Increased Action Potential Firing Research Articles

Adenosine A2B receptors differently modulate oligodendrogliogenesis and myelination depending on their cellular localization.

Differentiation of oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes (OLs) is a key event for axonal myelination in the brain; this process fails during demyelinating pathologies. Adenosine is emerging as an important player in oligodendrogliogenesis, by activating its metabotropic receptors (A1R, A2AR, A2BR, and A3R). We previously demonstrated that the Gs-coupled A2BR reduced differentiation of primary OPC cultures by inhibiting delayed rectifier (IK) as well as transient (IA) outward K+ currents. To deepen the unclear role of this receptor subtype in neuron-OL interplay and in myelination process, we tested the effects of different A2BR ligands in a dorsal root ganglion neuron (DRGN)/OPC cocultures, a corroborated in vitro myelination assay. The A2BR agonist, BAY60-6583, significantly reduced myelin basic protein levels but simultaneously increased myelination index in DRGN/OPC cocultures analyzed by confocal microscopy. The last effect was prevented by the selective A2BR antagonists, PSB-603 and MRS1706. To clarify this unexpected data, we wondered whether A2BRs could play a functional role on DRGNs. We first demonstrated, by immunocytochemistry, that primary DRGN monoculture expressed A2BRs. Their selective activation by BAY60-6583 enhanced DRGN excitability, as demonstrated by increased action potential firing, decreased rheobase and depolarized resting membrane potential and were prevented by PSB-603. Throughout this A2BR-dependent enhancement of neuronal activity, DRGNs could release factors to facilitate myelination processes. Finally, silencing A2BR in DRGNs alone prevents the increased myelination induced by BAY60-6583 in cocultures. In conclusion, our data suggest a different role of A2BR during oligodendrogliogenesis and myelination, depending on their activation on neurons or oligodendroglial cells.

Altered firing activity of intracardiac neurons isolated from mice carrying the Scn5a-1798insD mutation

Abstract Funding Acknowledgements None. Background Dysfunction of the cardiac sodium channel Nav1.5 (encoded by SCN5A) is associated with arrhythmias and sudden cardiac death, the mechanisms of which have been extensively studied in cardiomyocytes. However, patients carrying an SCN5A mutation often display clinical symptoms which are not readily explained solely by alterations in cardiomyocyte electrophysiology. For instance, carriers of the SCN5A-1795insD mutation, which is associated with features of Brugada syndrome and Long QT syndrome type 3 (LQT3), display severe bradycardia leading to excessive QT-prolongation and sudden death especially occurring at rest or during the night, suggesting autonomic triggers. Interestingly, SCN5A is not only present in cardiomyocytes, but also in intracardiac neurons (ICN). ICN modulate cardiac electrophysiological properties and their dysregulation may contribute to arrhythmogenesis. Purpose To explore whether ICN from mice carrying the Scn5a-1798insD mutation (the murine equivalent of SCN5A-1795insD) display electrophysiological alterations that may (in part) contribute to the arrhythmogenic phenotype. Methods and Results ICN were isolated by enzymatic dissociation from fat pads located in the pulmonary veins and sinoatrial node regions of the hearts of wild-type (WT) and Scn5a-1798insD mice. Immunodetection of the cholinergic marker ChAT confirmed the prominent cholinergic phenotype of ICN. In agreement with previous reports in other species, we also detected the presence of Nav1.5 in murine ICN. Nav1.5 expression was mainly observed in ChAT positive cells. To evoke repetitive action potential (AP) firing in single ICN, 500 ms-sustained depolarizing current pulses were applied of increasing amplitude (from 0 pA to 150 pA). Two types of neurons were identified on the basis of firing pattern characteristics: accommodating (neurons that discharged multiple APs decrementing in frequency during the pulse) and tonic (neurons that discharged multiple APs at a high frequency). APs firing frequency did not differ between WT and Scn5a-1798insD accommodating ICN, while it was significantly increased in Scn5a-1798insD tonic ICN as compared to WT (at 100 pA, AP count/500ms in WT CMs: 29.5±1.7, n=24; at 100 pA, AP count/500ms in Scn5a-1798insD: 36.2±2.0, n=18, p=0.02). Conclusions The impact of SCN5A mutations on ICN function are unexplored, but may play an essential role in arrhythmogenesis. The increased AP firing frequency observed in Scn5a-1798insD ICN may lead to increased release of neurotransmitters, such as acetylcholine, that in turn may contribute to the bradycardia observed in patients. Overall, further elucidation of the electrophysiological consequences of inherited Nav1.5 dysfunction in ICN might provide novel therapeutic targets to prevent lethal arrhythmias.

