Towards a European health research and innovation cloud (HRIC).

The European Union (EU) initiative on the Digital Transformation of Health and Care (Digicare) aims to provide the conditions necessary for building a secure, flexible, and decentralized digital health infrastructure. Creating a European Health Research and Innovation Cloud (HRIC) within this environment should enable data sharing and analysis for health research across the EU, in compliance with data protection legislation while preserving the full trust of the participants. Such a HRIC should learn from and build on existing data infrastructures, integrate best practices, and focus on the concrete needs of the community in terms of technologies, governance, management, regulation, and ethics requirements. Here, we describe the vision and expected benefits of digital data sharing in health research activities and present a roadmap that fosters the opportunities while answering the challenges of implementing a HRIC. For this, we put forward five specific recommendations and action points to ensure that a European HRIC: i) is built on established standards and guidelines, providing cloud technologies through an open and decentralized infrastructure; ii) is developed and certified to the highest standards of interoperability and data security that can be trusted by all stakeholders; iii) is supported by a robust ethical and legal framework that is compliant with the EU General Data Protection Regulation (GDPR); iv) establishes a proper environment for the training of new generations of data and medical scientists; and v) stimulates research and innovation in transnational collaborations through public and private initiatives and partnerships funded by the EU through Horizon 2020 and Horizon Europe.

Interoperable atlases of the human brain.

The last two decades have seen an unprecedented development of human brain mapping approaches at various spatial and temporal scales. Together, these have provided a large fundus of information on many different aspects of the human brain including micro- and macrostructural segregation, regional specialization of function, connectivity, and temporal dynamics. Atlases are central in order to integrate such diverse information in a topographically meaningful way. It is noteworthy, that the brain mapping field has been developed along several major lines such as structure vs. function, postmortem vs. in vivo, individual features of the brain vs. population-based aspects, or slow vs. fast dynamics. In order to understand human brain organization, however, it seems inevitable that these different lines are integrated and combined into a multimodal human brain model. To this aim, we held a workshop to determine the constraints of a multi-modal human brain model that are needed to enable (i) an integration of different spatial and temporal scales and data modalities into a common reference system, and (ii) efficient data exchange and analysis. As detailed in this report, to arrive at fully interoperable atlases of the human brain will still require much work at the frontiers of data acquisition, analysis, and representation. Among them, the latter may provide the most challenging task, in particular when it comes to representing features of vastly different scales of space, time and abstraction. The potential benefits of such endeavor, however, clearly outweigh the problems, as only such kind of multi-modal human brain atlas may provide a starting point from which the complex relationships between structure, function, and connectivity may be explored.

Vesicular glutamate transporter-3 in the rodent brain: vesicular colocalization with vesicular γ-aminobutyric acid transporter.

Vesicular glutamate transporters (VGLUT1-3) carry glutamate into synaptic vesicles. VGLUT3 has been reported to be localized in nonglutamatergic neuronal populations in the brain. However, detailed subcellular localization of VGLUT3 has not been shown. In particular, the identity of synaptic vesicles expressing VGLUT3 remains to be revealed. Here we present novel electron microscopic postembedding immunogold data from mouse and rat brains showing that small, clear, and round synaptic vesicles in γ-aminobutyric acid (GABA)-ergic nerve terminals contain labeling for both VGLUT3 and the vesicular GABA transporter (VGAT). Immunoisolation of synaptic vesicles confirmed the immunogold data and showed vesicular colocalization of VGLUT3 and VGAT. Moreover, we show that gold particles signaling VGLUT3 are present in synaptic vesicles in acetylcholinergic nerve terminals in the striatum. Quantitative immunogold analyses reveal that the density of VGLUT3 gold particles is similar in GABAergic terminals in the hippocampus and the neocortex to that in cholinergic terminals in the striatum. In contrast to in the hippocampus and the neocortex, VGLUT3 was absent from VGAT-positive terminals in the striatum. The labeling pattern produced by the VGLUT3 antibodies was found to be specific; there was no labeling in VGLUT3 knockout tissue, and the observed labeling throughout the rat brain corresponds to the known light-microscopic distribution of VGLUT3. From the present results, we infer that glutamate is released with GABA from inhibitory terminals and acetylcholine from cholinergic terminals.

The concentrations and distributions of three C-terminal variants of the GLT1 (EAAT2; slc1a2) glutamate transporter protein in rat brain tissue suggest differential regulation.

