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Keywords:

  • ABT-702;
  • electrical stimulation;
  • histidine;
  • propentofylline;
  • sensor;
  • striatum

Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Adenosine is an endogenous byproduct of metabolism that regulates cerebral blood flow and modulates neurotransmission. Four receptors, with affinities ranging from nanomolar to micromolar, mediate the effects of adenosine. Real-time measurements are needed to understand the extracellular adenosine concentrations available to activate these receptors. In this study, we measured the subsecond time course of adenosine efflux in the caudate–putamen of anesthetized rats after a 1 s, high-frequency stimulation of dopamine neurons in the substantia nigra. Fast-scan cyclic voltammetry at carbon-fiber microelectrodes was used for simultaneous detection of adenosine and dopamine, which have different oxidation potentials. While dopamine was immediately released after electrical stimulation, adenosine accumulation was slightly delayed and cleared in about 15 seconds. The concentration of adenosine measured after electrical stimulation was 0.94 ± 0.09 μM. An adenosine kinase inhibitor, adenosine transport inhibitor, and a histamine synthetic precursor were used to pharmacologically confirm the identity of the measured substance as adenosine. Adenosine efflux was also correlated with increases in oxygen, which occur because of changes in cerebral blood flow. This study shows that extracellular adenosine transiently increases after short bursts of neuronal activity in concentrations that can activate receptors.

Abbreviations used
CV

cyclic voltammogram

DMSO

dimethyl sulfoxide

DV

dorsoventral

FSCV

fast-scan cyclic voltammetry

SN

substantia nigra

Adenosine is an endogenous nucleoside, a byproduct of ATP metabolism, present in nearly all tissues in the body. In the brain, it acts not as a typical neurotransmitter, but as a neuromodulator. Adenosine exerts two parallel modulatory roles in the CNS: as a homeostatic modulator and a neuromodulator at the synaptic level (Cunha 2001). Adenosine can act pre- or post-synaptically on four different membrane bound G-protein coupled receptor subtypes (Fredholm et al. 2001) that have a wide range of affinities. A1 (3–30 nM) and A2A (1–20 nM) receptors are higher affinity while A2B (5–20 μM) and A3 (> 1 μM) receptors are lower affinity (Fredholm et al. 1994). The receptors are present in the entire brain, but with different abundance and effects. A1 receptors are mostly localized in the cerebral cortex and hippocampus and are primarily involved in inhibitory neurotransmission. A2A receptors modulate excitatory neurotransmission and are most abundant in the caudate–putamen. A1 and A2A receptors are thought to be activated under normal physiological conditions, because the basal level of adenosine is in the low nanomolar range (Cunha 2001). Low affinity A2B and A3 receptors would presumably be activated only under stressful or pathophysiological conditions such as hypoxia or ischemia. Variations in extracellular concentrations would affect the extent of adenosine receptor activation. Therefore, to understand the function and formation of adenosine, real-time measurements of adenosine concentrations are needed.

Adenosine is produced intracellularly and extracellularly, but by different mechanisms (Latini and Pedata 2001). Intracellularly, adenosine is mainly formed by catabolism of AMP and then transported through the membrane via bidirectional equilibrative nucleoside transporters. In the extracellular space, released nucleotides such as ATP are metabolized to adenosine by ecto-5′-nucleotidase. The time course of extracellular adenosine concentrations is expected to vary with the mechanism of formation, with direct transport after intracellular formation expected to be the fastest.

Basal levels and evoked concentrations of adenosine have been studied in vitro (in brain slices) as well in vivo. Microdialysis, coupled to HPLC, is the most common method for measurement of neurochemicals in vivo. It is useful for measuring basal adenosine levels and can be chronically used in awake, behaving animals. Inserting the relatively large microdialysis probe (250–500 μm) in the brain causes tissue damage that raises levels of adenosine immediately after implantation, so experiments are usually performed 24 h after probe implantation (Grabb et al. 1998). The temporal resolution of microdialysis is normally limited to sampling every 5–20 min, which does not allow real-time measurements of adenosine concentrations. Recently, new sensor techniques have emerged that provide better temporal resolution. Electrochemical sensors, 5–30 μm in diameter, are smaller than microdialysis probes and therefore cause less damage to the surrounding tissue as well as less disturbance to cellular metabolism (Peters et al. 2004). An enzyme-based electrochemical sensor has been developed over last decade for measurements of adenosine (Dale 1998; Dale et al. 2000; Llaudet et al. 2003; Frenguelli et al. 2007). The biosensor is small (25 μm) and responds to concentration changes in about 2 s. But the construction requires three active, entrapped enzymes (adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase) to detect adenosine and a second, null sensor (lacking adenosine deaminase) to distinguish adenosine from inosine.

Our laboratory has developed a method to directly detect adenosine at carbon-fiber microelectrodes using fast-scan cyclic voltammetry (FSCV) (Swamy and Venton 2007). The advantages of carbon-fiber microelectrodes in comparison with enzyme biosensors are their small size (5–10 μm), the easy fabrication (a second, null sensor is not required), and subsecond temporal resolution. The 15 nM limit of detection is less than the estimated basal adenosine level in the brain of 50–200 nM (Cunha 2001; Latini and Pedata 2001) and is more sensitive than other electrode methods. A linear response is obtained up to 20 μM adenosine. Dopamine and guanine, possible interferents for adenosine detection in biological samples, are distinguishable from adenosine using cyclic voltammetry and inosine is not detected. FSCV is a differential method; therefore, it cannot be used to measure basal adenosine levels, but it is the fastest method available for measuring changes in extracellular concentrations.

