Sustained Depolarization Research Articles

Adrenal fasciculata cells express T-type and rapidly and slowly activating L-type Ca2+ channels that regulate cortisol secretion.

In whole cell patch-clamp recordings, we characterized the L-type Ca(2+) currents in bovine adrenal zona fasciculata (AZF) cells and explored their role, along with the role of T-type channels, in ACTH- and angiotensin II (ANG II)-stimulated cortisol secretion. Two distinct dihydropyridine-sensitive L-type currents were identified, both of which were activated at relatively hyperpolarized potentials. One activated with rapid kinetics and, in conjunction with Northern blotting and PCR, was determined to be Cav1.3. The other, expressed in approximately one-half of AZF cells, activated with extremely slow voltage-dependent kinetics and combined properties not previously reported for an L-type Ca(2+) channel. The T-type Ca(2+) channel antagonist 3,5-dichloro-N-[1-(2,2-dimethyl-tetrahydro-pyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide (TTA-P2) inhibited Cav3.2 current in these cells, as well as ACTH- and ANG II-stimulated cortisol secretion, at concentrations that did not affect L-type currents. In contrast, nifedipine specifically inhibited L-type currents and cortisol secretion, but less effectively than TTA-P2. Diphenylbutylpiperidine Ca(2+) antagonists, including pimozide, penfluridol, and fluspirilene, and the dihydropyridine niguldipine blocked Cav3.2 and L-type currents and inhibited ACTH-stimulated cortisol secretion with similar potency. This study shows that bovine AZF cells express three Ca(2+) channels, the voltage-dependent gating and kinetics of which could orchestrate complex mechanisms linking peptide hormone receptors to cortisol secretion through action potentials or sustained depolarization. The function of the novel, slowly activating L-type channel is of particular interest in this respect. Regardless, the well-correlated selective inhibition of T- and L-type currents and ACTH- and ANG II-stimulated cortisol secretion by TTA-P2 and nifedipine establish the critical importance of these channels in AZF cell physiology.

How does spreading depression spread? Physiology and modeling.

Spreading depression (SD) is a wave phenomenon in gray matter tissue. Locally, it is characterized by massive redistribution of ions across cell membranes. As a consequence, there is sustained membrane depolarization and tissue polarization that depress any normal electrical activity. Despite these dramatic events, SD remains difficult to observe in humans noninvasively, which, for long, has slowed advances in this field. The growing appreciation of its clinical importance in migraine and stroke is therefore consistent with an increasing need for computational methods that tackle the complexity of the problem at multiple levels. In this review, we focus on mathematical tools to investigate the question of spread and its two complementary aspects: What are the physiological mechanisms and what is the spatial extent of SD in the cortex? This review discusses two types of models used to study these two questions, namely, Hodgkin-Huxley type and generic activator-inhibitor models, and the recent advances in techniques to link them.

NAVigating a transition from single action potential firing to bursting in chromaffin cells.

NAVigating a transition from single action potential firing to bursting in chromaffin cells.

Reduced availability of voltage-gated sodium channels by depolarization or blockade by tetrodotoxin boosts burst firing and catecholamine release in mouse chromaffin cells.

Mouse chromaffin cells (MCCs) of the adrenal medulla possess fast-inactivating Nav channels whose availability alters spontaneous action potential firing patterns and the Ca(2+)-dependent secretion of catecholamines. Here, we report MCCs expressing large densities of neuronal fast-inactivating Nav1.3 and Nav1.7 channels that carry little or no subthreshold pacemaker currents and can be slowly inactivated by 50% upon slight membrane depolarization. Reducing Nav1.3/Nav1.7 availability by tetrodotoxin or by sustained depolarization near rest leads to a switch from tonic to burst-firing patterns that give rise to elevated Ca(2+)-influx and increased catecholamine release. Spontaneous burst firing is also evident in a small percentage of control MCCs. Our results establish that burst firing comprises an intrinsic firing mode of MCCs that boosts their output. This occurs particularly when Nav channel availability is reduced by sustained splanchnic nerve stimulation or prolonged cell depolarizations induced by acidosis, hyperkalaemia and increased muscarine levels. Action potential (AP) firing in mouse chromaffin cells (MCCs) is mainly sustained by Cav1.3 L-type channels that drive BK and SK currents and regulate the pacemaking cycle. As secretory units, CCs optimally recruit Ca(2+) channels when stimulated, a process potentially dependent on the modulation of the AP waveform. Our previous work has shown that a critical determinant of AP shape is voltage-gated sodium channel (Nav) channel availability. Here, we studied the contribution of Nav channels to firing patterns and AP shapes at rest (-50 mV) and upon stimulation (-40 mV). Using quantitative RT-PCR and immunoblotting, we show that MCCs mainly express tetrodotoxin (TTX)-sensitive, fast-inactivating Nav1.3 and Nav1.7 channels that carry little or no Na(+) current during slow ramp depolarizations. Time constants and the percentage of recovery from fast inactivation and slow entry into closed-state inactivation are similar to that of brain Nav1.3 and Nav1.7 channels. The fraction of available Nav channels is reduced by half after 10 mV depolarization from -50 to -40 mV. This leads to low amplitude spikes and a reduction in repolarizing K(+) currents inverting the net current from outward to inward during the after-hyperpolarization. When Nav channel availability is reduced by up to 20% of total, either by TTX block or steady depolarization, a switch from tonic to burst firing is observed. The spontaneous occurrence of high frequency bursts is rare under control conditions (14% of cells) but leads to major Ca(2+)-entry and increased catecholamine release. Thus, Nav1.3/Nav1.7 channel availability sets the AP shape, burst-firing initiation and regulates catecholamine secretion in MCCs. Nav channel inactivation becomes important during periods of high activity, mimicking stress responses.

