Spontaneous Action Potential Firing Research Articles

Satellite glial contact enhances differentiation and maturation of human iPSC-derived sensory neurons.

Sensory neurons generated from induced pluripotent stem cells (iSNs) are used to model human peripheral neuropathies, however current differentiation protocols produce sensory neurons with an embryonic phenotype. Peripheral glial cells contact sensory neurons early in development and contribute to formation of the canonical pseudounipolar morphology, but these signals are not encompassed in current iSN differentiation protocols. Here, we show that terminal differentiation of iSNs in co-culture with rodent Dorsal Root Ganglion satellite glia (rSG) advances their differentiation and maturation. Co-cultured iSNs develop a pseudounipolar morphology through contact with rSGs. This transition depends on semaphorin-plexin guidance cues and on glial gap junction signaling. In addition to morphological changes, iSNs terminally differentiated in co-culture exhibit enhanced spontaneous action potential firing, more mature gene expression, and increased susceptibility to paclitaxel induced axonal degeneration. Thus, iSNs differentiated in coculture with rSGs provide a better model for investigating human peripheral neuropathies.

ZCCHC17 knockdown phenocopies Alzheimer's disease-related loss of synaptic proteins and hyperexcitability.

ZCCHC17 is a master regulator of synaptic gene expression and has recently been shown to play a role in splicing of neuronal mRNA. We previously showed that ZCCHC17 protein declines in Alzheimer's disease (AD) brain tissue before there is significant gliosis and neuronal loss, that ZCCHC17 loss partially replicates observed splicing abnormalities in AD brain tissue, and that maintenance of ZCCHC17 levels is predicted to support cognitive resilience in AD. Here, we assessed the functional consequences of reduced ZCCHC17 expression in primary cortical neuronal cultures using siRNA knockdown. Consistent with its previously identified role in synaptic gene expression, loss of ZCCHC17 led to loss of synaptic protein expression. Patch recording of neurons shows that ZCCHC17 loss significantly disrupted the excitation/inhibition balance of neurotransmission, and favored excitatory-dominant synaptic activity as measured by an increase in spontaneous excitatory post synaptic currents and action potential firing rate, and a decrease in spontaneous inhibitory post synaptic currents. These findings are consistent with the hyperexcitable phenotype seen in AD animal models and in patients. We are the first to assess the functional consequences of ZCCHC17 knockdown in neurons and conclude that ZCCHC17 loss partially phenocopies AD-related loss of synaptic proteins and hyperexcitability.

The mitochondrial calcium uniporter inhibitor Ru265 increases neuronal excitability and reduces neurotransmission via off-target effects.

Excitotoxicity due to mitochondrial calcium (Ca2+) overloading can trigger neuronal cell death in a variety of pathologies. Inhibiting the mitochondrial calcium uniporter (MCU) has been proposed as a therapeutic avenue to prevent calcium overloading. Ru265 (ClRu(NH3)4(μ-N)Ru(NH3)4Cl]Cl3) is a cell-permeable inhibitor of the mitochondrial calcium uniporter (MCU) with nanomolar affinity. Ru265 reduces sensorimotor deficits and neuronal death in models of ischemic stroke. However, the therapeutic use of Ru265 is limited by the induction of seizure-like behaviours. We examined the effect of Ru265 on synaptic and neuronal function in acute brain slices and hippocampal neuron cultures derived from mice, in control and where MCU expression was genetically abrogated. Ru265 decreased evoked responses from calyx terminals and induced spontaneous action potential firing of both the terminal and postsynaptic principal cell. Recordings of presynaptic Ca2+ currents suggested that Ru265 blocks the P/Q type channel, confirmed by the inhibition of currents in cells exogenously expressing the P/Q type channel. Measurements of presynaptic K+ currents further revealed that Ru265 blocked a KCNQ current, leading to increased membrane excitability, underlying spontaneous spiking. Ca2+ imaging of hippocampal neurons showed that Ru265 increased synchronized, high-amplitude events, recapitulating seizure-like activity seen in vivo. Importantly, MCU ablation did not suppress Ru265-induced increases in neuronal activity and seizures. Our findings provide a mechanistic explanation for the pro-convulsant effects of Ru265 and suggest counter screening assays based on the measurement of P/Q and KCNQ channel currents to identify safe MCU inhibitors.