The goal of this study was to determine the time course of stimulated adenosine efflux in vivo using carbon-fiber microelectrodes as adenosine sensors. These experiments, using FSCV, were the first real-time measurements of adenosine concentration changes in vivo on a subsecond time scale. We measured adenosine efflux in the rat caudate–putamen after electrical stimulation of dopaminergic neurons. Adenosine transiently increased and was cleared from the extracellular space in about 15 s. The identity of adenosine was confirmed by pharmacological studies. Correlation of adenosine and oxygen concentration changes in the brain was also demonstrated.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Fast-scan cyclic voltammetry

Cylindrical microelectrodes were constructed by aspirating a 7 μm diameter T-650 carbon-fiber (Cytec Engineering Materials, West Patterson, NJ, USA) into a glass capillary (1.2 × 0.68 mm; A-M Systems, Inc., Carlsborg, WA, USA). The capillary was then pulled on a vertical pipette puller (model PE-21; Narishige, Tokyo, Japan) to form two microelectrodes. The extended fiber was cut with a scalpel under a microscope to a length of approximately 50 μm. The interface between fiber and glass was sealed with epoxy [EPON resin 828; Miller-Stephenson chemical Co. Inc. (Danbury, CT, USA) mixed with 14% by weight m-phenylenediamine (Fluka, Milwaukee, WI, USA) and heated to 80°C). Electrodes were cured in an oven at 100°C for 2 h and then overnight at 150°C. Before use, electrodes were soaked in isopropanol for 10 min and then back-filled with an electrolyte solution (4 M potassium acetate and 150 mM potassium chloride).

Fast-scan cyclic voltammetry was used to detect dopamine, adenosine, and oxygen. Cyclic voltammograms (CVs) were collected using a Chem-Clamp voltammeter-amperometer (custom modified with lower gain settings; Dagan Corporation, Minneapolis, MN, USA). The microelectrode was attached to the headstage via a microelectrode holder (HB-120; Dagan Corporation). Data was collected using Tar Heel CV, written in LabVIEW (National Instruments, Austin, TX, USA) (Heien et al. 2003). Two National Instruments (Austin, TX, USA) computer interface boards (PCI 6052E and PCI 6711) and a homemade breakout box were used for to apply the waveform and collect the data.

For dopamine and adenosine detection, the electrode was linearly ramped from −0.4 V to +1.5 V and back at 400 V/s every 100 ms. The waveform was constantly applied to electrode, even if no data was being collected. To detect oxygen, the electrode was held at 0 V, scanned to +1.5 V, then −1.4 V and back to 0 V at 400 V/s every 125 ms. The reference electrode was a silver/silver chloride electrode. All color plots and voltammograms were background corrected, to remove the large electrode charging current, by subtracting the average of 10 CVs collected immediately before the signal was taken.

Chemicals and drugs

All components of the Tris buffer (in mM: 15 Tris-base, 140 NaCl, 3.25 KCl, 1.2 CaCl2, 1.25 NaH2PO4, 1.2 MgCl2, and 2.0 Na2SO4, pH 7.4) were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Adenosine and l-histamine were purchased from Sigma-Aldrich (Milwaukee, WI, USA) and dopamine (3-hydroxytyramine hydrochloride) from Acros Organics (Fair Lawn, NJ, USA). All aqueous solutions were made using deionized water (Milli-Q Biocel; Millipore, Billerica, MA, USA). Stock solutions (10 mM) of adenosine, dopamine, and histamine were made in 0.1 M perchloric acid (Fisher Scientific) and kept refrigerated for no longer than 1 month.

All drugs were administered intraperitoneally (i.p). Propentofylline (15 mg/kg; Sigma-Aldrich) and l-histidine (150 mg/kg; MP Biomedicals Inc., Solon, OH, USA) were dissolved in 0.5–1 mL saline (Baxter, Deerfield, IL). ABT-702 (5 mg/kg; Sigma-Aldrich) was dissolved in 0.2 mL dimethyl sulfoxide (DMSO; Fisher Scientific).

Calibrations

Before the experiment, the electrodes were calibrated using 1 μM dopamine and 5 μM adenosine. These concentrations were used because the response of our electrodes to 5 μM adenosine had been previously characterized (Swamy and Venton 2007) and 1 μM dopamine gave similar oxidation currents (see Fig. 1). The standard solutions were prepared fresh daily by diluting 10 mM stock solutions with Tris buffer. Calibrations were performed in a flow injection system. Standards were loaded into a 500-μL sample loop mounted on stainless steel HPLC loop injector (Swamy and Venton 2007). Buffer was constantly pumped through the flow cell at a rate of 2 mL/min and an air actuator switched the valve to make an injection. Three second long injections of neurochemicals were made to mimic fast concentration changes that occur in the brain. For each compound, at least six injections were performed, and the average peak oxidation current was calculated. This average current for a given concentration of analyte was used to convert measured currents in vivo to concentrations. The microelectrodes had reproducible responses to injections of adenosine or dopamine, with an average relative standard deviation for 10 repeated injections of only 3%.