Antinociceptive effects of lacosamide on spinal neuronal and behavioural measures of pain in a rat model of osteoarthritis.

IntroductionAlterations in voltage-gated sodium channel (VGSC) function have been linked to chronic pain and are good targets for analgesics. Lacosamide (LCM) is a novel anticonvulsant that enhances the slow inactivation state of VGSCs. This conformational state can be induced by repeated neuronal firing and/or under conditions of sustained membrane depolarisation, as is expected for hyperexcitable neurones in pathological conditions such as epilepsy and neuropathy, and probably osteoarthritis (OA). In this study, therefore, we examined the antinociceptive effect of LCM on spinal neuronal and behavioural measures of pain, in vivo, in a rat OA model.MethodsOA was induced in Sprague Dawley rats by intraarticular injection of 2 mg of monosodium iodoacetate (MIA). Sham rats received saline injections. Behavioural responses to mechanical and cooling stimulation of the ipsilateral hind paw and hindlimb weight-bearing were recorded. In vivo electrophysiology experiments were performed in anaesthetised MIA or sham rats, and we recorded the effects of spinal or systemic administration of LCM on the evoked responses of dorsal horn neurones to electrical, mechanical (brush, von Frey, 2 to 60 g) and heat (40°C to 50°C) stimulation of the peripheral receptive field. The effect of systemic LCM on nociceptive behaviours was assessed.ResultsBehavioural hypersensitivity ipsilateral to knee injury was seen as a reduced paw withdrawal threshold to mechanical stimulation, an increase in paw withdrawal frequency to cooling stimulation and hind limb weight-bearing asymmetry in MIA-treated rats only. Spinal and systemic administration of LCM produced significant reductions of the electrical Aβ- and C-fibre evoked neuronal responses and the mechanical and thermal evoked neuronal responses in the MIA group only. Systemic administration of LCM significantly reversed the behavioural hypersensitive responses to mechanical and cooling stimulation of the ipsilateral hind paw, but hind limb weight-bearing asymmetry was not corrected.ConclusionsOur in vivo electrophysiological results show that the inhibitory effects of LCM were MIA-dependent. This suggests that, if used in OA patients, LCM may allow physiological transmission but suppress secondary hyperalgesia and allodynia. The inhibitory effect on spinal neuronal firing aligned with analgesic efficacy on nociceptive behaviours and suggests that LCM may still prove worthwhile for OA pain treatment and merits further clinical investigation.

Frequency-dependent facilitation of synaptic throughput via postsynaptic NMDA receptors in the nucleus of the solitary tract.

