Structural annotation and literature comparison for the ligand-gated ion channel family

Structural annotation of ligand-gated ion channels in different brain regions and in discrete cortical layers

To assess the brain distribution of the ligand-gated ion channel family (as defined by the gene ontology of the Mouse Genome Informatics (MGI) web site an informatics approach was utilized to determine overall expression. Out of 67 total ligand-gated ion channel genes, data for 65 are present in the ABA database. The brain was parsed into 12 large anatomically distinct regions, and algorithms for expression level and relative density were used to determine relative distribution of each gene (see Supplemental document 4: Informatics Data Processing for precise variable calculations). Informatics values are represented by a color-coded score (Large expression tables are found in “Supplementary document 10b: Ion Channel Annotation”). The thresholds for each color bin were determined by analyzing the interquartile distribution across each structure. The lower quartile value defines the boundary between gray (no expression) and blue (very low expression). The median value defines the boundary between green (low-moderate) and yellow (moderate-high) categories. The boundary values for blue/green and yellow/red (high expression) were derived by interpolating the lower quartile and upper quartile with respect to the median. Informatics values were confirmed by a manual QC of all genes in all 12 structures examined, and discrepancies noted due to artifacts and tissue quality issues were adjusted accordingly. Of the 65 genes analyzed, 60/65 (92%) had detectable cellular expression in the brain. These included 2 well-described peripheral acetylcholine receptors where no previous brain expression has been reported Chrna91 and Chrnd2. The vast majority of the genes had a distinct combinatorial pattern of expression, with many genes being confined to a subset of brain regions, and most of the rest having differential levels or density of expression across the 12 structures. The major exceptions to this were in the glutamate sub-families, where several AMPA (Gria) and Kainate (Grik) receptor genes were highly expressed throughout the brain. Surprisingly, only one gene (Chrne) was confined to a single structure, with this gene showing light yet distinct expression in the septal region of the striatum. Overall, the complex distribution patterns and robust expression of the ligand-gated ion channel family underscores the importance of this gene family in the control of brain function.

To further define the distribution of the ligand-gated ion channels, detailed analysis oflaminar cortical expression was performed manually. Layers 1-6b (as delineated by the Allen Reference Atlas) were scored for both density and intensity of expression for each gene in each layer. Density values ranged from occasional cells (sparse-blue) to greater than 80% of cells in a layer with detectable signal (high-red). Intensity values ranged from very low punctuate precipitate per cell (lowest-blue) to very high precipitate levels per cell (red). Scoring of expression level was facilitated by the color-coded expression masks generated as part of the informatics signal quantification (“Supplemental document 4: Informatics Data Processing”). These masks provide a standardized means of visualizing the average pixel intensity per cell, and thereby scoring the mean intensity of labeled cells across a population of cells, and are available for side by side viewing on the online viewer application.

These data demonstrate that each cortical layer has a specific profile of ion channel expression. Out of 65 genes, 54/65 had detectable expression in the cortex. With few exceptions, expression was detected in all layers, with variance of expression seen between layers for each gene. Among the different subclasses, most of the layer to layer variance in expression level seems to occur within the glutamatergic ion channel family. However. scoring of density indicates that there is significant diversity within each layer, and that many channels are expressed in subpopulations of cortical neurons. In some cases, the expression density can predict cell class. For example, Htr3a is expressed at high levels with low density in what are very likely to be a subpopulation of GABAergic inhibitory interneurons. Channels expressed at high density must at least include expression in the predominant excitatory neuronal population (e.g. pyramidal neurons forlayers 2/3, 5 and 6).

Comparison of ligand-gated ion channel expression patterns between ABA and other data sources

As a control for accurate expression, ABAin situhybridization data for the ligand-gated ion channels was compared to both the published literature and to the Brain Gene Expression Map (BGEM) database ( Wherever possible, comparisons were made to published literature describing distribution in adult mouse brain using in situ hybridization techniques. However, in some cases, other methodologies such as immunohistochemistry, RT-PCR, Northern blots or microarray data were used for comparisonif ISH data was not available. Additionally, when adult mouse data was not available, juvenile mouse or other species data such as rat or primate was used for comparison analysis. Despite methodological and species differences, 60/65 genes examined showed broad general agreement for brain distribution between ABA data and the published literature (Table 1). In 3 of the 5 cases where major disagreement existed, the primary reason may be one of sensitivity, as in these cases either ABA showed no expression while published paper did, or vice versa. In one case (P2rx5), ABA analysis revealed widespread expression compared to regional expression in a published paper using rat data 3 and in another case (Glra3) regional patterns seen were fundamentally different between ABA data and published data4.

