Locus coeruleus neurones in vitro: pH-sensitive oscillations of membrane potential in an electrically coupled network

The response to hypercapnic acidosis (2-8% CO2, bath pH 7.8-7.2) was examined in whole cell recordings from neonatal (P1 to P5) rat Locus coeruleus (LC) neurones in the in vitro brainstem-spinal cord preparation exposed to low Ca2+ (0.2 mM)-high Mg2+ (5 mM). This medium suppressed chemical synaptic transmission and resulted in a pattern of subthreshold oscillations of membrane potential and rhythmic burst discharge which was synchronized throughout the network. The oscillation was suppressed, and the discharge of individual neurones desynchronized, by the gap junction uncoupler, carbenoxolone, indicating that in low Ca2+-high Mg2+ LC neurones form an electrically coupled network. Switching from 2 to 8% CO2 decreased the oscillation amplitude and increased its frequency. The oscillation was suppressed by external Cd2+ and by TTX. but persisted during injection into the cell soma of QX-314. We conclude that in LC neurones acidosis increases the frequency of a Ca2+- and Na+-dependent dendritic oscillator which is synchronized by gap junction coupling throughout the network. This coupling is retained during acidosis.     

Neuronal synchronization without calcium-dependent synaptic transmission in the hypothalamus.

A critical question in understanding the mammalian brain is how populations of neurons become synchronized. This is particularly important for the neurons and neuroendocrine cells of the hypothalamus, which are activated synchronously to control endocrine glands and the autonomic nervous system. It is widely accepted that communication between neurons of the adult mammalian brain is mediated primarily by Ca(2+)-dependent synaptic transmission. Here we report that synchronous neuronal activity can occur in the hypothalamic suprachiasmatic nucleus without active Ca(2+)-dependent synaptic transmission. Simultaneous extracellular recordings of neuronal activity in the suprachiasmatic nucleus, which contains the mammalian biological clock, confirmed a circadian rhythm of synchronized activity in hypothalamic slices. Ca(2+)-free medium, which blocks chemical synaptic transmission and increases membrane excitability, produced periodic and synchronized bursts of action potentials in a large population of suprachiasmatic nucleus neurons with diverse firing patterns. N-Methyl-D-aspartic acid, non-N-methyl-D-aspartic acid, and gamma-aminobutyric acid type A receptor antagonists had no effect on burst synchrony. Whole-cell patch-clamp recordings confirmed that the Ca(2+)-free solution blocked evoked postsynaptic potentials and that the mixture of antagonists blocked the remaining spontaneous postsynaptic potentials. Therefore, mechanisms other than Ca(2+)-dependent synaptic transmission can synchronize neurons in the mammalian hypothalamus and may be important wherever neuronal networks are synchronized.     

The Ca(V)3.3 calcium channel is the major sleep spindle pacemaker in thalamus

Low-threshold (T-type) Ca(2+) channels encoded by the Ca(V)3 genes endow neurons with oscillatory properties that underlie slow waves characteristic of the non-rapid eye movement (NREM) sleep EEG. Three Ca(V)3 channel subtypes are expressed in the thalamocortical (TC) system, but their respective roles for the sleep EEG are unclear. Ca(V)3.3 protein is expressed abundantly in the nucleus reticularis thalami (nRt), an essential oscillatory burst generator. We report the characterization of a transgenic Ca(V)3.3(-/-) mouse line and demonstrate that Ca(V)3.3 channels are indispensable for nRt function and for sleep spindles, a hallmark of natural sleep. The absence of Ca(V)3.3 channels prevented oscillatory bursting in the low-frequency (4-10 Hz) range in nRt cells but spared tonic discharge. In contrast, adjacent TC neurons expressing Ca(V)3.1 channels retained low-threshold bursts. Nevertheless, the generation of synchronized thalamic network oscillations underlying sleep-spindle waves was weakened markedly because of the reduced inhibition of TC neurons via nRt cells. T currents in Ca(V)3.3(-/-) mice were <30% compared with those in WT mice, and the remaining current, carried by Ca(V)3.2 channels, generated dendritic [Ca(2+)](i) signals insufficient to provoke oscillatory bursting that arises from interplay with Ca(2+)-dependent small conductance-type 2 K(+) channels. Finally, naturally sleeping Ca(V)3.3(-/-) mice showed a selective reduction in the power density of the σ frequency band (10-12 Hz) at transitions from NREM to REM sleep, with other EEG waves remaining unaltered. Together, these data identify a central role for Ca(V)3.3 channels in the rhythmogenic properties of the sleep-spindle generator and provide a molecular target to elucidate the roles of sleep spindles for brain function and development.     