The pain target NaV1.7 is expressed late during human iPS cell differentiation into sensory neurons as determined in high-resolution imaging

Human-induced pluripotent stem cells (iPS cells) are efficiently differentiated into sensory neurons. These cells express the voltage-gated sodium channel NaV1.7, which is a validated pain target. NaV1.7 deficiency leads to pain insensitivity, whereas NaV1.7 gain-of-function mutants are associated with chronic pain. During differentiation, the sensory neurons start spontaneous action potential firing around day 22, with increasing firing rate until day 40. Here, we used CRISPR/Cas9 genome editing to generate a HA-tag NaV1.7 to follow its expression during differentiation. We used two protocols to generate sensory neurons: the classical small molecule approach and a directed differentiation methodology and assessed surface NaV1.7 expression by Airyscan high-resolution microscopy. Our results show that maturation of at least 49 days is necessary to observe robust NaV1.7 surface expression in both protocols. Electric activity of the sensory neurons precedes NaV1.7 surface expression. A clinically effective NaV1.7 blocker is still missing, and we expect this iPS cell model system to be useful for drug discovery and disease modeling.

Phenotypic Screening of Prospective Analgesics Among FDA-Approved Compounds using an iPSC-Based Model of Acute and Chronic Inflammatory Nociception.

Classical target-based drug screening is low-throughput, largely subjective, and costly. Phenotypic screening based on in vitro models is increasingly being used to identify candidate compounds that modulate complex cell/tissue functions. Chronic inflammatory nociception, and subsequent chronic pain conditions, affect peripheral sensory neuron activity (e.g., firing of action potentials) through myriad pathways, and remain unaddressed in regard to effective, non-addictive management/treatment options. Here, a chronic inflammatory nociception model is demonstrated based on induced pluripotent stem cell (iPSC) sensory neurons and glia, co-cultured on microelectrode arrays (MEAs). iPSC sensory co-cultures exhibit coordinated spontaneous extracellular action potential (EAP) firing, reaching a stable baseline after ≈27 days in vitro (DIV). Spontaneous and evoked EAP metrics are significantly modulated by 24-h incubation with tumor necrosis factor-alpha (TNF-α), representing an inflammatory phenotype. Compared with positive controls (lidocaine), this model is identified as an "excellent" stand-alone assay based on a modified Z' assay quality metric. This model is then used to screen 15 cherry-picked, off-label, Food and Drug Administration (FDA)-approved compounds; 10 of 15 are identified as "hits". Both hits and "misses" are discussed in turn. In total, this data suggests that iPSC sensory co-cultures on MEAs may represent a moderate-to-high-throughput assay for drug discovery targeting inflammatory nociception.

Gonadal Feedback Alters the Relationship between Action Potentials and Hormone Release in Gonadotropin-Releasing Hormone Neurons in Male Mice.

In vertebrates, the pulsatile release of gonadotropin-releasing hormone (GnRH) from neurons in the hypothalamus triggers secretion of anterior pituitary gonadotropins, which activate steroidogenesis, and steroids in turn exert typically homeostatic negative feedback on GnRH release. Although long-term episodic firing patterns of GnRH neurons in brain slices resemble the pulsatile release of GnRH and LH in vivo, neither the relationship between GnRH neuron firing and release nor whether this relationship is influenced by gonadal feedback are known. We combined fast-scan cyclic voltammetry and patch-clamp to perform simultaneous measurements of neuropeptide release with either spontaneous action potential firing or in response to neuromodulator or action-potential-spike templates in brain slice preparations from male mice. GnRH release increased with higher frequency spontaneous firing to a point; release reached a plateau after which further increases in firing rate did not elicit further increased release. Kisspeptin, a potent GnRH neuron activator via a Gq-coupled signaling pathway, triggered GnRH release before increasing firing rate, whether globally perfused or locally applied. Increasing the number of spikes in an applied burst template increased release; orchidectomized mice had higher sensitivity to the increased action potential number than sham-operated mice. Similarly, Ca2+ currents triggered by these burst templates were increased in GnRH neurons of orchidectomized mice. These results suggest removal of gonadal feedback increases the efficacy of the stimulus-secretion coupling mechanisms, a phenomenon that may extend to other steroid-sensitive regions of the brain.SIGNIFICANCE STATEMENT Pulsatile secretion of GnRH plays a critical role in fertility. The temporal relationship between GnRH neuron action potential firing and GnRH release remains unknown as does whether this relationship is influenced by gonadal feedback. By combining techniques of fast-scan cyclic voltammetry and patch-clamp we, for the first time, monitored GnRH concentration changes during spontaneous and neuromodulator-induced GnRH neuron firing. We also made the novel observation that gonadal factors exert negative feedback on excitation-secretion coupling to reduce release in response to the same stimulus. This has implications for the control of normal fertility, central causes of infertility, and more broadly for the effects of sex steroids in the brain.

