Genetic enhancement of memory and long-term potentiation but not CA1 long-term depression in NR2B transgenic rats

Abstract

One major theory in learning and memory posits that the NR2B gene is a universal genetic factor that acts as rate-limiting molecule in controlling the optimal NMDA receptor's coincidence-detection property and subsequent learning and memory function across multiple animal species. If so, can memory function be enhanced via transgenic overexpression of NR2B in another species other than the previously reported mouse species? To examine these crucial issues, we generated transgenic rats in which NR2B is overexpressed in the cortex and hippocampus and investigated the role of NR2B gene in NMDA receptor-mediated synaptic plasticity and memory functions by combining electrophysiological technique with behavioral measurements. We found that overexpression of the NR2B subunit had no effect on CA1-LTD, but rather resulted in enhanced CA1-LTP and improved memory performances in novel object recognition test, spatial water maze, and delayed-to-nonmatch working memory test. Our slices recordings using NR2A- and NR2B-selective antagonists further demonstrate that the larger LTP in transgenic hippocampal slices was due to contribution from the increased NR2B-containing NMDARs. Therefore, our genetic experiments suggest that NR2B at CA1 synapses is not designated as a rate-limiting factor for the induction of long-term synaptic depression, but rather plays a crucial role in initiating the synaptic potentiation. Moreover, our studies provide strong evidence that the NR2B subunit represents a universal rate-limiting molecule for gating NMDA receptor's optimal coincidence-detection property and for enhancing memory function in adulthood across multiple mammalian species.

Figure 1. Production and Basic Characterizations of NR2B Transgenic Rats.

A: The construct for making NR2B transgenic rats. An 8.5 kb alpha-CaMKII promoter was used to drive NR2B overexpression. The small solid bar indicates the probes used for in situ hybridization, (*) indicates the probe for vector of 265-1 intron, (**) represents the probe within the NR2B encoding sequence. B: in situ hybridization using transgene-specific probe of 265-1. The transgene mRNA expression showed forebrain-specific expression pattern. No signal was detected in the wild-type brain. C: in situ hybridization using a probe detecting NR2B mRNA expression. D: Western blots measure the amount of NR2A, NR2B and NR1 expression in the rat brains. Hippocampus: HIP, Cortex: CX, Cerebellum: CB.

Figure 2. Enhancement of Novel Object Recognition Memory in Transgenic Rats.

A: Similar exploratory preference between transgenic and wild-type rats during training session of the novel object recognition tests. B: Enhanced exploratory preference in transgenic rats in 1-day and 3-day retention tests.

Figure 3. Enhanced Performance in the Hidden-platform Water Maze Task by NR2B Transgenic Rats.

A: Escape latency in water maze training. Transgenic rats spent the significant less time to find the platform during the second and third training sessions. B: At the end of the third training session, spatial memory function was tested under full cue condition. Transgenic and Wt rats exhibited equal amount of strong preference for the target quadrant under full-cue conditions (Student's t-test, *P<0.05). C: Place preference was further tested at the end of the third training session under the partial cue condition. Transgenic rats spent more time in the target quadrant than other quadrants, in comparison to that of wild-type rats.

Figure 4. Enhancement of Spatial Working Memory in Transgenic Rats.

A: NR2B transgenic rats exhibited the similar learning curve in comparison to that of controls during the T-maze spatial working memory test. The delay interval between the sample-run and trial-run is 15 seconds. B: Spatial working memories were tested using various intervals. Better performance in the transgenic rats was noted in the 3-min delay non-matching to place test. Data are expressed as mean±SEM. Asterisk, p<0.05; double asterisk, p<0.01; post hoc analysis.

Figure 5. Synaptic Transmission and Plasticity in the Transgenic Rat Hippocampus.

A: Normal CA3-CA1 input/output curve in Tg hippocampal slices. B: Normal paired-pulse facilitation at CA3-CA1 in Tg hippocampal slice. C: Tetanic stimulation induced larger LTP in transgenic slices than that of control. D: Low-frequency stimulation evoked the similar LTD between Tg and WT slices. Data were presented as the mean±SEM. Student's t-test was used for statistical analysis.

Figure 6. The Role of NR2B Subunit in Enhanced CA1-LTP in Transgenic Hippomcampal Slices.

A: Effects of NR2A-selective antagonist, NVP-AAM077, on wild-type slices. B: NVP-AAM077 also significantly reduced, but not completely blocked, CA1 LTP in transgenic slices. C: Statistical analysis shows the effects of NVP-AAM077 on LTP in both Tg and Wt slice. It indicates a significant involvement of NR2A-containing NMDARs in CA1-LTP induction in both Tg and WT slices. D: There was a significant, but small, effect of NR2B-selective antagonist, Ro25-6981, on CA1-LTP in wild-type slices. E: Ro25-6981 had much larger effect on CA1-LTP in transgenic slices, but it did not completely block LTP in transgenic slices. F: Summarized effects of Ro25-6981 on CA1-LTP in both Tg and Wt slice. G: Combination of NVP-AAM077 and Ro25-6981 completely blocked the formation of CA3-CA1 LTP completely in the wild-type slices. H: Complete blockage of CA1-LTP in transgenic slices by combined application of NVP-AAM077 and Ro25-6981. I: Statistical analysis shows the combined effects of NVP-AAM077 and Ro25-6981 on CA1-LTP. (Data were presented as the mean±SEM. Student's t-test was used for statistical analysis).