Saltatory Conduction along Myelinated Axons Involves a Periaxonal Nanocircuit.

The propagation of electrical impulses along axons is highly accelerated by the myelin sheath and produces saltating or "jumping" action potentials across internodes, from one node of Ranvier to the next. The underlying electrical circuit, as well as the existence and role of submyelin conduction in saltatory conduction remain, however, elusive. Here, we made patch-clamp and high-speed voltage-calibrated optical recordings of potentials across the nodal and internodal axolemma of myelinated neocortical pyramidal axons combined with electron microscopy and experimentally constrained cable modeling. Our results reveal a nanoscale yet conductive periaxonal space, incompletely sealed at the paranodes, which separates the potentials across the low-capacitance myelin sheath and internodal axolemma. The emerging double-cable model reproduces the recorded evolution of voltage waveforms across nodes and internodes, including rapid nodal potentials traveling in advance of attenuated waves in the internodal axolemma, revealing a mechanism for saltation across time and space.

Neuronal Inactivity Co-opts LTP Machinery to Drive Potassium Channel Splicing and Homeostatic Spike Widening.

Homeostasis of neural firing properties is important in stabilizing neuronal circuitry, but how such plasticity might depend on alternative splicing is not known. Here we report that chronic inactivity homeostatically increases action potential duration by changing alternative splicing of BK channels; this requires nuclear export of the splicing factor Nova-2. Inactivity and Nova-2 relocation were connected by a novel synapto-nuclear signaling pathway that surprisingly invoked mechanisms akin to Hebbian plasticity: Ca2+-permeable AMPA receptor upregulation, L-type Ca2+ channel activation, enhanced spine Ca2+ transients, nuclear translocation of a CaM shuttle, and nuclear CaMKIV activation. These findings not only uncover commonalities between homeostatic and Hebbian plasticity but also connect homeostatic regulation of synaptic transmission and neuronal excitability. The signaling cascade provides a full-loop mechanism for a classic autoregulatory feedback loop proposed ∼25 years ago. Each element of the loop has been implicated previously in neuropsychiatric disease.

A Platform for Generation of Chamber-Specific Cardiac Tissues and Disease Modeling.

Tissue engineering using cardiomyocytes derived from human pluripotent stem cells holds a promise to revolutionize drug discovery, but only if limitations related to cardiac chamber specification and platform versatility can be overcome. We describe here a scalable tissue-cultivation platform that is cell source agnostic and enables drug testing under electrical pacing. The plastic platform enabled on-line noninvasive recording of passive tension, active force, contractile dynamics, and Ca2+ transients, as well as endpoint assessments of action potentials and conduction velocity. By combining directed cell differentiation with electrical field conditioning, we engineered electrophysiologically distinct atrial and ventricular tissues with chamber-specific drug responses and gene expression. We report, for the first time, engineering of heteropolar cardiac tissues containing distinct atrial and ventricular ends, and we demonstrate their spatially confined responses to serotonin and ranolazine. Uniquely, electrical conditioning for up to 8 months enabled modeling of polygenic left ventricular hypertrophy starting from patient cells.

Ultrafast Two-Photon Imaging of a High-Gain Voltage Indicator in Awake Behaving Mice.

Optical interrogation of voltage in deep brain locations with cellular resolution would be immensely useful for understanding how neuronal circuits process information. Here, we report ASAP3, a genetically encoded voltage indicator with 51% fluorescence modulation by physiological voltages, submillisecond activation kinetics, and full responsivity under two-photon excitation. We also introduce an ultrafast local volume excitation (ULoVE) method for kilohertz-rate two-photon sampling in vivo with increased stability and sensitivity. Combining a soma-targeted ASAP3 variant and ULoVE, we show single-trial tracking of spikes and subthreshold events for minutes in deep locations, with subcellular resolution and with repeated sampling over days. In the visual cortex, we use soma-targeted ASAP3 to illustrate cell-type-dependent subthreshold modulation by locomotion. Thus, ASAP3 and ULoVE enable high-speed optical recording of electrical activity in genetically defined neurons at deep locations during awake behavior.

