RGS14 is a natural suppressor of both synaptic plasticity in CA2 neurons and hippocampal-based learning and memory

Abstract

Learning and memory have been closely linked to strengthening of synaptic connections between neurons (i.e., synaptic plasticity) within the dentate gyrus (DG)-CA3-CA1 trisynaptic circuit of the hippocampus. Conspicuously absent from this circuit is area CA2, an intervening hippocampal region that is poorly understood. Schaffer collateral synapses on CA2 neurons are distinct from those on other hippocampal neurons in that they exhibit a perplexing lack of synaptic long-term potentiation (LTP). Here we demonstrate that the signaling protein RGS14 is highly enriched in CA2 pyramidal neurons and plays a role in suppression of both synaptic plasticity at these synapses and hippocampal-based learning and memory. RGS14 is a scaffolding protein that integrates G protein and H-Ras/ERK/MAP kinase signaling pathways, thereby making it well positioned to suppress plasticity in CA2 neurons. Supporting this idea, deletion of exons 2-7 of the RGS14 gene yields mice that lack RGS14 (RGS14-KO) and now express robust LTP at glutamatergic synapses in CA2 neurons with no impact on synaptic plasticity in CA1 neurons. Treatment of RGS14-deficient CA2 neurons with a specific MEK inhibitor blocked this LTP, suggesting a role for ERK/MAP kinase signaling pathways in this process. When tested behaviorally, RGS14-KO mice exhibited marked enhancement in spatial learning and in object recognition memory compared with their wild-type littermates, but showed no differences in their performance on tests of nonhippocampal-dependent behaviors. These results demonstrate that RGS14 is a key regulator of signaling pathways linking synaptic plasticity in CA2 pyramidal neurons to hippocampal-based learning and memory but distinct from the canonical DG-CA3-CA1 circuit.

Fig. 1.

RGS14 is enriched in hippocampal CA2 neurons. (A and B) Fixed paraffin-embedded mouse brain sections stained with anti-RGS14 monoclonal antibody (brown, DAB staining with specific anti-RGS14 monoclonal antibody) and counterstained with hematoxylin (blue). (C) Elimination of RGS14 staining by preadsorption of antibody with purified recombinant RGS14 protein. (Scale bar, 400 μm.) (D) RGS14 expression in CA2 neurons, (E) in a discrete subset of CA1 neurons, and (F) in neurons within the fasciola cinerea (FC). (G–I) Electron micrographs of RGS14-immunoreactive (red arrows). (G) Dendritic shaft (de), (H) spine (sp) neck, and (I) spine head in the stratum oriens region of mouse CA2 hippocampus. (Scale bar, 0.2 μm.) DAB staining with RGS14 antibody. Other recognized structures are mitochondria (mit).

Fig. 2.

Loss of RGS14 allows for induction of nascent LTP in CA2 neurons following high-frequency stimulation that is blocked by a specific MEK inhibitor. (A Upper) PCR genotyping. Multiplex PCR shows single larger band for wild-type (WT) genomic DNA, two bands for heterozygous RGS14(+/−) genomic DNA, and a single lower band for knockout RGS14(+/−) (RGS14-KO) genomic DNA, indicating loss of RGS14 gene and insertion of lacZ/neo cassette. (A Lower) RT-PCR analysis of RGS14 mRNA. No mRNA product seen for any of the RT-PCR primers used in RGS14-KO. (B) Protein immunoblot for RGS14 protein using a specific anti-RGS14 monoclonal antibody. Lane 1, WT RGS14 (+/+) brain lysates; lane 2, RGS14-KO brain lysates. (C) For induction of LTP, CA2 neurons were stimulated (2 × 1 s, 100 Hz, 20-s intervals) and excitatory postsynaptic currents (EPSCs) measured WT (n = 5 mice, 22 neurons) and RGS14-KO (n = 6 mice, 24 neurons). Plotted are means ± SEM. (D) For inhibition of LTP in CA2 neurons by treatment with a MEK inhibitor, experiments on individual CA2 neurons from RGS14-KO mice were performed as in C except that 500 nM U0126 was included in the bath solution (n = 7 with U0126 and n = 9 without). Plotted are means ± SEM. (E) For induction of LTP in CA1, hippocampal slices from WT and RGS14-KO mice were stimulated (2 × 1 s, 100 Hz, 20-s intervals) and postsynaptic neurotransmission was monitored every 15 s for 180 min. Data are pooled (mean ± SD); WT (n = 10), RGS14-KO (n = 8). (F) For inhibition of LTP in CA1 neurons by treatment with a MEK inhibitor, experiments on individual CA1 neurons from RGS14-KO mice were performed as in C except that 500 nM U0126 was included in the bath solution (n = 7 with U0126 and n = 4 without). Plotted are means ± SEM.

Fig. 3.

Loss of RGS14 enhances hippocampal-based spatial learning and object working memory. (A and B) Novel object recognition task: (A) Percentage of total time spent exploring and (B) percentage of total contacts made on two objects for 5 min during training, and memory for object at 4 and 24 h after training (paired t test, *P < 0.01; n = 34, WT; n = 20, RGS14-KO). (C) Morris water maze task: latency for WT and RGS14-KO mice to reach a hidden platform in acquisition trials over 5 d (two factor, repeated measures ANOVA; *P < 0.01). (D) Probe trial on day 6 for WT and RGS14-KO mice; time spent in each quadrant with escape platform removed. (E) Average swim speed over 5 d of acquisition training (C–E: n = 17, WT; n = 16, RGS14-KO).