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Characterization of Spontaneous, Transient Adenosine Release in the Caudate-Putamen and Prefrontal Cortex

Characterization of Spontaneous, Transient Adenosine Release in the Caudate-Putamen and... Adenosine is a neuroprotective agent that inhibits neuronal activity and modulates neurotransmission. Previous research has shown adenosine gradually accumulates during pathologies such as stroke and regulates neurotransmission on the minute-to-hour time scale. Our lab developed a method using carbon-fiber microelectrodes to directly measure adenosine changes on a sub-second time scale with fast-scan cyclic voltammetry (FSCV). Recently, adenosine release lasting a couple of seconds has been found in murine spinal cord slices. In this study, we characterized spontaneous, transient adenosine release in vivo, in the caudate-putamen and prefrontal cortex of anesthetized rats. The average concentration of adenosine release was 0.1760.01 mM in the caudate and 0.1960.01 mM in the prefrontal cortex, although the range was large, from 0.04 to 3.2 mM. The average duration of spontaneous adenosine release was 2.960.1 seconds and 2.860.1 seconds in the caudate and prefrontal cortex, respectively. The concentration and number of transients detected do not change over a four hour period, suggesting spontaneous events are not caused by electrode implantation. The frequency of adenosine transients was higher in the prefrontal cortex than the caudate-putamen and was modulated by A receptors. The A 1 1 antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine, 6 mg/kg i.p.) increased the frequency of spontaneous adenosine release, while the A agonist CPA (N -cyclopentyladenosine, 1 mg/kg i.p.) decreased the frequency. These findings are a paradigm shift for understanding the time course of adenosine signaling, demonstrating that there is a rapid mode of adenosine signaling that could cause transient, local neuromodulation. Citation: Nguyen MD, Lee ST, Ross AE, Ryals M, Choudhry VI, et al. (2014) Characterization of Spontaneous, Transient Adenosine Release in the Caudate-Putamen and Prefrontal Cortex. PLoS ONE 9(1): e87165. doi:10.1371/journal.pone.0087165 Editor: Gilberto Fisone, Karolinska Inst, Sweden Received September 9, 2013; Accepted December 19, 2013; Published January 29, 2014 Copyright:  2014 Nguyen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This reasearch was supported by the National Institute of Health (R01NS076875). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared no competing interests exist. * E-mail: bjv2n@virginia.edu Recently, electrochemical sensors were developed for rapid Introduction measurements of changes in local adenosine concentration. Adenosine is an important neuroprotective modulator in the Enzyme biosensors can quantitate adenosine with a time brain that regulates neurotransmission and blood flow. Adenosine resolution of 2 seconds [8]. Using these biosensors, electrically- increases in the brain during pathological events, such as ischemia stimulated adenosine release in the cerebellum was measured that [1] and seizures [2], where it can act as a retaliatory metabolite. lasted 100 seconds [9]. Our lab developed fast-scan cyclic The increases in adenosine during these pathologies typically last voltammetry (FSCV) at carbon-fiber microelectrodes [10] to for minutes to hours [3]. For example, the concentration of directly detect electrically-stimulated adenosine release in the adenosine doubled one minute following ischemia and was ten- caudate-putamen that lasts only 10–40 seconds [11]. The fold larger twenty minutes afterwards [4]. During hypoxia, concentration of stimulated release was regulated by A receptors adenosine activates inhibitory A adenosine receptors a couple [12]. Using FSCV, the Zylka group recently discovered sponta- of minutes after onset, which decreases cAMP concentrations, neous adenosine transients. The adenosine lasted less than 2 hyperpolarizes neurons, and prevents excitatory firing [5,6]. While seconds in the extracellular space and was a result of extracellular these studies demonstrate adenosine signaling on a longer time ATP metabolism [13,14]. These studies establish that adenosine scale, there is growing evidence that adenosine also signals on a can be released on a more rapid time scale; however, the much shorter time scale. characteristics and regulation of rapid adenosine signaling in vivo Using electrophysiological techniques, Dunwiddie’s group are not well understood. explored a rapid modulatory role for adenosine in the brain. In this study, we measured spontaneous, transient adenosine During very short electrical stimulations (1–5 pulses at 100 Hz), release in vivo for the first time. Spontaneous adenosine release, not adenosine regulated glutamate receptor-mediated excitatory post- evoked by electrical stimulation, was measured in the caudate- synaptic potentials (EPSPs) in the hippocampus in an activity putamen and the prefrontal cortex of the anesthetized rat. The dependent manner [7]. The duration of change in EPSPs was only duration of direct adenosine release in vivo was only about 3 2 seconds, implying fast adenosine changes were responsible. seconds, a time scale that matches the previous electrophysiolog- However, the experiment did not directly measure adenosine ical study [15]. The concentration of adenosine release was on concentration on a rapid time scale. average 0.18 mM but a large range of concentrations was detected. PLOS ONE | www.plosone.org 1 January 2014 | Volume 9 | Issue 1 | e87165 Spontaneous, Transient Adenosine Release The frequency of spontaneous adenosine events was one transient electrodes with 1 M KCl. Bare electrodes were soaked in 2- propanol for at least 10 minutes before use. every 2–3 minutes and was modulated by A receptors; however, the events follow a random, not a regular firing pattern. The Fast-scan cyclic voltammetry (FSCV) was used to monitor spontaneous adenosine transients observed in vivo were similar in adenosine with sub-second temporal resolution [10]. Waveform concentration, duration, and frequency to what others have found generation and data collection was computer controlled by Tar in murine spinal cord slices [13], suggesting transient adenosine Heel CV (gift of Mark Wightman, UNC at Chapel Hill) [21]. A release is common in multiple regions in the nervous system and Dagan ChemClamp potentiostat (Dagan Corporation; Minneap- across different species. This study demonstrates that adenosine is olis, MN, USA) was used to collect data. Electrical stimulations rapidly released and cleared in the brain and may have a rapid were applied with a BSI-950 Bi-Phasic Stimulus Isolator (Dagan). neuromodulatory role in addition to the previously characterized Electrodes were continuously held at 20.40 V and scanned to function as a long-term modulator. 1.45 V and back every 100 milliseconds against a Ag/AgCl reference electrode, at a rate of 400 V/s. All data was background subtracted to remove any non-Faradic currents by averaging Experimental Methods 10 CVs from no more than 20 seconds before the analysis point. Ethics Electrodes were calibrated using flow injection analysis [22] with All animal experiments were carried out under strict accordance 1.0 mM adenosine in PBS made fresh daily from the 10 mM stock with the recommendations in the Guide for the Care and Use of solution. Pre- and post-calibrations were performed and no Laboratory Animals of the National Institutes of Health. The significant difference in adenosine oxidation current was found protocol was approved by the Institutional Animal Care and Use after electrode implantation (data not shown). Committee of the University of Virginia (Protocol Number 3517). All surgery was performed under urethane anesthesia and all Animals and Surgery efforts were made to minimize suffering. Male Sprague-Dawley Rats (250–350 g; Charles-River, Wil- mington, MA, USA) were housed in a vivarium with 12-h light/ Chemicals dark cycles and provided food and water ad libitum. The rats were All reagents were purchased from Fisher Scientific (Fair Lawn, anesthetized with 50% wt urethane (Sigma Aldrich) solution in saline (Baxter; Deerfield, IL, USA) (1.5 g/kg, i.p). The surgical site NJ, USA) unless otherwise stated. Phosphate buffered saline (PBS) was used to calibrate electrodes containing (in mM): 131.25 NaCl, was shaved and 0.25 mL of bupivicaine (SensorcaineH MPF, APP Pharmaceuticals, LLC; Schaumburg, IL, USA) was administered 3.0 KCl, 10.0 NaH PO , 1.2 MgCl , 2.0 Na SO , and 1.2 CaCl 2 4 2 2 4 2 with the pH adjusted to 7.4. Sodium phosphate was purchased subcutaneously for local analgesia. After exposing the skull, holes were drilled for placement of electrodes using a stereotaxic drill from RICCA Chemical Company (Arlington, TX, USA). All [23]. Adenosine transients were measured in either the caudate or aqueous solutions were prepared with deionized water (Milli-Q Biocel; Millipore, Billerica, MA, USA). Adenosine was prepared as the prefrontal cortex in each rat. The coordinates for the caudate- putamen are (in mm from bregma): anterior-posterior (AP): +1.2, a 10 mM stock solution in 0.1 M HClO and stored in the refrigerator. mediolateral (ML): +2.0, and dorsoventral (DV): 24.5. Coordi- nates for the prefrontal cortex are: AP: +2.7, ML: +0.8, and DV: DPCPX (6 mg/kg, i.p., 8-cyclopentyl-1,3-dipropylxanthine, 23.0. The Ag/AgCl reference electrode was inserted on the Sigma Aldrich) was dissolved in dimethylsulfoxide (DMSO). contralateral side of the brain. The rat’s body temperature was CPA (N -cyclopentyladenosine) was purchased from Tocris maintained at 37uC using a heating pad with a thermistor probe Bioscience (Ellisville, MO, USA), dissolved in saline, and (FHC, Bowdoin, ME, USA). administered at 1 mg/kg. These doses were chosen as large doses that were previously used in the literature [12,16,17]. Data Collection and Analysis Electrodes were implanted into the tissue and allowed to Electrodes and FSCV equilibrate with the waveform being applied for at least 30 minutes Carbon-fiber microelectrodes were prepared as previously before any data collection. After equilibration, data was contin- described [18]. Briefly, cylindrical microelectrodes were prepared uously collected and if no transients were found after 30 minutes with 7 mm diameter T-650 carbon-fibers (Cytec Engineering or if the electrode baseline was unstable, a new electrode was Materials, West Patterson, NJ, USA). Fibers were aspirated into inserted until transients were observed. Up to three electrodes per glass capillaries (1.2 mm60.68 mm; A-M Systems, Inc., Seqium, animal were inserted to search for transient adenosine release. Any WA, USA), pulled by a vertical pipette puller (model PE-21; animals with fewer than four transients in the hour long pre-drug Narishige, Tokyo, Japan) into two microelectrodes, and the time period were excluded. The overall success rate for finding extended fiber cut with a scalpel to about 50 mm. The fiber/glass transients was 80% in both the caudate-putamen and in the interface was sealed with epoxy [Epon resin 828] (Miller- prefrontal cortex. After electrode placement was deemed optimal, Stephenson Chemical Co. Inc.; Danbury, CT, USA) and 14% one hour of pre-drug data was collected and then drug was wt m-phenylenediamine heated to 80uC. Excess epoxy was rinsed injected. Nafion-CNT coated electrodes were placed in vivo and with acetone and electrodes were dried overnight at room data collected for one hour. temperature, then cured at 100uC for two hours, and then at 150uC overnight. The Nafion-CNT coated electrodes were prepared as previously described [19]. High pressure carbon Principal Component Analysis monoxide conversion single-walled CNTs (Carbon Nanotechnol- All adenosine transients were qualitatively and quantitatively ogies, Houston, TX, USA) were functionalized as described here analyzed using High Definition Cyclic Voltammetry (HDCV) [20]. The electrodes were dipped in 0.05 mg/mL CNTs Analysis software (from Mark Wightman, UNC at Chapel Hill). A suspended 5% wt Nafion in methanol (Ion Power, New Castle, training set was compiled for each rat of the five largest, most DE, USA) for 5 minutes, air dried for 10 seconds, placed in the definitive adenosine transients with a clear secondary peak. The oven for 10 minutes at 70uC, and stored at room temperature largest transients were chosen because they were easily identifiable, overnight. Electrical connections were created by backfilling the whereas smaller transients created poor principal component PLOS ONE | www.plosone.org 2 January 2014 | Volume 9 | Issue 1 | e87165 Spontaneous, Transient Adenosine Release correlations. Principal components were extracted from the training set and all data was analyzed using principal component regression [24]. This produced an adenosine concentration vs. time trace that was used to identify and determine the amount and duration of each transient. Every training set has residuals which account for currents