Effects of NALCN-Encoded Na+ Leak Currents on the Repetitive Firing Properties of SCN Neurons Depend on K+-Driven Rhythmic Changes in Input Resistance.

Neurons in the suprachiasmatic nucleus (SCN) generate circadian changes in the rates of spontaneous action potential firing that regulate and synchronize daily rhythms in physiology and behavior. Considerable evidence suggests that daily rhythms in the repetitive firing rates (higher during the day than at night) of SCN neurons are mediated by changes in subthreshold potassium (K+) conductance(s). An alternative "bicycle" model for circadian regulation of membrane excitability in clock neurons, however, suggests that an increase in NALCN-encoded sodium (Na+) leak conductance underlies daytime increases in firing rates. The experiments reported here explored the role of Na+ leak currents in regulating daytime and nighttime repetitive firing rates in identified adult male and female mouse SCN neurons: vasoactive intestinal peptide-expressing (VIP+), neuromedin S-expressing (NMS+) and gastrin-releasing peptide-expressing (GRP+) cells. Whole-cell recordings from VIP+, NMS+, and GRP+ neurons in acute SCN slices revealed that Na+ leak current amplitudes/densities are similar during the day and at night, but have a larger impact on membrane potentials in daytime neurons. Additional experiments, using an in vivo conditional knockout approach, demonstrated that NALCN-encoded Na+ currents selectively regulate daytime repetitive firing rates of adult SCN neurons. Dynamic clamp-mediated manipulation revealed that the effects of NALCN-encoded Na+ currents on the repetitive firing rates of SCN neurons depend on K+ current-driven changes in input resistances. Together, these findings demonstrate that NALCN-encoded Na+ leak channels contribute to regulating daily rhythms in the excitability of SCN neurons by a mechanism that depends on K+ current-mediated rhythmic changes in intrinsic membrane properties.SIGNIFICANCE STATEMENT Elucidating the ionic mechanisms responsible for generating daily rhythms in the rates of spontaneous action potential firing of neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals, is an important step toward understanding how the molecular clock controls circadian rhythms in physiology and behavior. While numerous studies have focused on identifying subthreshold K+ channel(s) that mediate day-night changes in the firing rates of SCN neurons, a role for Na+ leak currents has also been suggested. The results of the experiments presented here demonstrate that NALCN-encoded Na+ leak currents differentially modulate daily rhythms in the daytime/nighttime repetitive firing rates of SCN neurons as a consequence of rhythmic changes in subthreshold K+ currents.

Development of electrophysiological properties of fusiform neurons from the dorsal cochlear nucleus of mice before and after hearing onset.

The dorsal cochlear nucleus (DCN) in the auditory brainstem integrates auditory and somatosensory information. Mature DCN fusiform neurons fall into two qualitatively distinct types: quiet, with no spontaneous regular action potential firing, or active, with regular spontaneous action potential firing. However, how these firing states and other electrophysiological properties of fusiform neurons develop during early postnatal days to adulthood is not known. Thus, we recorded fusiform neurons from mice from P4 to P21 and analyzed their electrophysiological properties. In the prehearing phase (P4-P13), we found that most fusiform neurons are quiet, with active neurons emerging after hearing onset at P14. Subthreshold properties underwent significant changes before hearing onset, whereas changes to the action potential waveform occurred mainly after P14, with the depolarization and repolarization phases becoming markedly faster and half-width significantly decreased. The activity threshold in posthearing neurons was more negative than in prehearing cells. Persistent sodium current (INaP) was increased after P14, coinciding with the emergence of spontaneous firing. Thus, we suggest that posthearing expression of INaP leads to hyperpolarization of the activity threshold and the active state of the fusiform neuron. At the same time, other changes refine the passive membrane properties and increase the speed of action potential firing of fusiform neurons.NEW & NOTEWORTHY Auditory brainstem neurons express unique electrophysiological properties adapted for their complex physiological functions that develop before hearing onset. Fusiform neurons of the DCN present two firing states, quiet and active, but the origin of these states is not known. Here, we showed that the quiet and active states develop after hearing onset at P14, along with changes in action potentials, suggesting an influence of auditory input on the refining of fusiform neuron's excitability.

