Depolarizing Response Research Articles

A possible role for membrane depolarization in epithelial wound healing

Linear narrow wounds produced on cultured bovine corneal endothelial monolayers heal by actin cable formation at the wound border and lamellar crawling of cells into the injured area. We report the novel finding that membrane potential depolarization occurs at the leading edge of wounds and gradually extends inward toward the neighboring cells. We have determined that the replacement of extracellular Na(+) by choline and the incorporation of phenamil, an inhibitor of the epithelial Na(+) channel (ENaC), provoke a decrease in the actin cable and depolarization areas and in the lamellar activity of the wound edges. To the contrary, extracellular Li(+) can successfully replace Na(+) in the determination of the depolarization and cytoskeletal responses. This finding supports the idea that membrane depolarization, not the increase in intracellular Na(+) concentration, is responsible for the formation of the actin cable, a result that is in agreement with previous evidence showing that nonspecific depolarization of the plasma membrane potential (PMP) of epithelial cells may promote characteristic cytoskeletal rearrangements per se (Chifflet S, Hernandez JA, Grasso S, and Cirillo A. Exp Cell Res 282: 1-13, 2003). We suggest that spontaneous depolarization of the PMP of the cells at the wound borders determined by a rise in the ENaC activity of these cells constitutes an additional factor in the intermediate cellular processes leading to wound healing in some epithelia.

Integration Time in a Subset of Spinal Lamina I Neurons Is Lengthened by Sodium and Calcium Currents Acting Synergistically to Prolong Subthreshold Depolarization

Lamina I of the spinal dorsal horn plays an important role in processing and relaying nociceptive information to the brain. It comprises physiologically distinct cell types that process information in fundamentally different ways: tonic neurons fire repetitively during stimulation and display prolonged EPSPs, suggesting operation as integrators, whereas single-spike neurons act like coincidence detectors. Using whole-cell recordings from a rat spinal slice preparation, we set out to determine the basis for prolonged EPSPs in tonic cells and the implications for signal processing. Kinetics of synaptic currents could not explain differences in EPSP kinetics. Instead, tonic neurons were found to express a persistent sodium current, I(Na,P), that amplified and prolonged depolarization in response to brief stimulation. Tonic neurons also expressed a persistent calcium current, I(Ca,P), that contributed to prolongation but not to amplification. Simulations using NEURON software demonstrated that I(Na,P) was necessary and sufficient to explain amplification, whereas I(Na,P) and I(Ca,P) acted synergistically to prolong depolarization: initial activation of the slower current (I(Ca,P)) depended on the faster current (I(Na,P)) but maintained activation of the faster current likewise depended on the slower current. Additional investigation revealed that I(Na,P) and I(Ca,P) could dramatically increase integration time (>30x) and thereby encourage temporal summation but at the expense of spike time precision. Thus, by prolonging subthreshold depolarization, intrinsic inward currents allow tonic neurons in spinal lamina I to specialize as integrators that are optimally suited to encode stimulus intensity.

Postnatal development of carotid body glomus cell O2 sensitivity

In mammals, the main sensors of arterial oxygen level are the carotid chemoreceptors, which exhibit low sensitivity to hypoxia at birth and become more sensitive over the first few days or weeks of life. This postnatal increase in hypoxia sensitivity of the arterial chemoreceptors, termed "resetting", remains poorly understood. In the carotid body, hypoxia is transduced by glomus cells, which are secretory sensory neurons that respond to hypoxia at higher P(O2) levels than non-chemoreceptor cell types. Maturation or resetting of carotid body O2 sensitivity potentially involves numerous aspects of the O2 transduction cascade at the glomus cell level, including glomus cell neurotransmitter secretion, neuromodulator function, neurotransmitter receptor expression, glomus cell depolarization in response to hypoxia, [Ca2+]i responses to hypoxia, K+ and Ca2+ channel O2 sensitivity and K+ channel expression. However, although progress has been made in the understanding of carotid body development, the precise mechanisms underlying postnatal maturation of these numerous aspects of chemotransduction remain obscure.

