http://nova.newcastle.edu.au/vital/access/services/Feed ${session.getAttribute("locale")} 5 http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:12355 The present study compares the electrophysiological properties and the primary pacemaker currents that flow during the interspike interval in locus coeruleus (LC) neurons from infant (P7–12 days) and young adult (8–12 weeks) mice. The magnitude of the primary pacemaker currents, which consist of an excitatory TTX-sensitive Na⁺ current and an inhibitory voltage-dependent K⁺ current, increased in parallel during development. We found no evidence for the involvement of hyperpolarization-activated (IH) or Ca2⁺ currents in pacemaking in infant or adult LC neurons. The incidence of TTX-resistant spikes, observed during current clamp recordings, was greater in adult neurons. Neurons from adult animals also showed an increase in voltage fluctuations, during the interspike interval, as revealed in the presence of the K⁺ channel blocker, 4-AP (1 mM). In summary, our results suggest that mouse LC neurons undergo changes in basic electrophysiological properties during development that influence pacemaking and hence spontaneous firing in LC neurons. 2013-01-08T03:33:35.286Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:11513 We have characterized the currents that flow during the interspike interval in mouse locus coeruleus (LC) neurons, by application of depolarizing ramps and pulses, and compared our results with information available for rats. A tetrodotoxin (TTX)-sensitive current was the only inward conductance active during the interspike interval; no TTX-insensitive Na⁺ or oscillatory currents were detected. Ca2⁺-free and Ba2⁺-containing solutions failed to demonstrate a Ca2⁺ current during the interspike interval, although a Ca2⁺ current was activated at membrane potentials positive to −40 mV. A high- tetraethylammonium chloride (TEA) (15 mM) sensitive current accounted for almost all the K⁺ conductance during the interspike interval. Ca2⁺-activated K⁺, inward rectifier and low-TEA (10 μM) sensitive currents were not detected within the interspike interval. Comparison of these findings to those reported for neonatal rat LC neurons indicates that the pacemaker currents are similar, but not identical, in the two species with mice lacking a persistent Ca2⁺ current during the interspike interval. The net pacemaking current determined by differentiating the interspike interval from averaged action potential recordings closely matched the net ramp-induced currents obtained either under voltage clamp or after reconstructing this current from pharmacologically isolated currents. In summary, our results suggest the interspike interval pacemaker mechanism in mouse LC neurons involves a combination of a TTX-sensitive Na⁺ current and a high TEA-sensitive K⁺ current. In contrast with rats, a persistent Ca2⁺ current is not involved. 2012-09-12T05:52:36.926Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:9917 This study presents a theoretical analysis of the role of store Ca²⁺ uptake on sinoatrial node (SAN) cell pacemaking. Two mechanisms have been shown to be involved in SAN pacemaking, these being: 1) the membrane oscillator model where rhythm generation is based on the interaction of voltage-dependent membrane ion channels and, 2) the store oscillator model where cyclical release of Ca²⁺ from intracellular Ca²⁺ stores depolarizes the membrane through activation of the sodium-calcium exchanger (NCX). The relative roles of these oscillators in generation and modulation of pacemaker rate have been vigorously debated and have many consequences. The main new outcomes of our study are: 1) uptake of Ca²⁺ by intracellular Ca²⁺ stores increases the maximum diastolic potential (MDP) by reducing the cytosolic Ca²⁺ concentration [Ca²⁺]c and hence decreasing the NCX current; 2) this hyperpolarization enhances recruitment of key pacemaker currents (e.g. the hyperpolarization-activated HCN current (If) and T-type Ca²⁺ current (IT-Ca)); 3) the resultant enhanced Ca²⁺ entry during the pacemaker depolarization increases [Ca²⁺]c causing advancement of the store Ca²⁺ release cycle and increased NCX current. In overview, the novel feature of our study is an investigation of the role of store Ca²⁺ uptake on SAN pacemaking. This occurs during the early diastolic period and causes enhanced If, IT-Ca and store release (and hence INCX) during the later diastolic period. There is thus a symbiotic interaction between the two pacemaker “clocks” over the entire diastolic period, this providing robust and highly malleable SAN pacemaking. Accounting for store Ca²⁺ uptake also provides insight into hitherto unexplained SAN behaviour, as we exemplify for the sinus bradycardia exhibited in catecholaminergic polymorphic ventricular tachycardia (CPVT). 