Frequency-dependent impairment of hippocampal LTP from NMDA receptors with reduced calcium permeability.

Changes in postsynaptic Ca2+ levels are essential for alterations in synaptic strength. At hippocampal CA3-to-CA1 synapses, the Ca2+ elevations required for LTP induction are typically mediated by NMDA receptor (NMDAR) channels but a contribution of NMDAR-independent Ca2+ sources has been implicated. Here, we tested the sensitivity of different protocols modifying synaptic strength to reduced NMDAR-mediated Ca2+ influx by employing mice genetically programmed to express in forebrain principal neurons an NR1 form that curtails Ca2+ permeability. Reduced NMDAR-mediated Ca2+ influx did not facilitate synaptic depression in CA1 neurons of these genetically modified mice. However, we observed that LTP could not be induced by pairing low frequency synaptic stimulation (LFS pairing) with postsynaptic depolarization, a protocol that induced robust LTP in wild-type mice. By contrast to LFS pairing, similar LTP levels were generated in both genotypes when postsynaptic depolarization was paired with high frequency synaptic stimulation (HFS). This indicates that the postsynaptic Ca2+ elevation also reached threshold during HFS in the mutant, probably due to summation of NMDAR-mediated Ca2+ influx. However, only in wild-type mice did repeated HFS further enhance LTP. All tested forms of LTP were blocked by the NMDAR antagonist D-AP5. Collectively, our results indicate that only NMDAR-dependent Ca2+ sources (NMDARs and Ca2+-dependent Ca2+ release from intracellular stores) mediate LFS pairing-evoked LTP. Moreover, LTP induced by the first HFS stimulus train required lower Ca2+ levels than the additional LTP obtained by repeated trains.

Impaired synaptic scaling in mouse hippocampal neurones expressing NMDA receptors with reduced calcium permeability.

NMDA receptors (NMDARs) play a crucial role for the acquisition of functional AMPARs during Hebbian synaptic plasticity at cortical and hippocampal synapses over a short timescale of seconds to minutes. In contrast, homeostatic synaptic plasticity can occur over longer timescales of hours to days. The induction mechanisms of this activity-dependent synaptic scaling are poorly understood but are assumed to be independent of NMDAR signalling in the cortex. Here we investigated in the hippocampus a potential role of NMDAR-mediated Ca(2+) influx for synaptic scaling of AMPA currents by genetic means. The Ca(2+) permeability of NMDARs was reduced by selective postnatal expression in principal neurones of mouse forebrain half of the NR1 subunits with an amino acid substitution at the critical channel site (N598R). This genetic manipulation did not reduce the total charge transfer via NMDARs in nucleated patches (somatic) and at synaptic sites. In contrast, the current amplitude and the charge carried through AMPARs were substantially reduced at somatic and synaptic sites in juvenile and adult mutants, indicating persistent downscaling of AMPA responses. Smaller and less frequent AMPA miniature currents in the mutant demonstrated a postsynaptic locus of this down-regulation. Afferent innervation and release probability were unchanged at CA3-to-CA1 synapses of mutants, as judged from input-output and minimal stimulation experiments. Our results indicate that NMDAR-mediated Ca(2+) signalling is important for synaptic scaling of AMPA currents in the hippocampus in vivo.

Glutamatergic plasticity by synaptic delivery of GluR-B(long)-containing AMPA receptors.

Activity-driven delivery of AMPA receptors is proposed to mediate glutamatergic synaptic plasticity, both during development and learning. In hippocampal CA1 principal neurons, such trafficking is primarily mediated by the abundant GluR-A subunit. We now report a study of GluR-B(long), a C-terminal splice variant of the GluR-B subunit. GluR-B(long) synaptic delivery is regulated by two forms of activity. Spontaneous synaptic activity-driven GluR-B(long) transport maintains one-third of the steady-state AMPA receptor-mediated responses, while GluR-B(long) delivery following the induction of LTP is responsible for approximately 50% of the resulting potentiation at the hippocampal CA3 to CA1 synapses at the time of GluR-B(long) peak expression-the second postnatal week. Trafficking of GluR-B(long)-containing receptors thus mediates a GluR-A-independent form of glutamatergic synaptic plasticity in the juvenile hippocampus.

Importance of the gamma-aminobutyric acid(B) receptor C-termini for G-protein coupling.

Functional gamma-aminobutyric acid(B) (GABA(B)) receptors assemble from two subunits, GABA(B(1)) and GABA(B(2).) This heteromerization, which involves a C-terminal coiled-coil interaction, ensures efficient surface trafficking and agonist-dependent G-protein activation. In the present study, we took a closer look at the implications of the intracellular C termini of GABA(B(1)) and GABA(B(2)) for G-protein coupling. We generated a series of C-terminal mutants of GABA(B(1)) and GABA(B(2)) and tested them for physical interaction, surface trafficking, coupling to adenylyl cyclase, and G-protein-gated inwardly rectifying potassium channels in human embryonic kidney (HEK) 293 cells as well as on endogenous calcium channels in sympathetic neurons of the superior cervical ganglion (SCG). We found that the C-terminal interaction contributes only partly to the heterodimeric assembly of the subunits, indicating the presence of an additional interaction site. The described endoplasmic reticulum retention signal within the C terminus of GABA(B(1)) functioned only in the context of specific amino acids, which constitute part of the GABA(B(1)) coiled-coil sequence. This finding may provide a link between the retention signal and its shielding by the coiled coil of GABA(B(2).) In HEK293 cells, we observed that the two well-known GABA(B) receptor antagonists [S-(R*,R*)]-[3-[[1-(3,4-dichlorophenyl)ethyl]amino]-2-hydroxypropyl](cyclohexylmethyl) phosphinic acid (CGP54626) and (+)-(2S)-5,5-dimethyl-2-morpholineacetic acid (SCH50911) CGP54626 and SCH50911 function as inverse agonists. The C termini of GABA(B(1)) and GABA(B(2)) strongly influenced agonist-independent G-protein coupling, although they were not necessary for agonist-dependent G-protein coupling. The C-terminal GABA(B) receptor mutants described here demonstrate that the active receptor conformation is stabilized by the coiled-coil interaction. Thus, the C-terminal conformation of the GABA(B) receptor may determine its constitutive activity, which could be a therapeutic target for inverse agonists.