Sign-conserving amacrine neurons in the fly's external plexiform layer

Amacrine cells in the external plexiform layer of the fly's lamina have been intracellulary recorded and dye-filled for the first time. The recordings demonstrate that like the lamina's short photoreceptors R1-R6, type 1 lamina amacrine neurons exhibit nonspiking, "sign-conserving" sustained depolarizations in response to illumination. This contrasts with the sign-inverting responses that typify first-order retinotopic relay neurons: monopolar cells L1-L5 and the T1 efferent neuron. The contrast frequency tuning of amacrine neurons is similar to that of photoreceptors and large lamina monopolar cells. Initial observations indicate that lamina amacrine receptive fields are also photoreceptor-like, suggesting either that their inputs originate from a small number of neighboring visual sampling units (VSUs), or that locally generated potentials decay rapidly with displacement. Lamina amacrines also respond to motion, and in one recording these responses were selective for the orientation of moving edges. This functional organization corresponds to the anatomy of amacrine cells, in which postsynaptic inputs from several neighboring photoreceptor endings are linked by a network of very thin distal processes. In this way, each VSU can receive convergent inputs from a surround of amacrine processes. This arrangement is well suited for relaying responses to local intensity fluctuations from neighboring VSUs to a central VSU where amacrines are known to be presynaptic to the dendrites of the T1 efferent. The T1 terminal converges at a deeper level with that of the L2 monopolar cell relaying from the same optic cartridge. Thus, the localized spatial responses and receptor-like temporal response properties of amacrines are consistent with possible roles in lateral inhibition, motion processing, or orientation processing.

Sulfur‐containing amino acids block stretch‐dependent K+ channels and nitrergic responses in the murine colon

1. Efforts to determine the role of stretch-dependent K(+) (SDK) channels in enteric inhibitory neural responses in gastrointestinal muscles are difficult due to a lack of blocking drugs for SDK channels. 2. SDK channels are blocked by sulfur-containing amino acids. These compounds reduced the open probability of SDK channels in on and off-cell patches of murine colonic myocytes. L-Methionine was the most selective and had little or no effect on other known K(+) conductances in colonic myocytes. 3. Application of L-cysteine, L-methionine or DL-homocysteine depolarized intact muscles and enhanced spontaneous contractions. D-Stereoisomers of these amino acids were less effective than L-stereoisomers. 4. Pretreatment of muscles with tetrodotoxin, N(W)-nitro-L-arginine or 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one reduced the depolarization responses to these compounds, suggesting that spontaneous neural activity and release of NO tonically activates SDK channels. 5. Nitrergic responses to nerve stimulation were reduced by sulfur-containing amino acids. 6. These data suggest that nitrergic inhibitory junction potentials are mediated, in part, by activation of SDK channels in murine colonic muscles.

Responses of the ciliates Tetrahymena and Paramecium to external ATP and GTP.

The unicellular ciliates Paramecium and Tetrahymena are the simplest eukaryotic cells to show reliable depolarizing responses to micromolar concentrations of external ATP and GTP. Their simplicity allows for combined analysis of swimming behavior, electrophysiology, receptor binding, behavioral mutant and drug screens as well as molecular genetic approaches such as RNAi and gene knockouts experiments. ATP and GTP are depolarizing chemorepellents in both ciliates, producing measurable receptor potentials and Ca2+-based action potentials that are correlated with jerking behaviors called avoiding reactions (AR). GTP also causes repetitive continuous ciliary reversals (CCR) and oscillating plateau depolarizations in Paramecium. Both ciliates show high affinity, saturable external binding of 32P-GTP and 32P-ATP but GTP does not compete for ATP binding and vice versa. Chemosensory adaptation occurs after continued exposure (15 min) to these ligands, producing a loss of external binding and forward swimming. However, cells adapted to ATP still bind and respond to GTP and GTP-adapted cells still bind and respond to ATP. This, combined with pharmacological analyses, suggests that there are two separate receptor systems: A metabotropic ATP receptor pathway and a different, novel GTP receptor pathway. A Paramecium mutant (ginA) lacks the GTP-induced oscillating depolarizations but does show AR in GTP, unveiling isolated GTP-receptor potentials for study. An ecto-ATPase is also present that may be involved in inactivation of ATP and GTP signals. Gene knockout experiments are currently underway to determine the roles of the ecto-ATPase and a putative 7-transmembrane spanning receptor in these responses.

