Calcium-Dependent and Synapsin-Dependent Pathways for the Presynaptic Actions of BDNF

Abstract

We used cultured hippocampal neurons to determine the signaling pathways mediating brain-derived neurotrophic factor (BDNF) regulation of spontaneous glutamate and GABA release. BDNF treatment elevated calcium concentration in presynaptic terminals; this calcium signal reached a peak within 1 min and declined in the sustained presence of BDNF. This BDNF-induced transient rise in presynaptic calcium was reduced by SKF96365, indicating that BDNF causes presynaptic calcium influx via TRPC channels. BDNF treatment increased the frequency of miniature excitatory postsynaptic currents (mEPSCs). This response consisted of two components: a transient component that peaked within 1 min of initiating BDNF application and a second component that was sustained, at a lower mEPSC frequency, for the duration of BDNF application. The initial transient component was greatly reduced by removing external calcium or by treatment with SKF96365, as well as by Pyr3, a selective blocker of TRPC3 channels. In contrast, the sustained component was unaffected in these conditions but was eliminated by U0126, an inhibitor of the MAP kinase (MAPK) pathway, as well as by genetic deletion of synapsins in neurons from a synapsin triple knock-out (TKO) mouse. Thus, two pathways mediate the ability of BDNF to enhance spontaneous glutamate release: the transient component arises from calcium influx through TRPC3 channels, while the sustained component is mediated by MAPK phosphorylation of synapsins. We also examined the ability of these two BDNF-dependent pathways to regulate spontaneous release of the inhibitory neurotransmitter, GABA. BDNF had no effect on the frequency of spontaneous miniature inhibitory postsynaptic currents (mIPSCs) in neurons from wild-type (WT) mice, but surprisingly did increase mIPSC frequency in synapsin TKO mice. This covert BDNF response was blocked by removal of external calcium or by treatment with SKF96365 or Pyr3, indicating that it results from calcium influx mediated by TRPC3 channels. Thus, the BDNF-activated calcium signaling pathway can also enhance spontaneous GABA release, though this effect is suppressed by synapsins under normal physiological conditions.

Keywords: TRP channels; neurotransmitter release; neurotrophins; synapsins.

Figure 1

Brain-derived neurotrophic factor (BDNF) induced calcium rise at presynaptic terminals. (A) Representative images of presynaptic terminals from triple wild-type (TWT) neurons transfected with synaptophysin-mCherry (red) after Fluo4-AM loading (green). Images of Fluo4 fluorescence during 1st (left) and 5th minute (right) of BDNF application in the absence (B) and presence of SKF96365 (C). Scale at right indicates relative changes in Fluo4 fluorescence relative to baseline levels measured prior to BDNF treatment (ΔF/F₀). (D) Time course of BDNF-induced rise in presynaptic calcium, measured in control conditions (black) and in the presence of SKF96365 (red).

Figure 2

Role of calcium influx in BDNF enhancement of spontaneous glutamate release. (A) Representative traces of miniature EPSCs before and during BDNF application from a WT autaptic neuron. (B) Time course of changes in normalized miniature excitatory postsynaptic current (mEPSC) frequency during BDNF application (bar) in normal external solution (black) and calcium-free external solution (orange, n = 6). (C) Changes in mEPSC frequency during 1st (left) and 5th (right) minute of BDNF application. Throughout the article, values indicate mean ± standard error of the mean and *denotes a significant difference (p < 0.05).

Figure 3

TRPC3 mediates spontaneous glutamate release by BDNF. (A) Changes in mEPSC frequency caused by BDNF application in the absence (black, n = 13) and presence of SKF95365 (green, n = 6). (B) Changes in mEPSC frequency during 1st (left) and 5th (right) minute of BDNF application in control conditions and during treatment with SKF96365. (C) Changes in mEPSC frequency caused by BDNF application in the absence (black, n = 5) and presence of Pyr3 (sky blue, n = 5). (D) Changes in mEPSC frequency during 1st (left) and 5th (right) minute of BDNF application in control conditions and during treatment with Pyr3.

Figure 4

MAP kinase (MAPK)-dependent and calcium-dependent responses to BDNF. (A) Time course of changes in normalized mEPSC frequency during BDNF application in the absence and presence of the MAPK inhibitor, U0126 (20 μM). U0126 was bath perfused 5 min prior BDNF application. (B) Changes in mEPSC frequency during 1st (left) and 5th (right) minute of BDNF application, with and without U0126. (C) Time course of changes in normalized mEPSC frequency in neurons in normal external solution (black) or in calcium-free solution containing EGTA and U0126 (aqua). (D) Changes in mEPSC frequency during 1st (left) and 5th minute (right) of BDNF application in the indicated conditions.

