Simvastatin enhances hippocampal long-term potentiation in C57BL/6 mice

Abstract

Statins inhibit 3-hydroxy-3-methylglutaryl CoA reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway, and they are widely used to control plasma cholesterol levels and prevent cardiovascular disease. However, emerging evidence indicates that the beneficial effects of statins extend to the CNS. Statins have been shown to improve the outcome of stroke and traumatic brain injury, and statin use has been associated with a reduced prevalence of Alzheimer's disease (AD) and dementia. However, prospective studies with statins in AD have produced mixed results. Recently, we reported that simvastatin, a widely used statin in humans, enhances learning and memory in non-transgenic mice as well as in transgenic mice with AD-like pathology on a mixed genetic background. However, the cellular and molecular mechanisms underlying the beneficial effects of simvastatin on learning and memory remain elusive. The present study was undertaken to investigate the effect of acute simvastatin treatment on hippocampal long-term potentiation (LTP), a cellular model of learning and memory, in brain slices from C57BL/6 mice. Our results demonstrate that a prolonged in vitro simvastatin treatment for 2-4 h, but not a short-term 20-min exposure, significantly increases the magnitude of LTP at CA3-CA1 synapses without altering basal synaptic transmission or the paired-pulse facilitation ratio in hippocampal slices. Furthermore, we show that phosphorylation of Akt (protein kinase B) is increased significantly in the CA1 region following 2-hour treatment with simvastatin, and that inhibition of Akt phosphorylation suppresses the simvastatin-induced enhancement of LTP. These findings suggest activation of Akt as a molecular pathway for augmented hippocampal LTP by simvastatin treatment, and implicate enhancement of hippocampal LTP as a potential cellular mechanism underlying the beneficial effects of simvastatin on cognitive function.

Fig. 1. Basal synaptic transmission and early LTP are unaffected by a 20-minute exposure to simvastatin

Vehicle was bath applied to hippocampal slices for the first 10 min of baseline stimulation, and simvastatin (10μM) application ensued for the final 20 min of baseline prior to HFS (arrow) and continued for the duration of the experiment. A: Paired-pulse facilitation (PPF) ratio was unchanged during simvastatin exposure (1.7 ± 0.05) compared to vehicle (1.7 ± 0.06). B: Mean magnitude of LTP from slices treated with simvastatin or vehicle. Mean magnitude of LTP, averaged over the last 5 min of run-out (min 65-70), was unaffected by 20-min simvastatin treatment (P = 0.583). LTP from simvastatin-treated slices was 147 ± 11% (n = 7 slices/6 mice), and 137 ± 12% (n=7 slices/6 mice) from vehicle-treated slices. Also, fEPSP slope did not change after onset of drug application, indicating basal synaptic transmission was unaffected by simvastatin. Waveforms show fEPSPs during baseline (dotted) and 40-min post-tetanus (solid) from a pair of slices treated with vehicle or simvastatin.

Fig. 2. Prolonged incubation in simvastatin significantly enhances early LTP in slices prepared in sucrose-based aCSF

A: Stimulus response curves for slices prepared in sucrose-based aCSF and incubated in simvastatin or vehicle for 2-4 hours. Incubation in simvastatin (n = 5 slices) did not alter the input/output curves over the range of baseline stimulation intensities utilized for baseline compared to vehicle-treated slices (n = 4 slices). B: Paired-pulse facilitation (PPF) ratio from slices prepared in sucrose-based aCSF and incubated in simvastatin or vehicle. PPF ratio was unchanged during baseline stimulation after incubation in simvastatin (1.6 ± 0.06) compared to vehicle (1.6 ± 0.06). C: The mean magnitude of LTP from simvastatin-treated slices (149 ± 10%, n= 9 slices/9 animals) was significantly higher than that from vehicle-treated slices (128 ± 7%, n= 9 slices/9 animals) (**P = 0.007). For clarity purposes, error bars are shown in only one direction. Waveforms show fEPSPs during baseline (dotted) and 40-min post-tetanus (solid) from a pair of slices treated with vehicle or simvastatin.

Fig. 3. Slicing in regular aCSF followed by 2-4 hr incubation in simvastatin significantly enhances early LTP

A: Stimulus response curves for slices prepared in regular aCSF and incubated in simvastatin or vehicle for 2-4 hours. Incubation in simvastatin (18 slices/7 animals) did not alter basal transmission over the range of stimulus intensities utilized for baseline acquisition compared to slices incubated in vehicle (9 slices/5 animals). B: Paired-pulse facilitation (PPF) ratio after incubation in simvastatin or vehicle. PPF ratio was not affected by simvastatin incubation. C: Mean magnitude of LTP from slices incubated in simvastatin or vehicle. The magnitude of LTP from simvastatin-treated slices (148 ± 7%, n = 11 slices/8 animals) was significantly higher (*P = 0.025) than that from slices incubated in vehicle (127 ± 5%, n = 10 slices/9 animals). For clarity purposes, error bars are shown in only one direction. Waveforms show fEPSPs during baseline (dotted) and 40-min post-tetanus (solid) from slices treated with vehicle or simvastatin.

Fig. 4. Effect of simvastatin treatment on the level of p-Akt and total Akt

A: Representative immunoblot images of p-Akt and total Akt in the homogenate of CA1 region of hippocampal slices treated with simvastatin (SV) or vehicle (Veh) for 20 min or 2 hr. B: Densitometric analysis of immunoblots (normalized by the amount of tubulin) with the levels in vehicle-treated group set as 100%. Data represent means ± SEM (n=15 slices/5 mice per treatment). The results showed that the levels of p-Akt in the CA1 region were increased significantly (about 2.5 fold) in the hippocampal slices treated with simvastatin for 2 hr, but not for 20 min, compared to slices treated with vehicle. ** P = 0.008.

Fig. 5. PI3K inhibitor LY294002 suppresses simvastatin-induced enhancement of LTP

A. Mean magnitude of LTP from slices incubated in simvastatin or vehicle with LY294002 (LY) (20 μM) applied 20 min after LTP induction in simvastatin (SV) + LY and vehicle (Veh) + LY slices. The vehicle slices were treated with vehicle (containing 0.1% DMSO) with no application of LY and served as control. The magnitude of LTP from simvastatin + LY slices (141 ± 9%, n = 8 slices/8 animals) was significantly higher (*P = 0.038) than that from vehicle + LY slices (116 ± 4%, n = 8 slices/8 animals). The magnitude of LTP from vehicle + DMSO was 118 ± 6% (n = 6 slices/6 animals) similar to that from vehicle + LY slices. The results show LY does not affect the expression of LTP in either simvastatin-or vehicle-treated slices. B. Mean magnitude of LTP from slices incubated in simvastatin or vehicle and treated with LY294002 (20 μM) or DMSO for at least 40 min pre-induction and for the rest of the experiment. The LTP magnitude from simvastatin-treated slices was increased significantly compared to that from vehicle-treated slices (145 ± 9% vs. 129 ± 6%, n = 10 slices/animals, P = 0.025). From the slices treated with simvastatin + LY, the LTP magnitude was significantly decreased to 119 ± 7% (n = 7 slices/animals, *P = 0.045 compared with the LTP magnitude from simvastatin-treated slices). From slices treated with vehicle + LY (n = 10 slices/animals), the LTP magnitude was unchanged (129 ± 7%) compared to slices treated with vehicle + DMSO (129 ± 6%). For clarity purposes, error bars are shown in only one direction. Waveforms show fEPSPs during baseline (dotted) and 40-min post-tetanus (solid) from slices in different treatment groups.