Autocrine BDNF-TrkB signalling within a single dendritic spine.

Brain-derived neurotrophic factor (BDNF) and its receptor TrkB are crucial for many forms of neuronal plasticity, including structural long-term potentiation (sLTP), which is a correlate of an animal's learning. However, it is unknown whether BDNF release and TrkB activation occur during sLTP, and if so, when and where. Here, using a fluorescence resonance energy transfer-based sensor for TrkB and two-photon fluorescence lifetime imaging microscopy, we monitor TrkB activity in single dendritic spines of CA1 pyramidal neurons in cultured murine hippocampal slices. In response to sLTP induction, we find fast (onset < 1 min) and sustained (>20 min) activation of TrkB in the stimulated spine that depends on NMDAR (N-methyl-d-aspartate receptor) and CaMKII signalling and on postsynaptically synthesized BDNF. We confirm the presence of postsynaptic BDNF using electron microscopy to localize endogenous BDNF to dendrites and spines of hippocampal CA1 pyramidal neurons. Consistent with these findings, we also show rapid, glutamate-uncaging-evoked, time-locked BDNF release from single dendritic spines using BDNF fused to superecliptic pHluorin. We demonstrate that this postsynaptic BDNF-TrkB signalling pathway is necessary for both structural and functional LTP. Together, these findings reveal a spine-autonomous, autocrine signalling mechanism involving NMDAR-CaMKII-dependent BDNF release from stimulated dendritic spines and subsequent TrkB activation on these same spines that is crucial for structural and functional plasticity.

LTD at mossy fiber synapses onto stratum lucidum interneurons requires TrkB and retrograde endocannabinoid signaling.

Hippocampal mossy fiber axons simultaneously activate CA3 pyramidal cells and stratum lucidum interneurons (SLINs), the latter providing feedforward inhibition to control CA3 pyramidal cell excitability. Filopodial extensions of giant boutons of mossy fibers provide excitatory synaptic input to the SLIN. These filopodia undergo extraordinary structural plasticity causally linked to execution of memory tasks, leading us to seek the mechanisms by which activity regulates these synapses. High-frequency stimulation of the mossy fibers induces long-term depression (LTD) of their calcium-permeable AMPA receptor synapses with SLINs; previous work localized the site of induction to be postsynaptic and the site of expression to be presynaptic. Yet, the underlying signaling events and the identity of the retrograde signal are incompletely understood. We used whole cell recordings of SLINs in hippocampal slices from wild-type and mutant mice to explore the mechanisms. Genetic and pharmacologic perturbations revealed a requirement for both the receptor tyrosine kinase TrkB and its agonist, brain-derived neurotrophic factor (BDNF), for induction of LTD. Inclusion of inhibitors of Trk receptor kinase and PLC in the patch pipette prevented LTD. Endocannabinoid receptor antagonists and genetic deletion of the CB1 receptor prevented LTD. We propose a model whereby release of BDNF from mossy fiber filopodia activates TrkB and PLCγ1 signaling postsynaptically within SLINs, triggering synthesis and release of an endocannabinoid that serves as a retrograde signal, culminating in reduced glutamate release. Insights into the signaling pathways by which activity modifies function of these synapses will facilitate an understanding of their contribution to the local circuit and behavioral consequences of hippocampal granule cell activity. NEW & NOTEWORTHY We investigated signaling mechanisms underlying plasticity of the hippocampal mossy fiber filopodial synapse with interneurons in stratum lucidum. High-frequency stimulation of the mossy fibers induces long-term depression of this synapse. Our findings are consistent with a model in which brain-derived neurotrophic factor released from filopodia activates TrkB of a stratum lucidum interneuron; the ensuing activation of PLCγ1 induces synthesis of an endocannabinoid, which provides a retrograde signal leading to reduced release of glutamate presynaptically.

Long-Term Potentiation of Mossy Fiber Feedforward Inhibition of CA3 Pyramidal Cells Maintains E/I Balance in Epilepsy Model.

Insight into the cellular and circuit mechanisms underlying development of temporal lobe epilepsy (TLE) will provide a foundation for improved therapies. We studied a model in which an episode of prolonged seizures is followed by recovery lasting two weeks before emergence of spontaneous recurrent seizures. We focused on the interval between the prolonged seizures and the late onset recurrent seizures. We investigated the hippocampal mossy fiber CA3 pyramidal cell microcircuit in models spanning in vitro, in vivo, and ex vivo preparations. Expression of channelrhodopsin-2 in the dentate granule cells of DGC ChR mice enabled the selective activation of mossy fiber axons. In vivo studies revealed marked potentiation of mossy fiber evoked field potentials in hippocampal CA3 beginning within hours following seizures, a potentiation which persisted at least 7 d. Stimulation of mossy fibers in hippocampal slices in vitro using patterns of activity mimicking seizures induced LTP not only of the monosynaptic EPSC but also of the disynaptic IPSC of CA3 pyramidal cells. Ex vivo studies of slices isolated following seizures revealed evidence of LTP of mossy fiber evoked EPSC and disynaptic IPSC of CA3 pyramidal cells. We suggest that activation of dentate granule cells during seizures induces these plasticities in vivo and the retained balance of synaptic excitation and inhibition limits excessive activation of CA3 pyramidal cells, thereby protecting animals from spontaneous recurrent seizures at this interval following status epilepticus.