Figure 5

Role of synapsins in MAPK pathway. (A) Time course of changes in normalized mEPSC frequency during BDNF application for TWT (black, n = 13) and synapsin triple knock-out (TKO) neurons (red, n = 12). (B) Changes in mEPSC frequency during 1st (left) and 5th minute (right) of BDNF application in TWT and TKO neurons. (C) Time course of changes in normalized mEPSC frequency during BDNF application in TKO neurons in the absence (red) or presence (purple) of U0126. (D) Changes in mEPSC frequency during 1st (left) and 5th minute (right) of BDNF application in TKO neurons with and without U0126 treatment.

Figure 6

Role of calcium influx in the BDNF response of synapsin TKO neurons. Either removal of calcium (A) or inhibiting TRPC channels (C) abolished the residual increase in mEPSC frequency produced in TKO neurons by BDNF. Data from TKO neurons in normal (red, n = 12) and calcium-free external solution (brown) shown in (A); data from TKO neurons in the absence (red) and presence of SKF95365 (green, n = 6) shown in (C). (B) Changes in mEPSC frequency during 1st (left) and 5th minute (right) of BDNF application in TKO neurons in normal and calcium free solution. (D) Changes in mEPSC frequency during 1st (left) and 5th minute (right) of BDNF application in TKO neurons in the absence or presence of SKF95365 and Pyr3 (sky blue, n = 6).

Figure 7

Dual signaling pathway model for BDNF action at glutamatergic synapses.

Figure 8

BDNF regulation of spontaneous GABA release. (A) Representative miniature IPSCs before and during BDNF application, recorded in TWT neurons (left) and TKO neurons (right). (B) Time course of changes in normalized miniature inhibitory postsynaptic current (mIPSC) frequency during BDNF application for TWT (black, n = 21) and synapsin TKO neurons (red, n = 18). (C) Changes in mIPSC frequency during 1st (left) and 6th minute (right) of BDNF application.

Figure 9

MAPK-independent BDNF effects on spontaneous GABA release. (A) Time course of changes in normalized mIPSC frequency during BDNF application for TWT neurons without (black, n = 21) and with (orange, n = 10) MAPK inhibitor U0126 (20 μM). (B) Changes in mIPSC frequency during 6th minute of BDNF application in TWT neurons, with and without U0126 treatment. (C) Time course of changes in normalized mIPSC frequency in TKO neurons during BDNF application without (red, n = 18) and with (green, n = 10) U0126 treatment. (D) Changes in mIPSC frequency in TKO neurons during 6th minute of BDNF application, with and without U0126.

Figure 10

Role of calcium influx in presynaptic action of BDNF on spontaneous GABA release in TKO neurons. (A) Time course of changes in normalized mIPSC frequency during BDNF application for TWT neurons in normal (black, n = 21) and calcium-free gray, n = 14) external solution. (B) Changes in mIPSC frequency in TWT neurons during 6th minute of BDNF application in normal and calcium-free external solution. (C) Time course of changes in normalized mIPSC frequency in TKO neurons during BDNF application in normal (red, n = 18) and calcium-free (blue, n = 13) external solution. (D) Changes in mIPSC frequency in TKO neurons during 6th minute of BDNF application, in the absence and presence of external calcium.

Figure 11

Role of TRPC channels in presynaptic action of BDNF on spontaneous GABA release in TKO neurons. (A) Time course of changes in normalized mIPSC frequency during BDNF application for TWT neurons without (black, n = 21) and with (olive green, n = 12) SKF96365 (3 μM). (B) Changes in mIPSC frequency during 6th minute of BDNF application in TWT neurons, with and without SKF96365 treatment. (C) Time course of changes in normalized mIPSC frequency in TKO neurons during BDNF application without (red, n = 18) and with (turquoise, n = 12) SKF96365 treatment. (D) Changes in mIPSC frequency in TKO neurons during 6th minute of BDNF application, with and without SKF96365. (E) Time course of changes in normalized mIPSC frequency in TKO neurons during BDNF application without (red, n = 18) and with (sky blue, n = 8) Pyr3 treatment (3 μM). (F) Changes in mIPSC frequency in TKO neurons during 6th minute of BDNF application, with and without Pyr3.