C. elegans AWA Olfactory Neurons Fire Calcium-Mediated All-or-None Action Potentials.

Neurons in Caenorhabditis elegans and other nematodes have been thought to lack classical action potentials. Unexpectedly, we observe membrane potential spikes with defining characteristics of action potentials in C. elegans AWA olfactory neurons recorded under current-clamp conditions. Ion substitution experiments, mutant analysis, pharmacology, and modeling indicate that AWA fires calcium spikes, which are initiated by EGL-19 voltage-gated CaV1 calcium channels and terminated by SHK-1 Shaker-type potassium channels. AWA action potentials result in characteristic signals in calcium imaging experiments. These calcium signals are also observed when intact animals are exposed to odors, suggesting that natural odor stimuli induce AWA spiking. The stimuli that elicit action potentials match AWA's specialized function in climbing odor gradients. Our results provide evidence that C. elegans neurons can encode information through regenerative all-or-none action potentials, expand the computational repertoire of its nervous system, and inform future modeling of its neural coding and network dynamics.

A Population Representation of Absolute Light Intensity in the Mammalian Retina.

Environmental illumination spans many log units of intensity and is tracked for essential functions that include regulation of the circadian clock, arousal state, and hormone levels. Little is known about the neural representation of light intensity and how it covers the necessary range. This question became accessible with the discovery of mammalian photoreceptors that are required for intensity-driven functions, the M1 ipRGCs. The spike outputs of M1s are thought to uniformly track intensity over a wide range. We provide a different understanding: individual cells operate over a narrow range, but the population covers irradiances from moonlight to full daylight. The range of most M1s is limited by depolarization block, which is generally considered pathological but is produced intrinsically by these cells. The dynamics of block allow the population to code stimulus intensity with flexibility and efficiency. Moreover, although spikes are distorted by block, they are regularized during axonal propagation.

Cardiac transmembrane ion channels and action potentials: cellular physiology and arrhythmogenic behavior.

Cardiac arrhythmias are among the leading causes of mortality. They often arise from alterations in the electrophysiological properties of cardiac cells and their underlying ionic mechanisms. It is therefore critical to further unravel the pathophysiology of the ionic basis of human cardiac electrophysiology in health and disease. In the first part of this review, current knowledge on the differences in ion channel expression and properties of the ionic processes that determine the morphology and properties of cardiac action potentials and calcium dynamics from cardiomyocytes in different regions of the heart are described. Then the cellular mechanisms promoting arrhythmias in congenital or acquired conditions of ion channel function (electrical remodeling) are discussed. The focus is on human-relevant findings obtained with clinical, experimental, and computational studies, given that interspecies differences make the extrapolation from animal experiments to human clinical settings difficult. Deepening the understanding of the diverse pathophysiology of human cellular electrophysiology will help in developing novel and effective antiarrhythmic strategies for specific subpopulations and disease conditions.

Ion Channels Controlling Circadian Rhythms in Suprachiasmatic Nucleus Excitability.

Animals synchronize to the environmental day-night cycle by means of an internal circadian clock in the brain. In mammals, this timekeeping mechanism is housed in the suprachiasmatic nucleus (SCN) of the hypothalamus and is entrained by light input from the retina. One output of the SCN is a neural code for circadian time, which arises from the collective activity of neurons within the SCN circuit and comprises two fundamental components: 1) periodic alterations in the spontaneous excitability of individual neurons that result in higher firing rates during the day and lower firing rates at night, and 2) synchronization of these cellular oscillations throughout the SCN. In this review, we summarize current evidence for the identity of ion channels in SCN neurons and the mechanisms by which they set the rhythmic parameters of the time code. During the day, voltage-dependent and independent Na+ and Ca2+ currents, as well as several K+ currents, contribute to increased membrane excitability and therefore higher firing frequency. At night, an increase in different K+ currents, including Ca2+-activated BK currents, contribute to membrane hyperpolarization and decreased firing. Layered on top of these intrinsically regulated changes in membrane excitability, more than a dozen neuromodulators influence action potential activity and rhythmicity in SCN neurons, facilitating both synchronization and plasticity of the neural code.

Structural basis for severe pain caused by mutations in the S4-S5 linkers of voltage-gated sodium channel NaV1.7.

