Anal. Chem. 2003, 75, 4875-4881
Monitoring Hydrogen Peroxide in the Extracellular Space of the Brain with Amperometric Microsensors Nadezhda V. Kulagina† and Adrian C. Michael*
Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
Interest in the detection of hydrogen peroxide in living brain tissue is growing for several reasons. Peroxide and other reactive oxygen species are implicated in neurodegenerative disorders and appear to have neuromodulatory functions in the brain. Also, there is a need to measure peroxide levels as a companion to measurements with amperometric sensors that rely on enzymes to generate peroxide for the detection of glutamate, choline, and glucose. Herein, we report on measurements performed in the brain of anesthetized rats with carbon fiber amperometric sensors coated with a cross-linked redox polymer film that contains horseradish peroxidase. Prior work with these sensors has established that they are both sensitive and selective toward hydrogen peroxide. When implanted in the striatal region of the rat brain, a biphasic response is observed upon electrical stimulation of the dopaminergic pathway that innervates the striatal tissue. No response is observed at sensors lacking HRP, which are not sensitive to peroxide, suggesting that the biphasic response is due to the production of hydrogen peroxide by two separate mechanisms. Additional measurements of dopamine and oxygen, and the administration of two drugs with well-known effects on the biochemical kinetics of the dopamine neurons, are used to identify those mechanisms. One appears to be the production of peroxide upon the oxidation of dopamine by molecular oxygen. This occurs during the electrical stimulation itself, which elevates both dopamine and oxygen levels in the extracellular space. The other appears to be the production of peroxide as a byproduct in the oxidative metabolic conversion of dopamine to DOPAC by the mitochondrial enzyme, monoamine oxidase. The production of peroxide due to dopamine metabolism is also observed after rats receive a dose of L-DOPA, a drug used in the treatment of Parkinson’s disease. Hydrogen peroxide is a byproduct of numerous biological oxidation reactions. Due perhaps in part to this characterization, the in vivo detection of hydrogen peroxide in living tissues has not been a focus of intense effort. Interest is growing, however, * To whom correspondence should be addressed. Phone: (412) 624-8560. Fax: (412) 624-8611. E-mail:
[email protected]. † Current address: U.S. Naval Research Laboratory, Center for Bio/Molecular Science & Engineering, Washington, DC 20375. 10.1021/ac034573g CCC: $25.00 Published on Web 08/12/2003
© 2003 American Chemical Society
in the detection of hydrogen peroxide in living brain tissue. First, the production of hydrogen peroxide and other reactive oxygen species continues to be implicated in the etiology of neurodegenerative disorders such as Parkinson’s disease.1-3 Second, emerging evidence suggests that hydrogen peroxide, much like nitric oxide, plays an important role as a diffusible neuromodulator in various aspects of brain function.4 And third, amperometric sensors that rely on the detection of enzymatically produced hydrogen peroxide are finding increased application to studies of the brain,5-10 which requires the in vivo detection of hydrogen peroxide comparison signals. Two research strategies have been used lately to investigate the possible role of hydrogen peroxide as a neuromodulator in brain function. The first examines the effects of exogenous hydrogen peroxide added to the fluid in which the tissue is bathed.11 Exogenous peroxide has some effects that are irreversible and, so, are attributable to toxicity. On the other hand, some effects are reversible; i.e., they dissipate once the peroxide is washed out of the tissue, and appear unrelated to toxicity. The second strategy focuses on the effects of endogenous peroxide, i.e., peroxide formed within the tissue itself, by the use of catalase or inhibitors of endogenous enzymes, such as glutathione peroxidase, that consume peroxide.4 Together, these strategies have revealed that peroxide modulates several neurochemical processes, most likely by interacting with redox-sensitive sites on receptors and proteins. Hence, it would be interesting to know more about the concentration of endogenous peroxide in the brain, the rate and duration of changes in its concentration, and factors that control the rate and mechanism of its production. (1) Halliwell, B. J. Neurochem. 1992, 59, 1609-1623. (2) Simonian, N. A.; Coyle, J. T. Annu. Rev. Pharmacol. Toxicol. 1996, 36, 83106. (3) Stokes, A. H.; Hastings, T. G.; Vrana, K. E. J. Neurosci. Res. 1999, 55, 659665. (4) Avshalumov, M. V.; Chen, B. T.; Marshall, S. P.; Penˇa, D. M.; Rice, M. E. J. Neurosci. 2003, 23, 2744-2750. (5) Garguilo, M. G.; Michael, A. C. J. Neurosci. Methods 1996, 70, 73-82. (6) Kulagina, N. V.; Shankar, L.; Michael A. C. Anal. Chem. 1999, 71, 50935100. (7) Cui, J.; Kulagina, N. V.; Michael, A. C. J. Neurosci. Methods 2001, 104, 183-189. (8) Burmeister, J. J.; Gerhardt, G. A. Anal. Chem. 2001, 73, 1037-42. (9) Burmeister, J. J.; Pomerleau, F.; Palmer, M.; Day, B. K.; Huettl, P.; Gerhardt, G. A. J. Neurosci. Methods 2002, 119, 163-171. (10) You, T.; Niwa, O.; Tomita, M.; Hirono, S. Anal. Chem. 2003, 75, 20802085. (11) Avshalumov, M. V.; Chen, B. T.; Rice, M. E. Brain Res. 2000, 882, 86-94.