Hindbrain NMDA receptors play important roles in reflexive and behavioural responses to vagal activation. NMDA receptors have also been shown to contribute to the synaptic responses of neurons in the nucleus of the solitary tract (NTS), but their exact role remains unclear. In this study we used whole cell patch-clamping techniques in rat horizontal brain slice to investigate the role of NMDA receptors in the fidelity of transmission across solitary tract afferent-NTS neuron synapses. Results show that NMDA receptors contribute up to 70% of the charge transferred across the synapse at high (>5 Hz) firing rates, but have little contribution at lower firing frequencies. Results also show that NMDA receptors critically contribute to the fidelity of transmission across these synapses during high frequency (>5 Hz) afferent discharge rates. This novel role of NMDA receptors may explain in part how primary visceral afferents, including vagal afferents, can maintain fidelity of transmission across a broad range of firing frequencies. Neurons within the nucleus of the solitary tract (NTS) receive vagal afferent innervations that initiate gastrointestinal and cardiovascular reflexes. Glutamate is the fast excitatory neurotransmitter released in the NTS by vagal afferents, which arrive there via the solitary tract (ST). ST stimulation elicits excitatory postsynaptic currents (EPSCs) in NTS neurons mediated by both AMPA- and NMDA-type glutamate receptors (-Rs). Vagal afferents exhibit a high probability of vesicle release and exhibit robust frequency-dependent depression due to presynaptic vesicle depletion. Nonetheless, synaptic throughput is maintained even at high frequencies of afferent activation. Here we test the hypothesis that postsynaptic NMDA-Rs are essential in maintaining throughput across ST-NTS synapses. Using patch clamp electrophysiology in horizontal brainstem slices, we found that NMDA-Rs, including NR2B subtypes, carry up to 70% of the charge transferred across the synapse during high frequency stimulations (>5 Hz). In contrast, their relative contribution to the ST-EPSC is much less during low (<2 Hz) frequency stimulations. Afferent-driven activation of NMDA-Rs produces a sustained depolarization during high, but not low, frequencies of stimulation as a result of relatively slow decay kinetics. Hence, NMDA-Rs are critical for maintaining action potential generation at high firing rates. These results demonstrate a novel role for NMDA-Rs enabling a high probability of release synapse to maintain the fidelity of synaptic transmission during high frequency firing when glutamate release and AMPA-R responses are reduced. They also suggest why NMDA-Rs are critical for responses that may depend on high rates of afferent discharge.

Developmental increase in hyperpolarization-activated current regulates intrinsic firing properties in rat vestibular ganglion cells

The primary vestibular neurons convey afferent information from hair cells in the inner ear to the vestibular nuclei and the cerebellum. The intrinsic firing properties of vestibular ganglion cells (VGCs) are heterogeneous to sustained membrane depolarization, and undergo marked developmental changes from phasic to tonic types during the early postnatal period. Previous studies have shown that low-voltage-activated potassium channels, Kv1 and Kv7, play a critical role in determining the firing pattern of VGCs. In the present study, we explored the developmental changes in the properties of hyperpolarization-activated current (Ih) in rat VGCs and the role played by Ih in determining the firing properties of VGCs. Tonic firing VGCs showed a larger current density of Ih as compared to phasic firing VGCs, and tonic firing VGCs became phasic firing in the presence of ZD7288, an Ih channel blocker, indicating that Ih contributes to control the firing pattern of VGCs. The amplitude of Ih increased and the activation kinetics of Ih became faster during the developmental period. Analysis of developmental changes in the expression of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels revealed that expression of HCN1 protein and its mRNA increased during the developmental period, whereas expression of HCN2–4 protein and its mRNA did not change. Our results suggest that HCN1 channels as well as Kv1 channels are critical in determining the firing pattern of rat VGCs and that developmental up-regulation of HCN1 transforms VGCs from phasic to tonic firing phenotypes.

Spiny neurons of amygdala, striatum, and cortex use dendritic plateau potentials to detect network UP states.

Spiny neurons of amygdala, striatum, and cerebral cortex share four interesting features: (1) they are the most abundant cell type within their respective brain area, (2) covered by thousands of thorny protrusions (dendritic spines), (3) possess high levels of dendritic NMDA conductances, and (4) experience sustained somatic depolarizations in vivo and in vitro (UP states). In all spiny neurons of the forebrain, adequate glutamatergic inputs generate dendritic plateau potentials (“dendritic UP states”) characterized by (i) fast rise, (ii) plateau phase lasting several hundred milliseconds, and (iii) abrupt decline at the end of the plateau phase. The dendritic plateau potential propagates toward the cell body decrementally to induce a long-lasting (longer than 100 ms, most often 200–800 ms) steady depolarization (∼20 mV amplitude), which resembles a neuronal UP state. Based on voltage-sensitive dye imaging, the plateau depolarization in the soma is precisely time-locked to the regenerative plateau potential taking place in the dendrite. The somatic plateau rises after the onset of the dendritic voltage transient and collapses with the breakdown of the dendritic plateau depolarization. We hypothesize that neuronal UP states in vivo reflect the occurrence of dendritic plateau potentials (dendritic UP states). We propose that the somatic voltage waveform during a neuronal UP state is determined by dendritic plateau potentials. A mammalian spiny neuron uses dendritic plateau potentials to detect and transform coherent network activity into a ubiquitous neuronal UP state. The biophysical properties of dendritic plateau potentials allow neurons to quickly attune to the ongoing network activity, as well as secure the stable amplitudes of successive UP states.