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Figure 1.  Detection of dopamine and adenosine standards and their mixture using fast-scan cyclic voltammetry at a carbon-fiber microelectrode. The potential was scanned from −0.4 V to 1.5 V and back at 400 V/s every 100 ms. The reference electrode was Ag/AgCl. The top shows cyclic voltammograms for (a) 1 μM dopamine, (b) 5 μM adenosine, and (c) a mixture of 1 μM dopamine and 5 μM adenosine. The bottom shows color plots for the flow injection analysis experiments. The color plots display all the in vivo data in one plot: the scanned potential is plotted on y-axis, time on x-axis, and current detected is shown in pseudocolor. For the flow injection experiment, buffer was injected for 5 s, followed by a 3 s injection of the test compound before switching back to buffer. CVs are taken from color plots at 7.5 s [shown by the white dashed line on (a)] after the start of the recording.

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To calibrate oxygen concentrations, oxygen-, nitrogen-, and air-saturated Tris buffers were used. Nitrogen-saturated buffer, with no oxygen, was loaded as the run buffer and injections of air- or oxygen-saturated buffers were made. A calibration plot of reduction peak current for oxygen versus concentration of oxygen in solution was obtained. The concentration of oxygen in each buffer, which depends on the atmospheric pressure, temperature, and salt concentration, was calculated as previously described (Hitchman 1978; Zimmerman and Wightman 1991). A calibration CV for oxygen is shown in the supplemental information.

Microelectrodes were also calibrated in the presence of the drugs used for the pharmacological tests to ensure that the drugs did not interfere with adenosine detection. Propentofylline and l-histidine were not electroactive. ABT-702 was electroactive but did not have peaks at the same potentials as adenosine. Saline does not affect electrode calibrations, but the presence of 0.1% DMSO in the background buffer did reduce adenosine peak currents slightly. When ABT-702 was dissolved in DMSO, no change in adenosine peak current was observed compared with just DMSO. Because our control experiments in the rats with DMSO showed no change in the measured signal after just administration of DMSO, it appears that DMSO is not reaching the brain in sufficient concentrations to foul the electrode in vivo. Calibration data for the drugs and a more detailed explanation of the calibration experiments are given in the supplemental material.

Animals and surgery

All animal experimental procedures were approved by the Animal Care and Use Committee of University of Virginia. Male, Sprague–Dawley rats (250–300 g; Charles River, Wilmington, MA, USA) were anesthetized with urethane (1.5 g/kg, i.p). Hair on the head and neck was shaved and the animal was placed in a stereotaxic frame. The skull was exposed and holes drilled with a stereotaxic drill for the placement of electrodes (Paxinos and Watson 2005). The carbon-fiber microelectrode was lowered to the caudate–putamen (coordinates in mm from bregma): anterior–posterior: +1.2, mediolateral: +2.1, and from the top of the skull, dorsoventral (DV): −4.5. The bipolar stimulating electrode (metal MS 303/2; Plastics One Inc., Roanoke, VA, USA) was inserted to the substantia nigra (SN)/ventral tegmental area at anterior–posterior: −5.3 mm, mediolateral +1.2 mm, and DV −7.5 mm. The DV placement of the working and stimulating electrodes was adjusted slightly to ensure measurement of a robust signal. A silver/silver chloride wire was inserted on the contralateral side of brain as a reference electrode. The body temperature of the animal during the experiment was maintained at 37°C using a heating pad with a thermistor probe for temperature control (FHC, Bowdoin, ME, USA).

The carbon-fiber microelectrode was allowed to equilibrate in the brain for 30–40 min after implantation. During that period, the scanning potential was applied to obtain a stable background charging current at the microelectrode. In addition, this waiting period allowed trauma caused by electrode implantation time to heal. For each drug studied, at least four animals were used and only one drug was administered to each animal.

Data collection and analysis

Electrical stimulations were applied using a BSI-950 Biphasic Stimulus Isolator (Dagan Corporation). A stimulation train of 60 biphasic square pulses at 60 Hz (2 ms wide and 300 μA per phase) was used. With the oxygen waveform, a train of 60 pulses was delivered, but at 40 Hz. This frequency change was necessary to time-synchronize waveform application and stimulation delivery to prevent stimulations from occurring while the potential was being ramped and data collected. The voltage waveform was continually applied to the carbon-fiber microelectrode for the whole experiment; however data was only collected around the time of stimulation. Every 3 min, a 45-s long data file was collected. Stimulation pulses were applied 5 s after the beginning of the file. Ten baseline stimulation files were recorded before a drug was administered to the rat, then an additional thirty data files were recorded. Total acquisition time for data collection was 120 min (30 min before and 90 min after drug administration).

From each data file, the peak current for adenosine at 1.5 V and for dopamine at 0.6 V was converted to concentration using the averaged calibration values obtained before the experiment. Then, to calculate average stimulated release, the stimulated concentrations obtained from the last five data files before drug injection were averaged and compared with the average concentration obtained from five data files after drug administration. We considered the five files where the effect of each drug was the most visible. Usually this was about 20–45 min after drug administration. While the period varied for different drugs because of different rates of effects, the timing was kept constant in every animal for the same drug tested. For example, for propentofylline the files collected at 18, 21, 24, 27, and 30 min after administration were used to calculate the concentration after drug.

Statistical evaluation

Statistical analyses were performed using GraphPad PRISM (GraphPad Software Inc., San Diego, CA, USA). All data are presented as mean ± SEM for n numbers of animals (n = 4 if not otherwise given). To determine the effects of the drugs a paired t-test was performed which compared pre- and post-drug responses in the same animals. Data were considered statistically significant at the 95% confidence level.