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Herein, we describe an amperometric sensor suitable for the in vivo detection of hydrogen peroxide in the extracellular space of living brain tissue. We have used these sensors to monitor peroxide in the brain of anesthetized rats. The sensor is of the same design that we have used previously to monitor background signals during the in vivo detection of glutamate, choline, and glucose with enzyme-modified amperometric sensors.5-7 The sensor is based on a carbon fiber microelectrode coated with a cross-linked redox polymer (RP) film that contains pendant, nondiffusing, osmium-centered polypyridyl complexes12,13 and horseradish peroxidase (HRP). Two additional steps are taken to increase the selectivity and stability of the sensors. First, ascorbate oxidase is included in the RP film to prevent ascorbate from reducing the osmium complex. Ascorbate oxidase is effective in this application because is produces water rather peroxide upon the oxidation of ascorbate. Second, the sensors are coated with Nafion, which protects the sensor while it is implanted in brain tissue. The sensor operates at an applied potential of -0.1 V versus a Ag/AgCl reference electrode, which ensures that the several oxidizable compounds present in the extracellular space of the brain do not interfere with the detection of peroxide. In the majority of our previous in vivo studies, the signals at the peroxide-sensitive comparison sensors remained unchanged even while responses were observed at amperometric sensors that detect glutamate, choline, or glucose.5-7 This signifies that changes in possibly electroactive interfering substances, including peroxide, were not contributing to the observed responses. Herein, we report two exceptions to this case, in which robust changes in the signal at the peroxide-sensitive sensors occurred during manipulations of the neurochemical activity of the rat striatum, which is densely innervated with dopaminergic axon terminals. One of these manipulations involved the electrical stimulation of the medial forebrain bundle (MFB), which contains the dopaminergic input pathway to the striatum. The other manipulation was the administration of L-3,4-dihydroxyphenylalanine (L-DOPA), which is the biosynthetic precursor of dopamine and the most widely prescribed therapeutic agent for the treatment of Parkinson’s disease. To confirm that the signals observed during these manipulations were due to changes in hydrogen peroxide concentrations, measurements were also performed at microsensors lacking HRP, which do not respond to peroxide. To aid in identifying the mechanisms by which the observed peroxide was produced, extracellular levels of dopamine and oxygen were measured by fast-scan cyclic voltammetry.14,15 In addition, drugs with well-known effects on the biochemistry of dopamine neurons were used. Overall, the findings of this work confirm that it is possible to monitor the formation of hydrogen peroxide associated specifically with manipulations of neuronal brain activity. EXPERIMENTAL SECTION Reagents. Peroxidase type II (from horseradish, HRP), glutamate oxidase (GOx), ascorbate oxidase (from cucurbita), (12) Gregg, B. A.; Heller, A. J. Phys. Chem. 1991, 95, 59-76-5980. (13) Shankar, L.; Garguilo, M. G.; Michael, A. C. In Methods in Biotechnology, Vol 6: Enzyme and Microbial Sensors: Techniques and Protocols; Mulchandani, A., Rogers, K. R., Eds.; Humana Press: Totowa, NJ, 1998; pp 121132. (14) Zimmerman, J. B.; Wightman, R. M. Anal. Chem. 1991, 63, 24-28. (15) Kennedy, R. T.; Jones, S. R.; Wightman, R. M. Neuroscience 1992, 47, 603612.