from unknown signals, such as noise [25]. The sum of squares of the residuals for each variable, or the Q score, was calculated and any signal above Q failed and was not counted as an adenosine transient. With a limit of quantitation at ten times the noise for the secondary peak, the smallest transients that could be quantified were 40 nM. Any transient signal without a secondary peak or any sustained signal greater than 10 seconds, where the background could not be accurately subtracted out, was not counted. Statistics Statistics performed using GraphPad PRISM 6 (GraphPad Software Inc., San Diego, CA, USA), MatLABH (The Math- Works, Inc., Natick, MA, USA) and OriginPro 7.5 (OriginLab Corporation, Northampton, MA, USA). Data presented as mean 6 SEM with n number of animals. A Kolmogorov-Smirnov (KS) test was used to determine underlying distributions between inter- Figure 1. Detection of adenosine in vitro and in vivo using fast- scan cyclic voltammetry at the same carbon-fiber microelec- event times (time between consecutive transients). All data was trode. (A) In vitro calibration of adenosine. A 3-D color plot (bottom) considered significant at the 95% confidence level. depicts time on the x-axis, potential on the y-axis, and current in false color. The primary oxidation occurs at 1.4 V (large green oval in center Results of color plot) and the secondary oxidation occurs at 1.0 V (green/purple oval below center oval). The dashed white line on the color plot Adenosine Detection using FSCV denotes where the background subtracted cyclic voltammogram (CV, top) is obtained. (B) In vivo spontaneous, transient adenosine event. The Spontaneous adenosine transients were monitored using FSCV, 3-D color plot and CV shows primary and secondary oxidation peaks which has sub-second temporal resolution, allowing real time that match the in vitro calibration. measurements of adenosine changes in the brain. No information doi:10.1371/journal.pone.0087165.g001 about basal levels is obtained with FSCV because all data are background subtracted. The carbon-fiber microelectrode was from spontaneous adenosine release and not ATP release. Due to scanned from 20.40 V to 1.45 V and back at 10 Hz. Adenosine the labor intensive process of making Nafion-coated electrodes and undergoes two sequential, two electron oxidations [26] that the loss of temporal resolution, bare electrodes were used in all the produce two peaks in the background-subtracted cyclic voltam- other experiments. mogram (CV) [10] (Fig. 1A top). Both oxidation peaks are evident Representative color plots and matching CVs from the same in the color plot of an electrode calibration with 1.0 mM adenosine electrode are shown for an in vitro calibration of adenosine (Fig. 1A) (Fig. 1A). The primary oxidation occurs at 1.4 V and the and a spontaneous adenosine transient (Fig. 1B) in the caudate- secondary oxidation at 1.0 V is slightly delayed in time from the putamen. The cyclic voltammograms of in vitro adenosine and main oxidation peak because the secondary product forms after spontaneous adenosine release in vivo show a clear primary the primary product is produced. oxidation peak at 1.4 V and a definitive secondary peak at Spontaneous adenosine release was identified by its two 1.0 V. The CV for the calibration was similar to the in vivo oxidation peaks in the CVs and color plots, which help distinguish transient and there was an R correlation value of 0.81 between adenosine from other compounds with similar oxidation potentials the two CVs. The color plots illustrate that both have a minor including hydrogen peroxide [27], histamine [28], and hypoxan- delay in the secondary peak formation. Stimulated adenosine thine [29]. In addition, the slight delay in the formation of the release has been previously characterized in our laboratory [11], second peak helps identify the analyte as adenosine [30]. While but the secondary peak was not consistently observed due to the adenosine and ATP have similar cyclic voltammograms, carbon- overall small signals and other chemical changes. The CVs for fiber electrodes are more sensitive for adenosine than ATP [10] stimulated adenosine can be convoluted with residual dopamine, and ATP breaks down to adenosine in the extracellular space pH, and oxygen changes [33]. Thus, stimulated adenosine release within 200 milliseconds [31]. As an additional test to confirm was not a good comparison for identifying spontaneous adenosine adenosine was detected and not ATP, we used Nafion-CNT release. coated electrodes, which are three times more sensitive for adenosine than ATP compared to bare carbon-fiber microelec- Automated Identification of Spontaneous Adenosine trodes. Spontaneous adenosine transients were found with Nafion- CNT electrodes implanted in the caudate-putamen with similar Transients concentrations (Nafion-CNT average = 0.1360.01 mM, n=3, An automated system was needed to identify transient unpaired t-test, p = 0.1751). The duration of the Nafion-CNT adenosine release events without bias. Principal components transients were longer than those detected with bare electrodes analysis (PCA) [34] has been previously used to identify (average = 3.860.2 seconds, n = 3, unpaired t-test, p = 0.0002). spontaneous dopamine transients [35]; therefore we adapted Nafion is known to slow the temporal response of electrodes so PCA for use with adenosine. Unlike dopamine release [36], the longer durations are due to a slower electrode response [32]. The CV of adenosine is not constant over time, due to the secondary similar magnitude of transient release suggests that the signal is product having a delayed increase in current. Figure 2A shows a PLOS ONE | www.plosone.org 3 January 2014 | Volume 9 | Issue 1 | e87165 Spontaneous, Transient Adenosine Release color plot of a spontaneous adenosine transient with a current vs. 95% confidence level), and be above 40 nM, the quantitation time trace (top) through the primary (orange) and secondary limit. (black) oxidation peaks overlaid. The maximum signal at the second peak is 1.0 second after the first peak, demonstrating the Concentration of Spontaneous Adenosine Transients lag time between the primary and secondary oxidation peaks. Transient adenosine release was examined in the caudate- Cyclic voltammograms (Fig. 2B) were taken at sequential times putamen and prefrontal cortex. The color plots and concentration and show that the CV of adenosine release changes over time. The vs. time profiles of example adenosine transients show the first CV has a primary oxidation peak and no secondary peak. magnitude of concentration released, as well as the duration of Half a second later, the primary peak is more prominent while the adenosine in the extracellular space in the caudate-putamen secondary peak has grown, and in the third CV, the secondary and (Fig. 3A) and the prefrontal cortex (Fig. 3C). The average primary peak are almost equal in magnitude. The CVs for the adenosine concentration was 0.1760.01 mM in the caudate- training set of spontaneous adenosine release were taken at the putamen and 0.1960.01 mM in the prefrontal cortex, which is apex of the primary oxidation peak, which gives the most accurate significantly different (n= 30 and 29 rats, t-test, p = 0.0238). Table 1 calibration of adenosine. Although the residuals were higher for gives the average, SEM, and range for the concentration in each adenosine than for dopamine because of the variable CVs, brain region. The range of recorded adenosine transients was adenosine transients were still easily determined at a 95% large, spanning almost two orders of magnitude. While the confidence level. majority of adenosine release events were in the hundreds of nM A training set was created for each individual rat from five of the range, 1% of transients were greater than 1 mM, which largest, easily identifiable spontaneous adenosine transients, each demonstrates that large amounts of adenosine can be spontane- containing a characteristic secondary peak. From the training set, ously released. To further investigate spontaneous adenosine the eigenvalues were calculated for CVs of varying concentrations release, concentrations were placed in 0.05 mM bins and of adenosine. The highest eigenvalues correspond to the principal histograms examined for both brain regions. The histograms have components with the highest variance, and thus the best normal distributions and are overlaid with Gaussian fits with correlation to the data [37]. A residual Q-score from the training positive skews, which is expected when non-negative values are set was used to reject any data that did not significantly match the excluded. In the caudate-putamen (Fig. 3B) over half of the values principal components. After removing the residuals, the concen- fall between 0.05 and 0.15 mM, although the average value was tration and duration of spontaneous adenosine release was 0.17 mM, a reflection of the positive skew. Similarly, in the determined from a concentration vs. time plot. The limit of prefrontal cortex (Fig. 3D), a little less than half of the quantitation for our electrodes was 40 nM, so any transients with measurements fall between 0.05 and 0.15 mM concentrations concentrations below this value were excluded. Below 40 nM, the and the average is 0.19 mM. Both graphs show that although the secondary peak is difficult to identify and is obscured by noise. bulk of transient adenosine events are in the 0.10 mM range, about Thus, for a transient to be considered adenosine, it had to have a 4% of release is 0.50 mM and higher. secondary peak, be below the residual Q-score (corresponding to Figure 2. Detection of spontaneous, transient adenosine release in vivo. (A) In vivo spontaneous, transient adenosine release. The current vs. time plot has two traces, an orange line at 1.4 V for the primary oxidation and a black line at 1.2 V for the secondary oxidation. The dashed lines on st the color plot and current vs. time plots indicate where CVs were taken. (B) Cyclic voltammograms of adenosine over time. The top (1 ) is when nd rd adenosine first appears, the middle CV (2 ) is half a second later when the primary peak is at a maximum and the bottom (3 ) is half a second later when the secondary peak is at its maximum. The ratio of the primary and secondary oxidation peaks can change over time. doi:10.1371/journal.pone.0087165.g002 PLOS ONE | www.plosone.org 4 January 2014 | Volume 9 | Issue 1 | e87165 Spontaneous, Transient Adenosine Release Figure 3. Spontaneous adenosine transient concentration. (A) A color plot of a spontaneous adenosine transient in the caudate putamen with a corresponding concentration vs. time plot above. (B) Caudate-putamen concentration histogram. The y-axis is relative frequency. All concentrations from the first hour of data collection in the caudate putamen were placed into 0.05 mM bins (x-axis) and fit with a Gaussian (x{0:09549) {0:5 0:4588 distribution (red line). The Gaussian fit equation is y~0:3512 e (R = 0.9327, n= 30 rats). The mean and median are marked. The majority of transients are in the 100–200 nM range. (C) An example color plot and concentration vs. time plot in the prefrontal cortex. (D) The histogram of (x{0:1305) {0:5 0:07079 concentrations in the prefrontal cortex fit a Gaussian distribution with the equation: y~0:2538 e (R = 0.9505, n = 29 rats). doi:10.1371/journal.pone.0087165.g003 seconds (Table 1). Histograms of the duration of spontaneous Duration of Transient Adenosine Release adenosine transients were plotted using 0.5 second bins for the The duration of adenosine release provides information about caudate-putamen (Fig. 4B) and the prefrontal cortex (Fig. 4D). how long adenosine is available for signaling in the extracellular The plots are overlaid with a Gaussian distribution fit with positive space. Color plots of typical adenosine transients with correspond- skews. The majority of transients lasted only 2–4 seconds, but ing concentration vs. time plots from PCA (above) show adenosine outliers were present in both brain regions. Thus, adenosine is only is rapidly cleared in the caudate-putamen (Fig. 4A) and the available for signaling for a few seconds. prefrontal cortex (Fig. 4C). From the top plots the duration was calculated as the amount of time adenosine was over 10% (horizontal dashed line) of the peak concentration for each Frequency of Adenosine Transients transient to eliminate any effects from noise in the baseline. The On average, spontaneous, transient adenosine release occurs average duration of an adenosine transient was 2.960.1 seconds in once every several minutes (Table 1). The inter-event times, or the the caudate-putamen and 2.860.1 seconds in the prefrontal time between consecutive transients, were calculated to examine if cortex, which is not significantly different (n= 30 and 29 rats, t-test, the adenosine transients were regularly spaced. Histograms of p = 0.0826). The duration ranged from less than a second to ten inter-event times are plotted for the caudate (Fig. 5A) and PLOS ONE | www.plosone.org 