Hippocampal extracellular signal-regulated kinase signaling has a role in passive avoidance memory retrieval induced by GABAA Receptor modulation in mice

Abstract

Available evidence strongly suggests that the γ-aminobutyric acid type A (GABA(A)) receptor has a crucial role in memory retrieval. However, the signaling mechanisms underlying the role of GABA(A) receptor modulation in memory retrieval are unclear. We conducted one-trial passive avoidance task with pre-retention trial drug administration in the hippocampus to test the effects of GABA(A) receptor modulation on memory retrieval. We further tested the co-involvement of signaling molecules: extracellular signal-regulated kinase (ERK), Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), and cAMP responsive element-binding protein (CREB). First, we observed that the phosphorylation of hippocampal ERK was required for memory retrieval during the task. Accordingly, to investigate whether GABA(A) receptor activation or inhibition induces ERK phosphorylation during memory retrieval, drugs that target the GABA(A) receptor were administered into the hippocampus before the retention trial. Muscimol, a GABA(A) receptor agonist, and diazepam, an agonist to benzodiazepine-binding site of GABA(A) receptor, blocked retention trial-induced ERK phosphorylation and impaired memory retrieval. Furthermore, co-treatment with sub-effective dose of U0126, a mitogen-activated protein kinase inhibitor, blocked the upregulation of ERK phosphorylation and impaired memory retrieval, and bicuculline methiodide (BMI), a GABA(A) receptor antagonist, increased ERK phosphorylation induced by the retention trial and facilitated memory retrieval. Finally, the effects of BMI were blocked by the co-application of a sub-effective dose of U0126. These results suggest that GABA(A) receptor-mediated memory retrieval is closely related to ERK activity.

Figure 1

The site of intra-hippocampal microinjections. (a) Summary of the site of microinjection in the hippocampal CA3 region. Mice in which the cannulae were found to be misplaced were excluded. (b) Drug administration site confirmed by the infusion of 0.1 μl of 1% Evans Blue into the hippocampus after behavioral testing (coordinates: anteroposterior −2.10 mm from bregma; mediolateral ±2.50 mm from midline; and dorsoventral −2.00 mm from dura).

Figure 2

Memory retrieval-induced changes in the expression of memory-related signaling molecules in hippocampal tissues. (a) Photographs of immunohistochemical results showing memory retrieval-induced changes in the immunoreactivity of anti-phosphorylated CREB (pCREB), anti-phosphorylated ERK (pERK), or anti-phosphorylated CaMKII (pCaMKII) in the hippocampus. (b–d) Western blot analysis of pCREB (b), pERK (c), and pCaMKII (d). Mice were killed immediately after the retention trial of a one-trial passive avoidance task. Data are presented as the means±SEM (n=4). ^*p<0.05, compared with the control group. The control group was not subjected to a retention trial. The retrieval group was subjected to a retention trial. Magnification: × 50. Bar=200 μm.

Figure 3

Blockade of extracellular signal-regulated kinase (ERK) phosphorylation inhibits memory retrieval. (a) Latency times measured during the retention trial were reduced by the administration of U0126 but not KN62. (b), Dose-dependent effects of U0126 on memory retrieval. Fifteen minutes before the retention trial, KN62 (1 μg/μl/side) or U0126 (0.25, 0.5, or 1 nmol/μl/side) were administered bilaterally into the hippocampus. Data are presented as the means±SEM (n=8–10 per group). ^*p<0.05, compared with the vehicle-treated group. (c, d), Immunoblotting (c) and quantitative analysis (d) of pCREB, pCaMKII, and pERK levels in hippocampal tissues after the administration of KN62 or U0126 before the retention trial. Fifteen minutes before the retention trial, KN62 (1 μg/μl/side) or U0126 (1 nmol/μl/side) were administered bilaterally into the hippocampus. The mice were immediately killed after the retention trial. Data are presented as the means±SEM (n=4 per group). ^*p<0.05, compared with the control group; ^#p<0.05, compared with the vehicle-treated group. Control, animals that did not undergo a retention trial; Vehicle, animals that underwent a retention trial but were treated only with vehicle; KN62, animals treated with KN62 that underwent a retention trial; U0126, animals treated with U0126 that underwent a retention trial.

