Article Figures & Data
Figures
- Figure 1.
RGS14 KO mice exhibit plasticity of CA2 synapses into adulthood. A, Amigo2-EGFP fluorescence (green) labels CA2 pyramidal neurons as shown by overlap with another CA2 molecular marker PCP4 (red). Scale bar = 100 μm. B, Amigo2-EGFP fluorescence (green) does not colocalize with immunoreactivity for the CA1 pyramidal neuron marker WFS1 (magenta). Scale bar = 100 μm. C, Summary graph of field potential recordings from acute hippocampal slices prepared from adult RGS14 WT and KO mice (both Amigo2-EGFP+) validate that RGS14 KO mice possess a capacity for LTP in CA2 in adulthood (red), which is absent in WT mice (purple). RGS14 WT and KO mice do not differ in CA1 plasticity (green, gray). LTP was induced by high-frequency stimulation (HFS; 3 × 100 Hz) at time 0 (arrow). Data are represented as mean normalized fEPSP slope ± SEM (dotted reference line at 1.0). Sample sizes (in slices/animals) are WT CA2 n = 14/5; KO CA2 n = 16/4; WT CA1 n = 17/4; KO CA1 n = 12/5. Insets (top) are representative traces of field potentials recorded from areas CA2 and CA1 from slices of RGS14 WT and KO mice before (light line) and after (heavy line) LTP induction. D, Quantification of the mean normalized fEPSP slope 40-60 min following LTP induction (C) with error bars representing SEM. The difference in the degree of LTP induced in RGS14 WT and KO CA2 synapses was significant, whereas no difference was detected between RGS14 WT and KO synapses recorded in CA1. Sidak’s, **, p ≤ 0.01, ns = not significant.
- Figure 2.
The nascent CA2 LTP in RGS14 KO mice requires Ca2+-activated pathways. A, Summary graph of LTP induction experiments performed in area CA2 of RGS14 KO (Amigo2-EGFP+) mice either in the presence (blue) or absence (gray) of bath-applied NMDA receptor antagonist APV (50 μM). LTP was induced by HFS (3 × 100 Hz) in stratum radiatum at time 0 (arrow). Data are represented as mean normalized fEPSP slope ± SEM (dotted reference line at 1.0). Insets (top) are representative traces of field potentials recorded in CA2 stratum radiatum of RGS14 KO mice before (light line) and after (heavy line) LTP induction. B, Summary graph of LTP induction experiments performed in area CA2 of RGS14 KO (Amigo2-EGFP+) mice either in the presence (yellow) or absence (gray) of bath-applied CaMK inhibitor KN-62 (10 μM). C, Summary graph of LTP induction experiments performed in area CA2 of RGS14 KO (Amigo2-EGFP+) mice either in the presence (blue) or absence (gray) of bath applied PKA inhibitor PKI (1 μM). D, Bar graph displaying the mean normalized field potential slope (mV s−1) from data shown in A–C, at 40–60 min following LTP induction with error bars representing SEM. Sample sizes (in slices/animals) are for APV: drug n = 14/10, KO control n = 13/8. For KN-62: drug n = 19/9, KO control n = 16/9. For PKI: drug n = 18/10, KO control n = 16/10. Sample sizes are the same for averaged time courses of each group in A–C. Each inhibitor was compared with paired KO CA2 controls by unpaired two-tailed t test; **, p < 0.01; *, p < 0.05. E, Signaling diagram of a CA2 spine from a RGS14 KO mouse depicting mechanistic targets of the pharmacological inhibitors used in A–C.
- Figure 3.
