Theta patterns of stimulation induce synaptic and intrinsic potentiation in O-LM interneurons.

Brain oscillations have long-lasting effects on synaptic and cellular properties. For instance, synaptic stimulation at theta (θ) frequency induces persistent depression of both excitatory synaptic transmission and intrinsic excitability in CA1 principal neurons. However, the incidence of θ activity on synaptic transmission and intrinsic excitability in hippocampal GABAergic interneurons is unclear. We report here the induction of both synaptic and intrinsic potentiation in oriens-lacunosum moleculare (O-LM) interneurons following stimulation of afferent glutamatergic inputs in the θ frequency range (∼5 Hz). Long-term synaptic potentiation (LTP) is induced by synaptic activation of calcium-permeable AMPA receptors (CP-AMPAR), whereas long-term potentiation of intrinsic excitability (LTP-IE) results from the mGluR1-dependent down-regulation of Kv7 voltage-dependent potassium channel and hyperpolarization activated and cyclic nucleotide-gated (HCN) channel through the depletion of phosphatidylinositol-4,5-biphosphate (PIP2). LTP and LTP-IE are reversible, demonstrating that both synaptic and intrinsic changes are bidirectional in O-LM cells. We conclude that synaptic activity at θ frequency induces both synaptic and intrinsic potentiation in O-LM interneurons, i.e., the opposite of what is typically seen in glutamatergic neurons.

Rescue of Normal Excitability in LGI1-Deficient Epileptic Neurons.

Leucine-rich glioma inactivated 1 (LGI1) is a glycoprotein secreted by neurons, the deletion of which leads to autosomal dominant lateral temporal lobe epilepsy. We previously showed that LGI1 deficiency in a mouse model (i.e., knock-out for LGI1 or KO-Lgi1) decreased Kv1.1 channel density at the axon initial segment (AIS) and at presynaptic terminals, thus enhancing both intrinsic excitability and glutamate release. However, it is not known whether normal excitability can be restored in epileptic neurons. Here, we show that the selective expression of LGI1 in KO-Lgi1 neurons from mice of both sexes, using single-cell electroporation, reduces intrinsic excitability and restores both the Kv1.1-mediated D-type current and Kv1.1 channels at the AIS. In addition, we show that the homeostatic-like shortening of the AIS length observed in KO-Lgi1 neurons is prevented in neurons electroporated with the Lgi1 gene. Furthermore, we reveal a spatial gradient of intrinsic excitability that is centered on the electroporated neuron. We conclude that expression of LGI1 restores normal excitability through functional Kv1 channels at the AIS.SIGNIFICANCE STATEMENT The lack of leucine-rich glioma inactivated 1 (LGI1) protein induces severe epileptic seizures that leads to death. Enhanced intrinsic and synaptic excitation in KO-Lgi1 mice is because of the decrease in Kv1.1 channels in CA3 neurons. However, the conditions to restore normal excitability profile in epileptic neurons remain to be defined. We show here that the expression of LGI1 in KO-Lgi1 neurons in single neurons reduces intrinsic excitability, and restores both the Kv1.1-mediated D-type current and Kv1.1 channels at the axon initial segment (AIS). Furthermore, the homeostatic shortening of the AIS length observed in KO-Lgi1 neurons is prevented in neurons in which the Lgi1 gene has been rescued. We conclude that LGI1 constitutes a critical factor to restore normal excitability in epileptic neurons.

Neural excitability increases with axonal resistance between soma and axon initial segment.

The position of the axon initial segment (AIS) is thought to play a critical role in neuronal excitability. Previous experimental studies have found that a distal shift in AIS position correlates with a reduction in excitability. Yet theoretical work has suggested the opposite, because of increased electrical isolation. A distal shift in AIS position corresponds to an elevation of axial resistance Ra We therefore examined how changes in Ra at the axon hillock impact the voltage threshold (Vth) of the somatic action potential in L5 pyramidal neurons. Increasing Ra by mechanically pinching the axon between the soma and the AIS was found to lower Vth by ∼6 mV. Conversely, decreasing Ra by substituting internal ions with higher mobility elevated Vth All Ra -dependent changes in Vth could be reproduced in a Hodgkin-Huxley compartmental model. We conclude that in L5 pyramidal neurons, excitability increases with axial resistance and therefore with a distal shift of the AIS.