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R-methyl-DL-p-tyrosine methyl ester hydrochloride (RMPT), pargyline, L-DOPA, (S)-(-)-carbidopa, and chloral hydrate were obtained from Sigma (St. Louis, MO). Nafion (5% solution in a mixture of lower aliphatic alcohols and water, 1100 equiv wt) was purchased from Aldrich (Milwuakee, WI) and diluted with 2-propanol to make 0.5 and 1% solutions before use. [4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid] (HEPES) and HEPES sodium salt were obtained from Aldrich (Milwaukee, WI). Poly(ethylene glycol 400 diglycidyl ether) (PEGDGE) was purchased from Polysciences (Warrington, PA). All the reagents mentioned above were used as received without further purification. The cross-linkable redox polymer used for the preparation of the peroxide microsensors was synthesized according to procedures described in detail elsewhere.12,13 Artificial cerebrospinal fluid (aCSF: 145 mM Na+, 1.2 mM 2+ Ca , 2.7 mM K+, 1.0 mM Mg2+, 152 mM Cl-, 2.0 mM phosphate, pH 7.4) was used for the preparation of calibration standards. Enzymes used for preparation of the microsensors were dissolved in a HEPES buffer, pH 8.0, prepared by the addition of the HEPES sodium salt to a 10 mM solution of the acid. Ultrapure water (Nanopure, Barnstead, Dubuque, IA) was used for all solutions prepared in-house. Electrochemical Microsensors and Techniques. The principle of operation, the construction, the calibration, and the in vivo use of the enzyme-modified microsensors and associated electrochemical techniques have been described in detail in prior reports.5-7 Briefly, enzymes were immobilized within a crosslinked redox polymer gel that was deposited onto carbon fiber microelectrodes (P-55 fibers, 10 µm in diameter, 400 µm in length). The RP has a poly(vinyl pyridine) backbone with pendant, nondiffusing osmium-centered polypyridyl complexes that participate in both electron self-exchange (electron hopping) and cross-exchange with HRP. Hence, the polymer both immobilizes the enzymes and mediates electron transfer between the electrode and HRP. Three styles of microsensor were used for this study: GOx/HRP/RP microsensors were prepared by immobilization of GOx and HRP in the redox polymer film; HRP/RP microsensors were prepared by immobilizing only HRP; RP microsensors were coated with the redox polymer film with no GOx or HPR. Microsensors of all three types contained ascorbate oxidase and were coated with Nafion. The microsensors were operated in the amperometric (constant potential) mode with an applied potential of -0.1 V versus Ag/AgCl. Currents were amplified with picoammeters (model 428, Keithley Instruments, Cleveland, OH) set to a gain of 1010 V/A and a rise time of 300 ms. The output of the picoammeters was digitized via a Labmaster interface (Scientific Solutions, Solon, OH) with software developed in-house. Evoked changes in extracellular dopamine and oxygen were monitored by fast-scan cyclic voltammetry in conjunction with plain carbon fiber microelectrodes (T-300, 7 µm in diameter and 400 µm in length) using procedures exactly as described elsewhere.14,15 Fast-scan voltammetry was performed with a customdesigned potentiostat-amplifier system (EI 400, Ensman Instrumentation, Bloomington, IN) under computer control with software developed in-house. Voltammetric scans started at 0 V versus Ag/ AgCl and comprised sequential linear potential sweeps to 1 V, -1.4 V, and back to 0 V at a sweep rate of 300 V/s. Voltammetric scans were repeated at 200-ms intervals. The dopamine oxidation
L-ascorbate, dopamine, nomifensine,
signal was obtained by calculating the average current value in the 0.7-0.8 V potential window on the first sweep of each scan. The oxygen reduction signal was obtained by averaging the current in the -1.2 to -1.3 V potential window on the second sweep of each scan. Background-subtracted voltammograms were used to confirm that the voltammetric properties of the substances detected in vivo matched with those of dopamine and oxygen observed during calibration of the electrodes after they were removed from the animals. The appearance of these voltammograms was consistent with several previously published reports,14,15,17,18 so the voltammograms themselves are not reproduced here. The microelectrodes used during this work were calibrated both before and after the in vivo experiments. Calibration prior to the in vivo experiments served just as a performance check. Data obtained during postcalibration of the microsensors after the in vivo experiments were used for the conversion of amperometric signals recorded in the brain to units of peroxide concentration. The postcalibration