Effect of calcium on reactive oxygen species in isolated rat cardiomyocytes during hypoxia and reoxygenation

It has been suggested that calcium (Ca(2+)) overload and oxidative stress damage the myocardium during ischemia and reperfusion. We investigated the possible effect of varying extracellular Ca(2+)and total cell Ca(2+)on reactive oxygen species (ROS) levels in resting adult rat cardiomyocytes. Cardiomyocytes were isolated by trypsin/collagenase digestion and exposed to 1 h of hypoxia (H) (95% N(2)/5% CO(2), no glucose) and 2 h of reoxygenation (R) (95% air/5% CO(2), glucose 5.5 m M) in suspension. Cell Ca(2+)was measured by uptake of(45)Ca(2+). ROS was measured by flow cytometry of ethidium's red fluorescence formed by oxidation of dihydroethidium mostly by superoxide anion. Cell viability decreased during H and R, expressed as uptake of trypan blue, loss of rod shape morphology and release of lactate dehydrogenase. Rapidly exchangeable cell Ca(2+)was closely correlated with extracellular Ca(2+)concentration. Cell Ca(2+)was unchanged during H but increased three to four times after R. This increase was attenuated by adding 3,4-dichlorobenzamil, 10 microm at R, and amplified by adding ouabain 1 m M (from start), respectively. Levels of ROS in hypoxic cells were unchanged or slightly reduced at the end of H and increased significantly by 20% compared to control after R. Levels of ROS were significantly decreased by lowering total extracellular Ca(2+)from 1 m M to 0.1 m M or by decreasing free extracellular Ca(2+)with EGTA 0.9 m M at the onset of R. Keeping extracellular Ca(2+)constant, ROS levels were neither affected by attenuating the increase in cell Ca(2+)by DCB nor by amplifying the increase in cell Ca(2+)by ouabain. In conclusion, ROS (superoxide anion) levels increase rapidly after reoxygenation, are correlated with extracellular-free Ca(2+)and are reduced by lowering extracellular-free Ca(2+). Levels of ROS are apparently not consistently correlated with total cell Ca(2+).     

Development of in vivo ventilatory and single chemosensitive neuron responses to hypercapnia in rats

We used pressure plethysmography to study breathing patterns of neonatal and adult rats acutely exposed to elevated levels of CO2. Ventilation (VE) increased progressively with increasing inspired CO2. The rise in VE was associated with an increase in tidal volume, but not respiratory rate. In all animals studied, the CO2 sensitivity (determined from the slope of the VE vs. inspired % CO2 curve) was variable on a day to day basis. Chemosensitivity was high in neonates 1 day after birth (P1) and fell throughout the first week to a minimum at about P8. Chemosensitivity rose again to somewhat higher values in P10 through adult rats. The developmental pattern of these in vivo ventilatory responses was different than individual locus coeruleus (LC) neuron responses to increased CO2. The membrane potential (V(m)) of LC neurons was measured using perforated patch (amphotericin B) techniques in brain slices. At all ages studied, LC neurons increased their firing rate by approximately 44% in response to hypercapnic acidosis (10% CO2, pH 7.0). Thus the in vivo ventilatory response to hypercapnia was not correlated with the V(m) response of individual LC neurons to hypercapnic acidosis in neonatal rats. These data suggest that CO2 sensitivity of ventilation in rats may exist in two forms, a high-sensitivity neonatal (or fetal) form and a lower-sensitivity adult form, with a critical window of very low sensitivity during the period of transition between the two (approximately P8).     

