Expression of Kv4.2 and Kv4.3 potassium channels in human umbilical veins from normal, diabetic and hypertensive pregnancies

Objective: A substantial line of evidence indicates that Kv4.2 and Kv4.3 channels are the major components of rapid transient-outward potassium currents (A-type currents). It is speculated that those currents may be involved in the maintenance of the membrane potential, as well as in the regulation of propagation and frequency of action potentials. However, very little is known about the presence and function of A-type currents in human vascular smooth muscles such as human umbilical vein (HUV). Having in mind its crucial role in the proper fetal oxygenation the aim of the study was to determine whether Kv4.2 and Kv4.3 potassium channels are present in HUV smooth muscle and to investigate potential alterations of their expression during maternal pathological conditions - gestational diabetes mellitus (GDM) and pregnancy-induced hypertension (PIH). Materials and methods: Healthy, diabetic and hypertensive pregnancies were subjects of this investigation. Each group was consisted of 6 HUV samples obtained from 6 normal pregnancies, 6 pregnancies with GDM, and 6 pregnancies with PIH. After pharmacology analysis, immunohistochemistry and Western blot were performed. Results: Immunohistochemistry revealed similar expression pattern of both, Kv4.2 and Kv4.3 subunits, in HUV smooth muscle in all groups of patients. Results obtained by Western blot were in agreement with immunohistochemical staining. The expression of Kv4.2 and Kv4.3 subunits was not significantly different between the groups. Conclusion: Collectively, this is the first study that demonstrated presence of Kv4.2 and Kv4.3 potassium channels in the HUV smooth muscle and their preservation during the course of GDM and PIH. These channels are most likely major components of rapid A-type currents that may be relevant for maternal-fetus blood flow and hence fetal development. Also, they may represent sensors for detecting hemodynamic and/or metabolic changes in the local environment.

The effect of temperature on the activity of the trigeminal nerve in the rat meningeal sheath

Migraine is a debilitating neurological disorder that affects approximately 1 billion people worldwide. It is known that migraine is associated with the activity of the trigeminal nerve, therefore, many studies are aimed at studying changes in the activity of the meningeal nerve fibers. It is known that inflammatory processes accompanied by temperature rise are often accompanied by headaches. Therefore, we investigated the effect of temperature increase on trigeminal nerve activity. It turned out that temperature increase leads to a significant increase in the frequency of action potentials in the trigeminal nerve. Key words: migraine, trigeminal nerve, cluster analysis, action potential.

The Thermodynamically Expensive Contribution of Three Calcium Sources to Somatic Release of Serotonin

The soma, dendrites and axon of neurons may display calcium-dependent release of transmitters and peptides. Such release is named extrasynaptic for occurring in the absence of synaptic structures. This review describes cooperative actions of three calcium sources on somatic exocytosis. Emphasis is given to the release of serotonin by the classical serotonergic leech Retzius neuron, which has allowed detailed studies of each step between excitation and exoctytosis. Trains of action potentials induce transmembrane calcium entry through L-type channels. If the frequency of action potentials is above 5 Hz, summation of calcium transients upon individual action potentials increases the intracellular calcium concentration to activate calcium–induced calcium release. The amplified calcium wave activates motochondrial ATP synthesis that fuels the transport of vesicles to the plasma membrane. Serotonin that is released activates autoreceptors coupled to phospholipase C. Production of IP3 produces release of calcium that sustains the large-scale exocytosis. The swiss-clock workings of the release machinery for somatic exocytosis has a striking disadvantage. The essential calcium-releasing endoplasmic reticulum that lays between resting vesicles and the plasma membrane becomes an obstacle for the vesicle transport. Such architecture reduces drastically the thermodynamic efficiency of the vesicle transport and elevates its energy cost..

