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Voltammetry in brain tissue: Chronic recording of stimulated dopamine and 5-hydroxytryptamine release
Peragmon Press
Life Sciences, Vol . 23, pp . 2705-2716 Printed in the U .S .A .
VOLTAMMETRY IN BRAIN TISSUE : CHRONIC RECORDING OF STIMULATED DOPAMINE AND 5-HYDROXYTRYPTAMINE RELEASE J. C . Conti, E . Strope, R. N . Adams and C . A . Marsden* Department of Chemistry University of Kansas, Lawrence, Kansas 66045 and *Department of Physiology and Pharmacology University Hospital and Medical School Nottingham, NG7 2UH, Great Britain (Received in final form November 6,
1978)
SUMMARY Micro voltammetric electrodes which continuously monitor stimulated release of dopamine and 5-hydroxytryptamine in unanesthetized, essentially unrestrained rats are described . The results demonstrate that the electrochemical technique correctly follows dopamine efflux, especially in the case of amphetamine-stimulated dopamine release in the caudate . While the method is still exploratory, its value in pharmacological manipulations of neurotransmitter release, etc . is already clearly evident . Electrochemical measurements of the release of dopamine metabolites in ventricular fluid have .been described in detail (1,2) . A similar technique can be used to record directly from CNS tissue (3) . The results presented herein, together with recent reports from other laboratories--Lane and coworkers (4), Gonon et aZ . (5)--already show that the method can monitor the actual release of neurotransmitter substances (and appearance of their metabolites) following pharmacological manipulations of dopamine (DA) and serotonin (5-HT) pathways . The technique is still in its infancy and much needs to be done to improve its selectivity and quantitative aspects . However, it is presently ready to be used, especially for in situ measurements of drug effects in the CNS . This report deals with chronic electrode implants and experiments which verify that the release of neurotransmitter substances is being monitored . The experimental approach was designed to illustrate that the in vivo electrochemical technique can monitor the stimulated release of certain neurotransmitter substances and does not just respond to some electroactive species only circumstantially related to release phenomena . Accordingly, the experiments were patterned after established literature examples in which postmortem biochemical assays, perfusion or "cortical cup" type analyses had been used to illustrate electrical- or drug-stimulated neûrotransmitter release . While a very thorough and lengthy investigation was carried out, the results 0300-9653/78/1231-270502 .00/0 Copyright (c) 1978 Pergamon Press
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Vol . 23, Nos . 27 $ 28, 1978
can be summarized briefly since the neurobiology and pharmacology are well known to all readers . So fâr manipulations which are believed to act primarily on catecholamine and serotonin systems have been examined . METHODS AND PROCEDURE The electrochemical procedures were essentially identical with those employed for CSF monitoring (1,2) . Current-potential curves or voltammograms were used primarily to observe qualitative changes of electroactive species in the tissue near the electrode tip (1) . For quantitative results the applied potential was stepped to a fixed value (+0 .8 V vs . Ag/AgCl) and the currenttime response (chronoamperometric measurement) was recorded for a short period, typically 1 sec . A.
Microelectrod es for Ch ronic Implantation
The voltammetric or working electrode is encased in a glass micro capillary . A mixture of high purity graphite plus an inert liquid (Nujol) provides a carbon surface with very low residual current (that current obtained in the absence of any electrooxidizable materials near the electrode) . To make the surface rigid and capable of lasting for chronic measurements, the graphite paste was mixed with epoxy resin, forced into the capillaries and then allowed to harden before use . The graphite paste is prepared by dissolving 0 .9 g of Nujol (mineral oil commonly sold as a laxative at any pharmacy) in 15 ml of carbon tetrachloride . This solution is vigorously stirred and 2 .1 g of graphite powder (Ultra Carbon UCP-1-M, Ultra Carbon, Bay City, Michigan) is slowly mixed in and stirring continued for 5 minutes . The slurry is then gently evaporated until all carbon tetrachloride is volatilized . This graphite paste may be stored for long periods in a vial until ready to be mixed with epoxy resin . Fifty to seventy-five electrodes were usually prepared at a time . A batch of capillaries was pulled from ca . 75 mm lengths of 3 mm o .d . glass tubing in a commercial pulley . Tip diameters were ordinarily between 75-200 u . The necessary number of lead wires made from 6-8 cm lengths of fairly stiff, insulated X28 wire (c a . 0 .3 mm dia.), stripped at each end, were readied . The resin is prepared by thoroughly mixing 0 .45 g of triethylene tetramine (Fisher) with 3 .6 g of Shell Epon 815 resin . Then 1 .05 g of this mixture is added, in a small vial, to 1 .35 g of the graphite paste prepared as above . Thorough stirring with a glass rod is continued until the mixture appears homogeneous . A portion is then transferred to a glass slide . The capillary is inverted and a portion of the epoxy mixture pressed into the barrel end of the tube . A suitable plunger is used to force the paste down the tip . Care is taken to visuaYly inspect for holes or breaks in the graphite-epoxy pathway throughout the capillary length . The barrel is then cut off and one end of the stripped lead wire forced as far down the capillary as possible . About 1 hour is available for preparing the electrodes once the epoxy mixture is mixed . The electrodes are conveniently stored by attaching them to a board with masking tape until they are cured (24-48
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hr) and ready for testing and calibration prior to implantation . The Ag/AgCl reference is prepared by stripping a 2 mm length of teflon-coated silver wire (dia . 0 .25 mm, Medwire Corp ., Mt . Vernon, NY) and anodizing as usual in 1 M hydrochloric acid . A 108 (w/v) gelatin-salt solution is prepared by dissolving 1 q of gelatin in 9 ml of 3 M NaCl solution with heat and allowing it to cool somewhat . A 4 mm length of ca . 0 .4 mm i .d . teflon tubing is filled with the gelatin, the Aq/AgCl wire inserted and both ends crimped to minimize leakage . A stock of reference electrodes may be prepared and stored in 3 M NaCl solution . The auxiliary electrode is a stainless steel screw inserted through a burr hole in the skull until it touches the surface of the brain . Electrical connection can be made with fine wire around the screw. B.