P(O(2))-P(CO(2)) stimulus interaction in [Ca(2+)](i) and CSN activity in the adult rat carotid body

Since glomus cell intracellular calcium ([Ca(2+)](i)) plays a key role in generating carotid sinus nerve (CSN) discharge, we hypothesized that glomus cell [Ca(2+)](i) would correspond to CSN discharge rates during P(O(2))-P(CO(2)) stimulus interaction in adult rat carotid body (CB). Accordingly, we measured steady state P(O(2))-P(CO(2)) interaction in CSN discharge rates during hypocapnia (P(CO(2))=8-10 Torr), normocapnia (P(CO(2))=33-35 Torr) and hypercapnia (P(CO(2))=68-70 Torr) in normoxia (P(O(2)) approximately 130 Torr) and hypoxia (P(O(2)) approximately 36 Torr). The results showed P(O(2))-P(CO(2)) stimulus interaction in CSN responses. [Ca(2+)](i) levels were measured in isolated type I cells (2-3 cells/field), using Ca(2+) sensitive fluoroprobe indo-1AM. The [Ca(2+)](i) responses increased with increasing P(CO(2)) in normoxia. In hypoxia, [Ca(2+)](i) did not increase during hypocapnia but increased during normocapnia, showing P(O(2))-P(CO(2)) interaction. However, CSN response during hypoxia was far greater than that for [Ca(2+)](i) response, particularly during hypocapnic hypoxia. Thus, the [Ca(2+)](i) interaction cannot account for the whole CSN interaction. The origin of this CSN P(O(2)-)P(CO(2)) interaction must have occurred in part beyond cellular [Ca(2+)](i) interaction. Interactions at both sites (glomus cell membrane and sinus nerve endings) are reminiscent of reversible O(2)-heme protein reaction with a Bohr effect.     

Three distinct types of Ca(2+) waves in Langendorff-perfused rat heart revealed by real-time confocal microscopy

Although Ca(2+) waves in cardiac myocytes are regarded as arrhythmogenic substrates, their properties in the heart in situ are poorly understood. On the hypothesis that Ca(2+) waves in the heart behave diversely and some of them influence the cardiac function, we analyzed their incidence, propagation velocity, and intercellular propagation at the subepicardial myocardium of fluo 3-loaded rat whole hearts using real-time laser scanning confocal microscopy. We classified Ca(2+) waves into 3 types. In intact regions showing homogeneous Ca(2+) transients under sinus rhythm (2 mmol/L [Ca(2+)](o)), Ca(2+) waves did not occur. Under quiescence, the waves occurred sporadically (3.8 waves. min(-1) x cell(-1)), with a velocity of 84 microm/s, a decline half-time (t(1/2)) of 0.16 seconds, and rare intercellular propagation (propagation ratio <0.06) (sporadic wave). In contrast, in presumably Ca(2+)-overloaded regions showing higher fluorescent intensity (113% versus the intact regions), Ca(2+) waves occurred at 28 waves x min(-1) x cell(-1) under quiescence with a higher velocity (116 microm/s), longer decline time (t(1/2) = 0.41 second), and occasional intercellular propagation (propagation ratio = 0.23) (Ca(2+)-overloaded wave). In regions with much higher fluorescent intensity (124% versus the intact region), Ca(2+) waves occurred with a high incidence (133 waves x min(-1) x cell(-1)) and little intercellular propagation (agonal wave). We conclude that the spatiotemporal properties of Ca(2+) waves in the heart are diverse and modulated by the Ca(2+)-loading state. The sporadic waves would not affect cardiac function, but prevalent Ca(2+)-overloaded and agonal waves may induce contractile failure and arrhythmias.     

Inositol 1,4,5-trisphosphate receptors and pacemaker rhythms

Intracellular Ca(2+) plays an important role in the control of the heart rate through the interaction between Ca(2+) release by ryanodine receptors in the sarcoplasmic reticulum (SR) and the extrusion of Ca(2+) by the sodium-calcium exchanger which generates an inward current. A second type of SR Ca(2+) release channel, the inositol 1,4,5-trisphosphate receptor (IP(3)R), can release Ca(2+) from SR stores in many cell types, including cardiac myocytes. However, it is still uncertain whether IP(3)Rs play any functional role in regulating the heart rate. Accumulated evidence shows that IP(3) and IP(3)R are involved in rhythm control in non-cardiac pacemaker tissues and in the embryonic heart. In this review we focus on intracellular Ca(2+) oscillations generated by Ca(2+) release from IP(3)R that initiates membrane depolarization and provides a common mechanism producing spontaneous activity in a range of cells with pacemaker function. Emerging new evidence also suggests that IP(3)/IP(3)Rs play a functional role in normal and diseased hearts and in cardiac rhythm control. Several membrane currents, including a store-operated Ca(2+) current, might be activated by Ca(2+) release from IP(3)Rs. IP(3)/IP(3)R may thus add another dimension to the complex regulation of heart rate.     

