Mechanisms of mGluR-dependent plasticity in hippocampal area CA2

Abstract

Pyramidal cells in hippocampal area CA2 have synaptic properties that are distinct from the other CA subregions. Notably, this includes a lack of typical long-term potentiation of stratum radiatum synapses. CA2 neurons express high levels of several known and potential regulators of metabotropic glutamate receptor (mGluR)-dependent signaling including Striatal-Enriched Tyrosine Phosphatase (STEP) and several Regulator of G-protein Signaling (RGS) proteins, yet the functions of these proteins in regulating mGluR-dependent synaptic plasticity in CA2 are completely unknown. Thus, the aim of this study was to examine mGluR-dependent synaptic depression and to determine whether STEP and the RGS proteins RGS4 and RGS14 are involved. Using whole cell voltage-clamp recordings from mouse pyramidal cells, we found that mGluR agonist-induced long-term depression (mGluR-LTD) is more pronounced in CA2 compared with that observed in CA1. This mGluR-LTD in CA2 was found to be protein synthesis and STEP dependent, suggesting that CA2 mGluR-LTD shares mechanistic processes with those seen in CA1, but in addition, RGS14, but not RGS4, was essential for mGluR-LTD in CA2. In addition, we found that exogenous application of STEP could rescue mGluR-LTD in RGS14 KO slices. Supporting a role for CA2 synaptic plasticity in social cognition, we found that RGS14 KO mice had impaired social recognition memory as assessed in a social discrimination task. These results highlight possible roles for mGluRs, RGS14, and STEP in CA2-dependent behaviors, perhaps by biasing the dominant form of synaptic plasticity away from LTP and toward LTD in CA2.

Keywords: RGS14; hippocampus; long-term depression; social recognition memory; synaptic plasticity.

FIGURE 1

DHPG induces LTD in CA2 in P13‐P21 mice. (a) Normalized PSC amplitudes recorded in CA2 or CA1 following 100 μM DHPG application. Data points represent the mean PSC amplitude. Error bars represent the standard error of the mean. Red circles represent recordings from CA2 pyramidal cells (n = 10). Blue circles represent recordings from CA1 (n = 8). Gray shaded area indicates duration of DHPG application. (b) Example PSC traces from CA2 and CA1 pyramidal cells before and 40 min after completion of DHPG application. Black traces represent PSC response before DHPG application. Above: red trace indicates PSC response of CA2 after DHPG application. Below: blue trace represents PSC response of CA1 after DHPG application. (c) Normalized PSC amplitude of CA2 (red) and CA1 (blue) cells at 41–50 min during recovery. Asterisk denotes significant difference (p ≤ .05) when compared with mean PSC amplitude before DHPG application. Double asterisk denotes significant difference in PSC amplitude when comparing CA2 and CA1 pyramidal cells. (d) Normalized PSC amplitude recorded in CA2 or CA3 following 100 μM DHPG application. Red circles represent recordings from CA2 pyramidal cells (n = 9). Magenta circles represent recordings from CA3 (n = 6). (e) Example PSC traces from CA2 and CA3 pyramidal cells before and 40 min after completion of DHPG application. Black traces represent PSC response before DHPG application. Above: red trace indicates PSC response of CA2 after DHPG application. Below: Magenta trace represents PSC response of CA3 after DHPG application. (f) Normalized PSC amplitude of CA2 (red) and CA3 (magenta) cells at 41–50 min during recovery.

FIGURE 2

RGS14, but not RGS4, is required for group I mGluR LTD in CA2. (a) Normalized PSC amplitude following 100 μM DHPG application. Red circles represent PSC amplitude of CA2 pyramidal cells from C57BL/6J wildtype (WT) control mice (n = 8). Pink circles represent PSC amplitude of CA2 pyramidal cells in RGS14 KO mice (n = 7). Gray shaded area indicates duration of DHPG application. (b) Example PSC traces from CA2 pyramidal cell before and after DHPG application. Black traces represent PSC response before DHPG application. Above: red trace indicates PSC response after DHPG application in CA2 cell from a WT control mouse. Below: pink trace represents response after DHPG application from an RGS14 KO mouse. (c) Normalized PSC amplitude in CA2 WT control (red) and RGS14 KO mice (pink) at 41–50 min from drug onset. White circles represent mean PSC amplitude from individual cells. Single asterisks denote significant difference in PSC amplitude when compared with baseline (p < .05). Double asterisks denote significant difference in normalized PSC amplitude when comparing WT control (red) and RGS14 KO (pink) mice. Panels d–f as in a–c except with recordings from CA2 pyramidal cells from WT control mice (red, n = 6) and RGS4 KO mice (green, n = 5).

