Action Potential Discharge Research Articles

Induction of hypertension blunts baroreflex inhibition of vasopressin neurons in the rat.

Vasopressin secretion from the posterior pituitary gland is determined by action potential discharge of hypothalamic magnocellular neurosecretory cells. Vasopressin is a potent vasoconstrictor, but vasopressin levels are paradoxically elevated in some patients with established hypertension. To determine whether vasopressin neurons are excited in hypertension, extracellular single-unit recordings of vasopressin neurons from urethane-anaesthetized Cyp1a1-Ren2 rats with inducible angiotensin-dependent hypertension were made. The basal firing rate of vasopressin neurons was higher in hypertensive Cyp1a1-Ren2 rats than in non-hypertensive Cyp1a1-Ren2 rats. The increase in firing rate was specific to vasopressin neurons because oxytocin neuron firing rate was unaffected by the induction of hypertension. Intravenous injection of the α1-adrenoreceptor agonist, phenylephrine (2.5 μg/kg), transiently increased mean arterial blood pressure to cause a baroreflex-induced inhibition of heart rate and vasopressin neuron firing rate (by 52 ± 9%) in non-hypertensive rats. By contrast, intravenous phenylephrine did not inhibit vasopressin neurons in hypertensive rats, despite a similar increase in mean arterial blood pressure and inhibition of heart rate. Circulating angiotensin II can excite vasopressin neurons via activation of afferent inputs from the subfornical organ. However, the increase in vasopressin neuron firing rate and the loss of inhibition by intravenous phenylephrine were not blocked by intra-subfornical organ infusion of the angiotensin AT1 receptor antagonist, losartan. It can be concluded that increased vasopressin neuron activity at the onset of hypertension is driven, at least in part, by reduced baroreflex inhibition of vasopressin neurons and that this might exacerbate the increase in blood pressure at the onset of hypertension.

Neural processing of novel sounds in the rat’s inferior colliculus

A novel sound is an occasionally occurring acoustic event in a constant/repetitive acoustic environment. Auditory neurons display sensitivity to a novel sound by reducing action potential discharges over repetitive acoustic stimulation and restoring discharges upon presence of a novel sound. We used the rat as an animal model and conducted in vivo neurophysiological recordings to examine how novel sounds are processed in the midbrain auditory structure, the inferior colliculus (IC). The IC receives convergent inputs from structures including those with neurons sensitive to novel sounds. These inputs have diverse sensitivities to temporal, spectral, and directional acoustic cues and produce excitatory/inhibitory synaptic events with different time courses in IC neurons. Integration among these inputs in IC neurons provides a likely physiological basis for processing the novelty of a sound and other dynamic acoustic characteristics. We used both closed- and free-field stimuli to examine responses to oddball paradigms. Our results indicate that neurons sensitive to novel sounds existed in the IC. These neurons typically have onset firing patterns; and their sensitivity to a novel sound is affected by the spatial relationship between the novel sound and a repetitive standard sound in the environment. [Research supported by NSERC of Canada.]

Hydrogen sulfide induces hyperpolarization and decreases the exocytosis of secretory granules of rat GH3 pituitary tumor cells

The aim of the present study was to evaluate the effects of hydrogen sulfide (H2S) on the membrane potential, action potential discharge and exocytosis of secretory granules in neurosecretory pituitary tumor cells (GH3). The H2S donor – sodium hydrosulfide (NaHS) induced membrane hyperpolarization, followed by truncation of spontaneous electrical activity and decrease of the membrane resistance. The NaHS effect was dose-dependent with an EC50 of 152 μM (equals effective H2S of 16–19 μM). NaHS effects were not altered after inhibition of maxi conductance calcium-activated potassium (BK) channels by tetraethylammonium or paxilline, but were significantly reduced after inhibition or activation of ATP-dependent potassium channels (KATP) by glibenclamide or by diazoxide, respectively. In whole-cell recordings NaHS increased the amplitude of KATP currents, induced by hyperpolarizing pulses and subsequent application of glibenclamide decreased currents to control levels. Using the fluorescent dye FM 1–43 exocytosis of secretory granules was analyzed in basal and stimulated conditions (high K+ external solution). Prior application of NaHS decreased the fluorescence of the cell membrane in both conditions which links with activation of KATP currents (basal secretion) and activation of KATP currents and BK-currents (stimulated exocytosis). We suggest that H2S induces hyperpolarization of GH3 cells by activation of KATP channels which results in a truncation of spontaneous action potentials and a decrease of hormone release.

