Triggered Action Potentials Research Articles

Cortical neurons and networks are dormant but fully responsive during isoelectric brain state.

A continuous isoelectric electroencephalogram reflects an interruption of endogenously-generated activity in cortical networks and systematically results in a complete dissolution of conscious processes. This electro-cerebral inactivity occurs during various brain disorders, including hypothermia, drug intoxication, long-lasting anoxia and brain trauma. It can also be induced in a therapeutic context, following the administration of high doses of barbiturate-derived compounds, to interrupt a hyper-refractory status epilepticus. Although altered sensory responses can be occasionally observed on an isoelectric electroencephalogram, the electrical membrane properties and synaptic responses of individual neurons during this cerebral state remain largely unknown. The aim of the present study was to characterize the intracellular correlates of a barbiturate-induced isoelectric electroencephalogram and to analyse the sensory-evoked synaptic responses that can emerge from a brain deprived of spontaneous electrical activity. We first examined the sensory responsiveness from patients suffering from intractable status epilepticus and treated by administration of thiopental. Multimodal sensory responses could be evoked on the flat electroencephalogram, including visually-evoked potentials that were significantly amplified and delayed, with a high trial-to-trial reproducibility compared to awake healthy subjects. Using an analogous pharmacological procedure to induce prolonged electro-cerebral inactivity in the rat, we could describe its cortical and subcortical intracellular counterparts. Neocortical, hippocampal and thalamo-cortical neurons were all silent during the isoelectric state and displayed a flat membrane potential significantly hyperpolarized compared with spontaneously active control states. Nonetheless, all recorded neurons could fire action potentials in response to intracellularly injected depolarizing current pulses and their specific intrinsic electrophysiological features were preserved. Manipulations of the membrane potential and intracellular injection of chloride in neocortical neurons failed to reveal an augmented synaptic inhibition during the isoelectric condition. Consistent with the sensory responses recorded from comatose patients, large and highly reproducible somatosensory-evoked potentials could be generated on the inactive electrocorticogram in rats. Intracellular recordings revealed that the underlying neocortical pyramidal cells responded to sensory stimuli by complex synaptic potentials able to trigger action potentials. As in patients, sensory responses in the isoelectric state were delayed compared to control responses and exhibited an elevated reliability during repeated stimuli. Our findings demonstrate that during prolonged isoelectric brain state neurons and synaptic networks are dormant rather than excessively inhibited, conserving their intrinsic properties and their ability to integrate and propagate environmental stimuli.

Development of membrane ion channels during neural differentiation from human embryonic stem cells

ObjectiveFor human embryonic stem cells (hESCs) to differentiate into neurons, enormous changes has to occur leading to trigger action potential and neurotransmitter release. We attempt to determine the changes in expression of voltage gated channels (VGCs) and their electrophysiological properties during neural differentiation. Materials and methodsThe relative expressions of α-subunit of voltage gated potassium, sodium and calcium channels were characterized by qRT-PCR technique. Patch clamp recording was performed to characterize the electrophysiological properties of hESCs during their differentiation into neuron-like cells. ResultsRelative expression of α-subunit of channels changed significantly. 4-AP and TEA sensitive outward currents were observed in all stages, although TEA sensitive currents were recorded once in rosette structure. Nifedipine and QX314 sensitive inward currents were recorded only in neuron-like cells. ConclusionK+ currents were recorded in hESCs and rosette structure cells. Inward currents, sensitive to Nifedipine and QX314, were recorded in neuron-like cells.

Polarity of varicosity initiation in central neuron mechanosensation.

Little is known about mechanical regulation of morphological and functional polarity of central neurons. In this study, we report that mechanical stress specifically induces varicosities in the axons but not the dendrites of central neurons by activating TRPV4, a Ca2+/Na+-permeable mechanosensitive channel. This process is unexpectedly rapid and reversible, consistent with the formation of axonal varicosities in vivo induced by mechanical impact in a mouse model of mild traumatic brain injury. In contrast, prolonged stimulation of glutamate receptors induces varicosities in dendrites but not in axons. We further show that axonal varicosities are induced by persistent Ca2+ increase, disassembled microtubules (MTs), and subsequently reversible disruption of axonal transport, and are regulated by stable tubulin-only polypeptide, an MT-associated protein. Finally, axonal varicosity initiation can trigger action potentials to antidromically propagate to the soma in retrograde signaling. Therefore, our study demonstrates a new feature of neuronal polarity: axons and dendrites preferentially respond to physical and chemical stresses, respectively.

