Functional Uncoupling NMDAR NR2A Subunit from PSD-95 in the Prefrontal Cortex: Effects on Behavioral Dysfunction and Parvalbumin Loss after Early-Life Stress

Abstract

Exposure to early-life stress increases vulnerability to psychiatric disorders, including depression, schizophrenia, and anxiety. Growing evidence implicates aberrant development of the prefrontal cortex (PFC) in the effects of early-life stress, which often emerge in adolescence or young adulthood. Specifically, early-life stress in the form of maternal separation (MS) in rodents has been shown to decrease parvalbumin (PVB)-positive interneurons in the adolescent PFC; however, the mechanism underpinning behavioral dysfunction and PVB loss is not yet known. We recently reported that MS causes overexpression of the NMDA subunit NR2A in the PFC of adolescent rats. Elevated PFC NR2A is also found in developmental models of schizophrenia and is correlated with behavioral deficits, acting largely through its association with the postsynaptic protein PSD-95. In addition, adolescent maturation of PVB-positive interneurons relies on NR2A-driven NMDA activity. Therefore, it is possible that the NR2A/PSD-95 signaling complex has a role in adolescent MS effects. Here, we aimed to determine whether a discrete manipulation of PFC NR2A could prevent MS effects on PFC-controlled behaviors, including cognition, anxiety, and novelty-induced hyperlocomotion, as well as PVB loss in adolescence. We intracranially infused the NR2A-specific blocking peptide TAT2A in order to uncouple NR2A from PSD-95 in the early-adolescent PFC, without antagonizing the NMDA receptor. We demonstrated that MS rats treated with TAT2A during early adolescence were protected from MS-induced PVB loss and exhibited less anxious behavior than those infused with control peptide. These data implicate NR2A-related N-methyl-D-aspartate receptor development in adolescent behavioral and neural consequences of early-life stress.

Figure 1

Western blots show that maternal separation (MS) results in overexpression of PSD-95 (a) with a slight but not significant overexpression of NR2A (b) in the medial prefrontal cortex (PFC) during adolescence. *p<0.05 difference between groups; n=6. (c) NR2A optical density (OD) was positively correlated with PSD-95 expression in the medial PFC (R²=0.471; p=0.020). Subjects from control (CON) and MS are represented as different symbols. (d) Immunohistochemistry reveals that NR2A+ cells are increased in MS-exposed animals; *p<0.05 difference between groups; n=7. Representative photomicrographs are shown of an individual NR2A+ cell with NR2A alone, a DAPI-stained nucleus, and an overlay; scale bars, 10 μm. All photomicrographs are taken from the plPFC. (e) Treatment with TAT2A successfully uncoupled NR2A from PSD-95, as illustrated by representative co-immunoprecipitation blots from subjects treated with either 500 μM TAT2A, 100 μM TAT2A, or TAT-SDV CON peptide). Of note, TAT2A 500 and TAT2A 100 were run on separate gels, therefore CON peptides are shown from each respective gel. (f) Relative levels of co-immunoprecipitated PSD-95 (relative to NR2A) from TAT2A (100 and 500 μM)-treated subjects and TAT-SDV subjects; n=3. NR2A-IR, NR2A-immunoreactive.

Figure 2

(a) Treatment with 500 μM TAT2A, but not 100 μM TAT2A, prevented anxiety-like behavior in maternal separation (MS) adolescents, as illustrated by less time spent in the open arms of an elevated plus maze (EPM). *p<0.05 difference from control (CON) group with same treatment; n=7–8. (b) Number of arm crosses over 5 min in the EPM was not affected by rearing group or treatment. (c) Treatment with either 500 μM TAT2A or 100 μM TAT2A prevented anxiety-like behavior in MS adolescents, as illustrated by fewer visits to the center of an open field. *p<0.05 difference from CON group; letters (a,b) represent differences between treatments within the same Rearing group. n=7–8. (d) MS adolescents displayed less time spent in the center of an open field, which was driven by TAT-SDV-treated animals. However, no rearing group × treatment interaction was observed.

Figure 3

(a) Maternal separation (MS) results in increased novelty-induced locomotion during adolescence, which was not prevented by TAT2A treatment. *p<0.05 difference from control (CON) group. n=7–8 (b) General locomotion in a familiar field was not affected by either rearing condition or treatment.

Figure 4

Recognition of a novel object was not affected by rearing group or treatment. n=7–8. CON, control; MS, maternal separation.

Figure 5

Treatment with 500 μM TAT2A during early adolescence prevented an maternal separation (MS)-induced decrease in parvalbumin (PVB+) interneurons within the infralimbic prefrontal cortex (PFC) and prelimbic PFC. *p<0.05 difference from control (CON) group with same treatment. Data are presented as % difference from TAT-SDV-treated CON subjects. n=7–8. Bottom: representative photomicrographs of PVB-IR in CON and MS subjects treated with TAT-SDV and an MS subject treated with 500 μM TAT2A. Scale bar, 100 μm.