Blockade of protein phosphatase 2B activity in the amygdala increases anxiety- and depression-like behaviors in mice

Abstract

Background: Organ transplant patients receive chronic administration of the calcineurin inhibitor cyclosporin-A (CsA) and demonstrate increased incidence of mood disorders. Significant calcineurin expression can be observed with immunohistochemistry in the amygdala, a brain area important for behaviors related to mood disorders and anxiety. It is therefore important to determine whether chronic blockade of calcineurin might contribute to symptoms of anxiety and depression in these patients.

Methods: Pharmacological CsA and viral-mediated gene transfer (adeno-associated viral expression of short hairpin RNA [AAV-shRNA]) approaches were used to inhibit calcineurin activity globally and selectively in the amygdala of the mouse brain to determine the role of calcineurin in behaviors related to depression and anxiety.

Results: Systemic inhibition of calcineurin activity with CsA or local downregulation of calcineurin levels in the amygdala with AAV-delivered shRNAs targeting calcineurin A increased behavioral measures of anxiety in both the elevated plus maze and light/dark tests with no changes in locomotor activity. In the forced swim and tail suspension models of depression-like behavior, calcineurin blockade in the amygdala increased immobility similarly to manipulations that lead to a depression-like phenotype.

Conclusions: Taken together, these data demonstrate that decreasing calcineurin activity in the amygdala increases anxiety- and depression-like behaviors. These studies suggest that chronic administration of CsA to organ transplant patients could have significant effects on anxiety and mood and that this should be recognized as a clinical consequence of treatment to prevent transplant rejection.

Figure 1. Anxiety-like behavior in mice treated chronically with CsA

A) Percent time spent in and B) number of entries into open arms in the elevated plus maze test. C) Time spent in dark side and D) number of entries into the dark side in the light/dark test. Data are expressed as mean ± SEM, N = 12-15 per group. *** p<0.001.

Figure 2. Depression-like behavior in mice treated chronically with CsA

Immobility time in the A) forced swim and B) tail suspension tests. C) Distance traveled in a novel environment as measured by beam breaks. Data are expressed as mean ± SEM, N = 12-15 per group. *** p<0.001

Figure 3. Calcineurin A mRNA levels and activity in the amygdala, nucleus accumbens (17) and ventral tegmental area (VTA) of mice treated chronically with CsA

A) RT-PCR analysis of the mRNA encoding calcineurin, GAPDH and β-actin in the amygdala. B) Calcineurin activity in brain extracts from amygdala, VTA and NAc. Data are expressed as mean ± SEM, N = 9 per group. *** p<0.001.

Figure 4. Calcineurin A mRNA levels and activity in the amygdala following local knockdown of calcineurin in the amygdala, nucleus accumbens (17) or ventral tegmental area (VTA)

A) RT-PCR analysis of calcineurin, GAPDH and β-actin transcripts in the amygdala after stereotaxic injection of AAV-shCnA or AAV-GFP into amygdala, NAc or VTA. B) Calcineurin activity in brain extracts from amygdala, NAc and VTA following stereotaxic injection with AAV-shRNAs or AAV-GFP into the amygdala. Data are expressed as mean ± SEM, N = 9 per group. *** p<0.001.

Figure 5. Anxiety-like behavior following local knockdown of calcineurin A in the amygdala, nucleus accumbens (17) or ventral tegmental area (VTA)

A) Time spent and B) number of entries into open arms in the elevated plus maze test. C) Time spent in dark side and D) number of entries into the dark side in the light/dark test for groups of mice with local knockdown of calcineurin in the amygdala, NAc or VTA. Data are expressed as mean ± SEM, N = 12-15 per group. *** p<0.001.

Figure 6. Depression-like behavior following local knockdown of calcineurin A in the amygdala, nucleus accumbens (17) or ventral tegmental area (VTA)

Immobility time in the A) forced swim and B) tail suspension tests for groups of mice with local knockdown of calcineurin in the amygdala, NAc or VTA. C) Distance traveled in a novel environment as measured by beam breaks in each group. Data are expressed as mean ± SEM, N = 12-15 per group. *** p<0.001.

Figure 7. c-fos immunoreactivity in the BLA in response to an acute swim stress following local knockdown of calcineurin A in the amygdala

A) Average number of c-fos-positive cells per amygdala in mice injected with AAV-GFP or AAV-shCnA following an acute swim stress or no stress (Stress or Controls). Data are expressed as mean ± SEM. N = 12 to 24 per group. *** p<0.001. B) Representative photomicrographs of amygdalar sections stained for c-fos. CC: corpus callosum; BLA: basolateral amygdala; EN: endopiriform nucleus. C) Left: Microphotograph of GFP-positive neurons after viral infection in the amygdala; Right: mouse brain diagram from Franklin and Paxinos (45) indicating sites of viral injections (black ovals). Scale bar = 50 μm.