Morphology controls how hippocampal CA1 pyramidal neuron responds to uniform electric fields: a biophysical modeling study

Responses of different neurons to electric field (EF) are highly variable, which depends on intrinsic properties of cell type. Here we use multi-compartmental biophysical models to investigate how morphologic features affect EF-induced responses in hippocampal CA1 pyramidal neurons. We find that the basic morphologies of neuronal elements, including diameter, length, bend, branch, and axon terminals, are all correlated with somatic depolarization through altering the current sources or sinks created by applied field. Varying them alters the EF threshold for triggering action potentials (APs), and then determines cell sensitivity to suprathreshold field. Introducing excitatory postsynaptic potential increases cell excitability and reduces morphology-dependent EF firing threshold. It is also shown that applying identical subthreshold EF results in distinct polarizations on cell membrane with different realistic morphologies. These findings shed light on the crucial role of morphologies in determining field-induced neural response from the point of view of biophysical models. The predictions are conducive to better understanding the variability in modulatory effects of EF stimulation at the cellular level, which could also aid the interpretations of how applied fields activate central nervous system neurons and affect relevant circuits.

Role of mucosa in generating spontaneous activity in the guinea pig seminal vesicle.

The mucosa may have neuron-like functions as urinary bladder mucosa releases bioactive substances that modulate sensory nerve activity as well as detrusor muscle contractility. However, such mucosal function in other visceral organs remains to be established. The role of mucosa in generating spontaneous contractions in seminal vesicles (SVs), a paired organ in the male reproductive tract, was investigated. The intact mucosa is essential for the generation of spontaneous phasic contractions of SV smooth muscle arising from electrical slow waves and corresponding increases in intracellular Ca2+ . These spontaneous events primarily depend on Ca2+ handling by sarco-endoplasmic reticulum Ca2+ stores. A population of mucosal cells developed spontaneous rises in intracellular Ca2+ relying on sarco-endoplasmic reticulum Ca2+ handling. The spontaneously active cells in the SV mucosa appear to drive spontaneous activity in smooth muscle either by sending depolarizing signals and/or by releasing humoral substances. The role of the mucosa in generating the spontaneous activity of guinea-pig seminal vesicle (SV) was explored. Changes in contractility, membrane potential and intracellular Ca2+ dynamics of SV smooth muscle cells (SMCs) were recorded using isometric tension recording, intracellular microelectrode recording and epi-fluorescence Ca2+ imaging, respectively. Mucosa-intact but not mucosa-denuded SV preparations generated TTX- (1μm) resistant spontaneous phasic contractions that were abolished by nifedipine (3μm). Consistently, SMCs developed mucosa-dependent slow waves (SWs) that triggered action potentials and corresponding Ca2+ flashes. Nifedipine (10μm) abolished the action potentials and spontaneous contractions, while suppressing the SWs and Ca2+ flashes. Both the residual SWs and spontaneous Ca2+ transients were abolished by cyclopiazonic acid (CPA, 10μm), a sarco-endoplasmic reticulum Ca2+ -ATPase (SERCA) inhibitor. DIDS (300μm) and niflumic acid (100μm), blockers for Ca2+ -activated Cl- channels (CACCs), or low Cl- solution also slowed or prevented the generation of SWs. In SV mucosal preparations detached from the muscle layer, a population of mucosal cells generated spontaneous Ca2+ transients that were blocked by CPA but not nifedipine. These results suggested that spontaneous contractions and corresponding Ca2+ flashes in SV SMCs arise from action potential generation due to the opening of L-type voltage-dependent Ca2+ channels. Spontaneous Ca2+ transients appear to primarily result from Ca2+ release from sarco-endoplasmic reticulum Ca2+ stores to activate CACCs to develop SWs. The mucosal cells firing spontaneous Ca2+ transients may play a critical role in driving spontaneous activity of SV smooth muscle either by sending depolarizing signals or by releasing humoral substances.