Divergent electrophysiology between two cardiac vagal motor populations despite similar chronotropic effects

Cardiac vagal motor output is generated by brainstem cardiac vagal motorneurons (CVNs) that send axonal projections, through the vagus nerve, to cardiac ganglia. CVNs have a well-established cardioinhibitory influence through reductions in heart rate (HR) and studies using vagotomy confirm cardiac vagal drive originates in CVNs. CVNs can be found in two brainstem regions: nucleus ambiguus (NA) and dorsal motor nucleus of the vagus (DMV). Although the cardioinhibitory role of CVNs in NA (CVN NA ) is widely accepted, effects of CVNs in DMV (CVN DMV ) on HR regulation is controversial. The present study aims to provide evidence of CVN DMV existence and identify the unique role DMV plays in HR regulation. We hypothesized that CVN DMV cause robust bradycardias and associate with unique electrophysiological properties making them hyperexcitable compared to CVN NA . Experiments were performed on adult (9-11 week old) male mice. Using a transgenic mouse line where light activated channelrhodopsin (ChR) is expressed in choline acetyltransferase neurons, activation of ChR in DMV using fiber optic probes resulted in a 359±43 bpm bradycardia (n=8) in awake behaving animals. DMV-mediated ChR-dependent bradycardia remained after i.p. administration of atenolol (256±31 bpm). However, i.p. scopolamine-methyl nitrate abolished the ability of ChR activation of DMV to impact HR (12±2 bpm, p=0.0002). ChR stimulation of NA resulted in a similar bradycardia (447±6 bpm; p<0.0001) that was also blocked by scopolamine-methyl nitrate (9±7 bpm, p=0.005). Retrograde tracing from cardiac tissue using rhodamine and cholera-toxin B (CTB) confirmed CVNs in DMV (50±15 labeled neurons, n=6; CTB: 12±4 labeled neurons, n=8). However, the number of CVN DMV was significantly lower than CVN NA (rhodamine: 116±24 labeled neurons, n=6; CTB: 83.33±9.548 labeled neurons, n=6, p<0.0001 for both). Unilateral vagotomy eliminated tracer presence in DMV ipsilateral (2±1 neurons) compared to contralateral (32±8 neurons; n=4; p=0.048). Using whole cell patch clamp, CVN DMV demonstrated spontaneous action potential firing in vitro (0.7±0.3 Hz, n=14 neurons/10 mice) whereas CVN NA were devoid of spontaneous firing (0.0±0.0 Hz, n=10 neurons/9 mice). CVN DMV (-58.6±2.7 Hz, n=14 neurons/10 mice) and CVN NA (-62.2±1.8 mV, n=10 neurons/9 mice, p=0.3) demonstrated similar resting membrane potential. However, CVN DMV (492.0±22.0 MΩ, n=14 neurons/10 mice) had higher input resistance compared to CVN NA (158.2±10.6 MΩ, n=10 neurons/9 mice, p<0.0001). Taken together, these data support the hypothesis that DMV contains a population of CVNs capable of producing cardioinhibitory actions with unique hyperexcitability compared to CVN NA . Future studies will examine both intracellular mechanism(s) of these differences, but also unique microcircuit pathways which utilize CVN DMV , and not CVN NA . NIH T32 HL007446 for MS; NIH R01HL157366 to CRB This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Neuropathic pain changes the output of rat lamina I spino-parabrachial neurons

• Spared nerve injury (SNI) altered the action potential (AP) output of lamina I spino-parabrachial neurons (SPNs) without affecting their resting potential or membrane resistance.• In one-third of SPNs, high-threshold dorsal root stimulation elicited persistent AP firing which was never observed in cells from naïve animals.• 38% of SPNs from SNI rats showed spontaneous persistent AP firing.• After SNI low- and high-output SPNs were no longer nociceptive-specific as part of them responded with APs to low-threshold stimulation.• These SNI-induced changes of SPN output might represent cellular mechanisms for neuropathy-associated allodynia, hyperalgesia, and spontaneous pain.

Adenosine reduces sinoatrial node cell action potential firing rate by uncoupling its membrane and calcium clocks.

