Workflow and Atlas System for Brain-Wide Mapping of Axonal Connectivity in Rat

Detailed knowledge about the anatomical organization of axonal connections is important for understanding normal functions of brain systems and disease-related dysfunctions. Such connectivity data are typically generated in neuroanatomical tract-tracing experiments in which specific axonal connections are visualized in histological sections. Since journal publications typically only accommodate restricted data descriptions and example images, literature search is a cumbersome way to retrieve overviews of brain connectivity. To explore more efficient ways of mapping, analyzing, and sharing detailed axonal connectivity data from the rodent brain, we have implemented a workflow for data production and developed an atlas system tailored for online presentation of axonal tracing data. The system is available online through the Rodent Brain WorkBench (www.rbwb.org; Whole Brain Connectivity Atlas) and holds experimental metadata and high-resolution images of histological sections from experiments in which axonal tracers were injected in the primary somatosensory cortex. We here present the workflow and the data system, and exemplify how the online image repository can be used to map different aspects of the brain-wide connectivity of the rat primary somatosensory cortex, including not only presence of connections but also morphology, densities, and spatial organization. The accuracy of the approach is validated by comparing results generated with our system with findings reported in previous publications. The present study is a contribution to a systematic mapping of rodent brain connections and represents a starting point for further large-scale mapping efforts

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. J. Comp. Neurol. 521: 3042–3056, 2013. © 2013 Wiley Periodicals, Inc

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

Altered diffusion tensor imaging measurements in aged transgenic Huntington disease rats

Rodent models of Huntington disease (HD) are valuable tools for investigating HD pathophysiology and evaluating new therapeutic approaches. Non-invasive characterization of HD-related phenotype changes is important for monitoring progression of pathological processes and possible effects of interventions. The first transgenic rat model for HD exhibits progressive late-onset affective, cognitive, and motor impairments, as well as neuropathological features reflecting observations from HD patients. In this report, we contribute to the anatomical phenotyping of this model by comparing high-resolution ex vivo DTI measurements obtained in aged transgenic HD rats and wild-type controls. By region of interest analysis supplemented by voxel-based statistics, we find little evidence of atrophy in basal ganglia regions, but demonstrate altered DTI measurements in the dorsal and ventral striatum, globus pallidus, entopeduncular nucleus, substantia nigra, and hippocampus. These changes are largely compatible with DTI findings in preclinical and clinical HD patients. We confirm earlier reports that HD rats express a moderate neuropathological phenotype, and provide evidence of altered DTI measures in specific HD-related brain regions, in the absence of pronounced morphometric changes

Wistar rat brain fibre orientation model

The 3D fibre orientation model of a male Wistar rat brain was derived from 3D-PLI as described in Axer et al. 2011a [1]. The brain was immersion fixed in 4% paraformaldehyde. After cryoprotection (10% glycerin for 3 days, followed by 20% glycerin for 14 days at +4°C), the brain was deep frozen at -50°C and stored till further processing. The brain was serially sectioned in the coronal plane (section thickness 60 μm) using a large-scale cryostat microtome (Polycut CM 3500, Leica, Germany) and coverslipped with glycerin. Immediately after coverslipping, the sections were measured using the large-area polarimeter (LAP, pixel size: 64 μm x 64 μm, cf. [1]). During sectioning, each blockface was digitized using a CCD camera mounted above the brain in order to obtain an undistorted reference image of each section. Spatial resolution in the z-direction was 60 μm. No staining was applied. This procedure resulted in an uninterrupted series of 446 sections through the entire brain, which ultimately enabled the 3D reconstruction. The application of the Jones calculus [2] describes the light transmittance through the LAP and enables the calculation of the individual spatial fiber orientation in each voxel (defined by pixel size and section thickness). The fiber orientation is defined by the pair of angles (α, φ) = (inclination, direction) indicating the fiber axis orientation out of and within the section plane, respectively. Inclination and direction angles are encoded in RGB or HSV color space to provide one fiber orientation map (FOM) per section. The entire data set of aligned FOMs (i.e. the fibre orientation model) is assembled in a single NIfTI file (http://nifti.nimh.nih.gov). FOMs are the fundamental data structure provided by 3D-PLI and have an in-plane resolution of 64 μm×64 μm, and, since each section was 60 μm thick, a spatial resolution in the z-direction of 60 μm. They contain a single 3D fiber orientation vector per voxel that is interpreted as the spatial orientation of the fibers in this voxel. Non-linear deformations introduced by brain sectioning and mounting were corrected using blockface images as undistorted references for the spatial alignment of 3D-PLI FOMs. Hence, in a first step the blockface images had to be 3D reconstructed. The reconstruction method consisted of a two-phase registration: a marker-based alignment of the blockface images and a refinement of the pre-reconstructed volume using 3D information [3]. The 3D reconstruction of the FOMs was done in two steps: (i) a 3D affine registration ensured the correct spatial alignment of the brains and (ii) a subsequent 3D non-linear registration compensated non-linear distortions of the brain sections. Using segmented images the centers of gravity of the corresponding brain masks were calculated and aligned. Based on this initial transformation, an intensity based rigid registration was performed using mutual information as metric. The second step, the refinement, was done by means of a slice-by-slice B-Spline registration with sum of squared differences as metric and a grid size of 5 × 6 [4]. Afterwards the fibre orientation model was transferred into the common rodent reference space, the Waxholm Space atlas [5]. The transformation of the brains into the same space was also done in the two step strategy described above. **References** [1] Axer, M., Amunts, K., Gräßel, D., Palm, C., Dammers, J., Axer, H., et al. (2011a). A novel approach to the human connectome: ultra-high resolution mapping of fiber tracts in the brain. NeuroImage 54, 1091–1101. doi: 10.1016/j.neuroimage.2010.08.075 [2] Jones, RC. (1941) A new calculus for the treatment of optical systems. J. Opt. Soc. Am. 31, 488–503. doi:10.1364/JOSA.31.000488 [3] Schober, M., Schlömer, P., Cremer, M., Mohlberg, H., Huynh, A.-M., Schubert, N., et al. (2015). “Reference volume generation for subsequent 3D reconstruction of histological sections,” in Proceedings of Bildverarbeitung für die Medizin, (Lübeck), 143–148. [4] Schubert, N., Kirlangic, M. E., Schober, M., Huynh, A.-M., Amunts, K., Zilles, K., et al. (2016). 3D Reconstructed Cyto-, Muscarinic M2 Receptor, and Fiber Architecture of the Rat Brain Registered to the Waxholm Space Atlas. Frontiers Neuroanatomy 10, 1-13. doi: 10.3389/fnana.2016.00051 [5] Papp, E. A., Leergaard, T. B., Calabrese, E., Johnson, G. A., and Bjaalie, J. G. (2014). Waxholm space atlas of the Spraque Dawley rat brain. NeuroImage 97, 374–386. doi: 10.1016/j.neuroimage.2014.04.00