Myosin II motor activity in the lateral amygdala is required for fear memory consolidation

Abstract

Learning induces dynamic changes to the actin cytoskeleton that are required to support memory formation. However, the molecular mechanisms that mediate filamentous actin (F-actin) dynamics during learning and memory are poorly understood. Myosin II motors are highly expressed in actin-rich growth structures including dendritic spines, and we have recently shown that these molecular machines mobilize F-actin in response to synaptic stimulation and learning in the hippocampus. In this study, we report that Myosin II motors in the rat lateral amygdala (LA) are essential for fear memory formation. Pretraining infusions of the Myosin II inhibitor, blebbistatin (blebb), disrupted long term memory, while short term memory was unaffected. Interestingly, both post-training and pretesting infusions had no effect on memory formation, indicating that Myosin II motors operate during or shortly after learning to promote memory consolidation. These data support the idea that Myosin II motor-force generation is a general mechanism that supports memory consolidation in the mammalian CNS.

Figure 1.

Validation of the cued fear conditioning experimental paradigm. Cued fear conditioning of Sprague-Dawley rats, no cannulations. Paired animals froze more during the tone presentation than unpaired animals in a distinct context (F_(1,9) = 8.39, paired n = 5, unpaired n = 5; * denotes significant difference between paired and unpaired animals, P < 0.02). Differences between pretone and tone freezing demonstrate that freezing is tone-dependent in the paired group only (F_(1,4) = 34.10, P < 0.001; # denotes significant difference between pretone and tone-dependent freezing in the paired group; unpaired: F_(1,4) = 1.10, P > 0.05). Error bars represent SEM.

Figure 2.

Inhibition of Myosin II ATPase activity in the lateral amygdala blocks the formation, but not expression, of long-term fear memory. (A) Schematic of experimental design for LTM test. (B) Intra-LA infusions of the Myosin II ATPase inhibitor, blebb, prior to cued fear conditioning blocks memory formation (F_(1,10) = 26.90; n = 5, inactive; n = 6, active blebb; * denotes significantly different LTM at 24 h, P ≤ 0.001). (C) Location of needle tips for all intra-LA infusions. Diagram represents histology from animals whose behavioral data are depicted in B. (D) Schematic of experimental design for memory expression test. (E) Infusions of blebb into the LA 30 min prior to a LTM test have no effect on memory expression (F_(1,10) = 0.15, P > 0.05; n = 5, inactive; n = 6, active blebb). (F) Location of needle tips for all intra-LA infusions given to animals whose behavioral data are depicted in E. Because of the extensive overlap of the infusion needle tips between animals, not all tip locations are resolvable on the diagrams. Error bars represent SEM.

Figure 3.

Myosin II motor activity is required for consolidation, not acquisition, of fear memory. (A) Schematic of experimental design for post-training blebb infusions. (B) Intra-LA infusions of the Myosin II ATPase inhibitor, blebb, 30 min after cued fear conditioning has no effect on fear memory formation (F_(1,14) = 0.18, P > 0.05; n = 8, inactive; n = 7, active blebb). (C) Location of needle tips for all intra-LA infusions from animals whose behavioral data are depicted in B. (D) Schematic of experimental design to test the role of Myosin II in memory acquisition. (E) Pretraining intra-LA infusions of the Myosin II ATPase inhibitor blebb have no effect on STM (F_(1,11) = 0.004, P > 0.05; n = 6, inactive; n = 6, active blebb). (F) Location of needle tips for infusions depicted in E. Error bars represent SEM.

Figure 4.

Reducing Myosin IIB heavy chain expression in the lateral amygdala disrupts fear memory formation. (A) Schematic of experimental design for in vivo disruption of Myosin IIB. (B) In vivo knockdown of Myosin IIB disrupts fear memory formation (F_(1,15) = 7.32, P < 0.05; n = 7, NTC shRNA; n = 9, MYH10 hairpin; *denotes significantly different LTM at 24 h). (C) In vivo reduction of Myosin IIB does not affect amygdala-dependent associative learning or the ability to freeze (F_(2,15) = 0.37, P > 0.05). (D) Representative image of a brain section from an animal injected with rAAV NTC-GFP. Dashed line outlines the lateral and basolateral amygdala. GFP expression is limited to dorsolateral amygdala. (E,F) Western blot images (picture of the actual immunoblot) and quantifications show an ∼50% decrease in Myosin IIB expression in the LA of animals injected with rAAV expressing a shRNA against Myosin IIB. **Denotes significantly different from control, P ≤ 0.005. Error bars represent SEM.

Figure 5.

Actin polymerization is required in the lateral amygdala for cued fear memory. (A) Experimental design for intra-LA infusions of LatA. (B) Intra-LA infusions of LatA prior to cued fear conditioning block memory formation (F_(1,9) = 10.711, P < 0.05; veh, n = 5; LatA, n = 5; * denotes significantly different LTM at 24 h). (C) Location of needle tips for all intra-LA infusions depicted in B.

Figure 6.

Myosin II motor activity and actin polymerization are involved in a similar molecular process that supports memory consolidation in the lateral amygdala. (A,B) Schematic of experimental design for intra-LA infusions of blebb and LatA. Treatment groups shown in table. (C) Thirty-minute pretraining infusions of blebb and 15-min pretraining infusions of LatA block LTM (also demonstrated in Figs 2B, 5B), but showed no additive block when infused together (F_(1,22) = 8.33, P ≤ 0.005; n = 6, inactive/veh; n = 6, blebb/veh; n = 5, inactive/lat A; n = 6, blebb/lat A; P < 0.05 for all groups post hoc comparisons to inactive/veh; * denotes significantly different from vehicle at LTM test). LTM is not completely abolished in any treatment group when compared to pretone freezing (F_{(inactive/veh)} = 689.20, P ≤ 0.001; F_(blebb/veh) = 36.40, P ≤ 0.001; F_{(inactive/LatA)} = 10.55, P ≤ 0.01; F_(blebb/LatA) = 27.28, P ≤ 0.001, denoted by #). (D) Location of needle tips for all intra-LA infusions. Error bars represent SEM.