Test ing and Calibration of Electrodes
Just prior to implantation, the graphite electrodes are tested and calibrated in a small (ti5 ml) beaker . The electrode to be tested is snipped with scissors to expose a fresh tip and it is placed approximately vertically in the solution with the tip at least 2-3 mm below the solution surface . The micro Ag/AgCl reference and a platinum wire auxiliary make up the remainder of the electrochemical circuit . A suitable potential (+O .g V vs . Ag/AgCl in these studies) is applied and a series of chronoamperometric (i-t) responses are recorded . The residual i-t response in the absence of any electroactive materials in solution is first observed in pH 7 .4 buffer alone . Normally, electrodes with residual current values greater than 1-5 nanoamps (measured at 1 sec) are discarded as unsuitable . A standard solution of the catecholamine analog 4-methylcatechol (Aldrich) in pH 7 .4 buffer is then substituted and several i-t curves determined . The average value of the chronoamperometric response divided by the 4-methylcatechol concentration can be used later to estimate the in vivo concentrations . Since solutions of catecholamines and 4-methylcatechol are rapidly air oxidized, all standard solutions should be thoroughly deserated (preferably with argon) prior to dissolving any catechol compounds . The tested graphite electrodes are implanted along with the reference and auxiliary electrodes by standard stereotaxic procedures . Three or four graphite electrodes may be implanted in desired brain regions . Reference and auxiliary electrode placement is not critical, but they were usually placed in convenient locations within 1 cm of the detector electrodes . When implanted, all electrodes were fixed with cranioplastic cement and the leads were then attached to a 6-pin pedestal which mated to a flexible cable (Plastic Products, Roanoke, VA, parts MS363 and MS363-S1/6, respectively) . The pedestal was anchored with skull screws and the entire assembly firmly cemented in place. (This electrode pedestal is conventional and any similar arrangement for chronic recording is satisfactory .) For those experiments utilizing electrical stimulation of the brain, bipolar stimulating electrodes were implanted at appropriate coordinates . A separate 2-pin flexible connection was attached to a Grass stimulator or similar
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device . Some, but not all electrode placements were verified by standard histology . Experiments were carried out, ordinarily after 1-2 days surgical recovery, with the rats either free to move about the laboratory bench or in a large copper-screened box . The latter Faraday cage environment lessened noise problems . C.
in vivo Measurements
A typical experiment consisted of recording the i-t curve and measuring the current at 1 second with a 3 minute interval between measurements . This current is variously designated as i, signal, or response throughout these discussions . The first few measurements at all in vivo electrodes give large responses, but these decrease rapidly with repetition . Within 0 .5 to 1 hour the values stabilize at a baseline level which ordinarily is constant to t5-10$ and will remain so for long periods if no purposeful stimulation is applied to the animal . (Slow cyclic variations have been observed with 24 and 48 hour monitoring, but the significance of any rhythmic baseline changes has not been investigated .) The essentially unrestrained animals may move, groom, eat. and drink, and even scratch the head region with no apparent signal artifacts . When the baseline response is established, the stimulus (electrical, drug, or behavioral manipulation) is applied and the changes in the response are followed . All the experiments described herein, for convenience in data gathering, utilized a minicomputer which programmed and carried out the electrochemical measurements and data processing . How ever, it is possible to obtain similar results with far more simple instrumentaion . Several such arrangements and descriptions of measurement methods and calibrations are given in more detail elsewhere (Huff et aZ ., manuscript in preparation) . RESULTS AND DISCUSSION A.
Pharmacological Studies 1.
Amphetamine-induced Dopamine Release
Amphetamine-stimulated release of dopamine (DA) is well established in the literature (6) . Electrochemical measurements of this effect were carried out with working electrodes in the rat caudate nucleus . After baseline signals were stablized for at least 0 .5 hr, the animals received i .p . doses of dZ-amphetamine varying between 3-10 mg/kg and the electrochemical recording was continued . Approximately 15-20 minutes after the injection, signals rose by 20-408 and slowly returned to baseline within 1-2 hr . Figure 1 illustrates such an experiment in which electrodes in both left and right caudates were monitored . The onset of the electrochemical signal was always time-locked with the characteristic amphetamine-induced behavioral effects . These experiments have been repeated over a 1~ year period with more than 50 rats, using several batches of electrodes and three different types of measuring devices . Positive results were obtained in every case except those in which experimental problems (electrode breakage, equipment failure, etc .) readily explained the lack of signal increase . Similar amphetamine results have already been reported by Gonon et aL . using 8 u diameter graphite fiber electrodes in
Volta~etry in Brain Tissue
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anesthetized rats . These workers also illustrated the decrease or absence of amphetamine-induced electrochemical response following selective degeneration of DA terminals with 6-hydroxydopamine (5) . Lane and coworkers detected DA release electrochemically from intact caudate tissue ca . 30 sec after nearby microinjection of amphetamine (7) . Moat recently we have been able to electrochemically measure quantitative amphetamine dose-response curves in the range of 1-5 mg/kg of amphetamine (Huff et aZ ., manuscript in preparation) . These complementary results from several different laboratories convincingly demonstrate the electrochemical method readily follows amphetamine-induced DA release .
le 16 14 12
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FIG . 1 . Electrochemical Monitoring of Amphetamine Response Inj . oa . 4 mg/kg dZ-amphetamine i .p . L,R
voltammetric electrode recording in left and right caudate, respectively . current response in arbitrary units .
2.