Functional heterogeneity of calretinin-expressing neurons in the mouse superficial dorsal horn: implications for spinal pain processing.

The superficial spinal dorsal horn contains a heterogeneous population of neurons that process sensory inputs. Information on the properties of excitatory interneurons in this region is limited. As calretinin is a protein thought to be restricted to an excitatory population in this region, the aim of this study was to characterize calretinin-expressing neurons. Most calretinin cells (85%) exhibited large A-type potassium currents and delayed firing action potential discharge, and received strong excitatory synaptic input, whereas the remainder exhibited hyperpolarization-activated cation currents and low threshold T-type calcium currents, and tonic- or initial bursting firing patterns, and received weak excitatory synaptic input. These respective features are consistent with properties of excitatory and inhibitory interneuron populations in this region of the spinal cord. Our findings have resolved a previously unidentified population of inhibitory interneurons. Furthermore, the contrasting excitability patterns of excitatory and inhibitory calretinin-expressing neurons suggest that they play distinct roles in spinal sensory processing circuits. Neurons in the superficial dorsal horn (SDH) of the spinal cord play an important role in nociceptive, thermal, itch and light touch sensations. Excitatory interneurons comprise ∼65% of all SDH neurons but surprisingly few studies have investigated their role in spinal sensory processing. Here we use a transgenic mouse to study putative excitatory SDH neurons that express the calcium binding protein calretinin (CR). Our immunocytochemical, morphological and electrophysiological analysis identified two distinct populations of CR-expressing neurons, which we termed 'Typical' and 'Atypical'. Typical CR-expressing neurons comprised ∼85% of the population and exhibited characteristic excitatory interneuron properties including delayed firing discharge, large rapid A-type potassium currents, and central, radial or vertical cell morphologies. Atypical neurons exhibited properties consistent with inhibitory interneurons, including tonic firing or initial bursting discharge, Ih currents, and islet cell morphology. Although both Typical and Atypical CR-expressing neurons responded to noxious peripheral stimulation, the excitatory drive onto Typical CR-expressing neurons was much stronger. Furthermore, Atypical CR-expressing cells comprise at least two functionally distinct subpopulations based on their responsiveness to noxious peripheral stimulation and neurochemical profile. Together our data suggest CR expression is not restricted to excitatory neurons in the SDH. Under normal conditions, the contribution of 'Typical' excitatory CR-expressing neurons to overall SDH excitability may be limited by the presence of A-type potassium currents, which limit the effectiveness of their strong excitatory input. Their contribution may, however, be increased in pathological situations where A-type potassium currents are decreased. By contrast, 'Atypical' inhibitory neurons with their excitable phenotype but weak excitatory input may be more easily recruited during increased peripheral stimulation.

Oxidative Stress and Maxi Calcium-Activated Potassium (BK) Channels.

All cells contain ion channels in their outer (plasma) and inner (organelle) membranes. Ion channels, similar to other proteins, are targets of oxidative impact, which modulates ion fluxes across membranes. Subsequently, these ion currents affect electrical excitability, such as action potential discharge (in neurons, muscle, and receptor cells), alteration of the membrane resting potential, synaptic transmission, hormone secretion, muscle contraction or coordination of the cell cycle. In this chapter we summarize effects of oxidative stress and redox mechanisms on some ion channels, in particular on maxi calcium-activated potassium (BK) channels which play an outstanding role in a plethora of physiological and pathophysiological functions in almost all cells and tissues. We first elaborate on some general features of ion channel structure and function and then summarize effects of oxidative alterations of ion channels and their functional consequences.

Isoflurane causes concentration-dependent inhibition of medullary raphé 5-HT neurons in situ.

Anesthetics have a profound influence on a myriad of autonomic processes. Mechanisms of general anesthesia, and how these mechanisms give rise to the multifaceted state of anesthesia, are largely unknown. The ascending and descending serotonin (5-HT) networks are key modulators of autonomic pathways, and are critically involved in homeostatic reflexes across the motor, somatosensory, limbic and autonomic systems. These 5-HT networks are thought to contribute to anesthetic effects, but how anesthetics affect 5-HT neuron function remains a pertinent question. We hypothesized that the volatile anesthetic isoflurane inhibits action potential discharge of medullary raphé 5-HT neurons. We conducted extracellular recordings on individual neurons in the medullary raphé region of the unanesthetized in situ perfused brainstem preparation to determine how exposure to isoflurane affects 5-HT neurons. We examined changes in 5-HT neuron baseline firing in response to treatment with either 1, 1.5, or 2% isoflurane. We measured isoflurane concentrations by gas chromatography-mass spectrometry (GC-MS) analysis. Exposure to isoflurane inhibited action potential discharge in raphé 5-HT neurons. We document a concentration-dependent inhibition over a range of concentrations approximating isoflurane MAC (minimum alveolar concentration required for surgical anesthesia). Delivered concentrations of isoflurane were confirmed using GC-MS analysis. These findings illustrate that halogenated anesthetics greatly affect 5-HT neuron firing and suggest 5-HT neuron contributions to mechanisms of general anesthesia.

