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

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

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

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