Reduced Firing of Nucleus Accumbens Parvalbumin Interneurons Impairs Risk Avoidance in DISC1 Transgenic Mice

A strong animal survival instinct is to approach objects and situations that are of benefit and to avoid risk. In humans, a large proportion of mental disorders are accompanied by impairments in risk avoidance. One of the most important genes involved in mental disorders is disrupted-in-schizophrenia-1 (DISC1), and animal models in which this gene has some level of dysfunction show emotion-related impairments. However, it is not known whether DISC1 mouse models have an impairment in avoiding potential risks. In the present study, we used DISC1-N terminal truncation (DISC1-NTM) mice to investigate risk avoidance and found that these mice were impaired in risk avoidance on the elevated plus maze (EPM) and showed reduced social preference in a three-chamber social interaction test. Following EPM tests, c-Fos expression levels indicated that the nucleus accumbens (NAc) was associated with risk-avoidance behavior in DISC1-NTM mice. In addition, in vivo electrophysiological recordings following tamoxifen administration showed that the firing rates of fast-spiking neurons (FS) in the NAc were significantly lower in DISC1-NTM mice than in wild-type (WT) mice. In addition, in vitro patch clamp recording revealed that the frequency of action potentials stimulated by current injection was lower in parvalbumin (PV) neurons in the NAc of DISC1-NTM mice than in WT controls. The impairment of risk avoidance in DISC1-NTM mice was rescued using optogenetic tools that activated NAcPV neurons. Finally, inhibition of the activity of NAcPV neurons in PV-Cre mice mimicked the risk-avoidance impairment found in DISC1-NTM mice during tests on the elevated zero maze. Taken together, our findings confirm an impairment in risk avoidance in DISC1-NTM mice and suggest that reduced excitability of NAcPV neurons is responsible.

Cholinergic regulation of cardiac pacemaker activity by L-type Cav1.3 channels

Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Fondation Recherche Médicale Introduction The cholinergic regulation of heart rate (HR) is mediated by acetylcholine (ACh)-dependent activation of M2-receptors (M2R). Activated M2R promote release of the βγ-subunit of G-proteins to directly gate GIRK1/4 channels (underlying the cardiac IKACh current), while αi-subunits inhibit adenylate cyclase (AC) activity. AC inhibition reduces the intracellular concentration of cAMP, decreasing the activity of ion channels involved in pacemaking, including "funny" f-(HCN4) and L-type Cav1.3 calcium channels. Purpose To determine the role of L-type Cav1.3 channels in cholinergic regulation of heart rate. Methods We recorded the frequency of activation and position of pacemaker leading site in ex vivo sinus nodes and the HR of isolated Langendorff perfused hearts of mice at baseline or during ACh perfusion. We used control wild type (WT) mice, and five genetically modified mouse models: Cav1.3 knockout (KO, ablated Cav1.3-mediated L-type current), GIRK4KO (ablated IKACh current), HCN4-CNBD (selective deletion of cAMP-dependent regulation of HCN4), GIRK4KO/HCN4-CNBD and GIRK4KO/Cav1.3KO. We performed in vivo telemetric recordings of heart rate (HR) in WT and GIRK4KO/Cav1.3KO animals. Results Data from optical mapping experiments showed that, under basal conditions, perfusion of 3 μM ACh significantly reduced the frequency of action potentials in WT (44%), HCN4-CNBD (38%), Cav1.3KO (65%) and GIRK4KO (8%) isolated mouse sinus node tissues. ACh application did not significantly affect the frequency of action potentials recorded in tissue from GIRK4KO/HCN4-CNBD and GIRK4KO/Cav1.3KO animals. Furthermore, in all sinus nodes tested, regardless of genotype, ACh shifted the pacemaker leading site from its normal position by at least 0.7 mm. Upon stimulation of the β-adrenergic pathway by Isoproterenol, to reproduce conditions of accentuated antagonism, 3µM ACh reduced HR in isolated hearts from WT (43.8%), HCN4-CNBD (38.7%), Cav1.3KO (25,4%), GIRK4KO (16.9%) and GIRK4KO/HCN4-CNBD (16.4%) mice. No significant HR reduction was recorded in hearts from GIRK4KO/Cav1.3KO animals. In vivo data indicate that HR reduction induced by combined injection of Hexamethonium ( a Nicotinic acetylcholine receptor blocker) with Carbamoylcholine (CCH, M2 receptor agonist) or with 2-Chloro-N6-Cyclopentyladenosine (CCPA, A1 receptor agonist) is higher in WT than in GIRK4KO/Cav1.3KO animals (68% vs 48% CCH, and 79% vs 62% CCPA, respectively). Conclusion Our data indicate that L-type Cav1.3 channels are involved in cholinergic regulation of heart rate in mice. In addition, when the intracellular concentration of cAMP is elevated (i.e. under conditions of accentuated antagonism), cholinergic regulation of sinus node pacemaking is reliant on Cav1.3 and KACh channels.

