Langmuir 1990,6, 56-65
56
(DMF), 82614-58-2; [(TPP)Ru(CO)(DMF)]+, 82614-66-2; [ (TPP)Ru(CO)(DMF)]'+ 123332-77-4; [ (TPP)Ru(CO)(DMF)]-, 82614-74-2; [(TPP)Ru(CO)(DMF)]'-, 123332-82-1; (TPP)Ru(CO)(THF), 82614-57-1; [(TPP)Ru(CO)nTHF)]+,
82614-65-1; [(TPP)Ru(CO)(THF)]'+, 123332-78-5; [(TPP)Ru(CO)(THF)]-,82614-73-1; [(TPP)Ru(CO)(THF)]'-, 123332-832; CH,Cl,, 75-09-2; PhCN, 100-47-0; Py, 110-86-1;Me,SO, 6768-5; P t , 7440-06-4.
Detection of Catecholamines in Brain Tissue: Surface-Modified Electrodes Enabling in Vivo Investigations of Dopamine Function' Ross F. Lane*?$and Charles D. Blahas Department of Chemistry and Departments of Psychiatry and Psychology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1W5 Received J u n e 27, 1989. I n Final Form: August 17, 1989 Recent evidence has shown that graphite paste electrodes modified with stearic acid show high resolution for catecholamines, dopamine (DA), and norepinephrine (NE), when implanted into brain tissue. The extent of this resolution has now been examined in more detail both in vitro and in vivo. Voltammetry at the modified electrodes at physiological pH shows DA and NE can be resolved from their major metabolites and precursors: 5-hydroxytryptamine and its principal metabolite and precursors, ascorbic acid and uric acid. Measurements with electrodes chronically implanted in conscious, unrestrained rats give voltammograms for DA in brain regions rich in DA nerve terminals with a peak potential similar to that observed for DA in vitro. This voltammogram is completely eliminated by selective lesions of DA neurons by 6-hydroxydopamine, is abolished by y-butyrolactone (GBL), and is reduced to undetectable levels by the directly acting DA agonist apomorphine (APO). Combined, these findings demonstrate that the voltammograms represent DA efflux that originates from intact DA nerve terminals, depends on axonal conduction of nerve impulses (GBL), and is regulated by DA receptors controlling normal DA cell activity (APO). The electrodes exhibit reproducible, long-term stability in brain tissue, enabling continuous monitoring of DA efflux for periods of 2 months or more in individual animals. An example is provided demonstrating that chronic treatment with classical antipsychotic drugs decreases the basal efflux of DA in the striatum and nucleus accumbens, whereas "atypical" antipsychotic drugs decrease basal DA efflux only in the accumbens. Evidence is presented that the decreases, when observed, are due to the induction of depolarization block in DA neurons. These findings suggest that the inability of atypical antipsychotic drugs to decrease striatal DA efflux may be related to their low incidence of neurological side effects and that a decrease in limbic DA efflux may be involved in the delayed onset of therapeutic efficacy in man.
Introduction In recent years, there has been considerable interest in developing methods to measure the efflux of small molecules, known as neurotransmitters, from neurons in multiple brain areas of both anesthetized and freely moving animals (for reviews, see ref 1-4). The interaction of neuPresented at the symposium entitled "Photoelectrochemical and Electrochemical Surface Science: Microstructural Probes of Electrode Processes", sponsored jointly by the Divisions of Analytical Chemistry and Colloid and Surface Chemistry, 197th National Meeting of the American Chemical Society, Dallas, April 9-14, 1989. Department of Chemistry. 3 Departments of Psychiatry and Psychology. (1) Adams, R. N.; Marsden, C. A. In Handbook of Psychopharmacology; Iversen, L. L., Iversen, S. D., Snyder, S. H., Eds.; Plenum Press: New York, 1982; Vol. 15; p 1. (2) Measurement of Neurotransmitter Release I n Vivo; Marsden, C . A,, Ed.; IBRO Handbook Series; Wiley: New York, 1984. (3) Justice, J. B.; Michael, A. C.; Neill, D. B. In Neuromethods; Boulton, A. A,, Baker, G. B., Baker, J. M., Eds.; Humana Press: New Jersey, 1985; p 212. (4) Blaha, C. D.; Lane, R. F.; Phillips, A. G. In Advances in Behavioral Biology; Carpenter, M. B., Jayaraman, A,, Eds.; Plenum Press: New York, 1987; Vol. 32; p 115.