Multiscale Determinants of Delayed Afterdepolarization Amplitude in Cardiac Tissue

Spontaneous calcium (Ca) waves in cardiac myocytes underlie delayed afterdepolarizations (DADs) that trigger cardiac arrhythmias. How these subcellular/cellular events overcome source-sink factors in cardiac tissue to generate DADs of sufficient amplitude to trigger action potentials is not fully understood. Here, we evaluate quantitatively how factors at the subcellular scale (number of Ca wave initiation sites), cellular scale (sarcoplasmic reticulum (SR) Ca load), and tissue scale (synchrony of Ca release in populations of myocytes) determine DAD features in cardiac tissue using a combined experimental and computational modeling approach. Isolated patch-clamped rabbit ventricular myocytes loaded with Fluo-4 to image intracellular Ca were rapidly paced during exposure to elevated extracellular Ca (2.7 mmol/L) and isoproterenol (0.25 μmol/L) to induce diastolic Ca waves and subthreshold DADs. As the number of paced beats increased from 1 to 5, SR Ca content (assessed with caffeine pulses) increased, the number of Ca wave initiation sites increased, integrated Ca transients and DADs became larger and shorter in duration, and the latency period to the onset of Ca waves shortened with reduced variance. In silico analysis using a computer model of ventricular tissue incorporating these experimental measurements revealed that whereas all of these factors promoted larger DADs with higher probability of generating triggered activity, the latency period variance and SR Ca load had the greatest influences. Therefore, incorporating quantitative experimental data into tissue level simulations reveals that increased intracellular Ca promotes DAD-mediated triggered activity in tissue predominantly by increasing both the synchrony (decreasing latency variance) of Ca waves in nearby myocytes and SR Ca load, whereas the number of Ca wave initiation sites per myocyte is less important.

Effects of chronic abdominal vagal stimulation of small-diameter neurons on brain metabolism and food intake

BackgroundAbdominal bilateral vagal stimulation reduces food intake in animals. However, the classical square wave, mA range current generator is poorly effective to evoke action potentials on A∂ and C neurons that represent the majority of vagal neurons at the abdominal level. Objective/Hypothesis(i)To ascertain the capability of very high-frequency stimulation schemes (pulsons) to trigger action potentials in abdominal vagal neurons in anaesthetized pigs. (ii) To compare these stimulation schemes with classical ones using PET imaging of brain metabolism and food intake behaviour in conscious pigs. MethodsThe current thresholds for pulsons (S2 & S3) and millisecond pulses (S1) required to trigger action potentials were calculated in 5 anaesthetized pigs using single fibre recording. Similar stimulation protocols were compared chronically to sham stimulation in 24 pigs. After two weeks of chronic stimulation, food intake and brain metabolism were investigated. The electrical characteristics and histology of the vagus nerve were also studied. ResultsS3 stimulation required a lower amount of charges to trigger an action potential. Chronically applied S2 & S3 activated the dorsal vagal complex and increased the metabolism of its afferent cortical structures. They also reduced energy intake together with a reduced ingestion of high fat and high sugar diets. All these effects were not observed for the S1 group. The vagal histology for the S1, S2 and S3 groups was not different from that of the sham. ConclusionsThese findings demonstrate that pulsons applied bilaterally on the abdominal vagus reduced food intake as a consequence of the activation of the brainstem and higher-order brain areas.

Spatial patterning of excitatory and inhibitory neuropil territories during spinal circuit development.

To generate rhythmic motor behaviors, both single neurons and neural circuits require a balance between excitatory inputs that trigger action potentials and inhibitory inputs that promote a stable resting potential (E/I balance). Previous studies have focused on individual neurons and have shown that, over a short spatial scale, excitatory and inhibitory (E/I) synapses tend to form structured territories with inhibitory inputs enriched on cell bodies and proximal dendrites and excitatory inputs on distal dendrites. However, systems-level E/I patterns, at spatial scales larger than single neurons, are largely uncharted. We used immunostaining for PSD-95 and gephyrin postsynaptic scaffolding proteins as proxies for excitatory and inhibitory synapses, respectively, to quantify the numbers and map the distributions of E/I synapses in zebrafish spinal cord at both an embryonic stage and a larval stage. At the embryonic stage, we found that PSD-95 puncta outnumber gephyrin puncta, with the number of gephyrin puncta increasing to match that of PSD-95 puncta at the larval stage. At both stages, PSD-95 puncta are enriched in the most lateral neuropil corresponding to distal dendrites while gephyrin puncta are enriched on neuronal somata and in the medial neuropil. Significantly, similar to synaptic puncta, neuronal processes also exhibit medial-lateral territories at both developmental stages with enrichment of glutamatergic (excitatory) processes laterally and glycinergic (inhibitory) processes medially. This establishment of neuropil excitatory-inhibitory structure largely precedes dendritic arborization of primary motor neurons, suggesting that the structured neuropil could provide a framework for the development of E/I balance at the cellular level. J. Comp. Neurol. 525:1649-1667, 2017. © 2016 Wiley Periodicals, Inc.

