The role of ryanodine receptors in development

PhDCalcium ions (Ca2+) are fundamental to the regulation of many cellular processes; however, the coordination of these signals during embryogenesis is not well understood. Ryanodine receptors (RyR) are a family of important intracellular ion channels that are responsible for the release of Ca2+ and they regulate the cytosolic Ca2+ concentration. Humans have three differentially expressed ryr genes (ryr1, ryr2 and ryr3) and mutations can cause both skeletal and cardiac diseases. Although the primary function of RyR is to mediate excitation-contraction coupling in muscle, they may also regulate Ca2+ signalling during developmental processes. The project has addressed the role of RyR during embryonic development, using the zebrafish as an in vivo vertebrate model. Five zebrafish RyR genes (ryr1a, ryr1b, ryr2a, ryr2b and ryr3) were characterised and a comprehensive overview of their spatial and temporal expression in the embryo was determined. At 24 hours post-fertilisation (hpf), ryr1a, ryr1b and ryr3 are expressed in the skeletal muscle, ryr2a in specific neuronal populations and ryr2b in the cardiac muscle. Semi-quantitative PCR data and wholemount in situ hybridisation revealed strong maternal expression of ryr3 during the cleavage and blastula periods and into adulthood. The early expression of the ryr3 gene suggests that this receptor functions during the initial stages of development; a role that has not been described previously. The functional significance of RyR3 during early embryogenesis was investigated in a loss-of- 3 function model using antisense morpholino oligonucleotides. The ryr3 specific knockdown experiments appeared to affect the establishment of embryonic axis prior to the segmentation periods (before 10 hpf). In addition, by 19 to 20 hpf ryr3 morphants failed to exhibit spontaneous muscle contractions and displayed a defect in neuromuscular development. In conclusion, this study has characterised the ryr genes and provided an overview on their temporal and spatial expression. The work provides evidence that ryr3 expression provides the Ca2+ vital for myofibrils organisation and that is required for the spontaneous movements during zebrafish embryonic development. The knowledge of RyR tissue distribution in zebrafish has provided a strong foundation for loss-of-function studies aimed at addressing their role in development. In the long term, the work will also facilitate more focused studies on disease.School of Biological and Chemical Sciences Queen Mary University of London. Central Research Fund and Physiological Society Travel Grant

Sleep-wake sensitive mechanisms of adenosine release in the basal forebrain of rodents : an in vitro study

Adenosine acting in the basal forebrain is a key mediator of sleep homeostasis. Extracellular adenosine concentrations increase during wakefulness, especially during prolonged wakefulness and lead to increased sleep pressure and subsequent rebound sleep. The release of endogenous adenosine during the sleep-wake cycle has mainly been studied in vivo with microdialysis techniques. The biochemical changes that accompany sleep-wake status may be preserved in vitro. We have therefore used adenosine-sensitive biosensors in slices of the basal forebrain (BFB) to study both depolarization-evoked adenosine release and the steady state adenosine tone in rats, mice and hamsters. Adenosine release was evoked by high K+, AMPA, NMDA and mGlu receptor agonists, but not by other transmitters associated with wakefulness such as orexin, histamine or neurotensin. Evoked and basal adenosine release in the BFB in vitro exhibited three key features: the magnitude of each varied systematically with the diurnal time at which the animal was sacrificed; sleep deprivation prior to sacrifice greatly increased both evoked adenosine release and the basal tone; and the enhancement of evoked adenosine release and basal tone resulting from sleep deprivation was reversed by the inducible nitric oxide synthase (iNOS) inhibitor, 1400 W. These data indicate that characteristics of adenosine release recorded in the BFB in vitro reflect those that have been linked in vivo to the homeostatic control of sleep. Our results provide methodologically independent support for a key role for induction of iNOS as a trigger for enhanced adenosine release following sleep deprivation and suggest that this induction may constitute a biochemical memory of this state

Differential adenosine release in basal forebrain due to seasonal sleep patterns of Djungarian hamsters.

<p>Slices from Djungarian hamsters kept in either long photoperiod (n = 15) or short (n = 14) light cycles were sacrificed two hours after the end of the dark phase were used. Both AMPA-evoked ADO’ release (a) and basal ADO’ tone (b) in the BFB were significantly greater in long day cycle hamsters (Mann-Whitney test, p<0.05).</p

2h sleep deprivation causes an increase in basal forebrain adenosine release in rats.

