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The dopaminergic system undergoes major reorganization during development, a period especially vulnerable to mental disorders. Forebrain neurons expressing dopamine 1 and 2 receptors (D1R and D2R, respectively) play a key role in this system. However, neuroanatomical information about the typical development of these neurons is sparse and scattered across publications investigating one or a few brain regions. We here present a public online collection of microscopic images of immunohistochemically stained serial sections from male and female mice at five stages of development (postnatal day 17 (P17), P25, P35, P49, and adult), showing the distribution of D1R and D2R expressing neurons across the forebrain. All images from adult brains are registered to the Allen Mouse brain Common Coordinate Framework, while images from P17-P35 age groups are registered to spatially modified atlas versions matching the morphology of young brains. This online resource provides microscopic visualization of the developing dopaminergic system in mice, which is suitable as a benchmark reference for performing new experiments and building computational models of the brain.

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T cell-specific adapter protein (TSAd) encoded by the SH2D2A gene is expressed in activated T cells, NK cells and endothelial cells, but its tissue expression has not yet been mapped. Here, we have defined the specificity of two commercially available anti-TSAd monoclonal reagents using peptide arrays. We found them to bind separate epitopes in the C-terminal part of TSAd. We then used immunohistochemistry to examine TSAd expression in various human lymphoid and non-lymphoid tissues. Immunostaining of adjacent tissue sections revealed that a substantial fraction of CD3-positive cells in normal lymphoid and non-lymphoid tissues expressed TSAd. In particular, essentially all intra-epithelial T cells appeared to coexpress TSAd. In addition, TSAd expression was observed in endothelial cells of dermal microvessels, while it was not detected in endothelial cells of the other tested tissues. This work provides insight into the expression pattern of TSAd in various healthy human tissues.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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We have explored basic rules guiding the early development of topographically organized projections, employing the rat corticopontine projection as a model system. Using anterograde in vivo tracing with 1,1',dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), we studied the distribution of labelled fibers in the pontine nuclei in relation to cortical site of origin during the first postnatal week. Labelled corticopontine fibers enter the pontine nuclei in distinct, sharply defined zones. The putative terminal fibers typically occupy lamella-like subspaces. Related to changes in cortical site of origin, we describe mediolateral, internal to external, and caudorostral distribution gradients in the pontine nuclei. Fibers originating in the anterolateral cortex occupy an internal central core, while implantations at increasing distance from the anterolateral cortex produce 1) more externally located lamellae, and 2) a caudal to rostral shift in fiber location. Previous investigations have shown that pontocerebellar neurons migrate into the ventral pons in a temporal sequence (Altman and Bayer [1987] J. Comp. Neurol. 257:529). The earliest arriving neurons occupy the central core and later arriving neurons settle in more externally and rostrally located subspaces. We hypothesize that the earliest arriving corticopontine fibers grow into the then only available zone of pontocerebellar neurons (central core), attracted by a diffusible chemotropic cue. Later arriving fibers grow into correspondingly later and more externally and rostrally located contingents of pontocerebellar neurons. Thus, we propose that the topographical organization in the early postnatal corticopontine projection is determined by simple temporal and spatial gradients operative within source (cerebral cortex) and target region (pontine nuclei).

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We present a computer programme, MicroTrace, designed for user-guided digitisation of objects in biological sections. The programme is optimised for recording the spatial distribution of neuronal structures, such as large populations of tracer-labelled cell bodies or axonal plexuses, regional borders, and surfaces. System requirements are a PC running Microsoft Windows, a microscope equipped with stepping motors, and a drawing tube. A computer generated drawing area, surrounded by menus and icons, is projected into the microscope field of view via the drawing tube. Different 'object' icons are assigned to individual object categories (cell types, surfaces, etc.). Digitisation is performed by pointing the cursor at objects in the section. Computer graphical symbols are superimposed on the digitised objects. All object categories are digitised, before moving the stage to other fields of view by manipulating the joystick or scroll bars. Movement of the microscope stage is accompanied by a translation of the graphical image, so that continuous feedback on the progress of the digitisation is provided. MicroTrace can readily be adapted to the specific needs of the user. We show its use in different experimental neuroanatomical techniques. Two-dimensional images and three-dimensional reconstructions of neuronal distribution and surfaces are demonstrated.

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