Shear Stress Induces Transverse Ca2+ Waves via Autocrine Activation of P2X Purinoceptors in Rat Atrial Myocytes

Atrial myocytes are exposed to high shear stress during blood regurgitation and high intra-atrial pressure due to valve diseases and heart failure, since such disturbances disrupt endocardium. We have previously reported that shear stress induces two types of global Ca2+ waves in atrial myocytes, longitudinal and transverse Ca2+ waves (T-waves) (Biophys J 2012;102(3, Suppl 1);227a), and that the longitudinal wave is triggered by Ca2+ release via P2Y1 purinoceptor-inositol 1,4,5-trisphosphate receptor signaling (J Physiol 2015;593:5091-5109). Here, we investigated cellular mechanism for the generation of T-wave in atrial cells under shear stress. Shear stress of ∼16 dyn/cm2 was applied onto single myocytes using micro fluid-jet, and two-dimensional confocal Ca2+ imaging was performed. Shear stress-induced T-waves were observed repetitively under 3-4 min intervals between the stimuli, and occurred at ∼1 event per 10 s. They were eliminated by inhibition of the voltage-gated Na+ or Ca2+ channels, or ryanodine receptors, suggesting that the T-wave is mediated by action potential-triggered Ca2+ release. Blockades of key stretch signaling molecules, stretch-activated channel, Na+-Ca2+ exchange, and NADPH oxidase, did not suppress T-wave generation by shear. However, shear-induced T-wave generation was abolished by pre-incubation of cells with external ATP-metabolizing enzyme apyrase, the gap junction blocker carbenoxolone, or with P2X purinoceptor antagonist iso-PPADS. Inhibition of P2Y1 purinergic signaling that mediates the longitudinal Ca2+ wave under shear did not attenuate the occurrence of T-waves. Our data suggest that shear stress induces activation of P2X purinoceptors via gap junction-mediated ATP release, thereby triggering action potential with subsequent T-wave in atrial myocytes.

Extracellular ATP Triggers Action Potentials in Ventricular Cardiomyocytes

Myocardial ischemia causes a high frequency of ventricular arrhythmias. A long established metabolic response to hypoxia in the heart is the release of ATP. Here we tested the hypothesis that ATP released from cardiomyocytes during acute myocardial injury can contribute to ventricular ectopy by causing delayed afterdepolarizations and triggered action potentials. Isolated mouse ventricular cardiomyocytes were loaded with Fura2-AM to measure intracellular Ca. Spontaneous Ca waves (SCW) and triggered beats (TB) were quantified after a train of field-stimulated Ca transients (for 20s with 3 Hz) in presence of inotropic stimulation with isoproterenol (1 µM) and partial inhibition of the inward rectifier with Ba (5 µM) to facilitate TBs. Application of extracellular ATP (3-50 µM) significantly increased the incidence and frequency of TBs, whereas the rate of SCWs was not significantly changed. Notably, extracellular ATP application generated a different type of TBs that was not preceded by SCW, which were termed ATP-triggered beats (TBATP). Higher concentrations of ATP (> 50 µM) reduced the rates of TBATP. Simultaneous [Ca]i and membrane potential measurements using perforated patch method demonstrated that both regular TBs and TBATP were preceded by a delayed afterdepolarization (DAD) of similar shape. However, in presence of ATP, 34% of DAD that triggered beats occurred WITHOUT concomitant elevations of cytosolic [Ca]. In other words, general DADs and TBs were mediated by the classic Ca-dependent mechanism (i.e., Na-Ca exchange current in response to spontaneous SR Ca release), whereas ATP generated DADs occurred in the absence of the classic Ca-dependent pathway. In isolated Langendorff perfused mouse hearts, adding ATP (30 µM) to the perfusate triggered both atrial and ventricular premature beats. We conclude that low micromolar extracellular ATP activates an inward current that is sufficient to generate DADs and spontaneous action potentials in the absence of spontaneous SR Ca release. Although the molecular identity of ATP-activated inward current remains to be determined, the current study demonstrates that ATP release by acute ischemic stress could facilitate arrhythmia susceptibility via a Ca-independent mechanism.