Gain-of-function mutations in voltage-gated sodium channel NaV1.7 cause severe inherited pain syndromes, including inherited erythromelalgia (IEM). The structural basis of these disease mutations, however, remains elusive. Here, we focused on three mutations that all substitute threonine residues in the alpha-helical S4-S5 intracellular linker that connects the voltage sensor to the pore: NaV1.7/I234T, NaV1.7/I848T, and NaV1.7/S241T in order of their positions in the amino acid sequence within the S4-S5 linkers. Introduction of these IEM mutations into the ancestral bacterial sodium channel NaVAb recapitulated the pathogenic gain-of-function of these mutants by inducing a negative shift in the voltage dependence of activation and slowing the kinetics of inactivation. Remarkably, our structural analysis reveals a common mechanism of action among the three mutations, in which the mutant threonine residues create new hydrogen bonds between the S4-S5 linker and the pore-lining S5 or S6 segment in the pore module. Because the S4-S5 linkers couple voltage sensor movements to pore opening, these newly formed hydrogen bonds would stabilize the activated state substantially and thereby promote the 8 to 18 mV negative shift in the voltage dependence of activation that is characteristic of the NaV1.7 IEM mutants. Our results provide key structural insights into how IEM mutations in the S4-S5 linkers may cause hyperexcitability of NaV1.7 and lead to severe pain in this debilitating disease.

Human iPSC-Derived Neurons with Reliable Synapses and Large Presynaptic Action Potentials.

Understanding the function of the human brain requires determining basic properties of synaptic transmission in human neurons. One of the most fundamental parameters controlling neurotransmitter release is the presynaptic action potential, but its amplitude and duration remain controversial. Presynaptic action potentials have so far been measured with high temporal resolution only in a limited number of vertebrate but not in human neurons. To uncover properties of human presynaptic action potentials, we exploited recently developed tools to generate human glutamatergic neurons by transient expression of Neurogenin 2 (Ngn2) in pluripotent stem cells. During maturation for 3 to 9 weeks of culturing in different established media, the proportion of cells with multiple axon initial segments decreased, while the amount of axonal tau protein and neuronal excitability increased. Super-resolution microscopy revealed the alignment of the pre- and postsynaptic proteins, Bassoon and Homer. Synaptic transmission was surprisingly reliable at frequencies of 20, 50, and 100 Hz. The synchronicity of synaptic transmission during high-frequency transmission increased during 9 weeks of neuronal maturation. To analyze the mechanisms of synchronous high-frequency glutamate release, we developed direct presynaptic patch-clamp recordings from human neurons. The presynaptic action potentials had large overshoots to ∼25 mV and short durations of ∼0.5 ms. Our findings show that Ngn2-induced neurons represent an elegant model system allowing for functional, structural, and molecular analyses of glutamatergic synaptic transmission with high spatiotemporal resolution in human neurons. Furthermore, our data predict that glutamatergic transmission is mediated by large and rapid presynaptic action potentials in the human brain.

Impact of Extracellular Current Flow on Action Potential Propagation in Myelinated Axons.

Myelinated axons conduct action potentials, or spikes, in a saltatory manner. Inward current caused by a spike occurring at one node of Ranvier spreads axially to the next node, which regenerates the spike when depolarized enough for voltage-gated sodium channels to activate, and so on. The rate at which this process progresses dictates the velocity at which the spike is conducted and depends on several factors including axial resistivity and axon diameter that directly affect axial current. Here we show through computational simulations in modified double-cable axon models that conduction velocity also depends on extracellular factors whose effects can be explained by their indirect influence on axial current. Specifically, we show that a conventional double-cable model, with its outside layer connected to ground, transmits less axial current than a model whose outside layer is less absorptive. A more resistive barrier exists when an axon is packed tightly between other myelinated fibers, for example. We show that realistically resistive boundary conditions can significantly increase the velocity and energy efficiency of spike propagation, while also protecting against propagation failure. Certain factors like myelin thickness may be less important than typically thought if extracellular conditions are more resistive than normally considered. We also show how realistically resistive boundary conditions affect ephaptic interactions. Overall, these results highlight the unappreciated importance of extracellular conditions for axon function.

