Early presynaptic and late postsynaptic components contribute independently to brain-derived neurotrophic factor-induced synaptic plasticity

Abstract

Trophin-induced synaptic plasticity consists of both presynaptic and postsynaptic processes. The potential interdependence of these mechanisms and their temporal relationships are undefined. The synaptic vesicle protein Rab3A is required for the early, initial 10 min phase but not for the later phase of BDNF-enhanced transmission. We now examine the temporal distinction and mechanistic relationships between these phases of BDNF action. Rab3A mutant cells did not exhibit increased miniature EPSC frequency in response to BDNF in cell culture, indicating an absence of the presynaptic component. In contrast, BDNF enhanced postsynaptic glutamate-induced current in the mutant neurons as in the wild type, indicating that the postsynaptic component of the response was intact. Finally, the postsynaptic NMDA receptor subunit NR2B was phosphorylated at Tyr1472 by BDNF in Rab3A knock-outs, as shown previously in wild type. Our results are the first to demonstrate that presynaptic and postsynaptic components of BDNF-enhanced synaptic activity are independent and temporally distinct.

Figure 1.

Prolonged exposure to BDNF evokes a dual-component enhancement of synaptic activity in rat hippocampal cells. A, Sample sweeps taken before and during BDNF exposure at the indicated time points. Note that synaptic activity increases during the first 5 min of exposure and then declines to near-baseline levels but subsequently increases with continued exposure to BDNF. B, Time course of changes in synaptic activity from the experiment shown in A. The two components of the response are evident. C, Group time course of those experiments (13 of 22) that exhibited a two-component response. Points represent average charge ± SEM. In the remaining nine recordings, BDNF produced a persistent elevation in synaptic activity in which a clear separation between an early and late component could not be made.

Figure 2.

A, Representative traces of mEPSCs in wild-type or Rab3A knock-out cells before and ∼5 min after exposure to BDNF. Whole-cell voltage-clamp recordings (V_hold = -80 mV) were performed in the presence of TTX. An increase in mEPSC frequency was observed within 5 min of BDNF exposure in wild-type cells but not in Rab3A knock-out cells. B, Averaged mEPSCs from before and two time periods after BDNF treatment for wild-type and Rab3A cells. Individual mEPSCs were normalized to the same amplitude and averaged. Rise time was unchanged, but there was a trend toward longer decay times after BDNF treatment for both genotypes. Averaged mEPSCs from a second set of recordings gave similar results. C, Average mEPSC frequency in response to BDNF (bar) over the course of the recording. mEPSC frequency was normalized to baseline (-2 to 0 min). Wild-type cells displayed an immediate increase (∼1.7-fold) in mEPSC frequency in response to BDNF relative to Rab3A knock-out cells (p < 0.05; t test). Rab3A mutant cells did not respond to BDNF over the entire 20 min of recording and actually showed a small but insignificant decrease in mEPSC frequency relative to baseline (p > 0.05; t test). The dashed line indicates baseline. Error bars represent SEM. D, Cumulative mEPSC amplitude probability for wild-type and Rab3A mutant cells (n = 8 and 7, respectively). Errors bars represent SEM. No statistical differences were observed between the amplitudes before and two time periods after BDNF treatment for either wild-type or Rab3A mutant cells (p > 0.05; Kolmogorov-Smirnov test). Recordings were obtained from multiple platings.

Figure 3.

A, Example traces showing glutamate-induced current before and ∼20 min after exposure to BDNF. Whole-cell voltage-clamp recordings (V_hold = -40 mV) were obtained in the presence of TTX. The profiles before and after BDNF in wild-type and Rab3A mutant cells are similar. B, Average peak current in response to BDNF (horizontal bar) over the course of the recording. Peak current amplitude was normalized to baseline (-2 to 0 min). Note that the y-axis starts at 0.75, with 1 indicating no response to BDNF. There is an increase in current starting at ∼15 min. Wild-type and Rab3A knock-out cells are not statistically different from each other (p > 0.05; t test). The small, early increase in glutamate-induced current observed in wild-type cells was not statistically significant when compared with Rab3A at individual time points 0-5 min after BDNF (p > 0.05; t test). The dashed line indicates baseline. Error bars represent SEM. Recordings were obtained from multiple platings.

Figure 4.

A, Western blot of synaptoneurosomal proteins (100 μg) from adult wild-type and Rab3A knock-out mouse cortices. Synaptoneurosomes were incubated in the presence of 10 or 50 ng/ml BDNF for 10 min. Phospho-NR2B was detected using an antibody to Tyr¹⁴⁷², and total NR2B protein was detected on the same membrane. B, Quantitation of NR2B phosphorylation indicates that BDNF enhances NR2B phosphorylation by ∼1.5-fold. No difference in BDNF-induced phosphorylation levels was observed between wild-type and Rab3A knock-out samples. NR2B phosphorylation levels were similar in 10 and 50 ng/ml BDNF treatments. Phospho-NR2B was normalized to total NR2B protein, and data are expressed as a fold of phospho-NR2B relative to control, untreated samples (n = 3). Error bars represent SEM. ^*p < 0.05, significantly different from control samples (ANOVA).