Results

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Adenosine and dopamine cyclic voltammetry

Fast-scan cyclic voltammetry was used to measure fast changes in adenosine and dopamine concentrations. It has high temporal resolution and good chemical selectivity, because of the uniqueness of the CVs. Figure 1 shows typical CVs and color plots of dopamine, adenosine, and a mixture of the two in vitro. The color plots display the three components of data collected during a flow injection analysis experiment used to calibrate electrodes. In the color plots, time is plotted on the x-axis, electrode potential is on the y-axis and the color gradient represents current detected. For the flow injection experiments, buffer is flowed by the electrode for 5 s, then the solution changed to the calibration compound for 3 s before changing back to buffer. In Fig. 1a, the green oval at 0.6 V from 6 to 9 s is representative of the dopamine oxidation current, the yellow-dark blue oval at the top of the plot at −0.2 V is because of the dopamine reduction current. For adenosine, the main oxidation peak occurred close to the switching potential at 1.5 V (Fig. 1b). A secondary oxidation peak at 1.0 V is due to oxidation of a byproduct formed after previous scanning to 1.5 V (Swamy and Venton 2007). Because the oxidization potential for dopamine and adenosine are different, codetection in the same solution is possible. The adenosine and dopamine peaks are separated in the mixture CV (Fig. 1c).

Adenosine efflux in vivo

Figure 2 shows dopamine and adenosine efflux in the rat caudate–putamen after electrical stimulation of dopamine cell bodies in SN/ventral tegmental area. The train of 60 stimulation pulses at 60 Hz was delivered 5 s after data collection began. Stimulation duration is shown by a short black bar under the concentration versus time traces on all figures. On the color plot the stimulation duration is shown as a red bar between 5 and 6 s. The green oval at 0.6 V on the color plot is due to dopamine oxidation and the longer green feature at 1.5 V in the middle of the plot is due to oxidation of adenosine. Changes in current amplitude at other potentials are sometimes seen in color plots after the stimulation and are likely because of pH or ionic changes (Venton et al. 2003a).

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Figure 2.  Dopamine and adenosine efflux in vivo. After electrical stimulation (60 pulses at 60 Hz, 300 μA per pulse) of the substantia nigra/ventral tegmental area, dopamine and adenosine concentrations were measured in the caudate–putamen of the rat. Concentration versus time traces are shown for (a) dopamine (current taken at 0.6 V, red dashed line on color plot) and (b) adenosine (current taken at 1.5 V, yellow dashed line). The current was converted to concentration using calibration values for that microelectrode obtained before experiment. The duration of stimulation (1 s) is shown with black bar under the both traces. Cyclic voltammograms are shown at two different times (c) 6 s (white dashed line) and (d) 7.3 s (blue dashed line) after start of the recording (e) The color plot shows all the in vivo data in one plot. The red bar underneath shows the duration of stimulation.

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To examine the changes in concentration over time, the currents taken at the oxidation potentials for dopamine (0.6 V, red dashed line on color plot, Fig. 2) and adenosine (1.5 V, yellow dashed line) were converted to concentrations by using calibration values for the electrode obtained before the experiment (see the Calibration section in Experimental procedures). Dopamine is immediately released upon stimulation and is present for only 3–4 s because dopamine transporters quickly clear it from extracellular space (Fig. 2a) (Venton et al. 2003b). Adenosine efflux is a little delayed from the start of the stimulation as the oxidative current for adenosine rises slower compared with dopamine (Fig. 2b). Adenosine reaches its maximum later than dopamine and is present for a longer time until it is cleared from the extracellular space. The CVs taken at different times also reveal the different time courses of dopamine and adenosine concentrations. Six seconds after the start of the recording, which is 1 s after stimulation, the CV looks primarily like dopamine with only a small peak at 1.5 V (Fig. 2c, white dashed line on color plot). At 7.3 s, a clear peak is seen at 1.5 V because of adenosine oxidation and the dopamine peak at 0.6 V is diminished (blue dashed line on color plot).

The adenosine increase is sometimes biphasic. The first peak occurs 2–5 s after stimulation (7–10 s after the start of the recording), is cleared within about 15 s, and is always present. The second peak occurs 15–25 s after stimulation but is not always observed. Examples of biphasic release are seen in Figs 3–5, where the first and second peaks are labeled. A small increase during the stimulus is sometimes also present. It is not considered the first peak if it does not persist after stimulation and probably occurs as a stimulation interference with data collection. Because the first peak was more consistent, all reported concentrations were measured for the first peak.

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Figure 3.  Control experiments. Example concentration versus time traces for adenosine before (solid line) and 30 min after (dotted line) administration of (a) 0.2 mL DMSO and (b) 1 mL saline. Current versus time traces are taken from color plots at the adenosine oxidization potential and converted to concentration using calibration values. A short, black bar under the traces shows the duration of stimulation (1 s).

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Figure 4.  Adenosine transport inhibitor. Example concentration versus time trace for adenosine before (solid line) and 20 min after (dotted line) drug administration of propentofylline (15 mg/kg, i.p. in saline). A short, black bar under the traces shows the duration of the stimulation (1 s).

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Figure 5.  Adenosine metabolic inhibitor. Example trace for adenosine is shown before (solid line) and 25 min after (dotted line) drug administration of ABT-702 (5 mg/kg, i.p. in DMSO), an adenosine kinase inhibitor. The black bar under the traces shows the duration of the stimulation (1 s).

