Figure 1.

[³⁵S]GTPγS autoradiography of rat brain sections reveals LPA-mimicking and NEM-sensitive G protein activation in response to stimulation with c48/80. (a) [³⁵S]GTPγS autoradiography was conducted using a three-step protocol with DPCPX (1 μM) present throughout steps 2 and 3, as detailed in the Methods section. c48/80 (100 μg ml⁻¹) from two suppliers (MP Biomedicals and Sigma-Aldrich) or LPA (50 μM in 0.1% fatty acid-free BSA) was present during the [³⁵S]GTPγS labeling in step 3. Some sections were treated with the irreversible G_i/o protein inhibitor NEM (1 mM, included in step 1 of the protocol). In the control panel (left), the white matter anatomical loci where LPA typically activates G proteins are indicated. Note the highly restricted and LPA-mimicking distribution of c48/80-stimulated G protein activity throughout the white matter tracts, including the anterior commissure (aca), the corpus callosum (cca) and the fimbria of the hippocampus (fi). Other abbreviations: ctx, cerebral cortex; hip, hippocampal structures; str, striatum. Scale bar=5 mm. (b) Quantitative autoradiography data on selected brain regions and on whole brain for NEM-pretreated sections (inset). Autoradiography images were digitized and bound radioactivity values were obtained from the digitized images for two white matter regions (corpus callosum (cca) and the fimbria of the hippocampus (fi)), two gray matter regions (cerebral cortex (ctx) and hippocampus (hip)), an area containing both white and gray matter (striatum (str)) and for whole section area, as detailed in the Methods section. Values are mean+s.e.m. representing sections from four individual animals. Risk level: ^*P<0.05 compared to basal in each specified brain region and ^#P<0.05 compared to NEM-pretreated basal on whole section radioactivity (one-way ANOVA with Tukey's multiple comparison).