Inappropriate interpretation of surface EMG signals and muscle fiber characteristics impedes understanding of the control of neuromuscular function

as the final common pathway from the nervous system to muscle, the motor unit transmits an activation signal generated by the nervous system to engage the contractile proteins and produce the muscle forces needed for reflex responses, automatic behaviors, and voluntary actions ([11][1]). The net

Self-regulation of adult thalamocortical neurons.

The thalamus acts as a conduit for sensory and other information traveling to the cortex. In response to continuous sensory stimulation in vivo, the firing rate of thalamocortical neurons initially increases, but then within a minute firing rate decreases and T-type Ca(2+) channel-dependent action potential burst firing emerges. While neuromodulatory systems could play a role in this inhibitory response, we instead report a novel and cell-autonomous inhibitory mechanism intrinsic to the thalamic relay neuron. Direct intracellular stimulation of thalamocortical neuron firing initially triggered a continuous and high rate of action potential discharge, but within a minute membrane potential (Vm) was hyperpolarized and firing rate to the same stimulus was decreased. This self-inhibition was observed across a wide variety of thalamic nuclei, and in a subset firing mode switched from tonic to bursting. The self-inhibition resisted blockers of intracellular Ca(2+) signaling, Na(+)-K(+)-ATPases, and G protein-regulated inward rectifier (GIRK) channels as implicated in other neuron subtypes, but instead was in part inhibited by an ATP-sensitive K(+) channel blocker. The results identify a new homeostatic mechanism within the thalamus capable of gating excitatory signals at the single-cell level.

Effect Of Neuromuscular Electrical Stimulation On Contralateral Motor Unit Activity In Biceps Brachii

Neuromuscular electrical stimulation (NMES) elicits action potentials in intramuscular branches of peripheral nerves. Because the time constant for the strength-duration relation is longer for sensory axons than motor axons, narrow pulse widths (0.2-0.4 ms) preferentially activate motor axons, whereas wider pulse widths (0.5-1 ms) recruit relatively more sensory axons. PURPOSE: The purpose of the study was to determine the influence of NMES pulse width and frequency on the modulation of motor unit (MU) activity in a contralateral limb. The hypothesis was that wide-pulse NMES would cause greater modulation of MU activity due to augmented activation of sensory axons. METHODS: The discharge of action potentials by MUs in the left biceps brachii of young adults (18-35 yrs) was recorded with subcutaneous fine-wire electrodes. The protocol involved subjects performing submaximal isometric contractions to maintain the discharge of an isolated MU. In the middle 5 s of each contraction, NMES was applied to the right biceps brachii with either of two pulse durations (0.2 or 1 ms, each at 40 Hz) and three current intensities (80% motor threshold, 10%, or 20% of maximal voluntary contraction (MVC) force). In an additional group of 7 MUs (recruited between 4-22% MVC force), NMES was applied at both pulse widths and two stimulus frequencies (40 Hz and 100 Hz). The primary outcome was the influence of NMES on the discharge characteristics (mean and coefficient of variation (CV) of interspike interval (ISI)) of the isolated MUs before, during, and after stimulation. RESULTS: Forty-four MUs were discriminated, 16 of which had high recruitment thresholds (≥30% MVC force). Neither narrow- nor wide-pulse NMES elicited a significant change in mean ISI or CV for ISI. For example, mean ISI during an NMES-evoked contraction at 10% MVC force was 11.4 ms, 12.3 ms, and 11.8 ms before, during, and after stimulation (respectively) for narrow pulses and 11.4 ms, 12.3 ms, and 12.0 ms for wide pulses. Similarly, CV for ISI was 20.3%, 20.2%, and 21.2% for narrow pulses and 19.4%, 18.7%, and 19.9% for wide pulses. Moreover, stimulus frequency did not influence the discharge characteristics of the isolated MUs. CONCLUSION: NMES applied to the right biceps brachii did not influence the discharge characteristics of motor units in the left biceps brachii during brief voluntary contractions.