*
rotransmitters with specific receptors is one of the major modes of communication between neurons. The ability to detect neurotransmitters directly within the brain, but exterior to neurons, provides a direct method to understand this mode of chemical communication. The catecholamines (dopamine (DA) and norepinephrine (NE)) and their metabolites, as well as 5-hydroxytryptamine (5-HT, serotonin) and its metabolites, are easily oxidized. This enables voltammetric detection. Voltammetric probes of micrometer dimensions have been developed that cause minimal damage to tissue and are sufficiently large t h a t extracellular measurements are assured.'-* One aspect of particular interest to our investigations in this area is the monitoring of the neurotransmitter DA because of its established connection to Parkinson's disease and other movement impairments and its putative involvement in the pathology of several neuropsychiatric disorders. A necessary condition for the application of in vivo voltammetry to the study of the central nervous system (CNS) is the identification of neurochemicals responsible for changes in voltammetric oxidation currents. Oxidation currents measured in the brain extracellular fluid 0 1990 American Chemical Society
Langmuir, Vol. 6, No. 1, 1990 57
Surface-Modified Electrode Investigations
(ECF) represent the contributions of all electroactive molecules oxidized at a given potential. Compounds with similar oxidation potentials will produce a composite signal with nonspecific electrodes; thus, data interpretation becomes very A particular concern is the resolution of DA and NE from the DA metabolite 3,4-dihydroxyphenylaceticacid (DOPAC), ascorbic acid (AA), the 5-HT metabolite 5-hydroxyindoleacetic acid (5HIAA), and the purine metabolite uric acid (UA). All of these compounds oxidize within a narrow potential range of approximately +0.10 to +0.35 V, and their basal brain extracellular concentrations are 10-100 times higher than the concentrations of DA and NE.5 Consequently, a large proportion of the basal in vivo voltammetric signal can be due to the oxidation of DOPAC; AA;-' 5-HIAA,1° and UA,"*12 which can mask the currents due to DA and NE. In addition, there is good evidence that the concentrations of these chemicals can change in response to neuronal depolari~ation'~ and pharmacological manip~lations,6,~,'~'~ thus confusing in vivo voltammetric data still further. Strategies for the enhancement of voltammetric resolution include electrodes modified with iodide,I4 ascorbic acid oxidase,15 the anionic polymer electrochemical pretreatment?" and the implementation of pulse-scanning procedures in combination with graphite fiber electrodes.' More recent developments include the use of electrochemically pretreated, Nafion-electrocoated cylindrical graphite fiber electrode^'^ and the use of fast scan (high-speed) cyclic voltammetry in conjunction with disk-shaped, Nafioncoated graphite fibers.17 The use of graphite paste electrodes is especially attractive because of the ease of preparation, biocompatibility with tissue, and the fact that they have typically low background currents over a relatively wide potential range. The properties of these electrodes depend upon the nature of the pasting liquid, the graphite, and the composition ratio; thus each exhibits different voltammetric response^.'*^^.^^ Since the electrodes consist of a mixture of graphite and organic liquid, various organic compounds can be incorporated into the pasting medium, thus modifying the electrode characteristics still further. ( 5 ) Zetterstrom, T.; Sharp, T.; Marsden, C. A.; Ungerstedt, U. J. Neurochem. 1983,41,1769. (6) Gonon, F.; Buda, M.; Cespuglio, R.; Jouvet, J.; Pujol, J. F. Brain Res. 1981,223,69. (7) Lane. R. F.: Hubbard., A. T.:, Blaha. C. D. Bioelectrochem. Bioenerg: i978,5,'504. (8) Ewing, A. G.; Wightman, R. M.; Dayton, M. A. Brain Res. 1982, 249,361. (9) O'Neill, R. D.; Grunewald, R. A.; Fillenz, M. Neuroscience 1982, 7,1945. (10) Cespuglio, A,; Faradji, H.; Riou, F.; Gonon, F.; Pujol, J.-F.; Jouvet. M. Brain Res. 1981.223. 299. ill) Crespi, F.; Sharp, T:;Maidment, N.; Marsden, C. A. Neurosci. Lett. 1983,43,203. (12) Mueller, K.; Palmour, R.; Andrews, C.; Knott, P. J. Brain Res. 1985,335,231. (13) Milby, K. H.; Mefford, I. N.; Chey, W.; Adams, R. N. Brain Res. Bull. 1981,7,237.