5 January 2014 | Volume 9 | Issue 1 | e87165 Spontaneous, Transient Adenosine Release significantly longer than the durations of the first and second bins Table 1. Averages for spontaneous adenosine release. in the caudate-putamen (Bonferroni post-test, one-way ANOVA p.0.05); however, since the sixth bin was lower, we conclude there is no trend of increasing duration over time. Figures 6C and 6F Caudate-Putamen show the average number of transient release events normalized in Avg. SEM Range 30 minute time bins. The large error bars were due to the large variability in the number of transients from animal to animal. Concentration (mM) 0.17 0.01 0.04–2.5 There was no significant effect of time on the average number of Duration (s) 2.9 0.1 0.8–9.9 transients, indicating time after implantation had no effect in the Inter-event Time (s) *156 8 1.4–1405 caudate-putamen (n = 6, one-way ANOVA, p = 0.4782) or the prefrontal cortex (n = 6, one-way ANOVA, p = 0.9299). These Prefrontal Cortex results indicate that the number, concentration, or duration of adenosine transients does not change for at least three hours Avg. SEM Range following electrode implantation. Concentration (mM) 0.19 0.01 0.04–3.2 Duration (s) 2.8 0.1 0.3–10 A Receptor Modulation Previous studies found A receptors modulate stimulated Inter-event Time (s) *108 6 0.9–2069 adenosine release [12], so we tested the effects of A receptor Legend: Data from the caudate-putamen and prefrontal cortex. Concentration drugs on spontaneous adenosine release. For all drug experiments, was significantly different between the two regions (n= 588 transients in 30 rats after the electrode was equilibrated, one hour of baseline data was and 804 transients in 29 rats, t-test, p = 0.0238, respectively). The duration of collected and then the drug administered. DPCPX, an A receptor transient adenosine was not different between the caudate and prefrontal 1 cortex (n= 30 and 29 rats, t-test, p = 0.0826). * Inter-event times, or the time antagonist, was administered at 6 mg/kg, i.p. The CVs of between consecutive transients have significantly different underlying adenosine transients observed after DPCPX were similar to those distributions between the two regions (n = 30 and 29 rats, KS test, p = 0.0338). observed before drug, indicating that DPCPX did not interfere doi:10.1371/journal.pone.0087165.t001 with the detection of adenosine using FSCV. The concentration and duration of adenosine release were compared before and after prefrontal cortex (Fig. 5B), with the median and mean values DPCPX using paired t-tests to examine release in the same animal. marked. Relative frequency is plotted on the y-axis and 30 second DPCPX did not significantly affect the concentration of release in time bins are on the x-axis. The distribution was not Gaussian and the caudate (n= 6, paired t-test, p = 0.1186) or prefrontal cortex shows that adenosine transients occur closer together than (n= 5, paired t-test, p = 0.8547). However, in the caudate-putamen, expected from mean inter-event values. The average time between DPCPX significantly increased the duration from 2.6 to 3.0 transients for the caudate putamen was 156 seconds, however, the seconds (n = 6, paired t-test, p = 0.0092). The duration of adenosine majority of transients occurred within 2 minutes of each other. release in the prefrontal cortex increased from 2.4 to 2.9 seconds Similarly, in the prefrontal cortex, the mean inter-event time was after DPCPX, but the increase was not significant (n= 5, paired t- 108 seconds, but a majority of transients happened less than a test, p = 0.1300). Following DPCPX administration, there was a minute apart. Thus, adenosine release occurs randomly and is not decrease in the mean and median inter-event times and a a result of pacemaker firing. Color plots show that occasionally significant difference in the underlying frequency distribution in spontaneous release events occur within a couple of seconds of the caudate-putamen (n = 6 rats, KS test, p.0.0001) (Fig. 7A and each other (Fig. 5A inset) and the amount of adenosine release 7B) and the prefrontal cortex (n = 5 rats, KS test, p = 0.0286) does not always decrease with the sequential release (Fig. 5B inset). (Fig. 7C and 7D). The time between events decreased after The underlying distributions between the two brain regions DPCPX administration from a median of 141 seconds pre-drug to were compared and the patterns are significantly different (n=30 63 seconds in the caudate and from 80 seconds to 60 seconds in and 29 rats, Kolmogorov-Smirnov test, p = 0.0338). Thus, the time the prefrontal cortex. Therefore, blocking A receptors with between adenosine transient events was shorter in the prefrontal DPCPX in both brain regions increases the frequency of cortex than in the caudate-putamen. An exponential decay was fit spontaneous, transient adenosine release. to the data and plotted on the histogram. The exponential fit in 6 The A agonist, N -cyclopentyladenosine (CPA), was adminis- the caudate-putamen has a rate constant of 0.00891 s or a firing tered to determine if activation of A receptors affected rate of every 112 seconds. The rate constant in the prefrontal spontaneous adenosine transients. There was no significant change cortex is 0.0141 s or a firing rate of every 71 seconds. in adenosine concentration in either the caudate or the prefrontal cortex following administration of CPA (paired t-test, n= 6, Adenosine Transients Continue Over Time p = 0.9818; n= 6, p = 0.4607, respectively). However, CPA did To examine adenosine transients over time, data were collected increase the duration of adenosine transients in the caudate from continuously over three hours. Figures 6A and 6D display the 2.7 to 3.4 seconds (n= 6, paired t-test, p = 0.0195) and from 3.2 to average concentration of adenosine transients in 30 minute bins in 3.8 seconds in the prefrontal cortex (n = 6, paired t-test, the caudate and prefrontal cortex, respectively. The average p = 0.0259). The drugs may interfere with uptake or metabolism, concentration in both brain regions was around 0.13 mM and did thus increasing duration. In the caudate-putamen, the median and not significantly differ with time (one-way ANOVA, caudate: mean inter-event times increased after CPA (Fig. 8A and 8B) and n=6, p= 0.2013; prefrontal cortex: n=6, p= 0.6268). The the underlying distributions were significantly different than pre- duration of adenosine release was also binned into 30 minute drug (n = 6 rats, KS test, p = 0.0308). The time between events epochs for the caudate-putamen (Fig. 6B) and the prefrontal cortex increased from a median of 75 seconds to 98 seconds following (Fig. 6E). There was a significant effect of time on event duration CPA in the caudate putamen, the opposite effect on inter-event between the 30 minute bins in the caudate-putamen (n = 6, one- time as DPCPX. However, in the prefrontal cortex (Fig. 8C and way ANOVA, p = 0.0005) but not the prefrontal cortex (n = 6, one- 8D), CPA did not significantly change the underlying distribution way ANOVA, p = 0.8449). The duration of the fifth bin was and the inter-event times were not different (n = 6 rats, KS test, PLOS ONE | www.plosone.org 6 January 2014 | Volume 9 | Issue 1 | e87165 Spontaneous, Transient Adenosine Release Figure 4. Spontaneous adenosine duration. (A) A color plot and concentration vs. time plot of adenosine transients in the caudate-putamen. The horizontal dashed line is 10% of the concentration of the first peak and the vertical lines are when the baseline value crosses this value, showing the duration. (B) Caudate-putamen duration histogram. The y-axis is relative frequency and the x-axis shows 0.5 second bins. The Gaussian (x{2:554) {0:5 0:9908 distribution equation is y~0:1886 e (red line, R = 0.9712, n = 30 rats). (C) Color plot and concentration vs. time plot of an example spontaneous adenosine transient in the prefrontal cortex. The duration is marked with vertical lines. (D) Adenosine duration histogram for the (x{2:556) {0:5 0:1060 prefrontal cortex is plotted with a Gaussian distribution equation y~0:1823 e (red line, R = 0.9765, n = 29 rats). doi:10.1371/journal.pone.0087165.g004 p = 0.9299). Thus, A activation with CPA decreased the 1 Discussion frequency of spontaneous transient adenosine release in the Spontaneous, transient adenosine release occurs in both the caudate-putamen but not the prefrontal cortex. caudate-putamen and prefrontal cortex. Transients were on average a couple hundred nM, which is sufficient to activate adenosine receptors [38]. Adenosine was elevated for only a few PLOS ONE | www.plosone.org 7 January 2014 | Volume 9 | Issue 1 | e87165 Spontaneous, Transient Adenosine Release adenosine events but not the concentration. These studies reveal that large amounts of adenosine can be quickly released and cleared from the extracellular space, a contrast to previous studies which have documented a slower role for adenosine [39–41]. Thus, adenosine has a rapid signaling mode that may locally cause transient neuromodulation. The Concentration of Transient Release is Sufficient for Adenosine Receptor Activation The average concentration of adenosine released was 180 nM, but the amount varied widely from 40 nM to 3.2 mM. Sponta- neous adenosine release was the same order of magnitude as stimulated release in brain slices from the cerebellum [9], caudate- putamen [42] and prefrontal cortex [43]. However, evoked adenosine release in the caudate-putamen of anesthetized rats was typically 600–900 nM [11,12], larger than the average spontaneous transient. Electrical stimulation likely activates all cells but different firing rates or number of cells activated during spontaneous adenosine release could lead to lower release. In the prefrontal cortex, the concentration of adenosine release was significantly higher than in the caudate, although the difference was not large enough to suggest that different types of adenosine receptors are being activated in the two brain regions. Inhibitory A receptors and excitatory A receptors both have 1 2a affinities in the low nanomolar range [44] and have high to intermediate distributions in the caudate and prefrontal cortex [45]. About one percent of transients had concentrations greater than 1 mM, which could be sufficient to activate A and A 2b 3 receptors [46]. These large transients demonstrate the potential for transient, micromolar adenosine signaling and high amounts of receptor activation. Spontaneous Adenosine Release is Random and the Frequency is Regulated by A Receptors Adenosine release occurred on average once every 3–4 minutes; however, about half of the transients occurred within two minutes of each other in the caudate and one minute of each other in the prefrontal cortex. Spontaneous adenosine release is not periodic and consequently is unlikely to be caused by pacemaker firing or to directly modulate a rhythmic process such as breathing or tonic cell firing [47,48]. Instead, release was random and the inter-event times fit an exponential decay, which arises from a discrete Poisson process [49] where each transient is not dependent on the prior Figure 5. Histograms of inter-event times. (A) Inter-event adenosine event. The shortest time between transients was less histograms in the caudate-putamen. The time between consecutive transients (termed the inter-event time) was calculated and plotted for than one second, indicating a long time is not required to reset the first hour of data collection. The x-axis shows 30 second time bins adenosine release (Fig. 5A and 5B insets). Some of the larger and the y-axis is relative frequency of inter-event times. Median, mean transients may be two or more release events that occur 20.00891x 2 and exponential fit (y = 0.242 e (R = 0.9926)) are plotted on the simultaneously and cannot be temporally resolved. The frequency histogram. The inset plots show an example of three consecutive and concentration of release did not change over three hours, transients. (B) Inter-event histograms for the prefrontal cortex. The 20.0141x 2 demonstrating that transient adenosine continues for long periods exponential fit is y = 0.388 e (R = 0.9933). The inset color plot shows an example of two transients that occurred close together. The of time and that adenosine release is not just a response to underlying distribution of inter-event times was significantly different immediate damage after electrode implantation [9,13,50]. between the caudate-putamen and prefrontal cortex (n = 30 and 29 A receptors are inhibitory adenosine receptors with low nM animals, 588 and 804 inter-event times respectively, Kolmogorov- affinities that downregulate cAMP, hyperpolarize neurons, and Smirnov test, p = 0.0338). The time between transients is shorter in the can be neuroprotective during ischemia [38]. The cortex and prefrontal cortex. doi:10.1371/journal.pone.0087165.g005 striatum have intermediate to high levels of A receptor expression [45]. Previously, A receptors were shown to have autoreceptor characteristics