Optogenetic activation of melanin-concentrating hormone neurons increases non-rapid eye movement and rapid eye movement sleep during the night in rats.

Neurons containing melanin-concentrating hormone (MCH) are located in the hypothalamus. In mice, optogenetic activation of the MCH neurons induces both non-rapid eye movement (NREM) and rapid eye movement (REM) sleep at night, the normal wake-active period for nocturnal rodents [R. R. Konadhode etal. (2013) J. Neurosci., 33, 10257-10263]. Here we selectively activate these neurons in rats to test the validity of the sleep network hypothesis in another species. Channelrhodopsin-2 (ChR2) driven by the MCH promoter was selectively expressed by MCH neurons after injection of rAAV-MCHp-ChR2-EYFP into the hypothalamus of Long-Evans rats. An invitro study confirmed that the optogenetic activation of MCH neurons faithfully triggered action potentials. In the second study, in Long-Evans rats, rAAV-MCH-ChR2, or the control vector, rAAV-MCH-EYFP, were delivered into the hypothalamus. Three weeks later, baseline sleep was recorded for 48h without optogenetic stimulation (0Hz). Subsequently, at the start of the lights-off cycle, the MCH neurons were stimulated at 5, 10, or 30Hz (1mW at tip; 1min on - 4min off) for 24h. Sleep was recorded during the 24-h stimulation period. Optogenetic activation of MCH neurons increased both REM and NREM sleep at night, whereas during the day cycle, only REM sleep was increased. Delta power, an indicator of sleep intensity, was also increased. In control rats without ChR2, optogenetic stimulation did not increase sleep or delta power. These results lend further support to the view that sleep-active MCH neurons contribute to drive sleep in mammals.

In vivo matching of postsynaptic excitability with spontaneous synaptic inputs during formation of the rat calyx of Held synapse.

Neurons in the medial nucleus of the trapezoid body of anaesthetized rats of postnatal day (P)2-6 showed burst firing with a preferred interval of about 100ms, which was stable, and a second preferred interval of 5-30ms, which shortened during development. In 3 out of 132 cases, evidence for the presence of two large inputs was found. In vivo whole-cell recordings revealed that the excitability of the principal neuron and the size of its largest synaptic inputs were developmentally matched. At P2-4, action potentials were triggered by barrages of small synaptic events that summated to plateau potentials, while at later stages firing depended on a single, large and often prespike-associated input, which is probably the nascent calyx of Held. Simulations with a Hodgkin-Huxley-like model, which was based on fits of the intrinsic postsynaptic properties, suggested an essential role for the low-threshold potassium conductance in this transition. In the adult, principal neurons of the medial nucleus of the trapezoid body (MNTB) are typically contacted by a single, giant terminal called the calyx of Held, whereas during early development a principal neuron receives inputs from many axons. How these changes in innervation impact the postsynaptic activity has not yet been studied in vivo. We therefore recorded spontaneous inputs and intrinsic properties of principal neurons in anaesthetized rat pups during the developmental period in which the calyx forms. A characteristic bursting pattern could already be observed at postnatal day (P)2, before formation of the calyx. At this age, action potentials (APs) were triggered by barrages of summating EPSPs causing plateau depolarizations. In contrast, at P5, a single EPSP reliably triggered APs, resulting in a close match between pre- and postsynaptic firing. Postsynaptic excitability and the size of the largest synaptic events were developmentally matched. The developmental changes in intrinsic properties were estimated by fitting in vivo current injections to a Hodgkin-Huxley-type model of the principal neuron. Our simulations indicated that the developmental increases in Ih , low-threshold K+ channels and leak currents contributed to the reduction in postsynaptic excitability, but that low-threshold K+ channels specifically functioned as a dampening influence in the near-threshold range, thus precluding small inputs from triggering APs. Together, these coincident changes help to propagate bursting activity along the auditory brainstem, and are essential steps towards establishing the relay function of the calyx of Held synapse.