(14) Lane, R. F.; Hubbard, A. T.; Fukanaga, K.; Blanchard, R. J.
Brain Res. 1976,114,346.
(15) Nagy, G.; Rice, M. E.; Adams, R. N. Life Sci. 1982,31,2611. (16) Gerhardt, G. A.; Oke, A. F.; Nagy, G.; Moghaddam, B.; Adams, R. N. Brain Res. 1984,290,390. (17) Baur, J. E.; Kristensen, E. W.; May, L. J.; Wiedemann, D. J.; Wightman, R. M. Anal. Chem. 1988,60,1268. (18) Feng, J.-X.; Brazell, M.; Renner, K.; Kasser, R.; Adams, R. N. Anal. Chem. 1987,59,1863. (19) Brazell, M. P.; Kasser, R. J.; Renner, K. J.; Feng, J.; Moghaddam, B.; Adams, R. N. J. Neurosci. Methods 1987,22,167. (20) Sternson, A. W.; McCreery, R. L.; Feinberg, B.; Adams, R. N. J . Electroanal. Chem. 1973,46,313. (21) Rice, M. E.; Galus, Z.; Adams, R. N. J. Electroanal. Chem. 1983,143,89.
We have previously shown that graphite paste electrodes impregnated with stearic acid show high selectivity for DA and NE, which are protonated at physiological pH.22-25 Incorporation of stearic acid into the paste was designed to impart anionic (carboxylic) charge sites onto the electrode surface, retarding the rate of electron transfer of anionic metabolites such as DOPAC and 5HIAA, AA and UA (both anions at physiological pH) and, therefore, shifting the oxidation potential region to values more positive than that a t which DA and NE are normally observed. A description of this generalized approach to surface modification as it applies to electron transfer kinetics has been presented in detail previously.26 Pharmacological, anatomical, voltammetric, and postmortem data obtained from anesthetized and freely moving animals have provided evidence that DA and NE are the major substances detected in anatomically distinct brain areas, even under conditions where much larger, divergent changes in AA and DOPAC concentrations are occurring s i m u l t a n e o ~ s l y . ~ ~ In -~~~~'-~~ this paper, we present data which (1) further demonstrates the voltammetric properties of stearate-modified graphite paste electrodes and their ability to resolve DA from several electroactive and neurochemically important compounds, (2) demonstrates the utility of the electrodes to monitor extracellular DA levels for periods of months in the same animal, and (3) establishes the cellular origin of the voltammetric signal and its association with DA neuronal activity. In addition, an example of the usefulness of these electrodes in providing new insights into the sites and mechanisms underlying the therapeutic efficacy of antipsychotic drugs is described.