Manipulation of 5-HT via p-Chloroamphetamine
The compound p-chloroamphetamine (p-C1-AMP) is a very interesting drug for testing the in vivo electrochemical method . It is known from biochemical studies that p-C1-AMP first gives a rapid release of 5-HT from nerve endings followed by long-lasting depletion of brain 5-HT (8) . Thus, if the graphite working electrode were present in a tissue region known to contain 5-HT nerve terminals and p-C1-AMP were administered i .p ., one could expect a
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Voltammetry in Brain Tissue
rather rapid rise in signal followed by a gradual decrease . This decrease should fall to a level considerably lower than the starting baseline as the 5-HT synthesis is shut off and the endogenous extracellular 5-HT concentrations necessarily decrease . This is exactly the response obtained . Figure 2 illustrates the effect of 3 mg/kg p-C1-AMP on the signals obtained at working electrodes in both the left and right globus pallidus of the caudates (coordinates : AP+1 .0 from bregma ; Lat .±2 .8 ; C/V-7 .5 from skull surface) . While this region also contains much DA, it is known to be richer in 5-HT than the more anterior caudate (9) . The lack of identical responses at the left and right electrodes may be due to different effective electrode areas or perhaps to unequal 5-HT distribution at slightly dissimilar electrode placements . Such variability is to be expected and in no way invalidates the data-other experiments have given left responses larger than right . A variety of other pharmacological manipulations of the 5-HT system futher demonstrates that the electrochemical method can be utilized to measure 5-HT release (Maraden et al ., submitted for publication) .
36
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FIG : 2 . Electrochemical Response to p-Chloroamphetamine Inj . 3 mg/kg p-chloroamphetamine . voltammetric electrode recording in left L,R and right globus pallidus region, respectively (see text) . current response in arbitrary units .
Vol . 23, Nos . 27 & 28, 1978
H.
Volta~etry in Brain Tissue
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Electrical Stimulation of Neural Pathways
Many studies have shown that low level electrical stimulation of the substantia nigra (pars compacts) or the ascending nigrostriatal pathway results in enhanced DA release at nerve terminals in the ipsilateral caudate nucleus (6) . We mimicked this style of experiment by implanting bipolar stimulating electrodes in the substantia nigra (or ascending fiber tract) and monitoring the electrochemical response at graphite working electrodes in the caudate . After establishing a stable baseline i, stimulating currents between 70-200 microamps (60 Hz sinusoidal) were applied for very brief periods (1-5 seconds) . Four sets of stimulating electrodes gave electrochemical responses averaging 408 rise above baseline in the ipsilateral caudate in 14 different stimulations . The rises were very sharp and occurred as quickly as 15 seconds after the stimulus . However, in most experiments the rise was first seen 2-3 minutes after stimulation, due to the standard 3 minute interval between chronoamperometric measurements . The caudate signals returned to baseline within 4-60 minutes and, after this, stimulations often could The second and third such stimulations be repeated successfully . ordinarily gave caudate signals of lesser magnitude than the first . Essentially equivalent results were obtained when similar stimulation parameters were applied to the bipolar electrodes in the ascending nigrostriatal pathway. Nineteen successful Here responses were obtained and Figure 3 is representative . both caudates were monitored simultaneously with only the right nigrostriatal pathway being stimulated . The right, but not the left, caudate showed a sharp electrochemical response within 1 minute after stimulation . C.
Is the Electrochemical Method Detectin Related to Neurotransm tter Re ease?
S ecies not Directl
Each of the drug and electrical stimulations summarized above gives electrochemical results consistent with established DA and 5-HT release studies . Some of the electrochemical data have been available for two years, but we have tested and retested because we felt it was incumbent upon us to show, as definitively as possible, that the electrochemical results were not due to some extraneous electroactive species and thus only artifactually related to neurotransmitter release . From the beginning we have worried that the DA and 5-HT signals were contaminated by concurrent detection of ascorbic said (AA) . Ascorbate is present in all mammalian brains, and its total endogenous concentration in rat brain is ca . 2 .3 mmols/kg wet tissue, which corresponds to about 1-2 mM (11) . AA is oxidized at almost the same potential as DA or 5-HT and it cannot be separately distinguished by the electrochemical techniques we use . In fact, so far only Lane and coworkers have succeeded in obtaining separate 'oxidation signals for DA and AA in CNS tissue . This was accomplished by using iodine surfacetreated platinum electrodes which, however, are usable for only short periods of time tin situ (7) . Thus, one of the major efforts has been to show that the electrochemical responses obtained by drug or electrical atimulations are independent of, or only minimally influenced by, the presence of AA . A variety of data substantiate that the latter is true .
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Voltammetry in Brain Tissue
240
160 Current Response
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Time (min) FIG .. 3 . Caudate Response to Electrical Stimulation of Right Nigrostriatal Pathway Stim . 20 ua, 60 Hz, duration 4 .2 sec to right nigrostriatal pathway only . Although the real nature of the chronic electrode-tissue interface after implantation cannot be ascertained, it is clear from the tip dimensions (ca . 100 u) that our electrodes are pre dominantly in contact with extracellular fluid space . When a potential is applied, all freely diffusable electroactive species in the vicinity of the electrode can be oxidized . The stable baseline currents correspond (via in vitro calibration) to a total electroactive species concentration of about 100 uM . Since the endogenous AA "concentration" is approximately 1-2 mM, it is obvious no more than about 10$ of the brain AA is being detected in the extracellular fluid under the baseline conditions . Using a typical average value of 14,000 ng/g for the endogenous DA content of the rat caudate, its "concentration" in caudate is ca . 70 uM . The observed baseline signal thus probably corresponds to mostly AA plus a small extracellular level of spontaneously released DA (plus small amounts of other possible electroactive species which are presently not being considered) . In a typical amphetamine experiment the electrochemical response rises by a factor corresponding to an increase in electroactive species concentrations of 15-50 yM . The question is : does this increase correspond to DA release, or is it only circumstantially related because the extracellular fluid AA concentration rises concurrently? Actually, Lane et aZ . answered this question
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some time ago with their iodine-treated platinum in vivo electrode, With this electrode in rat caudate, nearby injection of 2 ug of amphetamine increased the DA current by 7- to 10-fold without any concomitant increase in the AA current (7) .
which could differentiate DA and AA oxidation currents .