Homeostatic regulation of axonal Kv1.1 channels accounts for both synaptic and intrinsic modifications in the hippocampal CA3 circuit.

Homeostatic plasticity of intrinsic excitability goes hand in hand with homeostatic plasticity of synaptic transmission. However, the mechanisms linking the two forms of homeostatic regulation have not been identified so far. Using electrophysiological, imaging, and immunohistochemical techniques, we show here that blockade of excitatory synaptic receptors for 2 to 3 d induces an up-regulation of both synaptic transmission at CA3-CA3 connections and intrinsic excitability of CA3 pyramidal neurons. Intrinsic plasticity was found to be mediated by a reduction of Kv1.1 channel density at the axon initial segment. In activity-deprived circuits, CA3-CA3 synapses were found to express a high release probability, an insensitivity to dendrotoxin, and a lack of depolarization-induced presynaptic facilitation, indicating a reduction in presynaptic Kv1.1 function. Further support for the down-regulation of axonal Kv1.1 channels in activity-deprived neurons was the broadening of action potentials measured in the axon. We conclude that regulation of the axonal Kv1.1 channel constitutes a major mechanism linking intrinsic excitability and synaptic strength that accounts for the functional synergy existing between homeostatic regulation of intrinsic excitability and synaptic transmission.

Patient-derived antibodies reveal the subcellular distribution and heterogeneous interactome of LGI1.

Autoantibodies against leucine-rich glioma-inactivated 1 (LGI1) occur in patients with encephalitis who present with frequent focal seizures and a pattern of amnesia consistent with focal hippocampal damage. To investigate whether the cellular and subcellular distribution of LGI1 may explain the localization of these features, and hence gain broader insights into LGI1's neurobiology, we analysed the detailed localization of LGI1 and the diversity of its protein interactome, in mouse brains using patient-derived recombinant monoclonal LGI1 antibodies. Combined immunofluorescence and mass spectrometry analyses showed that LGI1 is enriched in excitatory and inhibitory synaptic contact sites, most densely within CA3 regions of the hippocampus. LGI1 is secreted in both neuronal somatodendritic and axonal compartments, and occurs in oligodendrocytic, neuro-oligodendrocytic and astro-microglial protein complexes. Proteomic data support the presence of LGI1-Kv1-MAGUK complexes, but did not reveal LGI1 complexes with postsynaptic glutamate receptors. Our results extend our understanding of regional, cellular and subcellular LGI1 expression profiles and reveal novel LGI1-associated complexes, thus providing insights into the complex biology of LGI1 and its relationship to seizures and memory loss.

Synaptic plasticity rules with physiological calcium levels.

Spike-timing-dependent plasticity (STDP) is considered as a primary mechanism underlying formation of new memories during learning. Despite the growing interest in activity-dependent plasticity, it is still unclear whether synaptic plasticity rules inferred from in vitro experiments are correct in physiological conditions. The abnormally high calcium concentration used in in vitro studies of STDP suggests that in vivo plasticity rules may differ significantly from in vitro experiments, especially since STDP depends strongly on calcium for induction. We therefore studied here the influence of extracellular calcium on synaptic plasticity. Using a combination of experimental (patch-clamp recording and Ca2+ imaging at CA3-CA1 synapses) and theoretical approaches, we show here that the classic STDP rule in which pairs of single pre- and postsynaptic action potentials induce synaptic modifications is not valid in the physiological Ca2+ range. Rather, we found that these pairs of single stimuli are unable to induce any synaptic modification in 1.3 and 1.5 mM calcium and lead to depression in 1.8 mM. Plasticity can only be recovered when bursts of postsynaptic spikes are used, or when neurons fire at sufficiently high frequency. In conclusion, the STDP rule is profoundly altered in physiological Ca2+, but specific activity regimes restore a classical STDP profile.