in air-equilibrated aCSF at 37 °C. The microsensors exhibited a detection limit for hydrogen peroxide (S/N ) 3) of 285 ( 60 nM (mean ( SD, n ) 9), a sensitivity of 7.1 ( 3.2 pA/µM (mean ( SD, n ) 5), and a correlation coefficient of greater than 0.99 in peroxide concentrations up to 10 µM, the highest concentration routinely tested. Animals and Surgical Procedures. Experiments involving animals were carried out with approval from the Institutional Animal Care and Use Committee of the University of Pittsburgh. Male Sprague-Dawley rats, 250-350 g, were anesthetized with chloral hydrate (400 mg/kg ip), placed in a stereotaxic frame (Kopf, Tujunga, CA) with the incisor bar raised 5 mm above the intraural line,16 and kept unconscious with additional doses of chloral hydrate as determined by the reflexive withdrawal of the hind limb upon application of gentle pressure to the paw. Body temperature was monitored and maintained at 37 °C with a homeothermic blanket system (EKEG Electronics, Vancouver, BC). Small holes were drilled through the skull to allow insertion of microsensors and microinfusion pipets into the striatum and stimulating electrodes into the MFB. Electrical contact between brain tissue and a Ag/AgCl reference electrode was establish via a salt bridge fashioned from a plastic pipet tip plugged with a wick of tissue paper. Microsensors were mounted onto individual stereotaxic micromanipulators angled 8° from vertical in the coronal plane and were implanted into the striatum at a point 2.4 mm anterior to bregma, 2.5 mm lateral from the midline, and 4.5 mm below dura.16 The distance between the microsensors was ∼200 µm. Electrical Stimulation. Electrical stimulation of dopaminergic axons in the MFB was carried out as described before17,18 in order to evoke the release of dopamine from neuronal terminals in the striatum. Stimulation was delivered via a bipolar stainless steel stimulating electrode insulated everywhere except at the tips (MS303/1, Plastics One, Roanoke, VA). The stimulating electrode was placed in the MFB at a point 2.2 mm posterior to bregma, 1.6 mm from midline, and 7.5 mm below dura.16 The final vertical position of the electrode was adjusted until evoked dopamine (16) Pelligrino, L.; Pellegrino, A.; Cushman, A. A Stereotaxic Atlas of the Rat Brain, 2nd ed.; Plenum Press: New York, 1979. (17) Lu, Y.; Peters, J. L.; Michael, A. C. J. Neurochem. 1998, 70, 584-593. (18) Peters, J. L.; Michael, A. C. J. Neurochem. 2000, 74, 1563-1573.
release was observed by fast-scan cyclic voltammetry at a plain carbon fiber microelectrode implanted in the striatum using the same stereotaxic coordinates as above. The stimulus was an optically isolated (Neurolog 800, Medical Systems, Greenvale, NY), biphasic, constant current, square waveform (pulse frequency, 40 Hz; pulse duration 2 ms; pulse height, 280 µA; train length, 20 s; train interval, at least 30 min). At least three reproducible evoked dopamine responses were recorded before additional experiments were initiated. In some cases, recording of electrically evoked dopamine and oxygen responses was continued with the carbon fiber microelectrode. In other cases, the carbon fiber microelectrode was removed and replaced with a pair of microsensors, one with and one without HRP. To aid in identifying the source of the signals observed during and after evoked dopamine release, stimulation experiments were also performed in animals that received a single dose of one of two drugs with well-known actions in dopamine neurons. The first was RMPT, which is a selective inhibitor of tyrosine hydroxylase, the enzyme that catalyzes the rate-determining step in the biosynthesis of dopamine. The other drug was pargyline, which inhibits MAO, an enzyme that participates in the metabolism of dopamine to its principal metabolite, dihydroxyphenylacetic acid (DOPAC). When used, RMPT (250 mg/kg, ip) was administered 60 min before the next electrical stimulus, while pargyline (75 mg/kg, ip) was administered 40 min before the next electrical stimulus. The effect of drugs on the amplitude of the evoked response is represented as a percentage of the average of the magnitude of the three responses obtained just prior to drug administration. Direct Infusion of Dopamine into the Striatum. After implantation of GOx/HRP/RP and HRP/RP microsensors in the striatum, some rats received a microinfusion of dopamine (100 µM in aCSF, 200 nL) via a micropipet. The pipet was fashioned from a fused-silica capillary tube with an internal diameter of 25 µm: the capillary wall at the tip of the pipet was etched with HF to a diameter of 30 µm. For the infusion, the capillary inlet was attached to a