Intrinsic and Synaptic Contributions to Repetitive Spiking in Dentate Granule Cells.

Repetitive firing of granule cells (GCs) in the dentate gyrus (DG) facilitates synaptic transmission to the CA3 region. This facilitation can gate and amplify the flow of information through the hippocampus. High-frequency bursts in the DG are linked to behavior and plasticity, but GCs do not readily burst. Under normal conditions, a single shock to the perforant path in a hippocampal slice typically drives a GC to fire a single spike, and only occasionally more than one spike is seen. Repetitive spiking in GCs is not robust, and the mechanisms are poorly understood. Here, we used a hybrid genetically encoded voltage sensor to image voltage changes evoked by cortical inputs in many mature GCs simultaneously in hippocampal slices from male and female mice. This enabled us to study relatively infrequent double and triple spikes. We found GCs are relatively homogeneous and their double spiking behavior is cell autonomous. Blockade of GABA type A receptors increased multiple spikes and prolonged the interspike interval, indicating inhibitory interneurons limit repetitive spiking and set the time window for successive spikes. Inhibiting synaptic glutamate release showed that recurrent excitation mediated by hilar mossy cells contributes to, but is not necessary for, multiple spiking. Blockade of T-type Ca2+ channels did not reduce multiple spiking but prolonged interspike intervals. Imaging voltage changes in different GC compartments revealed that second spikes can be initiated in either dendrites or somata. Thus, pharmacological and biophysical experiments reveal roles for both synaptic circuitry and intrinsic excitability in GC repetitive spiking.

E3 ubiquitin ligase rififylin has yin and yang effects on rabbit cardiac transient outward potassium currents (Ito) and corresponding channel proteins.

Genome-wide association studies have reported a correlation between a SNP of the RING finger E3 ubiquitin protein ligase rififylin (RFFL) and QT interval variability in humans (Newton-Cheh et al., 2009). Previously, we have shown that RFFL downregulates expression and function of the human-like ether-a-go-go-related gene potassium channel and corresponding rapidly activating delayed rectifier potassium current (IKr) in adult rabbit ventricular cardiomyocytes. Here, we report that RFFL also affects the transient outward current (Ito), but in a peculiar way. RFFL overexpression in adult rabbit ventricular cardiomyocytes significantly decreases the contribution of its fast component (Ito,f) from 35% to 21% and increases the contribution of its slow component (Ito,s) from 65% to 79%. Since Ito,f in rabbits is mainly conducted by Kv4.3, we investigated the effect of RFFL on Kv4.3 expressed in HEK293A cells. We found that RFFL overexpression reduced Kv4.3 expression and corresponding Ito,f in a RING domain-dependent manner in the presence or absence of its accessory subunit Kv channel-interacting protein 2. On the other hand, RFFL overexpression in Kv1.4-expressing HEK cells leads to an increase in both Kv1.4 expression level and Ito,s, similarly in a RING domain-dependent manner. Our physiologically detailed rabbit ventricular myocyte computational model shows that these yin and yang effects of RFFL overexpression on Ito,f, and Ito,s affect phase 1 of the action potential waveform and slightly decrease its duration in addition to suppressing IKr. Thus, RFFL modifies cardiac repolarization reserve via ubiquitination of multiple proteins that differently affect various potassium channels and cardiac action potential duration.

Electrical excitability of cancer cells-CELEX model updated.

The normal functioning of every cell in the body depends on its bioelectric properties and many diseases are caused by genetic and/or epigenetic dysregulation of the underlying ion channels. Metastasis, the main cause of death from cancer, is a complex multi-stage process in which cells break away from a primary tumour, invade the surrounding tissues, enter the circulation by encountering a blood vessel and spread around the body, ultimately lodging in distant organs and reproliferating to form secondary tumours leading to devastating organ failure. Such cellular behaviours are well known to involve ion channels. The CELEX model offers a novel insight to metastasis where it is the electrical excitation of the cancer cells that is responsible for their aggressive and invasive behaviour. In turn, the hyperexcitability is underpinned by concomitant upregulation of functional voltage-gated sodium channels and downregulation of voltage-gated potassium channels. Here, we update the in vitro and in vivo evidence in favour of the CELEX model for carcinomas. The results are unequivocal for the sodium channel. The potassium channel arm is also broadly supported by existing evidence although these data are complicated by the impact of the channels on the membrane potential and consequent secondary effects. Finally, consistent with the CELEX model, we show (i) that carcinomas are indeed electrically excitable and capable of generating action potentials and (ii) that combination of a sodium channel inhibitor and a potassium channel opener can produce a strong, additive anti-invasive effect. We discuss the possible clinical implications of the CELEX model in managing cancer.

