Inhibition of the Na⁺-K⁺ ATPase by ouabain reduces AMPA-evoked adenosine release.

<p>Adenosine biosensor recordings from BFB and CTX from slices were perfused with zero Ca²⁺ aCSF. A) Comparison of 5 µM AMPA application (black lines) alone with simultaneous application of 100 µM ouabain and 5 µM AMPA (red lines). The data are presented as averages ±SD (thin lines) of 4 experiments for each condition. When applied simultaneously with AMPA the ouabain caused the adenosine release to be cut short and truncated compared to the adenosine release evoked by AMPA alone. B) Preincubation with 100 µM ouabain, once the adenosine levels had stabilized, greatly reduced the response to AMPA (5 µM). Inset: Summary graph showing that prior treatment with ouabain greatly inhibits the adenosine release evoked by 5 µM AMPA (mean ± sem).</p

Na⁺-dependent concentrative nucleoside transporters (CNTs) and equilibrative nucleoside transporters (ENTs) do not play a role in AMPA-evoked adenosine efflux.

<p>A) Preincubation of slices (for 1 h) with a competing substrate for the CNTs, 100 µM uridine, had no effect on adenosine release in basal forebrain (BFB) or cortex (CTX). B) Application of 200 µM phloridzin, a general blocker of Na⁺-dependent transporters including the CNTs had no effect on adenosine release either. C) Addition of the ENT blocker dipyramidole (DIPY; 20 µM) prior to AMPA application caused a significant reduction in BFB adenosine release but not CTX. D) Application of both 20 µM DIPY and 10 µM NBTI (another ENT blocker) however did not significantly reduce adenosine release in BFB or CTX.</p

Biosensor placement and example of SBFI loading.

<p>A) Photograph of the slice in the holding chamber demonstrating biosensor placement in basal forebrain (BFB) and cortex. B) Representative example of SBFI loading at 340 nm excitation, showing loaded neurons (red arrows) and astrocytes (green arrows).</p

AMPA receptor evoked adenosine release depends upon a Na⁺ influx in cortex and basal forebrain.

<p>A) Adenosine biosensor recording, showing that when 80% of Na⁺ was substituted with NMDG⁺, AMPA (5 µM) could not evoke adenosine release in basal forebrain (BFB) or cortex (CTX). Re-introduction of Na⁺ restored AMPA-evoked adenosine release. B) Summary data showing that AMPA-evoked adenosine release in BFB and cortex is completely dependent on extracellular Na⁺ (n = 6). C) High K⁺-evoked adenosine release depends upon extracellular Na⁺ in BFB but not in cortex (n = 6). D,E) AMPA-evoked adenosine release was substantially blocked by TTX. f) TTX also blocked high K⁺-evoked adenosine release in cortex (control, n = 10; TTX n = 12). Statistical comparisons via Student's unpaired t-test.</p

Adenosine release depends upon activation of the Na⁺-K⁺ ATPase via accumulation of intracellular Na⁺.

<p>A) Simultaneous measurement of intracellular concentration of Na⁺ with SBFI, and adenosine release during AMPA receptor activation (5 µM AMPA) in basal forebrain. Note the increase in intracellular Na⁺ precedes the adenosine release recorded by the biosensor (ADO). Black trace, neuron; green trace, astrocyte. B) Application of 100 µM ouabain caused adenosine release in BFB and cortex in presence of normal extracellular Ca²⁺. In the absence of extracellular Ca²⁺ ouabain caused either no change in extracellular adenosine or a reduction in adenosine. C) In BFB, application of zero Ca²⁺ aCSF caused an increase in intracellular Na⁺ in neurons (back, blue, red) and astrocytes (green) traces as measured by SBFI. Simultaneous recording with an adenosine biosensor showed an increase in extracellular adenosine following shortly after this increase in intracellular Na⁺. When ouabain was applied, intracellular Na⁺ levels increased in all cells, but the extracellular adenosine levels initially fell (arrow), before increasing as ouabain washed out. D) In cortex a similar pattern was seen: zero Ca²⁺ aCSF caused an increase in intracellular Na⁺ in neurons (back, blue, red) accompanied after a short delay by the release of adenosine measured by the nearby biosensor. When 100 µM ouabain was applied, adenosine levels fell (arrow) despite the increase in intracellular Na⁺.</p

Median data from Evolutionary adaptation of the sensitivity of Connexin26 hemichannels to CO₂

Median data for 4 species at 3 levels of PCO<sub>2</sub

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 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

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