Mechanisms of lidocaine's action on subtypes of spinal dorsal horn neurons subject to the diverse roles of Na(+) and K(+) channels in action potential generation.

Superficial dorsal horn neurons of the spinal cord receive sensory information from Aδ and C fibers. According to their response to sustained depolarization, these cells can be divided into 3 groups: tonic (TFN), adapting (AFN), and single spike firing (SSN) neurons. During spinal and systemic administration of lidocaine, these neurons are exposed to different concentrations of the local anesthetic lidocaine. In this study, we explored its effect on the excitability of sensory neurons. Whole-cell patch-clamp recordings from dorsal horn neurons of Wistar rats were used to study the action of lidocaine on firing properties. To estimate the impact of a blockade of voltage-gated potassium channels by lidocaine (100 μM) on the firing properties of different neurons, the sodium and potassium channel inhibition of lidocaine was investigated in the light of the effects of tetrodotoxin (TTX, 10 nM) and tetraethylammonium (10 mM). For statistical analysis, the Wilcoxon matched-pairs signed rank test was used throughout. All 3 types of neurons responded to lidocaine with changes in the shape of their action potentials. The peak amplitude of the single action potentials was decreased (P = 0.031, P = 0.013, and P = 0.014 for SSN, AFN, and TFN neurons, respectively), and the duration of the action potentials was increased (P = 0.016, P = 0.032, and P = 0.031 for SSN, AFN, and TFN neurons, respectively). The maximum positive slope (P = 0.016 and P = 0.0010 for SSN and AFN, respectively) and the negative slope (P = 0.016, P = 0.0025, and P = 0.020 for SSN, AFN, and TFN neurons, respectively) decreased after application of lidocaine. In tonically firing neurons, lidocaine reduced the repetitive firing (P = 0.0016), and this effect was mimicked by a combination of TTX and tetraethylammonium. In AFN, TTX mimicked the action of lidocaine. Lidocaine at low concentrations suppresses tonic firing neurons by interacting with voltage-gated potassium channels. The effects on adapting firing neurons can be explained by an interaction with voltage-gated sodium channels. In contrast, the firing pattern of SSN is not affected at the administered concentrations. This different sensitivity to low concentrations of sodium and particularly of potassium channel blockers might represent a novel approach for a differentiated blockade of different spinal dorsal horn neurons.

Depolarization of Cellular Resting Membrane Potential Promotes Neonatal Cardiomyocyte Proliferation In Vitro.

Cardiomyocytes (CMs) undergo a rapid transition from hyperplastic to hypertrophic growth soon after birth, which is a major challenge to the development of engineered cardiac tissue for pediatric patients. Resting membrane potential (Vmem) has been shown to play an important role in cell differentiation and proliferation during development. We hypothesized that depolarization of neonatal CMs would stimulate or maintain CM proliferation in vitro. To test our hypothesis, we isolated postnatal day 3 neonatal rat CMs and subjected them to sustained depolarization via the addition of potassium gluconate or Ouabain to the culture medium. Cell density and CM percentage measurements demonstrated an increase in mitotic CMs along with a ~2 fold increase in CM numbers with depolarization. In addition, depolarization led to an increase in cells in G2 and S phase, indicating increased proliferation, as measured by flow cytometry. Surprisingly depolarization of Vmem with either treatment led to inhibition of proliferation in cardiac fibroblasts. This effect is abrogated when the study was carried out on postnatal day 7 neonatal CMs, which are less proliferative, indicating that the likely mechanism of depolarization is the maintenance of the proliferating CM population. In summary, our findings suggest that depolarization maintains postnatal CM proliferation and may be a novel approach to encourage growth of engineered tissue and cardiac regeneration in pediatric patients.

Calpain activation and CaMKIV reduction in spinal cords from hSOD1G93A mouse model.

Amyotrophic Lateral Sclerosis (ALS), a severe neurodegenerative disease, affects the upper and lower motor neurons in the brain and spinal cord. In some studies, ALS disease progression has been associated with an increase in calcium-dependent degeneration processes. Motoneurons are specifically vulnerable to sustained membrane depolarization and excessive elevation of intracellular calcium concentration. The present study analyzed intracellular events in embryonic motoneurons and adult spinal cords of the hSOD1G93A ALS mouse model. We observed activation of calpain, a calcium-dependent cysteine protease that degrades a variety of substrates, and a reduction in calcium-calmodulin dependent protein kinase type IV (CaMKIV) levels in protein extracts from spinal cords obtained at several time-points of hSOD1G93A mice disease progression. However, in cultured embryonic motoneurons these differences between controls and hSOD1G93A mutants are not evident. Our results support the hypothesis that age-dependent changes in calcium homeostasis and resulting events, e.g., calpain activation and CaMKIV processing, are involved in ALS pathogenesis.