Nav1.2 channel mutations preventing fast inactivation lead to SCN2A encephalopathy.

SCN2A gene-related early-infantile developmental and epileptic encephalopathy (EI-DEE) is a rare and severe disorder that manifests in early infancy. SCN2A mutations affecting the fast inactivation gating mechanism can result in altered voltage dependence and incomplete inactivation of the encoded neuronal Nav1.2 channel and lead to abnormal neuronal excitability. In this study, we evaluated clinical data of seven missense Nav1.2 variants associated with DEE and performed molecular dynamics simulations, patch-clamp electrophysiology, and dynamic clamp real-time neuronal modelling to elucidate the molecular and neuron-scale phenotypic consequences of the mutations. The N1662D mutation almost completely prevented fast inactivation without affecting activation. The comparison of wild-type and N1662D channel structures suggested that the ambifunctional hydrogen bond formation between residues N1662 and Q1494 is essential for fast inactivation. Fast inactivation could also be prevented with engineered Q1494A or Q1494L Nav1.2 channel variants, whereas Q1494E or Q1494 K variants resulted in incomplete inactivation and persistent current. Molecular dynamics simulations revealed a reduced affinity of the hydrophobic IFM-motif to its receptor site with N1662D and Q1494L variants relative to wild-type. These results demonstrate that the interactions between N1662 and Q1494 underpin the stability and the orientation of the inactivation gate and are essential for the development of fast inactivation. Six DEE-associated Nav1.2 variants, with mutations mapped to channel segments known to be implicated in fast inactivation were also evaluated. Remarkably, the L1657P variant also prevented fast inactivation and produced biophysical characteristics that were similar to those of N1662D, whereas the M1501 V, M1501T, F1651C, P1658S, and A1659 V variants resulted in biophysical properties that were consistent with gain-of-function and enhanced action potential firing of hybrid neurons in dynamic action potential clamp experiments. Paradoxically, low densities of N1662D or L1657P currents potentiated action potential firing, whereas increased densities resulted in sustained depolarization. Our results provide novel structural insights into the molecular mechanism of Nav1.2 channel fast inactivation and inform treatment strategies for SCN2A-related EI-DEE. The contribution of non-inactivating Nav1.2 channels to neuronal excitability may constitute a distinct cellular mechanism in the pathogenesis of SCN2A-related DEE.

Ionic mechanisms involved in arginine vasopressin-mediated excitation of auditory cortical and thalamic neurons.

The axons containing arginine vasopressin (AVP) from the hypothalamus innervate a variety of structures including the cerebral cortex, thalamus, hippocampus and amygdala. A plethora amount of evidence indicates that activation of the V1a subtype of the vasopressin receptors facilitates anxiety-like and fear responses. As an essential structure involved in fear and anxiety responses, the amygdala, especially the lateral nucleus of amygdala (LA), receives glutamatergic innervations from the auditory cortex and auditory thalamus where high density of V1a receptors have been detected. However, the roles and mechanisms of AVP in these two important areas have not been determined, which prevents the understanding of the mechanisms whereby V1a activation augments anxiety and fear responses. Here, we used coronal brain slices and studied the effects of AVP on neuronal activities of the auditory cortical and thalamic neurons. Our results indicate that activation of V1a receptors excited both auditory cortical and thalamic neurons. In the auditory cortical neurons, AVP increased neuronal excitability by depressing multiple subtypes of inwardly rectifying K+ (Kir) channels including the Kir2 subfamily, the ATP-sensitive K+ channels and the G protein-gated inwardly rectifying K+ (GIRK) channels, whereas activation of V1a receptors excited the auditory thalamic neurons by depressing the Kir2 subfamily of the Kir channels as well as activating the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and a persistent Na+ channel. Our results may help explain the roles of V1a receptors in facilitating fear and anxiety responses. Categories: Cell Physiology.

