Search

We have previously demonstrated that satiety sensing vagal afferent neurons are less responsive to meal-related stimuli in obesity because of reduced electrical excitability. As leak K + currents are key determinants of membrane excitability, we hypothesized that leak K + currents are increased in vagal afferents during obesity. Diet-induced obesity was induced by feeding C57Bl/6J mice a high-fat diet (HFF) (60% energy from fat) for 8-10 wk. In vitro extracellular recordings were performed on jejunal afferent nerves. Whole cell patch-clamp recordings were performed on mouse nodose ganglion neurons. Leak K + currents were isolated using ion substitution and pharmacological blockers. mRNA for TWIK-related acid-sensitive K + (TASK) subunits was measured using quantitative real-time PCR. Intestinal afferent responses to nutrient (oleate) and non-nutrient (ATP) stimuli were significantly decreased in HFF mice. Voltage clamp experiments revealed the presence of a voltage-insensitive resting potassium conductance that was increased by external alkaline pH and halothane, known properties of TASK currents. In HFF neurons, leak K + current was approximately doubled and was reduced by TASK1 and TASK3 inhibitors. The halothane sensitive current was similarly increased. Quantitative PCR revealed the presence of mRNA encoding TASK1 (KCNK3) and TASK3 (KCNK9) channels in nodose neurons. TASK3 transcript was significantly increased in HFF mice. The reduction in vagal afferent excitability in obesity is due in part to an increase of resting (leak) K + conductance. TASK channels may account for the impairment of satiety signaling in diet-induced obesity and thus is a therapeutic target for obesity treatment. NEW & NOTEWORTHY This study characterized the electrophysiological properties and gene expression of the TWIK-related acid-sensitive K + (TASK) channel in vagal afferent neurons. TASK conductance was increased and contributed to decreased excitability in diet-induced obesity. TASK channels may account for the impairment of satiety signaling in diet-induced obesity and thus is a promising therapeutic target.

Sepsis is a severe systemic inflammatory disorder that rapidly activates the sympathetic nervous system to enhance catecholamine secretion from postganglionic sympathetic neurons and adrenal chromaffin cells. Although an increase in preganglionic drive to postganglionic sympathetic tissues has been known to contribute to this response for quite some time, only recently was it determined that sepsis also has direct effects on adrenal chromaffin cell Ca2+ signaling and epinephrine release. In the present study, we characterized the direct effects of sepsis on postganglionic sympathetic neuron function. Using the endotoxemia model of sepsis in mice, we found that almost a quarter of postganglionic neurons acquired the ability to fire spontaneous action potentials, which was absent in cells from control mice. Spontaneously firing neurons possessed significantly lower rheobases and fired a greater number of action potentials at twice the rheobase compared to neurons from control mice. Sepsis did not significantly affect voltage-gated Ca2+ currents. However, global Ca2+ signaling was enhanced in postganglionic neurons isolated from 1 to 24 h endotoxemic mice. A similar increase in the amplitude of high-K+-stimulated Ca2+ transients was observed during the cecal ligation and puncture model of sepsis. The enhanced excitability and Ca2+ signaling produced during sepsis likely amplify the effect of increased preganglionic drive on norepinephrine release from postganglionic neurons. This is important, as sympathetic neurons are integral to the anti-inflammatory autonomic reflex that is activated during sepsis., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

Neural activity is integral to the development of the enteric nervous system (ENS). A subpopulation of neural crest-derived cells expresses pan-neuronal markers at early stages of ENS development (at E10.5 in the mouse). However, the electrical activity of these cells has not been previously characterized, and it is not known whether all cells expressing neuronal markers are capable of firing action potentials (APs). In this study, we examined the activity of "neuron"-like cells (expressing pan-neuronal markers or with neuronal morphology) in the gut of E11.5 and E12.5 mice using whole-cell patch-clamp electrophysiology and compared them to the activity of neonatal and adult enteric neurons. Around 30-40% of neuron-like cells at E11.5 and E12.5 fired APs, some of which were very similar to those of adult enteric neurons. All APs were sensitive to tetrodotoxin (TTX), indicating that they were driven by voltage-gated Na+ currents. Expression of mRNA encoding several voltage-gated Na+ channels by the E11.5 gut was detected using RT-PCR. The density of voltage-gated Na+ currents increased from E11.5 to neonates. Immature active responses, mediated in part by TTX- and lidocaine-insensitive channels, were observed in most cells at E11.5 and E12.5, but not in P0/P1 or adult neurons. However, some cells expressing neuronal markers at E11.5 or E12.5 did not exhibit an active response to depolarization. Spontaneous depolarizations resembling excitatory postsynaptic potentials were observed at E12.5. The ENS is one of the earliest parts of the developing nervous system to exhibit mature forms of electrical activity.