We now have additional evidence that amphetamine does not cause any AA efflux at the same time it stimulates the release of DA . Using caudate brain slices, the release of endogenous DA and AA was analysed as a function of varying concentrations of amphetamine via liquid chromatography (Chey and Adams, unpublished data). While the absolute amount of AA spontaneously released from the slices was greater than that of DA, there was no change in the release~ AA with increasing concentrations of amphetamine from 5 x 10 - ° to 5 x 10 -3 M, i .e ., no observable amphetamine-stimulated release . Such results are certainly consistent with the currently accepted carrier-mediated release mechanisms for amphetamine action . Thus all the evidence supports the conclusion that the baseline electrochemical signals are a summed response due to extracellular AA, DA and other electroactive species . Amphetamine administration produces a well-defined increase "on top" of this stable baseline current and this increased signal is primarily du. to released DA . By analogy it would seem reasonable that the electrochemical signals for 5-HT release (by P-chloroamphetamine) are similarly free from drug-induced AA interference . More exact definition must await better methods of electrode calibration and measurement techniques . The brain slice technique was used in an attempt to show that electrical field stimulation of caudate tissue resulted in release of DA but not AA . Using caudate tissue in oxygenated Yamamoto buffer, field stimulation was applied and electrochemical responses in the brain slice almost identical to those seen in vivo with nigrostriatal stimulation (i .e ., like those of Fig . 3) were obtained . The region close to the voltammetric electrode was perfused with a push-pull cannula and serial samples of the effluent were analysed for release of DA and AA by liquid chromatography . In 8 of 10 experiments in which electrochemical response was obtained with field stimulation, perfusion analyses showed the expected rise in DA . In only 1 case was there a concomitant rise in AA and this amounted to only 308 of the DA rise . However, we cannot feel confident that these results are unequivocal due to possible losses of AA by air oxidation during handling of the perfusates . At present, while we have no positive evidence that AA is released along with DA upon electrical stimulation of caudate tissue, we are unable to prove the converse . D.
Present Limitations of the Method
At first glance, the specificity of the electrochemical method seems questionable since several species oxidize at or near the same potential . The situation may be assessed as follows : DA, norepinephrine (NE), 5-HT, and ascorbate all oxidize in the region of +0 .2 to +0 .3 V at the graphite-epoxy electrode and indeed cannot be distinguished by the simple electrochemical procedures alone. However, the brain slice and synaptosome studies mentioned earlier show that carrier-mediated release (e . g ., amphetamine-
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Volta~netry in Brain Tissue
induced DA release) does not involve concomitant efflux of secorbate . Hence, these electrochemical measurements are not "contaminated" by ascorbate--the latter contributes only to the baseline signals . Further, one can place detector electrodes in brain areas with high ratios of DA to 5-HT and use relatively low amphetamine dosages (ti3 mg/kg), thus ensuring that 5-HT contributes only minimally, if at all, to the amphetamine-induced DA signal . The methoxylated metabolites of catecholamines are easily distinguished from the parent compounds and non-methoxylated derivatives since the former oxidize at least 0 .3 V more anodic ally (ca . +0 .7 to +0 .8 V) . Thus, measuring current responses at +0 .8 V vs . those at +0 .5 V reveals methoxylated metabolites . (As an example, in the amphetamine studies described herein, measuring 1 1 sec at +0 .8V or +0 .5 V gave similar responses, showing that DA but not homovanillic acid release was being detected .) it is not possible to differentiate between the various intermediates (alcohols, aldehydes, acids) of the monoamine oxidase pathway . In summary, we feel confident that the electrochemical technique is monitoring in situ neurotransmitter efflux in the case of amphetamine-induced DA release (and, similarly, the p-chloro amphetamine-induced 5-HT release) . At the present time we must conservatively suggest that electrochemical signals resulting from electrical stimulation of DA neurons in the caudate may be "contaminated" by concomitant release of AA . In addition, the electrochemical technique shows large and sharp signals in caudate, cortex and other brain regions with a variety of behavioral stimuli such as low-level foot shock, forced restraint, cold stress and various anxiety-provoking stimuli . At present in these cases we can only state that these stimuli produce a rapid and large efflux of electroactive species into the brain extracellular fluid where they are detected by our electrodes . We hope soon to be able to define properly the electroactive components responsible for such signals . ACRNOWLEDGMENTS The support of the National Institutes of Health, The National Science Foundation, The Medical Research Council and The Wellcome Trust through research and travel grants is gratefully acknowledged . REFERENCES 1. 2. 3. 4. 5. 6.
R. M . WIGHTMAN, E . STROPE, P . M . PLOTSKY and R. N . ADAMS, Nature, 262, 145-146 (1976) . R. M . WIGHTMAN, E . STROPE, P . M . PLOTSRY and R . N . ADAMS, Brain Res ., in press . R. N . ADAMS, Trends in Neurosciences, in press . R . F . LANE, A . T . HUBBARD and C . D . BLAHA, Bioelectrochem . and Bioenerget ., 5, 506-527 (1978) . F . GONON, R . CESPLiGLIO, J .-L . PONCHON, M . BUDA, M . JOUVET, R. N . ADAMS and J .-F . PUJOL, C . R . Acad . Sc . Paris, _T _2 B6, Serie D, 1203-1206 (1978) . R . J. BALDESSARINI, in Handbook of Ps cho harmacolo , Vol . 3, L . L . IVERSEN, S . D . IVElt~SEN and H . SNYDE , e hors, Plenum Press, New York, 1975, pp . 37-137 .
S.
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7. 8. 9. 10 . 11 .
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R . F . LANE, A . T . HUBBARD, K . FUKUNAGA and R . J . BLANCHARD, Brain Res ., 114, 346-352 (1976) . E . SANDERS-BUSH and J . MASSARI, Fed . Proc ., 36, 2149-2153 (1977) . O . J . BROCH and C . A . MARSDEN, Hrain Res ., _38, 425-428 (1972) . L . J . PELLEGRINO and A . J . CUSHMAN, A Stereotaxic Atlas o_f the Rat Brain, Appleton-Century-Crofts, New York, 1967 . .H. J ALLISON and M. H. STEWART, Anal . Hiochem., _43, 401-409 (1971) .