FIGURE 3

NMDAR‐LTD in CA2 is unaffected by RGS14 knockout. (a) Normalized PSC amplitude following Low Frequency Stimulation (LFS; 900 pulses at 1 Hz, indicated by the bar). Red circles represent PSC amplitude of CA2 pyramidal cells from WT control mice (n = 6). Green circles represent PSC amplitude of CA2 pyramidal cells in WT control mice with inclusion of D‐AP5 in the bath (n = 7). (b) Example PSC traces from CA2 pyramidal cell before and after LFS. Black traces represent PSC response before LFS. Above: red trace indicates PSC response after LFS in a CA2 cell. Below: green trace represents response after LFS with D‐AP5. (c) Normalized PSC amplitude in CA2 WT control mice without D‐AP5 (red) or with D‐AP5 (green) at 46–55 min from LFS onset. White circles represent mean PSC amplitude from individual cells. Single asterisks denote significant difference in PSC amplitude when compared with baseline (p < .05). Double asterisks denote significant difference in normalized PSC amplitude when comparing without (red) and with (green) D‐AP5. Panels d–f as in a–c except with recordings from CA2 pyramidal cells from RGS14 KO mice without D‐AP5 (pink, n = 7) and RGS4 KO mice with D‐AP5 (green, n = 5).

FIGURE 4

mGluR LTD in CA2 requires STEP. (a) Normalized PSC amplitude following 100 μM DHPG application. Pink circles represent PSC amplitude of CA2 pyramidal cells from RGS14 KO mice with inactive STEP in the intracellular filling solution (n = 6). Gray circles represent PSC amplitude of CA2 pyramidal cells from RGS14 KO mice with active STEP in intracellular filling solution (n = 7). Gray shaded area indicates duration of DHPG application. (b) Example PSC traces from a CA2 pyramidal cell before and after DHPG application. Black traces represent PSC response before DHPG application. Above: Pink trace indicates PSC response after DHPG application in an RGS14 KO mice with inactive STEP. Below: Gray traces represents response after DHPG application in an RGS14 KO mice with active STEP in intracellular solution. (c) Normalized PSC amplitude in RGS14 KO (pink) and RGS14 KO with STEP in intracellular solution (gray) at 41–50 min after onset of DHPG application. White circles represent mean PSC amplitude from individual cells. Single asterisks denote significant difference in PSC amplitude when compared with baseline (p < .05). Double asterisks denote significant difference in normalized PSC amplitude when comparing RGS14 KO with inactive STEP with RGS14 KO with active STEP in the intracellular solution. Panels d–f as in a–c except with recordings from CA2 pyramidal cells from WT control mice (red, n = 7) and STEP KO mice (magenta, n = 8). Double asterisks denote significant difference in normalized PSC amplitude when comparing data from WT control mice with data from STEP KO mice.

