Michelle D. Amaral

TRPC3 Channels Are Necessary for Brain-Derived Neurotrophic Factor to Activate a Nonselective Cationic Current and to Induce Dendritic Spine Formation

Brain-derived neurotrophic factor (BDNF) exerts prominent effects on hippocampal neurons, but the mechanisms that initiate its actions are poorly understood. We report here that BDNF evokes a slowly developing and sustained nonselective cationic current (IBDNF) in CA1 pyramidal neurons. These responses require phospholipase C, IP3 receptors, Ca2+ stores, and Ca2+ influx, suggesting the involvement of transient receptor potential canonical subfamily (TRPC) channels. Indeed, IBDNF is absent after small interfering RNA-mediated TRPC3 knockdown. The sustained kinetics of IBDNF appears to depend on phosphatidylinositol 3-kinase-mediated TRPC3 membrane insertion, as shown by surface biotinylation assays. Slowly emerging membrane currents after theta burst stimulation are sensitive to the scavenger TrkB–IgG and TRPC inhibitors, suggesting IBDNF activation by evoked released of endogenous, native BDNF. Last, TRPC3 channels are necessary for BDNF to increase dendritic spine density. Thus, TRPC channels emerge as novel mediators of BDNF-mediated dendritic remodeling through the activation of a slowly developing and sustained membrane depolarization.

Activity-dependent release of endogenous BDNF from mossy fibers evokes a TRPC3 current and Ca2+ elevations in CA3 pyramidal neurons.

Multiple studies have demonstrated that brain-derived neurotrophic factor (BDNF) is a potent modulator of neuronal structure and function in the hippocampus. However, the majority of studies to date have relied on the application of recombinant BDNF. We herein report that endogenous BDNF, released via theta burst stimulation of mossy fibers (MF), elicits a slowly developing cationic current and intracellular Ca(2+) elevations in CA3 pyramidal neurons with the same pharmacological profile of the transient receptor potential canonical 3 (TRPC3)-mediated I(BDNF) activated in CA1 neurons by brief localized applications of recombinant BDNF. Indeed, sensitivity to both the extracellular BDNF scavenger tropomyosin-related kinase B (TrkB)-IgG and small hairpin interference RNA-mediated TRPC3 channel knockdown confirms the identity of this conductance as such, henceforth-denoted MF-I(BDNF). Consistent with such activity-dependent release of BDNF, these MF-I(BDNF) responses were insensitive to manipulations of extracellular Zn(2+) concentration. Brief theta burst stimulation of MFs induced a long-lasting depression in the amplitude of excitatory postsynaptic currents (EPSCs) mediated by both AMPA and N-methyl-d-aspartate (NMDA) receptors without changes in the NMDA receptor/AMPA receptor ratio, suggesting a reduction in neurotransmitter release. This depression of NMDAR-mediated EPSCs required activity-dependent release of endogenous BDNF from MFs and activation of Trk receptors, as it was sensitive to the extracellular BDNF scavenger TrkB-IgG and the tyrosine kinase inhibitor k-252b. These results uncovered the most immediate response to endogenously released--native--BDNF in hippocampal neurons and lend further credence to the relevance of BDNF signaling for synaptic function in the hippocampus.

Hyperforin modulates dendritic spine morphology in hippocampal pyramidal neurons by activating Ca2+-permeable TRPC6 channels

The standardized extract of the St. Johns wort plant (Hypericum perforatum) is commonly used to treat mild to moderate depression. Its active constituent is hyperforin, a phloroglucinol derivative that reduces the reuptake of serotonin and norepinephrine by increasing intracellular Na+ concentration through the activation of nonselective cationic TRPC6 channels. TRPC6 channels are also Ca2+‐permeable, resulting in intracellular Ca2+ elevations. Indeed, hyperforin activates TRPC6‐mediated currents and Ca2+ transients in rat PC12 cells, which induce their differentiation, mimicking the neurotrophic effect of nerve growth factor. Here, we show that hyperforin modulates dendritic spine morphology in CA1 and CA3 pyramidal neurons of hippocampal slice cultures through the activation of TRPC6 channels. Hyperforin also evoked intracellular Ca2+ transients and depolarizing inward currents sensitive to the TRPC channel blocker La3+, thus resembling the actions of the neurotrophin brain‐derived neurotrophic factor (BDNF) in hippocampal pyramidal neurons. These results suggest that the antidepressant actions of St. Johns wort are mediated by a mechanism similar to that engaged by BDNF.

Intracellular Ca2+ stores and Ca2+ influx are both required for BDNF to rapidly increase quantal vesicular transmitter release.

Brain-derived neurotrophic factor (BDNF) is well known as a survival factor during brain development as well as a regulator of adult synaptic plasticity. One potential mechanism to initiate BDNF actions is through its modulation of quantal presynaptic transmitter release. In response to local BDNF application to CA1 pyramidal neurons, the frequency of miniature excitatory postsynaptic currents (mEPSC) increased significantly within 30 seconds; mEPSC amplitude and kinetics were unchanged. This effect was mediated via TrkB receptor activation and required both full intracellular Ca2+ stores as well as extracellular Ca2+. Consistent with a role of Ca2+-permeable plasma membrane channels of the TRPC family, the inhibitor SKF96365 prevented the BDNF-induced increase in mEPSC frequency. Furthermore, labeling presynaptic terminals with amphipathic styryl dyes and then monitoring their post-BDNF destaining in slice cultures by multiphoton excitation microscopy revealed that the increase in frequency of mEPSCs reflects vesicular fusion events. Indeed, BDNF application to CA3-CA1 synapses in TTX rapidly enhanced FM1-43 or FM2-10 destaining with a time course that paralleled the phase of increased mEPSC frequency. We conclude that BDNF increases mEPSC frequency by boosting vesicular fusion through a presynaptic, Ca2+-dependent mechanism involving TrkB receptors, Ca2+ stores, and TRPC channels.

On the Role of Neurotrophins in Dendritic Calcium Signaling

In addition to its strong presynaptic actions on quantal neurotransmitter release, the regulation of the spatial and temporal patterns of postsynaptic Ca2+ elevations by BDNF is also a likely mechanism for its modulation of synaptic plasticity. The intracellular signaling cascades activated by TrkB receptors include several well-characterized protein kinases that target most of the routes of Ca2+ entry into hippocampal neurons. In addition, TrkB activation leads to IP3 formation, strongly arguing for direct Ca2+ mobilization from intracellular Ca2+ stores. Lastly, depletion of intracellular Ca2+ stores is associated with the activation of plasma membrane non-selective cationic currents thought to mediate Ca2+ store refilling. These membrane currents mediated by members of the TRPC family of ion channels not only represent novel downstream effects of neurotrophin action, but also are intriguing points of convergence with other intracellular signaling cascades, such as those triggered by group-I metabotropic glutamate receptors. The information gained from future experiments in this rapidly evolving field will integrate the actions of BDNF at synapses with the requirement of dendritic Ca2+ signals necessary for the induction of synaptic plasticity. Ultimately, the challenge ahead is to assimilate the varied functional and structural consequences of BDNF signaling through TrkB receptors at both sides of the synaptic cleft at excitatory synapses in the hippocampus with its intriguing role in the consolidation of hippocampal-dependent learning. Indeed, BDNF represents the prototypical example of a consolidation factor necessary for trans-synaptic plasticity at hippocampal synapses.