Rat odontoblasts may use glutamate to signal dentin injury

Accumulating evidence indicates that odontoblasts act as sensor cells, capable of triggering action potentials in adjacent pulpal nociceptive axons, suggesting a paracrine signaling via a currently unknown mediator. Since glutamate can mediate signaling by non-neuronal cells, and peripheral axons may express glutamate receptors (GluR), we hypothesized that the expression of high levels of glutamate, and of sensory receptors in odontoblasts, combined with an expression of GluR in adjacent pulpal axons, is the morphological basis for odontoblastic sensory signaling. To test this hypothesis, we investigated the expression of glutamate, the thermo- and mechanosensitive ion channels transient receptor potential vanilloid 1 (TRPV1), transient receptor potential ankyrin 1 (TRPA1), and TWIK-1-related K+channel (TREK-1), and the glutamate receptor mGluR5, in a normal rat dental pulp, and following dentin injury. We also examined the glutamate release from odontoblast in cell culture.Odontoblasts were enriched with glutamate, at the level as high as in adjacent pulpal axons, and showed immunoreactivity for TRPV1, TRPA1, and TREK-1. Pulpal sensory axons adjacent to odontoblasts expressed mGluR5. Both the levels of glutamate in odontoblasts, and the expression of mGluR5 in nearby axons, were upregulated following dentin injury. The extracellular glutamate concentration was increased significantly after treating of odontoblast cell line with calcium permeable ionophore, suggesting glutamate release from odontoblasts.These findings lend morphological support to the hypothesis that odontoblasts contain glutamate as a potential neuroactive substance that may activate adjacent pulpal axons, and thus contribute to dental pain and hypersensitivity.

Abstract 459: Oxidized CaMKII Causes Atrial Fibrillation Susceptibility in a Diabetic Mouse Model

Background: Atrial fibrillation (AF) and diabetes mellitus (DM) are major, unsolved public health problems. DM is a known risk factor for AF, and both are associated with increased reactive oxygen species (ROS), suggesting a ROS responsive disease signal could be a mechanistic link between them. The multifunctional Ca2+ and calmodulin-dependent protein kinase-II (CaMKII) is activated by oxidation of paired methionines. Oxidized CaMKII (ox-CaMKII) is increased in atria from DM patients and causes ryanodine receptor (RyR2) hyperphosphorylation that promotes pathological intracellular Ca2+ release and Ca2+ triggered arrhythmias. We hypothesize that DM increases myocardial ox-CaMKII, RyR2 hyperphosphorylation and AF. Methods and Results: C57BL/6J mice with streptozocin-induced type 1 DM had increased AF susceptibility following atrial burst pacing compared with citrate buffer-treated wild-type (WT) controls [70% (14/20) vs. 25% (5/20), p = 0.01]. Ox-CaMKII was increased in atrial tissue from diabetic mice compared to controls, consistent with a role for ox-CaMKII in this model. Diabetic ox-CaMKII resistant knock-in (MM-VV) mice (37.5% (9/24) [p < 0.05]) and diabetic mice with myocardium-restricted transgenic overexpression of methionine sulfoxide reductase A (25% (5/20) [p < 0.05]), which reverses ox-CaMKII, were protected from DM increased AF susceptibility compared to diabetic WT controls. Atrial myocytes from diabetic WT mice demonstrated increased RyR2 mediated Ca2+ spark frequency, triggered action potentials and delayed intracellular [Ca2+] decay compared to controls. Diabetic knock-in mice resistant to CaMKII-mediated RyR2 phosphorylation (S2814A) had decreased AF susceptibility (25% (5/20) [p < 0.05]), compared with diabetic WT mice. All groups of diabetic mice had similar increases in plasma glucose. Conclusions: Hyperglycemia increases AF susceptibility and increased ox-CaMKII is associated with increased AF in this diabetic mouse model. Genetic manipulation of an ox-CaMKII pathway can protect against AF susceptibility in DM. These findings suggest that ox-CaMKII is a critical proarrhythmic signal in DM and a potential therapeutic target for AF management in DM patients.

Current view on regulation of voltage-gated sodium channels by calcium and auxiliary proteins.

In cardiac and skeletal myocytes, and in most neurons, the opening of voltage-gated Na(+) channels (NaV channels) triggers action potentials, a process that is regulated via the interactions of the channels' intercellular C-termini with auxiliary proteins and/or Ca(2+) . The molecular and structural details for how Ca(2+) and/or auxiliary proteins modulate NaV channel function, however, have eluded a concise mechanistic explanation and details have been shrouded for the last decade behind controversy about whether Ca(2+) acts directly upon the NaV channel or through interacting proteins, such as the Ca(2+) binding protein calmodulin (CaM). Here, we review recent advances in defining the structure of NaV intracellular C-termini and associated proteins such as CaM or fibroblast growth factor homologous factors (FHFs) to reveal new insights into how Ca(2+) affects NaV function, and how altered Ca(2+) -dependent or FHF-mediated regulation of NaV channels is perturbed in various disease states through mutations that disrupt CaM or FHF interaction.