Predictive analytics identifies key factors driving hyperalgesic priming of muscle sensory neurons.

Hyperalgesic priming, a form of neuroplasticity induced by inflammatory mediators, in peripheral nociceptors enhances the magnitude and duration of action potential (AP) firing to future inflammatory events and can potentially lead to pain chronification. The mechanisms underlying the development of hyperalgesic priming are not well understood, limiting the identification of novel therapeutic strategies to combat chronic pain. In this study, we used a computational model to identify key proteins whose modifications caused priming of muscle nociceptors and made them hyperexcitable to a subsequent inflammatory event. First, we extended a previously validated model of mouse muscle nociceptor sensitization to incorporate Epac-mediated interaction between two G protein-coupled receptor signaling pathways commonly activated by inflammatory mediators. Next, we calibrated and validated the model simulations of the nociceptor's AP response to both innocuous and noxious levels of mechanical force after two subsequent inflammatory events using literature data. Then, by performing global sensitivity analyses that simulated thousands of nociceptor-priming scenarios, we identified five ion channels and two molecular processes (from the 18 modeled transmembrane proteins and 29 intracellular signaling components) as potential regulators of the increase in AP firing in response to mechanical forces. Finally, when we simulated specific neuroplastic modifications in Kv1.1 and Nav1.7 alone as well as with simultaneous modifications in Nav1.7, Nav1.8, TRPA1, and Kv7.2, we observed a considerable increase in the fold change in the number of triggered APs in primed nociceptors. These results suggest that altering the expression of Kv1.1 and Nav1.7 might regulate the neuronal hyperexcitability in primed mechanosensitive muscle nociceptors.

Dysfunctional sodium channel kinetics as a novel epilepsy mechanism in chromosome 15q11-q13 duplication syndrome.

Duplication of the maternal chromosome 15q11.2-q13.1 region causes Dup15q syndrome, a highly penetrant neurodevelopmental disorder characterized by severe autism and refractory seizures. Although UBE3A, the gene encoding the ubiquitin ligase E3A, is thought to be the main driver of disease phenotypes, the cellular and molecular mechanisms that contribute to the development of the syndrome are yet to be determined. We previously established the necessity of UBE3A overexpression for the development of cellular phenotypes in human Dup15q neurons, including increased action potential firing and increased inward current density, which prompted us to further investigate sodium channel kinetics. We used a Dup15q patient-derived induced pluripotent stem cell line that was CRISPR-edited to remove the supernumerary chromosome and create an isogenic control line. We performed whole cell patch clamp electrophysiology on Dup15q and corrected control neurons at two time points of in vitro development. Compared to corrected neurons, Dup15q neurons showed increased sodium current density and a depolarizing shift in steady-state inactivation. Moreover, onset of slow inactivation was delayed, and a faster recovery from both fast and slow inactivation processes was observed in Dup15q neurons. A fraction of sodium current in Dup15q neurons (~15%) appeared to be resistant to slow inactivation. Not unexpectedly, a higher fraction of persistent sodium current was also observed in Dup15q neurons. These phenotypes were modulated by the anticonvulsant drug rufinamide. Sodium channels play a crucial role in the generation of action potentials, and sodium channelopathies have been uncovered in multiple forms of epilepsy. For the first time, our work identifies in Dup15q neurons dysfunctional inactivation kinetics, which have been previously linked to multiple forms of epilepsy. Our work can also guide therapeutic approaches to epileptic seizures in Dup15q patients and emphasize the role of drugs that modulate inactivation kinetics, such as rufinamide.

WNK1 promotes water homeostasis by acting as a central osmolality sensor for arginine vasopressin release.

