Spontaneous adenosine release frequency and concentration in the caudate-putamen during stroke.

<p>All data and statistics are for n = 10 animals. (A) The average event adenosine concentration per transient for normoxia (n = 829) and ischemia-reperfusion (n = 1306) was not significantly different (unpaired t-test, n = 10 animals, p = 0.57) (B) Average event adenosine concentration per transient were divided into 30 min periods during normoxia, ischemia and reperfusion periods and event concentration did not show any significant difference (One-way ANOVA, p = 0.9354) (C) The cumulative adenosine concentration was significantly different after stroke compared to normoxia (paired t-test, n = 10 animals, p = 0.03). (D) Cumulative adenosine concentration was divided into 30 min periods. The first four bars are 30 min periods during normoxia, then one bar for the 30 min of ischemia, followed by three 30 min bars for the total 90 min of reperfusion. The dashed lines show the average for the normoxia and ischemia-reperfusion periods. Cumulative concentration during normoxia, ischemia and reperfusion was significantly different (One-way ANOVA, p = 0.0022).</p

Transient Adenosine Modulates Serotonin Release Indirectly in the Dorsal Raphe Nuclei

Rapid adenosine transiently regulates dopamine and glutamate via A1 receptors, but other neurotransmitters, such as serotonin, have not been studied. In this study, we examined the rapid modulatory effect of adenosine on serotonin release in the dorsal raphe nuclei (DRN) of mouse brain slices by using fast-scan cyclic voltammetry. To mimic adenosine release during damage, a rapid microinjection of adenosine at 50 pmol was applied before electrical stimulation of serotonin release. Transient adenosine significantly reduced electrically evoked serotonin release in the first 20 s after application, but serotonin release recovered to baseline as adenosine was cleared from the slice. The continuous perfusion of adenosine did not change the evoked serotonin release. Surprisingly, the modulatory effects of adenosine were not regulated by A1 receptors as adenosine still inhibited serotonin release in A1KO mice and also after perfusion of an A1 antagonist (8-cyclopentyl-1,3-dipropyl xanthine). The inhibition was also not regulated by A3 receptors as perfusion of the A3 antagonist (MRS 1220) in A1KO brain slices did not eliminate the inhibitory effects of transient adenosine. In addition, adenosine also inhibited serotonin release in A2AKO mice, showing that A2A did not modulate serotonin. However, perfusion of a selective 5HT1A autoreceptor antagonist drug [(S)-WAY 100135 dihydrochloride] abolished the inhibitory effect of transient adenosine on serotonin release. Thus, the transient neuromodulatory effect of adenosine on DRN serotonin release is regulated by serotonin autoreceptors and not by adenosine receptors. Rapid, transient adenosine modulation of neurotransmitters such as serotonin may have important implications for diseases such as depression and brain injury

TEM images of control rat, ischemia induced and A_2A antagonist administered brain slices.

<p>TEM images of the nucleus from rat brain caudate-putamen. Cerebral neurons in sham rats showed (a) normal cell nucleus (arrow) and (b) normal mitochondria (*). Rats after 30 min of BCCAO and 8h of reperfusion led to substantial changes to organelle structure. After BCCAO, (c) cell nucleus appears to have shrunken with condensed chromatin (arrow). (d) Mitochondria are swollen (*) and with disorganized cristae. Rats after the administration A_2A antagonist SCH 442416 prior to 30 min of BCCAO and 8h of reperfusion showed (e) normal cell nucleus (arrow) and (f) normal mitochondria (*) with no disruption to cell membrane. This indicates that A_2A antagonist administration prior to the cerebral ischemia proven to be neuroprotective.</p

Rapid, Sensitive Detection of Neurotransmitters at Microelectrodes Modified with Self-assembled SWCNT Forests

Carbon nanotube (CNT) modification of microelectrodes can result in increased sensitivity without compromising time response. However, dip coating CNTs is not very reproducible and the CNTs tend to lay flat on the electrode surface which limits access to the electroactive sites on the ends. In this study, aligned CNT forests were formed using a chemical self-assembly method, which resulted in more exposed CNT ends to the analyte. Shortened, carboxylic acid functionalized single-walled CNTs were assembled from a dimethylformamide (DMF) suspension onto a carbon-fiber disk microelectrode modified with a thin iron hydroxide-decorated Nafion film. The modified electrodes were highly sensitive, with 36-fold higher oxidation currents for dopamine using fast-scan cyclic voltammetry than bare electrodes and 34-fold more current than electrodes dipped in CNTs. The limit of detection (LOD) for dopamine was 17 ± 3 nM at a 10 Hz repetition rate and 65 ± 7 nM at 90 Hz. The LOD at 90 Hz was the same as a bare electrode at 10 Hz, allowing a 9-fold increase in temporal resolution without a decrease in sensitivity. Similar increases were observed for other cationic catecholamine neurotransmitters, and the increases in current were greater than for anionic interferents such as ascorbic acid and 3,4-dihydroxyphenylacetic acid (DOPAC). The CNT forest electrodes had high sensitivity at 90 Hz repetition rate when stimulated dopamine release was measured in Drosophila. The sensitivity, temporal resolution, and spatial resolution of these CNT forest modified disk electrodes facilitate enhanced electrochemical measurements of neurotransmitter release in vivo

Rat schematic diagram and experimental timeline of normoxia, ischemia and reperfusion.