(287) Anoctamin-1 Cl- channel in nociception: inhibition by T16[inh]-A01 and activation by an N-aroylaminothiazole and capsaicin

Anoctamin 1 (ANO1 or TMEM16A) Ca2+−gated Cl− channels of nociceptor sensory neurons are emerging as important molecular components of peripheral pain transduction. At physiological intracellular Cl− concentrations ([Cl−]i) sensory neuronal Cl− channels, such as ANO1, are excitatory. ANO1 channels are directly activated by an N-aroylaminothiazole (E-act). At physiological [Cl−]i (40mM), E-act (10μM) direct activation of ANO1 increased current sizes (in voltage-clamp) and action potential firing (in current-clamp) recorded using whole-cell electrophysiology of mouse dorsal root ganglia (DRG) sensory neurons. Nocifensive responses of mice hind paws were also dramatically induced by subcutaneous injections of E-act (5mM), which was attenuated by co-injection of ANO1-inhibitor T16A[inh]-A01 (1.3mM). ANO1 channels are also activated by intracellular Ca2+ ([Ca2+]i) from sensory neuronal TRPV1 (transient-receptor-potential vallinoid 1) ion channels and other noxicant receptors. At physiological [Cl−]i, capsaicin (15μM)-induced action potential firing was diminished by co-application with ANO1-inhibitor T16A[inh]-A01 (20μM) To not disrupt TRPV1 carried-Ca2+ activation of ANO1 in DRG neurons. (To not disrupt TRPV1 carried-Ca2+ activation of ANO1 in DRG neurons, ANO1 modulation of capsaicin-induced action potentials was measured by perforated-patch (Amphotericin−B) current-clamp technique). Under conditions of low [Cl−]i (10mM), ANO1 actions were reversed. Specifically, E-act did not trigger action potentials; however, capsaicin-induced action potential firing was inhibited by co-application of E-act, but was unaffected by co-application of T16A[inh]-A01. Nocifensive responses of mice hind paws were dramatically induced by subcutaneous injections of E-actcapsaicin (50μM). The nocifensive responses were attenuated by co-injection with T16A[inh]-A01 (1.3mM). In summary, an ANO1-activator induced [Cl−]i-dependent sensory neuronal action potentials and mouse nocifensive behaviors; thus, direct ANO1 activation can induce pain perception. ANO1-inhibition attenuated capsaicin-triggering of action potentials and capsaicin-induced nocifensive behaviors. The results indicate that sensory neuronal ANO1 facilitates TRPV1 triggering of action potentials, resulting in enhanced nociception and inhibition of ANO1 could be a novel means of inducing analgesia.

Predicting binaural responses from monaural responses in the gerbil medial superior olive.

Accurate sound source localization of low-frequency sounds in the horizontal plane depends critically on the comparison of arrival times at both ears. A specialized brainstem circuit containing the principal neurons of the medial superior olive (MSO) is dedicated to this comparison. MSO neurons are innervated by segregated inputs from both ears. The coincident arrival of excitatory inputs from both ears is thought to trigger action potentials, with differences in internal delays creating a unique sensitivity to interaural time differences (ITDs) for each cell. How the inputs from both ears are integrated by the MSO neurons is still debated. Using juxtacellular recordings, we tested to what extent MSO neurons from anesthetized Mongolian gerbils function as simple cross-correlators of their bilateral inputs. From the measured subthreshold responses to monaural wideband stimuli we predicted the rate-ITD functions obtained from the same MSO neuron, which have a damped oscillatory shape. The rate of the oscillations and the position of the peaks and troughs were accurately predicted. The amplitude ratio between dominant and secondary peaks of the rate-ITD function, captured in the width of its envelope, was not always exactly reproduced. This minor imperfection pointed to the methodological limitation of using a linear representation of the monaural inputs, which disregards any temporal sharpening occurring in the cochlear nucleus. The successful prediction of the major aspects of rate-ITD curves supports a simple scheme in which the ITD sensitivity of MSO neurons is realized by the coincidence detection of excitatory monaural inputs.