Ca2+ sparks and secretion in dorsal root ganglion neurons

Ca(2+) sparks as the elementary intracellular Ca(2+) release events are instrumental to local control of Ca(2+) signaling in many types of cells. Here, we visualized neural Ca(2+) sparks in dorsal root ganglion (DRG) sensory neurons and investigated possible role of DRG sparks in the regulation of secretion from the somata of the cell. DRG sparks arose mainly from type 3 ryanodine receptor Ca(2+) release channels on subsurface cisternae of the endoplasmic reticulum, rendering a striking subsurface localization. Caffeine- or 3,7-dimethyl-1-(2-propynyl)xanthine-induced store Ca(2+) release, in the form of Ca(2+) sparks, triggered exocytosis, independently of membrane depolarization and external Ca(2+). The spark-secretion coupling probability was estimated to be between 1 vesicle per 6.6 sparks and 1 vesicle per 11.4 sparks. During excitation, subsurface sparks were evoked by physiological Ca(2+) entry via the Ca(2+)-induced Ca(2+) release mechanism, and their synergistic interaction with Ca(2+) influx accounted for approximately 60% of the Ca(2+)-dependent exocytosis. Furthermore, inhibition of Ca(2+)-induced Ca(2+) release abolished endotoxin-induced secretion of pain-related neuropeptides. These findings underscore an important role for Ca(2+) sparks in the amplification of surface Ca(2+) influx and regulation of neural secretion.     

In situ Ca2+ dynamics of Purkinje fibers and its interconnection with subjacent ventricular myocytes

Purkinje fibers play essential roles in impulse propagation to the ventricles, and their functional impairment can become arrhythmogenic. However, little is known about precise spatiotemporal pattern(s) of interconnection between Purkinje-fiber network and the underlying ventricular myocardium within the heart. To address this issue, we simultaneously visualized intracellular Ca(2+) dynamics at Purkinje fibers and subjacent ventricular myocytes in Langendorff-perfused rat hearts using multi-pinhole type, rapid-scanning confocal microscopy. Under recording of electrocardiogram at room temperature spatiotemporal changes in fluo3-fluorescence intensity were visualized on the subendocardial region of the right-ventricular septum. Staining of the heart with either fluo3, acetylthiocholine iodide (ATCHI), or di-4-ANEPPS revealed characteristic structures of Purkinje fibers. During sinus rhythm (about 60 bpm) or atrial pacing (up to 3 Hz) each Purkinje-fiber exhibited spatiotemporally synchronous Ca(2+) transients nearly simultaneously to ventricular excitation. Ca(2+) transients in individual fibers were still synchronized within the Purkinje-fiber network not only under high-K(+) (8 mM) perfusion-induced Purkinje-to-ventricular (P-V) conduction delay, but also under unidirectional, orthodromic P-V block produced by 10-mM K(+) perfusion. While spontaneous, asynchronous intracellular Ca(2+) waves were identified in injured fibers of Purkinje network locally, surrounding fibers still exhibited Ca(2+) transients synchronously to ventricular excitation. In summary, these results are the first demonstration of intracellular Ca(2+) dynamics in the Purkinje-fiber network in situ. The synchronous Ca(2+) transients, preserved even under P-V conduction disturbances or under emergence of Ca(2+) waves, imply a syncytial role of Purkinje fibers as a specialized conduction system, whereas unidirectional block at P-V junctions indicates a substrate for reentrant arrhythmias.     