The spontaneous action potential (AP) firing rate of sinoatrial nodal cells (SANC) is regulated by a system of intracellular Ca2+ and membrane ion current clocks driven by Ca2+-calmodulin-activated adenylyl cyclase-protein kinase-A signaling. The mean AP-cycle length (APCL) and APCL variability inform on the effectiveness of clock coupling. Endogenous ATP metabolite adenosine binds to adenosine receptors (A1, A3) that couple to Gi protein-coupled receptors, reducing spontaneous AP firing rate via Gβγ signaling that activates IKAch,Ado. Adenosine also inhibits adenylyl cyclase activity via Gαi signaling, impacting cAMP-mediated protein kinase-A-dependent protein phosphorylation. We hypothesize that in addition to IKAch,Ado activation, adenosine impacts also Ca2+ via Gαi signaling and that both effects reduce AP firing rate by reducing the effectiveness of the Ca2+ and membrane clock coupling. To this end, we measured Ca2+ and membrane potential characteristics in enzymatically isolated single rabbit SANC. 10 µM adenosine substantially increased both the mean APCL (on average by 43%, n = 10) and AP beat-to-beat variability from 5.1 ± 1.7% to 7.2 ± 2.0% (n = 10) measured via membrane potential and 5.0 ± 2.2% to 10.6 ± 5.9% (n = 40) measured via Ca2+ (assessed as the coefficient of variability = SD/mean). These effects were mediated by hyperpolarization of the maximum diastolic membrane potential (membrane clock effect) and suppression of diastolic local Ca2+releases (LCRs) (Ca2+-clock effect): as LCR size distributions shifted to smaller values, the time of LCR occurrence during diastolic depolarization (LCR period) became prolonged, and the ensemble LCR signal became reduced. The tight linear relationship of coupling between LCR period to the APCL in the presence of adenosine "drifted" upward and leftward, i.e. for a given LCR period, APCL was prolonged, becoming non-linear indicating clock uncoupling. An extreme case of uncoupling occurred at higher adenosine concentrations (>100µM): small stochastic LCRs failed to self-organize and synchronize to the membrane clock, thus creating a failed attempt to generate an AP resulting in arrhythmia and cessation of AP firing. Thus, the effects of adenosine to activate Gβγ and IKACh,Ado and to activate Gαi, suppressing adenylyl cyclase activity, both contribute to the adenosine-induced increase in the mean APCL and APCL variability by reducing the fidelity of clock coupling and AP firing rate.

Action potential firing rhythms in the suprachiasmatic nucleus of the diurnal grass rat, Arvicanthis niloticus

In mammals, daily physiological activities are regulated by a central circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). Recently, an increasing number of studies have used diurnal grass rats to analyze neuronal mechanisms regulating diurnal behavior. However, spontaneous action potential firing rhythms in SCN neurons have not been demonstrated clearly in diurnal grass rats. Therefore, the present study examined extracellular single-unit recordings from SCN neurons in acute hypothalamic slices of Arvicanthis niloticus (Nile grass rats). The results of this study found that circadian firing rhythms with the highest frequency occurred at dusk (6.4 Hz at zeitgeber time (ZT)10–12), while the secondary peak occurred at dawn (5.6 Hz at ZT0-2), and the lowest frequency took place in the middle of the night (3.6 Hz at ZT14-16). Locomotor activity recordings from a separate group of animals demonstrated that the Nile grass rats of the laboratory colony used in this study displayed diurnal behaviors that coincided with large crepuscular peaks under 12:12 h light–dark cycles and bimodal rhythms under constant dim red light. Thus, a positive correlation between SCN firing frequencies and locomotor activity levels was observed in the Nile grass rats. Previously, behavioral coupling of action potential firings in SCN neurons has been suggested by in vivo recordings while the present study demonstrates that the sustenance of bimodal firing rhythms in grass rat SCN neurons can last at least one day in vitro.

The funny current If is essential for the fight-or-flight response in cardiac pacemaker cells.

The sympathetic nervous system fight-or-flight response is characterized by a rapid increase in heart rate, which is mediated by an increase in the spontaneous action potential (AP) firing rate of pacemaker cells in the sinoatrial node. Sympathetic neurons stimulate sinoatrial myocytes (SAMs) by activating β adrenergic receptors (βARs) and increasing cAMP. The funny current (If) is among the cAMP-sensitive currents in SAMs. If is critical for pacemaker activity, however, its role in the fight-or-flight response remains controversial. In this study, we used AP waveform analysis, machine learning, and dynamic clamp experiments in acutely isolated SAMs from mice to quantitatively define the AP waveform changes and role of If in the fight-or-flight increase in AP firing rate. We found that while βAR stimulation significantly altered nearly all AP waveform parameters, the increase in firing rate was only correlated with changes in a subset of parameters (diastolic duration, late AP duration, and diastolic depolarization rate). Dynamic clamp injection of the βAR-sensitive component of If showed that it accounts for ∼41% of the fight-or-flight increase in AP firing rate and 60% of the decrease in the interval between APs. Thus, If is an essential contributor to the fight-or-flight increase in heart rate.

Effects of Oxaliplatin on Facial Sensitivity to Cool Temperatures and TRPM8 Expressing Trigeminal Ganglion Neurons in Mice.

