Image analysis for quantification of GluA1 intensity

All fluorescently labeled brain sections were examined under an LSM700 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany) with diode lasers at 488 and 555 nm. Images were obtained at a resolution of 1,024 × 1,024 pixels per x-y plane, and stacks spaced by 0.5 µm along the z-axis were acquired with 1 Airy unit of optical thickness using a 40× (or 63×) oil immersion objective (numerical aperture 1.4) depending on the purpose of the experiment. Scan averaging was set to 4 to improve the signal-to-noise ratio of each optical section. All images were acquired with identical settings for laser intensity, photomultiplier voltages, and scan speed.

On each section, one or two square regions from the core and shell, respectively, were randomly selected. The GluA1 intensity for a selected single square region (101.52 × 101.52 µm), which was obtained with raw image stacks, was quantified using ZEN image analysis software (Carl Zeiss). First, the brightest optical image stack along the z-axis was designated as the reference image. Then, the analysis was performed with three consecutive images, including the reference itself and two adjacent images (above and below, spaced 0.5 µm apart). The mean value of the GluA1 intensity readings obtained from these three images was used as a value representing the GluA1 intensity for the selected single region. This procedure normalizes the variability of GluA1 intensity due to different antibody penetration and light scattering effects along the depth of the tissue. In order to distinguish GluA1 expression in PSD from non-PSD areas, the intensity of co-localized PSD95 was also analyzed. Considering the spreading effect of fluorescence and the relatively small size of PSD (mean diameter of approximately 300–400 nm) [25], we defined the PSD area as displaying a PSD95 intensity at least two times higher than the mean intensity of total PSD95, and the rest of the area (with a lower PSD95 intensity) as being extra-PSD.

The mean values of the GluA1 intensity from selected regions per slice were averaged for each experimental group and compared for PSD vs. extra-PSD areas, the core vs. shell of the NAcc, and saline vs. cocaine treatment groups, respectively.

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