Positive emotional learning is regulated in the medial prefrontal cortex by GluN2B-containing NMDA receptors

Abstract

In rats, hedonic ultrasonic vocalizations (USVs) is a validated model of positive affect and is best elicited by rough-and-tumble play. Here we report that modulation of GluN2B-containing NMDA receptors (NMDAR) in the medial prefrontal cortex (MPFC) is involved in positive emotional learning. Rough and tumble play increased both GluN1 and GluN2B NMDAR subunit mRNA and protein levels in the frontal cortex. GLYX-13, a GluN2B-preferring, NMDAR glycine-site partial agonist (1 mg/kg, i.v.) significantly increased positive emotional learning whereas the GluN2B receptor-specific antagonist, ifenprodil (10 mg/kg, i.p.), inhibited positive emotional learning. Animals selectively bred for low rates of hedonic USVs were returned to wild-type levels of positive emotional learning following GLYX-13 treatment. MPFC microinjections of GLYX-13 (0.1-10 μg/side) significantly increased rates of positive emotional learning. Thus GluN2B-containing NMDARs may be involved in positive emotional learning in the MPFC by similar mechanisms as spatial/temporal learning in the hippocampus.

Figure 1. Both rough-and-tumble play and trace-eyeblink conditioning upregulate NMDA receptors in rats

(A) Venn diagram of significantly changed genes in the frontal and posterior cortex 6 hrs after play, with NMDA receptors being significantly enriched in the 17 shared genes. Mean ± SEM mRNA levels (top panels) or protein levels (bottom panels) of GluN1, GluN2A and GluN2B NMDA receptor subunits following (B, D) conspecific rough-and-tumble play, (E) heterospecific rough-and-tumble play, or (C) trace eyeblink conditioning. (F) Representative Western analyses depicting GluN1, GluN2A and GluN2B NMDA receptor subunit expression following rough-and-tumble play. N = 6–12 per group. * P < .05 Fisher PLSD post hoc test vs. control.

Figure 2. The GluN2B subunit plays a functional role in positive emotional learning

Mean ± SEM rates of hedonic 50-kHz USVs in response to a conditioned stimulus that predicts heterospecific rough-and-tumble play in adult male rats pretreated with (A-B) the GluN2B preferring glycine site functional partial agonist GLYX-13 (1, 3, 10 mg/kg i.v.), (C) the NMDA receptor antagonist MK-801 (0.025, 0.1, .25 mg / kg i.p.), or (D) the GluN2B selective antagonist ifenprodil (10 mg/kg i.p.). Inset to Figure B depicts a sonogram of a representative hedonic 50-kHz USVs. N = 7-10 per group. * P < .05 Fisher PLSD post test vs. vehicle.

Figure 3. Animals selectively bred for low rates of hedonic 50-kHz USVs show deficits in positive emotional learning that are rescued by GLYX-13

Mean ± SEM rates of hedonic 50-kHz USVs in response to a conditioned stimulus that predicts heterospecific rough-and-tumble play in adolescent rats (A) selectively bred from low rates of 50-kHz USVs or wild type (random line) controls; (B) Low line rats pretreated with GLYX-13 (10, 50 mg/kg i.p.). N = 9–10 per group. * P < .05 Fisher PLSD post test vs. vehicle.

Figure 4. Medial prefrontal cortex injections of GLYX-13 increase positive emotional learning

Mean ± SEM Rates of Hedonic 50-kHz USVs in response to a conditioned stimulus that predicts heterospecific rough-and-tumble play in adult male rats pretreated with infralimbic / prelimbic cortex injections of (A) GLYX-13 (0.1, 1, 10 μg / side), (B) injection of a scrambled peptide variant of GLYX-13 (1 μg / side), or (C) injection of GLYX-13 (1 μg / side) into the primary or secondary motor cortex. (D) Anatomical location of the cannulae tips in the rat brain using the atlas of (Paxinos and Watson, 2007); medial prefrontal cortex, mostly prelimbic region (● ), Motor cortex (□).N = 9 - 11 per group. * P < .05 Fisher PLSD post test vs. vehicle.