Role and mechanism of EphB3 in epileptic seizures and epileptogenesis through Kalirin.

BACKGROUND: The EphB receptor tyrosine kinase family participates in intricate signaling pathways that orchestrate neural networks, guide neuronal axon development, and modulate synaptic plasticity through interactions with surface-bound ephrinB ligands. Additionally, Kalirin, a Rho guanine nucleotide exchange factor, is notably expressed in the postsynaptic membrane of excitatory neurons and plays a role in synaptic morphogenesis. This study postulates that Kalirin may act as a downstream effector of EphB3 in epilepsy. This investigation focuses on understanding the link between EphB3 and epilepsy. MATERIALS AND METHODS: Chronic seizure models using LiCl-pilocarpine (LiCl/Pilo) and pentylenetetrazol were developed in rats. Neuronal excitability was gauged through whole-cell patch clamp recordings on rat hippocampal slices. Real-time PCR determined Kalirin's mRNA expression, and Western blotting was employed to quantify EphB3 and Kalirin protein levels. Moreover, dendritic spine density in epileptic rats was evaluated using Golgi staining. RESULTS: Modulation of EphB3 functionality influenced acute seizure severity, latency duration, and frequency of spontaneous recurrent seizures. Golgi staining disclosed an EphB3-driven alteration in dendritic spine density within the hippocampus of epileptic rats, underscoring its pivotal role in the reconfiguration of hippocampal neural circuits. Furthermore, our data propose Kalirin as a prospective downstream mediator of the EphB3 receptor. CONCLUSIONS: Our findings elucidate that EphB3 impacts the action potential dynamics in isolated rat hippocampal slices and alters dendritic spine density in the inner molecular layer of epileptic rat hippocampi, likely through Kalirin-mediated pathways. This hints at EphB3's significant role in shaping excitatory circuit loops and recurrent seizure activity via Kalirin.

Analysis of changes in the action potential morphology of the mouse sinoatrial node true pacemaker cells during ontogenetic development in vitro and in silico.

BACKGROUND: Maturation of the mouse is accompanied by the increase in heart rate. However, the mechanisms underlying this process remain unclear. We performed an action potentials (APs) recordings in mouse sinoatrial node (SAN) true pacemaker cells and in silico analysis to clarify the mechanisms underlying pre-postnatal period heart rate changes. RESULTS: The APs of true pacemaker cells at different stages had similar configurations and dV/dtmax values. The cycle length, action potential duration (APD90 ), maximal diastolic potential (MDP), and AP amplitude decreased, meanwhile the velocity of diastolic depolarization (DDR) increased from E12.5 stage to adult. Using a pharmacological approach we found that in SAN true pacemaker cells ivabradine reduces the DDR and the cycle length significantly stronger in E12.5 than in newborn and adult mice, whereas the effects of Ni2+ and nifedipine were significantly stronger in adult mice. Computer simulations further suggested that the density of the hyperpolarization-activated pacemaker сurrent (If ) decreased during development, whereas transmembrane and intracellular Ca2+ flows increased. CONCLUSIONS: The ontogenetic decrease in IK1 density from E12.5 to adult leads to depolarization of MDP to the voltage range in which calcium currents are activated, thereby shifting the balance from the "membrane-clock" to the "calcium-clock."

Contributions of the Sodium Leak Channel NALCN to Pacemaking of Medial Ventral Tegmental Area and Substantia Nigra Dopaminergic Neurons.

