Figure 8.

Array tomography analysis of AMPAR and NMDAR subunits in spines of NAc core MSN after saline or cocaine self-administration and protracted withdrawal. A, Representative labeling for each epitope in MSN dendrites after saline or cocaine self-administration. B, Spine density, determined from biocytin labeling, did not differ between groups (unpaired t test, t₍₁₉₎ = 0.43). C, Average number of spots/spine was elevated for GluN3B (unpaired t test, t₍₁₉₎ = 3.20, p = 0.0047) but not for other subunits (p > 0.05). A spot is defined as a continuous collection of immunopositive pixels that colocalizes with the spine. D, Colocalization of spots (defined as spots within 0.2 µm of each other) was altered for some NMDAR subunit pairs in the cocaine group (unpaired t tests, saline vs cocaine: GluA1/GluA2, t₍₁₉₎ = 2.686, p = 0.0146; GluN2B/3B, t₍₁₉₎ = 2.137, p = 0.046; GluN3A/3B, t₍₁₉₎ = 2.283, p = 0.0340) but interestingly was not altered for GluN2B/3A (t₍₁₉₎ = 0.4658, p = 0.6467). Other subunit pairs were analyzed but are not shown (all p values > 0.05). E, As spot analysis is not sensitive to changes in spot size, we also measured volume of immunoreactive puncta for each subunit. Shown here is the average puncta volume for each subunit. Far-right bar represents the average for all antibodies (All ABS), which provides an estimate of spine volume (unpaired t tests, saline vs cocaine: GluA2, t₍₁₉₎ = 2.496, p = 0.0219; All ABS, t₍₁₀₃₎ = 2.002, p = 0.0479; for all other subunits, p > 0.05). F-J, Frequency distribution plots of puncta volume for each subunit. X² tests were used for statistical comparison of saline and cocaine relative frequency: GluA1, X²(5) = 6.22, p = 0.2859.; GluA2, X²(5) = 25.81, p = 0.000098; GluN2B, X²(4) = 7.94, p = 0.0938; GluN3A, X²(5) = 15.91, p = 0.0071; GluN3B, X²(5) = 28.24, p = 0.000033. *p < 0.05. **p < 0.01. ****p < 0.0001. Saline group (>WD38): 3 rats, 4 cells, 12 dendrites, 825 spines. Cocaine group (WD46-WD47): 2 rats, 4 cells, 9 dendrites, 965 spines.