Life Sciences, Vol . 23, pp . 2705-2716 Printed in the U .S .A .
VOLTAMMETRY IN BRAIN TISSUE : CHRONIC RECORDING OF STIMULATED DOPAMINE AND 5-HYDROXYTRYPTAMINE RELEASE J. C . Conti, E . Strope, R. N . Adams and C . A . Marsden* Department of Chemistry University of Kansas, Lawrence, Kansas 66045 and *Department of Physiology and Pharmacology University Hospital and Medical School Nottingham, NG7 2UH, Great Britain (Received in final form November 6,
1978)
SUMMARY Micro voltammetric electrodes which continuously monitor stimulated release of dopamine and 5-hydroxytryptamine in unanesthetized, essentially unrestrained rats are described . The results demonstrate that the electrochemical technique correctly follows dopamine efflux, especially in the case of amphetamine-stimulated dopamine release in the caudate . While the method is still exploratory, its value in pharmacological manipulations of neurotransmitter release, etc . is already clearly evident . Electrochemical measurements of the release of dopamine metabolites in ventricular fluid have .been described in detail (1,2) . A similar technique can be used to record directly from CNS tissue (3) . The results presented herein, together with recent reports from other laboratories--Lane and coworkers (4), Gonon et aZ . (5)--already show that the method can monitor the actual release of neurotransmitter substances (and appearance of their metabolites) following pharmacological manipulations of dopamine (DA) and serotonin (5-HT) pathways . The technique is still in its infancy and much needs to be done to improve its selectivity and quantitative aspects . However, it is presently ready to be used, especially for in situ measurements of drug effects in the CNS . This report deals with chronic electrode implants and experiments which verify that the release of neurotransmitter substances is being monitored . The experimental approach was designed to illustrate that the in vivo electrochemical technique can monitor the stimulated release of certain neurotransmitter substances and does not just respond to some electroactive species only circumstantially related to release phenomena . Accordingly, the experiments were patterned after established literature examples in which postmortem biochemical assays, perfusion or "cortical cup" type analyses had been used to illustrate electrical- or drug-stimulated neûrotransmitter release . While a very thorough and lengthy investigation was carried out, the results 0300-9653/78/1231-270502 .00/0 Copyright (c) 1978 Pergamon Press
2706
Voltammetry in Brain Tissue
Vol . 23, Nos . 27 $ 28, 1978
can be summarized briefly since the neurobiology and pharmacology are well known to all readers . So fâr manipulations which are believed to act primarily on catecholamine and serotonin systems have been examined . METHODS AND PROCEDURE The electrochemical procedures were essentially identical with those employed for CSF monitoring (1,2) . Current-potential curves or voltammograms were used primarily to observe qualitative changes of electroactive species in the tissue near the electrode tip (1) . For quantitative results the applied potential was stepped to a fixed value (+0 .8 V vs . Ag/AgCl) and the currenttime response (chronoamperometric measurement) was recorded for a short period, typically 1 sec . A.
Microelectrod es for Ch ronic Implantation
The voltammetric or working electrode is encased in a glass micro capillary . A mixture of high purity graphite plus an inert liquid (Nujol) provides a carbon surface with very low residual current (that current obtained in the absence of any electrooxidizable materials near the electrode) . To make the surface rigid and capable of lasting for chronic measurements, the graphite paste was mixed with epoxy resin, forced into the capillaries and then allowed to harden before use . The graphite paste is prepared by dissolving 0 .9 g of Nujol (mineral oil commonly sold as a laxative at any pharmacy) in 15 ml of carbon tetrachloride . This solution is vigorously stirred and 2 .1 g of graphite powder (Ultra Carbon UCP-1-M, Ultra Carbon, Bay City, Michigan) is slowly mixed in and stirring continued for 5 minutes . The slurry is then gently evaporated until all carbon tetrachloride is volatilized . This graphite paste may be stored for long periods in a vial until ready to be mixed with epoxy resin . Fifty to seventy-five electrodes were usually prepared at a time . A batch of capillaries was pulled from ca . 75 mm lengths of 3 mm o .d . glass tubing in a commercial pulley . Tip diameters were ordinarily between 75-200 u . The necessary number of lead wires made from 6-8 cm lengths of fairly stiff, insulated X28 wire (c a . 0 .3 mm dia.), stripped at each end, were readied . The resin is prepared by thoroughly mixing 0 .45 g of triethylene tetramine (Fisher) with 3 .6 g of Shell Epon 815 resin . Then 1 .05 g of this mixture is added, in a small vial, to 1 .35 g of the graphite paste prepared as above . Thorough stirring with a glass rod is continued until the mixture appears homogeneous . A portion is then transferred to a glass slide . The capillary is inverted and a portion of the epoxy mixture pressed into the barrel end of the tube . A suitable plunger is used to force the paste down the tip . Care is taken to visuaYly inspect for holes or breaks in the graphite-epoxy pathway throughout the capillary length . The barrel is then cut off and one end of the stripped lead wire forced as far down the capillary as possible . About 1 hour is available for preparing the electrodes once the epoxy mixture is mixed . The electrodes are conveniently stored by attaching them to a board with masking tape until they are cured (24-48
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hr) and ready for testing and calibration prior to implantation . The Ag/AgCl reference is prepared by stripping a 2 mm length of teflon-coated silver wire (dia . 0 .25 mm, Medwire Corp ., Mt . Vernon, NY) and anodizing as usual in 1 M hydrochloric acid . A 108 (w/v) gelatin-salt solution is prepared by dissolving 1 q of gelatin in 9 ml of 3 M NaCl solution with heat and allowing it to cool somewhat . A 4 mm length of ca . 0 .4 mm i .d . teflon tubing is filled with the gelatin, the Aq/AgCl wire inserted and both ends crimped to minimize leakage . A stock of reference electrodes may be prepared and stored in 3 M NaCl solution . The auxiliary electrode is a stainless steel screw inserted through a burr hole in the skull until it touches the surface of the brain . Electrical connection can be made with fine wire around the screw. B.