TRPM7 channels in hippocampal neurons detect levels of extracellular divalent cations

Exposure to low Ca(2+) and/or Mg(2+) is tolerated by cardiac myocytes, astrocytes, and neurons, but restoration to normal divalent cation levels paradoxically causes Ca(2+) overload and cell death. This phenomenon has been called the "Ca(2+) paradox" of ischemia-reperfusion. The mechanism by which a decrease in extracellular Ca(2+) and Mg(2+) is "detected" and triggers subsequent cell death is unknown. Transient periods of brain ischemia are characterized by substantial decreases in extracellular Ca(2+) and Mg(2+) that mimic the initial condition of the Ca(2+) paradox. In CA1 hippocampal neurons, lowering extracellular divalents stimulates a nonselective cation current. We show that this current resembles TRPM7 currents in several ways. Both (i) respond to transient decreases in extracellular divalents with inward currents and cell excitation, (ii) demonstrate outward rectification that depends on the presence of extracellular divalents, (iii) are inhibited by physiological concentrations of intracellular Mg(2+), (iv) are enhanced by intracellular phosphatidylinositol 4,5-bisphosphate (PIP(2)), and (v) can be inhibited by Galphaq-linked G protein-coupled receptors linked to phospholipase C beta1-induced hydrolysis of PIP(2). Furthermore, suppression of TRPM7 expression in hippocampal neurons strongly depressed the inward currents evoked by lowering extracellular divalents. Finally, we show that activation of TRPM7 channels by lowering divalents significantly contributes to cell death. Together, the results demonstrate that TRPM7 contributes to the mechanism by which hippocampal neurons "detect" reductions in extracellular divalents and provide a means by which TRPM7 contributes to neuronal death during transient brain ischemia.     

Functional dependence of Ca(2+)-activated K+ current on L- and N-type Ca2+ channels: differences between chicken sympathetic and parasympathetic neurons suggest different regulatory mechanisms.

The influx of Ca2+ ions controls many important processes in excitable cells, including the regulation of the gating of Ca(2+)-activated K+ channels (the current IK[Ca]). Various IK[Ca] channels contribute to the regulation of the action-potential waveform, the repetitive discharge of spikes, and the secretion of neurotransmitters. It is thought that large-conductance IK[Ca] channels must be closely colocalized with Ca2+ channels (ICa) to be gated by Ca2+ influx. We now report that IK[Ca] channels can be preferentially colocalized with pharmacologically distinct subtypes of voltage-activated Ca2+ channel and that this occurs differently in embryonic chicken sympathetic and parasympathetic neurons. The effects of various dihydropyridines and omega-conotoxin on voltage-activated Ca2+ currents (ICa) and Ca(2+)-activated K+ currents (IK[Ca]) were examined by using perforated-patch whole-cell recordings from embryonic chicken ciliary and sympathetic ganglion neurons. Application of nifedipine or omega-conotoxin each caused a 40-60% reduction in ICa, whereas application of S-(-)-BAY K 8644 potentiated ICa in ciliary ganglion neurons. But application of omega-conotoxin had little or no effect on IK[Ca], whereas nifedipine and S-(-)-BAY K 8644 inhibited and potentiated IK[Ca], respectively. These results indicate that IK[Ca] channels are preferentially coupled to L-type, but not to N-type, Ca2+ channels on chicken ciliary ganglion neurons. Chicken sympathetic neurons also express dihydropyridine-sensitive and omega-conotoxin-sensitive components of ICa. However, in those cells, application of omega-conotoxin caused a 40-60% reduction in IK[Ca], whereas nifedipine reduced IK[Ca] but only in a subpopulation of cells. Therefore, IK[Ca] in sympathetic neurons is either coupled to N-type Ca2+ channels or is not selectively coupled to a single Ca(2+)-channel subtype. The preferential coupling of IK[Ca] channels with distinct ICa subtypes may be part of a mechanism to allow for selective modulation of neurotransmitter release. Preferential coupling may also be important for the differentiation and development of vertebrate neurons.     