and regulate the amount of stimulated adenosine seconds and these are the most rapid adenosine changes that have release in the caudate in vivo [12]. Here, A receptors modulated been measured in vivo. While adenosine transients occurred on 1 the frequency of spontaneous adenosine release but not the average once every several minutes, at least 30% of transients were concentration. DPCPX, an A antagonist, decreased the inter- less than one minute apart and there was no regularity to event times in both brain regions. CPA, an A agonist, had the adenosine release. A receptors modulated the time between 1 opposite effect and increased the inter-event times in the caudate- PLOS ONE | www.plosone.org 8 January 2014 | Volume 9 | Issue 1 | e87165 Spontaneous, Transient Adenosine Release Figure 6. Spontaneous transient adenosine over time. All x-axes are time in 30 minute bins and all y-axes were normalized to the first bin. (A) Normalized concentrations of adenosine transients in the caudate putamen. There was no significant effect of time on the concentration (n=6 animals, one-way ANOVA, p = 0.2013). (B) Duration of adenosine release from the caudate-putamen. There was a main effect of time on durations (n = 6 animals, one-way ANOVA, p = 0.0005). (C) Number of transients in the caudate-putamen. There was no significant effect of time on the number of transients (n = 6 animals, one-way ANOVA, p = 0.4782). (D) Concentrations in the prefrontal cortex. There was no significant effect of time on the concentration (n = 6 animals, one-way ANOVA, p = 0.6268). (E) Durations of transient adenosine in the prefrontal cortex. There was no significant effect of time on the duration (n = 6 animals, one-way ANOVA, p = 0.8449). (F) Number of transients from the prefrontal cortex. There was no significant effect of time on the number of transients (n = 6 animals, one-way ANOVA, p = 0.9299). doi:10.1371/journal.pone.0087165.g006 putamen but did not have a significant effect in the prefrontal cies of spontaneous adenosine transients were also similar, with cortex. A receptors are part of a feedback loop controlling events occurring every two to three minutes. The comparable transient adenosine release in the brain, where activation of A adenosine transients in different regions of rats and mice, along receptors decreases transient adenosine events. A receptor with previous electrophysiology work in the hippocampus impli- modulation does not affect concentration suggesting that the cating fast adenosine release regulating glutamate receptor receptors do not control synthesis or the amount packaged into excitability [7], show that transient adenosine release is a common vesicles. Instead, A receptors modulate event frequency, such as feature in the nervous system. These transients could be an regulating the number of docking or release events. important mechanism of adenosine signaling, facilitating rapid neuromodulation throughout the brain. Spontaneous Adenosine Release Occurs in Multiple Brain Spontaneous Adenosine Release Provides Local, Regions Transient Modulation Spontaneous, transient adenosine release was measured in both the caudate-putamen and prefrontal cortex. Basal adenosine levels Spontaneous adenosine signaling was fast, with the average are higher in the prefrontal cortex than the caudate-putamen [51– transient lasting only about three seconds. This mode of transient 53]. Similarly, the concentration of transient adenosine release is signaling is a contrast to gradual adenosine buildup for minutes in larger in the prefrontal cortex. However, transient adenosine the extracellular space during pathological events, such as release is unlikely to contribute to the basal levels of adenosine ischemia [54]. Spontaneous adenosine transients were even more because of the rapid clearance. Spontaneous release was more rapid than electrically-stimulated adenosine release, which elevat- frequent in the prefrontal cortex than in the caudate, which is ed adenosine for up to 20 seconds in the caudate-putamen [11] or similar to electrically-evoked release which was also more 100 seconds in the cerebellum [9]. Our time course matched well frequently observed in the prefrontal cortex [43]. The higher with previous measurement of transient adenosine release in spinal frequency of adenosine release in the prefrontal cortex could be cord slices [13] and electrophysiology studies that observed a due to either additional release sites or more release events per site. transient, 2 second variation in glutamate-evoked EPSPs after The spontaneous adenosine transients measured here in the rat stimulated adenosine release [15]. The short duration demon- strates that the adenosine clearance mechanism from the caudate and cortex were remarkably similar to the spontaneous adenosine transients measured in murine slices from lamina II of extracellular space is very rapid, likely due to uptake [44]. Future the spinal cord by Zylka’s group [13]. Despite the differences in studies will examine the effects of adenosine clearance mecha- nisms. Since the duration of adenosine release was the same in brain region, preparation (slices vs. in vivo), and species, the average concentrations were the same order of magnitude both brain regions, the mechanism of clearance is expected to be conserved between the caudate and prefrontal cortex. (180 nM in rats compared to 570 nM in mouse slices). Differences could be a result of different numbers of cells activated or the The time course of spontaneous adenosine signaling is similar to amount of adenosine available for release. The average frequen- that of transient, exocytotic release events of neurotransmitters. PLOS ONE | www.plosone.org 9 January 2014 | Volume 9 | Issue 1 | e87165 Spontaneous, Transient Adenosine Release Figure 7. Effect of the A antagonist, DPCPX (6 mg/kg, i.p.), on adenosine transients. (A) Inter-event time histograms of first hour of pre- 20.0186 2 drug in caudate. Median, mean and exponential fit (green line) (y = 0.295 e (R = 0.7889)) are plotted on the frequency distribution. (B) Inter- 20.0101x 2 event time histogram for transients in the first hour post-DPCPX in caudate. The exponential fit (red line) is y = 0.318 e (R = 0.9490). In the caudate, there was a significant difference between the underlying distributions before and after DPCPX (n = 6 animals, KS-test, p,0.0001). (C) Inter- 20.0101x 2 event histograms pre- and (D) post-DPCPX in the prefrontal cortex. The exponential fit equations are y = 0.260 e (R = 0.9124) pre-drug and 20.0111x 2 y = 0.340 e (R = 0.9851) after DPCPX. In the prefrontal cortex, there was a significant difference between underlying distributions pre- and post-DPCPX (n = 5 animals, KS-test, p = 0.0287). doi:10.1371/journal.pone.0087165.g007 For example, dopamine signaling after phasic firing lasts only [56]. Therefore, only receptors close to the release event would be three to four seconds and dopamine is rapidly cleared from the activated and transient adenosine release would provide local extracellular space [55]. While our study did not address the neuromodulation, close to the site of release. In addition, there pathway of extracellular adenosine formation, the mechanism is could be heterogeneity within a region for receptor activation if likely breakdown of exocytotically released ATP or direct, activity- not all areas experienced adenosine transients at the same time. dependent release of adenosine. Recent evidence shows transient, The local variation of release was not directly assessed in these electrically-stimulated adenosine is activity dependent and a studies, but moving the electrode did change the number of portion is not dependent on extracellular breakdown of ATP adenosine transients detected suggesting a spatial effect on release [9,42]. Spontaneous adenosine release in slices of the spinal cord events. of mice is ATP-dependent [13,14]. While the mechanism of The function of rapid adenosine release is likely transient spontaneous transient release is difficult to elucidate in vivo, future neuromodulation in the brain. Although most studies of adenosine experiments could be performed in brain slices to determine if function have not been performed on the seconds time scale, transient adenosine release is exocytotic or a downstream product adenosine is known to regulate cerebral blood flow and of ATP. neurotransmission on a longer time scale. It is likely that faster The rapid release and clearance of adenosine has two regulation of these processes is caused by transient adenosine consequences: adenosine can only signal locally and receptors will release. Adenosine increases cerebral vasodilation in less than 60 be activated transiently. The distance a molecule can travel in seconds [57] and also increases cerebral blood flow [58]. Transient three seconds in the extracellular space is only 10–20 micrometers adenosine release could increase the flow of blood to localized PLOS ONE | www.plosone.org 10 January 2014 | Volume 9 | Issue 1 | e87165 Spontaneous, Transient Adenosine Release Figure 8. Effect of the A agonist, CPA (1 mg/kg, i.p.), on adenosine transients. (A) Inter-event time histograms for the caudate. Median, 20.0115x 2 mean and exponential fit (green line) (y = 0.298 e (R = 0.9636)) are plotted on the frequency distribution. (B) Inter-event time histograms for 20.00787x 2 the first hour after CPA in caudate. The exponential fit (blue line) is y = 0.226 e (R = 0.9528). There was a significant difference between the underlying distributions before and after CPA (n = 6 animals, KS-test, p = 0.0308). (C) Inter-event histograms pre- and (D) post-CPA in the prefrontal 20.00983x 2 20.00938x 2 cortex. Exponential equations are y = 0.280 e (R = 0.9430) pre-drug and y = 0.264 e (R = 0.9337) after CPA. In the prefrontal cortex, there was no significant difference in the underlying distributions before and after CPA (n = 6 animals, KS-test, p = 0.9299). doi:10.1371/journal.pone.0087165.g008 areas in the brain that require an immediate boost in oxygen and Conclusions nutrients. Adenosine regulation of neurotransmitter release can be Spontaneous transient adenosine release was characterized for inhibitory; for example, adenosine inhibits acetylcholine, gluta- the first time in vivo. The spontaneous release in the caudate and mate, serotonin, dopamine, noradrenaline signaling by activation prefrontal cortex is fast, lasting only a few seconds, and large, in of A receptors [59]. Adenosine can also increase the release of the hundred nM range. The frequency of release was random, acetylcholine and glutamate through A receptor activation [59]. 2a higher in the prefrontal cortex, and modulated by A receptors. Although A and A adenosine receptor affinities are in the low 1 2a These findings are a paradigm shift for understanding the time nanomolar range, the effective in vivo EC is estimated to be course of adenosine signaling. Previous studies have documented a 600 nM [60], so changes on the order of hundreds of nanomolar long-term modulatory effect of adenosine during pathologies but concentrations could have significant physiological effects. This here we demonstrate a new mode of transient adenosine signaling study shows that A receptors control the frequency of adenosine that could lead to rapid, local modulation. Future studies transients, but the downstream modulatory effects of the transient investigating the formation and function of this new type of adenosine activation of A receptors are not known and should be adenosine signaling will reveal how adenosine modulates on the explored in the future. A rapid, transient mode of adenosine second time scale and regulates local brain function. signaling would provide discrete, local neuromodulation, which could facilitate fine control of neurotransmission. PLOS ONE | www.plosone.org 11 January 2014 | Volume 9 | Issue 1 | e87165 Spontaneous, Transient Adenosine Release Acknowledgments Author Contributions Conceived and designed the experiments: BJV. Performed the experi- We would like to thank University of Virginia Alliance for Computational ments: MDN AER. 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Ann Biomed Eng 13: 321–328. hippocampal brain slices and the tonic inhibitory modulation of evoked 58. Soricelli A, Postiglione A, Cuocolo A, De CS, Ruocco A, et al. (1995) Effect of excitatory responses. J Pharmacol Exp Ther 268: 537–545. adenosine on cerebral blood flow as evaluated by single-photon emission PLOS ONE | www.plosone.org 13 January 2014 | Volume 9 | Issue 1 | e87165 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png PLoS ONE Pubmed Central

Characterization of Spontaneous, Transient Adenosine Release in the Caudate-Putamen and Prefrontal Cortex

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Abstract

