Completeness of Data. The Practice shall ensure that:
Completeness of Data. These datasets exceed in scope and size the datasets we had anticipated producing, in that we captured more cell types and subtypes using the shotgun capture approach than we could have obtained with a more targeted approach. In addition to the mouse CA1 transcriptomes reported in Zeisel et al. (see below), there are a couple of ongoing efforts to produce single-cell transcriptomes in hippocampus. As far as we know, this is the only dataset reporting full-length whole-cell single-cell transcriptomes in mouse hippocampus. We are also not aware of efforts to produce such data in cerebellum, making this particularly interesting. Using this approach we detect more genes expressed in a cell of any given type than Zeisel et al. There are two especially relevant datasets generated by the community over the past 30 months. The first, from HBP collaborator Xxxx XXXXXXXXXX, has been discussed in detail in previous reports and is published in Zeisel et al. (Science, 2015). Briefly, this dataset represents shotgun captured cells with sequenced tags to count transcripts from juvenile mouse somatosensory cortex and CA1. The second, from the Xxxxx Institute, has been published in Tasic et al. (2016) and describes single cells (especially neurons) targeted in young adult mouse primary visual cortex. These are excellent cortical complements to the data generated here in whole hippocampus and cerebellum.
Completeness of Data. Four cell-types specific synaptic proteomes have been isolated and analysed, which exceeds the anticipated dataset of one. These datasets are the first known cell type specific synapse proteomes. There have been no other reports of cell type specific synapse proteomes, primarily because the state-of-the-art transgenic technology is the only known way to obtain this data from intact mouse brain. Various indirect approaches involving ribosome tagging have been developed but these do not measure synaptic proteins directly. The technology is now poised to be exploited for comprehensive studies across an expanded number of cell types.
Completeness of Data. Dataset generated in T1.2.4 (neocortex) could be considered as the final dataset.
Completeness of Data. Although not specifically stated as one or “data production” goals, it must be pointed out a large part of the effort by our group during the Ramp-Up Phase has been invested to testing and developing methods and equipment for efficient and reliable labelling and 3D reconstruction of individual long-range projection neurons across large tissue volumes, potentially the entire brain. Developing these methodologies and workflows is crucial for the future phases of the project. In this regard, we have succeeded in establishing two new protocols for single-cell labelling with high spatial precision based on the electroporation of RNA Sidbis or DNA AAv vectors. A paper reporting the RNA protocol is currently under review in Frontiers in Neuronatomy. In addition, we have labelled a total of 20 (out of 30 planned for M30) cells, of which 11 were found to be of sufficient quality and appropriate location for full 3D reconstructions. During the M1-M30 period a paper has been published (Nakamura et al., 2015) by the laboratory of Xxxxxxx XXXXXX (Kyoto University) reporting the single cell architecture of thalamocortical projection cells of the lateral posterior complex of rats also using the Sindbis vector. We collaborate with Xxxx. XXXXXX’x group, and originally obtained from them the vector. Although, because of the species and size difference, the quantitative data from Xxxxxxx-Xxxxxx rats cannot be used for the HBP atlas and modelling, the study findings validate our approach and provide a framework for reference and interspecies comparison.
Completeness of Data. The brain region synapse protome data collection is completed for the seven regions and a manuscript is in preparation. The synaptome mapping has produced the first whole-brain scale synaptome maps in the mouse and these maps are completed. A manuscript is in preparation. There have been no mouse synapse proteome maps produced in the community to our knowledge.
Completeness of Data. The dataset anticipated at the beginning of the project falls a bit short with the dataset delivered by M30 in regards to the use of the immunogold FIB/SEM technique. We could only generate around 80% of the data anticipated using such technique due to the lack of specific software that allows generation of fast 3D reconstruction of long series of serial sections and particularly the quantitative analysis in three dimensions. Regarding the SDS- FRL technique the anticipated dataset fits exactly with the dataset delivered by M30. However, we also generated 2D mapping of GABAB receptors, GIRK, SK and Cav2.1 in Purkinje cells of the cerebellum, not included in the original grant.
Completeness of Data. The algorithm for automatic segmentation is in a very advanced stage of development. Results have been already obtained and validated and most of the problems identified have been corrected and validated. Six datasets have been generated (including data from every cortical layer in each case), detailing the cell density and distribution of GABAergic interneurons in the somatosensory cortex. Quantitative data on the number and 3D distribution of GABAergic cells have been extracted from the segmentation. The two-steps binarisation part of the algorithm has been completed and the algorithm can combine information of different channels (Figure 6). 3D data have been used by WP5.4 /T5.4.2 (Neuronal Structural Design and Predictions) by spatial statistical techniques to characterise the density and principle patterns of spatial distribution of GABAergic interneurons (currently ongoing). These data are essential to build the models of the cortical column.
Completeness of Data. We have obtained 3D confocal digital reconstructions of 60 dendrites, including main apical, colateral and basal dendrites from 20 individual identified pyramidal neurons in the hippocampus (n=10) and somatosensory cortex (n=10) together with the distribution of PSD95 puncta in the same stacks of images. We are currently generating the synaptic maps of the 60 dendrites by co-registration of both channels (Alexa and GFP). Unfortunately, we have detected a problem with the data generated with the confocal microscope. The images that were obtained during the last 12 months show a distortion in the z-axis. Image processing to reconstruct dendritic spines is very time consuming because there are not automatic software tools to obtain an accurate 3D reconstruction of the dendritic spine. Thus, the reconstruction of these structures is mainly based on manual tracing using informatic tools. Therefore, it will take longer time than expected to generate the final maps because we have to acquire again the 3D confocal digital reconstructions of 46 dendrites and re-analyse the previous data. In addition to the goal of the generation of synaptic maps on identified neurons, we have developed a new tool called PyramidalExplorer (Toharia et al., 2016) to interactively explore and reveal the detailed organisation of the microanatomy of pyramidal neurons with functionally related models (Figure 9). This tool consists of a set of functionalities that allow possible regional differences in the pyramidal cell architecture to be interactively discovered by combining quantitative morphological information about the structure of the cell with implemented functional models. We are planning to use this tool to analyse the synaptic maps to discover new aspects of the morpho-functional organization of the pyramidal neuron. Figure 9: Pyramidal Cell Explorer Graphical user interface (GUI) of PyramidalExplorer showing the cell comparison query result concerning Spine Area, from a pyramidal neuron in a global view (A) and in a zoom in view (B). Values are represented by colour code. Red colours represent highest values whereas blue colours represent the lowest values. White frame in the main window highlights the spine visualized in the Detail viewer widget. Taken from Toharia et al. (Front Neuroanat. 9:159, 2016) As far as we know, this is the only dataset reporting large-scale synaptic maps of individual neurons from intact mouse brain. The technology is now available to be exploited fo...
Completeness of Data. 3.9.1.4.1 High resolution optical synaptic maps We have obtained 10 optical synaptic maps, including all hippocampal regions and all layers of the somatosensory.