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We converted the peak currents measured after electrical stimulation to concentration for each animal and then averaged these for all animals. The averaged stimulated concentration before drug administration was 0.94 ± 0.09 μM (n = 31) for adenosine and 0.19 ± 0.02 μM (n = 17) for dopamine. The range of values for adenosine was from 0.1 to 2.7 μM, illustrating that there is large biological variability between animals. Variability between subjects was also observed in the time course of adenosine efflux and might be because of electrode placement or biological differences. Within one animal, changing the placement of the electrode also leads to differences in observed stimulated release. To further confirm the identity of signal measured at 1.5 V as adenosine, we studied the pharmacological effects of drugs that modulate the adenosine system.

Control experiments

Figure 3a and b shows examples of concentration versus time traces for stimulated adenosine efflux after administration of vehicles, saline and DMSO, used for the pharmacology experiments. Current versus time traces at the adenosine oxidation potential were converted to concentration based on electrode calibration. The solid lines represent the traces obtained before vehicle administration; the dotted lines show stimulated adenosine 30 min after vehicle administration in the same animal. The data for administration of DMSO and saline in vivo confirm that the vehicles do not alter the adenosine signal. Adenosine concentrations were calculated using the first peak, directly after the stimulation. For each animal, the five stimulated adenosine concentrations directly before vehicle administration were averaged to obtain a pre-drug value and then five stimulations starting 30 min after vehicle administration were averaged for a post-drug concentration. Table 1 summarizes the averaged data. The concentrations of stimulated adenosine measured before and after DMSO or saline injections are not statistically different (> 0.05, Table 1). A percentage of pre-drug value was also calculated for each animal by dividing the average adenosine concentration after vehicle by the average concentration before vehicle administration and multiplying by 100. Those percentage of pre-drug values were then averaged for all animals and are also given in Table 1. A value near 100% indicates that adenosine levels did not change after vehicle administration.

Table 1.   Concentration of stimulated adenosine efflux before and after drug administration
AdenosinePropentofyllineABT-702l-HistidineDMSOSaline
  1. Pre- and post-drug concentrations and percentage of pre-drug values are given as mean ± SEM for five animals, except for saline, which is for four animals. Percentage of pre-drug value was calculated for each animal and then averaged; p-value is given for a paired t-test comparing pre- and post-drug concentrations in the same animal. *Significantly different at the 95% confidence level. DMSO, dimethyl sulfoxide.

Pre-drug [μM] 1.17 ± 0.18  0.69 ± 0.07  0.7 ± 0.1  0.8 ± 0.1 0.9 ± 0.1
Post-drug [μM] 0.90 ± 0.15  0.80 ± 0.08  0.7 ± 0.1  0.8 ± 0.1 0.9 ± 0.2
p-value 0.047*  0.036*  0.751  0.771 0.756
Percentage of pre-drug (%)73 ± 6124 ± 7101 ± 4103 ± 490 ± 10

Pharmacological effects of an adenosine transport inhibitor

Propentofylline (15 mg/kg in 0.5 mL saline, i.p.), a xanthine derivative, was administered to inhibit adenosine transport. Adenosine is transported across the cell membrane by equilibrative nucleoside transporters, which carry nucleosides across cell membranes in either direction, according to their concentration gradients (Thorn and Jarvis 1996). Propentofylline can cross the blood–brain barrier (Noji et al. 2004), and thus can be administered effectively i.p. The dose, 15 mg/kg, was chosen based on a literature search for intraperitoneal administration which found doses between 10 and 30 mg/kg to be effective at eliciting neuroprotective effects, with lower doses of 10–15 mg/kg most commonly used (Turcani and Turcani 2001; Noji et al. 2004).

Propentofylline significantly decreased the first phase of stimulated adenosine efflux. Figure 4 shows an example of adenosine concentration versus time trace before (solid line) and 20 min after (dotted line) the administration of propentofylline. For this animal, the averaged stimulated adenosine concentration before and after administration was 0.69 ± 0.05 and 0.53 ± 0.06 μM, respectively, a decrease to 77% of pre-drug levels. For all five animals, the results are summarized in Table 1. On average, stimulated adenosine efflux was 73% of pre-drug values after propentofylline.

Pharmacological effect of an adenosine kinase inhibitor

Another strategy to prove that the measured signal was adenosine was to inhibit its metabolism, thereby increasing its concentration. Inhibition of adenosine kinase, which phosphorylates adenosine to AMP in cells, has been shown to increase the extracellular level of adenosine in the brain (Sciotti and van Wylen 1993; Pazzagli et al. 1995). In our study, we used ABT-702 (5 mg/kg in 0.2 mL DMSO, i.p.) a novel and selective non-nucleoside adenosine kinase inhibitor (Jarvis et al. 2000). This compound increases endogenous extracellular adenosine when given i.p. (Kowaluk et al. 2000). Usual doses for i.p. injection are in the range of 5–15 mg/kg (Radek et al. 2004).

