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

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 (a) GABA_AR α1, (b) GABA_AR α2, or (c) 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 Materials and methods for details. (mean ± SD; n = 16–31 images from N = 3 to 4 independent experiments). Underlying data can be found in S2 Data. DIV, days in vitro; GABA, γ-aminobutyric acid; MPP2, membrane protein palmitoylated 2.