@article{326, abstract = {Three-dimensional (3D) super-resolution microscopy technique structured illumination microscopy (SIM) imaging of dendritic spines along the dendrite has not been previously performed in fixed tissues, mainly due to deterioration of the stripe pattern of the excitation laser induced by light scattering and optical aberrations. To address this issue and solve these optical problems, we applied a novel clearing reagent, LUCID, to fixed brains. In SIM imaging, the penetration depth and the spatial resolution were improved in LUCID-treated slices, and 160-nm spatial resolution was obtained in a large portion of the imaging volume on a single apical dendrite. Furthermore, in a morphological analysis of spine heads of layer V pyramidal neurons (L5PNs) in the medial prefrontal cortex (mPFC) of chronic dexamethasone (Dex)-treated mice, SIM imaging revealed an altered distribution of spine forms that could not be detected by high-NA confocal imaging. Thus, super-resolution SIM imaging represents a promising high-throughput method for revealing spine morphologies in single dendrites.}, author = {Sawada, Kazuaki and Kawakami, Ryosuke and Shigemoto, Ryuichi and Nemoto, Tomomi}, journal = {European Journal of Neuroscience}, number = {9}, pages = {1033 -- 1042}, publisher = {Wiley}, title = {{Super resolution structural analysis of dendritic spines using three-dimensional structured illumination microscopy in cleared mouse brain slices}}, doi = {10.1111/ejn.13901}, volume = {47}, year = {2018}, } @article{705, abstract = {Although dopamine receptors D1 and D2 play key roles in hippocampal function, their synaptic localization within the hippocampus has not been fully elucidated. In order to understand precise functions of pre- or postsynaptic dopamine receptors (DRs), the development of protocols to differentiate pre- and postsynaptic DRs is essential. So far, most studies on determination and quantification of DRs did not discriminate between subsynaptic localization. Therefore, the aim of the study was to generate a robust workflow for the localization of DRs. This work provides the basis for future work on hippocampal DRs, in light that DRs may have different functions at pre- or postsynaptic sites. Synaptosomes from rat hippocampi isolated by a sucrose gradient protocol were prepared for super-resolution direct stochastic optical reconstruction microscopy (dSTORM) using Bassoon as a presynaptic zone and Homer1 as postsynaptic density marker. Direct labeling of primary validated antibodies against dopamine receptors D1 (D1R) and D2 (D2R) with Alexa Fluor 594 enabled unequivocal assignment of D1R and D2R to both, pre- and postsynaptic sites. D1R immunoreactivity clusters were observed within the presynaptic active zone as well as at perisynaptic sites at the edge of the presynaptic active zone. The results may be useful for the interpretation of previous studies and the design of future work on DRs in the hippocampus. Moreover, the reduction of the complexity of brain tissue by the use of synaptosomal preparations and dSTORM technology may represent a useful tool for synaptic localization of brain proteins.}, author = {Miklosi, Andras and Del Favero, Giorgia and Bulat, Tanja and Höger, Harald and Shigemoto, Ryuichi and Marko, Doris and Lubec, Gert}, journal = {Molecular Neurobiology}, number = {6}, pages = {4857 – 4869}, publisher = {Springer}, title = {{Super resolution microscopical localization of dopamine receptors 1 and 2 in rat hippocampal synaptosomes}}, doi = {10.1007/s12035-017-0688-y}, volume = {55}, year = {2018}, } @article{163, abstract = {For ultrafast fixation of biological samples to avoid artifacts, high-pressure freezing (HPF) followed by freeze substitution (FS) is preferred over chemical fixation at room temperature. After HPF, samples are maintained at low temperature during dehydration and fixation, while avoiding damaging recrystallization. This is a notoriously slow process. McDonald and Webb demonstrated, in 2011, that sample agitation during FS dramatically reduces the necessary time. Then, in 2015, we (H.G. and S.R.) introduced an agitation module into the cryochamber of an automated FS unit and demonstrated that the preparation of algae could be shortened from days to a couple of hours. We argued that variability in the processing, reproducibility, and safety issues are better addressed using automated FS units. For dissemination, we started low-cost manufacturing of agitation modules for two of the most widely used FS units, the Automatic Freeze Substitution Systems, AFS(1) and AFS2, from Leica Microsystems, using three dimensional (3D)-printing of the major components. To test them, several labs independently used the modules on a wide variety of specimens that had previously been processed by manual agitation, or without agitation. We demonstrate that automated processing with sample agitation saves time, increases flexibility with respect to sample requirements and protocols, and produces data of at least as good quality as other approaches.