Disruption of TrkB-mediated phospholipase Cgamma signaling inhibits limbic epileptogenesis.

The BDNF receptor, TrkB, is critical to limbic epileptogenesis, but the responsible downstream signaling pathways are unknown. We hypothesized that TrkB-dependent activation of phospholipase Cgamma1 (PLCgamma1) signaling is the key pathway and tested this in trkB(PLC/PLC) mice carrying a mutation (Y816F) that uncouples TrkB from PLCgamma1. Biochemical measures revealed activation of both TrkB and PLCgamma1 in hippocampi in the pilocarpine and kindling models in wild-type mice. PLCgamma1 activation was decreased in hippocampi isolated from trkB(PLC/PLC) compared with control mice. Epileptogenesis assessed by development of kindling was inhibited in trkB(PLC/PLC) compared with control mice. Long-term potentiation of the mossy fiber-CA3 pyramid synapse was impaired in slices of trkB(PLC/PLC) mice. We conclude that TrkB-dependent activation of PLCgamma1 signaling is an important molecular mechanism of limbic epileptogenesis. Elucidating signaling pathways activated by a cell membrane receptor in animal models of CNS disorders promises to reveal novel targets for specific and effective therapeutic intervention.

Intravenous kainic acid induces status epilepticus and late onset seizures in mice.

We set out to establish a novel model of temporal lobe epilepsy (TLE) in a mouse. We sought to induce TLE through the injection of kainic acid (KA) into the tail vein with subsequent development of status epilepticus (SE). Using C57BL/6 mice, we implanted hippocampal EEG recording electrodes before or after injection of KA or phosphate buffered saline (PBS). Video and EEG analysis were conducted to evaluate for SE and development of recurrent seizures, the hallmark of TLE. All mice injected with KA developed SE while those who were injected with PBS did not. Of the animals injected with KA monitored for recurrent seizures following SE, 33% developed spontaneous recurrent seizures while those injected with PBS did not. Injection of KA through the tail vein of a mouse reliably and rapidly induces SE which remits spontaneously and leads to the development of TLE in a subset of mice.

Ion channel properties underlying axonal action potential initiation in pyramidal neurons.

A high density of Na+ channels in the axon hillock, or initial segment, is believed to determine the threshold for action potential initiation in neurons. Here we report evidence for an alternative mechanism that lowers the threshold in the axon. We investigated properties and distributions of ion channels in outside-out patches from axons and somata of layer 5 pyramidal neurons in rat neocortical slices. Na+ channels in axonal patches (<30 microm from the soma) were activated by 7 mV less depolarization than were somatic Na+ channels. A-type K+ channels, which were prominent in somatic and dendritic patches, were rarely seen in axonal patches. We incorporated these findings into numerical simulations which indicate that biophysical properties of axonal channels, rather than a high density of channels in the initial segment, are most likely to determine the lowest threshold for action potential initiation.

Vesicular zinc promotes presynaptic and inhibits postsynaptic long-term potentiation of mossy fiber-CA3 synapse.

The presence of zinc in glutamatergic synaptic vesicles of excitatory neurons of mammalian cerebral cortex suggests that zinc might regulate plasticity of synapses formed by these neurons. Long-term potentiation (LTP) is a form of synaptic plasticity that may underlie learning and memory. We tested the hypothesis that zinc within vesicles of mossy fibers (mf) contributes to mf-LTP, a classical form of presynaptic LTP. We synthesized an extracellular zinc chelator with selectivity and kinetic properties suitable for study of the large transient of zinc in the synaptic cleft induced by mf stimulation. We found that vesicular zinc is required for presynaptic mf-LTP. Unexpectedly, vesicular zinc also inhibits a form of postsynaptic mf-LTP. Because the mf-CA3 synapse provides a major source of excitatory input to the hippocampus, regulating its efficacy by these dual actions, vesicular zinc is critical to proper function of hippocampal circuitry in health and disease.

Zinc-mediated transactivation of TrkB potentiates the hippocampal mossy fiber-CA3 pyramid synapse.

The receptor tyrosine kinase, TrkB, is critical to diverse functions of the mammalian nervous system in health and disease. Evidence of TrkB activation during epileptogenesis in vivo despite genetic deletion of its prototypic neurotrophin ligands led us to hypothesize that a non-neurotrophin, the divalent cation zinc, can transactivate TrkB. We found that zinc activates TrkB through increasing Src family kinase activity by an activity-regulated mechanism independent of neurotrophins. One subcellular locale at which zinc activates TrkB is the postsynaptic density of excitatory synapses. Exogenous zinc potentiates the efficacy of the hippocampal mossy fiber (mf)-CA3 pyramid synapse by a TrkB-requiring mechanism. Long-term potentiation of this synapse is impaired by deletion of TrkB, inhibition of TrkB kinase activity, and by CaEDTA, a selective chelator of zinc. The activity-dependent activation of synaptic TrkB in a neurotrophin-independent manner provides a mechanism by which this receptor can regulate synaptic plasticity.