Figure 5 shows an example of concentration versus time traces before and 25 min after administration of ABT-702. Both the first and second peaks of adenosine increased after ABT-702 administration. For the example animal in Fig. 5, the stimulated concentration is 140% of the pre-drug value for the first peak, and 180% of pre-drug values for the second peak after ABT-702. The data for five animals, as measured for the first peak, are summarized in Table 1. The starting concentration varied from 0.3 to 1.5 μM, because of biological variability and placement of the electrodes, leading to the error values. However, every animal showed an increase in the first peak after ABT-702 administration. Therefore, the increase was statistically significant using the paired t-test (Table 1). The average increase for the second adenosine peak is even larger, to 176% of pre-drug value.

L-Histamine and adenosine

l-Histamine is a neurotransmitter abundantly present in the brain (Ito 2000). The oxidation of histamine occurs around the same potential as adenosine and their CVs collected in vitro are very similar (Fig. 6a and b). Therefore, histamine could be a potential interferent to adenosine detection. To confirm that signal at 1.5 V is adenosine and not histamine, we administered the synthetic precursor of histamine, l-histidine (150 mg/kg in 1 mL saline, i.p.) (Yoshimatsu et al. 2002).

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Figure 6. l-Histamine and adenosine. Cyclic voltammograms of (a) 5 μM l-histamine and (b) 5 μM adenosine collected during in vitro calibrations are very similar. (c) Concentration versus time traces for adenosine in vivo before (solid line) and 30 min after administration (dotted line) of the l-histamine synthetic precursor, l-histidine (150 mg/kg, i.p. in saline). No change in the traces confirms that the signal measured at 1.5 V is not l-histamine.

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Figure 6c shows example concentration versus time traces for adenosine before and after the administration of histidine. If the signal at 1.5 V was due to histamine, a substantial increase in stimulated efflux should have been observed after administration of the synthetic precursor. No change in the stimulated efflux was observed, indicating that the signal is not because of histamine. The pharmacological data summarized in Table 1 indicate that adenosine concentrations were not significantly different after histidine.

Correlation of oxygen and adenosine signals

To measure adenosine and oxygen signals simultaneously, we used a different applied waveform that scanned to a potential low enough to reduce oxygen. Oxygen can be reduced at carbon electrodes around −1.2 V (Zimmerman and Wightman 1991; Lowry et al. 1997). A FSCV waveform which scans to −1.4 V has previously been used to detect oxygen changes after stimulated dopaminergic terminal activity (Venton et al. 2003a). We adapted this previous waveform slightly to detect both oxygen and adenosine. The electrode potential was initially held at 0 V, then linearly ramped to 1.5 V to oxidize adenosine and down to −1.4 V to reduce oxygen, then scanned back to 0 V. The voltammetric wave observed for oxygen reduction at carbon electrodes is stable and reproducible and provides a characteristic identifier for molecular oxygen. The role of adenosine to regulate cerebral blood flow and act as a vasodilator is well established (Winn et al. 1981; Phillis 1989; O’Regan 2005). Therefore, we expected that if adenosine efflux were inhibited, the increase in oxygen after stimulated neuronal activity because of increased blood flow would also be inhibited. To inhibit stimulated adenosine efflux, we administered the adenosine transport inhibitor propentofylline.

Figure 7 shows example concentration versus time traces before and after propentofylline (15 mg/kg in 0.5 mL saline, i.p.) administration for adenosine and oxygen. Both adenosine and oxygen concentrations increased after stimulation. As before (Fig. 4), propentofylline significantly decreased the stimulated adenosine concentration. Stimulated adenosine efflux was 2.5 ± 0.7 μM pre-drug and decreased to 1.4 ± 0.1 μM after propentofylline (n = 4 animals), which is 59 ± 4% of the pre-drug level (p = 0.002). Color plots and CVs for this experiment are given in the supplemental material. The averaged stimulated oxygen concentration decreased as well after propentofylline, from 9.3 ± 1 to 5.8 ± 0.6 μM, which is 64 ± 4% of the pre-drug level (p < 0.001). The magnitude of these decreases is similar, suggesting that adenosine is one of the molecules that control the increase in oxygen because of vasodilation after cellular activity. A correlation plot was constructed of oxygen peak current versus adenosine peak current, using both pre-drug and propentofylline data (Fig. 7c). In general, greater adenosine signals correlate with increased oxygen release. The R2 value for this plot is 0.78, which indicates a pretty good correlation, as other molecules could also be acting as vasodilators.

image

Figure 7.  Correlation of adenosine and oxygen changes. Concentration versus time traces are shown for (a) adenosine and (b) oxygen before (solid line) and 20 min after (dotted line) administration of 15 mg/kg propentofylline, an adenosine transport inhibitor. The black bar under the traces shows the duration of the stimulation (1 s). (c) A correlation plot of oxygen peak current versus adenosine peak current using both pre-drug and propentofylline data. Ten pre-drug and 20 post-drug current pair values were used from each rat experiment (n = 4).

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Discussion

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

In this study, we demonstrated for the first time that transient adenosine efflux occurs in the caudate–putamen after stimulation of dopamine cell bodies in the SN. FSCV allowed adenosine and dopamine codetection with subsecond temporal resolution. The increase in adenosine after a short, high-frequency stimulation was slightly delayed from the stimulation and more prolonged than dopamine release. Pharmacological agents were used to confirm the identity of the measured species as adenosine.

