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Fig. 4. Effects of DA-GFP on the density of GABA and glutamate receptors and synaptic proteins. Transfected neurons were recorded in whole-cell configuration at –60 mV for GABA_A- or AMPA-generated responses and –20 mV for NMDA-mediated current in the presence of TTX and strychnine. (A) Example of current responses induced by bath application of receptor agonists, Isoguvacine (20 µM), AMPA (100 µM) and NMDA (50 µM, with 10 µM glycine, NMDA coactivator) to activate GABA_A, AMPA and NMDA receptors, respectively, 10 seconds after starting perfusion (double arrows) with agonist-containing solution. (B) Quantitative analysis revealed that DA overexpression did not modify significantly the average current density of the agonist-mediated responses compared with that of GFP neurons. (C) Mixed hippocampal cultures were transfected at 21 DIV with GFP (C_1,E₁ and insets C_1',E_1') or DA-GFP (D₁,F₁ and insets D_1',F_1'), and 1 day after transfection, cells were double immunostained for synaptophysin (C₂,D₂) and the subunit β2,3 of GABA_A receptors (C₄,D₄) or for Gad-65 (E₂,F₂) and vGlut1 (E₄,F₄). All the synaptic markers were located either on the dendritic shafts or on dendritic protrusions. Interestingly, we observed in DA neurons that several dendritic spines share the same presynaptic terminal (see arrowheads in D_3' vs C_3' and F_3' vs E_3'). Scale bars: 10 µm in C₁-F₆ and 5 µm in insets in right column. (G) The average density of syn, β2,3, syn + β2,3, Gad-65 and vGlut1 clusters along dendrites of DA-GFP were not different from those of GFP neurons. Note that in addition to dendrites of transfected neurons, all synaptic markers also stained dendrites of nontransfected neurons.

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