Maintaining internal osmolality constancy is essential for life. Release of arginine vasopressin (AVP) in response to hyperosmolality is critical. Current hypotheses for osmolality sensors in circumventricular organs (CVOs) of the brain focus on mechanosensitive membrane proteins. The present study demonstrated that intracellular protein kinase WNK1 was involved. Focusing on vascular-organ-of-lamina-terminalis (OVLT) nuclei, we showed that WNK1 kinase was activated by water restriction. Neuron-specific conditional KO (cKO) of Wnk1 caused polyuria with decreased urine osmolality that persisted in water restriction and blunted water restriction-induced AVP release. Wnk1 cKO also blunted mannitol-induced AVP release but had no effect on osmotic thirst response. The role of WNK1 in the osmosensory neurons in CVOs was supported by neuronal pathway tracing. Hyperosmolality-induced increases in action potential firing in OVLT neurons was blunted by Wnk1 deletion or pharmacological WNK inhibitors. Knockdown of Kv3.1 channel in OVLT by shRNA reproduced the phenotypes. Thus, WNK1 in osmosensory neurons in CVOs detects extracellular hypertonicity and mediates the increase in AVP release by activating Kv3.1 and increasing action potential firing from osmosensory neurons.

Identification of key factors driving inflammation-induced sensitization of muscle sensory neurons.

Sensory neurons embedded in muscle tissue that initiate pain sensations, i.e., nociceptors, are temporarily sensitized by inflammatory mediators during musculoskeletal trauma. These neurons transduce peripheral noxious stimuli into an electrical signal [i.e., an action potential (AP)] and, when sensitized, demonstrate lower activation thresholds and a heightened AP response. We still do not understand the relative contributions of the various transmembrane proteins and intracellular signaling processes that drive the inflammation-induced hyperexcitability of nociceptors. In this study, we used computational analysis to identify key proteins that could regulate the inflammation-induced increase in the magnitude of AP firing in mechanosensitive muscle nociceptors. First, we extended a previously validated model of a mechanosensitive mouse muscle nociceptor to incorporate two inflammation-activated G protein-coupled receptor (GPCR) signaling pathways and validated the model simulations of inflammation-induced nociceptor sensitization using literature data. Then, by performing global sensitivity analyses that simulated thousands of inflammation-induced nociceptor sensitization scenarios, we identified three ion channels and four molecular processes (from the 17 modeled transmembrane proteins and 28 intracellular signaling components) as potential regulators of the inflammation-induced increase in AP firing in response to mechanical forces. Moreover, we found that simulating single knockouts of transient receptor potential ankyrin 1 (TRPA1) and reducing the rates of Gαq-coupled receptor phosphorylation and Gαq subunit activation considerably altered the excitability of nociceptors (i.e., each modification increased or decreased the inflammation-induced fold change in the number of triggered APs compared to when all channels were present). These results suggest that altering the expression of TRPA1 or the concentration of intracellular Gαq might regulate the inflammation-induced increase in AP response of mechanosensitive muscle nociceptors.

Central Basis For MDMA (ecstasy) Induced Hyponatremia

Clinical studies have shown that hyponatremia can be induced by the ingestion of the recreational drug MDMA (3,4-Methyl​enedioxy​methamphetamine, ecstasy) when combined with a water intake (Baggot et al., 2016). However, the mechanisms by which MDMA induces hyponatremia is unknown. Hyponatremia is a life-threatening condition that causes rapid swelling of the brain and is commonly encountered in critical care medicine (emergency) (Woods et al., 1993 & Kirch, Bigelow, Weinberger, 1985). Under the Controlled Drugs and Substances Act (CDSA), MDMA is a schedule 1 drug (illegal) and consumption usually occurs at dance parties or raves. Nonetheless, despite MDMA’s scheduling, the Multidisciplinary Association for Psychedelic Studies (MAPS) has headed the movement toward using MDMA for assisted psychotherapy for PTSD (Sessa, Higbed, & Nutt, 2019). In summary, with a prevalence of 3.5% of young adults 18-25 years old using MDMA in the United States in 2014 (Betzler, Viohl, & Romanczuk-Seiferth, 2017), along with the mainstream marketing of MDMA for PTSD, combined with very serious and potentially deathly side effects makes this substance worthwhile to investigate. Findings could not only aid in MDMA associated hyponatremia but could also provide the foundation clinically for fluid regulation during MDMA assisted psychotherapy. Clinically, ingestion of MDMA has been shown to cause an increase in the hormones vasopressin and oxytocin (Wolf et al., 2006). Therefore, we examined cFos activity in male rats, we show that there is an increase in both cFos activation of vasopressin as well as oxytocin neurons of the supra optic nucleus. Next, we wanted to test the notion that MDMA causes an increase in thirst. Our data shows that an injection of MDMA does not increase drinking behavior. Combatting the conventional thinking that MDMA causes an increase in thirst. Interestingly, in line with the human literature, when you combine MDMA with a gastric water load, it is sufficient to drop serum sodium levels to hyponatremic levels i.e. <135mmol/L. Indicating that a water load combined with MDMA is necessary for MDMA induced hyponatremia. Conventionally, MDMA has been shown to increase the amount of serotonin within the synaptic cleft, via the inhibition of uptake via the serotonin transporter (Oeri, 2021). To determine if MDMA is working via the release of 5HT in a temperature-controlled environment we utilized whole cell patch clamp slice electrophysiology of male double transgenic Wistar rats enabling fluorescent identification of vasopressin and oxytocin neurons. Interestingly, at a bath temperature of a constant 30°C, a bath application of 5HT was sufficient in causing an enhancement of action potential firing, and this could be blocked by a 5HT receptor antagonist. Moreover, a bath application of MDMA also caused an increase in action potential firing, which too could be blocked by a 5HT receptor antagonist. CIHR. HBHL, McGill Faculty of Medicine 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.