Dynamics of adenosine efflux in vivo

Fast-scan cyclic voltammetry is a differential technique, best used for measuring changes in analyte concentration. The 7-μm diameter microelectrodes allow high spatial resolution and cause minimal tissue damage (Peters et al. 2004). While basal levels cannot be determined (Michael et al. 1998), voltammetry has a temporal resolution of 0.1 s and is the fastest method available for measuring adenosine changes in the brain. Microdialysis can measure basal levels, but is much slower, with a typical temporal resolution of 10 min. Enzyme sensors take a few seconds to respond (Dale et al. 2002). Our data show that adenosine increased within 2–3 s after stimulation and the first phase of adenosine efflux was cleared within 15 s. Microdialysis would be too slow to measure the rapid time course and our voltammetric sensor would give a more accurate picture of the accumulation time than an enzyme sensor.

Adenosine efflux in the caudate–putamen was measured after electrical stimulation of dopaminergic neurons in the SN. The stimulation we chose (60 pulses at 60 Hz) had been previously shown to cause adenosine receptor-dependent oxygen changes (Venton et al. 2003a). In addition, behaviorally evoked dopamine transients have a similar intensity and duration compared to electrically stimulated dopamine release elicited with a similar pulse train (Robinson et al. 2002). Therefore, our pulse train is physiologically relevant and represents a stressed situation, such as a reaction to strong environmental stimuli. Other studies have also observed stimulated adenosine efflux in brain slices of different regions including the cortex (Pedata et al. 1990), the hippocampus (Lloyd et al. 1993; Cunha et al. 1996), and the striatum (Sciotti et al. 1993). The stimulations in those studies were much longer (1–5 min at 10–100 Hz). Long stimulations were needed to elicit measurable concentration changes on the order of minutes, because the studies had 3 min temporal resolution at best. Our observed adenosine concentration changes after short stimulations were much faster than those previously observed in slices.

The concentration of adenosine elicited by electrical stimulation ranged from 0.1 to 2.7 μM (average: 0.94 ± 0.09 μM), but was consistent for repeated stimulations in an individual animal. The levels of stimulated adenosine efflux would be sufficient to activate high affinity A1 and A2A receptors in all animals, and might also be able to activate some lower affinity receptors at their peak concentrations. However, this activation would be transient, lasting only a few seconds. The time course of adenosine efflux and clearance after the stimulation also varied. Sometimes, one peak in adenosine was observed immediately following the stimulation, but in other animals, two peaks were observed, with the second peak occurring 15–25 s after the stimulation. The variation is likely because of biological variability and positioning of the electrodes, for example how close the carbon-fiber electrode is to sites of adenosine production or which neurons are activated by the stimulating electrode. The different peaks may also arise from different sources of adenosine. Similarly, variations in the time course of oxygen changes have been observed after evoked dopamine activity (Venton et al. 2003a).

Stimulated adenosine efflux was sometimes observed when dopamine release was not detected. Therefore, the adenosine efflux is not because of a direct action of dopamine but is likely an indirect effect of increased activity after axonal stimulation. Activity would increase ATP use and metabolism, leading to adenosine formation.

Verification of the measured signal as adenosine

Pharmacological experiments were used to confirm the identity of signal as adenosine. Drugs were administered that were known to modulate adenosine signaling, allowing a closer look at the regulation of extracellular adenosine concentrations. Adenosine transport and metabolic inhibitors affected the adenosine signal, while a histamine precursor did not.

Propentofylline is an adenosine transport inhibitor that inhibits both rENT1 and rENT2 transporters (Parkinson et al. 1993). It has been shown to have a neuroprotective effect in different models of cerebral ischemia (Parkinson et al. 1994; Sweeney 1997). The nucleoside transporters are bidirectional, so transporter inhibition would have different effects depending on the mechanism of formation. If adenosine is formed in the intracellular compartment and then transported out of the cell, transport inhibition would decrease adenosine levels; but if adenosine was formed after extracellular metabolism of released nucleotides, then extracellular concentrations should increase because of slower clearance. Administration of propentofylline decreased the first peak of stimulated adenosine efflux. Therefore, adenosine efflux after short, high frequency stimulations is likely because of transport of intracellularly formed adenosine out of the cell. Propentofylline also has other effects on the adenosine system, acting as a weak adenosine receptor antagonist and a very weak indirect agonist (Parkinson et al. 1994). While these receptor interactions are low affinity, future studies are needed to assess the efficacy of propentofylline at different doses for acting as a transport inhibitor and receptor antagonist.

After uptake across the membrane of cells, one of the main intracellular metabolic pathways of adenosine is the formation of AMP by adenosine kinase. Previous studies have shown that inhibition of adenosine kinase increased extracellular adenosine levels in vivo (Ballarin et al. 1991) as well in vitro (Sciotti and van Wylen 1993; Pazzagli et al. 1995). We used ABT-702, a selective non-nucleoside adenosine kinase inhibitor, which has improved cellular and tissue penetration compared with tubercidin-analog adenosine kinase inhibitors (McGaraughty et al. 2001). In our study, administration of ABT-702 increased stimulated adenosine efflux. Both peaks of adenosine were increased, with the second one increasing the most. The increase in adenosine efflux after inhibiting an intracellular metabolism step also suggested that the primary method of adenosine formation was intracellular. While the uptake inhibition data suggest the transporters are involved in adenosine efflux, the accumulation of adenosine is so fast that direct, vesicular release of adenosine cannot be excluded.