The General Anesthetic Isoflurane Depresses Synaptic Vesicle Exocytosis

General anesthetics have marked effects on synaptic transmission, but the mechanisms of their presynaptic actions are unclear. We used quantitative laser-scanning fluorescence microscopy to analyze the effects of the volatile anesthetic isoflurane on synaptic vesicle cycling in cultured neonatal rat hippocampal neurons monitored using either transfection of a pH-sensitive form of green fluorescent protein fused to the luminal domain of VAMP (vesicle-associated membrane protein), (synapto-pHluorin) or vesicle loading with the fluorescent dye FM 1-43. Isoflurane reversibly inhibited action potential-evoked exocytosis over a range of concentrations, with little effect on vesicle pool size. In contrast, exocytosis evoked by depolarization in response to an elevated extracellular concentration of KCl, which is insensitive to the selective Na+ channel blocker tetrodotoxin, was relatively insensitive to isoflurane. Inhibition of exocytosis by isoflurane was resistant to bicuculline, indicating that this presynaptic effect is not caused by the well known GABA(A) receptor modulation by volatile anesthetics. Depression of exocytosis was mimicked by a reduction in stimulus frequency, suggesting a reduction in action potential initiation, conduction, or coupling to Ca2+ channel activation. There was no evidence for a direct effect on endocytosis. The effects of isoflurane on synaptic transmission are thus caused primarily by inhibition of action potential-evoked synaptic vesicle exocytosis at a site upstream of Ca2+ entry and exocytosis, possibly as a result of Na+ channel blockade and/or K+ channel activation, with the possibility of lesser contributions from Ca2+ channel blockade and/or soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated vesicle fusion.

MHC class II signaling function is regulated during maturation of plasmacytoid dendritic cells

Dendritic cells (DC) play a central role in the immune response, linking innate and adaptative responses to pathogens. Myeloid DC (MDC) produce interleukin-12 in response to bacterial stimuli, whereas plasmacytoid DC (PDC) produce high levels of type I interferon upon viral infection. Human leukocyte antigen (HLA)-DR engagement has been shown to induce apoptosis in various antigen-presenting cells (APC). We now report the consequences of HLA-DR molecule engagement in human PDC, which had thus far not been studied as a result of the difficulty in isolating such cells. HLA-DR engagement on PDC, obtained using a two-step, immunomagnetic separation, led to recruitment of HLA-DR molecules at the site of engagement in mature but not immature PDC. In contrast, relocalization of protein kinase C (PKC) isoenzymes, indicating PKC activation, was observed at the site of HLA-DR engagement and was accompanied by relocalization of a lipid raft marker, the ganglioside M1 staining, in immature and mature PDC. Similar to MDC, HLA-DR-mediated apoptosis was regulated throughout PDC maturation. Freshly isolated PDC were resistant, whereas CD40 ligand-matured PDC were sensitive to HLA-DR-mediated apoptosis. Neither caspase activation nor PKC activation was required for HLA-DR-mediated apoptosis. However, the intrinsic pathway of apoptosis was implicated as mature PDC underwent mitochondrial depolarization in response to HLA-DR engagement. These data provide further arguments for considering HLA-DR-mediated apoptosis as a conserved mechanism of regulating survival of diverse APC and support the ongoing development of humanized ligands for HLA class II molecules as therapeutic tools for use in lymphoproliferative disease.