The neurotransmitter glutamate is inactivated by cellular uptake; mostly catalyzed by the glutamate transporter GLT1 (slc1a2, excitatory amino acid transporter [EAAT2]) subtype which is expressed at high levels in brain astrocytes and at lower levels in neurons. Three coulombs-terminal variants of GLT1 exist (GLT1a, GLT1b and GLT1c). Their cellular distributions are currently being debated (that of GLT1b in particular). Here we have made antibodies to the variants and produced pure preparations of the individual variant proteins. The immunoreactivities of each variant per amount of protein were compared to that of total GLT1 immunoisolated from Wistar rat brains. At eight weeks of age GLT1a, GLT1b and GLT1c represented, respectively 90%+/-1%, 6+/-1% and 1%+/-0.5% (mean+/-SEM) of total hippocampal GLT1. The levels of all three variants were low at birth and increased towards adulthood, but GLT1a increased relatively more than the other two. At postnatal day 14 the levels of GLT1b and GLT1c relative to total GLT1 were, respectively, 1.7+/-0.1 and 2.5+/-0.1 times higher than at eight weeks. In tissue sections, antibodies to GLT1a gave stronger labeling than antibodies to GLT1b, but the distributions of GLT1a and GLT1b were similar in that both were predominantly expressed in astroglia, cell bodies as well as their finest ramifications. GLT1b was not detected in nerve terminals in normal brain tissue. The findings illustrate the need for quantitative measurements and support the notion that the importance of the variants may not be due to the transporter molecules themselves, but rather that their expression represents the activities of different regulatory pathways.

Database and tools for analysis of topographic organization and map transformations in major projection systems of the brain.

Integration of dispersed and complicated information collected from the brain is needed to build new knowledge. But integration may be hampered by rigid presentation formats, diversity of data formats among laboratories, and lack of access to lower level data. We have addressed some of the fundamental issues related to this challenge at the level of anatomical data, by producing a coordinate based digital atlas and database application for a major projection system in the rat brain: the cerebro-ponto-cerebellar system. This application, Functional Anatomy of the Cerebro-Cerebellar System in rat (FACCS), is available via the Rodent Brain WorkBench (http://www.rbwb.org). The data included are x,y,z-coordinate lists describing exact distributions of tissue elements (axonal terminal fields of axons, or cell bodies) that are labeled with axonal tracing techniques. All data are translated to a common local coordinate system to facilitate across animal comparison. A search capability allows queries based on, e.g. location of tracer injection sites, tracer category, size of the injection sites, and contributing author. A graphic search tool allows the user to move a volume cursor inside a coordinate system to detect particular injection sites having connections to a specific tissue volume at chosen density levels. Tools for visualization and analysis of selected data are included, as well as an option to download individual data sets for further analysis. With this application, data and metadata from different experiments are mapped into the same information structure and made available for re-use and re-analysis in novel combinations. The application is prepared for future handling of data from other projection systems as well as other data categories.

Three-dimensional chemoarchitecture of the basal forebrain: spatially specific association of cholinergic and calcium binding protein-containing neurons.

The basal forebrain refers to heterogeneous structures located close to the medial and ventral surfaces of the cerebral hemispheres. It contains diverse populations of neurons, including the cholinergic cortically projecting cells that show severe loss in Alzheimer's and related neurodegenerative diseases. The basal forebrain does not display any cytoarchitectural or other structural features that make it easy to demarcate functional boundaries, a problem that allowed different investigators to propose different organizational schemes. The present paper uses novel three-dimensional reconstructions and numerical analyses for studying the spatial organization of four major basal forebrain cell populations, the cholinergic, parvalbumin, calbindin and calretinin containing neurons in the rat. Our analyses suggest that the distribution of these four cell populations is not random but displays a general pattern of association. Within the cholinergic space (i.e. the volume occupied by the cortically projecting cholinergic cell bodies) the three other cell types form twisted bands along the longitudinal axis of a central dense core of cholinergic cells traversing the traditionally defined basal forebrain regions, (i.e. the medial septum, diagonal bands, the substantia innominata, pallidal regions and the bed nucleus of the stria terminalis). At a smaller scale, the different cell types within the cholinergic space occupy overlapping high-density cell clusters that are either chemically uniform or mixed. However, the cell composition of these high-density clusters is regionally specific. The proposed scheme of basal forebrain organization, using cell density or density relations as criteria, offers a new perspective on structure-function relationship, unconstrained by traditional region boundaries.

Three-dimensional computerised atlas of the rat brain stem precerebellar system: approaches for mapping, visualization, and comparison of spatial distribution data.