Test ing and Calibration of Electrodes
Just prior to implantation, the graphite electrodes are tested and calibrated in a small (ti5 ml) beaker . The electrode to be tested is snipped with scissors to expose a fresh tip and it is placed approximately vertically in the solution with the tip at least 2-3 mm below the solution surface . The micro Ag/AgCl reference and a platinum wire auxiliary make up the remainder of the electrochemical circuit . A suitable potential (+O .g V vs . Ag/AgCl in these studies) is applied and a series of chronoamperometric (i-t) responses are recorded . The residual i-t response in the absence of any electroactive materials in solution is first observed in pH 7 .4 buffer alone . Normally, electrodes with residual current values greater than 1-5 nanoamps (measured at 1 sec) are discarded as unsuitable . A standard solution of the catecholamine analog 4-methylcatechol (Aldrich) in pH 7 .4 buffer is then substituted and several i-t curves determined . The average value of the chronoamperometric response divided by the 4-methylcatechol concentration can be used later to estimate the in vivo concentrations . Since solutions of catecholamines and 4-methylcatechol are rapidly air oxidized, all standard solutions should be thoroughly deserated (preferably with argon) prior to dissolving any catechol compounds . The tested graphite electrodes are implanted along with the reference and auxiliary electrodes by standard stereotaxic procedures . Three or four graphite electrodes may be implanted in desired brain regions . Reference and auxiliary electrode placement is not critical, but they were usually placed in convenient locations within 1 cm of the detector electrodes . When implanted, all electrodes were fixed with cranioplastic cement and the leads were then attached to a 6-pin pedestal which mated to a flexible cable (Plastic Products, Roanoke, VA, parts MS363 and MS363-S1/6, respectively) . The pedestal was anchored with skull screws and the entire assembly firmly cemented in place. (This electrode pedestal is conventional and any similar arrangement for chronic recording is satisfactory .) For those experiments utilizing electrical stimulation of the brain, bipolar stimulating electrodes were implanted at appropriate coordinates . A separate 2-pin flexible connection was attached to a Grass stimulator or similar
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device . Some, but not all electrode placements were verified by standard histology . Experiments were carried out, ordinarily after 1-2 days surgical recovery, with the rats either free to move about the laboratory bench or in a large copper-screened box . The latter Faraday cage environment lessened noise problems . C.
in vivo Measurements
A typical experiment consisted of recording the i-t curve and measuring the current at 1 second with a 3 minute interval between measurements . This current is variously designated as i, signal, or response throughout these discussions . The first few measurements at all in vivo electrodes give large responses, but these decrease rapidly with repetition . Within 0 .5 to 1 hour the values stabilize at a baseline level which ordinarily is constant to t5-10$ and will remain so for long periods if no purposeful stimulation is applied to the animal . (Slow cyclic variations have been observed with 24 and 48 hour monitoring, but the significance of any rhythmic baseline changes has not been investigated .) The essentially unrestrained animals may move, groom, eat. and drink, and even scratch the head region with no apparent signal artifacts . When the baseline response is established, the stimulus (electrical, drug, or behavioral manipulation) is applied and the changes in the response are followed . All the experiments described herein, for convenience in data gathering, utilized a minicomputer which programmed and carried out the electrochemical measurements and data processing . How ever, it is possible to obtain similar results with far more simple instrumentaion . Several such arrangements and descriptions of measurement methods and calibrations are given in more detail elsewhere (Huff et aZ ., manuscript in preparation) . RESULTS AND DISCUSSION A.
Pharmacological Studies 1.
Amphetamine-induced Dopamine Release
Amphetamine-stimulated release of dopamine (DA) is well established in the literature (6) . Electrochemical measurements of this effect were carried out with working electrodes in the rat caudate nucleus . After baseline signals were stablized for at least 0 .5 hr, the animals received i .p . doses of dZ-amphetamine varying between 3-10 mg/kg and the electrochemical recording was continued . Approximately 15-20 minutes after the injection, signals rose by 20-408 and slowly returned to baseline within 1-2 hr . Figure 1 illustrates such an experiment in which electrodes in both left and right caudates were monitored . The onset of the electrochemical signal was always time-locked with the characteristic amphetamine-induced behavioral effects . These experiments have been repeated over a 1~ year period with more than 50 rats, using several batches of electrodes and three different types of measuring devices . Positive results were obtained in every case except those in which experimental problems (electrode breakage, equipment failure, etc .) readily explained the lack of signal increase . Similar amphetamine results have already been reported by Gonon et aL . using 8 u diameter graphite fiber electrodes in
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anesthetized rats . These workers also illustrated the decrease or absence of amphetamine-induced electrochemical response following selective degeneration of DA terminals with 6-hydroxydopamine (5) . Lane and coworkers detected DA release electrochemically from intact caudate tissue ca . 30 sec after nearby microinjection of amphetamine (7) . Moat recently we have been able to electrochemically measure quantitative amphetamine dose-response curves in the range of 1-5 mg/kg of amphetamine (Huff et aZ ., manuscript in preparation) . These complementary results from several different laboratories convincingly demonstrate the electrochemical method readily follows amphetamine-induced DA release .
le 16 14 12
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(min)
FIG . 1 . Electrochemical Monitoring of Amphetamine Response Inj . oa . 4 mg/kg dZ-amphetamine i .p . L,R
voltammetric electrode recording in left and right caudate, respectively . current response in arbitrary units .
2.