<p>Adenosine release evoked by AMPA in slices from 2 h SD rats was compared to control rats sacrificed at the same point in the diurnal cycle without sleep deprivation. (a) Raw data traces of representative experiments of sleep deprived and control rats adjusted to the adenosine calibration. (b) Slices from 2 h SD rats (n = 13) had greater ADO’ responses than controls (n = 14) with the asterisk indicating significance (Mann-Whitney test, p<0.05). (c) The cumulative probability graph illustrates the distribution of individual ADO’ responses for control (black fill) and 2 h SD (white fill).</p

6h sleep deprivation causes iNOS-dependent increases in basal forebrain adenosine release in mice.

<p>Comparison of adenosine release and tone following 6 h sleep deprivation in mice either with or without 1400 W. (a) AMPA-evoked release was significantly higher in slices after 6 h SD (n = 15) than control (n = 10), but was not significantly greater than 6 h SD incubated with 1400 W. (b) Basal tone however was significantly greater in 6 h SD mice than when incubated with 1400 W and also controls (b). Asterisks indicate significant difference (Mann-Whitney test, p<0.05). (c) Raw data traces from representative experiments for control (black) 6 h SD (light grey) and 6 h SD +1400 W (dark grey) normalised to 10 µM ADO’ calibration for tone measurements are shown (c). Bold arrows indicate the point of sensor removal from the slice causing artefacts, and those on the right tone measured by difference before and after removal.</p

6h sleep deprivation causes an iNOS-dependent increase in adenosine release in rats.

<p>Adenosine release was compared between 6 h sleep deprived and non-sleep deprived rats sacrificed at the same point in the diurnal cycle in the presence and absence of 1400 W. (a) Raw data from representative experiments of non-SD controls (black), 6 h SD (light grey) and 6 h SD +1400 W (dark grey) experiments, normalized to 10 µM ADO calibration. (b) In the BFB, slices from 6 h SD (n = 14) showed greater ADO’ release than those from control rats (n = 13) and 6 h SD rats +1400 W (n = 14), and non-sleep deprived rats +1400 W (n = 8) rats were not significantly different from controls. (c) Cumulative probability distribution of individual peak ADO’ responses for control (black fill), 6 h SD (white fill) and 6 h SD +1400 W (grey fill) in BFB. (d) No conventional significant difference (6 h SD v. control p = 0.09) was observed in basal adenosine tone for the same experiments in BFB. In the cortex, there were no significant differences in either the AMPA-evoked release (e) or basal tone (f). Asterisks indicate statistical significance (Mann Whitney U test, p<0.05).</p

Adenosine release occurs independently of calcium.

<p>Adenosine release was evoked by 5 µM AMPA in Ca²⁺-free aCSF (0 mM Ca²⁺ and 1 mM EGTA) with 20 µM CPA added to drain internal calcium stores, and then again after following wash in of normal 2 mM Ca²⁺ aCSF. (a) Raw data traces from a representative experiment in cortex (top) and BFB (bottom). Adenosine responses were not significantly different in Ca²⁺ -free conditions in either BFB (b) or cortex (c) by the Mann-Whitney U-test, n = 6.</p

6h sleep deprivation causes an increase in iNOS expression in BFB and cortex.

<p>iNOS Immunofluorescence (green) was largely absent in BFB slices from rats not sleep deprived after 1 h post-sacrifice incubation in aCSF (a), but strong after 6 h SD with 1 h (b) and 4 h (c) post-sacrifice incubation. The blue immunfluorescence is DAPI, showing nuclei. Double immunfluorescence staining for iNOS and ChaT (red) in BFB for a 6 h SD rat after 1 h incubation is shown in (d), single arrows indicate examples of somata with colocalised ChaT and iNOS, double arrows somata with iNOS but no ChaT. In the cortex, iNOS immunfluorescence after 4 h incubation was present in non-SD rats (e), but less strong than those after 6 h SD (f).</p

AMPA-evoked adenosine release varies with diurnal cycle in the basal forebrain.

<p>Animals were sacrificed at various times in the diurnal cycle and adenosine release from slices evoked by 5 µM AMPA. (a) In rat BFB, adenosine release varied with time of sacrifice, n values for each point as indicated in the figure. (b) Cumulative probability distributions of individual peak ADO’ responses at ZT 2 and 14 in rats. (c) ADO’ responses were not affected by the time slices were left to incubate following sacrifice and preparation, illustrated for slices used at ZT 6. (d) In mice, greater BFB ADO’ tone was also observed after wake periods as indicated by recordings at ZT 2 and ZT 14. Asterisks indicate significant differences, p<0.05, Mann-Whitney U test.</p