Two developmental switches in GABAergic signalling: the K+–Cl− cotransporter KCC2 and carbonic anhydrase CAVII

GABAergic signalling has the unique property of 'ionic plasticity', which is based on short-term and long-term changes in the Cl(-) and HCO(3)(-) ion concentrations in the postsynaptic neurones. While short-term ionic plasticity is caused by activity-dependent, channel-mediated anion shifts, long-term ionic plasticity depends on changes in the expression patterns and kinetic regulation of molecules involved in anion homeostasis. During development the efficacy and also the qualitative nature (depolarization/excitation versus hyperpolarization/inhibition) of GABAergic transmission is influenced by the neuronal expression of two key molecules: the chloride-extruding K(+)-Cl(-) cotransporter KCC2, and the cytosolic carbonic anhydrase (CA) isoform CAVII. In rat hippocampal pyramidal neurones, a steep up-regulation of KCC2 accounts for the 'developmental switch', which converts depolarizing and excitatory GABA responses of immature neurones to classical hyperpolarizing inhibition by the end of the second postnatal week. The immature hippocampus generates large-scale network activity, which is abolished in parallel by the up-regulation of KCC2 and the consequent increase in the efficacy of neuronal Cl(-) extrusion. At around postnatal day 12 (P12), an abrupt, steep increase in intrapyramidal CAVII expression takes place, promoting excitatory responses evoked by intense GABAergic activity. This is largely caused by a GABAergic potassium transient resulting in spatially widespread neuronal depolarization and synchronous spike discharges. These facts point to CAVII as a putative target of CA inhibitors that are used as antiepileptic drugs. KCC2 expression in adult rat neurones is down-regulated following epileptiform activity and/or neuronal damage by BDNF/TrkB signalling. The lifetime of membrane-associated KCC2 is very short, in the range of tens of minutes, which makes KCC2 ideally suited for mediating GABAergic ionic plasticity. In addition, factors influencing the trafficking and kinetic modulation of KCC2 as well as activation/deactivation of CAVII are obvious candidates in the ionic modulation of GABAergic responses. The down-regulation of KCC2 under pathophysiological conditions (epilepsy, damage) in mature neurones seems to reflect a 'recapitulation' of early developmental mechanisms, which may be a prerequisite for the re-establishment of connectivity in damaged brain tissue.

Postnatal development of E-4031-sensitive potassium current in rat carotid chemoreceptor cells

The O2 sensitivity of dissociated type I cells from rat carotid body increases with age until approximately 14-16 days. Hypoxia-induced depolarization appears to be mediated by an O2-sensitive K+ current, but other K+ currents may modulate depolarization. We hypothesized that membrane potential may be stabilized in newborn type I cells by human ether-a-go-go-related gene (HERG)-like K+ currents that inhibit hypoxia-induced depolarization and that a decrease in this current with age could underlie, in part, the developmental increase in type I cell depolarization response to hypoxia. In dissociated type I cells from 0- to 1- and 11- to 16-day-old rats, using perforated patch-clamp and 70 mM K+ extracellular solution, we measured repolarization-induced inward K+ tail currents in the absence and presence of E-4031, a specific HERG channel blocker. This allowed isolation of the E-4031-sensitive HERG-like current. E-4031-sensitive peak currents in type I cells from 0- to- 1-day-old rats were 2.5-fold larger than in cells from 11- to 16-day-old rats. E-4031-sensitive current density in newborn type I cells was twofold greater than in cells from 11- to 16-day-old rats. Under current clamp conditions, E-4031 enhanced hypoxia-induced depolarization in type I cells from 0- to- 1-day-old but not 11- to 16-day-old rats. With use of fura 2 to measure intracellular Ca2+, E-4031 increased the cytosolic Ca2+ concentration response to anoxia in cells from 0- to- 1-day-old but not cells from 11- to 16-day-old rats. E-4031-sensitive K+ currents are present in newborn carotid body type I cells and decline with age. These findings are consistent with a role for E-4031-sensitive K+ current, and possibly HERG-like K+ currents, in the type I cell hypoxia response maturation.