Endocannabinoids Tune Intrinsic Excitability in O-LM Interneurons by Direct Modulation of Postsynaptic Kv7 Channels.

KCNQ-Kv7 channels are found at the axon initial segment of pyramidal neurons, where they control cell firing and membrane potential. In oriens lacunosum moleculare (O-LM) interneurons, these channels are mainly expressed in the dendrites, suggesting a peculiar function of Kv7 channels in these neurons. Here, we show that Kv7 channel activity is upregulated following induction of presynaptic long-term synaptic depression (LTD) in O-LM interneurons from rats of both sex, thus resulting in a synergistic long-term depression of intrinsic excitability (LTD-IE). Both LTD and LTD-IE involve endocannabinoid (eCB) biosynthesis for induction. However, although LTD is dependent on cannabinoid type 1 receptors, LTD-IE is not. Molecular modeling shows a strong interaction of eCBs with Kv7.2/3 channel, suggesting a persistent action of these lipids on Kv7 channel activity. Our data thus unveil a major role for eCB synthesis in triggering both synaptic and intrinsic depression in O-LM interneurons.SIGNIFICANCE STATEMENT In principal cells, Kv7 channels are essentially located at the axon initial segment. In contrast, in O-LM interneurons, Kv7 channels are highly expressed in the dendrites, suggesting a singular role of these channels in O-LM cell function. Here, we show that LTD of excitatory inputs in O-LM interneurons is associated with an upregulation of Kv7 channels, thus resulting in a synergistic LTD of LTD-IE. Both forms of plasticity are mediated by the biosynthesis of eCBs. Stimulation of CB1 receptors induces LTD, whereas the direct interaction of eCBs with Kv7 channels induces LTD-IE. Our results thus provide a previously unexpected involvement of eCBs in long-lasting plasticity of intrinsic excitability in GABAergic interneurons.

LGI1 tunes intrinsic excitability by regulating the density of axonal Kv1 channels.

Autosomal dominant epilepsy with auditory features results from mutations in leucine-rich glioma-inactivated 1 (LGI1), a soluble glycoprotein secreted by neurons. Animal models of LGI1 depletion display spontaneous seizures, however, the function of LGI1 and the mechanisms by which deficiency leads to epilepsy are unknown. We investigated the effects of pure recombinant LGI1 and genetic depletion on intrinsic excitability, in the absence of synaptic input, in hippocampal CA3 neurons, a classical focus for epileptogenesis. Our data indicate that LGI1 is expressed at the axonal initial segment and regulates action potential firing by setting the density of the axonal Kv1.1 channels that underlie dendrotoxin-sensitive D-type potassium current. LGI1 deficiency incurs a >50% down-regulation of the expression of Kv1.1 and Kv1.2 via a posttranscriptional mechanism, resulting in a reduction in the capacity of axonal D-type current to limit glutamate release, thus contributing to epileptogenesis.

The contribution of ion channels in input-output plasticity.

Persistent changes that occur in brain circuits are classically thought to be mediated by long-term modifications in synaptic efficacy. Yet, many studies have shown that voltage-gated ion channels located at the input and output side of the neurons are also the subject to persistent modifications. These channels are thus responsible for intrinsic plasticity that is expressed in many different neuronal types including glutamatergic principal neurons and GABAergic interneurons. As for synaptic plasticity, activation of synaptic glutamate receptors initiate persistent modification in neuronal excitability. We review here how synaptic input can be efficiently altered by activity-dependent modulation of ion channels that control EPSP amplification, spike threshold or resting membrane potential. We discuss the nature of the learning rules shared by intrinsic and synaptic plasticity, the mechanisms of ion channel regulation and the impact of intrinsic plasticity on induction of synaptic modifications.

