Distinct roles for ephrinB3 in the formation and function of hippocampal synapses

Abstract

The transmembrane ephrinB ligands and their Eph receptor tyrosine kinases are known to regulate excitatory synaptic functions in the hippocampus. In the CA3-CA1 synapse, ephrinB ligands are localized to the post-synaptic membrane, while their cognate Eph receptors are presumed to be pre-synaptic. Interaction of ephrinB molecules with Eph receptors leads to changes in long-term potentiation (LTP), which has been reported to be mediated by reverse signaling into the post-synaptic membrane. Here, we demonstrate that the cytoplasmic domain of ephrinB3 and hence reverse signaling is not required for ephrinB dependent learning and memory tasks or for LTP of these synapses. Consistent with previous reports, we find that ephrinB3(KO) null mutant mice exhibit a striking reduction in CA3-CA1 LTP that is associated with defective learning and memory tasks. We find the null mutants also show changes in both pre- and post-synaptic proteins including increased levels of synapsin and synaptobrevin and reduced levels of NMDA receptor subunits. These abnormalities are not observed in ephrinB3(lacZ) reverse signaling mutants that specifically delete the ephrinB3 intracellular region, supporting a cytoplasmic domain-independent forward signaling role for ephrinB3 in these processes. We also find that both ephrinB3(KO) and ephrinB3(lacZ) mice show an increased number of excitatory synapses, demonstrating a cytoplasmic-dependent reverse signaling role of ephrinB3 in regulating synapse number. Together, these data suggest that ephrinB3 may act like a receptor to transduce reverse signals to regulate the number of synapses formed in the hippocampus, and that it likely acts to stimulate forward signaling to modulate a number of other proteins involved in synaptic activity and learning/memory.

Bidirectional ephrin/Eph signaling in synaptic functions

“…2). It could act at the postsynaptic side by clustering NMDA receptors at the synapse [17], or at the presynaptic side by activating postsynaptic ephrinB reverse signaling, which is implicated in this form of LTP [31,70]. A regional-specific (CA1 or CA3) knock-in of EphB2 LacZ in the EphB2 null mouse would directly address this issue.…”

“…EphrinB ligands are expressed in both pre-and post-synaptic neurons in the hippocampus [32] and are capable of reverse signaling [41]. Although ephrinB reverse signaling is not implicated in spine formation, recent evidence suggests that postsynaptic ephrinB3 may regulate synapse formation in CA1 neurons [70]. In the ephrinB3 knockout mouse, the number of excitatory CA1 synapses was increased while the size of the postsynaptic-density (PSD) was decreased, which may explain their normal basal synaptic transmission [31,70].…”

“…Although ephrinB reverse signaling is not implicated in spine formation, recent evidence suggests that postsynaptic ephrinB3 may regulate synapse formation in CA1 neurons [70]. In the ephrinB3 knockout mouse, the number of excitatory CA1 synapses was increased while the size of the postsynaptic-density (PSD) was decreased, which may explain their normal basal synaptic transmission [31,70]. In the signaling-deficient ephrinB3 lacZ knock-in mouse, in which the ephrinB3 cytoplasmic domain was replaced by β-galactosidase, the PSD size but not synaptic density is restored to the wildtype level [70].…”

“…In the ephrinB3 knockout mouse, the number of excitatory CA1 synapses was increased while the size of the postsynaptic-density (PSD) was decreased, which may explain their normal basal synaptic transmission [31,70]. In the signaling-deficient ephrinB3 lacZ knock-in mouse, in which the ephrinB3 cytoplasmic domain was replaced by β-galactosidase, the PSD size but not synaptic density is restored to the wildtype level [70]. Furthermore, relative to wild type animals, hippocampal synaptosomal preparations from ephrinB3 null mice showed altered levels of various synaptic proteins, most of which can be restored to normal levels in the ephrinB3 lacZ knock-in mouse [70].…”

“…In the signaling-deficient ephrinB3 lacZ knock-in mouse, in which the ephrinB3 cytoplasmic domain was replaced by β-galactosidase, the PSD size but not synaptic density is restored to the wildtype level [70]. Furthermore, relative to wild type animals, hippocampal synaptosomal preparations from ephrinB3 null mice showed altered levels of various synaptic proteins, most of which can be restored to normal levels in the ephrinB3 lacZ knock-in mouse [70]. Although developmental compensation for the loss of synaptic function cannot be formally excluded, the observed phenotypes nonetheless provide important insights in signaling-dependent and signaling-independent functions of postsynaptic ephrinB3 in synapse formation.…”