FIGURE 5

STEP is constitutively active and can regulate synaptic strength in CA2. (a,b) Coronal sections from a C57BL/6J mouse showing the hippocampus stained with total STEP or with active, non‐phosphorylated STEP. Scale bars = 100 μm. (c) Normalized PSC amplitude following 15 μM PAO application. Data points represent the mean PSC amplitude. Error bars represent the standard error of the mean. Red circles represent PSC amplitude of CA2 pyramidal cells (n = 8) and blue circles represent PSC amplitude of CA1 pyramidal cells (n = 6) from C57BL/6J mice. Black line indicates duration of PAO application. (d) Example PSC traces from CA2 and CA1 pyramidal cells before and after PAO application. Black traces represent PSC response before PAO application. Above: Red trace indicates PSC response of CA2 cell after 30 min of PAO application. Below: Blue trace represents response of CA1 cell after PAO application. (e) Normalized PSC amplitude in CA2 (red) and CA1 (blue) at 21–30 min after start of PAO. White circles represent mean PSC amplitude from individual cells. Single asterisk denotes significant difference when compared with baseline (p ≤ .05). Panels f–h as in c–e, but with recordings from CA2 pyramidal cells from WT control and STEP KO mice. Red circles represent PSC amplitude of CA2 pyramidal cells from WT control mice (n = 5). Magenta circles represent PSC amplitude of CA2 pyramidal cells from STEP KO mice (n = 4).

FIGURE 6

Group I mGluR LTD in CA2 requires protein synthesis. (a) Normalized PSC amplitude following 100 μM DHPG application. Red circles represent PSC amplitude of CA2 pyramidal cells from C57BL/6J mice (n = 9). Green circles represent PSC amplitude of CA2 pyramidal cells in the presence of protein synthesis inhibitor anisomycin (n = 6). Gray shaded area indicates duration of DHPG application. (b) Example PSC traces from CA2 pyramidal cell before and after DHPG application. Black traces represent PSC response before DHPG application. Above: Red trace indicates PSC response after DHPG application in CA2 cells with vehicle only. Below: Green traces represents response after DHPG application in the presence of anisomycin. (c) Normalized PSC amplitude in CA2 with either vehicle (0.1% DMSO) (red) or anisomycin (green) at 50–59 min after DHPG onset. Single asterisk denotes significant difference when compared with baseline (p ≤ .05). Double asterisks denote significant difference in normalized PSC amplitude when comparing without (red) and with (green) anisomycin.

FIGURE 7

CaMKII interacts with RGS14, but does not prevent mGluR‐LTD in RGS14 KO. (a), Coronal section from a C57BL/6J mouse showing the hippocampus co‐stained with RGS14 (pink) and CaMKII alpha (green). The merged image is shown on the right. Scale bar = 100 μm. (b–d) Coronal section from a C57BL/6J mouse showing duolink staining where signal (red) denotes interaction between two proteins targeted with primary antibodies: (b) negative control using primary antibodies targeting NeuN and Arc (proteins with minimal interaction); (c) positive control primary antibodies targeting β‐actin and Arc (proteins known to interact); and (d), using primary antibodies targeting RGS14 and CaMKII to test for protein interaction. Images in b and c taken from the CA1 region of hippocampus, and the image in d was taken from the CA2 region. Scale bar = 25 μm. (e) Normalized PSC amplitude following 100 μM DHPG application. Pink circles represent PSC amplitude of CA2 pyramidal cells in RGS14 KO mice (n = 7). Brown circles represent PSC amplitude of CA2 pyramidal cells in RGS14 KO mice with 10 μM of the CaMKII inhibitor KN‐62 in the bath (n = 4). Gray shaded area indicates duration of DHPG application. (f) Example PSC traces from a CA2 pyramidal cell before and after DHPG application. Black traces represent PSC response before DHPG application. Above: Pink trace indicates PSC response after DHPG application in the RGS14 KO mice. Below: Brown trace is a response after DHPG application in the RGS14 KO mice with KN‐62. (g) Normalized PSC amplitude in RGS14 KO (pink) and RGS14 KO with KN‐62 (brown) at 41–50 min after DHPG onset. White circles represent mean PSC amplitude from individual cells.

FIGURE 8

Social discrimination is impaired in RGS14 KO mice. (a) Illustration showing the experimental layout. Mice are presented with either a novel mouse or a familiar cagemate and allowed to freely investigate within the arena. (b) Example heatmaps from an individual WT control mouse or an RGS14 KO mouse. Red represents greater time spent interacting. (c) Discrimination ratios from WT control mice (red) and RGS14 KO mice (pink). White circles represent discrimination ratios from individual animals. Asterisk denotes significant difference between WT control and RGS14 KO (p ≤ .05).