GABA_AR subunits colocalise with MPP2 in a subset of dendritic spines.

<p>Primary E18 rat hippocampal neurons were fixed at DIV21 and immunostained for the endogenous proteins MAP2 (microtubule-associated protein 2, grey), MPP2 (cyan), and Homer1 (yellow) together with either <b>(a)</b> GABA_AR α1, <b>(b)</b> GABA_AR α2, or <b>(c)</b> GABA_AR β2/3 (all magenta) using respective primary and Alexa fluorophore-coupled secondary antibodies, and visualised by confocal microscopy. (i) Maximum projected overview of primary to secondary dendrite branches. Box indicates location of detail image in ii. Scale bar: 10 μm. (ii) Maximum projection composite of 4-colour confocal immunofluorescence image of a primary to secondary dendrite branch point of a mature (DIV21) hippocampal neuron. MPP2 is located in the majority of dendritic spines marked with Homer1, while GABA_AR subunits colocalise with a subset of these spines. Additionally, solely GABA_AR α1-, α2- or β2/3- positive puncta likely represent inhibitory synapses at the dendrite. Box indicates location of detail image in iii. Scale bar: 5 μm. (iii) Enlarged single plane image with corresponding orthogonal views of a dendritic spine exhibiting immunofluorescence staining for Homer1, MPP2, and GABA_AR α1, α2 or β2/3, respectively. Scale bar: 1 μm. (iv) Quantification of the fraction of excitatory synapses marked by Homer1, which also show MPP2 expression together with GABA_AR α1, α2, or β2/3 immunofluorescence. Please see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3001503#sec010" target="_blank">Materials and methods</a> for details. (mean ± SD; <i>n</i> = 16–31 images from <i>N</i> = 3 to 4 independent experiments). Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3001503#pbio.3001503.s016" target="_blank">S2 Data</a>. DIV, days in vitro; GABA, γ-aminobutyric acid; MPP2, membrane protein palmitoylated 2.</p>