Effects of CIS on spiny BLA neurons. (A) Low-power photomicrograph of a Golgi stain-impregnated pyramidal neuron in the BLA. (Scale bar, 20 μm.) (Inset) High-power image of spines on a secondary dendrite from the same neuron. (Scale bar, 20 μm.) (B) Schematic drawing classifying types of apical dendrites selected for spine density analysis. In our analysis, a dendritic branch emanating directly from the cell soma (▴) was defined as a primary branch, whereas a dendrite originating from a primary branch was defined as a secondary branch. Spines were counted, starting from the origin of a branch, in 10 consecutive segments of 8 μm each (small tick marks). (C) Photomicrographs of representative segments of primary (Left) and secondary (Right) branches from control and CIS neurons, demonstrating an increase in the number of spines. (Scale bar, 5 μm.) (D) Mean (±SEM) values for spine-density (calculated as the average number of spines per 10 μm) of primary (Left) and secondary (Right) branches of spiny BLA neurons from CIS and control groups. *, P < 0.05, compared with control, Student's t test; control, n = 20 neurons; CIS, n = 20 neurons. (E) Segmental analysis of the mean (±SEM) number of spines in each successive 8-μm segment along primary (Left) and secondary (Right) branches as a function of the distance of that segment from the origin of the branch. *, P < 0.05; **, P < 0.01, compared with control, Student's t test.

Effects of AIS on spine density, dendritic arborization, and anxiety-like behavior. For all graphs in A–D, summarizing changes in spine density or dendritic arbors, the ordinate depicts mean (±SEM) values for BLA spiny neurons from AIS-1 and AIS-10 animals, normalized to their respective control values (100%) for that parameter. (A) Percentage changes in spine density, relative to normalized control (100%), calculated from the total number of spines along an 80-μm segment of primary branch in spiny BLA neurons from AIS-1 and AIS-10 animals. Control, n = 18 neurons; AIS-1, n = 18 neurons; AIS-10, n = 30 neurons. One-way ANOVA; F_{(2, 63)} = 19.2, P < 0.001 for differences between groups; least significant difference (LSD) posthoc test; ***, P < 0.001. (B) Percentage changes in spine density, relative to normalized control (100%), calculated from the total number of spines along an 80-μm segment of secondary branch in spiny BLA neurons from AIS-1 and AIS-10 animals. One-way ANOVA; F_{(2, 63)} = 1.1, P > 0.3 for differences between groups. (C) Analysis of the percentage changes in spine density for each segment (mean ± SEM, normalized to respective control) for each successive 8-μm segment along primary branch as a function of the distance of that segment from the origin of the branch. Statistical significance was computed for each segment independently by using one-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001 between control and AIS-10; LSD posthoc test. ¥¥, P < 0.01 between control and AIS-1. (D) Percentage changes, relative to normalized control (100%), in total dendritic length (Left) and number of branch points (Right) of BLA spiny neurons from control (n = 18 neurons), AIS-1 (n = 18 neurons), and AIS-10 (n = 30 neurons) groups. One-way ANOVA; total dendritic length, F_{(2, 63)} = 2.7 and P > 0.2 for differences between groups; number of branch points, F_{(2, 63)} = 0.6 and P > 0.5 for differences between groups. (E) Anxiety-like behavior on the elevated plus-maze for all three experimental groups (control, AIS, and AIS-10). Changes in open-arm exploration were measured as the percentage of open-arm entries (Left) and the percentage of time spent in open arm (Right). (F) No effect on locomotor activity as measured by entries in enclosed arm in the plus-maze. Control, N = 10 animals; AIS-1, N = 4 animals; AIS-10, N = 8 animals. Kruskal–Wallis test for differences between three independent samples; P < 0.01 for the percentage of open-arm entries, P < 0.001 for the percentage of open-arm time, and P > 0.2 for the number of entries in enclosed arms. Significant effects were further analyzed by Mann–Whitney test; *, P < 0.05; ***, P < 0.001; relative to control.