The chemotherapeutic agent oxaliplatin is commonly used to treat colorectal cancer. Although effective as a chemotherapeutic, it frequently produces painful peripheral neuropathies. These neuropathies can be divided into an acute sensitivity to cool temperatures in the mouth and face, and chronic neuropathic pain in the limbs and possible numbness. The chronic neuropathy also includes sensitivity to cool temperatures. Neurons that detect cool temperatures are reported to utilize Transient Receptor Potential Cation Channel, Subfamily M, Member 8 (TRPM8). Therefore, we investigated the effects of oxaliplatin on facial nociception to cool temperatures (18°C) in mice and on TRPM8 expressing trigeminal ganglion (TRG) neurons. Paclitaxel, a chemotherapeutic that is used to treat breast cancer, was included for comparison because it produces neuropathies, but acute cool temperature sensitivity in the oral cavity or face is not typically reported. Behavioral testing of facial sensitivity to 18°C indicated no hypersensitivity either acutely or chronically following either chemotherapeutic agent. However, whole cell voltage clamp experiments in TRPM8 expressing TRG neurons indicated that both oxaliplatin and paclitaxel increased Hyperpolarization-Activated Cyclic Nucleotide-Gated channel (HCN), voltage gated sodium channel (Nav), and menthol evoked TRPM8 currents. Voltage gated potassium channel (Kv) currents were not altered. Histological examination of TRPM8 fibers in the skin of the whisker pads demonstrated that the TRPM8 expressing axons and possible Merkel cell-neurite complexes were damaged by oxaliplatin. These findings indicate that oxaliplatin induces a rapid degeneration of TRG neuron axons that express TRPM8, which prevents evoked activation of the sensitized neurons and likely leads to reduced sensitivity to touch and cool temperatures. The changes in HCN, Nav, and TRPM8 currents suggest that spontaneous firing of action potentials may be increased in the deafferented neurons within the ganglion, possibly producing spontaneously induced cooling or nociceptive sensations.

The Roles of BK Potassium Channels in Neuronal Excitability Differ in Excitatory and Inhibitory Hippocampal Neurons During Early Development

BK potassium channels are are activated by intracellular Ca+ and depolarization, opening during action potential firing and speeding repolarization. Because of this, they are key regulators of fast spiking activity and important determinants of neuronal excitability. Due to their role in regulating excitability as well as their broad expression in both excitable and non‐excitable tissues, BK channels are implicated in many pathological states including epilepsy. The BK channel antagonist, paxilline, has been shown to delay seizure induction in adult rodents that have had prior seizures, but not in seizure naïve rodents. History of seizures has been shown induce expression of more Ca2+ sensitive BK channel splice variants, which may underlie the difference in paxilline efficacy between the two states. Because expression of more Ca2+ sensitive isoforms predominates in neonatal rodents ‐similar to in the epileptic adults, we hypothesize that paxilline may be an effective inhibitor of seizure activity even in seizure‐naïve neonatal rodents. Our first goal is to understand how developmental shifts in BK channel expression and function map to changes in neuronal and circuit excitability. Our second goal is to test whether BK channel blockade can modulate seizure activity in neonates, as there is a need to identify pharmacological targets for seizures in neonates and children, which are often intractable to conventional treatments.To test how BK channel function changes with age and by cell type we measured the effect of paxilline on action potential (AP) firing ‐both on single spike kinetics and prolonged firing‐ in two classes of hippocampal neurons, CA1 pyramidal neurons and parvalbumin interneurons (PVIns), a key inhibitory component of local hippocampal circuits. We used P4‐5, P9‐10, and P14‐15 C57BL/6J mice with labelled PVIns. We also tested the efficacy of paxilline in reducing hyperexcitability evoked by application of 0 Mg2+ aCSF in brain slices, an established seizure model.We patched pyramidal neurons and PVIns in whole‐cell current‐clamp configuration and evoked APs using square depolarizing pulses. BK channel blockade with 1mM paxilline lengthens duration of APs in P4‐5 and P9‐10 pyramidal neurons and P4‐5 PVIns but does not affect P9‐10 PVIn AP duration (paired t‐tests, p<0.05). While BK blockade has the least effect on single APs in the P9‐10 PVIns, it has the most effect on the input‐output relationship and spike‐frequency accommodation in these neurons (3‐way ANOVA, p<0.001 for interaction of age and cell type in input‐output; 3‐way ANOVA p<0.05 for main effect of age in frequency adaptation).We measured the effect of BK channel blockade in controlling hyperexcitability in P4‐15 mice. We found that 0 Mg2+ aCSF application induced spontaneous AP firing in patched hippocampal pyramidal neurons within 3.86±0.87 minutes but when 0 Mg2+ aCSF was coapplied with 1 mM paxilline the average onset of spontaneous activity occurred at 8.43±1.40 minutes (t‐test p<0.05).For the first goal, we show that BK channels’ relative importance in regulating AP firing changes with age and by neuronal population. Toward the second goal we report that BK channel blockade can reduce hyperexcitability in a brain slice model of epilepsy.