RGS14 suppresses CA2 spine structural plasticity. A, Representative post hoc immunostaining to delineate hippocampal region CA2 region after imaging biolistically transfected neurons. Left: Organotypic hippocampus slice culture stained for the DG- and CA2-enriched gene PCP4 (red). Scale bar = 100 µm. Right: Magnified view of area CA2 in PCP4 immunostained (red) hippocampus on left with a biolistically labeled CA2 pyramidal neuron expressing mEGFP (green). Scale bar = 50 µm. B, Averaged time course of spine volume change during the induction of spine structural plasticity (sLTP) by repetitive two-photon glutamate uncaging (top bar; 30 pulses at 0.5 Hz) in the absence of extracellular Mg2+. The number of samples (spines/neurons/animals) for stimulated spines are 19/6/5 for WT CA2, 15/8/6 for KO CA2, 23/7/5 for WT CA1, and 16/6/5 for KO CA1. Sample size applies to B and C. Error bars denote SEM. C, Quantification of the average volume change for stimulated spines during the transient (1–3-min) and sustained (21–25-min) phases of sLTP induction. Sidak’s, *, p < 0.05. D, Representative two-photon fluorescence images of dendritic spines during sLTP induction in mEGFP-expressing hippocampal pyramidal neurons. Arrowheads indicate location of glutamate uncaging. Scale bar = 1 µm.
- Figure 4.
RGS14 restricts Ca2+ levels in CA2 spines. A, Averaged time courses of spine Ca2+ transients measured with Fluo-4FF during two-photon glutamate uncaging to induce sLTP (30 pulses, 0.5 Hz). Data are shown as the average change in spine Ca2+ concentration (Δ[Ca2+]) from CA2 pyramidal neurons. B, Averaged time courses of spine Ca2+ transients measured with Fluo-4FF and Alexa Fluor 594 during two-photon glutamate uncaging to induce sLTP (30 pulses, 0.5 Hz). Data are shown as the average change in spine Ca2+ (Δ[Ca2+]) from CA1 pyramidal neurons. C, Uncaging-triggered averages for the change in spine Ca2+ concentration during sLTP induction. Error bars denote SEM. D, RGS14 limits CA2 spine Ca2+ transients. Bar graphs displaying the average peak Δ[Ca2+] ± SEM (****, p < 0.0001). The number of samples (spines/neurons/animals) are 40/5/4 for WT CA2, 17/3/3 for KO CA2, 16/3/3 for WT CA1, and 10/2/2 for KO CA1. E, Normalized uncaging-triggered averages for the change in spine Ca2+ during sLTP induction reveal similar Ca2+ decay kinetics for RGS14 WT and KO CA2 neurons. Error bars denote SEM. WT CA2 average tau is 0.212 s; KO CA2 average tau is 0.240 s.
- Figure 5.
RGS14 expression blocks long-term spine plasticity in CA2 and CA1 neurons lacking RGS14, and high extracellular Ca2+ restores structural plasticity to RGS14-expressing neurons. A, Averaged time course of spine volume change in the presence or absence of RGS14 during the induction of spine structural plasticity by repetitive two-photon glutamate uncaging in the absence of extracellular Mg2+ in CA2 pyramidal neurons from RGS14 KO mice. The number of samples (spines/neurons/animals) for stimulated spines are 11/4/3 for GFP (black) and 11/4/3 for RGS14-GFP (red). B, Quantification of the average volume change for stimulated spines either in the presence or absence of RGS14 during the transient (1–3-min) and sustained (21–25-min) phases of sLTP induction for CA2 neurons. Unpaired t test, **, p = 0.01. C, Averaged time course of spine volume change during the induction of spine structural plasticity in the presence or absence of RGS14 by repetitive two-photon glutamate uncaging in the absence of extracellular Mg2+ in CA1 pyramidal neurons from RGS14 KO mice. The number of samples (spines/neurons/animals) for stimulated spines are 11/4/4 for GFP (black) and 23/9/6 for RGS14-GFP 4mM external [Ca2+] (red), and 9/4/4 for RGS14-GFP external 8mM [Ca2+] (blue). D, Quantification of the average volume change for stimulated spines in the presence or absence of RGS14 during the transient and sustained phases of sLTP induction for CA1 neurons. Fisher’s LSD test, *, p < 0.05; **, p < 0.01; ***, p < 0.001. Error bars, SEM. Dotted reference line drawn at 0% volume change.
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