An imaging system for di-4-ANEPPS (4-[beta-[2-(di-n-butylamino)-6-naphthylvinyl]pyridinium]) voltage-sensitive dye recordings has been adapted for recording from an in vitro mouse heart preparation that consists of both atria in isolation. This approach has been used to study inter- and intra-atrial activation and conduction and to monitor action potential durations (APDs) in the left and right atrium. The findings from this study confirm some of our previous findings in isolated mouse atrial myocytes and demonstrate that many electrophysiological properties of mouse atria closely resemble those of larger mammals. Specifically, we made the following observations: 1) Activation in mouse atria originates in the sinoatrial node and spreads into the right atrium and, after a delay, into the left atrium. 2) APD in the left atrium is shorter than in the right atrium. 3) Sites in the posterior walls have longer APDs than sites in the atrial appendages. 4) Superfusion of this preparation with 4-aminopyridine and tetraethylammonium resulted in increases in APD, consistent with their inhibitory effects on the K+ currents known to be expressed in mouse atria. 5) The muscarinic agonist carbachol shortened APD in all areas of the preparation, except the left atrial appendage, in which carbachol had no statistically significant effect on APD. These results validate a new approach for monitoring activation, conduction, and repolarization in mouse atria and demonstrate that the physiological and pharmacological properties of mouse atria are sufficiently similar to those of larger animals to warrant further studies using this preparation.

Consistent differences in K+ currents in left and right atria of adult mouse hearts have been identified by the application of current- and voltage-clamp protocols to isolated single myocytes. Left atrial myocytes had a significantly (P < 0.05) larger peak outward K+ current density than myocytes from the right atrium. Detailed analysis revealed that this difference was due to the rapidly activating sustained K+ current, which is inhibited by 100 muM 4-aminopyridine (4-AP); this current was almost three times larger in the left atrium than in the right atrium. Accordingly, 100 muM 4-AP caused a significantly (P < 0.05) larger increase in action potential duration in left than in right atrial myocytes. Inward rectifier K+ current density was also significantly (P < 0.05) larger in left atrial myocytes. There was no difference in the voltage-dependent L-type Ca2+ current between left and right atria. As expected from this voltage-clamp data, the duration of action potentials recorded from single myocytes was significantly (P < 0.05) shorter in myocytes from left atria, and left atrial tissue was found to have a significantly (P < 0.05) shorter effective refractory period than right atrial tissue. These results reveal similarities between mice and other mammalian species where the left atrium repolarizes more quickly than the right, and provide new insight into cellular electrophysiological mechanisms responsible for this difference. These findings, and previous results, suggest that the atria of adult mice may be a suitable model for detailed studies of atrial electrophysiology and pharmacology under control conditions and in the context of induced atrial rhythm disturbances.

Whole cell current and voltage clamp techniques were used to examine the properties of acetylcholine-sensitive K+ current (IKACh) in myocytes from adult mouse atrium. Superfusion of a maximal dose of carbachol (CCh; 10 microM) caused a substantial increase in K+ current in all myocytes examined. The current-voltage (I-V) relation of maximally activated IKACh exhibited weak inward rectification. Consequently, CCh increased the amount of depolarising current necessary to evoke action potentials (APs), and APs evoked in CCh had significantly shorter durations than control APs (P<0.05). The effects of CCh on K+ current and on AP properties were blocked by the muscarinic receptor antagonist methoctramine (1 microM). ACh (10 microM) activated a K+ current with identical properties to that activated by CCh, as did the A1 receptor agonist adenosine (100 microM). Right atrial myocytes had significantly more IKACh than left atrial myocytes (P<0.05), regardless of whether IKACh was evoked by superfusion of muscarinic or A1 receptor agonists. IKACh current density was significantly higher in SA node myocytes than either right or left atrial myocytes. These data identify a gradient of IKACh current density across the supraventricular structures of mouse heart. This gradient, combined with the heterogeneous distribution of parasympathetic innervation of the atria, may contribute to the proarrhythmic ability of vagal nerve stimulation to augment dispersion of atrial refractoriness.