In Situ Confocal Imaging in Intact Heart Reveals Stress-Induced Ca2+ Release Variability in a Murine Catecholaminergic Polymorphic Ventricular Tachycardia Model of Type 2 Ryanodine ReceptorR4496C+/- Mutation

Background- Catecholaminergic polymorphic ventricular tachycardia is directly linked to mutations in proteins (eg, type 2 ryanodine receptor [RyR2](R4496C)) responsible for intracellular Ca(2+) homeostasis in the heart. However, the mechanism of Ca(2+) release dysfunction underlying catecholaminergic polymorphic ventricular tachycardia has only been investigated in isolated cells but not in the in situ undisrupted myocardium. Methods and Results- We investigated in situ myocyte Ca(2+) dynamics in intact Langendorff-perfused hearts (ex vivo) from wild-type and RyR2(R4496C+/-) mice using laser scanning confocal microscopy. We found that myocytes from both wild-type and RyR2(R4496C+/-) hearts displayed uniform, synchronized Ca(2+) transients. Ca(2+) transients from beat to beat were comparable in amplitude with identical activation and decay kinetics in wild-type and RyR2(R4496C+/-) hearts, suggesting that excitation-contraction coupling between the sarcolemmal Ca(2+) channels and mutated RyR2(R4496C+/-) channels remains intact under baseline resting conditions. On adrenergic stimulation, RyR2(R4496C+/-) hearts exhibited a high degree of Ca(2+) release variability. The varied pattern of Ca(2+) release was absent in single isolated myocytes, independent of cell cycle length, synchronized among neighboring myocytes, and correlated with catecholaminergic polymorphic ventricular tachycardia. A similar pattern of action potential variability, which was synchronized among neighboring myocytes, was also revealed under adrenergic stress in intact hearts but not in isolated myocytes. Conclusions- Our studies using an in situ confocal imaging approach suggest that mutated RyR2s are functionally normal at rest but display a high degree of Ca(2+) release variability on intense adrenergic stimulation. Ca(2+) release variability is a Ca(2+) release abnormality, resulting from electric defects rather than the failure of the Ca(2+) release response to action potentials in mutated ventricular myocytes. Our data provide important insights into Ca(2+) release and electric dysfunction in an established model of catecholaminergic polymorphic ventricular tachycardia.     

In vivo mouse inferior olive neurons exhibit heterogeneous subthreshold oscillations and spiking patterns

In vitro whole-cell recordings of the inferior olive have demonstrated that its neurons are electrotonically coupled and have a tendency to oscillate. However, it remains to be shown to what extent subthreshold oscillations do indeed occur in the inferior olive in vivo and whether its spatiotemporal firing pattern may be dynamically generated by including or excluding different types of oscillatory neurons. Here, we did whole-cell recordings of olivary neurons in vivo to investigate the relation between their subthreshold activities and their spiking behavior in an intact brain. The vast majority of neurons (85%) showed subthreshold oscillatory activities. The frequencies of these subthreshold oscillations were used to distinguish four main olivary subtypes by statistical means. Type I showed both sinusoidal subthreshold oscillations (SSTOs) and low-threshold Ca(2+) oscillations (LTOs) (16%); type II showed only sinusoidal subthreshold oscillations (13%); type III showed only low-threshold Ca(2+) oscillations (56%); and type IV did not reveal any subthreshold oscillations (15%). These subthreshold oscillation frequencies were strongly correlated with the frequencies of preferred spiking. The frequency characteristics of the subthreshold oscillations and spiking behavior of virtually all olivary neurons were stable throughout the recordings. However, the occurrence of spontaneous or evoked action potentials modified the subthreshold oscillation by resetting the phase of its peak toward 90 degrees . Together, these findings indicate that the inferior olive in intact mammals offers a rich repertoire of different neurons with relatively stable frequency settings, which can be used to generate and reset temporal firing patterns in a dynamically coupled ensemble.     

CO(2) signaling in guard cells: calcium sensitivity response modulation, a Ca(2+)-independent phase, and CO(2) insensitivity of the gca2 mutant