We tested the role of the sodium leak channel, NALCN, in pacemaking of dopaminergic neuron (DAN) subpopulations from adult male and female mice. In situ hybridization revealed NALCN RNA in all DANs, with lower abundance in medial ventral tegmental area (VTA) relative to substantia nigra pars compacta (SNc). Despite lower relative abundance of NALCN, we found that acute pharmacological blockade of NALCN in medial VTA DANs slowed pacemaking by 49.08%. We also examined the electrophysiological properties of projection-defined VTA DAN subpopulations identified by retrograde labeling. Inhibition of NALCN reduced pacemaking in DANs projecting to medial nucleus accumbens (NAc) and others projecting to lateral NAc by 70.74% and 31.98%, respectively, suggesting that NALCN is a primary driver of pacemaking in VTA DANs. In SNc DANs, potentiating NALCN by lowering extracellular calcium concentration speeded pacemaking in wildtype but not NALCN conditional knockout mice, demonstrating functional presence of NALCN. In contrast to VTA DANs, however, pacemaking in SNc DANs was unaffected by inhibition of NALCN. Instead, we found that inhibition of NALCN increased the gain of frequency-current plots at firing frequencies slower than spontaneous firing. Similarly, inhibition of the hyperpolarization-activated cyclic nucleotide-gated (HCN) conductance increased gain but had little effect on pacemaking. Interestingly, simultaneous inhibition of NALCN and HCN resulted in significant reduction in pacemaker rate. Thus, we found NALCN makes substantial contributions to driving pacemaking in VTA DAN subpopulations. In SNc DANs, NALCN is not critical for pacemaking but inhibition of NALCN makes cells more sensitive to hyperpolarizing stimuli.SIGNIFICANCE STATEMENT Pacemaking in midbrain dopaminergic neurons (DAN) relies on multiple subthreshold conductances, including a sodium leak. Whether the sodium leak channel, NALCN, contributes to pacemaking in DANs located in the VTA and the SNc has not yet been determined. Using electrophysiology and pharmacology, we show that NALCN plays a prominent role in driving pacemaking in projection-defined VTA DAN subpopulations. By contrast, pacemaking in SNc neurons does not rely on NALCN. Instead, the presence of NALCN regulates the excitability of SNc DANs by reducing the gain of the neuron's response to inhibitory stimuli. Together, these findings will inform future efforts to obtain DAN subpopulation-specific treatments for use in neuropsychiatric disorders.

The Amyloid Precursor Protein Modulates the Position and Length of the Axon Initial Segment.

The amyloid precursor protein (APP) is linked to the genetics and pathogenesis of Alzheimer's disease (AD). It is the parent protein of the ß-amyloid (Aß) peptide, the main constituent of the amyloid plaques found in an AD brain. The pathways from APP to Aß are intensively studied, yet the normal functions of APP itself have generated less interest. We report here that glutamate stimulation of neuronal activity leads to a rapid increase in App gene expression. In mouse and human neurons, elevated APP protein changes the structure of the axon initial segment (AIS) where action potentials are initiated. The AIS is shortened in length and shifts away from the cell body. The GCaMP8f Ca2+ reporter confirms the predicted decrease in neuronal activity. NMDA antagonists or knockdown of App block the glutamate effects. The actions of APP on the AIS are cell-autonomous; exogenous Aß, either fibrillar or oligomeric, has no effect. In culture, APPSwe (a familial AD mutation) induces larger AIS changes than wild type APP. Ankyrin G and ßIV-spectrin, scaffolding proteins of the AIS, both physically associate with APP, more so in AD brains. Finally, in humans with sporadic AD or in the R1.40 AD mouse model, both females and males, neurons have elevated levels of APP protein that invade the AIS. In vivo as in vitro, this increased APP is associated with a significant shortening of the AIS. The findings outline a new role for the APP and encourage a reconsideration of its relationship to AD.SIGNIFICANCE STATEMENT While the amyloid precursor protein (APP) has long been associated with Alzheimer's disease (AD), the normal functions of the full-length Type I membrane protein have been largely unexplored. We report here that the levels of APP protein increase with neuronal activity. In vivo and in vitro, modest amounts of excess APP alter the properties of the axon initial segment. The ß-amyloid peptide derived from APP is without effect. Consistent with the observed changes in the axon initial segment which would be expected to decrease action potential firing, we show that APP expression depresses neuronal activity. In mouse AD models and human sporadic AD, APP physically associates with the scaffolding proteins of the axon initial segment, suggesting a relationship with AD dementia.