Neuromedin B excites central lateral amygdala neurons and reduces cardiovascular output and fear-potentiated startle.

Neuromedin B (NMB) and gastrin-releasing peptide (GRP) are the two mammalian analogs in the bombesin peptide family that exert a variety of actions including emotional processing, appetitive behaviors, cognition, and tumor growth. The bombesin-like peptides interact with three receptors: the NMB-preferring bombesin 1 (BB1) receptors, the GRP-preferring bombesin 2 (BB2) receptors and the orphan bombesin 3 (BB3) receptors. Whereas, injection of bombesin into the central amygdala reduces satiety and modulates blood pressure, the underlying cellular and molecular mechanisms have not been determined. As administration of bombesin induces the expression of Fos in the lateral nucleus of the central amygdala (CeL) which expresses BB1 receptors, we probed the effects of NMB on CeL neurons using in vitro and in vivo approaches. We showed that activation of the BB1 receptors increased action potential firing frequency recorded from CeL neurons via inhibition of the inwardly rectifying K+ (Kir) channels. Activities of phospholipase Cβ and protein kinase C were required, whereas intracellular Ca2+ release was unnecessary for BB1 receptor-elicited potentiation of neuronal excitability. Application of NMB directly into the CeA reduced blood pressure and heart rate and significantly reduced fear-potentiated startle. We may provide a cellular and molecular mechanism whereby bombesin-like peptides modulate anxiety and fear responses in the amygdala.

Osmotically evoked PLCδ1-dependent translocation of ΔN-TRPV1 channels in rat supraoptic neurons.

Osmoregulation is an essential homeostatic process that requires constant release of vasopressin during sustained increases in plasma osmolality. The magnocellular neurosecretory cells (MNCs) respond to increases in external osmolality through increases in the activity of ΔN-TRPV1 channels, which leads to increased action potential firing and vasopressin release. We show that sustained exposure of acutely isolated rat and mouse MNCs to hypertonic solutions (90min) causes a reversible translocation of ΔN-TRPV1 channels from internal stores to the plasma membrane that depends on the activation of phospholipase C and on SNARE-dependent exocytosis. ΔN-TRPV1 channel translocation is absent in MNCs isolated from transgenic mice lacking the PLCδ1 isoform, suggesting that PLCδ1 is essential for triggering this process. The translocation of ΔN-TRPV1 channels to the cell surface could contribute to the maintenance of MNC excitability during sustained increases in osmolality. Our data therefore have important implications for the mechanisms underlying mammalian osmoregulation.

Oxytocin mediated excitation of hypoglossal motoneurons: implications for treating obstructive sleep apnea.