2012-02-08T01:20:04.698Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:6550 In ascidians, as in mammals, sperm trigger repetitive Ca²⁺-waves that originate from cortical pacemakers situated in the vegetal hemisphere of the zygotes. In ascidians, a vegetal protrusion termed the contraction pole (CP) acts as the Ca²⁺-wave pacemaker, but the mechanism that underlies the generation of a Ca²⁺-wave pacemaker is not known. Here, we tested four hypotheses to determine which factors at the CP are involved in setting the pace of the ascidian Ca²⁺-wave pacemaker: (1) localized Ca²⁺ influx; (2) accumulation of phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P₂]; (3)accumulation of cortical endoplasmic reticulum (cER); and (4) enrichment of the sperm activating factor. We developed a method of dynamically monitoring the location of the CP during fertilization using a plekstrin homology (PH) domain from phospholipase Cδ1 coupled to green fluorescent protein (GFP) that binds PtdIns(4,5)P₂. We found that eggs in Ca²⁺-free sea water displayed Ca²⁺ waves that originated from the CP, showing that enhanced CP Ca²⁺ influx does not determine the origin of the pacemaker. Also, disruption of the H::GFP-labelled CP once it had formed did not dislodge the Ca²⁺-wave pacemaker from that site. Next, when we prevented the accumulation of cER at the CP, all of the Ca²⁺ waves came from the site of sperm-egg fusion and the frequency of Ca²⁺ oscillations was unaltered. These data show that local Ca²⁺ influx, the accumulation of PtdIns(4,5)P₂ and cER at the CP are not required for Ca²⁺-wave pacemaker function and instead suggest that a factor associated with the sperm determines the site of the Ca²⁺-wave pacemaker. Finally, when we injected ascidian sperm extract into the centre of unfertilized ascidian eggs that had been treated with microfilament- and microtubuledisrupting drugs, all the Ca²⁺ waves still originated from near the plasma membrane, showing that the sperm factor does not require an intact cortex if it is enriched near the plasma membrane (PM). We suggest that the Ca²⁺- releasing sperm factor might be tethered near or on the PM and that following the cortical contraction, it is translocated to the vegetal CP, thus making that site act as a Ca²⁺-wave pacemaker. 2011-08-30T05:50:05.192Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:1733 Ca²⁺ imaging and multiple microelectrode recording procedures were used to investigate a slow wave-like electrical rhythmicity in single bundle strips from the circular muscle layer of the guinea-pig gastric pylorus. The 'slow waves' (SWs) consisted of a pacemaker and regenerative component, with both potentials composed of more elementary events variously termed spontaneous transient depolarizations (STDs) or unitary potentials. STDs and SW pacemaker and regenerative potentials exhibited associated local and distributed Ca²⁺ transients, respectively. Ca²⁺ transients were often larger in cellular regions that exhibited higher basal Ca²⁺ indicator-associated fluorescence, typical of regions likely to contain intramuscular interstitial cells of Cajal (ICCIM). The emergence of rhythmicity arose through entrainment of STDs resulting in pacemaker Ca²⁺ transients and potentials, events that exhibited considerable spatial synchronicity. Application of ACh to strips exhibiting weak rhythmicity caused marked enhancement of SW synchronicity. SWs and underlying Ca²⁺ increases exhibited very high 'apparent conduction velocities' ('CVs') orders of magnitude greater than for sequentially conducting Ca²⁺ waves. Central interruption of either intercellular connectivity or inositol 1,4,5-trisphosphate receptor (IP₃R)-mediated store Ca²⁺ release in strips caused SWs at the two ends to run independently of each other, consistent with a coupled oscillator-based mechanism. Central inhibition of stores required much wider regions of blockade than inhibition of connectivity indicating that stores were voltage-coupled. Simulations, made using a conventional store array model but now including depolarization coupled to IP₃R-mediated Ca²⁺ release, predicted the experimental findings. The linkage between membrane voltage and Ca²⁺ release provides a means for stores to interact as strongly coupled oscillators, resulting in the emergence of Ca²⁺ phase waves and associated pacemaker potentials. This distributed pacemaker triggers regenerative Ca²⁺ release and resultant SWs. 2010-04-27T06:10:13.658Z ]]>