Melamine disrupts spatial reversal learning and learning strategy via inhibiting hippocampal BDNF-mediated neural activity

Abstract

Although several studies showed adverse neurotoxic effects of melamine on hippocampus (HPC)-dependent learning and reversal learning, the evidence for this mechanism is still unknown. We recently demonstrated that intra-hippocampal melamine injection affected the induction of long-term depression, which is associated with novelty acquisition and memory consolidation. Here, we infused melamine into the HPC of rats, and employed behavioral tests, immunoblotting, immunocytochemistry and electrophysiological methods to sought evidence for its effects on cognitive flexibility. Rats with intra-hippocampal infusion of melamine displayed dose-dependent increase in trials to the criterion in reversal learning, with no locomotion or motivation defect. Compared with controls, melamine-treated rats avoided HPC-dependent place strategy. Meanwhile, the learning-induced BDNF level in the HPC neurons was significantly reduced. Importantly, bilateral intra-hippocampal BDNF infusion could effectively mitigate the suppressive effects of melamine on neural correlate with reversal performance, and rescue the strategy bias and reversal learning deficits. Our findings provide first evidence for the effect of melamine on cognitive flexibility and suggest that the reversal learning deficit is due to the inability to use place strategy. Furthermore, the suppressive effects of melamine on BDNF-mediated neural activity could be the mechanism, thus advancing the understanding of compulsive behavior in melamine-induced and other neuropsychiatric disorders.

Fig 1. Intra-hippocampal melamine impairs reversal learning and cognitive strategy.

(A) Schematic representation of the cannulae placements presented for the low dse (200 mM/μL) of melamine group (gray), for the high dose (400 mM/μL) of melamine group (black) and or the control group (hollow). (B) The total trails to the criterion in the reversal learning of Y-maze task. Both low and high dose of melamine induced reversal impairment, with a dose-dependent manner. n = 8 per group. Rats infused with melamine did not affect locomotion in open field task (C) or motivation behavior in level-press task (D). n = 6 per group. (E) Learning strategy was tested in a probe trial of the cross-maze task and the number of rats that used each learning strategy was presented. Melamine-treated rats showed a learning strategy preference but avoid using place strategy. n = 20 per group. *P<0.05, vs. Control group; #P<0.05, vs. Melamine (200 mM/μL) group.

Fig 2. Melamine reduces learning-induced BDNF expression in the HPC neurons.

(A) Representative micrographs showing labeling of NeuN (green), BDNF (red), and NeuN/BDNF overlap (yellow) in pre-reversal (Top) and post-reversal (Bottom) trained rats. After training in the learning stage of Y-maze task, the basal levels of BDNF in the neuron (B) and total BDNF (C) were tested 30 min following melamine treatment. No statistical difference was found before the reversal learning. n = 8 per group. (D) The BDNF level in the HPC neurons was significantly lower compared melamine to control groups. n = 8 per group. (E) A strong correlation between the trails to criterion during reversal learning and hippocampal BDNF expression. *P<0.05, vs. Good group. n = 8 per group. (F) After the reversal learning, there was no statistical difference in the total BDNF expression between melamine and control groups. n = 8 per group. *P<0.05, vs. Control group.

Fig 3. Intra-hippocampal BDNF infusion mitigates the disruptive effects of melamine on neural correlate of reversal performance and cognitive strategy.

(A) Exogenous BDNF effectively reduced the total trials to the criterion in the reversal learning of Y-maze task. n = 8 per group. (B) Infusion BDNF into the HPC of melamine-treated rats could rescue the learning strategy bias. n = 10 per group. BDNF-mediated neural activity of pyramidal neurons (C), but not FS interneurons (D), was inhibited by melamine but reversed following BDNF treatment. *P<0.05, Melamine group vs. other groups.