Group I metabotropic glutamate receptor signaling via Galpha q/Galpha 11 secures the induction of long-term potentiation in the hippocampal area CA1

Abstract

Heterotromeric G-proteins of the Gq family are thought to transduce signals from group I metabotropic glutamate receptors (mGluRs) in central neurons. We investigated roles of this cascade in hippocampal long-term potentiation (LTP) by using null-mutant mice lacking the alpha subunit of Gq (Galphaq) or G11 (Galpha11). We found no obvious abnormalities in the morphology, layer structure, expression of NMDA receptors, and basic parameters of excitatory synaptic transmission in the hippocampus of Galphaq mutant mice. We used theta burst stimulation (TBS) (3-10 burst trains at 5 Hz; each train consisted of five stimuli at 100 Hz) to induce LTP at Schaffer collateral to CA1 pyramidal cell synapses. Conventional TBS with 10 burst trains induced robust LTP in wild-type, Galphaq mutant, and Galpha11 mutant mice. Weak TBS with three burst trains consistently induced LTP in wild-type mice. In contrast, the same weak TBS was insufficient to induce LTP in Galphaq and Galpha11 mutant mice. In wild-type mice, the LTP by weak TBS was abolished by inhibiting group I mGluR or protein kinase C (PKC) but not by blocking muscarinic acetylcholine receptors. Prior activation of group I mGluR by an agonist significantly enhanced the LTP by weak TBS in wild-type mice. However, this priming effect was absent in Galphaq mutant mice. These results indicate that the signaling from group I mGluR to PKC involving Galphaq/Galpha11 does not constitute the main pathway for LTP, but it secures LTP induction by lowering its threshold in the hippocampal area CA1.

Fig. 1.

Histology and distributions of NMDA receptor subunits are normal in the Gαq mutant hippocampus.A–D, Wild-type mice; E–H, Gαq mutants. A, E, Hematoxylin staining. B, F, GluRε1 subunit. C, G, GluRε2 subunit.D, H, GluRξ1 subunit. CA1, CA1 region;DG, dentate gyrus; Gr, granule cell layer; LM, stratum lacunosum-moleculare;Mo, molecular layer; Or, stratum oriens;Py, pyramidal cell layer; Ra, stratum radiatum. Scale bars, 100 μm.

Fig. 2.

Basic properties of excitatory synaptic transmission of Gαq mutant are normal. A, The input–output relationships of fEPSPs in wild-type (○;n = 7) and Gαq mutant (●; n= 5) mice. The fEPSPs were evoked by single stimulation of the Schaffer collateral. Insets are typical responses. Calibration: 2 mV, 5 msec. B, Paired-pulse ratio of fEPSPs in wild-type (○; n = 5) and Gαq mutant (●;n = 5) mice. The initial slope of the fEPSP was measured to quantify the strength of the synaptic response. The ratio of the second to the first response is plotted against the interstimulus interval. Calibration: 1 mV, 20 msec. C, The ratio of amplitudes of the NMDA EPSC (recorded at a holding potential of +40 mV) to those of the AMPA EPSC (recorded at a holding potential of −70 mV) in wild-type (open column;n = 10) and Gαq mutant (filled column; n = 7) mice. The NMDA EPSC was recorded in the presence of CNQX (25 μm). Sample traces are shown in the top panel. Calibration: 20 pA, 20 msec.D, The current–voltage relationship of NMDA synaptic currents in wild-type (○; n = 8) and Gαq mutant (●; n = 6) mice. The amplitude of the EPSC is normalized to the value obtained at −30 mV.

Fig. 3.

Normal LTP induced by conventional stimulation protocols in the Gαq mutant hippocampus.A, The averaged time course of LTP induced by 10 trains of TBS in the area CA1 of wild-type (n = 10) and Gαq mutant (n = 10) mice. Initial EPSP slopes were measured, and the values were normalized in each experiment using the averaged slope value measured during the control period (time, −10 to 0 min). The TBS was applied at time 0 (downward arrow). In the following figures, the averaged time course of LTP is illustrated similarly. B, The averaged time course of LTP induced by tetanic stimulation (100 Hz, 1 sec) at the associational/commissural (asoc/com)-CA3 synapses in wild-type (n = 10) and Gαq mutant (n = 8) mice. C, The averaged time course of LTP induced by tetanic stimulation (100 Hz, 1 sec) at the mo–CA3 synapses in wild-type (n = 9) and Gαq mutant (n = 8) mice. Records were taken in the presence of AP-5 (25 μm) to block NMDA receptors. Mossy fibers were stimulated via a bipolar electrode placed in the dentate hilus.

Fig. 4.

The threshold for LTP induction is elevated in the area CA1 of Gαq mutant mice. A weak TBS (3 trains) applied at time 0 (downward arrows) induced a clear LTP in wild-type (n = 13) but not Gαq mutant (n = 9) mice. ∗∗p < 0.01.

Fig. 5.

The threshold for LTP induction is elevated in the area CA1 of Gα11 mutant mice. A, A TBS with 10 burst trains (downward arrow) induced a robust and stable LTP in both wild-type (n = 7) and Gα11 mutant (n = 8) mice. B, A weak TBS of three trains (downward arrow) induced a clear LTP in wild-type (n = 10) but not Gα11 mutant (n = 10) mice; ∗∗p < 0.01.C, The average increase in initial slopes of fEPSPs (50–60 min after TBS); ∗∗p < 0.01.

Fig. 6.

LTP induced by the weak TBS in the area CA1 is abolished by MCPG but not by atropine. A, The weak TBS (downward arrow) induced LTP in the area CA1 in control saline (n = 7). MCPG bath applied before and during the TBS (500 μm, horizontal bar) abolished LTP (n = 6); ∗∗p < 0.01. B, Atropine (1 μm, horizontal bar) bath applied before and during the TBS (downward arrow) enhanced post-tetanic potentiation but did not affect the level of LTP. n= 7 for control; n = 5 for atropine.C, The average increase in initial slopes of fEPSPs (50–60 min after TBS); ∗∗p < 0.01.

Fig. 7.

The ACPD-induced priming effect is deficient in Gαq mutant mice. A, Bath application of ACPD (20 μm, 10 min, horizontal bars) enhanced the subsequent LTP induced by the weak TBS of three trains (downward arrow) in wild-type mice (control,n = 6; ACPD, n = 7).B, A TBS of five trains (downward arrow) induced LTP in Gαq mutant mice (control, n = 6). Bath application of ACPD (20 μm, 10 min,horizontal bars) did not enhance LTP (ACPD,n = 6). C, The average increase in initial slopes of fEPSPs (50–60 min after TBS). ∗∗p < 0.01.

Fig. 8.

LTP induced by the weak TBS is abolished by a PKC inhibitor, chelerythrine. A, In wild-type mice, a PKC inhibitor, chelerythrine (2 μm, bath applied during the recoding period indicated with the horizontal bar), abolished the LTP induced by the TBS of three trains (downward arrow) in normal control saline (control, n = 9; chelerythrine, n = 9; ∗∗p < 0.01). B, In Gαq mutant mice, chelerythrine had no blocking effect on the LTP induced by the TBS of five trains (downward arrow) (control,n = 8; chelerythrine, n = 9).C, The average increase in initial slopes of fEPSPs (50–60 min after TBS); ∗∗p < 0.01.