Adenosine is a neuroprotective agent that inhibits neuronal activity and modulates neurotransmission. Previous research has shown adenosine gradually accumulates during pathologies such as stroke and regulates neurotransmission on the minute-to-hour time scale. Our lab developed a method using carbon-fiber microelectrodes to directly measure adenosine changes on a sub-second time scale with fast-scan cyclic voltammetry (FSCV). Recently, adenosine release lasting a couple of seconds has been found in murine spinal cord slices. In this study, we characterized spontaneous, transient adenosine release in vivo, in the caudate-putamen and prefrontal cortex of anesthetized rats. The average concentration of adenosine release was 0.1760.01 mM in the caudate and 0.1960.01 mM in the prefrontal cortex, although the range was large, from 0.04 to 3.2 mM. The average duration of spontaneous adenosine release was 2.960.1 seconds and 2.860.1 seconds in the caudate and prefrontal cortex, respectively. The concentration and number of transients detected do not change over a four hour period, suggesting spontaneous events are not caused by electrode implantation. The frequency of adenosine transients was higher in the prefrontal cortex than the caudate-putamen and was modulated by A receptors. The A 1 1 antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine, 6 mg/kg i.p.) increased the frequency of spontaneous adenosine release, while the A agonist CPA (N -cyclopentyladenosine, 1 mg/kg i.p.) decreased the frequency. These findings are a paradigm shift for understanding the time course of adenosine signaling, demonstrating that there is a rapid mode of adenosine signaling that could cause transient, local neuromodulation. Citation: Nguyen MD, Lee ST, Ross AE, Ryals M, Choudhry VI, et al. (2014) Characterization of Spontaneous, Transient Adenosine Release in the Caudate-Putamen and Prefrontal Cortex. PLoS ONE 9(1): e87165. doi:10.1371/journal.pone.0087165 Editor: Gilberto Fisone, Karolinska Inst, Sweden Received September 9, 2013; Accepted December 19, 2013; Published January 29, 2014 Copyright:  2014 Nguyen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This reasearch was supported by the National Institute of Health (R01NS076875). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared no competing interests exist. * E-mail: bjv2n@virginia.edu Recently, electrochemical sensors were developed for rapid Introduction measurements of changes in local adenosine concentration. Adenosine is an important neuroprotective modulator in the Enzyme biosensors can quantitate adenosine with a time brain that regulates neurotransmission and blood flow. Adenosine resolution of 2 seconds [8]. Using these biosensors, electrically- increases in the brain during pathological events, such as ischemia stimulated adenosine release in the cerebellum was measured that [1] and seizures [2], where it can act as a retaliatory metabolite. lasted 100 seconds [9]. Our lab developed fast-scan cyclic The increases in adenosine during these pathologies typically last voltammetry (FSCV) at carbon-fiber microelectrodes [10] to for minutes to hours [3]. For example, the concentration of directly detect electrically-stimulated adenosine release in the adenosine doubled one minute following ischemia and was ten- caudate-putamen that lasts only 10–40 seconds [11]. The fold larger twenty minutes afterwards [4]. During hypoxia, concentration of stimulated release was regulated by A receptors adenosine activates inhibitory A adenosine receptors a couple [12]. Using FSCV, the Zylka group recently discovered sponta- of minutes after onset, which decreases cAMP concentrations, neous adenosine transients. The adenosine lasted less than 2 hyperpolarizes neurons, and prevents excitatory firing [5,6]. While seconds in the extracellular space and was a result of extracellular these studies demonstrate adenosine signaling on a longer time ATP metabolism [13,14]. These studies establish that adenosine scale, there is growing evidence that adenosine also signals on a can be released on a more rapid time scale; however, the much shorter time scale. characteristics and regulation of rapid adenosine signaling in vivo Using electrophysiological techniques, Dunwiddie’s group are not well understood. explored a rapid modulatory role for adenosine in the brain. In this study, we measured spontaneous, transient adenosine During very short electrical stimulations (1–5 pulses at 100 Hz), release in vivo for the first time. Spontaneous adenosine release, not adenosine regulated glutamate receptor-mediated excitatory post- evoked by electrical stimulation, was measured in the caudate- synaptic potentials (EPSPs) in the hippocampus in an activity putamen and the prefrontal cortex of the anesthetized rat. The dependent manner [7]. The duration of change in EPSPs was only duration of direct adenosine release in vivo was only about 3 2 seconds, implying fast adenosine changes were responsible. seconds, a time scale that matches the previous electrophysiolog- However, the experiment did not directly measure adenosine ical study [15]. The concentration of adenosine release was on concentration on a rapid time scale. average 0.18 mM but a large range of concentrations was detected. PLOS ONE | www.plosone.org 1 January 2014 | Volume 9 | Issue 1 | e87165 Spontaneous, Transient Adenosine Release The frequency of spontaneous adenosine events was one transient electrodes with 1 M KCl. Bare electrodes were soaked in 2- propanol for at least 10 minutes before use. every 2–3 minutes and was modulated by A receptors; however, the events follow a random, not a regular firing pattern. The Fast-scan cyclic voltammetry (FSCV) was used to monitor spontaneous adenosine transients observed in vivo were similar in adenosine with sub-second temporal resolution [10]. Waveform concentration, duration, and frequency to what others have found generation and data collection was computer controlled by Tar in murine spinal cord slices [13], suggesting transient adenosine Heel CV (gift of Mark Wightman, UNC at Chapel Hill) [21]. A release is common in multiple regions in the nervous system and Dagan ChemClamp potentiostat (Dagan Corporation; Minneap- across different species. This study demonstrates that adenosine is olis, MN, USA) was used to collect data. Electrical stimulations rapidly released and cleared in the brain and may have a rapid were applied with a BSI-950 Bi-Phasic Stimulus Isolator (Dagan). neuromodulatory role in addition to the previously characterized Electrodes were continuously held at 20.40 V and scanned to function as a long-term modulator. 1.45 V and back every 100 milliseconds against a Ag/AgCl reference electrode, at a rate of 400 V/s. All data was background subtracted to remove any non-Faradic currents by averaging Experimental Methods 10 CVs from no more than 20 seconds before the analysis point. Ethics Electrodes were calibrated using flow injection analysis [22] with All animal experiments were carried out under strict accordance 1.0 mM adenosine in PBS made fresh daily from the 10 mM stock with the recommendations in the Guide for the Care and Use of solution. Pre- and post-calibrations were performed and no Laboratory Animals of the National Institutes of Health. The significant difference in adenosine oxidation current was found protocol was approved by the Institutional Animal Care and Use after electrode implantation (data not shown). Committee of the University of Virginia (Protocol Number 3517). All surgery was performed under urethane anesthesia and all Animals and Surgery efforts were made to minimize suffering. Male Sprague-Dawley Rats (250–350 g; Charles-River, Wil- mington, MA, USA) were housed in a vivarium with 12-h light/ Chemicals dark cycles and provided food and water ad libitum. The rats were All reagents were purchased from Fisher Scientific (Fair Lawn, anesthetized with 50% wt urethane (Sigma Aldrich) solution in saline (Baxter; Deerfield, IL, USA) (1.5 g/kg, i.p). The surgical site NJ, USA) unless otherwise stated. Phosphate buffered saline (PBS) was used to calibrate electrodes containing (in mM): 131.25 NaCl, was shaved and 0.25 mL of bupivicaine (SensorcaineH MPF, APP Pharmaceuticals, LLC; Schaumburg, IL, USA) was administered 3.0 KCl, 10.0 NaH PO , 1.2 MgCl , 2.0 Na SO , and 1.2 CaCl 2 4 2 2 4 2 with the pH adjusted to 7.4. Sodium phosphate was purchased subcutaneously for local analgesia. After exposing the skull, holes were drilled for placement of electrodes using a stereotaxic drill from RICCA Chemical Company (Arlington, TX, USA). All [23]. Adenosine transients were measured in either the caudate or aqueous solutions were prepared with deionized water (Milli-Q Biocel; Millipore, Billerica, MA, USA). Adenosine was prepared as the prefrontal cortex in each rat. The coordinates for the caudate- putamen are (in mm from bregma): anterior-posterior (AP): +1.2, a 10 mM stock solution in 0.1 M HClO and stored in the refrigerator. mediolateral (ML): +2.0, and dorsoventral (DV): 24.5. Coordi- nates for the prefrontal cortex are: AP: +2.7, ML: +0.8, and DV: DPCPX (6 mg/kg, i.p., 8-cyclopentyl-1,3-dipropylxanthine, 23.0. The Ag/AgCl reference electrode was inserted on the Sigma Aldrich) was dissolved in dimethylsulfoxide (DMSO). contralateral side of the brain. The rat’s body temperature was CPA (N -cyclopentyladenosine) was purchased from Tocris maintained at 37uC using a heating pad with a thermistor probe Bioscience (Ellisville, MO, USA), dissolved in saline, and (FHC, Bowdoin, ME, USA). administered at 1 mg/kg. These doses were chosen as large doses that were previously used in the literature [12,16,17]. Data Collection and Analysis Electrodes were implanted into the tissue and allowed to Electrodes and FSCV equilibrate with the waveform being applied for at least 30 minutes Carbon-fiber microelectrodes were prepared as previously before any data collection. After equilibration, data was contin- described [18]. Briefly, cylindrical microelectrodes were prepared uously collected and if no transients were found after 30 minutes with 7 mm diameter T-650 carbon-fibers (Cytec Engineering or if the electrode baseline was unstable, a new electrode was Materials, West Patterson, NJ, USA). Fibers were aspirated into inserted until transients were observed. Up to three electrodes per glass capillaries (1.2 mm60.68 mm; A-M Systems, Inc., Seqium, animal were inserted to search for transient adenosine release. Any WA, USA), pulled by a vertical pipette puller (model PE-21; animals with fewer than four transients in the hour long pre-drug Narishige, Tokyo, Japan) into two microelectrodes, and the time period were excluded. The overall success rate for finding extended fiber cut with a scalpel to about 50 mm. The fiber/glass transients was 80% in both the caudate-putamen and in the interface was sealed with epoxy [Epon resin 828] (Miller- prefrontal cortex. After electrode placement was deemed optimal, Stephenson Chemical Co. Inc.; Danbury, CT, USA) and 14% one hour of pre-drug data was collected and then drug was wt m-phenylenediamine heated to 80uC. Excess epoxy was rinsed injected. Nafion-CNT coated electrodes were placed in vivo and with acetone and electrodes were dried overnight at room data collected for one hour. temperature, then cured at 100uC for two hours, and then at 150uC overnight. The Nafion-CNT coated electrodes were prepared as previously described [19]. High pressure carbon Principal Component Analysis monoxide conversion single-walled CNTs (Carbon Nanotechnol- All adenosine transients were qualitatively and quantitatively ogies, Houston, TX, USA) were functionalized as described here analyzed using High Definition Cyclic Voltammetry (HDCV) [20]. The electrodes were dipped in 0.05 mg/mL CNTs Analysis software (from Mark Wightman, UNC at Chapel Hill). A suspended 5% wt Nafion in methanol (Ion Power, New Castle, training set was compiled for each rat of the five largest, most DE, USA) for 5 minutes, air dried for 10 seconds, placed in the definitive adenosine transients with a clear secondary peak. The oven for 10 minutes at 70uC, and stored at room temperature largest transients were chosen because they were easily identifiable, overnight. Electrical connections were created by backfilling the whereas smaller transients created poor principal component PLOS ONE | www.plosone.org 2 January 2014 | Volume 9 | Issue 1 | e87165 Spontaneous, Transient Adenosine Release correlations. Principal components were extracted from the training set and all data was analyzed using principal component regression [24]. This produced an adenosine concentration vs. time trace that was used to identify and determine the amount and duration of each transient. Every training set has residuals which account for currents from unknown signals, such as noise [25]. The sum of squares of the residuals for each variable, or the Q score, was calculated and any signal above Q failed and was not counted as an adenosine transient. With a limit of quantitation at ten times the noise for the secondary peak, the smallest transients that could be quantified were 40 nM. Any transient signal without a secondary peak or any sustained signal greater than 10 seconds, where the background could not be accurately subtracted out, was not