Forebrain NR2B overexpression facilitating the prefrontal cortex long-term potentiation and enhancing working memory function in mice

Abstract

Prefrontal cortex plays an important role in working memory, attention regulation and behavioral inhibition. Its functions are associated with NMDA receptors. However, there is little information regarding the roles of NMDA receptor NR2B subunit in prefrontal cortical synaptic plasticity and prefrontal cortex-related working memory. Whether the up-regulation of NR2B subunit influences prefrontal cortical synaptic plasticity and working memory is not yet clear. In the present study, we measured prefrontal cortical synaptic plasticity and working memory function in NR2B overexpressing transgenic mice. In vitro electrophysiological data showed that overexpression of NR2B specifically in the forebrain region resulted in enhancement of prefrontal cortical long-term potentiation (LTP) but did not alter long-term depression (LTD). The enhanced LTP was completely abolished by a NR2B subunit selective antagonist, Ro25-6981, indicating that overexpression of NR2B subunit is responsible for enhanced LTP. In addition, NR2B transgenic mice exhibited better performance in a set of working memory paradigms including delay no-match-to-place T-maze, working memory version of water maze and odor span task. Our study provides evidence that NR2B subunit of NMDA receptor in prefrontal cortex is critical for prefrontal cortex LTP and prefrontal cortex-related working memory.

Figure 1. Diagram of the Odor Span Task.

At span 0, mice are first presented with a random scented cup buried 2 pellets (e.g., A+). After consumption of the reward, the mouse and the cup were removed. At span 1, a second new scented cup with 2 pellets (e.g., B+) and another cup refilled with A odor woodchip without reward (e.g., A-) was pseudo-randomly relocated in the box. Mice were return to the box and were required to remember odor A and to dig at the cup with the new B odor. Then, additional cups of woodchip scented with different odors were placed in the same manner until 12 cups (span 11) were presented.

Figure 2. Synaptosome NR2B-receptor Protein in Prefrontal Cortex.

Analysis of OD value show that the relative quantity of NR2B-receptor protein of prefrontal cortex in Tg and Wt mice is 0.5±0.1 and 0.13±0.02, respectively (p<0.05, Student's t-test).

Figure 3. NR2B Overexpression Enhanced LTP but not Basal Transmission and LTD in prefrontal cortex.

A: No significant difference in input-output curve between Tg and Wt slices. B: No significant difference in pair-pulse responses between Tg and Wt slices. C: LTP induced by tetanic stimulations in Tg slices were significantly larger than that of Wt slices. D: LTD induced by a low frequency stimulation in Tg slices were not significant different from that of Wt slices. All data are presented as mean ± SEM. Statistical differences were evaluated with student's t -test.

Figure 4. The Role of NR2B Subunit in Enhanced Prefrontal-LTP in Transgenic Slices.

A: NR2A-selectice antagonist (NVP-AAM077) reduced prefrontal cortex l LTP in Wt slices. B: NR2B-selectice antagonist (Ro25-6981) also reduced prefrontal cortex LTP in Wt slices. C: Effect of NVP-AAM077 on prefrontal cortex LTP in Tg slices. D: Ro25-6981 had much larger effect on prefrontal cortex LTP in Tg slices. E: Statistical analysis shows the effects of NVP-AAM077 on prefrontal cortical LTP in both Tg and Wt slice, it indicates a significant involvement of NR2A subunits in prefrontal cortex LTP of both Tg and Wt slices. F: Statistical analysis shows the effects of Ro25-6981 on prefrontal cortex LTP in both Tg and Wt slice, suggesting a significant involvement of NR2B subunits in prefrontal cortex LTP of both Tg and Wt slices. All values are mean ± SEM. Statistical differences were evaluated with Student's t –test (A, B, C, D) and Tukey's HSD post-hoc test (E, F)(*denotes p<0.05, **denotes P<0.01).

Figure 5. Enhancement of Spatial Working Memory in NR2B Transgenic Mice.

A–B: Performance of mice in T-maze task. A: There was no difference in accuracy between Wt and Tg mice in training session. B: Tg mice exhibited superior performance both in 1- and 3 min-delay retention test. C–D: Performance of mice in the working memory version of water maze task. C: There was no difference in swim speed between Wt and Tg mice in pre-training. D: In the 2^nd trial of training, the latency of transgenic mice was significantly shorter than that of wild type. All values are mean ± SEM (**denotes p<0.01 when compared to Wt controls).

Figure 6. Enhancement of Non-spatial Working Memory in NR2B Transgenic Mice.

A: Performance of the odor span task by transgenic mice and their control littlemate was compared over successive training 16 days. The Tg mice showed significantly improved performance on training days 7,10,11,12 (*p<0.05). B–E: the effect of genotype on stable performance (sessions 13–16) was assessed after Wt and Tg mice reached to a stable performance at sessions 13–16, a significant difference between the two groups was observed in span length (B), % accuracy (C) and error (D), but not in mean span length (E). F: In the no reward probe, the mean span length of each group was comparable with the mean span length of each group across across 11 sessions after the acquisition period (sessions 6–16). All values are mean ± SEM (*denotes p<0.05 when compared to Wt controls).