To further our comparison of ABA gene expression for ligand-gated ion channels, we compared our data to the BGEM data set. 25 of 67 ligand-gated ion channel genes were found on the BGEM web site for adult mouse data sets with images. Of the genes in the BGEM set, all 25 exhibited broad agreement with the ABA data set (Table 1). While there were some subtle differences in apparent expression between the 2 data sets, overall the excellent correspondence between BGEM and ABA represents the most valid control of experimental accuracy, given that both methodologies use in situ hybridization in the same species and strain (C57BL/6).

GENE / ABA / Literature / BGEM
Acetylcholine / Chrna1 / Widespread / Widespread5 / No Data
Chrna2 / Widespread / Widespread6 / No Data
Chrna3 / Regional / Regional6 / Regional
Chrna4 / Regional / Regional7 / Regional
Chrna5 / Regional / Regional8 / Regional
Chrna6 / Regional / Regional9 / Regional
Chrna7 / Regional / Regional10 / Regional
Chrna9 / Non Detectable / Non Detectable1 / No Data
Chrna10 / No Data / No Data / No Data
Chrnb1 / Non Detectable / Non Detectable11 / No Data
Chrnb2 / Widespread / Widespread6 / Widespread
Chrnb3 / Regional / Regional12 / Regional
Chrnb4 / Regional / Regional13 / Regional
Chrnd / Non Detectable / Non Detectable2 / No Data
Chrne / Regional / Non Detectable2 / No Data
Chrng / Widespread / Widespread14 / No Data
Gaba / Gabra1 / Widespread / Widespread15 / Widespread
Gabra2 / Regional / Regional16 / No Data
Gabra3 / Widespread / Widespread16 / No Data
Gabra4 / Regional / Regional17 / No Data
Gabra5 / Regional / Regional17 / No Data
Gabra6 / Regional / Regional17 / Regional
Gabrb1 / Widespread / Widespread18 / No Data
Gabrb2 / Widespread / Widespread19 / Widespread
Gabrb3 / Widespread / Widespread20 / No Data
Gabrd / Regional / Regional21 / No Data
Gabre / Regional / Regional22 / No Data
Gabrg1 / Regional / Regional23 / Regional
Gabrg2 / Widespread / Widespread21 / Widespread
Gabrg3 / Widespread / Widespread24 / No Data
Gabrp / Non Detectable / Non Detectable25 / No Data
Gabrq / Regional / Regional26 / No Data
Gabrr1 / Regional / Regional27 / No Data
Gabrr2 / Widespread / Widespread27 / Widespread
Gabrr3 / No Data / No Data / No Data
Glycine / Glra1 / Regional / Regional28 / No Data
Glra2 / Widespread / Widespread28 / No Data
Glra3 / Regional / Regional4, 28 / No Data
Glra4 / Regional / Regional29 / No Data
Glrb / Widespread / Widespread30 / Widespread
Glutamate / Gria1 / Regional / Regional31 / No Images
Gria2 / Widespread / Widespread31 / Widespread
Gria3 / Widespread / Widespread32 / No Data
Gria4 / Regional / Regional33 / No Data
Grid1 / Widespread / Widespread34 / Widespread
Grid2 / Widespread / Widespread35 / No Data
Grik1 / Widespread / Widespread36 / No Images
Grik2 / Widespread / Widespread37 / No Data
Grik3 / Regional / Regional38 / No Data
Grik4 / Regional / Regional39 / Regional
Grik5 / Widespread / Widespread37 / No Data
Grin1 / Widespread / Widespread40 / Widespread
Grin2a / Widespread / Widespread40 / Widespread
Grin2b / Widespread / Widespread40 / Widespread
Grin2c / Regional / Regional41 / Regional
Grin2d / Widespread / Widespread40 / No Data
Grin3a / Regional / Regional42 / No Data
Grin3b / Regional / Regional43 / Regional
Htr / Htr3a / Regional / Regional44 / Regional
Htr3b / Non Detectable / Non Detectable45 / No Data
Purinergic / P2rx1 / Regional / Regional46 / No Data
P2rx2 / Regional / Regional47 / No Data
P2rx3 / Non Detectable / Regional48 / No Data
P2rx4 / Widespread / Widespread47 / Widespread
P2rx5 / Widespread / Regional47 / No Data
P2rx7 / Regional / Regional49 / No Data
P2rxl1 / Regional / Regional50 / No Data

Table 1: Comparison of ligand-gated ion channel expression patterns between the Allen Brain Atlas, BGEM and the published literature. Expression was scored as Widespread (Green), Regional (Yellow), or Non Detectable (Light blue). Expression patterns that do not match ABA data are shown in red.
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