Expression of type 1 corticotropin‐releasing factor receptor in the guinea pig enteric nervous system

Reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, electrophysiological recording, and intraneuronal injection of the neuronal tracer biocytin were integrated in a study of the functional expression of corticotropin-releasing factor (CRF) receptors in the guinea pig enteric nervous system. RT-PCR revealed expression of CRF1 receptor mRNA, but not CRF2, in both myenteric and submucosal plexuses. Immunoreactivity for the CRF1 receptor was distributed widely in the myenteric plexus of the stomach and small and large intestine and in the submucosal plexus of the small and large intestine. CRF1 receptor immunoreactivity was coexpressed with calbindin, choline acetyltransferase, and substance P in the myenteric plexus. In the submucosal plexus, CRF1 receptor immunoreactivity was found in neurons that expressed calbindin, substance P, choline acetyltransferase, or neuropeptide Y. Application of CRF evoked slowly activating depolarizing responses associated with elevated excitability in both myenteric and submucosal neurons. Histological analysis of biocytin-filled neurons revealed that both uniaxonal neurons with S-type electrophysiological behavior and neurons with AH-type electrophysiological behavior and Dogiel II morphology responded to CRF. The CRF-evoked depolarizing responses were suppressed by the CRF1/CRF2 receptor antagonist astressin and the selective CRF1 receptor antagonist NBI27914 and were unaffected by the selective CRF2 receptor antagonist antisauvagine-30. The findings support the hypothesis that the CRF1 receptor mediates the excitatory actions of CRF on neurons in the enteric nervous system. Actions on enteric neurons might underlie the neural mechanisms by which stress-related release of CRF in the periphery alters intestinal propulsive motor function, mucosal secretion, and barrier functions.

Supplementation with gliadin‐combined plant superoxide dismutase extract promotes antioxidant defences and protects against oxidative stress

The potential benefits to health of antioxidant enzymes supplied either through dietary intake or supplementation is still a matter of controversy. The development of dietary delivery systems using wheat gliadin biopolymers as a natural carrier represents a new alternative. Combination of antioxidant enzymes with this natural carrier not only delayed their degradation (i.e. the superoxide dismutase, SOD) during the gastrointestinal digestive process, but also promoted, in vivo, the cellular defences by strengthening the antioxidant status. The effects of supplementation for 28 days with a standardized melon SOD extract either combined (Glisodin) or not with gliadin, were evaluated on various oxidative-stress biomarkers. As already described there was no change either in superoxide dismutase, catalase or glutathione peroxidase activities in blood circulation or in the liver following non-protected SOD supplementation. However, animals supplemented with Glisodin showed a significant elevation in circulated antioxidant enzymes activities, correlated with an increased resistance of red blood cells to oxidative stress-induced hemolysis. In the presence of Sin-1, a chemical donor of peroxynitrites, mitochondria from hepatocytes regularly underwent membrane depolarization as the primary biological event of the apoptosis cascade. Hepatocytes isolated from animals supplemented with Glisodin presented a delayed depolarization response and an enhanced resistance to oxidative stress-induced apoptosis. It is concluded that supplementation with gliadin-combined standardized melon SOD extract (Glisodin) promoted the cellular antioxidant status and protected against oxidative stress-induced cell death.