Genomic Action of Sigma-1 Receptor Chaperone Relates to Neuropathic Pain

Sigma-1 receptors (Sig-1Rs) are endoplasmic reticulum (ER) chaperones implicated in neuropathic pain. Here we examine if the Sig-1R may relate to neuropathic pain at the level of dorsal root ganglia (DRG). We focus on the neuronal excitability of DRG in a “spare nerve injury” (SNI) model of neuropathic pain in rats and find that Sig-1Rs likely contribute to the genesis of DRG neuronal excitability by decreasing the protein level of voltage-gated Cav2.2 as a translational inhibitor of mRNA. Specifically, during SNI, Sig-1Rs translocate from ER to the nuclear envelope via a trafficking protein Sec61β. At the nucleus, the Sig-1R interacts with cFos and binds to the promoter of 4E-BP1, leading to an upregulation of 4E-BP1 that binds and prevents eIF4E from initiating the mRNA translation for Cav2.2. Interestingly, in Sig-1R knockout HEK cells, Cav2.2 is upregulated. In accordance with those findings, we find that intra-DRG injection of Sig-1R agonist (+)pentazocine increases frequency of action potentials via regulation of voltage-gated Ca2+ channels. Conversely, intra-DRG injection of Sig-1R antagonist BD1047 attenuates neuropathic pain. Hence, we discover that the Sig-1R chaperone causes neuropathic pain indirectly as a translational inhibitor.

Assembly and Function of the Juxtaparanodal Kv1 Complex in Health and Disease

The precise axonal distribution of specific potassium channels is known to secure the shape and frequency of action potentials in myelinated fibers. The low-threshold voltage-gated Kv1 channels located at the axon initial segment have a significant influence on spike initiation and waveform. Their role remains partially understood at the juxtaparanodes where they are trapped under the compact myelin bordering the nodes of Ranvier in physiological conditions. However, the exposure of Kv1 channels in de- or dys-myelinating neuropathy results in alteration of saltatory conduction. Moreover, cell adhesion molecules associated with the Kv1 complex, including Caspr2, Contactin2, and LGI1, are target antigens in autoimmune diseases associated with hyperexcitability such as encephalitis, neuromyotonia, or neuropathic pain. The clustering of Kv1.1/Kv1.2 channels at the axon initial segment and juxtaparanodes is based on interactions with cell adhesion molecules and cytoskeletal linkers. This review will focus on the trafficking and assembly of the axonal Kv1 complex in the peripheral and central nervous system (PNS and CNS), during development, and in health and disease.

Cholinergic regulation of cardiac pacemaker activity by l-type cav1.3 channels

Introduction The cholinergic regulation of heart rate (HR) is mediated by acetylcholine (ACh)-dependent activation of M2-receptors (M2R). Activated M2R promote release of the βγ-subunit of G-proteins to directly gate GIRK1/4 channels (underlying the cardiac IKACh current), while αi-subunits inhibit adenylate cyclase (AC) activity. AC inhibition reduces the intracellular concentration of cAMP, decreasing the activity of ion channels involved in pacemaking, including “funny” f-(HCN4) and L-type Cav1.3 calcium channels. Purpose To determine the importance of L-type Cav1.3 channels in the cholinergic regulation of heart rate. Methods We recorded the frequency and the position of the pacemaker leading site in ex vivo sinus nodes and the HR of isolated Langendorff perfused hearts of mice in control or during ACh perfusion. We used control wild type (WT) mice, and five genetically modified mouse models: Cav1.3 knockout (KO, ablated Cav1.3-mediated L-type current), GIRK4KO (ablated IKACh current), HCN4-CNBD (selective deletion of cAMP-dependent regulation of HCN4), GIRK4KO/HCN4-CNBD and GIRK4KO/Cav1.3KO. Results Data from optical mapping experiments showed that, under basal conditions, perfusion of 3 μM ACh significantly reduced the frequency of action potentials in WT (44%), HCN4-CNBD (38%), Cav1.3KO (65%) and GIRK4KO (8%) isolated mouse sinus node tissues. ACh application did not significantly affect the frequency of action potentials recorded in tissue from GIRK4KO/HCN4-CNBD and GIRK4KO/Cav1.3KO animals. Furthermore, in all the sinus node tissues tested, regardless of the genotypes, ACh shifted the pacemaker leading site from its normal position by at least 0.7 mm. Upon stimulation of the β-adrenergic pathway by Isoproterenol, to reproduce conditions of accentuated antagonism, 3μM ACh reduced HR in isolated hearts from WT (43.8%), HCN4-CNBD (38.7%), Cav1.3KO (25,4%), GIRK4KO (16.9%) and GIRK4KO/HCN4-CNBD (16.4%) mice. No significant HR reduction was recorded in hearts from GIRK4KO/Cav1.3KO animals. Conclusion Our data indicate that L-type Cav1.3 channels are involved in cholinergic regulation of heart rate in mice. In addition, when the intracellular concentration of cAMP is elevated (i.e. under conditions of accentuated antagonism), the cholinergic regulation of sinus node pacemaking is predominantly ensured by Cav1.3 and KACh channels. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): “Fondation pour la recherche medicale” FRM

