In Vivo Sub-chronic Treatment with Dichlorvos in Young Rats Promotes Synaptic Plasticity and Learning by a Mechanism that Involves Acylpeptide Hydrolase Instead of Acetylcholinesterase Inhibition. Correlation with Endogenous β-Amyloid Levels

Abstract

Acylpeptide hydrolase (APEH) is a serine hydrolase that displays two catalytic activities, acting both as an exopeptidase toward short N-acylated peptides and as an endopeptidase toward oxidized peptides or proteins. It has been demonstrated that this enzyme can degrade monomers, dimers, and trimers of the Aβ_1-40 peptide in the conditioned media of neuroblastoma cells. In a previous report, we showed that the specific inhibition of this enzyme by the organophosphate molecule dichlorvos (DDVP) triggers an enhancement of long-term potentiation in rat hippocampal slices. In this study, we demonstrate that the same effect can be accomplished in vivo by sub-chronic treatment of young rats with a low dose of DDVP (0.1 mg/kg). Besides exhibiting a significant enhancement of LTP, the treated animals also showed improvements in parameters of spatial learning and memory. Interestingly, higher doses of DDVP such as 2 mg/kg did not prove to be beneficial for synaptic plasticity or behavior. Due to the fact that at 2 mg/kg we observed inhibition of both APEH and acetylcholinesterase, we interpret that in order to achieve positive effects on the measured parameters only APEH inhibition should be obtained. The treatment with both DDVP doses produced an increase in the endogenous concentration of Aβ_1-40, although this was statistically significant only at the dose of 0.1 mg/kg. We propose that APEH represents an interesting pharmacological target for cognitive enhancement, acting through the modulation of the endogenous concentration of Aβ_1-40.

Keywords: acetylcholinesterase; acylpeptide hydrolase; dichlorvos; hippocampus; learning; synaptic plasticity.

FIGURE 1

Effect of sub-chronic DDVP treatment on LTP elicited in the stratum pyramidale and stratum radiatum of the rat hippocampus. For each experiment, the percentage potentiation was calculated with respect to the mean of the baseline response. The number of slices and animals analyzed are indicated in parentheses besides each treatment condition. (A) LTP recordings in the stratum pyramidale of slices obtained from control animals (open circles), and animals treated with 0.1 mg/kg (black squares), or 2 mg/kg (open triangles) of DDVP. The animals treated with 0.1 mg/kg DDVP showed an enhanced LTP relative to the control animals (^∗p < 0.05, unpaired t-test). (B) Representative traces of population spikes (PS) before (gray traces) and after TBS (black traces) for each condition. (C,D) Long-term potentiation recordings in the stratum radiatum. The symbols used are similar to those in (A,B).

FIGURE 2

Quantification of specific activities of APEH and AChE. (A) Dose–response curves for the inhibition of hippocampal specific activities of APEH (gray circles) and AChE (black circles) upon administration of various doses of DDVP to different groups of animals. Significant percentages of inhibition are showed above each activity point. The dose–response curves of inhibition were adjusted to a Hill equation (the IC₅₀ values for APEH and AChE were 0.234 ± 0.092 mg/kg and 0.546 ± 0.146 mg/kg, respectively. (B) Specific activities for the two enzymes under control conditions and at a DDVP dose of 0.1 mg/kg. Only APEH was significantly inhibited at this dose. ^∗∗∗p < 0.001, unpaired t-test.

FIGURE 3

Differential effects of low and high DDVP doses on learning and memory. (A,B) Graphs of the escape latency (time to reach the submerged platform) for groups of rats treated with (A) 0.1 mg/kg DDVP (black squares), (B) 2 mg/kg DDVP (black squares), and their control groups (open circles). Rats treated with the low DDVP dose showed an improved escape latency starting from the 2nd day of training. (C,D) Results of the memory tests performed 24 h after the learning test. (C) Rats treated with 0.1 mg/kg DDVP (black columns) showed a significantly higher number of target crossings and quadrant entries compared to (D) rats treated with 2.0 mg/kg DDVP (black columns), which did not show significant differences with respect to the controls. (E,F) Representative trajectories of rats swimming for each set of conditions. Rats treated with 0.1 mg/kg DDVP showed improved escape latency and quickly found the platform location. ^∗p < 0.05, ^∗∗p < 0.01. See the text in Results section for details.

FIGURE 4

Effect of 0.1 mg/kg DDVP on synaptic plasticity parameters. (A) Effect of DDVP on synaptic efficacy by the measurement of input/output curves obtained from hippocampal tissues of control rats (open circles) and those treated with 0.1 mg/kg DDVP (black squares). The treatment with DDVP did not produce any change in the synaptic efficacy of the Schaffer collateral → CA1 pathway. (B) Interstimulus interval (ISI) curves for hippocampal slices obtained from control (black squares) and DDVP-treated animals (open circles). Paired-pulse facilitation was observed for both conditions at interstimulus intervals below 160 ms. The insets show representative traces of fEPSPs for the control and DDVP-treated animals obtained at a interstimulus interval of 320 ms. Black traces indicate the first pulse (P1) and gray traces indicate the second pulse (P2). (C) Paired-pulse inhibition (PPI) experiments for control conditions (open circles) and DDVP treatment (black squares). The interstimulus interval for these experiments was 13 ms. The PS amplitudes obtained for the first and second pulses are shown. For both conditions, the means of P1 and P2 (gray circles and gray squares for control and DDVP treatment, respectively) are shown. The slopes of the lines connecting the means of P1 and P2 for the two conditions do not show statistical differences. The insets show representative PS traces. Black traces indicate the first pulse (P1) and gray traces indicate the second pulse (P2).

FIGURE 5

Effect of sub-chronic treatments with low and high DDVP doses on the endogenous concentrations of Ab_1-40 and Ab_1-42. Both DDVP doses produced increases in the endogenous concentration of Aβ_1-40, which were statistically significant for the low-dose condition. ^∗p < 0.05.