OVLT Neurons Exhibit Exquisite Osmosensitivity to Elevated NaCl Concentrations

The brain responds to changes in osmolarity through specialized osmosensitive neurons of the organum vasculosum of the lamina terminalis (OVLT). However, prior studies assessed OVLT neuronal responses to supraphysiologic changes in osmolarity (~5 ‐ 15%). Therefore, this study aimed to establish that OVLT neurons are sensitive to physiologic 5 ‐ 10mOsm elevations (~1.5 ‐ 3.5%) in bath NaCl using whole‐cell recordings of adult OVLT neurons in slices. We measured changes in action potential (AP) discharge frequency to 5 mOsm increases in NaCl. 56% of OVLT neurons (9/16) were osmosensitive as defined by a 25% increase in discharge frequency. Osmosensitive neurons' (n = 9) peak AP discharge increased significantly from a 295 mOsm baseline (0.88±0.25 Hz) by 154% to 300 mOsm (1.66±0.33 Hz; p < 0.05) and by 252% to 305 mOsm (1.97±0.34 Hz; p < 0.05) stimuli. Conversely, non‐osmosensitive neurons' (n = 7) peak AP discharge did not significantly change from a 295 mOsm baseline (0.51±0.16 Hz) to 300 mOsm (0.73±0.24 Hz) and 305 mOsm (0.57±0.22 Hz) stimuli (p > 0.05). Linear regression analysis showed that osmosensitive neurons exhibited concentration‐dependent increases in discharge frequency to hyperosmotic challenges (r2 = 0.284), but non‐osmosensitive neurons did not (r2 = 0.00374). Still, osmosensitive and non‐osmosensitive neurons did not differ with regard to input resistance, AP threshold, AP duration, afterhyperpolarization magnitude or repolarization rate. These data demonstrate, for the first time, that OVLT neurons exhibit exquisite sensitivity to 1‐3% increases in osmolarity, and may initiate homeostasis to physiologic elevations in circulating sodium.

202 Omeprazole and Fluticasone Inhibit IL-13-Stimulated Eotaxin-3 Expression by Esophageal Epithelial Cells Through Different Mechanisms and With Additive Effects: Rationale for Combining PPIs With Topical Steroids for EoE Patients

Introduction: Eosinophilic esophagitis (EoE) often presents with symptoms that are considered to relate to esophageal dysfunctions. Our previous study demonstrated that TRPA1 plays important role in tissue mast cell activation-induced increase in the excitability of esophageal vagal nodose C fibers. The present study aims to determine whether prolonged antigen exposure in vivo sensitizes TRPA1 in esophageal vagal afferents in a guinea pig model of EoE. Method: Antigen challenge-induced responses in esophageal mucosa were first studied by histological assessments and Ussing chamber methods. TRPA1 functions in vagal sensory neurons were then studied by calcium imaging and by whole-cell patch clamp recordings in DiI-labeled esophageal nodose and jugular neurons. Extracellular single-fiber recordings were performed in nodose and jugular C-fiber neurons using ex vivo esophagealvagal preparations with intact nerve endings in the esophagus. Results: (1) Prolonged antigen challenge (0.1% Ovalbumin, 30-second/day for 2-week) in antigen-sensitized guinea pigs increased inflammation scores from 2.0±0.47 (naive) to 4.33±0.27 (OVA-2w) in the esophagus (p<0.05). OVA challenge also increased the infiltrations of both mast cells (from 8.2±2.1 to 63.5±4.5 in mucosa, and from 14.0±2.4 to 43.1±4.5 in muscle layers, both p<0.05) and eosinophils (from 2.6±0.9 to 63.5±20.0 in mucosa, and from 0 to 5.2±2.55/ in muscle layers, both p<0.05) in the cross-sections of the esophagus. (2) Antigen challenge decreased the transepithelial resistance in esophageal epithelium from 564.7±63.4 V·cm2 to 356.0±45.5V·cm2 (p<0.05). (3) Antigen challenge increased TRPA1 agonist AITC-induced calcium influx in both nodose (from 43.5% to 66.7%) and jugular neurons (from 38.9% to 51.9%)(both p<0.05). (4) Antigen challenge increased current density elicited by TRPA1 agonist AITC in DiI-labeled esophageal nodose neurons (naive vs OVA-2w: 24.3±5.4 vs 59.7 ±4.7 pA/pF, p<0.05) and jugular neurons (naive vs OVA-2w: 31.5±5.3 vs 65.8 ±6.2 pA/pF, p<0.05). (5) In naive animals, intra-esophageal infusion of TRPA1 agonist AITC did not evoke action potential discharge in either esophageal nodose or jugular C fibers. After OVA challenge, intra-esophageal infusion of AITC was able to evoke action potential discharges in both nodose C fibers (baseline vs AITC: 0.75±0.25 Hz vs 4.63±1.03 Hz, p<0.01, n=8) and Jugular C fibers (baseline vs AITC: 0.88±0.25 Hz vs 2.5±0.38Hz, p<0.01, n=8). Conclusion: These results demonstrated that prolonged antigen challenge induced allergic inflammation, decreased epithelial barrier resistance, and sensitized TRPA1 in vagal nociceptive neurons and afferent C fibers in the esophagus. These changes enabled intra-esophageal noxious chemical to activate esophageal nociceptor. This novel finding may help to better understand esophageal dysfunction in EoE.

