Brain region-specific effects of cGMP-dependent kinase II knockout on AMPA receptor trafficking and animal behavior

Abstract

Phosphorylation of GluA1, a subunit of AMPA receptors (AMPARs), is critical for AMPAR synaptic trafficking and control of synaptic transmission. cGMP-dependent protein kinase II (cGKII) mediates this phosphorylation, and cGKII knockout (KO) affects GluA1 phosphorylation and alters animal behavior. Notably, GluA1 phosphorylation in the KO hippocampus is increased as a functional compensation for gene deletion, while such compensation is absent in the prefrontal cortex. Thus, there are brain region-specific effects of cGKII KO on AMPAR trafficking, which could affect animal behavior. Here, we show that GluA1 phosphorylation levels differ in various brain regions, and specific behaviors are altered according to region-specific changes in GluA1 phosphorylation. Moreover, we identified distinct regulations of phosphatases in different brain regions, leading to regional heterogeneity of GluA1 phosphorylation in the KO brain. Our work demonstrates region-specific changes in GluA1 phosphorylation in cGKII KO mice and corresponding effects on cognitive performance. We also reveal distinct regulation of phosphatases in different brain region in which region-specific effects of kinase gene KO arise and can selectively alter animal behavior.

Figure 1.

Reduction of GluA1 phosphorylation in the cGKII KO amygdala and impaired cued fear conditioning. (A) Representative immunoblots and quantitative analysis of whole amygdala lysates from the WT and KO animals showing significant reduction of GluA1-pS845 in the KO amygdala (n = 4 experiments, (*) P < 0.05, unpaired two-tailed student's t-test). (B) Diagram of auditory cued fear-conditioning protocol and summary of average percentage freezing during the 30 sec prior to the initial tone as well as the average freezing during each of the two tones for each genotype showing reduced freezing levels for KO mice during presentation of auditory cue (n = 20 WT and 21 KO mice, (*) P < 0.05, unpaired two-tailed Student's t-test).

Figure 2.

Enhanced contextual fear conditioning in cGKII KO animals. (A) Average percentage freezing 24 h after the initial training for WT and KO mice during each minute of the 4-min exposure to the training chamber showing increased freezing behavior in the KO mice during the final 2 min of the fear-conditioning test (n = 28 WT and 28 KO mice, (*) P < 0.05 and (**) P < 0.01, unpaired two-tailed student's t-test). (B) Average percentage freezing for WT and KO mice during reexposure to the training context 1 and 3 d after training showing no change in rate of extinction after day 3 (n = 28 WT and 28 KO mice). (C) Average percentage freezing 1 h after the initial training for WT and KO mice during each minute of the 4-min exposure to the training chamber showing no significant difference (n = 9 WT and 8 KO mice).

Figure 3.

Reduction of AMPAR levels in the NAc of KO animals and KO-mediated antidepressant effects. (A) Representative immunoblots and quantitative analysis of PSD from the NAc of the WT and KO animals showing significant reduction of GluA1, GluA1-pS845, and GluA2/3 in the KO NAc (n = 7 experiments, (****) P < 0.0001, unpaired two-tailed Student's t-test). (B) Percentage of sucrose water consumed out of total liquid consumption overnight for both WT and KO mice during the sucrose preference test showing significant increase of sucrose consumption for the KO mice (n = 15 WT and 15 KO mice, (*) P < 0.05, unpaired two-tailed Student's t-test). (C) Average of the total time WT and KO mice spent immobile during the 6-min tail suspension test showing significant reduction in immobility for KO mice (n = 16 WT and 16 KO mice, (*) P < 0.05 and (**) P < 0.01, unpaired two-tailed Student's t-test).

Figure 4.

No alteration in GluA1 phosphorylation in the KO olfactory bulb and normal olfactory function in KO animals. (A) Representative immunoblots and quantitative analysis of PSD from the olfactory bulb of the WT and KO animals showing no alteration of AMPAR trafficking in the KO olfactory bulb. (B) Average latency in seconds of the amount of time it took WT and KO mice to find and commence consumption of the hidden food showing no difference between WT and KO mice. (n = 13 WT and 15 KO mice).

Figure 5.

Distinct regulation of phosphatases in the hippocampus and PFC affects GluA1 phosphorylation differently in the region-specific manner. (A) Representative immunoblots showing that DARPP-32 expression is higher in the PFC than the hippocampus. (B) Representative immunoblots and quantitative analysis of hippocampal neurons treated with FK506, OA, or FK506 and OA showing that inhibition of phosphatases is sufficient to increase GluA1-pS845 in cultured hippocampal neurons (n = 10 experiments, (**) P < 0.01 and (***) P < 0.001, one-way analysis of variance (ANOVA) followed by Fisher's Least Significant Difference (LSD) test). (C) Representative immunoblots and quantitative analysis of PFC neurons treated with FK506, OA, or FK506 and OA showing no alteration of GluA1 phosphorylation (n = 10 experiments). (D) Representative immunoblots showing that DARPP-32 phosphorylation is not altered in the KO PFC. (E) In the hippocampus, calcineurin directly regulates I-1 phosphorylation, so that calcineurin and PP1 work in the same Ca²⁺ pathway to control GluA1 phosphorylation and trafficking. (F) In the PFC, unlike the hippocampus, DARPP-32 would play a central role in control of PP1 activity. Because DARPP-32 is mainly regulated by the dopamine signaling pathway, Ca²⁺-dependent calcineurin activity and dopamine-mediated PP1 activity regulate GluA1 phosphorylation independently in the PFC.