<p>Top: Rat schematic diagram showing the placement of the occluders around the common carotid artery and placement of carbon fiber microelectrode into the rat brain. Bottom: Timeline diagram of normoxia, ischemia and reperfusion periods.</p

Effect of stroke on the number of adenosine transients in the caudate-putamen.

<p>All data and statistics are for n = 10 animals. Example concentration traces and false color plots showing the number of transients during (A) normoxia (B) ischemia and (C) reperfusion over 180 s time window. There are more transients during ischemia and reperfusion than during normoxia. (D) The average number of adenosine transients during normoxia (2 h) and I-R periods (2 h) is significantly different (paired t-test, n = 10 animals, p = 0.0138). (E) Average number of adenosine transients, divided into 30 min periods. The first four bars are 30 min time periods during normoxia, then one bar for the 30 min of ischemia, followed by three 30 min bars for reperfusion. The dashed lines show the average for the normoxia and ischemia-reperfusion periods. The number of transients during normoxia, ischemia and reperfusion were significantly different (One-way ANOVA, p < 0.0001).</p

Effect of the A_2A antagonist, SCH442416 (3 mg/kg. i.p), on adenosine on adenosine transients during stroke.

<p>All data and statistics are for n = 8 animals. (A) Number of adenosine transients decreased significantly after SCH442416 during stroke (paired t-test, p = 0.01). (B) Inter-event time of all adenosine transients. The exponential fit (black line) during normoxia (predrug) is y = 0.5981e^-0.0207x (R² = 0.99) and SCH442416 + ischemia-reperfusion (red line) is y = 0.4491e^-0.0155x (R² = 0.99). After SCH442416 treatment, the inter-event time was significantly longer during stroke, with a median inter-event time change from 39 s (normoxia) to 48 s (I-R). There was a significant difference between the distributions before and after stroke (KS-test, p = p < 0.0001). (C) The average event adenosine concentration per transient after SCH442416 administration during ischemia and reperfusion 0.13 ± 0.01 μM (n = 759 transients) compared to normoxia 0.14 ± 0.01 μM (n = 1030 transients) was not significantly different (unpaired t-test, n = 8 animals, p = 0.05) (D) There was no significant change in the median cumulative concentration after SCH442416 during stroke compared to normoxia (p = 0.11).</p

Control experiments with no ischemia.

<p>All data and statistics are for n = 7 animals (A) Number of adenosine transients did not change between the 1^st and 2^nd 2 hour periods (paired t-test, p = 0.48). (B) Inter-event time of all adenosine transients. The exponential fit (black line) in the first 2 h is y = 0.5592e^-0.0207x (R² = 0.99) and in the second 2 h (grey line) is y = 0.5195e^-0.0172x (R² = 0.99). There was no significant difference between the underlying distributions in the first 2 h and second 2 h (KS-test, n = 7 animals, p = 0.6). (C) The average event adenosine concentration per transient. There was no significant change in the average adenosine concentration per transient for first 2 hrs (n = 1084) and second 2 hrs (n = 1064) (unpaired t-test, n = 7 animals, p = 0.12). (D) There was no significant change in the mean cumulative concentration between the 1^st and 2^nd 2 hour periods (paired t-test, p = 0.55).</p

Detection of Adenosine <i>in vitro</i> and <i>in vivo</i> using fast scan cyclic voltammetry.

<p>(A) <i>In vitro</i> calibration of adenosine. A 3-D color plot (middle) depicts the time on the x-axis, potential on the y-axis, and current in false color. The primary oxidation at +1.4 V (large green oval in center of color plot) and the secondary oxidation at +1.0 V (green/purple oval below center oval). The current vs time plot (top) shows the change in current in the presence of adenosine. (B) <i>In vivo</i> spontaneous, transient adenosine event, detected in rat caudate-putamen. The 3-D color plot shows primary and secondary oxidation peaks that match the in vitro calibration. The current vs time plot (top) shows the change in current due to the spontaneous adenosine transient.</p

Sawhorse Waveform Voltammetry for Selective Detection of Adenosine, ATP, and Hydrogen Peroxide

Fast-scan cyclic voltammetry (FSCV) is an electrochemistry technique which allows subsecond detection of neurotransmitters <i>in vivo</i>. Adenosine detection using FSCV has become increasingly popular but can be difficult because of interfering agents which oxidize at or near the same potential as adenosine. Triangle shaped waveforms are traditionally used for FSCV, but modified waveforms have been introduced to maximize analyte sensitivity and provide stability at high scan rates. Here, a modified sawhorse waveform was used to maximize the time for adenosine oxidation and to manipulate the shapes of cyclic voltammograms (CVs) of analytes which oxidize at the switching potential. The optimized waveform consists of scanning at 400 V/s from −0.4 to 1.35 V and holding briefly for 1.0 ms followed by a ramp back down to −0.4 V. This waveform allows the use of a lower switching potential for adenosine detection. Hydrogen peroxide and ATP also oxidize at the switching potential and can interfere with adenosine measurements <i>in vivo</i>; however, their CVs were altered with the sawhorse waveform and they could be distinguished from adenosine. Principal component analysis (PCA) was used to determine that the sawhorse waveform was better than the triangle waveform at discriminating between adenosine, hydrogen peroxide, and ATP. In slices, mechanically evoked adenosine was identified with PCA and changes in the ratio of ATP to adenosine were observed after manipulation of ATP metabolism by POM-1. The sawhorse waveform is useful for adenosine, hydrogen peroxide, and ATP discrimination and will facilitate more confident measurements of these analytes <i>in vivo</i>