Electrophysiological Properties of Induced Pluripotent Stem Cell-Derived Midbrain Dopaminergic Neurons Correlate With Expression of Tyrosine Hydroxylase.

Induced pluripotent stem cell (iPSC)-based generation of tyrosine hydroxylase-positive (TH+) dopaminergic neurons (DNs) is a powerful method for creating patient-specific in vitro models to elucidate mechanisms underlying Parkinson’s disease (PD) at the cellular and molecular level and to perform drug screening. However, currently available differentiation paradigms result in highly heterogeneous cell populations, often yielding a disappointing fraction (<50%) of the PD-relevant TH+ DNs. To facilitate the targeted analysis of this cell population and to characterize their electrophysiological properties, we employed CRISPR/Cas9 technology and generated an mCherry-based human TH reporter iPSC line. Subsequently, reporter iPSCs were subjected to dopaminergic differentiation using either a “floor plate protocol” generating DNs directly from iPSCs or an alternative method involving iPSC-derived neuronal precursors (NPC-derived DNs). To identify the strategy with the highest conversion efficiency to mature neurons, both cultures were examined for a period of 8 weeks after triggering neuronal differentiation by means of immunochemistry and single-cell electrophysiology. We confirmed that mCherry expression correlated with the expression of endogenous TH and that genetic editing did neither affect the differentiation process nor the endogenous TH expression in iPSC- and NPC-derived DNs. Although both cultures yielded identical proportions of TH+ cells (≈30%), whole-cell patch-clamp experiments revealed that iPSC-derived DNs gave rise to larger currents mediated by voltage-gated sodium and potassium channels, showed a higher degree of synaptic activity, and fired trains of mature spontaneous action potentials more frequently compared to NPC-derived DNs already after 2 weeks in differentiation. Moreover, spontaneous action potential firing was more frequently detected in TH+ neurons compared to the TH– cells, providing direct evidence that these two neuronal subpopulations exhibit different intrinsic electrophysiological properties. In summary, the data reveal substantial differences in the electrophysiological properties of iPSC-derived TH+ and TH– neuronal cell populations and that the “floor plate protocol” is particularly efficient in generating electrophysiologically mature TH+ DNs, which are the most vulnerable neuronal subtype in PD.

HDAC6 Inhibition Corrects Electrophysiological and Axonal Transport Deficits in a Human Stem Cell-Based Model of Charcot-Marie-Tooth Disease (Type 2D).

Charcot-Marie-Tooth disease type 2D (CMT2D), is a hereditary peripheral neuropathy caused by mutations in the gene encoding glycyl-tRNA synthetase (GARS1). Here, human induced pluripotent stem cell (hiPSC)-based models of CMT2D bearing mutations in GARS1 and their use for the identification of predictive biomarkers amenable to therapeutic efficacy screening is described. Cultures containing spinal cord motor neurons generated from this line exhibit network activity marked by significant deficiencies in spontaneous action potential firing and burst fire behavior. This result matches clinical data collected from a patient bearing a GARS1P724H mutation and is coupled with significant decreases in acetylated α-tubulin levels and mitochondrial movement within axons. Treatment with histone deacetylase 6 inhibitors, tubastatin A and CKD504, improves mitochondrial movement and α-tubulin acetylation in these cells. Furthermore, CKD504 treatment enhances population-level electrophysiological activity, highlighting its potential as an effective treatment for CMT2D.

Phosphoprotein Phosphatase 1 but Not 2A Activity Modulates Coupled-Clock Mechanisms to Impact on Intrinsic Automaticity of Sinoatrial Nodal Pacemaker Cells.