Supervised learning with complex spikes and spike-timing-dependent plasticity.

One distinctive feature of Purkinje cells is that they have two types of discharge: in addition to simple spikes they fire complex spikes in response to input from the climbing fibers. These complex spikes have an initial rapid burst of spikes and spikelets followed by a sustained depolarization; in some models of cerebellar function this climbing fiber input supervises learning in Purkinje cells. On the other hand, synaptic plasticity is often thought to rely on the timing of pre-synaptic and post-synaptic spikes. It is suggested here that the period of depolarization following a complex spike, combined with a simple spike-timing-dependent plasticity rule, gives a mechanism for the climbing fiber to supervise learning in the Purkinje cell. This proposal is illustrated using a simple simulation in which it is seen that the climbing fiber succeeds in supervising the learning.

Alternative splicing alterations of Ca2+handling genes are associated with Ca2+signal dysregulation in myotonic dystrophy type 1 (DM1) and type 2 (DM2) myotubes

Aims The pathogenesis of myotonic dystrophy type 1 (DM1) and type 2 (DM2) has been related to the aberrant splicing of several genes, including those encoding for ryanodine receptor 1 (RYR1), sarcoplasmatic/endoplasmatic Ca2+-ATPase (SERCA) and α1S subunit of voltage-gated Ca2+ channels (Cav1.1). The aim of this study is to determine whether alterations of these genes are associated with changes in the regulation of intracellular Ca2+ homeostasis and signalling. Methods We analysed the expression of RYR1, SERCA and Cav1.1 and the intracellular Ca2+ handling in cultured myotubes isolated from DM1, DM2 and control muscle biopsies by semiquantitative RT-PCR and confocal Ca2+ imaging respectively. Results (i) The alternative splicing of RYR1, SERCA and Cav1.1 was more severely affected in DM1 than in DM2 myotubes; (ii) DM1 myotubes exhibited higher resting intracellular Ca2+ levels than DM2; (iii) the amplitude of intracellular Ca2+ transients induced by sustained membrane depolarization was higher in DM1 myotubes than in controls, whereas DM2 showed opposite behaviour; and (iv) in both DM myotubes, Ca2+ release from sarcoplasmic reticulum through RYR1 was lower than in controls. Conclusion The aberrant splicing of RYR1, SERCA1 and Cav1.1 may alter intracellular Ca2+ signalling in DM1 and DM2 myotubes. The differing dysregulation of intracellular Ca2+ handling in DM1 and DM2 may explain their distinct sarcolemmal hyperexcitabilities.

CrossTalk proposal: The late sodium current is an important player in the development of diastolic heart failure (heart failure with a preserved ejection fraction)