Axon physiology.

Axons are generally considered as reliable transmission cables in which stable propagation occurs once an action potential is generated. Axon dysfunction occupies a central position in many inherited and acquired neurological disorders that affect both peripheral and central neurons. Recent findings suggest that the functional and computational repertoire of the axon is much richer than traditionally thought. Beyond classical axonal propagation, intrinsic voltage-gated ionic currents together with the geometrical properties of the axon determine several complex operations that not only control signal processing in brain circuits but also neuronal timing and synaptic efficacy. Recent evidence for the implication of these forms of axonal computation in the short-term dynamics of neuronal communication is discussed. Finally, we review how neuronal activity regulates both axon morphology and axonal function on a long-term time scale during development and adulthood.

What are the mechanisms for analogue and digital signalling in the brain?

Synaptic transmission in the brain generally depends on action potentials. However, recent studies indicate that subthreshold variation in the presynaptic membrane potential also determines spike-evoked transmission. The informational content of each presynaptic action potential is therefore greater than initially expected. The contribution of this synaptic property, which is a fast (from 0.01 to 10 s) and state-dependent modulation of functional coupling, has been largely underestimated and could have important consequences for our understanding of information processing in neural networks. We discuss here how the membrane voltage of the presynaptic terminal might modulate neurotransmitter release by mechanisms that do not involve a change in presynaptic Ca(2+) influx.

Modulation of spike-evoked synaptic transmission: The role of presynaptic calcium and potassium channels.

Action potentials are usually considered as the smallest unit of neuronal information conveyed by presynaptic neurons to their postsynaptic target. Thus, neuronal signaling in brain circuits is all-or-none or digital. However, recent studies indicate that subthreshold analog variation in presynaptic membrane potential modulates spike-evoked transmission. The informational content of each presynaptic action potential is therefore greater than initially expected. This property constitutes a form of fast activity-dependent modulation of functional coupling. Therefore, it could have important consequences on information processing in neural networks in parallel with more classical forms of presynaptic short-term facilitation based on repetitive stimulation, modulation of presynaptic calcium or modifications of the release machinery. We discuss here how analog voltage shift in the presynaptic neuron may regulate spike-evoked release of neurotransmitter through the modulation of voltage-gated calcium and potassium channels in the axon and presynaptic terminal. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

ATP-P2X7 Receptor Modulates Axon Initial Segment Composition and Function in Physiological Conditions and Brain Injury.

Axon properties, including action potential initiation and modulation, depend on both AIS integrity and the regulation of ion channel expression in the AIS. Alteration of the axon initial segment (AIS) has been implicated in neurodegenerative, psychiatric, and brain trauma diseases, thus identification of the physiological mechanisms that regulate the AIS is required to understand and circumvent AIS alterations in pathological conditions. Here, we show that the purinergic P2X7 receptor and its agonist, adenosine triphosphate (ATP), modulate both structural proteins and ion channel density at the AIS in cultured neurons and brain slices. In cultured hippocampal neurons, an increment of extracellular ATP concentration or P2X7-green fluorescent protein (GFP) expression reduced the density of ankyrin G and voltage-gated sodium channels at the AIS. This effect is mediated by P2X7-regulated calcium influx and calpain activation, and impaired by P2X7 inhibition with Brilliant Blue G (BBG), or P2X7 suppression. Electrophysiological studies in brain slices showed that P2X7-GFP transfection decreased both sodium current amplitude and intrinsic neuronal excitability, while P2X7 inhibition had the opposite effect. Finally, inhibition of P2X7 with BBG prevented AIS disruption after ischemia/reperfusion in rats. In conclusion, our study demonstrates an involvement of P2X7 receptors in the regulation of AIS mediated neuronal excitability in physiological and pathological conditions.

Homeostatic regulation of h-conductance controls intrinsic excitability and stabilizes the threshold for synaptic modification in CA1 neurons.