Comparisons of microscopical neuroanatomic data from different experiments and investigators are typically hampered by the use of different section planes and dissimilar techniques for data documentation. We have developed a framework for visualization and comparison of section-based, spatial distribution data, in brain stem nuclei. This framework provides opportunities for harmonized data presentation in neuroinformatics databases. Three-dimensional computerized reconstructions of the rat brain stem and precerebellar nuclei served as a basis for establishing internal coordinate systems for the pontine nuclei and the precerebellar divisions of the sensory trigeminal nuclei. Coordinate based diagrams were used for presentation of experimental data (spatial distribution of labelled neurons and axonal plexuses) from standard angles of view. Each nuclear coordinate system was based on a cuboid bounding box with a defined orientation. The bounding box was size-adjusted to touch cyto- and myeloarchitectonically defined boundaries of the individual nuclei, or easily identifiable nearby landmarks. We exemplify the use of these internal coordinate systems with dual retrograde neural tracing data from pontocerebellar and trigeminocerebellar systems. The new experimental data were combined, in the same coordinate based diagrams, with previously published data made available via a neuroinformatics data repository (www.nesys.uio.no/Database, see also www.cerebellum.org). Three-dimensional atlasing, internal nuclear coordinate systems, and consistent formats for presentation of neuroanatomic data in web-based data repositories, offer new opportunities for efficient analysis and re-analysis of neuroanatomic data.

A dendrodendritic reciprocal synapse provides a recurrent excitatory connection in the olfactory bulb.

Neuronal synchronization in the olfactory bulb has been proposed to arise from a diffuse action of glutamate released from mitral cells (MC, olfactory bulb relay neurons). According to this hypothesis, glutamate spills over from dendrodendritic synapses formed between MC and granule cells (GC, olfactory bulb interneurons) to activate neighboring MC. The excitation of MC is balanced by a strong inhibition from GC. Here we show that MC excitation is caused by glutamate released from bulbar interneurons located in the GC layer. These reciprocal synapses depend on an unusual, 2-amino-5-phosphonovaleric acid-resistant, N-methyl-d-aspartate receptor. This type of feedback excitation onto relay neurons may strengthen the original sensory input signal and further extend the function of the dendritic microcircuit within the main olfactory bulb.

Three-dimensional topography of corticopontine projections from rat barrel cortex: correlations with corticostriatal organization.

Subcortical re-entrant projection systems connecting cerebral cortical areas with the basal ganglia and cerebellum are topographically specific and therefore considered to be parallel circuits or "closed loops." The precision of projections within these circuits, however, has not been characterized sufficiently to indicate whether cortical signals are integrated within or among presumed compartments. To address this issue, we studied the first link of the rat cortico-ponto-cerebellar pathway with anterograde axonal tracing from physiologically defined, individual whisker "barrels" of the primary somatosensory cortex (SI). The labeled axons in the pontine nuclei formed several, sharply delineated clusters. Dual tracer injections into different SI whisker barrels gave rise to partly overlapping, paired clusters, indicating somatotopic specificity. Three-dimensional reconstructions revealed that the clusters were components of concentrically organized lamellar subspaces. Whisker barrels in the same row projected to different pontine lamellae (side by side), the somatotopic representation of which followed an inside-out sequence. By contrast, whisker barrels from separate rows projected to clusters located within the same lamellar subspace (end to end). In the neostriatum, this lamellar topography was the opposite, with barrels in the same row contacting different parts of the same lamellar subspace (end to end). The degree of overlap among pontine clusters varied as a function of the proximity of the cortical injections. Furthermore, corticopontine overlap was higher among projections from barrels in the same row than among projections from different whisker barrel rows. This anisotropy was the same in the corticostriatal projection. These findings have important implications for understanding convergence and local integration in somatosensory-related subcortical circuits.

Rat somatosensory cerebropontocerebellar pathways: spatial relationships of the somatotopic map of the primary somatosensory cortex are preserved in a three-dimensional clustered pontine map.

In the primary somatosensory cortex (SI), the body surface is mapped in a relatively continuous fashion, with adjacent body regions represented in adjacent cortical domains. In contrast, somatosensory maps found in regions of the cerebellar hemispheres, which are influenced by the SI through a monosynaptic link in the pontine nuclei, are discontinuous ("fractured") in organization. To elucidate this map transformation, the authors studied the organization of the first link in the SI-cerebellar pathway, the SI-pontine projection. After injecting anterograde axonal tracers into electrophysiologically defined parts of the SI, three-dimensional reconstruction and computer-graphic visualization techniques were used to analyze the spatial distribution of labeled fibers. Several target regions in the pontine nuclei were identified for each major body representation. The labeled axons formed sharply delineated clusters that were distributed in an inside-out, shell-like fashion. Upper lip and other perioral representations were located in a central core, whereas extremity and trunk representations were found more externally. The multiple clusters suggest that the pontine nuclei contain several representations of the SI map. Within each representation, the spatial relationships of the SI map are largely preserved. This corticopontine projection pattern is compatible with recently proposed principles for the establishment of subcortical topographic patterns during development. The largely preserved spatial relationships in the pontine somatotopic map also suggest that the transformation from an organized topography in SI to a fractured map in the cerebellum takes place primarily in the mossy fiber pontocerebellar projection.