Leaf stomata close in response to high carbon dioxide levels and open at low CO(2). CO(2) concentrations in leaves are altered by daily dark/light cycles, as well as the continuing rise in atmospheric CO(2). Relative to abscisic acid and blue light signaling, little is known about the molecular, cellular, and genetic mechanisms of CO(2) signaling in guard cells. Interestingly, we report that repetitive Ca(2+) transients were observed during the stomatal opening stimulus, low [CO(2)]. Furthermore, low/high [CO(2)] transitions modulated the cytosolic Ca(2+) transient pattern in Arabidopsis guard cells (Landsberg erecta). Inhibition of cytosolic Ca(2+) transients, achieved by loading guard cells with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and not adding external Ca(2+), attenuated both high CO(2)-induced stomatal closing and low CO(2)-induced stomatal opening, and also revealed a Ca(2+)-independent phase of the CO(2) response. Furthermore, the mutant, growth controlled by abscisic acid (gca2) shows impairment in [CO(2)] modulation of the cytosolic Ca(2+) transient rate and strong impairment in high CO(2)-induced stomatal closing. Our findings provide insights into guard cell CO(2) signaling mechanisms, reveal Ca(2+)-independent events, and demonstrate that calcium elevations can participate in opposed signaling events during stomatal opening and closing. A model is proposed in which CO(2) concentrations prime Ca(2+) sensors, which could mediate specificity in Ca(2+) signaling.     

Requirement for the synaptic protein interaction site for reconstitution of synaptic transmission by P/Q-type calcium channels

Ca(v)2.1 channels, which conduct P/Q-type Ca(2+) currents, were expressed in superior cervical ganglion neurons in cell culture, and neurotransmission initiated by these exogenously expressed Ca(2+) channels was measured. Deletions in the synaptic protein interaction (synprint) site in the intracellular loop between domains II and III of Ca(v)2.1 channels reduced their effectiveness in synaptic transmission. Surprisingly, this effect was correlated with loss of presynaptic localization of the exogenously expressed channels. Ca(v)1.2 channels, which conduct L-type Ca(2+) currents, are ineffective in supporting synaptic transmission, but substitution of the synprint site from Ca(v)2.1 channels in Ca(v)1.2 was sufficient to establish synaptic transmission initiated by L-type Ca(2+) currents through the exogenous Ca(v)1.2 channels. Substitution of the synprint site from Ca(v)2.2 channels, which conduct N-type Ca(2+) currents, was even more effective than Ca(v)2.1. Our results show that localization and function of exogenous Ca(2+) channels in nerve terminals of superior cervical ganglion neurons require a functional synprint site and suggest that binding of soluble NSF attachment protein receptor (SNARE) proteins to the synprint site is a necessary permissive event for nerve terminal localization of presynaptic Ca(2+) channels.     

Two endogenous gonadotropin-releasing hormones generate dissimilar Ca(2+) signals in identified goldfish gonadotropes

Ca(2+) signals are involved in the signal transduction of neuroendocrine regulators. In goldfish, two endogenous gonadotropin-releasing hormones, salmon (s)GnRH and chicken (c)GnRH-II, control maturational gonadotropin secretion. Although considerable evidence suggests that sGnRH and cGnRH-II exert their activity on goldfish gonadotropes through a single population of receptors, differences in signal transduction mechanisms between these peptides have been demonstrated. We used ratiometric Fura-2 Ca(2+) imaging of single morphologically identified gonadotropes to quantitatively compare the Ca(2+) signals evoked by sGnRH and cGnRH-II. The amplitude and the rate of rise of sGnRH- and cGnRH-II-evoked Ca(2+) signals increased with concentration. At maximal concentrations, Ca(2+) signals generated by cGnRH-II rose significantly faster than those elicited by sGnRH, while other parameters such as the maximum amplitude, average Ca(2+) increase, and latency did not differ between the two peptides. Ca(2+) signals evoked by sGnRH or cGnRH-II were often spatially restricted to one part of the cell over the duration of the response. We provide a comprehensive account of the spatial and temporal aspects, including calculated kinetics, of GnRH-evoked Ca(2+) signals in single identified gonadotropes. This is the first report of quantified differences in Ca(2+) signals generated by two endogenous GnRH neuropeptides, which may act through the same receptor population in this cell type.     

Switch in glutamate receptor subunit gene expression in CA1 subfield of hippocampus following global ischemia in rats.