counted. Statistics Statistics performed using GraphPad PRISM 6 (GraphPad Software Inc., San Diego, CA, USA), MatLABH (The Math- Works, Inc., Natick, MA, USA) and OriginPro 7.5 (OriginLab Corporation, Northampton, MA, USA). Data presented as mean 6 SEM with n number of animals. A Kolmogorov-Smirnov (KS) test was used to determine underlying distributions between inter- Figure 1. Detection of adenosine in vitro and in vivo using fast- scan cyclic voltammetry at the same carbon-fiber microelec- event times (time between consecutive transients). All data was trode. (A) In vitro calibration of adenosine. A 3-D color plot (bottom) considered significant at the 95% confidence level. depicts time on the x-axis, potential on the y-axis, and current in false color. The primary oxidation occurs at 1.4 V (large green oval in center Results of color plot) and the secondary oxidation occurs at 1.0 V (green/purple oval below center oval). The dashed white line on the color plot Adenosine Detection using FSCV denotes where the background subtracted cyclic voltammogram (CV, top) is obtained. (B) In vivo spontaneous, transient adenosine event. The Spontaneous adenosine transients were monitored using FSCV, 3-D color plot and CV shows primary and secondary oxidation peaks which has sub-second temporal resolution, allowing real time that match the in vitro calibration. measurements of adenosine changes in the brain. No information doi:10.1371/journal.pone.0087165.g001 about basal levels is obtained with FSCV because all data are background subtracted. The carbon-fiber microelectrode was from spontaneous adenosine release and not ATP release. Due to scanned from 20.40 V to 1.45 V and back at 10 Hz. Adenosine the labor intensive process of making Nafion-coated electrodes and undergoes two sequential, two electron oxidations [26] that the loss of temporal resolution, bare electrodes were used in all the produce two peaks in the background-subtracted cyclic voltam- other experiments. mogram (CV) [10] (Fig. 1A top). Both oxidation peaks are evident Representative color plots and matching CVs from the same in the color plot of an electrode calibration with 1.0 mM adenosine electrode are shown for an in vitro calibration of adenosine (Fig. 1A) (Fig. 1A). The primary oxidation occurs at 1.4 V and the and a spontaneous adenosine transient (Fig. 1B) in the caudate- secondary oxidation at 1.0 V is slightly delayed in time from the putamen. The cyclic voltammograms of in vitro adenosine and main oxidation peak because the secondary product forms after spontaneous adenosine release in vivo show a clear primary the primary product is produced. oxidation peak at 1.4 V and a definitive secondary peak at Spontaneous adenosine release was identified by its two 1.0 V. The CV for the calibration was similar to the in vivo oxidation peaks in the CVs and color plots, which help distinguish transient and there was an R correlation value of 0.81 between adenosine from other compounds with similar oxidation potentials the two CVs. The color plots illustrate that both have a minor including hydrogen peroxide [27], histamine [28], and hypoxan- delay in the secondary peak formation. Stimulated adenosine thine [29]. In addition, the slight delay in the formation of the release has been previously characterized in our laboratory [11], second peak helps identify the analyte as adenosine [30]. While but the secondary peak was not consistently observed due to the adenosine and ATP have similar cyclic voltammograms, carbon- overall small signals and other chemical changes. The CVs for fiber electrodes are more sensitive for adenosine than ATP [10] stimulated adenosine can be convoluted with residual dopamine, and ATP breaks down to adenosine in the extracellular space pH, and oxygen changes [33]. Thus, stimulated adenosine release within 200 milliseconds [31]. As an additional test to confirm was not a good comparison for identifying spontaneous adenosine adenosine was detected and not ATP, we used Nafion-CNT release. coated electrodes, which are three times more sensitive for adenosine than ATP compared to bare carbon-fiber microelec- Automated Identification of Spontaneous Adenosine trodes. Spontaneous adenosine transients were found with Nafion- CNT electrodes implanted in the caudate-putamen with similar Transients concentrations (Nafion-CNT average = 0.1360.01 mM, n=3, An automated system was needed to identify transient unpaired t-test, p = 0.1751). The duration of the Nafion-CNT adenosine release events without bias. Principal components transients were longer than those detected with bare electrodes analysis (PCA) [34] has been previously used to identify (average = 3.860.2 seconds, n = 3, unpaired t-test, p = 0.0002). spontaneous dopamine transients [35]; therefore we adapted Nafion is known to slow the temporal response of electrodes so PCA for use with adenosine. Unlike dopamine release [36], the longer durations are due to a slower electrode response [32]. The CV of adenosine is not constant over time, due to the secondary similar magnitude of transient release suggests that the signal is product having a delayed increase in current. Figure 2A shows a PLOS ONE | www.plosone.org 3 January 2014 | Volume 9 | Issue 1 | e87165 Spontaneous, Transient Adenosine Release color plot of a spontaneous adenosine transient with a current vs. 95% confidence level), and be above 40 nM, the quantitation time trace (top) through the primary (orange) and secondary limit. (black) oxidation peaks overlaid. The maximum signal at the second peak is 1.0 second after the first peak, demonstrating the Concentration of Spontaneous Adenosine Transients lag time between the primary and secondary oxidation peaks. Transient adenosine release was examined in the caudate- Cyclic voltammograms (Fig. 2B) were taken at sequential times putamen and prefrontal cortex. The color plots and concentration and show that the CV of adenosine release changes over time. The vs. time profiles of example adenosine transients show the first CV has a primary oxidation peak and no secondary peak. magnitude of concentration released, as well as the duration of Half a second later, the primary peak is more prominent while the adenosine in the extracellular space in the caudate-putamen secondary peak has grown, and in the third CV, the secondary and (Fig. 3A) and the prefrontal cortex (Fig. 3C). The average primary peak are almost equal in magnitude. The CVs for the adenosine concentration was 0.1760.01 mM in the caudate- training set of spontaneous adenosine release were taken at the putamen and 0.1960.01 mM in the prefrontal cortex, which is apex of the primary oxidation peak, which gives the most accurate significantly different (n= 30 and 29 rats, t-test, p = 0.0238). Table 1 calibration of adenosine. Although the residuals were higher for gives the average, SEM, and range for the concentration in each adenosine than for dopamine because of the variable CVs, brain region. The range of recorded adenosine transients was adenosine transients were still easily determined at a 95% large, spanning almost two orders of magnitude. While the confidence level. majority of adenosine release events were in the hundreds of nM A training set was created for each individual rat from five of the range, 1% of transients were greater than 1 mM, which largest, easily identifiable spontaneous adenosine transients, each demonstrates that large amounts of adenosine can be spontane- containing a characteristic secondary peak. From the training set, ously released. To further investigate spontaneous adenosine the eigenvalues were calculated for CVs of varying concentrations release, concentrations were placed in 0.05 mM bins and of adenosine. The highest eigenvalues correspond to the principal histograms examined for both brain regions. The histograms have components with the highest variance, and thus the best normal distributions and are overlaid with Gaussian fits with correlation to the data [37]. A residual Q-score from the training positive skews, which is expected when non-negative values are set was used to reject any data that did not significantly match the excluded. In the caudate-putamen (Fig. 3B) over half of the values principal components. After removing the residuals, the concen- fall between 0.05 and 0.15 mM, although the average value was tration and duration of spontaneous adenosine release was 0.17 mM, a reflection of the positive skew. Similarly, in the determined from a concentration vs. time plot. The limit of prefrontal cortex (Fig. 3D), a little less than half of the quantitation for our electrodes was 40 nM, so any transients with measurements fall between 0.05 and 0.15 mM concentrations concentrations below this value were excluded. Below 40 nM, the and the average is 0.19 mM. Both graphs show that although the secondary peak is difficult to identify and is obscured by noise. bulk of transient adenosine events are in the 0.10 mM range, about Thus, for a transient to be considered adenosine, it had to have a 4% of release is 0.50 mM and higher. secondary peak, be below the residual Q-score (corresponding to Figure 2. Detection of spontaneous, transient adenosine release in vivo. (A) In vivo spontaneous, transient adenosine release. The current vs. time plot has two traces, an orange line at 1.4 V for the primary oxidation and a black line at 1.2 V for the secondary oxidation. The dashed lines on st the color plot and current vs. time plots indicate where CVs were taken. (B) Cyclic voltammograms of adenosine over time. The top (1 ) is when nd rd adenosine first appears, the middle CV (2 ) is half a second later when the primary peak is at a maximum and the bottom (3 ) is half a second later when the secondary peak is at its maximum. The ratio of the primary and secondary oxidation peaks can change over time. doi:10.1371/journal.pone.0087165.g002 PLOS ONE | www.plosone.org 4 January 2014 | Volume 9 | Issue 1 | e87165 Spontaneous, Transient Adenosine Release Figure 3. Spontaneous adenosine transient concentration. (A) A color plot of a spontaneous adenosine transient in the caudate putamen with a corresponding concentration vs. time plot above. (B) Caudate-putamen concentration histogram. The y-axis is relative frequency. All concentrations from the first hour of data collection in the caudate putamen were placed into 0.05 mM bins (x-axis) and fit with a Gaussian (x{0:09549) {0:5 0:4588 distribution (red line). The Gaussian fit equation is y~0:3512 e (R = 0.9327, n= 30 rats). The mean and median are marked. The majority of transients are in the 100–200 nM range. (C) An example color plot and concentration vs. time plot in the prefrontal cortex. (D) The histogram of (x{0:1305) {0:5 0:07079 concentrations in the prefrontal cortex fit a Gaussian distribution with the equation: y~0:2538 e (R = 0.9505, n = 29 rats). doi:10.1371/journal.pone.0087165.g003 seconds (Table 1). Histograms of the duration of spontaneous Duration of Transient Adenosine Release adenosine transients were plotted using 0.5 second bins for the The duration of adenosine release provides information about caudate-putamen (Fig. 4B) and the prefrontal cortex (Fig. 4D). how long adenosine is available for signaling in the extracellular The plots are overlaid with a Gaussian distribution fit with positive space. Color plots of typical adenosine transients with correspond- skews. The majority of transients lasted only 2–4 seconds, but ing concentration vs. time plots from PCA (above) show adenosine outliers were present in both brain regions. Thus, adenosine is only is rapidly cleared in the caudate-putamen (Fig. 4A) and the available for signaling for a few seconds. prefrontal cortex (Fig. 4C). From the top plots the duration was calculated as the amount of time adenosine was over 10% (horizontal dashed line) of the peak concentration for each Frequency of Adenosine Transients transient to eliminate any effects from noise in the baseline. The On average, spontaneous, transient adenosine release occurs average duration of an adenosine transient was 2.960.1 seconds in once every several minutes (Table 1). The inter-event times, or the the caudate-putamen and 2.860.1 seconds in the prefrontal time between consecutive transients, were calculated to examine if cortex, which is not significantly different (n= 30 and 29 rats, t-test, the adenosine transients were regularly spaced. Histograms of p = 0.0826). The duration ranged from less than a second to ten inter-event times are plotted for the caudate (Fig. 5A) and PLOS ONE | www.plosone.org 5 January 2014 | Volume 9 | Issue 1 | e87165 Spontaneous, Transient Adenosine Release significantly longer than the durations of the first and second bins Table 1. Averages for spontaneous adenosine release. in the caudate-putamen (Bonferroni post-test, one-way ANOVA p.0.05); however, since the sixth bin was lower, we conclude there is no trend of increasing duration over time. Figures 6C and 6F Caudate-Putamen show the average number of transient release events normalized in Avg. SEM Range 30 