Histamine has a similar CV to adenosine, with an oxidation peak at 1.5 V, so it could be a possible interferent for detection of adenosine. Administration of l-histidine, a histamine synthetic precursor, failed to alter the measured oxidation peak at 1.5 V. Therefore, the measured signal is not histamine. Other electrochemically active compounds could also be possible interferents, like ATP and AMP. We have previously shown that voltammetry gives much smaller oxidation currents for these adenosine nucleotides because the negative holding potential of the electrode repels them (Swamy and Venton 2007). Inosine, a metabolite of adenosine, is not detected at these potentials. This is an advantage over the adenosine enzyme sensor, which is equally sensitive to adenosine and inosine, thus requiring a second, null sensor to be used simultaneously (Llaudet et al. 2003).

Correlation of adenosine and oxygen

After positive identification of the adenosine signal, FSCV was used to simultaneously measure the concentration changes of adenosine and oxygen in brain. Adenosine acts as an endogenous vasodilator, acting on A2A receptors (Ngai et al. 2001) to regulate cerebral blood flow (Winn et al. 1981). Stimulated activity can increase the concentration of chemical messengers such as adenosine or nitric oxide that act as vasodilators. Vasodilation results in an increase in oxygen, as more oxygen is delivered than can be used. This increase in oxygen after neuronal activation is used as a signal for neuronal activation in imaging studies, such as functional magnetic resonance imaging. Carbon-fiber microelectrodes can be used to measure changes in oxygen concentrations by scanning to potentials negative enough to reduce oxygen. Electrochemical measurements of oxygen have been validated by showing that regional increases in tissue oxygen, measured electrochemically, parallel the increases in cerebral blood flow measured by hydrogen clearance during behavioral activation (Lowry et al. 1997). Previous studies using cyclic voltammetry have shown that electrical stimulation of dopamine neurons increased oxygen concentrations and that this increase was blocked by the adenosine receptor antagonist theophylline (Venton et al. 2003a).

We chose to repeat the propentofylline experiment using a different potential waveform that would allow the detection of oxygen as well as adenosine. We hypothesized that since propentofylline decreased adenosine efflux, stimulated oxygen levels would also decrease, because less adenosine would be available to activate receptors and cause vasodilation. A correlation was observed between the adenosine and oxygen decrease after propentofylline administration, as both decreased by about 40%. These studies show that carbon-fiber electrodes are useful for observing both adenosine concentrations and its downstream effects.

The dynamic changes in adenosine concentrations suggest that adenosine receptors might be differentially activated during a stressful situation that leads to a burst of neuronal activity. Because both excitatory and inhibitory adenosine receptors are located in the caudate, extracellular adenosine efflux could have multiple effects (Cunha 2001, 2005). For example, activation of A1 receptors is inhibitory, which could lead to decreased glutamate release. However, A1 and A2A receptors are colocalized in the striatum on glutamate neurons and the excitatory A2A receptors are usually dominant, overriding the A1 effects (Borycz et al. 2007). Similarly, the increased oxygen concentrations we observed after neuronal activation could be because of direct activation of A2A receptors on endothelial cells that are vasodilatory. Or an indirect mechanism, because of reduced metabolism after activation of A1 receptors could lead to reduced oxygen consumption. Determining the mechanism and function of extracellular adenosine in activating receptors regulating neurotransmission and blood flow will be a fruitful area of further research.

Conclusions

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

In this study, we demonstrated that extracellular adenosine concentration transiently increased after stimulation of dopaminergic axons. Stimulated adenosine efflux was sufficient to activate high affinity receptors and might also transiently activate low affinity receptors. The use of this sensor in physiological experiments will allow new insight into the mechanism of adenosine signaling. For example, adenosine receptors can form heteromers with other metabotropic receptors such as dopamine receptors (Fuxe et al. 1998; Ciruela et al. 2006) and our methods could be used to determine how adenosine modulates dopamine neurotransmission. Adenosine is also thought to be protective in pathologies such as stroke and our sensor would be useful in determining the time course of adenosine efflux in models of ischemia. Previously, fast-scan voltammetry has been used to measured dopamine in freely moving animals, so our techniques could also be used to monitoring behaviorally evoked adenosine changes in the future. The knowledge of the real-time changes in adenosine concentrations will lead to a better understanding of the time course of adenosine receptor activation and its modulatory effects.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was supported by Grants to BJV from the American Heart Association (0765318U) and the NIH (R21-EB007830).

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  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Fig. S1 Cyclic voltammograms for A.) 1 μM Propentofylline, B.) 1 μM Histidine and C.) 1 μM ABT-702. Propentofylline and histidine were dissolved in Tris-buffer. ABT-702 was dissolved in 1% DMSO in Tris-buffer.

Fig. S2 Cyclic voltammograms for Adenosine (black), Adenosine and 0.1% DMSO (red) and Adenosine and 0.1% DMSO and 0.1 μM ABT-702 (blue). The DMSO slightly decreases electrode sensitivity to adenosine, but no further decreases were seen with addition of ABT-702.

Fig. S3 Calibration of oxygen saturated Tris-buffer in a flow injection analysis system. The oxygen saturated buffer contained 870 μM dissolved oxygen. The reduction peak for oxygen around -1.3 V is clearly visible in the CV.

Fig. S4 Color plots and cyclic voltammograms of adenosine and oxygen changes in vivo (A) before and (B) 30 min. after propentofylline (15 mg/kg). The potentials for oxygen and adenosine are labeled in the color plot of panel (A). The cyclic voltammograms were taken 3 seconds after the stimulus was administered and the adenosine and oxygen peaks are labeled in panel (A).

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