Parvalbumin interneurons in the nucleus accumbens regulate impairment of risk avoidance in DISC1 transgenic mice

One strong survival instinct in animals is to approach things that are of benefit and avoid risk. In humans, a large portion of mental disorders are accompanied by cognition-related impairments including the inability to recognize potential risks. One of the most important genes involved in risk behavior is disrupted-in-schizophrenia-1 (DISC1), and animal models where this gene has some dysfunction show cognitive impairments. However, whether DISC1 mice models have an impairment in avoiding potential risks is still not fully understood. In the present study, we used DISC1-N terminal truncation (DISC1-NTM) mice to study cognitive abilities related to potential risks. We found that DISC1-NTM mice were impaired in risk avoidance on the elevated plus maze (EPM) test, and showed impairment in social preference in a three-chamber social interaction test. Staining for c-Fos following the EPM indicated that the nucleus accumbens (NAc) was associated with risk avoidance behavior in DISC1-NTM mice. Meanwhile, in vivo electrophysiological recordings showed that firing rates of fast spiking neurons (FS) in the NAc significantly decreased in DISC1-NTM mice following tamoxifen administration. In addition, theta band power was lower when mice shuttled from the safe (closed) arms to the risky (open) arms, an effect which disappeared after induction of the truncated DISC1 gene. Furthermore, we found through in vitro patch clamp recording that the frequency of action potentials stimulated by current injection was lower in parvalbumin (PV) neurons in the NAc of DISC1-NTM mice than their wild-type littermates. Risk-avoidance impairments in DISC1-NTM mice were rescued using optogenetic tools that activated NAcPV neurons. Finally, we inhibited activitiy of NAcPV neurons in PV-Cre mice, which mimicked the risk-avoidance impairment found in the DISC1-NTM mice during tests on the elevated zero maze. Taken together, our findings confirmed a cognitive impairment in DISC1-NTM mice related to risk recognition and suggests that reduced excitability of NAcPV neurons may be responsible.

Ecm29-mediated proteasomal distribution modulates excitatory GABA responses in the developing brain

Neuronal GABAergic responses switch from excitatory to inhibitory at an early postnatal period in rodents. The timing of this switch is controlled by intracellular Cl− concentrations, but factors determining local levels of cation-chloride cotransporters remain elusive. Here, we report that local abundance of the chloride importer NKCC1 and timely emergence of GABAergic inhibition are modulated by proteasome distribution, which is mediated through interactions of proteasomes with the adaptor Ecm29 and the axon initial segment (AIS) scaffold protein ankyrin G. Mechanistically, both the Ecm29 N-terminal domain and an intact AIS structure are required for transport and tethering of proteasomes in the AIS region. In mice, Ecm29 knockout (KO) in neurons increases the density of NKCC1 protein in the AIS region, a change that positively correlates with a delay in the GABAergic response switch. Phenotypically, Ecm29 KO mice showed increased firing frequency of action potentials at early postnatal ages and were hypersusceptible to chemically induced convulsive seizures. Finally, Ecm29 KO neurons exhibited accelerated AIS developmental positioning, reflecting a perturbed AIS morphological plastic response to hyperexcitability arising from proteasome inhibition, a phenotype rescued by ectopic Ecm29 expression or NKCC1 inhibition. Together, our findings support the idea that neuronal maturation requires regulation of proteasomal distribution controlled by Ecm29.