minute time bins. The large error bars were due to the large variability in the number of transients from animal to animal. Concentration (mM) 0.17 0.01 0.04–2.5 There was no significant effect of time on the average number of Duration (s) 2.9 0.1 0.8–9.9 transients, indicating time after implantation had no effect in the Inter-event Time (s) *156 8 1.4–1405 caudate-putamen (n = 6, one-way ANOVA, p = 0.4782) or the prefrontal cortex (n = 6, one-way ANOVA, p = 0.9299). These Prefrontal Cortex results indicate that the number, concentration, or duration of adenosine transients does not change for at least three hours Avg. SEM Range following electrode implantation. Concentration (mM) 0.19 0.01 0.04–3.2 Duration (s) 2.8 0.1 0.3–10 A Receptor Modulation Previous studies found A receptors modulate stimulated Inter-event Time (s) *108 6 0.9–2069 adenosine release [12], so we tested the effects of A receptor Legend: Data from the caudate-putamen and prefrontal cortex. Concentration drugs on spontaneous adenosine release. For all drug experiments, was significantly different between the two regions (n= 588 transients in 30 rats after the electrode was equilibrated, one hour of baseline data was and 804 transients in 29 rats, t-test, p = 0.0238, respectively). The duration of collected and then the drug administered. DPCPX, an A receptor transient adenosine was not different between the caudate and prefrontal 1 cortex (n= 30 and 29 rats, t-test, p = 0.0826). * Inter-event times, or the time antagonist, was administered at 6 mg/kg, i.p. The CVs of between consecutive transients have significantly different underlying adenosine transients observed after DPCPX were similar to those distributions between the two regions (n = 30 and 29 rats, KS test, p = 0.0338). observed before drug, indicating that DPCPX did not interfere doi:10.1371/journal.pone.0087165.t001 with the detection of adenosine using FSCV. The concentration and duration of adenosine release were compared before and after prefrontal cortex (Fig. 5B), with the median and mean values DPCPX using paired t-tests to examine release in the same animal. marked. Relative frequency is plotted on the y-axis and 30 second DPCPX did not significantly affect the concentration of release in time bins are on the x-axis. The distribution was not Gaussian and the caudate (n= 6, paired t-test, p = 0.1186) or prefrontal cortex shows that adenosine transients occur closer together than (n= 5, paired t-test, p = 0.8547). However, in the caudate-putamen, expected from mean inter-event values. The average time between DPCPX significantly increased the duration from 2.6 to 3.0 transients for the caudate putamen was 156 seconds, however, the seconds (n = 6, paired t-test, p = 0.0092). The duration of adenosine majority of transients occurred within 2 minutes of each other. release in the prefrontal cortex increased from 2.4 to 2.9 seconds Similarly, in the prefrontal cortex, the mean inter-event time was after DPCPX, but the increase was not significant (n= 5, paired t- 108 seconds, but a majority of transients happened less than a test, p = 0.1300). Following DPCPX administration, there was a minute apart. Thus, adenosine release occurs randomly and is not decrease in the mean and median inter-event times and a a result of pacemaker firing. Color plots show that occasionally significant difference in the underlying frequency distribution in spontaneous release events occur within a couple of seconds of the caudate-putamen (n = 6 rats, KS test, p.0.0001) (Fig. 7A and each other (Fig. 5A inset) and the amount of adenosine release 7B) and the prefrontal cortex (n = 5 rats, KS test, p = 0.0286) does not always decrease with the sequential release (Fig. 5B inset). (Fig. 7C and 7D). The time between events decreased after The underlying distributions between the two brain regions DPCPX administration from a median of 141 seconds pre-drug to were compared and the patterns are significantly different (n=30 63 seconds in the caudate and from 80 seconds to 60 seconds in and 29 rats, Kolmogorov-Smirnov test, p = 0.0338). Thus, the time the prefrontal cortex. Therefore, blocking A receptors with between adenosine transient events was shorter in the prefrontal DPCPX in both brain regions increases the frequency of cortex than in the caudate-putamen. An exponential decay was fit spontaneous, transient adenosine release. to the data and plotted on the histogram. The exponential fit in 6 The A agonist, N -cyclopentyladenosine (CPA), was adminis- the caudate-putamen has a rate constant of 0.00891 s or a firing tered to determine if activation of A receptors affected rate of every 112 seconds. The rate constant in the prefrontal spontaneous adenosine transients. There was no significant change cortex is 0.0141 s or a firing rate of every 71 seconds. in adenosine concentration in either the caudate or the prefrontal cortex following administration of CPA (paired t-test, n= 6, Adenosine Transients Continue Over Time p = 0.9818; n= 6, p = 0.4607, respectively). However, CPA did To examine adenosine transients over time, data were collected increase the duration of adenosine transients in the caudate from continuously over three hours. Figures 6A and 6D display the 2.7 to 3.4 seconds (n= 6, paired t-test, p = 0.0195) and from 3.2 to average concentration of adenosine transients in 30 minute bins in 3.8 seconds in the prefrontal cortex (n = 6, paired t-test, the caudate and prefrontal cortex, respectively. The average p = 0.0259). The drugs may interfere with uptake or metabolism, concentration in both brain regions was around 0.13 mM and did thus increasing duration. In the caudate-putamen, the median and not significantly differ with time (one-way ANOVA, caudate: mean inter-event times increased after CPA (Fig. 8A and 8B) and n=6, p= 0.2013; prefrontal cortex: n=6, p= 0.6268). The the underlying distributions were significantly different than pre- duration of adenosine release was also binned into 30 minute drug (n = 6 rats, KS test, p = 0.0308). The time between events epochs for the caudate-putamen (Fig. 6B) and the prefrontal cortex increased from a median of 75 seconds to 98 seconds following (Fig. 6E). There was a significant effect of time on event duration CPA in the caudate putamen, the opposite effect on inter-event between the 30 minute bins in the caudate-putamen (n = 6, one- time as DPCPX. However, in the prefrontal cortex (Fig. 8C and way ANOVA, p = 0.0005) but not the prefrontal cortex (n = 6, one- 8D), CPA did not significantly change the underlying distribution way ANOVA, p = 0.8449). The duration of the fifth bin was and the inter-event times were not different (n = 6 rats, KS test, PLOS ONE | www.plosone.org 6 January 2014 | Volume 9 | Issue 1 | e87165 Spontaneous, Transient Adenosine Release Figure 4. Spontaneous adenosine duration. (A) A color plot and concentration vs. time plot of adenosine transients in the caudate-putamen. The horizontal dashed line is 10% of the concentration of the first peak and the vertical lines are when the baseline value crosses this value, showing the duration. (B) Caudate-putamen duration histogram. The y-axis is relative frequency and the x-axis shows 0.5 second bins. The Gaussian (x{2:554) {0:5 0:9908 distribution equation is y~0:1886 e (red line, R = 0.9712, n = 30 rats). (C) Color plot and concentration vs. time plot of an example spontaneous adenosine transient in the prefrontal cortex. The duration is marked with vertical lines. (D) Adenosine duration histogram for the (x{2:556) {0:5 0:1060 prefrontal cortex is plotted with a Gaussian distribution equation y~0:1823 e (red line, R = 0.9765, n = 29 rats). doi:10.1371/journal.pone.0087165.g004 p = 0.9299). Thus, A activation with CPA decreased the 1 Discussion frequency of spontaneous transient adenosine release in the Spontaneous, transient adenosine release occurs in both the caudate-putamen but not the prefrontal cortex. caudate-putamen and prefrontal cortex. Transients were on average a couple hundred nM, which is sufficient to activate adenosine receptors [38]. Adenosine was elevated for only a few PLOS ONE | www.plosone.org 7 January 2014 | Volume 9 | Issue 1 | e87165 Spontaneous, Transient Adenosine Release adenosine events but not the concentration. These studies reveal that large amounts of adenosine can be quickly released and cleared from the extracellular space, a contrast to previous studies which have documented a slower role for adenosine [39–41]. Thus, adenosine has a rapid signaling mode that may locally cause transient neuromodulation. The Concentration of Transient Release is Sufficient for Adenosine Receptor Activation The average concentration of adenosine released was 180 nM, but the amount varied widely from 40 nM to 3.2 mM. Sponta- neous adenosine release was the same order of magnitude as stimulated release in brain slices from the cerebellum [9], caudate- putamen [42] and prefrontal cortex [43]. However, evoked adenosine release in the caudate-putamen of anesthetized rats was typically 600–900 nM [11,12], larger than the average spontaneous transient. Electrical stimulation likely activates all cells but different firing rates or number of cells activated during spontaneous adenosine release could lead to lower release. In the prefrontal cortex, the concentration of adenosine release was significantly higher than in the caudate, although the difference was not large enough to suggest that different types of adenosine receptors are being activated in the two brain regions. Inhibitory A receptors and excitatory A receptors both have 1 2a affinities in the low nanomolar range [44] and have high to intermediate distributions in the caudate and prefrontal cortex [45]. About one percent of transients had concentrations greater than 1 mM, which could be sufficient to activate A and A 2b 3 receptors [46]. These large transients demonstrate the potential for transient, micromolar adenosine signaling and high amounts of receptor activation. Spontaneous Adenosine Release is Random and the Frequency is Regulated by A Receptors Adenosine release occurred on average once every 3–4 minutes; however, about half of the transients occurred within two minutes of each other in the caudate and one minute of each other in the prefrontal cortex. Spontaneous adenosine release is not periodic and consequently is unlikely to be caused by pacemaker firing or to directly modulate a rhythmic process such as breathing or tonic cell firing [47,48]. Instead, release was random and the inter-event times fit an exponential decay, which arises from a discrete Poisson process [49] where each transient is not dependent on the prior Figure 5. Histograms of inter-event times. (A) Inter-event adenosine event. The shortest time between transients was less histograms in the caudate-putamen. The time between consecutive transients (termed the inter-event time) was calculated and plotted for than one second, indicating a long time is not required to reset the first hour of data collection. The x-axis shows 30 second time bins adenosine release (Fig. 5A and 5B insets). Some of the larger and the y-axis is relative frequency of inter-event times. Median, mean transients may be two or more release events that occur 20.00891x 2 and exponential fit (y = 0.242 e (R = 0.9926)) are plotted on the simultaneously and cannot be temporally resolved. The frequency histogram. The inset plots show an example of three consecutive and concentration of release did not change over three hours, transients. (B) Inter-event histograms for the prefrontal cortex. The 20.0141x 2 demonstrating that transient adenosine continues for long periods exponential fit is y = 0.388 e (R = 0.9933). The inset color plot shows an example of two transients that occurred close together. The of time and that adenosine release is not just a response to underlying distribution of inter-event times was significantly different immediate damage after electrode implantation [9,13,50]. between the caudate-putamen and prefrontal cortex (n = 30 and 29 A receptors are inhibitory adenosine receptors with low nM animals, 588 and 804 inter-event times respectively, Kolmogorov- affinities that downregulate cAMP, hyperpolarize neurons, and Smirnov test, p = 0.0338). The time between transients is shorter in the can be neuroprotective during ischemia [38]. The cortex and prefrontal cortex. doi:10.1371/journal.pone.0087165.g005 striatum have intermediate to high levels of A receptor expression [45]. Previously, A receptors were shown to have autoreceptor characteristics and regulate the amount of stimulated adenosine seconds and these are the most rapid adenosine changes that have release in the caudate in vivo [12]. Here, A receptors modulated been measured in vivo. While adenosine transients occurred on 1 the frequency of spontaneous adenosine release but not the average once every several minutes, at least 30% of transients were concentration. DPCPX, an A antagonist, decreased the inter- less than one minute apart and there was no regularity to event times