201 Claudin-7 Dysregulation in Pediatric Eosinophilic Esophagitis: A Role for TGF-β in Esophageal Epithelial Barrier Dysfunction

Introduction: Eosinophilic esophagitis (EoE) often presents with symptoms that are considered to relate to esophageal dysfunctions. Our previous study demonstrated that TRPA1 plays important role in tissue mast cell activation-induced increase in the excitability of esophageal vagal nodose C fibers. The present study aims to determine whether prolonged antigen exposure in vivo sensitizes TRPA1 in esophageal vagal afferents in a guinea pig model of EoE. Method: Antigen challenge-induced responses in esophageal mucosa were first studied by histological assessments and Ussing chamber methods. TRPA1 functions in vagal sensory neurons were then studied by calcium imaging and by whole-cell patch clamp recordings in DiI-labeled esophageal nodose and jugular neurons. Extracellular single-fiber recordings were performed in nodose and jugular C-fiber neurons using ex vivo esophagealvagal preparations with intact nerve endings in the esophagus. Results: (1) Prolonged antigen challenge (0.1% Ovalbumin, 30-second/day for 2-week) in antigen-sensitized guinea pigs increased inflammation scores from 2.0±0.47 (naive) to 4.33±0.27 (OVA-2w) in the esophagus (p<0.05). OVA challenge also increased the infiltrations of both mast cells (from 8.2±2.1 to 63.5±4.5 in mucosa, and from 14.0±2.4 to 43.1±4.5 in muscle layers, both p<0.05) and eosinophils (from 2.6±0.9 to 63.5±20.0 in mucosa, and from 0 to 5.2±2.55/ in muscle layers, both p<0.05) in the cross-sections of the esophagus. (2) Antigen challenge decreased the transepithelial resistance in esophageal epithelium from 564.7±63.4 V·cm2 to 356.0±45.5V·cm2 (p<0.05). (3) Antigen challenge increased TRPA1 agonist AITC-induced calcium influx in both nodose (from 43.5% to 66.7%) and jugular neurons (from 38.9% to 51.9%)(both p<0.05). (4) Antigen challenge increased current density elicited by TRPA1 agonist AITC in DiI-labeled esophageal nodose neurons (naive vs OVA-2w: 24.3±5.4 vs 59.7 ±4.7 pA/pF, p<0.05) and jugular neurons (naive vs OVA-2w: 31.5±5.3 vs 65.8 ±6.2 pA/pF, p<0.05). (5) In naive animals, intra-esophageal infusion of TRPA1 agonist AITC did not evoke action potential discharge in either esophageal nodose or jugular C fibers. After OVA challenge, intra-esophageal infusion of AITC was able to evoke action potential discharges in both nodose C fibers (baseline vs AITC: 0.75±0.25 Hz vs 4.63±1.03 Hz, p<0.01, n=8) and Jugular C fibers (baseline vs AITC: 0.88±0.25 Hz vs 2.5±0.38Hz, p<0.01, n=8). Conclusion: These results demonstrated that prolonged antigen challenge induced allergic inflammation, decreased epithelial barrier resistance, and sensitized TRPA1 in vagal nociceptive neurons and afferent C fibers in the esophagus. These changes enabled intra-esophageal noxious chemical to activate esophageal nociceptor. This novel finding may help to better understand esophageal dysfunction in EoE.