}, author = {Reipert, Siegfried and Goldammer, Helmuth and Richardson, Christine and Goldberg, Martin and Hawkins, Timothy and Hollergschwandtner, Elena and Kaufmann, Walter and Antreich, Sebastian and Stierhof, York}, issn = {0022-1554}, journal = {Journal of Histochemistry and Cytochemistry}, number = {12}, pages = {903--921}, publisher = {SAGE Publications}, title = {{Agitation modules: Flexible means to accelerate automated freeze substitution}}, doi = {10.1369/0022155418786698}, volume = {66}, year = {2018}, } @phdthesis{51, abstract = {Asymmetries have long been known about in the central nervous system. From gross anatomical differences, such as the presence of the parapineal organ in only one hemisphere of the developing zebrafish, to more subtle differences in activity between both hemispheres, as seen in freely roaming animals or human participants under PET and fMRI imaging analysis. The presence of asymmetries has been demonstrated to have huge behavioural implications, with their disruption often leading to the generation of neurological disorders, memory problems, changes in personality, and in an organism's health and well-being. For my Ph.D. work I aimed to tackle two important avenues of research. The first being the process of input-side dependency in the hippocampus, with the goal of finding a key gene responsible for its development (Gene X). The second project was to do with experience-induced laterality formation in the hippocampus. Specifically, how laterality in the synapse density of the CA1 stratum radiatum (s.r.) could be induced purely through environmental enrichment. Through unilateral tracer injections into the CA3, I was able to selectively measure the properties of synapses within the CA1 and investigate how they differed based upon which hemisphere the presynaptic neurone originated. Having found the existence of a previously unreported reversed (left-isomerism) i.v. mutant, through morpholocal examination of labelled terminals in the CA1 s.r., I aimed to elucidate a key gene responsible for the process of left or right determination of inputs to the CA1 s.r.. This work relates to the previous finding of input-side dependent asymmetry in the wild-type rodent, where the origin of the projecting neurone to the CA1 will determine the morphology of a synapse, to a greater degree than the hemisphere in which the projection terminates. Using left- and right-isomerism i.v. mice, in combination with whole genome sequence analysis, I highlight Ena/VASP-like (Evl) as a potential target for Gene X. In relation to this topic, I also highlight my work in the recently published paper of how knockout of PirB can lead to a lack of input-side dependency in the murine hippocampus. For the second question, I show that the environmental enrichment paradigm will lead to an asymmetry in the synapse densities in the hippocampus of mice. I also highlight that the nature of the enrichment is of less consequence than the process of enrichment itself. I demonstrate that the CA3 region will dramatically alter its projection targets, in relation to environmental stimulation, with the asymmetry in synaptic density, caused by enrichment, relying heavily on commissural fibres. I also highlight the vital importance of input-side dependent asymmetry, as a necessary component of experience-dependent laterality formation in the CA1 s.r.. However, my results suggest that it isn't the only cause, as there appears to be a CA1 dependent mechanism also at play. Upon further investigation, I highlight the significant, and highly important, finding that the changes seen in the CA1 s.r. were predominantly caused through projections from the left-CA3, with the right-CA3 having less involvement in this mechanism.}, author = {Case, Matthew J}, issn = {2663-337X}, pages = {186}, publisher = {Institute of Science and Technology Austria}, title = {{From the left to the right: A tale of asymmetries, environments, and hippocampal development}}, doi = {10.15479/AT:ISTA:th_1032}, year = {2018}, } @article{612, abstract = {Metabotropic GABAB receptors mediate slow inhibitory effects presynaptically and postsynaptically through the modulation of different effector signalling pathways. Here, we analysed the distribution of GABAB receptors using highly sensitive SDS-digested freeze-fracture replica labelling in mouse cerebellar Purkinje cells. Immunoreactivity for GABAB1 was observed on presynaptic and, more abundantly, on postsynaptic compartments, showing both scattered and clustered distribution patterns. Quantitative analysis of immunoparticles revealed a somato-dendritic gradient, with the density of immunoparticles increasing 26-fold from somata to dendritic spines. To understand the spatial relationship of GABAB receptors with two key effector ion channels, the G protein-gated inwardly rectifying K+ (GIRK/Kir3) channel and the voltage-dependent Ca2+ channel, biochemical and immunohistochemical approaches were performed. Co-immunoprecipitation analysis demonstrated that GABAB receptors co-assembled with GIRK and CaV2.1 channels in the cerebellum. Using double-labelling immunoelectron microscopic techniques, co-clustering between GABAB1 and GIRK2 was detected in dendritic spines, whereas they were mainly segregated in the dendritic shafts. In contrast, co-clustering of GABAB1 and CaV2.1 was detected in dendritic shafts but not spines. Presynaptically, although no significant co-clustering of GABAB1 and GIRK2 or CaV2.1 channels was detected, inter-cluster distance for GABAB1 and GIRK2 was significantly smaller in the active zone than in the dendritic shafts, and that for GABAB1 and CaV2.1 was significantly smaller in the active zone than in the dendritic shafts and spines. Thus, GABAB receptors are associated with GIRK and CaV2.1 channels in different subcellular compartments. These data provide a better framework for understanding the different roles played by GABAB receptors and their effector ion channels in the cerebellar network.}, author = {Luján, Rafael and Aguado, Carolina and Ciruela, Francisco and Cózar, Javier and Kleindienst, David and De La Ossa, Luis and Bettler, Bernhard and Wickman, Kevin and Watanabe, Masahiko and Shigemoto, Ryuichi and Fukazawa, Yugo}, journal = {Brain Structure and Function}, number = {3}, pages = {1565 -- 1587}, publisher = {Springer}, title = {{Differential association of GABAB receptors with their effector ion channels in Purkinje cells}}, doi = {10.1007/s00429-017-1568-y}, volume = {223}, year = {2018}, }