Figure 4

γ-Aminobutyric acid type A (GABA_A) receptor activation detrimentally affects memory retrieval. (a, b) Fifteen minutes before the retention trial, (a) muscimol (0.25, 0.5, or 1 μg/μl/side) or (b) diazepam (10, 20, or 40 μg/μl/side) were administered bilaterally into the hippocampus. Latency times measured during the retention trial were reduced by muscimol or diazepam administration in a dose-dependent manner. (c) To test state-dependency of GABAergic drugs, muscimol (1 μg/μl/side) or diazepam (40 μg/μl/side) was administered bilaterally 15 min before both acquisition and retention trial. Data are presented as the means±SEM (n=8–10 per group). ^*p<0.05, compared with the vehicle-treated control group. (d) The effect of muscimol (1 μg/μl/side) or diazepam (40 μg/μl/side) on memory retrieval-induced ERK activation. The mice were killed immediately after the retention trial of passive avoidance task. Data are presented as the means±SEM (n=4 per group). ^*p<0.05, compared with the control group; ^#p<0.05, compared with the vehicle-treated retrieval group. Control, animals exposed to acquisition but not retention trial. (e, f) The motoric effects of muscimol (0.25, 0.5, or 1 μg/μl/side, (e) or diazepam (10, 20, or 40 μg/μl/side, f) in the open field test. Drugs were injected 15 min before the test, and the distance that each mouse moved was recorded for 5 min. Data are presented as the means±SEM (n=10 per group).

Figure 5

γ-Aminobutyric acid type A (GABA_A) receptor activation-induced memory retrieval impairment correlates with extracellular signal-regulated kinase (ERK) phosphorylation. Fifteen minutes before the retention trial, muscimol (0.25 μg/μl/side) with U0126 (0.25 nmol/μl/side) or diazepam (10 μg/μl/side) with U0126 (0.25 nmol/μl/side) was administered bilaterally into the hippocampus. (a), Latency times measured during the retention trial were reduced by muscimol and U0126 or diazepam and U0126 administration. Data are presented as the means±SEM (n=8 per group). ^*p<0.05, compared with the vehicle-treated control group. (b), Immediately after the retention trial, the mice were killed for western blotting to measure pERK level in the hippocampus. Data are presented as the means±SEM (n=4 per group). ^*p<0.05, compared with the vehicle only-treated retrieval group.

Figure 6

γ-Aminobutyric acid type A (GABA_A) receptor blockade facilitates memory retrieval. (a, b), Fifteen minutes before the retention trial, bicuculline methiodide (BMI: 0.25, 0.5, or 1 μg/μl/side, a) or flumazenil (0.5, 1, or 2 μg/μl/side, b) was administered bilaterally into the hippocampus. The latency times that were measured during retention trial were increased by BMI administration in a dose-dependent manner. (c), To test state-dependency of BMI (1 μg/μl/side), the mice were treated with BMI bilaterally 15 min before both the acquisition and the retention trial. Data are presented as the means±SEM (n=8–10 per group). ^*p<0.05, compared with the vehicle-treated group. (d), The effect of BMI (1 μg/μl/side) on memory retrieval-induced ERK activation. The mice were killed immediately after the retention trial. Data are presented as means±SEM (n=4 per group). ^*p<0.05, compared with the control group; ^#p<0.05, compared with the vehicle-treated group. Control, animals exposed to the acquisition but not the retention trial. (e), The motoric effect of BMI (0.25, 0.5, or 1 μg/μl/side) in the open field test. Drugs were injected 15 min before the test and the distance each mouse moved was recorded for 5 min. Data are presented as means±SEM (n=10 per group).

Figure 7

γ-Aminobutyric acid type A (GABA_A) receptor blockade-induced memory retrieval enhancement correlates with ERK phosphorylation. Fifteen minutes before the retention trial, BMI (1 μg/μl/side) with U0126 (0.25 nmol/μl/side) was administered bilaterally into the hippocampus. (a) BMI-induced increases in the latency time during the retention trial were reduced by U0126 administration. Data are presented as the means±SEM (n=8 per group). ^*p<0.05. (b) Immediately after the retention trial, the mice were killed for Western blotting to measure pERK levels in the hippocampus. Data are presented as the means±SEM (n=4 per group). ^*p<0.05.