Vagotomy reverses established allergen-induced airway hyperreactivity to methacholine in the mouse

We evaluated the role of vagal reflexes in a mouse model of allergen-induced airway hyperreactivity. Mice were actively sensitized to ovalbumin then exposed to the allergen via inhalation. Prior to ovalbumin inhalation, mice also received intratracheally-instilled particulate matter in order to boost the allergic response. In control mice, methacholine (i.v.) caused a dose-dependent increase in respiratory tract resistance (RT) that only modestly decreased if the vagi were severed bilaterally just prior to the methacholine challenge. Sensitized and challenged mice, however, manifested an airway reactivity increase that was abolished by severing the vagi prior to methacholine challenge. In an innervated ex vivo mouse lung model, methacholine selectively evoked action potential discharge in a subset of distension-sensitive A-fibers. These data support the hypothesis that the major component of the increased airway reactivity in inflamed mice is due to a vagal reflex initiated by activation of afferent fibers, even in response to a direct (i.e., smooth muscle)-acting muscarinic agonist.

Hydrogen Peroxide (H 2 O 2 ) Augments Intracellular Ca ++ via Voltage‐Gated Ca ++ Channels in Isolated Nucleus Tractus Solitarii (nTS) Neurons

Central neuronal activity and its modulation via 2nd messengers within the nTS is fundamental for cardiorespiratory control. We have previously shown that H2O2 in the nTS augments action potential discharge. Here we examined the role of H2O2 on Ca++ homeostasis in primary nTS cultures using the ratiometric dye Fura 2-AM. H2O2 dose dependently (100-500µM H2O2, 1 min) augmented intracellular Ca++. The majority of nTS cells (79% of 24 cells) responded to 200µM H2O2 with an increase in cytosolic Ca++ of 16.0 ±2.8% (p蠄0.01 vs. vehicle). To identify the source for the Ca++ rise we tested 200µM H2O2 in the presence of Thapsigargin (TG; 1µM) to deplete internal Ca++ stores. In the presence of TG, there was a reduction in the number of H2O2 responding cells (45% of 46 cells), yet those that did respond increased cytosolic Ca++ by 72.5 ±21.7% (p蠄0.05 vs. vehicle & 200µM H2O2 alone). Removing extracellular Ca++ (0 Ca++ with 1mM EDTA) strongly decreased the number of H2O2 responders (10% of 23) as well as the augmentation of Ca++ in the remaining responders. To examine the potential Ca++ channel responsible we included specific Ca++ channel blockers during H2O2. The number of responders to H2O2 was reduced to 40%, 40%, 34% and 20% by blocking L-type (5µM Nifedipine), N-Type (0.1µM ω-conotoxin GVIA), T-type (5µM Mibefradil) and P/Q/N-type (ω-conotoxin MVIIIC) Ca++ channels, respectively (N=20-28). Altogether, elevations in cytosolic Ca++ in nTS cells by H2O2 likely occurs through several available Ca++ channels that contribute to the cellular hyperexcitability. Supported by: RO1 HL098602, AHA 12POST11670002

203 A STAT6 Inhibitor (AS1517499) and a Tyrosine Kinase Inhibitor (Leflunomide) Block IL-13/STAT6 Signaling and Eotaxin-3 Expression in Esophageal Fibroblasts and Epithelial Cells: Promising Agents to Improve Inflammation and Prevent Fibrostenosis in EoE