Experimental Section Chemicals a n d Reagents. DA-HC1, NE-HCl, DOPAC, 5HT-HCl, 5-HIAA, epinephrine (E), L-ascorbic acid (AA), uric acid (UA), ~-3,4-dihydroxyphenylalanine(L-DOPA), 3,4dihydroxyphenylglycol (DOPEG), homovanillic acid (HVA), (3methoxy-4-hydroxypheny1)ethylamine(3-MT), (3-methoxy-4hydroxypheny1)glycol (MOPEG), vanillylmandelic acid (VMA), 5-hydroxytryptophan (5-HTP), tyrosine, tryptophan, tyramine, tryptamine, 6-hydroxydopamine-HBr (6-OHDA), 7-butyrolactone (GBL), apomorphineHC1, metoclopramide-HC1, and chloral hydrate were obtained from Sigma Chemical Co. (St. Louis, MO). Pentobarbital (Nembutal) was purchased from Abbott Laboratories (Abbott Park, IL). All chemicals and drugs were used as received. All other chemicals were reagent grade, and all solutions were prepared from triply distilled water. For evaluating electrode performance in vitro, the compounds were dissolved in phosphate-buffered saline solution (PBS, 0.16 M, pH 7.4), which is the supporting electrolyte of choice for in vivo voltammetric studies. Electrodes a n d A p p a r a t u s . Working (recording) electrodes were constructed from 2-2.5-cm lengths of Teflon-insulated stainless steel wire with an outer (tip) diameter of 175 pm (Leico Industries, New York). One end of the wire was carefully cut perpendicular to the wire axis with a fine-edged razor blade, and the Teflon insulation was extruded over this end to form a cylindrical well 0.3-0.5 mm in depth. A single loop of the insulated wire was cemented with dental acrylic near the (22) (23) (24) 19. (25) 2155. (26) (27)
Blaha, C. D.; Lane, R. F. Brain Res. Bull. 1983,10, 861. Blaha, C. D.; Lane, R. F. Eur. J. Pharmacol. 1984,98,113. Lane, R. F.; Blaha, C. D.; Hari, S. P. Brain Res. Bull. 1987,19, Yamamoto, B. K.; Lane, R. F.; Freed, C. R. Life Sci. 1982,30, Lane, R. F.; Hubbard, A. T. J . Phys. Chem. 1973,77,1411. Howard-Butcher, S.; Blaha, C. D.; Lane, R. F. J . Pharmacol.
Exp. Ther. 1985,233,58.
(28) Lane, R. F.; Blaha, C. D. Ann. N . Y . Acad. Sci. 1986,473,50. (29) Lane, R. F.; Blaha, C. D. Brain Res. 1987, 408, 317. (30) Marrocco, R. T.; Lane, R. F.; McClurkin, J. W.; Blaha, C. D.; Alkire, M. T. J. Neurosci. 1987,7,2756.
58 Langmuir, Vol. 6, No. 1, 1990 top of the opposite end of the exposed wire to prevent sleeving of the Teflon and to hold the well firmly in place. The well was tightly packed with either stearate-modified or unmodified (conventional) graphite paste, and the electrode tip was gently polished on a strip of Teflon tape to form a clean and reproducible surface. Prior to use, each electrode was examined under a stereomicroscope to ascertain that no striations were visible on the Teflon and that the paste was smooth and flush across the well surface, with no irregularities apparent. The stearate-modified graphite paste was prepared by dissolving 100 mg of stearic acid (ICN Pharmaceuticals, Irvine, CA, 99% purity) in 1 mL of mineral oil (Nujol, light weight) warmed t o about 40 "C t o aid the dissolution process. Higher temperatures were not employed to prevent decomposition of the stearic acid in the Nujol. While still warm, the solution was thoroughly mixed with 1.5 g of graphite powder (UCP-1M, Ultra Carbon, Bay City, MI), with a glass mortar and pestle, until a homogeneous paste was formed. Mixing the Nujolstearate solution with the graphite powder after cooling to room temperature had no effect on the performance of the electrodes. Pastes incorporating organic acids with shorter chain lengths (Cs-C13) yielded unstable responses and proved unsuitable for our purposes. This instability can be attributed to the increased solubility of the shorter chain length acids relative to longer hydrocarbons in aqueous medium." In contrast, stearic acid is held firmly in the pasting medium and is not leached from the paste in aqueous solution or in the brain ECF. Ag/AgCl reference