Manipulation of 5-HT via p-Chloroamphetamine
The compound p-chloroamphetamine (p-C1-AMP) is a very interesting drug for testing the in vivo electrochemical method . It is known from biochemical studies that p-C1-AMP first gives a rapid release of 5-HT from nerve endings followed by long-lasting depletion of brain 5-HT (8) . Thus, if the graphite working electrode were present in a tissue region known to contain 5-HT nerve terminals and p-C1-AMP were administered i .p ., one could expect a
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Voltammetry in Brain Tissue
rather rapid rise in signal followed by a gradual decrease . This decrease should fall to a level considerably lower than the starting baseline as the 5-HT synthesis is shut off and the endogenous extracellular 5-HT concentrations necessarily decrease . This is exactly the response obtained . Figure 2 illustrates the effect of 3 mg/kg p-C1-AMP on the signals obtained at working electrodes in both the left and right globus pallidus of the caudates (coordinates : AP+1 .0 from bregma ; Lat .±2 .8 ; C/V-7 .5 from skull surface) . While this region also contains much DA, it is known to be richer in 5-HT than the more anterior caudate (9) . The lack of identical responses at the left and right electrodes may be due to different effective electrode areas or perhaps to unequal 5-HT distribution at slightly dissimilar electrode placements . Such variability is to be expected and in no way invalidates the data-other experiments have given left responses larger than right . A variety of other pharmacological manipulations of the 5-HT system futher demonstrates that the electrochemical method can be utilized to measure 5-HT release (Maraden et al ., submitted for publication) .
36
30
24
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FIG : 2 . Electrochemical Response to p-Chloroamphetamine Inj . 3 mg/kg p-chloroamphetamine . voltammetric electrode recording in left L,R and right globus pallidus region, respectively (see text) . current response in arbitrary units .
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H.
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Electrical Stimulation of Neural Pathways
Many studies have shown that low level electrical stimulation of the substantia nigra (pars compacts) or the ascending nigrostriatal pathway results in enhanced DA release at nerve terminals in the ipsilateral caudate nucleus (6) . We mimicked this style of experiment by implanting bipolar stimulating electrodes in the substantia nigra (or ascending fiber tract) and monitoring the electrochemical response at graphite working electrodes in the caudate . After establishing a stable baseline i, stimulating currents between 70-200 microamps (60 Hz sinusoidal) were applied for very brief periods (1-5 seconds) . Four sets of stimulating electrodes gave electrochemical responses averaging 408 rise above baseline in the ipsilateral caudate in 14 different stimulations . The rises were very sharp and occurred as quickly as 15 seconds after the stimulus . However, in most experiments the rise was first seen 2-3 minutes after stimulation, due to the standard 3 minute interval between chronoamperometric measurements . The caudate signals returned to baseline within 4-60 minutes and, after this, stimulations often could The second and third such stimulations be repeated successfully . ordinarily gave caudate signals of lesser magnitude than the first . Essentially equivalent results were obtained when similar stimulation parameters were applied to the bipolar electrodes in the ascending nigrostriatal pathway. Nineteen successful Here responses were obtained and Figure 3 is representative . both caudates were monitored simultaneously with only the right nigrostriatal pathway being stimulated . The right, but not the left, caudate showed a sharp electrochemical response within 1 minute after stimulation . C.
Is the Electrochemical Method Detectin Related to Neurotransm tter Re ease?
S ecies not Directl
Each of the drug and electrical stimulations summarized above gives electrochemical results consistent with established DA and 5-HT release studies . Some of the electrochemical data have been available for two years, but we have tested and retested because we felt it was incumbent upon us to show, as definitively as possible, that the electrochemical results were not due to some extraneous electroactive species and thus only artifactually related to neurotransmitter release . From the beginning we have worried that the DA and 5-HT signals were contaminated by concurrent detection of ascorbic said (AA) . Ascorbate is present in all mammalian brains, and its total endogenous concentration in rat brain is ca . 2 .3 mmols/kg wet tissue, which corresponds to about 1-2 mM (11) . AA is oxidized at almost the same potential as DA or 5-HT and it cannot be separately distinguished by the electrochemical techniques we use . In fact, so far only Lane and coworkers have succeeded in obtaining separate 'oxidation signals for DA and AA in CNS tissue . This was accomplished by using iodine surfacetreated platinum electrodes which, however, are usable for only short periods of time tin situ (7) . Thus, one of the major efforts has been to show that the electrochemical responses obtained by drug or electrical atimulations are independent of, or only minimally influenced by, the presence of AA . A variety of data substantiate that the latter is true .
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240
160 Current Response
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Time (min) FIG .. 3 . Caudate Response to Electrical Stimulation of Right Nigrostriatal Pathway Stim . 20 ua, 60 Hz, duration 4 .2 sec to right nigrostriatal pathway only . Although the real nature of the chronic electrode-tissue interface after implantation cannot be ascertained, it is clear from the tip dimensions (ca . 100 u) that our electrodes are pre dominantly in contact with extracellular fluid space . When a potential is applied, all freely diffusable electroactive species in the vicinity of the electrode can be oxidized . The stable baseline currents correspond (via in vitro calibration) to a total electroactive species concentration of about 100 uM . Since the endogenous AA "concentration" is approximately 1-2 mM, it is obvious no more than about 10$ of the brain AA is being detected in the extracellular fluid under the baseline conditions . Using a typical average value of 14,000 ng/g for the endogenous DA content of the rat caudate, its "concentration" in caudate is ca . 70 uM . The observed baseline signal thus probably corresponds to mostly AA plus a small extracellular level of spontaneously released DA (plus small amounts of other possible electroactive species which are presently not being considered) . In a typical amphetamine experiment the electrochemical response rises by a factor corresponding to an increase in electroactive species concentrations of 15-50 yM . The question is : does this increase correspond to DA release, or is it only circumstantially related because the extracellular fluid AA concentration rises concurrently? Actually, Lane et aZ . answered this question
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some time ago with their iodine-treated platinum in vivo electrode, With this electrode in rat caudate, nearby injection of 2 ug of amphetamine increased the DA current by 7- to 10-fold without any concomitant increase in the AA current (7) .
which could differentiate DA and AA oxidation currents .