Clinical studies have shown that oxytocin administered intranasally (IN) decreased the incidence and duration of obstructive events in patients with obstructive sleep apnea (OSA). Although the mechanisms by which oxytocin promotes these beneficial effects are unknown, one possible target of oxytocin could be the excitation of tongue-projecting hypoglossal motoneurons in the medulla, that exert central control of upper airway patency. This study tested the hypothesis that IN oxytocin enhances tongue muscle activity via the excitation of hypoglossal motoneurons projecting to tongue protrudor muscles (PMNs). To test this hypothesis we performed in vivo and in vitro electrophysiological studies in C57BL6/J mice as well as fluorescent imaging studies in transgenic mice in which neurons that express oxytocin receptors co-express fluorescent protein. IN oxytocin significantly increased the amplitude of inspiratory-related tongue muscle activity. This effect was abolished by severing the medial branch of hypoglossal nerve that innervates PMNs of the tongue. Oxytocin receptor-positive neurons were more prevalent in the population of PMNs than in retractor-projecting hypoglossal motoneurons (RMNs). Oxytocin administration increased action potential firing in PMNs, but had no significant effect on firing activity in RMNs. In conclusion, IN oxytocin stimulates respiratory-relating tongue muscle activity likely acting on central hypoglossal motoneurons that provide tongue protrusion and upper airway opening. This mechanism may play a role in oxytocin-induced reductions in upper airway obstructions in patients with OSA.

Calcium inhibits the funny current (If)in sinoatrial myocytes.

Hyperpolarization-activated cyclic nucleotide sensitive (HCN4) channels are the molecular correlate of the funny current (If), which is required for spontaneous action potentials in cardiac pacemaker cells. Activation of β-adrenergic receptors on pacemaker cells increases cAMP, which shifts HCN4 activation to more depolarized potentials. The resulting increase in current, along with activation of Ca2+ cycling proteins, increases action potential firing rate and, thus, heart rate. Here we show that Mg2+ and Ca2+ regulate the effects of cAMP on If and HCN4. We found that physiological concentrations of intracellular Ca2+ inhibited cAMP-dependent potentiation of native If channels in excised membrane patches from sinoatrial node myocytes (IC50 ≈ 9 μM). This inhibition was recapitulated in heterologously expressed HCN4 channels, where intracellular Ca2+ inhibited the channels with a similar IC50 (≈ 5 μM) and limited the cAMP-dependent shift in channel activation (4 mV in 100 µM Ca2+ vs. 12 mV in zero Ca2+). Interestingly, Mg2+ had opposing effects to Ca2+. Mg2+ has been shown to interact with cAMP-bound HCN4 channels at a metal coordination site composed of two residues in the C-linker and two residues in the S4-S5 linker. We found that Mg2+ is also required to functionally couple the inhibitory, unbound CNBD to the pore in the absence of cAMP — removal of Mg2+ mimicked the effects of cAMP-dependent disinhibition. Although the metal coordination site is conserved among mammalian HCN channel isoforms, Mg2+ and Ca2+ regulation appear to be specific for HCN4 — cAMP regulation of HCN2 was insensitive to divalents. These results suggest that divalent cations directly regulate HCN4, raising the possibility that If in pacemaker cells is dynamically regulated in response to beat-to-beat changes in intracellular Ca2+.

Glucagon-like peptide-1 receptor differentially controls mossy cell activity across the dentate gyrus longitudinal axis.

Understanding the role of dentate gyrus (DG) mossy cells (MCs) in learning and memory has rapidly evolved due to increasingly precise methods for targeting MCs and for in vivo recording and activity manipulation in rodents. These studies have shown MCs are highly active in vivo, strongly remap to contextual manipulation, and that their inhibition or hyperactivation impairs pattern separation and location or context discrimination. Less well understood is how MC activity is modulated by neurohormonal mechanisms, which might differentially control the participation of MCs in cognitive functions during discrete states, such as hunger or satiety. In this study, we demonstrate that glucagon-like peptide-1 (GLP-1), a neuropeptide produced in the gut and the brain that regulates food consumption and hippocampal-dependent mnemonic function, might regulate MC function through expression of its receptor, GLP-1R. RNA-seq demonstrated that most, though not all, Glp1r in hippocampal principal neurons is expressed in MCs, and in situ hybridization revealed strong expression of Glp1r in hilar neurons. Glp1r-ires-Cre mice crossed with Ai14D reporter mice followed by co-labeling for the MC marker GluR2/3 revealed that almost all MCs in the ventral DG expressed Glp1r and that almost all Glp1r-expressing hilar neurons were MCs. However, only ~60% of dorsal DG MCs expressed Glp1r, and Glp1r was also expressed in small hilar neurons that were not MCs. Consistent with this expression pattern, peripheral administration of the GLP-1R agonist exendin-4 (5μg/kg) increased cFos expression in ventral but not dorsal DG hilar neurons. Finally, whole-cell patch-clamp recordings from ventral MCs showed that bath application of exendin-4 (200 nM) depolarized MCs and increased action potential firing. Taken together, this study adds to known MC activity modulators a neurohormonal mechanism that may preferentially affect ventral DG physiology and may potentially be targetable by several GLP-1R pharmacotherapies already in clinical use.