electrodes were used throughout. For studies in vitro and in anesthetized animals, the reference electrode was constructed by anodizing a bare silver wire and placing it into a pulled plastic micropipet tip filled with physiological (0.9% w/v, 0.16 M) NaCl solution. A length of the wire was passed through a small hole in the side near the top end of the micropipette and sealed in place with epoxy. A 35-gauge platinum wire was used as the auxiliary electrode. For recordings with electrodes chronically implanted in freely moving animals, the Ag/ AgCl reference electrode was prepared by stripping 1 mm of the Teflon sheath from both ends of a length of 30-gauge Tefloncoated silver wire and anodizing one end. The auxiliary electrode was a stainless steel skull screw with a length of stainless steel wire wound around the screw. Before implantation, goldplated miniature pins (Amphenol Relia-tac, Wyle Industries, Hillsboro, OR) were soldered onto the free ends of the wire leads of all electrodes. Linear sweep voltammetry was performed with a locally constructed low-current multipurpose electrochemical circuit similar in design to that used p r e v i ~ u s l y .The ~ current output was electronically semidifferentiated, as described elsewhere,31 to achieve better resolution of the voltammograms caused by the different substances present. The electrochemical cell used for in vitro studies consisted of a 20-mL glass vial with four holes drilled in a Teflon cap to accommodate the three-electrode system and a Teflon nitrogen delivery tube. All solutions were thoroughly deoxygenated with prepurified nitrogen and kept under nitrogen during voltammetric scans. Voltammetry was conducted a t a scan rate of 10 mV/s in all experiments. Since evidence has been presented that graphite electrodes are subject to changes in the nature of the electrode surface when implanted into brain tissue?"" d voltammograms were recorded after use in vivo. Subjects. Experiments were performed on 300-400-g male Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA) housed individually in an animal vivarium maintained a t 22 f 2 "C, 60% relative humidity, and on a lightdark cycle of 12-12 h (lights on a t 0700 h). The animals were adapted to the colony for a t least 2 weeks prior to use. All surgical and voltammetric recordings were performed during the light portion of this cycle. Animal S u r g e r y a n d Experimental Procedures. For voltammetry in conscious, behaving animals, rats were anesthetized with a pentobarbital-chioral hydrate combination (30 and 160 mg/kg ip, respectively) and mounted in a stereotaxic frame (31) Lane, R. F.; Hubbard, A. T.; Blaha, C. D. J . Electroanal. Chem. 1979, 95, 117.
Lane and Blaha (David Kopf) with the upper incisor bar set 5 mm above the interaural line. The skull was exposed and cleaned thoroughly, and a thin coating of bone wax was applied over the cranial surface. Burr holes approximately 1.0 mm in diameter were drilled for implantation of the electrodes and also for anchoring skull screws. After careful excision of the dura, recording electrodes were slowly lowered into either the anterior striatum or the nucleus accumbens, two brain regions rich in DA nerve terminals. A more detailed description of the anatomy of these DA systems is given below. In most cases, two electrodes were lowered into two brain regions, one on each side of the brain, and secured in place with dental cement. The combinations used were striatum-striatum, striatum-accumbens, and accumbal nuclei-accumbal nuclei. The implantation rate never exceeded 0.1 mm/min. Stereotaxic coordinates were as described p r e v i o ~ s l yand , ~ ~all~ electrode ~~ placements were verified histologically. The reference electrode was positioned 1 mm into the cortex just caudal to the working electrodes, and the auxiliary electrode was screwed into the skull in contact with