KEY POINTS: We determined the contribution of the hyperpolarization-activated cationic (h) current (Ih ) to the homeostatic regulation of CA1 pyramidal cells in vitro using chronic treatments (48 h) that either increase (picrotoxin) or decrease (kynurenate) neuronal activity. The h-conductance was found to be up- or down-regulated following chronic activity enhancement or activity deprivation, respectively. This bidirectional plasticity of Ih was found to subsequently alter both apparent input resistance and intrinsic neuronal excitability. Bidirectional homeostatic plasticity of Ih also determined EPSP waveform and EPSP summation tested at 5-30 Hz. Long-term synaptic modification induced by repetitive stimulation of the Schaffer collaterals was found to be constant across treatments in the presence of Ih but not when Ih was blocked pharmacologically. Thus, bidirectional homeostatic regulation of Ih stabilizes induction of long-term synaptic modification in CA1 pyramidal neurons that depends on EPSP summation. ABSTRACT: The hyperpolarization-activated cationic (h) current is a voltage-shock absorber, highly expressed in the dendrites of CA1 pyramidal neurons. Up-regulation of Ih has been reported following episodes of intense network activity but the effect of activity deprivation on Ih and the functional consequence of homeostatic regulation of Ih remain unclear. We determined here the contribution of Ih to the homeostatic regulation of CA1 pyramidal cell excitability. Intrinsic neuronal excitability was decreased in neurons treated for 2-3 days with the GABAA channel blocker picrotoxin (PiTx) but increased in neurons treated (2-3 days) with the glutamate receptor antagonist kynurenate (Kyn). Membrane capacitance remained unchanged after treatment but the apparent input resistance was reduced for PiTx-treated neurons and enhanced for Kyn-treated neurons. Maximal Ih conductance was up-regulated after chronic hyperactivity but down-regulated following chronic hypoactivity. Up-regulation of Ih in PiTx-treated cultures was found to accelerate EPSP kinetics and reduce temporal summation of EPSPs whereas opposite effects were observed in Kyn-treated cultures, indicating that homeostatic regulation of Ih may control the induction of synaptic modification depending on EPSP summation. In fact, stimulation of the Schaffer collaterals at 3-10 Hz induced differential levels of plasticity in PiTx-treated and Kyn-treated neurons when Ih was blocked pharmacologically but not in control conditions. These data indicate that homeostatic regulation of Ih normalizes the threshold for long-term synaptic modification that depends on EPSP summation. In conclusion, bidirectional homeostatic regulation of Ih not only controls spiking activity but also stabilizes the threshold for long-term potentiation induced in CA1 pyramidal neurons by repetitive stimulation.

Analog modulation of spike-evoked transmission in CA3 circuits is determined by axonal Kv1.1 channels in a time-dependent manner.

Synaptic transmission usually depends on action potentials (APs) in an all-or-none (digital) fashion. Recent studies indicate, however, that subthreshold presynaptic depolarization may facilitate spike-evoked transmission, thus creating an analog modulation of spike-evoked synaptic transmission, also called analog-digital (AD) synaptic facilitation. Yet, the underlying mechanisms behind this facilitation remain unclear. We show here that AD facilitation at rat CA3-CA3 synapses is time-dependent and requires long presynaptic depolarization (5-10 s) for its induction. This depolarization-induced AD facilitation (d-ADF) is blocked by the specific Kv1.1 channel blocker dendrotoxin-K. Using fast voltage-imaging of the axon, we show that somatic depolarization used for induction of d-ADF broadened the AP in the axon through inactivation of Kv1.1 channels. Somatic depolarization enhanced spike-evoked calcium signals in presynaptic terminals, but not basal calcium. In conclusion, axonal Kv1.1 channels determine glutamate release in CA3 neurons in a time-dependent manner through the control of the presynaptic spike waveform.

GSK3 and ß-catenin determines functional expression of sodium channels at the axon initial segment.