Spatial correspondence between tactile projection patterns and the distribution of the antigenic Purkinje cell markers anti-zebrin I and anti-zebrin II in the cerebellar folium crus IIA of the rat.

We have compared the band-like distribution of the Purkinje cell-specific polypeptides zebrin I and zebrin II with the spatial organization of tactile projections to crus IIa in the cerebellar hemisphere of the rat. Maps of tactile responses in the granular layer of the cerebellar hemispheres are fractured into discontinuous regions, termed "patches". High-density micromapping was used to identify specific patches and their boundaries within this fractured somatotopic map. In one series of experiments, medial and lateral boundaries of the large central ipsilateral upper lip-related patch were identified and labeled with either Fast Blue or India Ink. Following immunocytochemical processing, the band-like distribution of immunostained Purkinje cells (zebrin-positive bands) and the identified patch boundaries were digitized and reconstructed in three dimensions. Comparisons between these two features demonstrate a spatial correspondence between zebrin transitions and the boundaries of the electrophysiologically defined upper lip-related patch. In another series of experiments, we outlined the boundaries or centers of several smaller patches consistently located in the medial portion of the folium. Again, we found a correspondence between the distribution of granule cell layer tactile patches and the zebrin staining pattern. The correspondence between tactile projection patterns and molecular features demonstrated in the present study implies that there is a distinct and largely fixed spatial pattern of organization in the cerebellar hemispheres. We discuss possible causal connections and developmental determinates, as well as the physiological significance of the correspondence between the two features.

Topographic organization of the dorsal nucleus of the lateral lemniscus in the cat.

The dorsal nucleus of the lateral lemniscus (DNLL) is an auditory structure of the brainstem. It plays an important role in binaural processing and sound localization and it provides the inferior colliculus with an inhibitory projection. The DNLL is a highly conserved auditory structure across mammals, but differences among species in its detailed organization have been reported. The main goal of this study was to analyze the topographic organization of the cat DNLL. Single, small iontophoretic injections of biotinylated dextran amine were made at different loci in the central nucleus of the inferior colliculus (CNIC). The distribution of the labeled structures in the ipsi- and contralateral DNLL was computer reconstructed in three dimensions. In individual sections, a band of labeling is seen in the DNLL on both sides. These two labeled bands occupy symmetric locations and are made of retrogradely labeled neurons with flattened dendritic arbors oriented parallel to each other. Moreover, the ipsilateral labeled band contains labeled terminal fibers parallel to the labeled dendrites. With three-dimensional reconstructions, it becomes evident that the labeled band seen in each individual DNLL section represents a slice through a rostrocaudally oriented lamina. The shape, size, orientation, and location of this lamina change as the injection site is shifted along the tonotopic axis of the CNIC. An injection in the low-frequency region of the CNIC, produces a lamina that resembles a flattened tube located in the dorsolateral corner of the DNLL. An injection in the high-frequency region of the CNIC, by contrast, results in a lamina that is an elongated sheet located at the ventromedial surface of the DNLL. The laminae of the DNLL might constitute the structural basis for its tonotopical organization. Previous studies (Merchan MA, et al. 1994. J Comp Neurol 342:259-278) in conjunction with our current results suggest that the laminar organization in the DNLL might be common among mammals.

Monkey somatosensory cerebrocerebellar pathways: uneven densities of corticopontine neurons in different body representations of areas 3b, 1, and 2.

We have studied the anatomic organization of corticopontine neurons in the monkey cytoarchitectonic areas 3a, 3b, 1, and 2. The purpose was to provide information about the composition of somatosensory cortical influence on cerebellar operations. Large tracer injections were made in the pontine nuclei. Retrogradely labeled neurons were confined to cortical layer 5, with the largest cell bodies located in area 3a and the smallest in area 3b. The distribution of labeled cells was quantitatively recorded and displayed in three-dimensional reconstructions and in flat maps. We have: (1) compared the average densities of labeled cells among the cytoarchitectonic areas, and (2) outlined the distribution of labeled cells within the cortical map of the body surface representation. The average density of labeled cells was considerably higher in areas 3a, 1, and 2, compared to area 3b. This finding suggests that areas 3a, 1, and 2 are more engaged in cerebellar operations than area 3b. We found marked density gradients of labeled cells within areas 3b, 1, and 2, but not within area 3a. When the density maps from areas 3b, 1, and 2 were superimposed on previously published somatotopic maps, we found higher average densities of corticopontine neurons in regions representing the trunk and proximal limbs, than in regions representing the distal forelimb. Thus, the distal forelimb representation, which is known to be strongly emphasized in terms of cortical volume, appears not to be correspondingly emphasized in the corticopontine projection.