the dura contralateral to the reference electrode. While the position of the reference and auxiliary electrodes was not critical, it was general practice to implant them as close to the recording electrodes as possible. The Amphenol pins from the electrode leads were placed into a miniature series strip connector (Wyle Ind.), and the entire assembly was anchored with skull screws and firmly cemented to the animal's skull. After a postoperative period of a t least 2 days, the animals were placed in electrically shielded, ventilated chambers and connected to the electrochemical instrumentation by a flexible, coaxialshielded cable and low-noise mercury swivel commutator such that they were essentially unrestrained and free-ranging during periods of measurements. All experiments were conducted in a Faraday cage situated in a sound-proofed room. Electrochemical measurements were performed using semidifferential linear sweep ~ o l t a m m e t r y . Voltammetry ~~ was peformed by applying linear potential ramps from -0.10 to +0.25 V vs Ag/AgCl a t a scan rate of 10 mV/s. For dual-site recording, scanning was controlled automatically with each electrode scanned alternately a t 5- or 10-min intervals. The oxidation peak currents of the voltammograms are directly proportional to the concentration of DA,22,23,27,30 and thus a change in current reflects a change in extracellular DA. Measurements were taken for each experiment until stable base-line values were obtained (approximately three to five scans) and were continued for the times specified. When drug manipulation was the experiment of choice, drugs were injected, and changes in peak currents were monitored until the signals returned to base line. Unless noted otherwise, all time-course data were expressed as a percentage change in peak current. The 100% value represents the mean peak current determined from the three voltammograms obtained before drug administration. All data analyses were carried out on the absolute values (peak currents) prior to conversion to percentage changes. Voltammetry in anesthetized rats followed the procedures described above with the following exceptions. Animals were anesthetized with chloral hydrate (400 mg/kg ip) alone and given supplemental doses as needed to maintain a constant level of anesthesia throughout the experiment. Body temperature was strictly monitored and maintained during each recording session a t 37 "C with a heating pad and temperature control module (American Hospital Supply, McGraw Park, IL) in conjunction with a rectal probe (Yellow Springs Instruments, Yellow Springs, OH). Burr holes were drilled for placement of the electrodes only. The recording electrodes were positioned and held firmly on stereotaxic electrode holders. The Ag/AgCl reference and Pt auxiliary electrodes were implanted along the midline so as to make contact with the dura. Chronic Antipsychotic D r u g Studies. Rats (experimental groups of six) received a single subcutaneous (sc) injection of either haloperidol (0.5 mg/kg), chlorpromazine (10 mg/kg), (-)-sulpiride (25 mg/kg), clozapine (20 mg/kg), thioridazine (20 mg/kg), or metoclopramide ( 5 mg/kg) a t 1O:OO a.m. each day for 2 1 consecutive days. Stock solutions of drugs used for daily treatments were prepared by dissolving each drug in 1% lactic acid and adjusting the pH to 6 with a minimal amount of 0.1 M NaOH. Control groups ( n = 6) received identical treat-
Langmuir, Vol. 6, No. I, 1990 59
Surface-Modified Electrode Investigations CONVENTIONAL ELECTRODE
MODIFIED ELECTRODE
A
k D! I T
OOPAC
1.0
DOPAC
0.0
0.1
0.2
E (VOLTS
VO.
0.3
0.4
AgAgC1)
0.5
0.2 0 . 4 0 . B 0 . 8 E (VOLTS vm. A,g/AgCl)
modified graphite paste electrodes in phosphate-buffered saline, pH 7.4. Concentrations: (A) 10 pM DA, 10 pM DOPAC, 200 pM AA; (B) 1.5 pM DA, 400 pM DOPAC, 400 pM AA. Experimental conditions as described in text.