Chronic, episodic developmental nicotine exposure alters GABAergic synaptic transmission to hypoglossal motor neurons at a critical developmental age in rats

The adverse effects of nicotine exposure on brain structural and functional development is attributed to its action on the nicotinic acetylcholine receptors (nAChRs). nAChRs are expressed post‐synaptically on hypoglossal motor neurons (XIIMNs), the neurons that innervate the tongue. We recently showed that chronic, episodic developmental nicotine exposure alters the intrinsic properties of these neurons, which may be one mechanism to explain impaired tongue muscle function that is commonly observed in nicotine‐ exposed human and animal neonates. Importantly, along with the intrinsic properties of XIIMNs, the balance of excitatory and inhibitory fast‐synaptic inputs to these neurons is important in dictating the strength of motor output to the tongue. nAChRs are also located on the soma, dendrites, and axon end terminals of excitatory and inhibitory interneurons that synapse onto XIIMNs, activation of which increases action potential firing and neurotransmitter release. Here, we tested the hypothesis that chronic, episodic developmental nicotine exposure alters GABAergic transmission, a major source of fast‐synaptic inhibition, to XIIMNs. XIIMNs were recorded in brainstem slices from control and nicotine‐ exposed rats in two age groups: postnatal days (P)1‐5 and P10‐12, which is considered a critical period of brain development. In voltage‐ clamp mode, the frequency of GABAergic spontaneous inhibitory post‐synaptic currents (sIPSCs) was recorded at baseline and in response to activation of nAChRs with acute, bath‐applied nicotine. The baseline frequency of GABAergic sIPSCs was not different between control and nicotine‐exposed neurons at either age. In the P1‐5 age group, acute nicotine increased the frequency of GABAergic sIPSCs in both control and nicotine‐exposed neurons. However, at ages P10‐12, acute nicotine increased the frequency of GABAergic sIPSCs in nicotine‐exposed neurons, but had no effect in control neurons. These findings indicate that chronic, episodic nicotine exposure alters GABAergic synaptic transmission to XIIMNs at a critical developmental age, and may contribute to excitatory/inhibitory imbalance that underlies impaired control of the muscles of the tongue.

Activation of TGR5 Ameliorates Streptozotocin-Induced Cognitive Impairment by Modulating Apoptosis, Neurogenesis, and Neuronal Firing.

Takeda G protein-coupled receptor 5 (TGR5) is the first known G protein-coupled receptor specific for bile acids and is recognized as a new and critical target for type 2 diabetes and metabolic syndrome. It is expressed in many brain regions associated with memory such as the hippocampus and frontal cortex. Here, we hypothesize that activation of TGR5 may ameliorate streptozotocin- (STZ-) induced cognitive impairment. The mouse model of cognitive impairment was established by a single intracerebroventricular (ICV) injection of STZ (3.0 mg/kg), and we found that TGR5 activation by its agonist INT-777 (1.5 or 3.0 μg/mouse, ICV injection) ameliorated spatial memory impairment in the Morris water maze and Y-maze tests. Importantly, INT-777 reversed STZ-induced downregulation of TGR5 and glucose usage deficits. Our results further showed that INT-777 suppressed neuronal apoptosis and improved neurogenesis which were involved in tau phosphorylation and CREB-BDNF signaling. Moreover, INT-777 increased action potential firing of excitatory pyramidal neurons in the hippocampal CA3 and medial prefrontal cortex of ICV-STZ groups. Taken together, these findings reveal that activation of TGR5 has a neuroprotective effect against STZ-induced cognitive impairment by modulating apoptosis, neurogenesis, and neuronal firing in the brain and TGR5 might be a novel and potential target for Alzheimer's disease.

