Hippocampal long-term potentiation is supported by presynaptic and postsynaptic tyrosine receptor kinase B-mediated phospholipase Cgamma signaling

Abstract

Neurotrophins have been shown to play a critical role in activity-dependent synaptic plasticity such as long-term potentiation (LTP) in the hippocampus. Although the role of brain-derived neurotrophic factor (BDNF) and its tyrosine kinase receptor [tyrosine receptor kinase B (TrkB)] is well documented, it still remains unresolved whether presynaptic or postsynaptic activation of TrkB is involved in the induction of LTP. To address this question, we locally and specifically interfered with a downstream target of the TrkB receptor, phospholipase Cgamma (PLCgamma). We prevented PLCgamma signaling by overexpression of the PLCgamma pleckstrin homology (PH) domain with a Sindbis virus vector. The isolated PH domain has an inhibitory effect and thereby blocks endogenous PLCgamma signaling and consequently also IP3 production. Surprisingly, concurrent presynaptic and postsynaptic blockade of PLCgamma signaling was required to reduce LTP to levels comparable with those in TrkB and BDNF knock-out mice. Blockade of presynaptic or postsynaptic signaling alone did not result in a significant reduction of LTP.

Figure 1.

Sindbis viral vectors specifically target neurons in dissociated hippocampal cultures and acute slices. Dissociated hippocampal cultures (A–F) were transduced with Sin–GFP for 24 h. Only MAP2-positive cells (i.e., neurons) express GFP (C), whereas the glia-specific marker GFAP never labeled GFP-positive cells (F). The same specificity was seen in locally injected acute hippocampal slices (G–I) using the neuronal marker MAP2 (I) or by observing the very characteristic neuronal morphology of GFP-expressing cells (G, H). The arrowheads in G point to selected interneurons.

Figure 2.

The PLCγ1PH domain is associated to intracellular and extracellular membranes. A, B, Single confocal planes of dissociated hippocampal cells transduced by Sin–GFP (A) or Sin–PHGFP (B). The PHGFP domain shows a characteristic patchy distribution of a membrane associated protein, whereas Sin–GFP is evenly distributed in the cytoplasm and the nucleus. C, The PLCγ1PH domain is associated to intramembrane and plasma membrane. Two individual confocal planes of one hippocampal neuron expressing the GFP-tagged PLCPH domain are shown. In the middle plane, a clear accumulation in intracellular membranes is visible, whereas a plane through the bottom of the cell demonstrates the accumulation of the protein in the plasma membrane. D, The PLCγ1PH domain is expressed and transported into tips of axons (arrowhead) and dendrites (arrow) already 6 h after Sindbis viral-mediated transduction. In the top panel, the staining with the axonal marker tau-1 is shown, and in the bottom panel, the patchy GFP fluorescence of the GFP-tagged PH domain is shown. Scale bars, 10 μm.

Figure 3.

Local expression of the PLCγPH domain exclusively in area CA1 or exclusively CA3 does not decrease LTP significantly. Schematic and representative images of acute hippocampal slices injected in area CA1 (A–C) or CA3 (E–G) with either Sin–GFP (B, F) or Sin–PHGFP (C, G) are shown. No apparent difference was observable between extent and intensity of transgene expression in Sin–GFP- or Sin–PHGFP-transduced slices. After at least 6 h of incubation, Schaffer collaterals were stimulated and fEPSPs recorded in area CA1. EPSP slope size is shown before and after tetanic stimulation (3 × 30 pulses; 100 Hz; arrow) for Sin–PHGFP and Sin–GFP control slices in CA1 (D) and CA3 (H). There is no significant difference between Sin–GFP and Sin–PHGFP 55–60 min after tetanic stimulation (p > 0.1 for CA1 and CA3, t test). D, H, Insets show original sweeps from representative individual experiments. Letters correspond to the time point when traces were taken. F, G, Insets show axonal labeling of Sin–GFP and Sin–PHGFP, respectively. Error bars represent SEM. DG, Dentate gyrus; mf, mossy fibers; sc, Schaffer collaterals.

Figure 4.

Expression of the PLCγ1PH domain in both the CA1 and CA3 region significantly decreases LTP. A–C, Schematic and representative images of acute hippocampal slices injected simultaneously in area CA3 and CA1 with either Sin–GFP (B) or Sin–PHGFP (C). After at least 6 h of incubation, synaptic field potentials in area CA1 were elicited through stimulation of Schaffer collaterals. D, E, Grouped recordings are shown before and after tetanic stimulation (3 × 30 pulses; 100 Hz; arrow) for double area injected Sin–PHGFP and Sin–GFP wild-type (WT) control slices and in TrkB^PLC/PLC mice. The difference in LTP magnitude at 55–60 min after tetanic stimulation is significantly different from controls (p = 0.012, t test), whereas injection of Sin–PHGFP into TrkB^PLC/PLC slice leads to the same low level of potentiation as Sin–PHGFP in wild-type slices (p = 0.72, t test). It is important to note that Sin–PHGFP injection into TrkB^PLC/PLC slice did not further decrease the remaining low level of potentiation. D, E, Insets show original sweeps from representative individual experiments. Letters correspond to the time point when traces were taken. Error bars represent SEM. DG, Dentate gyrus; mf, mossy fibers; sc, Schaffer collaterals.

Figure 5.

LTP is strongly reduced in hippocampal slices pretreated with PLC inhibitors or with the IP₃ receptor blocker 2-APB. A, Slices treated with the selective membrane-permeable PLC inhibitor U-73122 (10 μm) show a significantly lower LTP (tetanic stimulation, 3 × 30, 100 Hz; arrow) than slices treated with the ineffective control substance U-73343 (10 μm). p = 0.003, t test. B, After tetanic stimulation (3 × 30 pulses; 100 Hz; arrow) of the Schaffer collaterals, control slices express pronounced potentiation, whereas slices incubated in 2-APB show no LTP (p = 0.0024, t test). Error bars represent SEM.