in both brain regions. CPA, an A agonist, had the adenosine release. A receptors modulated the time between 1 opposite effect and increased the inter-event times in the caudate- PLOS ONE | www.plosone.org 8 January 2014 | Volume 9 | Issue 1 | e87165 Spontaneous, Transient Adenosine Release Figure 6. Spontaneous transient adenosine over time. All x-axes are time in 30 minute bins and all y-axes were normalized to the first bin. (A) Normalized concentrations of adenosine transients in the caudate putamen. There was no significant effect of time on the concentration (n=6 animals, one-way ANOVA, p = 0.2013). (B) Duration of adenosine release from the caudate-putamen. There was a main effect of time on durations (n = 6 animals, one-way ANOVA, p = 0.0005). (C) Number of transients in the caudate-putamen. There was no significant effect of time on the number of transients (n = 6 animals, one-way ANOVA, p = 0.4782). (D) Concentrations in the prefrontal cortex. There was no significant effect of time on the concentration (n = 6 animals, one-way ANOVA, p = 0.6268). (E) Durations of transient adenosine in the prefrontal cortex. There was no significant effect of time on the duration (n = 6 animals, one-way ANOVA, p = 0.8449). (F) Number of transients from the prefrontal cortex. There was no significant effect of time on the number of transients (n = 6 animals, one-way ANOVA, p = 0.9299). doi:10.1371/journal.pone.0087165.g006 putamen but did not have a significant effect in the prefrontal cies of spontaneous adenosine transients were also similar, with cortex. A receptors are part of a feedback loop controlling events occurring every two to three minutes. The comparable transient adenosine release in the brain, where activation of A adenosine transients in different regions of rats and mice, along receptors decreases transient adenosine events. A receptor with previous electrophysiology work in the hippocampus impli- modulation does not affect concentration suggesting that the cating fast adenosine release regulating glutamate receptor receptors do not control synthesis or the amount packaged into excitability [7], show that transient adenosine release is a common vesicles. Instead, A receptors modulate event frequency, such as feature in the nervous system. These transients could be an regulating the number of docking or release events. important mechanism of adenosine signaling, facilitating rapid neuromodulation throughout the brain. Spontaneous Adenosine Release Occurs in Multiple Brain Spontaneous Adenosine Release Provides Local, Regions Transient Modulation Spontaneous, transient adenosine release was measured in both the caudate-putamen and prefrontal cortex. Basal adenosine levels Spontaneous adenosine signaling was fast, with the average are higher in the prefrontal cortex than the caudate-putamen [51– transient lasting only about three seconds. This mode of transient 53]. Similarly, the concentration of transient adenosine release is signaling is a contrast to gradual adenosine buildup for minutes in larger in the prefrontal cortex. However, transient adenosine the extracellular space during pathological events, such as release is unlikely to contribute to the basal levels of adenosine ischemia [54]. Spontaneous adenosine transients were even more because of the rapid clearance. Spontaneous release was more rapid than electrically-stimulated adenosine release, which elevat- frequent in the prefrontal cortex than in the caudate, which is ed adenosine for up to 20 seconds in the caudate-putamen [11] or similar to electrically-evoked release which was also more 100 seconds in the cerebellum [9]. Our time course matched well frequently observed in the prefrontal cortex [43]. The higher with previous measurement of transient adenosine release in spinal frequency of adenosine release in the prefrontal cortex could be cord slices [13] and electrophysiology studies that observed a due to either additional release sites or more release events per site. transient, 2 second variation in glutamate-evoked EPSPs after The spontaneous adenosine transients measured here in the rat stimulated adenosine release [15]. The short duration demon- strates that the adenosine clearance mechanism from the caudate and cortex were remarkably similar to the spontaneous adenosine transients measured in murine slices from lamina II of extracellular space is very rapid, likely due to uptake [44]. Future the spinal cord by Zylka’s group [13]. Despite the differences in studies will examine the effects of adenosine clearance mecha- nisms. Since the duration of adenosine release was the same in brain region, preparation (slices vs. in vivo), and species, the average concentrations were the same order of magnitude both brain regions, the mechanism of clearance is expected to be conserved between the caudate and prefrontal cortex. (180 nM in rats compared to 570 nM in mouse slices). Differences could be a result of different numbers of cells activated or the The time course of spontaneous adenosine signaling is similar to amount of adenosine available for release. The average frequen- that of transient, exocytotic release events of neurotransmitters. PLOS ONE | www.plosone.org 9 January 2014 | Volume 9 | Issue 1 | e87165 Spontaneous, Transient Adenosine Release Figure 7. Effect of the A antagonist, DPCPX (6 mg/kg, i.p.), on adenosine transients. (A) Inter-event time histograms of first hour of pre- 20.0186 2 drug in caudate. Median, mean and exponential fit (green line) (y = 0.295 e (R = 0.7889)) are plotted on the frequency distribution. (B) Inter- 20.0101x 2 event time histogram for transients in the first hour post-DPCPX in caudate. The exponential fit (red line) is y = 0.318 e (R = 0.9490). In the caudate, there was a significant difference between the underlying distributions before and after DPCPX (n = 6 animals, KS-test, p,0.0001). (C) Inter- 20.0101x 2 event histograms pre- and (D) post-DPCPX in the prefrontal cortex. The exponential fit equations are y = 0.260 e (R = 0.9124) pre-drug and 20.0111x 2 y = 0.340 e (R = 0.9851) after DPCPX. In the prefrontal cortex, there was a significant difference between underlying distributions pre- and post-DPCPX (n = 5 animals, KS-test, p = 0.0287). doi:10.1371/journal.pone.0087165.g007 For example, dopamine signaling after phasic firing lasts only [56]. Therefore, only receptors close to the release event would be three to four seconds and dopamine is rapidly cleared from the activated and transient adenosine release would provide local extracellular space [55]. While our study did not address the neuromodulation, close to the site of release. In addition, there pathway of extracellular adenosine formation, the mechanism is could be heterogeneity within a region for receptor activation if likely breakdown of exocytotically released ATP or direct, activity- not all areas experienced adenosine transients at the same time. dependent release of adenosine. Recent evidence shows transient, The local variation of release was not directly assessed in these electrically-stimulated adenosine is activity dependent and a studies, but moving the electrode did change the number of portion is not dependent on extracellular breakdown of ATP adenosine transients detected suggesting a spatial effect on release [9,42]. Spontaneous adenosine release in slices of the spinal cord events. of mice is ATP-dependent [13,14]. While the mechanism of The function of rapid adenosine release is likely transient spontaneous transient release is difficult to elucidate in vivo, future neuromodulation in the brain. Although most studies of adenosine experiments could be performed in brain slices to determine if function have not been performed on the seconds time scale, transient adenosine release is exocytotic or a downstream product adenosine is known to regulate cerebral blood flow and of ATP. neurotransmission on a longer time scale. It is likely that faster The rapid release and clearance of adenosine has two regulation of these processes is caused by transient adenosine consequences: adenosine can only signal locally and receptors will release. Adenosine increases cerebral vasodilation in less than 60 be activated transiently. The distance a molecule can travel in seconds [57] and also increases cerebral blood flow [58]. Transient three seconds in the extracellular space is only 10–20 micrometers adenosine release could increase the flow of blood to localized PLOS ONE | www.plosone.org 10 January 2014 | Volume 9 | Issue 1 | e87165 Spontaneous, Transient Adenosine Release Figure 8. Effect of the A agonist, CPA (1 mg/kg, i.p.), on adenosine transients. (A) Inter-event time histograms for the caudate. Median, 20.0115x 2 mean and exponential fit (green line) (y = 0.298 e (R = 0.9636)) are plotted on the frequency distribution. (B) Inter-event time histograms for 20.00787x 2 the first hour after CPA in caudate. The exponential fit (blue line) is y = 0.226 e (R = 0.9528). There was a significant difference between the underlying distributions before and after CPA (n = 6 animals, KS-test, p = 0.0308). (C) Inter-event histograms pre- and (D) post-CPA in the prefrontal 20.00983x 2 20.00938x 2 cortex. Exponential equations are y = 0.280 e (R = 0.9430) pre-drug and y = 0.264 e (R = 0.9337) after CPA. In the prefrontal cortex, there was no significant difference in the underlying distributions before and after CPA (n = 6 animals, KS-test, p = 0.9299). doi:10.1371/journal.pone.0087165.g008 areas in the brain that require an immediate boost in oxygen and Conclusions nutrients. Adenosine regulation of neurotransmitter release can be Spontaneous transient adenosine release was characterized for inhibitory; for example, adenosine inhibits acetylcholine, gluta- the first time in vivo. The spontaneous release in the caudate and mate, serotonin, dopamine, noradrenaline signaling by activation prefrontal cortex is fast, lasting only a few seconds, and large, in of A receptors [59]. Adenosine can also increase the release of the hundred nM range. The frequency of release was random, acetylcholine and glutamate through A receptor activation [59]. 2a higher in the prefrontal cortex, and modulated by A receptors. Although A and A adenosine receptor affinities are in the low 1 2a These findings are a paradigm shift for understanding the time nanomolar range, the effective in vivo EC is estimated to be course of adenosine signaling. Previous studies have documented a 600 nM [60], so changes on the order of hundreds of nanomolar long-term modulatory effect of adenosine during pathologies but concentrations could have significant physiological effects. This here we demonstrate a new mode of transient adenosine signaling study shows that A receptors control the frequency of adenosine that could lead to rapid, local modulation. Future studies transients, but the downstream modulatory effects of the transient investigating the formation and function of this new type of adenosine activation of A receptors are not known and should be adenosine signaling will reveal how adenosine modulates on the explored in the future. A rapid, transient mode of adenosine second time scale and regulates local brain function. signaling would provide discrete, local neuromodulation, which could facilitate fine control of neurotransmission. PLOS ONE | www.plosone.org 11 January 2014 | Volume 9 | Issue 1 | e87165 Spontaneous, Transient Adenosine Release Acknowledgments Author Contributions Conceived and designed the experiments: BJV. Performed the experi- We would like to thank University of Virginia Alliance for Computational ments: MDN AER. 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PLOS ONE | www.plosone.org 12 January 2014 | Volume 9 | Issue 1 | e87165 Spontaneous, Transient Adenosine Release 55. Wightman RM, Robinson DL (2002) Transient changes in mesolimbic computed tomography in normal subjects and in patients with occlusive carotid dopamine and their association with ‘reward’. J Neurochem 82: 721–735. disease. A comparison with acetazolamide. Stroke 26: 1572–1576. 56. Wightman RM, Amatore C, Engstrom RC, Hale PD, Kristensen EW, et al. 59. Sperlagh B, Vizi ES (2011) The role of extracellular adenosine in chemical (1988) Real-time characterization of dopamine overflow and uptake in the rat neurotransmission in the hippocampus and Basal Ganglia: pharmacological and striatum. Neuroscience 25: 513–523. clinical aspects. Curr Top Med Chem 11: 1034–1046. 57. Winn HR, Morii S, Berne RM (1985) The role of adenosine in autoregulation of 60. Dunwiddie TV, Diao L (1994) Extracellular adenosine concentrations in cerebral blood flow. Ann Biomed Eng 13: 321–328. hippocampal brain slices and the tonic inhibitory modulation of evoked 58. Soricelli A, Postiglione A, Cuocolo A, De CS, Ruocco A, et al. (1995) Effect of excitatory responses. J Pharmacol Exp Ther 268: 537–545. adenosine on cerebral blood flow as evaluated by single-photon emission PLOS ONE | www.plosone.org 13 January 2014 | Volume 9 | Issue 1 | e87165

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