Anatomic evidence of a three-dimensional mosaic pattern of tonotopic organization in the ventral complex of the lateral lemniscus in cat.

The ventral complex of the lateral lemniscus (VCLL, i.e., the ventral and intermediate nuclei) is composed of cells embedded in the fibers of the lateral lemniscus. These cells are involved in the processing of monaural information and receive input from the collaterals of the fibers ascending to the inferior colliculus. Whereas tonotopic organization is a feature of all other nuclei of the auditory system, this functional principle is debated in the VCLL. We have made focal injections of the tracer biotinylated dextran amine into different frequency band representations of the inferior colliculus in cat. Retrogradely labeled cells and terminal fibers (collaterals of efferent local axons and other ascending lemniscal fibers) were found in the ipsilateral VCLL. The spatial distribution of the labeling was analyzed using three-dimensional (3-D) reconstruction and computer graphical visualization techniques. A complex topographic organization was found. In all cases, labeled fibers and cells were distributed in multiple clusters throughout the dorsoventral extent of the VCLL. The shape, size, and location of the labeled clusters suggest an interdigitation of clusters assigned to different frequency-band representations. But an overall mediolateral distribution gradient was observed, with high frequencies represented medially and lower frequencies progressively more laterally. We conclude that the clusters may represent discontinuous frequency-band compartments as a counterpart to the continuous laminar compartments in the remaining auditory nuclei. The 3-D orderly mosaic pattern indicates that the VCLL preserves the spectral decomposition originated in the cochlea in a way that facilitates across-frequency integration.

Synaptic vesicular localization and exocytosis of L-aspartate in excitatory nerve terminals: a quantitative immunogold analysis in rat hippocampus.

To elucidate the role of aspartate as a signal molecule in the brain, its localization and those of related amino acids were examined by light and electron microscopic quantitative immunocytochemistry using antibodies specifically recognizing the aldehyde-fixed amino acids. Rat hippocampal slices were incubated at physiological and depolarizing [K+] before glutaraldehyde fixation. At normal [K+], aspartate-like and glutamate-like immunoreactivities were colocalized in nerve terminals forming asymmetrical synapses on spines in stratum radiatum of CA1 and the inner molecular layer of fascia dentata (i.e., excitatory afferents from CA3 and hilus, respectively). During K+ depolarization there was a loss of aspartate and glutamate from these terminals. Simultaneously the immunoreactivities strongly increased in glial cells. These changes were Ca2+-dependent and tetanus toxin-sensitive and did not comprise taurine-like immunoreactivity. Adding glutamine at CSF concentration prevented the loss of aspartate and glutamate and revealed an enhancement of aspartate in the terminals at moderate depolarization. In hippocampi from animals perfused with glutaraldehyde during insulin-induced hypoglycemia (to combine a strong aspartate signal with good ultrastructure) aspartate was colocalized with glutamate in excitatory terminals in stratum radiatum of CA1. The synaptic vesicle-to-cytoplasmic matrix ratios of immunogold particle density were similar for aspartate and glutamate, significantly higher than those observed for glutamine or taurine. Similar results were obtained in normoglycemic animals, although the nerve terminal contents of aspartate were lower. The results indicate that aspartate can be concentrated in synaptic vesicles and subject to sustained exocytotic release from the same nerve endings that contain and release glutamate.

Corticopontine terminal fibres form small scale clusters and large scale lamellae in the cat.

We investigated whether terminal fibres in the pontine nuclei are arranged in a lamellar pattern like that demonstrated earlier for pontocerebellar neurones. Following tracer injections in visual and parietal cortices and subsequent computer-based 3-D analysis, we found that labelled corticopontine terminal fibres form numerous sharply delimited aggregates of variable shape. Several of the aggregates are cylindroids (diameter 200-300 microns, length 1-3 mm). The aggregates are confined to a lamellar subspace, the position of which depends on the anteroposterior location of the cortical injections. These findings suggest that the cerebroponto-cerebellar system may be organized according to fairly simple, topographical rules. We discuss the implications of our results in relation to the development of corticopontine topographical organization.