gastight syringe mounted in a computer-controlled driver (Sutter Instruments, Novato, CA). To prevent the rapid uptake of the infused dopamine by neuronal terminals surrounding the infusion site (see ref 19, for example), animals were pretreated with the dopamine uptake inhibitor, nomifensine (20 mg/kg ip), 30 min prior to the infusion. Experiments Involving L-DOPA Administration. After implantation of the HRP/RP and RP microsensors in the striatum, some rats received a dose of L-DOPA (200 mg/kg ip) either alone or in conjunction with pargyline (75 mg/kg ip). Since L-DOPA is susceptible to enzymatic oxidation before it reaches the brain, animals were pretreated 30 min prior to receiving L-DOPA with (S)-(-)-carbidopa (100 mg/kg ip), which inhibits peripheral decarboxylase enzymes. RESULTS AND DISCUSSION Electrical Stimulation Experiments. Figure 1 shows that electrical stimulation of dopaminergic axons in the medial forebrain bundle evoked a rapid transient response at the amperometric HRP/RP microsensor (Figure 1A) but not at the amperometric RP sensor that lacks HRP (Figure 1B). The signal traces (19) Cass, W. A.; Gerhardt, G. A. J. Neurochem. 1995, 65, 201-207.
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Figure 2. Amperometric traces recorded in the rat striatum with HRP/RP sensors (a) and RP sensors (b) during repeated electrical stimulation of dopaminergic axons in the medial forebrain bundle. The electrical stimulus was 20 s long and was delivered at 1-h intervals. The open circles mark the beginning and the end of each stimulus. The vertical concentration scale bar was determined by postcalibration of the HRP/RP sensor after its removal from the brain.
Figure 1. Amperometric (A, B) and voltammetric (C, D) recordings in the striatum of anesthetized rats during electrical stimulation of dopaminergic axons in the medial forebrain bundle. The signals reflect the stimulus-evoked change in the concentration of hydrogen peroxide (A), dopamine (C), and oxygen (D). The trace in (B) is the background amperometric response at an RP sensor. The open symbols in each trace mark the beginning and the end of the electrical stimulus.
reported in Figure 1A and B were obtained simultaneously with the two microsensors placed side by side in the same animal. The absence of a stimulus-evoked response in Figure 1B shows that the response in Figure 1A is due to a change in the extracellular concentration of a reducible substrate of HRP, which, for reasons discussed further below, is most likely to be hydrogen peroxide. Figure 1 also shows that, in a different animal, electrical stimulation evoked rapid increases in the extracellular concentrations of dopamine (Figure 1C) and oxygen (Figure 1D) as measured by fast-scan cyclic voltammetry at a single carbon fiber microelectrode. Hence, as reported before, the electrical stimulus evoked a simultaneous transient elevation in the extracellular concentration of both dopamine and oxygen, the latter attributable to a rapid increase in blood flow to the striatal region.14,15 Figure 2 shows signal traces recorded in the striatum with side-by-side HRP/RP and RP amperometric sensors, the latter lacking HRP. These traces show that the electrical stimulus actually evoked a biphasic response at the HRP/RP amperometric sensors. The initial rapid transient response returned to baseline within a few seconds after the end of the stimulus. A few seconds later, a second slow phase of the response began. During the second phase of the response, the signal increased over a period of 4-6 min to a maximum amplitude corresponding to an increase in the extracellular peroxide concentration of 2.0 ( 0.2 µM, (mean ( SD, n ) 3 rats). After reaching a maximum, the signal slowly decreased, returning to baseline levels by ∼20 min after the stimulus. Figure 2 also shows that the biphasic response was highly reproducible when the electrical stimulus was repeated in individual animals at an interval of 60 min. During the second phase of the stimulus-evoked response, no response was observed at the amperometric sensor lacking HRP (Figure 2). The dopa4878 Analytical Chemistry, Vol. 75, No. 18, September 15, 2003