Post-Inhibitory Rebound Spikes in Rat Medial Entorhinal Layer II/III Principal Cells: In Vivo, In Vitro, and Computational Modeling Characterization.

Medial entorhinal cortex Layer-II stellate cells (mEC-LII-SCs) primarily interact via inhibitory interneurons. This suggests the presence of alternative mechanisms other than excitatory synaptic inputs for triggering action potentials (APs) in stellate cells during spatial navigation. Our intracellular recordings show that the hyperpolarization-activated cation current (Ih) allows post-inhibitory-rebound spikes (PIRS) in mEC-LII-SCs. In vivo, strong inhibitory-post-synaptic potentials immediately preceded most APs shortening their delay and enhancing excitability. In vitro experiments showed that inhibition initiated spikes more effectively than excitation and that more dorsal mEC-LII-SCs produced faster and more synchronous spikes. In contrast, PIRS in Layer-II/III pyramidal cells were harder to evoke, voltage-independent, and slower in dorsal mEC. In computational simulations, mEC-LII-SCs morphology and Ih homeostatically regulated the dorso-ventral differences in PIRS timing and most dendrites generated PIRS with a narrow range of stimulus amplitudes. These results suggest inhibitory inputs could mediate the emergence of grid cell firing in a neuronal network.

Biophysical Factors that Promote Mechanically-Induced Action Potentials in Neocortical and Hippocampal Pyramidal Neurons

Pressure activation of single mechanosensitive (MS) cation channel currents can trigger action potentials in neocortical and hippocampal pyramidal neurons (Nikolaev et al., 2015). Here we describe several factors that underlie this powerful influence on neuronal activity. First, cell-attached patch recordings indicate the MS cation channels are located on the soma and therefore inject a depolarizing current close to the axonal initial segment/spike initiation zone (SIZ) where action potential threshold is lowest. Second, the rapid rise of the single MS channel current (< 50 µs) generates a rapid depolarizing (dV/dt) event that lowers spike threshold by favoring fast Na+ channel activation over slower inactivation. Finally, with pressure activation the gating of the MS channel switches from single brief spontaneous openings (< 1 ms) to longer bursts of openings (∼ 10 ms) during which a spike may be triggered by each stochastic opening within the burst. Thus, the combination of SIZ proximity and rapid, burst gating underlies the powerful influence of single MS channels on central neuronal activity. For comparison, whereas a unitary excitatory post-synaptic current activated on a distal dendrite typically fails to trigger an action potential even when generating as much as 1000 fC charge transfer across the synaptic membrane, a single MS channel current that generates less than 200 fC can trigger repetitive spiking.Nikolaev, Y.A. et al., Single mechanically-gated cation channel currents can trigger action potentials in neocortical and hippocampal pyramidal neurons (2015) Brain Research. 1608: 1-13.

CA2+ Tides in Cardiomyocytes Under Mechanical Loading

AbstractCa2+ signaling is central to cardiac excitation-contraction coupling and extensive studies have revealed various Ca2+ signaling events in ventricular myocytes. In systole, the action potential opens L-type Ca2+ channels and triggers a synchronous release of Ca2+ from SR, which causes a global Ca2+ transient and whole cell contraction. During diastole, Ca2+ is sequestered back into SR and the cytosolic Ca2 concentration is kept low. However, pathological conditions can cause localized and spontaneous Ca2+ release from SR seen as Ca2+ sparks, puffs, embers, and waves. Spontaneous Ca2+ waves can drive Na+/Ca2+ exchange current to depolarize the membrane potential, manifesting as delayed afterdepolarizations and triggered action potentials that are arrhythmogenic. Here we report a new type of Ca2+ release event that is distinct from the previously known forms, named Ca2+ tide. A Ca2+ tide contains many discernable Ca2+ sparks that occur near synchronously, but does not show a propagating wave front. Ca2+ tides spontaneously arise after the systolic Ca2+ transient in a contracting myocyte and can happen several times during diastole. Interestingly and importantly, the Ca2+ tides are induced by mechanical loading of cardiomyocytes (by embedding cells in our Cell-in-Gel elastic matrix). Moreover, the cardiomyocytes isolated from TAC pressure-overload mouse model exhibited pronounced Ca2+ tides under mechanical loading. In contrast, cardiomyocytes under load-free conditions did not show any Ca2+ tides. The mechanisms and conditions that give rise to the ‘new kid on the block’ – Ca2+ tides – will be discussed.