Figure 3

Molecular identification of glutamatergic synapses in dendrites of the SNc dopamine neurons.

(a) Double immunostaining with antibodies specific for TH and PSD-95 in very gently dissociated dopamine neurons. Top, TH-positive (green), PSD-95-labeled (red) and overlapped images of a dopamine neuron (merge). Second to fourth lanes, expanded fluorescence images from a representative dendritic region. Bottom, images showing a dendritic shaft and PSD-95 synaptic sites. Arrowheads indicate PSD-95 fluorescence puncta in spines (blue) and shafts (yellow). Right, high-magnification images of the dendritic segment marked by a white box (left) show PSD-95 puncta in both dendritic spines and shafts. (b) PSD-95 density in the dendritic shaft, calculated from 12 dendrites in 10 cells. Error bar indicates SEM. Right, the spine density was redrawn from Fig. 2b,c for comparison. (c) Densities of PSD-95 puncta in the proximal dendritic shafts (<80 μm from the soma; n = 12) and distal dendritic shafts (>80 μm from the soma, n = 12). Error bars indicate SEM. (d) Distribution of AMPA receptors and NMDA receptors in the dendritic spines and shafts of SNc dopamine neurons. Double immunostaining of dissociated dopamine neurons with TH and either GluR1 for AMPAR or GluN1 for NMDAR. TH (green), GluR1 or GluN1 (red) and their overlapped images (merge) are presented. Arrowheads indicate GluR1 and GluN1 fluorescence puncta in spines (blue) and dendritic shafts (yellow). Dendritic regions containing a single spine marked by the white boxes are presented on the right. (e) GluR1 puncta (from 27 dendrites of 18 cells) and GluN1 puncta (from 17 dendrites of 10 cells) measured only in the dendritic shaft. *p < 0.05 by unpaired t-tests. Error bars indicate SEM.