GABA Provides a Swell Route to Calcium

In addition to its role as neurotransmitter, γ-aminobutyric acid (GABA) mediates trophic effects during neuronal development. These long-term actions have been hypothesized to depend on Ca 2+ influx through voltage-dependent Ca 2+ channels, a consequence of activation of GABA A receptors (ligand-gated ion channels that are permeable to Cl − and HCO 3 − ), which can lead to depolarization in developing neurons. Chavas et al. loaded interneurons in cerebellar slices from juvenile rats with a calcium indicator and observed that application of the GABA agonist muscimol elicited a rise in [Ca 2+ ] i , which was greater in dendrites than in the cell body. The authors used cell-attached recordings in which they measured currents through single BK channels to simultaneously evaluate membrane potential and [Ca 2+ ] i , and they observed that a late phase of increased [Ca 2+ ] i did not correlate with membrane depolarization. Moreover, the [Ca 2+ ] i increase was not blocked by cadmium, and the threshold for calcium channel activation was higher than the peak depolarization in response to muscimol. Both changes in membrane potential and changes in [Ca 2+ ] i required HCO 3 − ; the late phase of increased [Ca 2+ ] i was inhibited by ryanodine. Dendrites expanded in size during muscimol application, and hypoosmotic solutions mimicked, whereas hyperosmotic solutions inhibited, the [Ca 2+ ] i response to muscimol. Thus, the authors proposed that the rise in [Ca 2+ ] i that occurs following GABA A receptor activation depends on increased osmotic tension following bicarbonate-dependent influx of Cl − . The sensitivity to ryanodine suggested that this might involve release of Ca 2+ from intracellular stores, linking ion flux through a ligand-gated channel to a metabotropic receptor-like response. J. Chavas, M. E. Forero, T. Collin, I. Llano, A. Marty, Osmotic tension as a possible link between GABA A receptor activation and intracellular calcium elevation. Neuron 44 , 701-713 (2004). [Online Journal]

Pressure-induced smooth muscle cell depolarization in pulmonary arteries from control and chronically hypoxic rats does not cause myogenic vasoconstriction

Chronic obstructive pulmonary diseases, as well as prolonged residence at high altitude, can result in generalized airway hypoxia, eliciting an increase in pulmonary vascular resistance. We hypothesized that a portion of the elevated pulmonary vascular resistance following chronic hypoxia (CH) is due to the development of myogenic tone. Isolated, pressurized small pulmonary arteries from control (barometric pressure congruent with 630 Torr) and CH (4 wk, barometric pressure = 380 Torr) rats were loaded with fura 2-AM and perfused with warm (37 degrees C), aerated (21% O(2)-6% CO(2)-balance N(2)) physiological saline solution. Vascular smooth muscle (VSM) intracellular Ca(2+) concentration ([Ca(2+)](i)) and diameter responses to increasing intraluminal pressure were determined. Diameter and VSM cell [Ca(2+)](i) responses to KCl were also determined. In a separate set of experiments, VSM cell membrane potential responses to increasing luminal pressure were determined in arteries from control and CH rats. VSM cell membrane potential in arteries from CH animals was depolarized relative to control at each pressure step. VSM cells from both groups exhibited a further depolarization in response to step increases in intraluminal pressure. However, arteries from both control and CH rats distended passively to increasing intraluminal pressure, and VSM cell [Ca(2+)](i) was not affected. KCl elicited a dose-dependent vasoconstriction that was nearly identical between control and CH groups. Whereas KCl administration resulted in a dose-dependent increase in VSM cell [Ca(2+)](i) in arteries taken from control animals, this stimulus elicited only a slight increase in VSM cell [Ca(2+)](i) in arteries from CH animals. We conclude that the pulmonary circulation of the rat does not demonstrate pressure-induced vasoconstriction.

Physiological and morphological characterization of dentate granule cells in the p35 knock-out mouse hippocampus: evidence for an epileptic circuit.

There is a high correlation between pediatric epilepsies and neuronal migration disorders. What remains unclear is whether there are intrinsic features of the individual dysplastic cells that give rise to heightened seizure susceptibility, or whether these dysplastic cells contribute to seizure activity by establishing abnormal circuits that alter the balance of inhibition and excitation. Mice lacking a functional p35 gene provide an ideal model in which to address these questions, because these knock-out animals not only exhibit aberrant neuronal migration but also demonstrate spontaneous seizures. Extracellular field recordings from hippocampal slices, characterizing the input-output relationship in the dentate, revealed little difference between wild-type and knock-out mice under both normal and elevated extracellular potassium conditions. However, in the presence of the GABA(A) antagonist bicuculline, p35 knock-out slices, but not wild-type slices, exhibited prolonged depolarizations in response to stimulation of the perforant path. There were no significant differences in the intrinsic properties of dentate granule cells (i.e., input resistance, time constant, action potential generation) from wild-type versus knock-out mice. However, antidromic activation (mossy fiber stimulation) evoked an excitatory synaptic response in over 65% of granule cells from p35 knock-out slices that was never observed in wild-type slices. Ultrastructural analyses identified morphological substrates for this aberrant excitation: recurrent axon collaterals, abnormal basal dendrites, and mossy fiber terminals forming synapses onto the spines of neighboring granule cells. These studies suggest that granule cells in p35 knock-out mice contribute to seizure activity by forming an abnormal excitatory feedback circuit.