Introduction: Eosinophilic esophagitis (EoE) often presents with symptoms that are considered to relate to esophageal dysfunctions. Our previous study demonstrated that TRPA1 plays important role in tissue mast cell activation-induced increase in the excitability of esophageal vagal nodose C fibers. The present study aims to determine whether prolonged antigen exposure in vivo sensitizes TRPA1 in esophageal vagal afferents in a guinea pig model of EoE. Method: Antigen challenge-induced responses in esophageal mucosa were first studied by histological assessments and Ussing chamber methods. TRPA1 functions in vagal sensory neurons were then studied by calcium imaging and by whole-cell patch clamp recordings in DiI-labeled esophageal nodose and jugular neurons. Extracellular single-fiber recordings were performed in nodose and jugular C-fiber neurons using ex vivo esophagealvagal preparations with intact nerve endings in the esophagus. Results: (1) Prolonged antigen challenge (0.1% Ovalbumin, 30-second/day for 2-week) in antigen-sensitized guinea pigs increased inflammation scores from 2.0±0.47 (naive) to 4.33±0.27 (OVA-2w) in the esophagus (p<0.05). OVA challenge also increased the infiltrations of both mast cells (from 8.2±2.1 to 63.5±4.5 in mucosa, and from 14.0±2.4 to 43.1±4.5 in muscle layers, both p<0.05) and eosinophils (from 2.6±0.9 to 63.5±20.0 in mucosa, and from 0 to 5.2±2.55/ in muscle layers, both p<0.05) in the cross-sections of the esophagus. (2) Antigen challenge decreased the transepithelial resistance in esophageal epithelium from 564.7±63.4 V·cm2 to 356.0±45.5V·cm2 (p<0.05). (3) Antigen challenge increased TRPA1 agonist AITC-induced calcium influx in both nodose (from 43.5% to 66.7%) and jugular neurons (from 38.9% to 51.9%)(both p<0.05). (4) Antigen challenge increased current density elicited by TRPA1 agonist AITC in DiI-labeled esophageal nodose neurons (naive vs OVA-2w: 24.3±5.4 vs 59.7 ±4.7 pA/pF, p<0.05) and jugular neurons (naive vs OVA-2w: 31.5±5.3 vs 65.8 ±6.2 pA/pF, p<0.05). (5) In naive animals, intra-esophageal infusion of TRPA1 agonist AITC did not evoke action potential discharge in either esophageal nodose or jugular C fibers. After OVA challenge, intra-esophageal infusion of AITC was able to evoke action potential discharges in both nodose C fibers (baseline vs AITC: 0.75±0.25 Hz vs 4.63±1.03 Hz, p<0.01, n=8) and Jugular C fibers (baseline vs AITC: 0.88±0.25 Hz vs 2.5±0.38Hz, p<0.01, n=8). Conclusion: These results demonstrated that prolonged antigen challenge induced allergic inflammation, decreased epithelial barrier resistance, and sensitized TRPA1 in vagal nociceptive neurons and afferent C fibers in the esophagus. These changes enabled intra-esophageal noxious chemical to activate esophageal nociceptor. This novel finding may help to better understand esophageal dysfunction in EoE.

Excitatory Amino Acid Transporters (EAATs) Modulate Synaptic Properties of nTS Cardiorespiratory Neurons

Glutamate (Glu) is the primary excitatory neurotransmitter in the nTS, the central termination and integration site for visceral afferent reflexes. Extracellular Glu is tightly regulated via astrocytic EAATs that fine tune synaptic signaling and prevent excitotoxicity. The role of EAATs in the nTS is relatively unknown. Our goal was to identify the expression, role and mechanism by which nTS astrocyte EAATs influence synaptic and neuronal activity. Astrocytes were pre-labeled with SR-101. nTS neurons and astrocytes were recorded in acute slices by patch clamp. Immunohistochemistry showed EAAT positive astrocytes tightly associated with nTS neurons and glutamatergic synaptic terminals. TBOA (general EAAT antagonist) and DHK (EAAT2 blocker) dose-dependently induced inward neuronal currents that were attenuated by AP-5 (NMDA receptor blocker). TBOA reduced spontaneous EPSC amplitude and frequency, whereas DHK only reduced amplitude suggesting differential roles for EAATs. DHK significantly decreased neuronal input resistance, and tended to depolarize membrane potential and enhance action potential discharge. DHK did not alter solitary tract evoked current amplitude but significantly increased EPSC decay time. Compared to nTS neurons, astrocytes had a hyperpolarized membrane potential (-53±4 vs -72±2mV), and their activation by 500µM D-Aspartate induced an inward current that was attenuated by DHK. Microinjection of DHK in vivo decreased blood pressure and sympathetic nerve activity. These data suggest astrocyte EAATs tonically uptake Glu to modulate synaptic transmission and neuronal activity, which influence cardiorespiratory regulation. (HL085108, HL098602)