ments with the inactive isomer of (-)-sulpiride, (+)-sulpiride (25 mg/kg), the non-neuroleptic phenothiazine promethazine (20 mg/kg), the antidepressant desipramine (10 mg/kg), and the drug vehicle injected in a volume (1 mL/kg) equal to that used in the experimental groups. Doses were chosen on the basis of clinical doses equivalent to haloperidol at 0.5 mg/kg.32 Twenty-four hours following the last injection of drug or vehicle, each rat was anesthetized with chloral hydrate and implanted with stearate-modified electrodes into both striatum and nucleus accumbens to the coordinates described above. An Ag/ AgC1 reference and a Pt auxiliary electrode were positioned along the midline in contact with the dura. Linear sweep voltammetry with semidifferentiationwas performed according to the protocol described above. In these experiments, base-line release of DA in both drug-treated and vehicle-treated control groups was assessed by measuring the peak current of the DA voltammogram centered at +0.1 V. After two additional scans, apomorphine (100 pg/kg iv) was administered, and changes in oxidation currents were monitored until signals returned to preinjection (base line) values. All changes in DA release in each brain region were expressed as a percentage of the mean (hSEM) of the single base-line value taken in vehicle-treated controls 30 min prior to injection of apomorphine. This value was also designated as 100%. Statisticalcomparisons were made with a two-tailed Student's t test for independent groups.
Results and Discussion Voltammetry in Aqueous Solution. Representative semidifferentiated voltammograms for the oxidation of DA, DOPAC, and AA in p H 7.4 PBS (buffer) a t unmodified graphite paste electrodes are shown in Figure 1A. At these electrodes, DA is oxidized a t the least positive potentials. In addition, the voltammogram is relatively symmetrical. In contrast, voltammograms for DOPAC and AA are less symmetrical, are broader in shape, are shifted to more positive potentials, and, when normalized to concentration, exhibit lower oxidation currents, especially for AA. This is consistent with the irreversible nature of DOPAC and AA oxidation reported previously for paste electrodes of similar composition." The linear scan oxidation peaks for DA, DOPAC, and AA are characterized by average peak potentials centered at +0.12, +0.20, and +0.22 V, respectively. Considerable overlap of the signals is apparent, which leads to the difficulties that exist in interpreting electrochemical results obtained in vivo. This lack of discrimination was recognized in the first papers on in vivo ~ o l t a m m e t r y ' ~and ' ~ ~was pointed out as a primary dis(32) Baldessarini, R.J. Chemotherapy in Psychiatry: Principles and Practice, 2nd ed; Harvard University Press: Cambridge, 1985. (33) Kissinger, P.T.;Hart, J. B.; Adams, R. N. Brain Res. 1973,55, 209.
-
r?l
*
1.0
Figure 1. Semidifferentiated voltammograms of dopamine (DA), 3,4-dihydroxyphenylaceticacid (DOPAC),and ascorbic acid (AA) obtained at (A) unmodified (conventional) and (B) stearate-
m m m
*
I I I I I I I I I I J I 0.0
OA NE E L-DOPA L OOPAC DOPEG AA UA * HVA 3-MT MHPG VMA 5-HT 5-HIAA 5-HTP TYROSINE TRYPTOPHAN TYRAMINE I TRYPTAMINE
I , ~
,
I
0.0 0 . 1 E
;
I
0.2 0.3 0 . 4 0.5 0.e (
0.7 0 . t
VOLTS v s . A q / A g C l )
Figure 2. Voltammetric characteristicsof several endogenous
neurochemicals measured at stearate-modifiedgraphite paste electrodes in phosphate-buffered saline, pH 7.4. Concentration of each compound was 100 pM. The center line is placed at the peak potential, and the ends of the rectangles are placed at the potentials where the current value is half the peak current. Asterisks denote compounds with no discernible peak over the potential range -0.1 to +0.9 V. Abbreviations and experimental conditions as stated in text. Adapted from ref 65. advantage of electrochemical measurements in brain tissue. Dramatic differences are observed when stearic acid is incorporated into the paste. Voltammetry is shown for DA, DOPAC, and AA in Figure 1B. The average peak potential for DA occurs at approximately +0.1 V, and the magnitude of the peak current normalized t o concentration is approximately the same. Significantly, the voltammogram for DA is completely separated from those due to DOPAC and AA, although the concentration of DOPAC and AA is approximately 400 times higher than the DA concentration. The voltammograms for DOPAC and AA are shifted to more positive potentials and exhibit no distinct features over the potential range from -0.1 to +0.9 V. The modified electrodes are insensitive to both compounds a t potentials less than about +0.4 V for concentrations approximating those present in the brain ECF under physiological condition^.