mine and oxygen levels as measured by fast-scan cyclic voltammetry also remained unchanged during the second phase of the response at the HRP/RP sensor (data not shown). A possible source of the initial rapid transient response at the HRP/RP sensor is hydrogen peroxide formed upon the oxidation of dopamine in the hyperbaric extracellular environment that exists during the electrical stimulus (Figure 1D). A possible source of the second slower phase of the response at the HRP/RP sensor is hydrogen peroxide formed by the actions of MAO as it metabolizes the dopamine that was released by the stimulus and is subsequently taken back up into the cytoplasm of dopamine terminals by the dopamine reuptake mechanism. Once in the cytoplasm, dopamine becomes a target of MAO, which is present in the mitochondria. Further experiments were undertaken to test these hypotheses. Several drugs are well known for their ability to change the amount of dopamine released to the extracellular space of the striatum during electrical stimulation of the dopamine pathway that innervates this brain region. One of these is RMPT, which blocks the synthesis of dopamine and decreases the amount of stimulus-evoked dopamine release.20 Figure 3 shows that RMPT, when administered 1 h prior to stimulation, decreased the amplitude of both phases of the stimulus-evoked response at the HRP/RP amperometric sensors. The traces in Figure 3A and B were obtained in the same animal before and after, respectively, RMPT administration. Figure 4A, which summarizes the results from several animals, shows that RMPT caused a similar decrease in the amplitude of the rapid initial stimulus-evoked peroxide response and the stimulus-evoked dopamine response. Figure 4B shows that RMPT decreased the amplitude of the slower second phase of the stimulus-evoked response at the HRP/RP amperometric sensors. The drug, however, had no effect on the amplitude of the oxygen response. The MAO inhibitor, pargyline, is well known for its ability to block the intraneuronal metabolism of dopamine to DOPAC, thereby causing an accumulation of dopamine in striatal terminals and increasing the amount of dopamine released during electrical (20) Kulagina, N. V.; Zigmond, M. J.; Michael, A. C. Neuroscience 2001, 102, 121-128.
Figure 3. Amperometric traces recorded in the rat striatum with HRP/RP sensors (a) and RP sensors (b) during repeated electrical stimulation of dopaminergic axons in the medial forebrain bundle. Traces are shown without drug treatment (A), after treatment with RMPT (B), and after treatment with pargyline (C). Traces in (A) and (B) are from the same animal. The open circles mark the beginning and the end of each stimulus. The vertical concentration scale bars were determined by postcalibration of the HRP/RP sensors after they were removed from the brain. The scale bars in each panel correspond to 1.2 µM peroxide.
stimulation.20 Figure 3C shows amperometric traces recorded in an animal that received pargyline 40 min prior to stimulation. The trace from the HRP/RP sensor clearly shows that the amplitude of the second slow phase of the response is decreased compared to the rapid initial phase. Data from several animals are summarized in Figure 4A, which shows that pargyline increased the amplitude of the first phase of the amperometric response and the dopamine response to a similar degree. Again, pargyline had no effect on the oxygen response. On the other hand, pargyline almost completely abolished the second phase of the amperometric response. The results in Figures 3 and 4 are consistent with the idea that the first phase of the stimulus-evoked amperometric response is due to the production of hydrogen peroxide by the oxidation of dopamine under the hyperbaric conditions created during the stimulus. The time courses of the stimulus-evoked peroxide and dopamine signals were well correlated. Changes in the amplitudes of the two signals after the administration of RMPT or pargyline were also very well correlated. Both of these correlations are consistent with the idea that the peroxide response is proportional to the availability of dopamine. Neither drug affected the stimulusevoked increase in the availability of oxygen, suggesting that the stimulus evoked similar hyperbaric conditions during experiments with each drug. It is not possible that the amperometric response is due somehow to the detection of dopamine itself, since thorough in vitro calibration experiments have confirmed that these sensors
Figure 4. (A) Effects of drug treatments on the amplitude of the initial rapid response at the HRP/RP sensors, the voltammetric dopamine signal, and the voltammetric oxygen signal recorded during electrical stimulation of the medial forebrain bundle. (B) Effects of drug treatments on the amplitude of the second slow phase of the stimulus-evoked response at HRP/RP sensors. In both panels, the response amplitudes have been normalized to the average of the amplitude of the three responses recorded prior to drug treatment. The control drug treatment was the ip injection of saline. Statistical analysis was performed with one-way ANOVA and Duncan’s multiple range test (*, p
