Inhibition of hippocampal Cdk5 activity facilitates extinction. (a) Mice (ten per group) were subjected to our extinction procedure and injected (intrahippocampal) with butyrolactone I (50 ng) immediately after E1–E3. When compared with the vehicle group, butyrolactone I–injected mice showed reduced freezing behavior on E2 and E3, indicating facilitated extinction. (b) The experiment was carried out as described in a, but a lower concentration (25 ng) of butyrolactone I was injected after E1–E3. Extinction was similar in vehicle and butyrolactone I–injected mice. (c) Similar results were obtained when mice were administered 12.5 ng of butyrolactone I. (d) Consistent with the data shown in a, a single injection of 50 ng of butyrolactone immediately after E1 facilitated extinction, as indicated by a significant reduction of freezing behavior on E2 when compared with a vehicle group. The reduced freezing behavior persisted throughout extinction trials in the absence of further butyrolactone I injection. (e) Mice were subjected to our extinction procedure and injected with butyrolactone I (50 ng) 1 h after E1. This facilitated extinction, as indicated by a significant reduction of freezing behavior on E2 when compared with a vehicle group. (f) Intrahippocampal injection of butyrolactone I 3 h after E1 had no significant effect on extinction when compared with the vehicle group. *P < 0.05 versus vehicle group, n = 9–10 mice per group. Error bars represent s.e.m.

Membrane association of Cdk5 and p35 during extinction is regulated by Rac-1. (a) Membrane and cytosolic fractions were prepared from the hippocampus of mice 0.5 h after exposure to E1, E3 or from naive animals. We observed a significant reduction of membrane-associated p35 that was accompanied by increased cytosolic p35 levels after E3. Synaptophysin (SVP) served as a loading control for the membrane fraction, whereas GAPDH was employed to verify equal loading in the cytosolic fraction. (b) Membrane lysates prepared from mice 0.5 h after E3 and from naive mice were used in a Rac-1 kinase assay. Rac-1 activity was significantly decreased after E3. (c) Hippocampal cytosolic and membrane fractions were prepared on E3 0.5 h after mice were injected (ICV) with either vehicle or Rac-1 inhibitor. Immunoblot analysis showed a significant reduction of membrane-associated Cdk5/p35 when compared with vehicle-injected mice. (d) Consistent application of Rac-1 inhibitor facilitated extinction. Mice (10 per group) were subjected to our extinction procedure and injected (ICV) with Rac-1 inhibitor (NSC23760; 10 μg μl⁻¹) immediately after E1–E3. When compared with the vehicle group, Rac-1 inhibitor injected mice showed reduced freezing, indicating facilitated extinction. n = 3–5 for biochemical analysis. ***P < 0.0001 versus naive/vehicle group, *P < 0.05 versus naive/vehicle group. Error bars represent s.e.m.

Cdk5 affects PAK-1 activity during extinction. (a) Experimental design. Microcannula were implanted into the hippocampus of mice that were subjected to contextual fear conditioning. The Cdk5 inhibitor butyrolactone I (50 ng) was injected immediately after exposure to E1 and hippocampal cytosolic and membrane fractions were prepared 0.5 h later. (b) Mice injected with butyrolactone I (But) showed significantly reduced levels of PAK-1_T212 in the cytosolic fraction. (c) Similarly, PAK-1_T212 levels were reduced in the membrane fraction. Conversely, levels of pPAK-1_T423 were significantly increased. (d) Notably, butyrolactone-injected mice also showed a significant redistribution of PAK-1 from the membrane to the cytosol when compared with vehicle-injected mice. Error bars represent s.e.m.

Increased Cdk5 activity in CK-p25 Tg mice impairs extinction. (a) Experimental design. CK-p25 Tg (n = 9) and control mice (n = 9) were trained by contextual fear conditioning, and p25 expression was induced 24 h later for 1 week. Afterwards, all mice were exposed to six extinction trials before repression of p25 production in CK-p25 Tg by reintroduction of doxycycline to the diet. After 1 week, all mice were subjected to another five extinction trials on consecutive days. (b) Immunoblot analysis of hippocampal lysates from CK-p25 Tg and control mice. Antibody to p35 (C-19) was employed to detected endogenous p25 and p25-GFP in CK-p25 Tg mice 1 week after p25 induction and 1 week after p25 repression, respectively. (c) Cdk5 kinase activity was analyzed by p35 (C-19) immunoprecipitation assays using histone H1 as an in vitro substrate. Cdk5 activity was significantly increased after 1 week of induction, and decreased to baseline levels after 1 week of p25 repression. (d) CK-p25 Tg and control mice were subjected to the experimental procedure described in a. As long as p25 was expressed in CK-p25 Tg, leading to increased Cdk5 activity, the extinction of freezing behavior was abolished. When p25 production was repressed and Cdk5 activity was reduced to baseline levels, extinction occurred in the same mice. ***P < 0.0001, n = 3–5 for biochemical analysis. Error bars represent s.e.m.

Inhibition of Rac-1 facilitates extinction. (a) Membrane and cytosolic fractions were prepared from the hippocampus of mice 0.5 h after exposure to E1, E3 or from naive animals and analyzed for PAK-1 by immunoblotting. Levels of PAK-1_Thr212 were significantly decreased in the membrane fraction after E3. (b) The same lysates as in a were used to analyze the levels of pPAK-1_T423, a non-Cdk5 phosphorylation site that indicates active PAK-1. pPAK-1_T423 levels were significantly increased after E3 in the membrane fraction. In the cytosolic fraction, pPAK-1_T423 levels decreased after E1 when compared with naive mice, but increased back to baseline levels after E3. (c) Hippocampal cytosolic and membrane fractions were prepared on E3 0.5h after mice were injected (ICV) with either vehicle or Rac-1 inhibitor. Immunoblot analysis showed a significant reduction of membrane-associated p35/Cdk5, pPAK-1_T212 and PAK-1 levels. (d) The same lysates as described in c were used to immunoprecipitate PAK-1. A hemagglutinin antibody served as a control for nonspecific binding of proteins to the protein A–agarose beads. The precipitates were immunoblotted with antibodies to PAK-1 and p35. There was a substantial reduction in the amount of p35 that co-immunoprecipitated with PAK-1 in lysates from mice that were administered with Rac-1 inhibitor. Error bars represent s.e.m.

PAK-1 activity promotes extinction. (a) Experimental design. Mice were implanted with microcannula in the dorsal hippocampus and subjected to contextual fear conditioning. After 24 h, mice were injected with either HSV-GFP or HSV-GFP– dnPAK-1 virus into the dorsal hippocampus. As of the next day, all mice were subjected to our extinction procedure. (b) Herpes virus expressing GFP and a dnPAK-1 construct or only GFP were injected into the dorsal hippocampus of mice. Upper, DAB staining for GFP of mice injected with dnPAK-1–GFP. Lower, high-magnification pictures of the hippocampal region showing staining for Hoechst, NeuN and GFP. The transfection efficiency was about 30%. Analysis was carried out 48 h after injection. (c) Mice (n = 10 per group) expressing dnPAK-1 in the hippocampus show a significantly impaired reduction of freezing behavior indicating impaired extinction. Error bars represent s.e.m.