Severe, transient global ischemia of the brain induces delayed damage to specific neuronal populations. Sustained Ca2+ influx through glutamate receptor channels is thought to play a critical role in postischemic cell death. Although most kainate-type glutamate receptors are Ca(2+)-impermeable, Ca(2+)-permeable kainate receptors have been reported in specific kinds of neurons and glia. Recombinant receptors assembled from GluR1 and/or GluR3 subunits in exogenous expression systems are permeable to Ca2+; heteromeric channels containing GluR2 subunits are Ca(2+)-impermeable. Thus, altered expression of GluR2 in development or following a neurological insult or injury to the brain can act as a switch to modify Ca2+ permeability. To investigate the molecular mechanism underlying delayed postischemic cell death, GluR1, GluR2, and GluR3 gene expression was examined by in situ hybridization in postischemic rats. Following severe, transient forebrain ischemia GluR2 gene expression was preferentially reduced in CA1 hippocampal neurons at a time point that preceded their degeneration. The switch in expression of kainate/AMPA receptor subunits coincided with the previously reported increase in Ca2+ influx into CA1 cells. Timing of the switch indicates that it may play a causal role in postischemic cell death.     

Calcium and small-conductance calcium-activated potassium channels in gonadotropin-releasing hormone neurons before, during, and after puberty

The pubertal increase in GnRH secretion resulting in sexual maturation and reproductive competence is a complex process involving kisspeptin stimulation of GnRH neurons and requiring Ca(2+) and possibly other intracellular messengers. To determine whether the expression of Ca(2+) channels, or small-conductance Ca(2+)-activated K(+) (SK) channels, whose activity reflects cytoplasmic free Ca(2+) concentration, changes at puberty in GnRH neurons, Ca(2+) and SK currents in GnRH neurons were recorded in brain slices of juvenile [postnatal day (P) 10-21], pubertal (P28-P42), and adult (> or =P56) male GnRH-green fluorescent protein transgenic mice using perforated-patch and whole-cell techniques. Ca(2+) currents were inhibited by the Ca(2+) channel blocker Cd(2+) and showed marked heterogeneity but were on average similar in juvenile, pubertal, and adult GnRH neurons. SK currents, which were inhibited by the SK channel blocker apamin and enhanced by the SK and intermediate-conductance Ca(2+)-activated K(+) channel activator 1-ethyl-2-benzimidazolinone, were also on average similar in juvenile, pubertal, and adult GnRH neurons. These findings suggest that whereas Ca(2+) and SK channels may participate in the pubertal increase in GnRH secretion and there may be changes in Ca(2+) or SK channel subtypes, overall Ca(2+) and SK channel expression in GnRH neurons remains relatively constant across pubertal development. Hence, the expected increase in GnRH neuron cytoplasmic free Ca(2+) concentration required for increased GnRH secretion at puberty appears to be due to mechanisms other than altered Ca(2+) or SK channel expression, e.g. increased membrane depolarization and subsequent activation of preexisting Ca(2+) channels after increased excitatory synaptic input.     

Extracellular acidosis increases neuronal cell calcium by activating acid-sensing ion channel 1a

Acid-sensing ion channel (ASIC) 1a subunit is expressed in synapses of central neurons where it contributes to synaptic plasticity. However, whether these channels can conduct Ca(2+) and thereby raise the cytosolic Ca(2+) concentration, [Ca(2+)](c), and possibly alter neuronal physiology has been uncertain. We found that extracellular acidosis opened ASIC1a channels, which provided a pathway for Ca(2+) entry and elevated [Ca(2+)](c) in wild-type, but not ASIC1(-/-), hippocampal neurons. Acid application also raised [Ca(2+)](c) and evoked Ca(2+) currents in heterologous cells expressing ASIC1a. Although ASIC2a is also expressed in central neurons, neither ASIC2a homomultimeric channels nor ASIC1a/2a heteromultimers showed H(+)-activated [Ca(2+)](c) elevation or Ca(2+) currents. Because extracellular acidosis accompanying cerebral ischemia contributes to neuronal injury, we tested the effect of acidosis on cell death measured as lactate dehydrogenase release. Eliminating ASIC1a from neurons or treating ASIC1a-expressing cells with the ASIC blocker amiloride attenuated acidosis-induced cell injury. These results indicate that ASIC1a provides a non-voltage-gated pathway for Ca(2+) to enter neurons. Thus, it may provide a target for modulation of [Ca(2+)](c).