Spontaneous AP (action potential) firing of sinoatrial nodal cells (SANC) is critically dependent on protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent protein phosphorylation, which are required for the generation of spontaneous, diastolic local Ca2+ releases (LCRs). Although phosphoprotein phosphatases (PP) regulate protein phosphorylation, the expression level of PPs and phosphatase inhibitors in SANC and the impact of phosphatase inhibition on the spontaneous LCRs and other players of the oscillatory coupled-clock system is unknown. Here, we show that rabbit SANC express both PP1, PP2A, and endogenous PP inhibitors I-1 (PPI-1), dopamine and cyclic adenosine 3′,5′-monophosphate (cAMP)-regulated phosphoprotein (DARPP-32), kinase C-enhanced PP1 inhibitor (KEPI). Application of Calyculin A, (CyA), a PPs inhibitor, to intact, freshly isolated single SANC: (1) significantly increased phospholamban (PLB) phosphorylation (by 2–3-fold) at both CaMKII-dependent Thr17 and PKA-dependent Ser16 sites, in a time and concentration dependent manner; (2) increased ryanodine receptor (RyR) phosphorylation at the Ser2809 site; (3) substantially increased sarcoplasmic reticulum (SR) Ca2+ load; (4) augmented L-type Ca2+ current amplitude; (5) augmented LCR’s characteristics and decreased LCR period in intact and permeabilized SANC, and (6) increased the spontaneous basal AP firing rate. In contrast, the selective PP2A inhibitor okadaic acid (100 nmol/L) had no significant effect on spontaneous AP firing, LCR parameters, or PLB phosphorylation. Application of purified PP1 to permeabilized SANC suppressed LCR, whereas purified PP2A had no effect on LCR characteristics. Our numerical model simulations demonstrated that PP inhibition increases AP firing rate via a coupled-clock mechanism, including respective increases in the SR Ca2+ pumping rate, L-type Ca2+ current, and Na+/Ca2+-exchanger current. Thus, PP1 and its endogenous inhibitors modulate the basal spontaneous firing rate of cardiac pacemaker cells by suppressing SR Ca2+ cycling protein phosphorylation, the SR Ca2+ load and LCRs, and L-type Ca2+ current.

Distinct Effects of Ibrutinib and Acalabrutinib on Mouse Atrial and Sinoatrial Node Electrophysiology and Arrhythmogenesis.

BackgroundIbrutinib and acalabrutinib are Bruton tyrosine kinase inhibitors used in the treatment of B‐cell lymphoproliferative disorders. Ibrutinib is associated with new‐onset atrial fibrillation. Cases of sinus bradycardia and sinus arrest have also been reported following ibrutinib treatment. Conversely, acalabrutinib is less arrhythmogenic. The basis for these different effects is unclear.Methods and ResultsThe effects of ibrutinib and acalabrutinib on atrial electrophysiology were investigated in anesthetized mice using intracardiac electrophysiology, in isolated atrial preparations using high‐resolution optical mapping, and in isolated atrial and sinoatrial node (SAN) myocytes using patch‐clamping. Acute delivery of acalabrutinib did not affect atrial fibrillation susceptibility or other measures of atrial electrophysiology in mice in vivo. Optical mapping demonstrates that ibrutinib dose‐dependently impaired atrial and SAN conduction and slowed beating rate. Acalabrutinib had no effect on atrial and SAN conduction or beating rate. In isolated atrial myocytes, ibrutinib reduced action potential upstroke velocity and Na+ current. In contrast, acalabrutinib had no effects on atrial myocyte upstroke velocity or Na+ current. Both drugs increased action potential duration, but these effects were smaller for acalabrutinib compared with ibrutinib and occurred by different mechanisms. In SAN myocytes, ibrutinib impaired spontaneous action potential firing by inhibiting the delayed rectifier K+ current, while acalabrutinib had no effects on SAN myocyte action potential firing.ConclusionsIbrutinib and acalabrutinib have distinct effects on atrial electrophysiology and ion channel function that provide insight into the basis for increased atrial fibrillation susceptibility and SAN dysfunction with ibrutinib, but not with acalabrutinib.

Activity of Cerebellar Nuclei Neurons Correlates with ZebrinII Identity of Their Purkinje Cell Afferents.

Purkinje cells (PCs) in the cerebellar cortex can be divided into at least two main subpopulations: one subpopulation that prominently expresses ZebrinII (Z+), and shows a relatively low simple spike firing rate, and another that hardly expresses ZebrinII (Z–) and shows higher baseline firing rates. Likewise, the complex spike responses of PCs, which are evoked by climbing fiber inputs and thus reflect the activity of the inferior olive (IO), show the same dichotomy. However, it is not known whether the target neurons of PCs in the cerebellar nuclei (CN) maintain this bimodal distribution. Electrophysiological recordings in awake adult mice show that the rate of action potential firing of CN neurons that receive input from Z+ PCs was consistently lower than that of CN neurons innervated by Z– PCs. Similar in vivo recordings in juvenile and adolescent mice indicated that the firing frequency of CN neurons correlates to the ZebrinII identity of the PC afferents in adult, but not postnatal stages. Finally, the spontaneous action potential firing pattern of adult CN neurons recorded in vitro revealed no significant differences in intrinsic pacemaking activity between ZebrinII identities. Our findings indicate that all three main components of the olivocerebellar loop, i.e., PCs, IO neurons and CN neurons, operate at a higher rate in the Z– modules.