Angiotensin II Excites Paraventricular Nucleus Neurons That Innervate the Rostral Ventrolateral Medulla: An In Vitro Patch-Clamp Study in Brain Slices

Neurons of the hypothalamic paraventricular nucleus (PVN) are key controllers of sympathetic nerve activity and receive input from angiotensin II (ANG II)-containing neurons in the forebrain. This study determined the effect of ANG II on PVN neurons that innervate in the rostral ventrolateral medulla (RVLM)-a brain stem site critical for maintaining sympathetic outflow and arterial pressure. Using an in vitro brain slice preparation, whole cell patch-clamp recordings were made from PVN neurons retrogradely labeled from the ipsilateral RVLM of rats. Of 71 neurons tested, 62 (87%) responded to ANG II. In current-clamp mode, bath-applied ANG II (2 muM) significantly (P < 0.05) depolarized membrane potential from -58.5 +/- 2.5 to -54.5 +/- 2.0 mV and increased the frequency of action potential discharge from 0.7 +/- 0.3 to 2.8 +/- 0.8 Hz (n = 4). Local application of ANG II by low-pressure ejection from a glass pipette (2 pmol, 0.4 nl, 5 s) also elicited rapid and reproducible excitation in 17 of 20 cells. In this group, membrane potential depolarization averaged 21.5 +/- 4.1 mV, and spike activity increased from 0.7 +/- 0.4 to 21.3 +/- 3.3 Hz. In voltage-clamp mode, 41 of 47 neurons responded to pressure-ejected ANG II with a dose-dependent inward current that averaged -54.7 +/- 3.9 pA at a maximally effective dose of 2.0 pmol. Blockade of ANG II AT1 receptors significantly reduced discharge (P < 0.001, n = 5), depolarization (P < 0.05, n = 3), and inward current (P < 0.01, n = 11) responses to locally applied ANG II. In six of six cells tested, membrane input conductance increased (P < 0.001) during local application of ANG II (2 pmol), suggesting influx of cations. The ANG II current reversed polarity at +2.2 +/- 2.2 mV (n = 9) and was blocked (P < 0.01) by bath perfusion with gadolinium (Gd(3+), 100 muM, n = 8), suggesting that ANG II activates membrane channels that are nonselectively permeable to cations. These findings indicate that ANG II excites PVN neurons that innervate the ipsilateral RVLM by a mechanism that depends on activation of AT1 receptors and gating of one or more classes of ion channels that result in a mixed cation current.

Persistent electrical coupling and locomotory dysfunction in the zebrafish mutant shocked.

On initial formation of neuromuscular junctions, slow synaptic signals interact through an electrically coupled network of muscle cells. After the developmental onset of muscle excitability and the transition to fast synaptic responses, electrical coupling diminishes. No studies have revealed the functional importance of the electrical coupling or its precisely timed loss during development. In the mutant zebrafish shocked (sho) electrical coupling between fast muscle cells persists beyond the time that it would normally disappear in wild-type fish. Recordings from sho indicate that muscle depolarization in response to motor neuron stimulation remains slow due to the low-pass filter characteristics of the coupled network of muscle cells. Our findings suggest that the resultant prolonged muscle depolarizations contribute to the premature termination of swimming in sho and the delayed acquisition of the normally rapid touch-triggered movements. Thus the benefits of gap junctions during early synapse development likely become a liability if not inactivated by the time that muscle would normally achieve fast autonomous function.

