in layer III of both the rat somatosensory cortex and of several cortical areas of the mouse. Finally, we study the branching angle differences between the humans that compose our data. ; This work has been partially supported by the Spanish Ministry of Economy and Competitiveness through the Cajal Blue Brain (C080020-09; the Spanish partner of the Blue Brain initiative from EPFL) and TIN2013-41592-P projects, by the Regional Government of Madrid through the S2013/ICE-2845-CASI-CAM-CM project, by the European Union's Seventh Framework Programme (FP7/2007-2013) under Grant agreement no. 604102 (Human Brain Project), and by the Spanish Ministry of Education, Culture, and Sport Fellowship (FPU13/01941).

© The Author(s) 2017. ; Understanding the human brain is the ultimate goal in neuroscience, but this is extremely challenging in part due to the fact that brain tissue obtained from autopsy is practically the only source of normal brain tissue and also since changes at different levels of biological organization (genetic, molecular, biochemical, anatomical) occur after death due to multiple mechanisms. Here we used metabolomic and anatomical techniques to study the possible relationship between post-mortem time (PT)-induced changes that may occur at both the metabolomics and anatomical levels in the same brains. Our experiments have mainly focused on the hippocampus of the mouse. We found significant metabolomic changes at 2 h PT, whereas the integrity of neurons and glia, at the anatomical/ neurochemical level, was not significantly altered during the first 5 h PT for the majority of histological markers. ; This work was supported by grants from the following entities: the Spanish Ministerio de Economía y Competitividad (Grants CTQ2014-55279-R to CB and AG and Grant SAF 2015-66603-P to JD); the European Union's Horizon 2020 research and innovation programme under Grant Agreement No. 720270 (Human Brain Project) to JD; and Centro de Investigación en Red sobre Enfermedades Neurodegenerativas (CIBERNED, CB06/05/0066, Spain) to JD. CGR was awarded a research fellowship from the University CEU San Pablo.

The definition of the soma is fuzzy, as there is no clear line demarcating the soma of the labeled neurons and the origin of the dendrites and axon. Thus, the morphometric analysis of the neuronal soma is highly subjective. In this paper, we provide a mathematical definition and an automatic segmentation method to delimit the neuronal soma. We applied this method to the characterization of pyramidal cells, which are the most abundant neurons in the cerebral cortex. Since there are no benchmarks with which to compare the proposed procedure, we validated the goodness of this automatic segmentation method against manual segmentation by neuroanatomists to set up a framework for comparison. We concluded that there were no significant differences between automatically and manually segmented somata, i.e., the proposed procedure segments the neurons similarly to how a neuroanatomist does. It also provides univocal, justifiable and objective cutoffs. Thus, this study is a means of characterizing pyramidal neurons in order to objectively compare the morphometry of the somata of these neurons in different cortical areas and species. ; This work has been supported by grants from the following entities: the Spanish Ministry of Economics and Competitiveness (grant BFU2012-34963 the Cajal Blue Brain Project, Spanish partner of the Blue Brain Project initiative from EPFL); Centro de Investigación en Red sobre Enfermedades Neurodegenerativas (CIBERNED, CB06/05/0066, Spain); TIN2013-41592-P projects, by the Regional Government of Madrid through the S2013/ICE-2845-CASI-CAM-CM project; and the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 604102 (Human Brain Project). ; Peer reviewed ; Peer Reviewed

The hippocampus plays a key role in contextual conditioning and has been proposed as an important component of the cocaine addiction brain circuit. To gain knowledge about cocaine-induced alterations in this circuit, we used focused ion beam milling/scanning electron microscopy to reveal and quantify the three-dimensional synaptic organization of the neuropil of the stratum radiatum of the rat CA1, under normal circumstances and after cocaine-self administration (SA). Most synapses are asymmetric (excitatory), macular-shaped, and in contact with dendritic spine heads. After cocaine-SA, the size and the complexity of the shape of both asymmetric and symmetric (inhibitory) synapses increased but no changes were observed in the synaptic density. This work constitutes the first detailed report on the 3D synaptic organization in the stratum radiatum of the CA1 field of cocaine-SA rats. Our data contribute to the elucidation of the normal and altered synaptic organization of the hippocampus, which is crucial for better understanding the neurobiological mechanisms underlying cocaine addiction. ; Ministerio de Ciencia e Innovación (PSI2016-80541-P to E.A.); Ministerio de Sanidad, Servicios Sociales e Igualdad (Red de Trastornos Adictivos [RTA-RD16/0017/0022 del Instituto de Salud Carlos III to E.A.] and Plan Nacional sobre Drogas [2016I073 to E.A.]); UNED (Plan de Promoción de la Investigación, 2014-040-UNED-POST to L.B.-L.); UNED-Banco Santander ("Independent Thinking", 2017-VICE-0012 to L.B.- L.); European Union (JUST-2017-AG-DRUGS-806996-JUSTSO to E.A.); Centro de Investigación en Red sobre Enfermedades Neurodegenerativas (CIBERNED, CB06/05/0066 to J.D.); Ministerio de Ciencia, Innovación y Universidades (PGC2018-094307-B-I00 to J.D. and the Cajal Blue Brain Project [the Spanish partner of the Blue Brain Project initiative from EPFL, Switzerland] to J.D.); and Ministerio de Educación, Cultura y Deporte (contract FPU14/02245 to M.M.-C.).

The hippocampus plays a key role in contextual conditioning and has been proposed as an important component of the cocaine addiction brain circuit. To gain knowledge about cocaine-induced alterations in this circuit, we used focused ion beam milling/scanning electron microscopy to reveal and quantify the three-dimensional synaptic organization of the neuropil of the stratum radiatum of the rat CA1, under normal circumstances and after cocaine-self administration (SA). Most synapses are asymmetric (excitatory), macular-shaped, and in contact with dendritic spine heads. After cocaine-SA, the size and the complexity of the shape of both asymmetric and symmetric (inhibitory) synapses increased but no changes were observed in the synaptic density. This work constitutes the first detailed report on the 3D synaptic organization in the stratum radiatum of the CA1 field of cocaine-SA rats. Our data contribute to the elucidation of the normal and altered synaptic organization of the hippocampus, which is crucial for better understanding the neurobiological mechanisms underlying cocaine addiction. ; Ministerio de Ciencia e Innovación (PSI2016-80541-P to E.A.); Ministerio de Sanidad, Servicios Sociales e Igualdad (Red de Trastornos Adictivos [RTA-RD16/0017/0022 del Instituto de Salud Carlos III to E.A.] and Plan Nacional sobre Drogas [2016I073 to E.A.]); UNED (Plan de Promoción de la Investigación, 2014-040-UNED-POST to L.B.-L.); UNED-Banco Santander ("Independent Thinking", 2017-VICE-0012 to L.B.- L.); European Union (JUST-2017-AG-DRUGS-806996-JUSTSO to E.A.); Centro de Investigación en Red sobre Enfermedades Neurodegenerativas (CIBERNED, CB06/05/0066 to J.D.); Ministerio de Ciencia, Innovación y Universidades (PGC2018-094307-B-I00 to J.D. and the Cajal Blue Brain Project [the Spanish partner of the Blue Brain Project initiative from EPFL, Switzerland] to J.D.); and Ministerio de Educación, Cultura y Deporte (contract FPU14/02245 to M.M.-C.).