Approximately half of all patients with heart failure have an ejection fraction greater than 40–50% and may be diagnosed as having Heart Failure with preserved Ejection Fraction (HFpEF). Diastolic dysfunction is central to the pathophysiology of HFpEF (Borlaug & Paulus, 2011), and describes the slowing of ventricular relaxation and increased diastolic stiffness which ultimately impairs ventricular filling. The mechanistic basis of this impairment is complex and not yet well understood. Structural remodelling undoubtedly plays an important role in increasing left ventricular stiffness. However, the acute worsening of diastolic dysfunction during stress or exercise characteristic of HFpEF suggests an important contribution from dynamic changes in left ventricular (LV) functional properties. Frequency-dependent elevation of diastolic tension and intracellular Ca2+ ([Ca2+]i) has been observed in cardiac muscle strips from patients with left ventricular hypertrophy and diastolic dysfunction, or heart failure (Sossalla et al. 2008; Selby et al. 2011), implying that dysregulation of [Ca2+]i homeostasis of the cardiomyocyte contributes to diastolic dysfunction. Intracellular Ca2+ regulation is closely linked to intracellular Na+ homeostasis, through the Na+–Ca2+ exchanger (NCX). Intracellular Na+ of cardiomyocytes from failing hearts is increased and associated with elevated diastolic tension (Pieske et al. 2002). An important mechanism underlying this observation may be an increase in the late sodium current (INa,L). The Na+ conductance responsible for rapid depolarization of cardiomyocytes does not completely inactivate during the action potential. (Noble & Noble, 2006; Maier, 2012) Some channels continue to conduct, or even reactivate at relatively positive membrane potentials during the plateau and repolarization phases. This is INa,L (Zaza et al. 2008). Consequently, about half of the myocyte Na+ entry occurs during the initial 2–3 ms, and about half during the remainder of the action potential (Makielski & Farley, 2006). At the molecular level, INa,L results from channel reopening during sustained depolarization by two different modes of gating: burst openings and late scattered openings (Maltsev & Undrovinas, 2008). As outlined in Fig. 1, increased Na+ entry through INa,L increases intracellular Na+ ([Na+]i), which reduces the driving force for extrusion of Ca2+ and favours Ca2+ influx via the Na+–Ca2+ exchanger (NCX). This leads to increased [Ca2+]i. Elevated [Ca2+]i eventually increases actin–myosin filament interaction during diastole and thus increases diastolic tension. This mechanism of Ca2+ overload has been demonstrated in numerous animal studies, and in strips of ventricular muscle or myocytes isolated from patients with failing hearts (Valdivia et al. 2005; Makielski & Farley, 2006; Maltsev & Undrovinas, 2008; Sossalla et al. 2008; Selby et al. 2011; Coppini et al. 2013). Further, specific augmentation of INa,L with the sea anemone toxin ATXII in isolated myocytes and perfused hearts results in Na+ and Ca2+ overload (Fraser et al. 2006; Sossalla et al. 2008) and impaired diastolic function. Diastolic dysfunction with preserved systolic function has also been described in LQT syndrome type 3 patients, where INa,L is enhanced due to a Na+ channel mutation (Moss et al. 2008; Hummel et al. 2013). Figure 1 A pathological enhanced INa,L contributes to Na+-dependent Ca2+ overload, diastolic dysfunction We propose that a pathological increase in Na+ influx through cardiac Na+ channels, specifically due to enhanced INa,L is a major contributor to Ca2+ overload and diastolic dysfunction in HFpEF. Key evidence to support this hypothesis is outlined below.

Sonoporation-Induced Depolarization of Plasma Membrane Potential: Analysis of Heterogeneous Impact

Disrupting plasma membrane integrity would inevitably promote anomalous ion fluxes across the membrane and thereby upset the trans-membranous potential. In this article, we report new findings on how sonoporation as a physical membrane perforation strategy would lead to different forms of plasma membrane potential disruption. Our investigation was conducted with a customized fluorescence imaging platform that enabled live monitoring of plasma membrane potential in relation to individual sonoporation events triggered on HeLa cervical cancer cells. Sonovue microbubbles were used as sonoporation agents (added at a 4:3 cell-to-bubble ratio), and they were activated by 1-MHz pulsed ultrasound with 0.35-MPa peak negative pressure, 20-cycle pulse duration, 20-Hz pulse repetition frequency and 1-s total exposure duration. Results indicate that the plasma membrane potential response was heterogeneous among sonoporated cells: (i) membrane potential of irreversibly sonoporated cells was permanently depolarized; (ii) reversibly sonoporated cells exhibited either transient or sustained membrane depolarization; (iii) intact cells adjacent to sonoporated ones underwent transitory membrane depolarization. These findings effectively serve to substantiate the causal relationship between sonoporation and plasma membrane potential.

Disrupted in Schizophrenia 1 Modulates Medial Prefrontal Cortex Pyramidal Neuron Activity Through cAMP Regulation of Transient Receptor Potential C and Small-Conductance K+ Channels

Disrupted in schizophrenia 1 (DISC1) is a protein implicated in schizophrenia, bipolar disorder, major depressive disorder, and autism. To date, most of research examining DISC1 function has focused on its role in neurodevelopment, despite its presence throughout life. DISC1 also regulates cyclic adenosine monophosphate (cAMP) signaling by increasing type 4 phosphodiesterase catabolism of cAMP when cAMP concentrations are high. In this study, we tested the hypothesis that DISC1, through its regulation of cAMP, modulates I-SK and I-TRPC channel-mediated ionic currents that we have shown previously to regulate the activity of mature prefrontal cortical pyramidal neurons. We used patch-clamp recordings in prefrontal cortical slices from adult rats in which DISC1 function was reduced in vivo by short hairpin RNA viral knockdown or in vitro by dialysis of DISC1 antibodies. We found that DISC1 disruption resulted in an increase of metabotropic glutamate receptor-induced intracellular calcium (Ca2+) waves, small-conductance K+ (SK)-mediated hyperpolarization and a decrease of transient receptor potential C (TRPC)-mediated sustained depolarization. Consistent with a role for DISC1 in regulation of cAMP signaling, forskolin-induced cAMP production also increased intracellular Ca2+ waves, I-SK and decreased I-TRPC. Lastly, inhibiting cAMP generation with guanfacine, an α2A-noradrenergic agonist, normalized the function of SK and TRPC channels. Based on our findings, we propose that diminished DISC1 function, such as occurs in some mental disorders, can lead to the disruption of normal patterns of prefrontal cortex activity through the loss of cAMP regulation of metabotropic glutamate receptor-mediated intracellular Ca2+ waves, SK and TRPC channel activity.