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.

Stimulation of motilin secretion by bile, free fatty acids, and acidification in human duodenal organoids

ObjectiveMotilin is a proximal small intestinal hormone with roles in gastrointestinal motility, gallbladder emptying, and hunger initiation. In vivo motilin release is stimulated by fats, bile, and duodenal acidification but the underlying molecular mechanisms of motilin secretion remain poorly understood. This study aimed to establish the key signaling pathways involved in the regulation of secretion from human motilin-expressing M-cells. MethodsHuman duodenal organoids were CRISPR-Cas9 modified to express the fluorescent protein Venus or the Ca2+ sensor GCaMP7s under control of the endogenous motilin promoter. This enabled the identification and purification of M-cells for bulk RNA sequencing, peptidomics, calcium imaging, and electrophysiology. Motilin secretion from 2D organoid-derived cultures was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS), in parallel with other gut hormones. ResultsHuman duodenal M-cells synthesize active forms of motilin and acyl-ghrelin in organoid culture, and also co-express cholecystokinin (CCK). Activation of the bile acid receptor GPBAR1 stimulated a 3.4-fold increase in motilin secretion and increased action potential firing. Agonists of the long-chain fatty acid receptor FFA1 and monoacylglycerol receptor GPR119 stimulated secretion by 2.4-fold and 1.5-fold, respectively. Acidification (pH 5.0) was a potent stimulus of M-cell calcium elevation and electrical activity, an effect attributable to acid-sensing ion channels, and a modest inducer of motilin release. ConclusionsThis study presents the first in-depth transcriptomic and functional characterization of human duodenal motilin-expressing cells. We identify several receptors important for the postprandial and interdigestive regulation of motilin release.

Altered neuronal physiology, development, and function associated with a common chromosome 15 duplication involving CHRNA7

BackgroundCopy number variants (CNVs) linked to genes involved in nervous system development or function are often associated with neuropsychiatric disease. While CNVs involving deletions generally cause severe and highly penetrant patient phenotypes, CNVs leading to duplications tend instead to exhibit widely variable and less penetrant phenotypic expressivity among affected individuals. CNVs located on chromosome 15q13.3 affecting the alpha-7 nicotinic acetylcholine receptor subunit (CHRNA7) gene contribute to multiple neuropsychiatric disorders with highly variable penetrance. However, the basis of such differential penetrance remains uncharacterized. Here, we generated induced pluripotent stem cell (iPSC) models from first-degree relatives with a 15q13.3 duplication and analyzed their cellular phenotypes to uncover a basis for the dissimilar phenotypic expressivity.ResultsThe first-degree relatives studied included a boy with autism and emotional dysregulation (the affected proband-AP) and his clinically unaffected mother (UM), with comparison to unrelated control models lacking this duplication. Potential contributors to neuropsychiatric impairment were modeled in iPSC-derived cortical excitatory and inhibitory neurons. The AP-derived model uniquely exhibited disruptions of cellular physiology and neurodevelopment not observed in either the UM or unrelated controls. These included enhanced neural progenitor proliferation but impaired neuronal differentiation, maturation, and migration, and increased endoplasmic reticulum (ER) stress. Both the neuronal migration deficit and elevated ER stress could be selectively rescued by different pharmacologic agents. Neuronal gene expression was also dysregulated in the AP, including reduced expression of genes related to behavior, psychological disorders, neuritogenesis, neuronal migration, and Wnt, axonal guidance, and GABA receptor signaling. The UM model instead exhibited upregulated expression of genes in many of these same pathways, suggesting that molecular compensation could have contributed to the lack of neurodevelopmental phenotypes in this model. However, both AP- and UM-derived neurons exhibited shared alterations of neuronal function, including increased action potential firing and elevated cholinergic activity, consistent with increased homomeric CHRNA7 channel activity.ConclusionsThese data define both diagnosis-associated cellular phenotypes and shared functional anomalies related to CHRNA7 duplication that may contribute to variable phenotypic penetrance in individuals with 15q13.3 duplication. The capacity for pharmacological agents to rescue some neurodevelopmental anomalies associated with diagnosis suggests avenues for intervention for carriers of this duplication and other CNVs that cause related disorders.