Neuronal action potentials are generated through voltage-gated sodium channels, which are tethered by ankyrinG at the membrane of the axon initial segment (AIS). Despite the importance of the AIS in the control of neuronal excitability, the cellular and molecular mechanisms regulating sodium channel expression at the AIS remain elusive. Our results show that GSK3α/ß and ß-catenin phosphorylated by GSK3 (S33/37/T41) are localized at the AIS and are new components of this essential neuronal domain. Pharmacological inhibition of GSK3 or ß-catenin knockdown with shRNAs decreased the levels of phosphorylated-ß-catenin, ankyrinG, and voltage-gated sodium channels at the AIS, both "in vitro" and "in vivo", therefore diminishing neuronal excitability as evaluated via sodium current amplitude and action potential number. Thus, our results suggest a mechanism for the modulation of neuronal excitability through the control of sodium channel density by GSK3 and ß-catenin at the AIS.

Enhanced Release Probability without Changes in Synaptic Delay during Analogue-Digital Facilitation.

Neuronal timing with millisecond precision is critical for many brain functions such as sensory perception, learning and memory formation. At the level of the chemical synapse, the synaptic delay is determined by the presynaptic release probability (Pr) and the waveform of the presynaptic action potential (AP). For instance, paired-pulse facilitation or presynaptic long-term potentiation are associated with reductions in the synaptic delay, whereas paired-pulse depression or presynaptic long-term depression are associated with an increased synaptic delay. Parallelly, the AP broadening that results from the inactivation of voltage gated potassium (Kv) channels responsible for the repolarization phase of the AP delays the synaptic response, and the inactivation of sodium (Nav) channels by voltage reduces the synaptic latency. However, whether synaptic delay is modulated during depolarization-induced analogue-digital facilitation (d-ADF), a form of context-dependent synaptic facilitation induced by prolonged depolarization of the presynaptic neuron and mediated by the voltage-inactivation of presynaptic Kv1 channels, remains unclear. We show here that despite Pr being elevated during d-ADF at pyramidal L5-L5 cell synapses, the synaptic delay is surprisingly unchanged. This finding suggests that both Pr- and AP-dependent changes in synaptic delay compensate for each other during d-ADF. We conclude that, in contrast to other short- or long-term modulations of presynaptic release, synaptic timing is not affected during d-ADF because of the opposite interaction of Pr- and AP-dependent modulations of synaptic delay.

High-Resolution Proteomics Unravel a Native Functional Complex of Cav1.3, SK3, and Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in Midbrain Dopaminergic Neurons.

Pacemaking activity in substantia nigra dopaminergic neurons is generated by the coordinated activity of a variety of distinct somatodendritic voltage- and calcium-gated ion channels. We investigated whether these functional interactions could arise from a common localization in macromolecular complexes where physical proximity would allow for efficient interaction and co-regulations. For that purpose, we immunopurified six ion channel proteins involved in substantia nigra neuron autonomous firing to identify their molecular interactions. The ion channels chosen as bait were Cav1.2, Cav1.3, HCN2, HCN4, Kv4.3, and SK3 channel proteins, and the methods chosen to determine interactions were co-immunoprecipitation analyzed through immunoblot and mass spectrometry as well as proximity ligation assay. A macromolecular complex composed of Cav1.3, HCN, and SK3 channels was unraveled. In addition, novel potential interactions between SK3 channels and sclerosis tuberous complex (Tsc) proteins, inhibitors of mTOR, and between HCN4 channels and the pro-degenerative protein Sarm1 were uncovered. In order to demonstrate the presence of these molecular interactions in situ, we used proximity ligation assay (PLA) imaging on midbrain slices containing the substantia nigra, and we could ascertain the presence of these protein complexes specifically in substantia nigra dopaminergic neurons. Based on the complementary functional role of the ion channels in the macromolecular complex identified, these results suggest that such tight interactions could partly underly the robustness of pacemaking in dopaminergic neurons.