Intracellular calcium stores drive slow non-ribbon vesicle release from rod photoreceptors

Rods are capable of greater slow release than cones contributing to overall slower release kinetics. Slow release in rods involves Ca2+-induced Ca2+ release (CICR). By impairing release from ribbons, we found that unlike cones where release occurs entirely at ribbon-style active zones, slow release from rods occurs mostly at ectopic, non-ribbon sites. To investigate the role of CICR in ribbon and non-ribbon release from rods, we used total internal reflection fluorescence microscopy as a tool for visualizing terminals of isolated rods loaded with fluorescent Ca2+ indicator dyes and synaptic vesicles loaded with dextran-conjugated pH-sensitive rhodamine. We found that rather than simply facilitating release, activation of CICR by ryanodine triggered release directly in rods, independent of plasma membrane Ca2+ channel activation. Ryanodine-evoked release occurred mostly at non-ribbon sites and release evoked by sustained depolarization at non-ribbon sites was mostly due to CICR. Unlike release at ribbon-style active zones, non-ribbon release did not occur at fixed locations. Fluorescence recovery after photobleaching of endoplasmic reticulum (ER)-tracker dye in rod terminals showed that ER extends continuously from synapse to soma. Release of Ca2+ from terminal ER by lengthy depolarization did not significantly deplete Ca2+ from ER in the perikaryon. Collectively, these results indicate that CICR-triggered release at non-ribbon sites is a major mechanism for maintaining vesicle release from rods and that CICR in terminals may be sustained by diffusion of Ca2+ through ER from other parts of the cell.

Brimonidine tartrate effect on retinal spreading depression depends on Müller cells

Objective: Demonstrate the Brimonidine effect over Retinal Spreading Depression (SD). Brimonidine is an alpha-2–adrenergic receptor agonist, used in the management of glaucoma. Alpha2-agonists have been shown to be neuroprotective in various experimental models, however the molecular and cellular targets leading to these actions are still poorly defined. The SD of neuronal electric activity is a wave of cellular massive sustained depolarization that damages the nervous tissue. Local trauma, pressure, ischemic injuries and other chemical agents as high extracellular potassium concentration or glutamate, can trigger SD, leading to exaggerated focal electrical followed by an electrical silence. Methods: Using chicken retina as model, we performed alpha2-receptor detection by Western Blotting and Immunohistochemistry. After that we obtained electrical signals of SD by microelectrodes on retina in the absence or presence of Brimonidine. For in vivo visualization we observed retina with optical coherence tomography on normal state, with SD passing, and with SD + Brimonidine. Results: Our data demonstrated that: (1) alpha2-adrenergic receptors are present in Muller cells, (2) the treatment with Brimonidine decreases the SD‘s velocity as well as the voltage of SD waves and (3) OCT revealed that SD creates a hyper reflectance at inner plexiform layer, but on retinal treatment with brimonidine, SD was not visualized. Conclusions: Our study about brimonidine possible pathways of neuroprotection we observed it reduces SD (a neuronal damage wave), identified a new cellular target – the Muller cells, as well as, firstly demonstrated SD on OCT, showing that the inner plexiform layer is the main optically affected layer on SD.

Editors’ Pick: What a pain…, or not!