Hyperexcitable Phenotypes in Induced Pluripotent Stem Cell–Derived Neurons From Patients With 15q11-q13 Duplication Syndrome, a Genetic Form of Autism

BackgroundChromosome 15q11-q13 duplication syndrome (Dup15q) is a neurogenetic disorder caused by duplications of the maternal copy of this region. In addition to hypotonia, motor deficits, and language impairments, patients with Dup15q commonly meet the criteria for autism spectrum disorder and have a high prevalence of seizures. It is known from mouse models that synaptic impairments are a strong component of Dup15q pathophysiology; however, cellular phenotypes that relate to seizures are less clear. The development of patient-derived induced pluripotent stem cells provides a unique opportunity to study human neurons with the exact genetic disruptions that cause Dup15q. MethodsHere, we explored electrophysiological phenotypes in induced pluripotent stem cell–derived neurons from 4 patients with Dup15q compared with 6 unaffected control subjects, 1 patient with a 15q11-q13 paternal duplication, and 3 patients with Angelman syndrome. ResultsWe identified several properties of Dup15q neurons that could contribute to neuronal hyperexcitability and seizure susceptibility. Compared with control neurons, Dup15q neurons had increased excitatory synaptic event frequency and amplitude, increased density of dendritic protrusions, increased action potential firing, and decreased inhibitory synaptic transmission. Dup15q neurons also showed impairments in activity-dependent synaptic plasticity and homeostatic synaptic scaling. Finally, Dup15q neurons showed an increased frequency of spontaneous action potential firing compared with control neurons, in part due to disruption of KCNQ2 potassium channels. ConclusionsTogether, these data point to multiple electrophysiological mechanisms of hyperexcitability that may provide new targets for the treatment of seizures and other phenotypes associated with Dup15q.

Enhanced excitability of cortical neurons in low-divalent solutions is primarily mediated by altered voltage-dependence of voltage-gated sodium channels

Increasing extracellular [Ca2+] ([Ca2+]o) strongly decreases intrinsic excitability in neurons but the mechanism is unclear. By one hypothesis, [Ca2+]o screens surface charge, reducing voltage-gated sodium channel (VGSC) activation and by another [Ca2+]o activates Calcium-sensing receptor (CaSR) closing the sodium-leak channel (NALCN). Here we report that neocortical neurons from CaSR-deficient (Casr-/-) mice had more negative resting potentials and did not fire spontaneously in reduced divalent-containing solution (T0.2) in contrast with wild-type (WT). However, after setting membrane potential to -70 mV, T0.2 application similarly depolarized and increased action potential firing in Casr-/- and WT neurons. Enhanced activation of VGSCs was the dominant contributor to the depolarization and increase in excitability by T0.2 and occurred due to hyperpolarizing shifts in VGSC window currents. CaSR deletion depolarized VGSC window currents but did not affect NALCN activation. Regulation of VGSC gating by external divalents is the key mechanism mediating divalent-dependent changes in neocortical neuron excitability.

Impaired pattern separation in Tg2576 mice is associated with hyperexcitable dentate gyrus caused by Kv4.1 downregulation

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that causes memory loss. Most AD researches have focused on neurodegeneration mechanisms. Considering that neurodegenerative changes are not reversible, understanding early functional changes before neurodegeneration is critical to develop new strategies for early detection and treatment of AD. We found that Tg2576 mice exhibited impaired pattern separation at the early preclinical stage. Based on previous studies suggesting a critical role of dentate gyrus (DG) in pattern separation, we investigated functional changes in DG of Tg2576 mice. We found that granule cells in DG (DG-GCs) in Tg2576 mice showed increased action potential firing in response to long depolarizations and reduced 4-AP sensitive K+-currents compared to DG-GCs in wild-type (WT) mice. Among Kv4 family channels, Kv4.1 mRNA expression in DG was significantly lower in Tg2576 mice. We confirmed that Kv4.1 protein expression was reduced in Tg2576, and this reduction was restored by antioxidant treatment. Hyperexcitable DG and impaired pattern separation in Tg2576 mice were also recovered by antioxidant treatment. These results highlight the hyperexcitability of DG-GCs as a pathophysiologic mechanism underlying early cognitive deficits in AD and Kv4.1 as a new target for AD pathogenesis in relation to increased oxidative stress.