200 Allergen Challenge Sensitizes TRPA1 in Vagal Sensory Neurons and Afferent C-Fibers in Guinea Pig Esophagus

Introduction: Eosinophilic esophagitis (EoE) often presents with symptoms that are considered to relate to esophageal dysfunctions. Our previous study demonstrated that TRPA1 plays important role in tissue mast cell activation-induced increase in the excitability of esophageal vagal nodose C fibers. The present study aims to determine whether prolonged antigen exposure in vivo sensitizes TRPA1 in esophageal vagal afferents in a guinea pig model of EoE. Method: Antigen challenge-induced responses in esophageal mucosa were first studied by histological assessments and Ussing chamber methods. TRPA1 functions in vagal sensory neurons were then studied by calcium imaging and by whole-cell patch clamp recordings in DiI-labeled esophageal nodose and jugular neurons. Extracellular single-fiber recordings were performed in nodose and jugular C-fiber neurons using ex vivo esophagealvagal preparations with intact nerve endings in the esophagus. Results: (1) Prolonged antigen challenge (0.1% Ovalbumin, 30-second/day for 2-week) in antigen-sensitized guinea pigs increased inflammation scores from 2.0±0.47 (naive) to 4.33±0.27 (OVA-2w) in the esophagus (p<0.05). OVA challenge also increased the infiltrations of both mast cells (from 8.2±2.1 to 63.5±4.5 in mucosa, and from 14.0±2.4 to 43.1±4.5 in muscle layers, both p<0.05) and eosinophils (from 2.6±0.9 to 63.5±20.0 in mucosa, and from 0 to 5.2±2.55/ in muscle layers, both p<0.05) in the cross-sections of the esophagus. (2) Antigen challenge decreased the transepithelial resistance in esophageal epithelium from 564.7±63.4 V·cm2 to 356.0±45.5V·cm2 (p<0.05). (3) Antigen challenge increased TRPA1 agonist AITC-induced calcium influx in both nodose (from 43.5% to 66.7%) and jugular neurons (from 38.9% to 51.9%)(both p<0.05). (4) Antigen challenge increased current density elicited by TRPA1 agonist AITC in DiI-labeled esophageal nodose neurons (naive vs OVA-2w: 24.3±5.4 vs 59.7 ±4.7 pA/pF, p<0.05) and jugular neurons (naive vs OVA-2w: 31.5±5.3 vs 65.8 ±6.2 pA/pF, p<0.05). (5) In naive animals, intra-esophageal infusion of TRPA1 agonist AITC did not evoke action potential discharge in either esophageal nodose or jugular C fibers. After OVA challenge, intra-esophageal infusion of AITC was able to evoke action potential discharges in both nodose C fibers (baseline vs AITC: 0.75±0.25 Hz vs 4.63±1.03 Hz, p<0.01, n=8) and Jugular C fibers (baseline vs AITC: 0.88±0.25 Hz vs 2.5±0.38Hz, p<0.01, n=8). Conclusion: These results demonstrated that prolonged antigen challenge induced allergic inflammation, decreased epithelial barrier resistance, and sensitized TRPA1 in vagal nociceptive neurons and afferent C fibers in the esophagus. These changes enabled intra-esophageal noxious chemical to activate esophageal nociceptor. This novel finding may help to better understand esophageal dysfunction in EoE.

Enrichment of human embryonic stem cell derived motor neuron cultures using arabinofuranosyl cytidine

ABSTRACT Aim: Human embryonic stem cell (hESC)-derived motor neurons (MNs) can model neurodegenerative diseases, in other words, for the development of cell-based drug screening assays. However, efficient MN generation is challenging and high level of contamination of undifferentiated cells remains a significant problem when culturing hESC-derived MNs. We describe a protocol for the removal of undifferentiated cells using the DNA synthesis inhibitor arabinofuranosyl cytidine (Ara-C). Materials &amp; methods: hESC-MNs were treated with Ara-C after the last step of differentiation. FACS analysis and fluorescence microscopy were used to identify and quantify MNs. Results: HB9/ChAT-positive live mature MNs were enriched 3.9-times in Ara-C-treated cultures when compared with untreated cells. The Ara-C-treated MNs are electrophysiologically functional and discharge action potentials. Conclusion: Ara-C selection of MNs can be combined with stem cell differentiation protocols to enrich MNs in culture.

Sympathy for the ganglion.

Sympathy for the ganglion.

Aberrant synaptic integration in adult lamina I projection neurons following neonatal tissue damage.

Mounting evidence suggests that neonatal tissue damage evokes alterations in spinal pain reflexes which persist into adulthood. However, less is known about potential concomitant effects on the transmission of nociceptive information to the brain, as the degree to which early injury modulates synaptic integration and membrane excitability in mature spinal projection neurons remains unclear. Here we demonstrate that neonatal surgical injury leads to a significant shift in the balance between synaptic excitation and inhibition onto identified lamina I projection neurons of the adult mouse spinal cord. The strength of direct primary afferent input to mature spino-parabrachial neurons was enhanced following neonatal tissue damage, whereas the efficacy of both GABAergic and glycinergic inhibition onto the same population was compromised. This was accompanied by reorganization in the pattern of sensory input to adult projection neurons, which included a greater prevalence of monosynaptic input from low-threshold A-fibers when preceded by early tissue damage. In addition, neonatal incision resulted in greater primary afferent-evoked action potential discharge in mature projection neurons. Overall, these results demonstrate that tissue damage during early life causes a long-term increase in the gain of spinal nociceptive circuits, and suggest that the prolonged consequences of neonatal trauma may not be restricted to the spinal cord but rather include excessive ascending signaling to supraspinal pain centers.