Optical mapping of pontine chemosensitive regions of neonatal rat

We analyzed the neuronal response to hypercapnic acidosis, using an optical recording technique with a fluorescent voltage-sensitive dye (di-4-ANEPPS), in pontine slice preparations of neonatal rats, containing the locus coeruleus (LC), which has been electrophysiologically demonstrated to be chemosensitive. The dye-stained preparation was continuously superfused with artificial cerebrospinal fluid. Epifluorescence of the slice was detected using a high-sensitivity optical recording system. Changes in the intensity of fluorescence were serially analyzed while switching artificial cerebrospinal fluid from control to hypercapnic acidosis, or vice versa. The optical recording method revealed that the LC, as reported in previous studies, reversibly showed a depolarizing response to hypercapnic acidosis in 56% of the examined preparations. The A5 area (56%) also exhibited a reversible, depolarizing response to hypercapnic acidosis. The response was preserved under conditions in which chemical synaptic transmission was blocked by low Ca 2+-high Mg 2+ solution. These results suggest that the optical recording method is applicable to identification of potentially chemosensitive areas, which deserve further electrophysiological analysis, and that the A5 area could be chemosensitive.

How retinal ganglion cells prevent synaptic noise from reaching the spike output.

Synaptic vesicles are released stochastically, and therefore stimuli that increase a neuron's synaptic input might increase noise at its spike output. Indeed this appears true for neurons in primary visual cortex, where spike output variability increases with stimulus contrast. But in retinal ganglion cells, although intracellular recordings (with spikes blocked) showed that stronger stimuli increase membrane fluctuations, extracellular recordings showed that noise at the spike output is constant. Here we show that these seemingly paradoxical findings occur in the same cell and explain why. We made intracellular recordings from ganglion cells, in vitro, and presented periodic stimuli of various contrasts. For each stimulus cycle, we measured the response at the stimulus frequency (F1) for both membrane potential and spikes as well as the spike rate. The membrane and spike F1 response increased with contrast, but noise (SD) in the F1 responses and the spike rate was constant. We also measured membrane fluctuations (with spikes blocked) during the response depolarization and found that they did increase with contrast. However, increases in fluctuation amplitude were small relative to the depolarization (<10% at high contrast). A model based on estimated synaptic convergence, release rates, and membrane properties accounted for the relative magnitudes of fluctuations and depolarization. Furthermore, a cell's peak spike response preceded the peak depolarization, and therefore fluctuation amplitude peaked as the spike response declined. We conclude that two extremely general properties of a neuron, synaptic convergence and spike generation, combine to minimize the effects of membrane fluctuations on spiking.

Glucose and insulin treatment of insulinoma cells results in transcriptional regulation of a common set of genes.

Glucose and insulin are important regulators of islet beta-cell growth and function by activating signaling pathways resulting in transcriptional changes that lead to adaptive responses. Several immediate early genes have been shown to be rapidly induced by glucose-activated depolarization in islet beta-cells. The current studies address aspects of glucose-regulated transcription: 1) the number and characteristics of these genes, 2) if depolarization is the major mechanism, and 3) if glucose-stimulated insulin secretion is responsible, because insulin per se can activate transcription. Here, the expression profiles of glucose-responsive insulinoma cells 45 min after the addition of glucose, KCl to induce depolarization, or insulin were assessed by endocrine pancreas cDNA microarrays. Glucose activated more than 90 genes, representing diverse gene ontology functions, and most were not previously known to be glucose responsive. KCl activated 80% of these same glucose-regulated genes and, along with the effects of pretreatment with diazoxide, suggested that glucose signaling is mediated primarily via depolarization. There were >150 genes activated by insulin, and remarkably 71% were also regulated by glucose. Preincubation with a phosphatidylinositol (PI) 3-kinase inhibitor resulted in almost total inhibition of depolarization and insulin-activated transcriptional responses. Thus, through gene expression profiling, these data demonstrate that glucose and insulin rapidly activate a PI 3-kinase pathway, resulting in transcription of a common set of genes. This is consistent with glucose activation of gene transcription either directly or indirectly through a paracrine/autocrine effect via insulin release. These results illustrate that expression gene profiling can contribute to the elucidation of important beta-cell biological functions.