We now have additional evidence that amphetamine does not cause any AA efflux at the same time it stimulates the release of DA . Using caudate brain slices, the release of endogenous DA and AA was analysed as a function of varying concentrations of amphetamine via liquid chromatography (Chey and Adams, unpublished data). While the absolute amount of AA spontaneously released from the slices was greater than that of DA, there was no change in the release~ AA with increasing concentrations of amphetamine from 5 x 10 - ° to 5 x 10 -3 M, i .e ., no observable amphetamine-stimulated release . Such results are certainly consistent with the currently accepted carrier-mediated release mechanisms for amphetamine action . Thus all the evidence supports the conclusion that the baseline electrochemical signals are a summed response due to extracellular AA, DA and other electroactive species . Amphetamine administration produces a well-defined increase "on top" of this stable baseline current and this increased signal is primarily du. to released DA . By analogy it would seem reasonable that the electrochemical signals for 5-HT release (by P-chloroamphetamine) are similarly free from drug-induced AA interference . More exact definition must await better methods of electrode calibration and measurement techniques . The brain slice technique was used in an attempt to show that electrical field stimulation of caudate tissue resulted in release of DA but not AA . Using caudate tissue in oxygenated Yamamoto buffer, field stimulation was applied and electrochemical responses in the brain slice almost identical to those seen in vivo with nigrostriatal stimulation (i .e ., like those of Fig . 3) were obtained . The region close to the voltammetric electrode was perfused with a push-pull cannula and serial samples of the effluent were analysed for release of DA and AA by liquid chromatography . In 8 of 10 experiments in which electrochemical response was obtained with field stimulation, perfusion analyses showed the expected rise in DA . In only 1 case was there a concomitant rise in AA and this amounted to only 308 of the DA rise . However, we cannot feel confident that these results are unequivocal due to possible losses of AA by air oxidation during handling of the perfusates . At present, while we have no positive evidence that AA is released along with DA upon electrical stimulation of caudate tissue, we are unable to prove the converse . D.
Present Limitations of the Method
At first glance, the specificity of the electrochemical method seems questionable since several species oxidize at or near the same potential . The situation may be assessed as follows : DA, norepinephrine (NE), 5-HT, and ascorbate all oxidize in the region of +0 .2 to +0 .3 V at the graphite-epoxy electrode and indeed cannot be distinguished by the simple electrochemical procedures alone. However, the brain slice and synaptosome studies mentioned earlier show that carrier-mediated release (e . g ., amphetamine-
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induced DA release) does not involve concomitant efflux of secorbate . Hence, these electrochemical measurements are not "contaminated" by ascorbate--the latter contributes only to the baseline signals . Further, one can place detector electrodes in brain areas with high ratios of DA to 5-HT and use relatively low amphetamine dosages (ti3 mg/kg), thus ensuring that 5-HT contributes only minimally, if at all, to the amphetamine-induced DA signal . The methoxylated metabolites of catecholamines are easily distinguished from the parent compounds and non-methoxylated derivatives since the former oxidize at least 0 .3 V more anodic ally (ca . +0 .7 to +0 .8 V) . Thus, measuring current responses at +0 .8 V vs . those at +0 .5 V reveals methoxylated metabolites . (As an example, in the amphetamine studies described herein, measuring 1 1 sec at +0 .8V or +0 .5 V gave similar responses, showing that DA but not homovanillic acid release was being detected .) it is not possible to differentiate between the various intermediates (alcohols, aldehydes, acids) of the monoamine oxidase pathway . In summary, we feel confident that the electrochemical technique is monitoring in situ neurotransmitter efflux in the case of amphetamine-induced DA release (and, similarly, the p-chloro amphetamine-induced 5-HT release) . At the present time we must conservatively suggest that electrochemical signals resulting from electrical stimulation of DA neurons in the caudate may be "contaminated" by concomitant release of AA . In addition, the electrochemical technique shows large and sharp signals in caudate, cortex and other brain regions with a variety of behavioral stimuli such as low-level foot shock, forced restraint, cold stress and various anxiety-provoking stimuli . At present in these cases we can only state that these stimuli produce a rapid and large efflux of electroactive species into the brain extracellular fluid where they are detected by our electrodes . We hope soon to be able to define properly the electroactive components responsible for such signals . ACRNOWLEDGMENTS The support of the National Institutes of Health, The National Science Foundation, The Medical Research Council and The Wellcome Trust through research and travel grants is gratefully acknowledged . REFERENCES 1. 2. 3. 4. 5. 6.
R. M . WIGHTMAN, E . STROPE, P . M . PLOTSKY and R. N . ADAMS, Nature, 262, 145-146 (1976) . R. M . WIGHTMAN, E . STROPE, P . M . PLOTSRY and R . N . ADAMS, Brain Res ., in press . R. N . ADAMS, Trends in Neurosciences, in press . R . F . LANE, A . T . HUBBARD and C . D . BLAHA, Bioelectrochem . and Bioenerget ., 5, 506-527 (1978) . F . GONON, R . CESPLiGLIO, J .-L . PONCHON, M . BUDA, M . JOUVET, R. N . ADAMS and J .-F . PUJOL, C . R . Acad . Sc . Paris, _T _2 B6, Serie D, 1203-1206 (1978) . R . J. BALDESSARINI, in Handbook of Ps cho harmacolo , Vol . 3, L . L . IVERSEN, S . D . IVElt~SEN and H . SNYDE , e hors, Plenum Press, New York, 1975, pp . 37-137 .
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7. 8. 9. 10 . 11 .
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R . F . LANE, A . T . HUBBARD, K . FUKUNAGA and R . J . BLANCHARD, Brain Res ., 114, 346-352 (1976) . E . SANDERS-BUSH and J . MASSARI, Fed . Proc ., 36, 2149-2153 (1977) . O . J . BROCH and C . A . MARSDEN, Hrain Res ., _38, 425-428 (1972) . L . J . PELLEGRINO and A . J . CUSHMAN, A Stereotaxic Atlas o_f the Rat Brain, Appleton-Century-Crofts, New York, 1967 . .H. J ALLISON and M. H. STEWART, Anal . Hiochem., _43, 401-409 (1971) .