How nice, I thought as a child, would it be to not feel physical pain. Later I realized that sense of pain may exist for a reason; it was suggested that physical pain has evolved as an alarm system to protect the body from serious damage. Much later I read about human disorders involving severe insensitivity to physical pain. Still, the idea of not feeling pain remains appealing (especially anticipating my coming years when some painful age-related health problems are likely to kick in). Previously, it was demonstrated that a loss-of-function mutation in the sodium ion channel gene SCN9A leads to the inability to experience pain [1]. In a more recent study, Leipold et al.[2] identified another sodium ion channel gene, SCN11A, which, when harboring a particular missense mutation, causes loss of pain perception. SCN11A encodes the voltage-gated sodium ion channel NAv1.9, which is highly expressed in nociceptors (i.e., neurons that transmit sensory information from the body periphery to the spinal cord). The authors started out with whole-exome sequencing of healthy German parents and their child diagnosed with the congenital inability to experience pain. A heterozygote non-synonymous mutation in SCN11A was identified as the only de novo event detected in this affected child. Aiming to confirm this finding in independent samples, the authors performed Sanger sequencing of SCN11A exons in 58 additional individuals diagnosed with early-onset severe sensory loss, most of them representing sporadic cases. One Swedish individual showed the very same heterozygous SCN11A mutation, and the clinical history of both individuals was also similar. From these findings, the authors hypothesized that the identified mutation that leads to a replacement of leucine with proline (p.Leu811Pro) at the distal end of one of the transmembrane segments in domain II of NAv1.9 is disease causing and leads to loss of pain perception. Seeking molecular proof, the authors introduced the orthologous alteration (Leu799Pro) into the mouse Scn11a gene, but found no obvious morphological changes in sensory axons or in nerve fibers in the skin of the viable heterozygote knock-in mice. However, 11% of the knock-in mice had severe lesions that were not observed in the wild-type mice. Since the lesions also appeared in knock-in mice housed solitarily, it was assumed that they were self-inflicted. Also other experimental evidence pointed towards reduced pain sensitivity in these knock-in mice. Because NAv1.9 had been suggested to influence the excitability of dorsal root ganglia (DRG) neurons (i.e., their property to react to stimulation), the authors then investigated the electrical potential of DRG neurons isolated from wild-type and mutant mice. They found that a significant fraction of the mutant channels were active under resting conditions, and that the duration of action potentials was significantly reduced in the mutant neurons compared with wild-type neurons. In knock-out mice, however, only a minor effect on action potentials was observed. Additional experiments showed that the excessive activity at resting conditions caused sustained depolarization of nociceptors, impaired generation of action potentials and aberrant synaptic transmission. To study the effect of the mutation further, the authors heterologously expressed human NAv1.9 and NAv1.9 Leu811Pro mutant channels in ND7/23 cells (i.e., a mouse neuroblastoma x rat neuron hybrid cell line). They found that the phenotype of human NAv1.9 Leu811Pro mutant channels resembled that of mouse NAv1.9 Leu799Pro mutant channels, except in terms of steady-state inactivation. From the weak influence of the mutation on inactivation in the human channel, they concluded a stronger gain-of-function effect in humans than in knock-in mice. Besides impressively demonstrating the power of whole-exome sequencing to find rare candidate disease causing mutations, this study provides a new gain-of-function view on the molecular basis of pain loss, which was previously linked to loss-of-function mutations such as in the NAv1.7 channel encoded by SCN9A[1]. Furthermore, this finding may open up new avenues for the development of new painkilling drugs [3].

Increased Activity of Nav1.9 Sodium Channels Causes Loss of Pain Perception

Nociceptive sensory neurons transmit painful stimuli from the periphery to the central nervous system. Voltage-gated sodium (NaV) channels are instrumental for the generation of the corresponding electrical signals, but - so far - the sensory neuron-specific NaV1.9 only appeared to play a minor role. Recently, we identified a de novo heterozygous mutation in the SCN11A gene encoding for NaV1.9 and demonstrated the implication of NaV1.9 in human pain perception (Leipold et al., Nature Genetics, DOI 10.1038/ng.2767). Surprisingly, in affected individuals the mutation (L811P) leads to an inability to experience pain by conferring gain-of-function (GOF) properties to NaV1.9. Mutant channels activate at hyperpolarized voltages and display a slow-down of channel inactivation and deactivation, thereby causing sustained depolarization of nociceptor cells and alterations of action potential characteristics. This new channelopathy is different from other pain-related NaV channel disorders: a GOF of homologous NaV1.7 channels is associated with chronic pain, and loss of functional NaV1.7 channels causes congenital indifference to pain. To gain further insight into the mechanisms underlying the mutation we introduced the homologous L-to-P mutation into channel isoforms NaV1.4 (L802P), NaV1.7 (L957P) and NaV1.8 (L890P) and compared the functional parameters of wild-type and mutant channels by means of the whole-cell patch-clamp technique. The L-to-P mutation shifted the activation in all channel subtypes to hyperpolarized potentials in the order NaV1.9 > NaV1.7 > NaV1.4 > NaV1.8. Fast channel inactivation was slowed down most prominently in NaV1.9 and NaV1.8, followed by NaV1.7 and NaV1.4. These results show that a homologous L-to-P mutation affects channel activation and inactivation in a subtype-specific manner.