Archaerhodopsin Selectively and Reversibly Silences Synaptic Transmission through Altered pH.

Tools that allow acute and selective silencing of synaptic transmission in vivo would be invaluable for understanding the synaptic basis of specific behaviors. Here, we show that presynaptic expression of the proton pump archaerhodopsin enables robust, selective, and reversible optogenetic synaptic silencing with rapid onset and offset. Two-photon fluorescence imaging revealed that this effect is accompanied by a transient increase in pH restricted to archaerhodopsin-expressing boutons. Crucially, clamping intracellular pH abolished synaptic silencing without affecting the archaerhodopsin-mediated hyperpolarizing current, indicating that changes in pH mediate the synaptic silencing effect. To verify the utility of this technique, we used trial-limited, archaerhodopsin-mediated silencing to uncover a requirement for CA3-CA1 synapses whose afferents originate from the left CA3, but not those from the right CA3, for performance on a long-term memory task. These results highlight optogenetic, pH-mediated silencing of synaptic transmission as a spatiotemporally selective approach to dissecting synaptic function in behaving animals.Biotechnology and Biological Sciences Research Council (BBSRC)This is the final version of the article. It first appeared from Elsevier(Cell Press) via http://dx.doi.org/10.1016/j.celrep.2016.07.05

Pharmacological changes in cellular Ca2+ homeostasis parallel initiation of atrial arrhythmogenesis in murine langendorff-perfused hearts

Intracellular Ca2+ overload has been associated with established atrial arrhythmogenesis. The present experiments went on to correlate acute initiation of atrial arrhythmogenesis in Langendorff-perfused mouse hearts with changes in Ca2+ homeostasis in isolated atrial myocytes following pharmacological procedures that modified the storage or release of sarcoplasmic reticular (SR) Ca2+ or inhibited entry of extracellular Ca2+.Caffeine (1mmol/L) elicited diastolic Ca2+ waves in regularly stimulated atrial myocytes immediately following addition. This was followed by a decline in the amplitude of the evoked transients and the disappearance of such diastolic events, suggesting partial SR Ca2+ depletion.Cyclopiazonic acid (CPA; 0.15µmol/L) produced more gradual reductions in evoked Ca2+ transients and abolished diastolic Ca2+ events produced by the further addition of caffeine.Nifedipine (0.5µmol/L) produced immediate reductions in evoked Ca2+ transients. Further addition of caffeine produced an immediate increase followed by a decline in the amplitude of the evoked Ca2+ transients, without eliciting diastolic Ca2+ events.These findings correlated with changes in spontaneous and provoked atrial arrhythmogenecity in mouse isolated Langendorf-perfused hearts. Thus, caffeine was pro-arrhythmogenic immediately following but not >5min after application and both CPA and nifedipine pretreatment inhibited such arrhythmogenesis.Together, these findings relate acute atrial arrhythmogenesis in intact hearts to diastolic Ca2+ events in atrial myocytes that, in turn, depend upon a finite SR Ca2+ store and diastolic Ca2+ release following Ca2+-induced Ca2+ release initiated by the entry of extracellular Ca2+

Reduced Na+ and higher K+ channel expression and function contribute to right ventricular origin of arrhythmias in Scn5a+/− mice

Brugada syndrome (BrS) is associated with ventricular tachycardia originating particularly in the right ventricle (RV). We explore electrophysiological features predisposing to such arrhythmic tendency and their possible RV localization in a heterozygotic Scn5a+/− murine model. Nav1.5 mRNA and protein expression were lower in Scn5a+/− than wild-type (WT), with a further reduction in the RV compared with the left ventricle (LV). RVs showed higher expression levels of Kv4.2, Kv4.3 and KChIP2 in both Scn5a+/− and WT. Action potential upstroke velocity and maximum Na+ current (INa) density were correspondingly decreased in Scn5a+/−, with a further reduction in the RV. The voltage dependence of inactivation was shifted to more negative values in Scn5a+/−. These findings are predictive of a localized depolarization abnormality leading to slowed conduction. Persistent Na+ current (IpNa) density was decreased in a similar pattern to INa. RV transient outward current (Ito) density was greater than LV in both WT and Scn5a+/−, and had larger time constants of inactivation. These findings were also consistent with the observation that AP durations were smallest in the RV of Scn5a+/−, fulfilling predictions of an increased heterogeneity of repolarization as an additional possible electrophysiological mechanism for arrhythmogenesis in BrS

Protons released during pancreatic acinar cell secretion acidify the lumen and contribute to pancreatitis in mice

Secretory granules are acidic; cell secretion will therefore lead to extracellular acidification. We propose that during secretion, protons co-released with proteins from secretory granules of pancreatic acinar cells acidify the restricted extracellular space of the pancreatic lumen to regulate normal physiological and pathophysiological functions in this orga

Archaerhodopsin Selectively and Reversibly Silences Synaptic Transmission through Altered pH

Tools that allow acute and selective silencing of synaptic transmission in vivo would be invaluable for understanding the synaptic basis of specific behaviors. Here, we show that presynaptic expression of the proton pump archaerhodopsin enables robust, selective, and reversible optogenetic synaptic silencing with rapid onset and offset. Two-photon fluorescence imaging revealed that this effect is accompanied by a transient increase in pH restricted to archaerhodopsin-expressing boutons. Crucially, clamping intracellular pH abolished synaptic silencing without affecting the archaerhodopsin-mediated hyperpolarizing current, indicating that changes in pH mediate the synaptic silencing effect. To verify the utility of this technique, we used trial-limited, archaerhodopsin-mediated silencing to uncover a requirement for CA3-CA1 synapses whose afferents originate from the left CA3, but not those from the right CA3, for performance on a long-term memory task. These results highlight optogenetic, pH-mediated silencing of synaptic transmission as a spatiotemporally selective approach to dissecting synaptic function in behaving animals

Electrically evoked dendritic pH transients in rat cerebellar Purkinje cells

Our aim was to test the hypothesis that depolarization-induced intracellular pH (pHi) shifts in restricted regions (dendrites) of mammalian neurones might be larger and faster than those previously reported from the cell soma. We used confocal imaging of the pH-sensitive dye, HPTS, to measure pH changes in both the soma and dendrites of whole-cell patch-clamped rat cerebellar Purkinje cells. In the absence of added CO2–HCO3−, depolarization to +20 mV for 1 s caused large (≈0.14 pH units) and fast dendritic acid shifts, whilst the somatic acidifications were significantly smaller (≈0.06 pH units) and slower. The pHi shifts were smaller in the presence of 5 % CO2–25 mm HCO3−-buffered saline (≈0.08 pH units in the dendrites and ≈0.03 pH units in the soma), although a clear spatiotemporal heterogeneity remained. Acetazolamide (50 μM) doubled the size of the dendritic acid shifts in the presence of CO2–HCO3−, indicating carbonic anhydrase activity. Removal of extracellular calcium or addition of the calcium channel blocker lanthanum (0.5 mm) inhibited the depolarization-evoked acid shifts. We investigated more physiological pHi changes by evoking modest bursts of action potentials (≈10 s duration) in CO2–HCO3−-buffered saline. Such neuronal firing induced an acidification of ≈0.11 pH units in the fine dendritic regions, but only ≈0.03 pH units in the soma. There was considerable variation in the size of the pHi shifts between cells, with dendritic acid shifts as large as 0.2-0.3 pH units following a 10 s burst of action potentials in some Purkinje cells. We postulate that these large dendritic pHi changes (pH microdomains) might act as important signals in synaptic function