Figure 2. Cholinergic interneurons identified in the NAc-S contain a preferential distribution of DORs in the plasma membrane of the somatic region. A–D, Cellular physiological characteristics of a CIN identified in the NAc-S of DOR-eGFPki mice: a sag Ih-mediated I–V (A), a broad action potential (B), and a lack of plateau low-threshold spiking (C, D). E, Single-plane analysis of DOR distribution in soma, proximal, and distal dendrites of the neuron in A–D after biocytin injection and confocal reconstruction. Note how the membrane DOR is clearly distributed in the somatic region and how the signal is gradually reduced from the soma to distal dendrites (right). F, G, Quantification of eGFP fluorescence contained in the somatic membrane of CINs in DOR-eGFPki mice. F, In individual confocal images, mean gray value line profiles were plotted from 10 to 20 segments distributed along the somatic area of the ChAT image (visible channel). Segments were 2 μm long, comprised a continuous line of 35 pixels, and were placed perpendicular to the edge and with the center (red) located in the intracellular–extracellular interphase defined by the ChAT staining. Values for each segment were then collected from the overlapped eGFP/A488/FITC image (masked channel) and plotted individually (gray traces, right). The mean trace is indicated in black. Ext, Extracellular; M, membrane; Int, intracellular. G, A second type of analysis was performed on raw 12-bit images, where two different regions of interest were defined in the ChAT image (visible channel) of each neuron: ROI 1 (red) comprised the somatic region (located in the intracellular–extracellular interphase defined by the ChAT staining), whereas ROI 2 (yellow) was used as a background correction and comprised the nuclear region (as defined by the central region devoid of ChAT staining). The mean gray value for each ROI was then collected from the overlapped eGFP/A488/FITC image and expressed, for each neuron, as ROI 1–ROI 2 (right). This straightforward method allowed large-scale quantification of individual neurons (a total of 3118 CINs were quantified with this method in the present study).