SK2 potassium channel overexpression in basolateral amygdala reduces anxiety, stress-induced corticosterone secretion and dendritic arborization

Abstract

The basolateral amygdala is critical for generation of anxiety. In addition, exposure to both stress and glucocorticoids induces anxiety. Demonstrated ability of the amygdala to change in response to stress and glucocorticoids could thus be important therapeutic target for anxiety management. Several studies have reported a relationship between anxiety and dendritic arborization of the amygdaloid neurons. In this study we employed a gene therapeutic approach to reduce anxiety and dendritic arborization of the amygdala neurons. Specifically, we overexpressed SK2 potassium channel in the basolateral amygdala using a herpes simplex viral system. Our choice of therapeutic cargo was guided by the indications that activation of the amygdala might underlie anxiety and that SK2 could reduce neuronal activation by exerting inhibitory influence on action potentials. We report that SK2 overexpression reduced anxiety and stress-induced corticosterone secretion at a systemic level. SK2 overexpression also reduced dendritic arborization of the amygdala neurons. Hence, SK2 is a potential gene therapy candidate molecule that can be used against stress-related neuropsychiatric disorders such as anxiety.

Figure 1

SK2 viral vector resulted in over expression of reporter gene β-gal, stained with X-gal (blue), in the BLA, 48 hours post-infusion. (A) β-gal staining in BLA (next to RF, an identifiable anatomical landmark); scale = 2.5 mm (B) Diagram depicting coronal section of rat brain from comparable level as shown in A (C) β-gal stained cells of BLA at higher magnification; scale = 250 μm. (D) scale = 125 μm (E) Single cell stained with β-gal in BLA; scale = 20 μm. Inset: Coronal section depicting BLA and PC in rat brain.

Figure 2

Effect of SK2 over-expression on anxiety and locomotion on EPM. SK2 infusion enhanced percentage open-arm entries (A) and percentage open-arm time (B). This enhancement was not accompanied by differences in entries in enclosed arm (C) Mean ± SEM, n = 9 animals each group; * p < 0.001, Student's t-test. Inset: Time profile of experiment and endpoint.

Figure 3

Effect of SK2 over-expression on anxiety and locomotion as measured in open field. SK2 over-expression enhanced time spent away from wall in a big open arena (A) without affecting the total distance traveled (B) Mean ± SEM. n = 12 animals per group. SK2 over-expression also increased the number of crossings made at the center of a smaller open field (C) Mean ± SEM. n = 5-6 animals per group; * p < 0.001, Student's t-test. Inset: Time profile for infusion and behavior.

Figure 4

SK2 reduced secretion of CORT when measured after a 30-minute session of stress (B) SK2 did not affect basal CORT at 3 days (A) or 12 days post infusion (C) Mean ± SEM. n = 4-6 animals, * p < 0.01, Student's t-test. Inset: Time profile for infusion and CORT measurement. Note: The ordinate scale in panel A, B and C are different.

Figure 5

SK2 over-expression decreased dendritic arborization of neurons in the BLA. The decrease in dendritic arborization was reflected in the total dendritic length (A) but not in the number of branch points (B). (C) Camera Lucida drawings of representative neuron from β-gal-expressing (upper) or SK2-over-expressing (lower) animals. Scale bar = 100 μm. (D) Segmental analysis of dendritic length in SK2-infused animals showing reduction in segments 150-200 μm away from cell body. * p < 0.05; Student's t-test; n= 40-50 neurons from 4-5 animals per group. Inset (D): Sholl's analysis of a typical pyramidal neuron of BLA. Inset: Time profile for morphological measurement post infusion.

Figure 6

Stress and CORT-treatment enhanced dendritic length, and SK2 over-expression decreased dendritic length in Control, Stress and CORT-treated animals, compared to animals of each group expressing β-gal (A). * p < 0.05; planned comparison using Student's t-test; n = 40-50 neurons from 5-6 animals per group. Control data in Fig 6A is same as in Fig 5A. (B) Segmental analysis showed a reduction in segments 100-150 μm away from cell body in stress-treated animals. Inset: Time profile for morphological measurement post infusion.

Figure 7

SK2 did not change anxiety in stress or CORT-treated animals in either open-arm entries (A) or number of center crossings (B); n = 8-10 animals per group. Control data in Fig 7 has been shown earlier in Fig 2 and 3. Inset: Time profile for infusion and behavior.