^^^^ The positive displacement of the voltammograms for AA and DOPAC and their drawn-out character are indicative of the fact that the rates of electron transfer for these compounds are impaired at the modified electrodes. These results clearly demonstrate the ability of the modified electrodes to resolve DA from AA and DOPAC. A comparison of the voltammetric characteristics of several other electrooxidizable, plausible contributors to the DA signal obtained at stearate-modified graphite paste electrodes is shown in Figure 2. Although there is no significant difference between the voltammogram for DA and those due t o NE and E, DA is clearly resolved from all of the other compounds tested. When compared with Figure 1 and previous observations made with graphite the paste electrodes with Nujol as pasting voltammograms for all anions are shifted to more positive potentials than those observed a t the unmodified electrodes, which implies an effect of surface anionic groups on the heterogenous electron transfer. Since the voltammograms for UA and HVA are nondescript a t these electrodes, assignments of peak potentials cannot be made in these cases. Electrode modification does not appreciably affect the voltammograms of neutral substances
60 Langmuir, Vol. 6, No. 1, 1990
and has only a slight effect on the zwitterions L-DOPA and 5-HTP. One interpretation of the ability of the modified electrodes to resolve the catecholamines from all anions is that the changes in the voltammetric characteristics of the anionic compounds are due to reduced charge transfer rates attributed to electrostatic effects. For each anion, peak potentials are shifted to more positive values, which is what is expected if the observed behavior is influenced by electrostatic sites on the surface of the electrode and by the charge in the compact double layer, resulting in a change in the potential across the diffuse double layer.26 Although the magnitudes of the shifts in peak potential for some anions (e.g., VMA and 5-HIAA) could be due solely to potential field effects in the diffuse layer, the differences in the shapes of the voltammograms and magnitudes of the shifts in peak position for AA, DOPAC, and UA appear too large to be explained by electrostatics alone. That the anionic species are kinetically hindered by the hydrophobic nature of the electrode surface, such that the surface is partially blocked, constitutes an additional consideration. At a partially blocked surface comprised of closely spaced sites, rate constants are impeded because of the reduced area available for electron transfer. Rice et al.'l have shown that mixing dry graphite with a wide variety of hydrocarbon pasting liquids, which are adsorbed onto the graphite, always decreases charge-transfer rates. In particular, markedly reduced charge-transfer rates were observed for DOPAC at pH 7.4 at electrodes with Nujol as pasting liquid when compared to those observed at electrodes prepared with dry graphite. The fact that AA, the most polar compound examined in our experiment, is one of the most kinetically hindered anions supports the proposal that the hydrophobic nature of the stearate-modified electrodes contributes to the observed results. For other anions, e.g., 5-HIAA, the hydrophobic nature of the indole moiety would be expected to be partially offset by the anionic rejection by electrostatic effects, resulting in a voltammogram that is less shifted to more positive potentials. Consequently, it is likely that a combination of both of the above effects contributes to the voltammetry observed. Alternatively, the markedly different behavior of AA, DOPAC, and UA at the modified electrodes may indicate that there is some overt change in the electrode mechanisms of these anions. In contrast to the behavior of the anionic species, AA, DOPAC, and UA, the peak potentials for the cations such as DA, NE, E, 5-HT, 3-MT, and tyramine are shifted to more negative potentials than those observed a t unmodified electrode^^,^^^^^^^^ whereas the neutral species are relatively unaffected. Because the direction and magnitude of these shifts correlate with reactant charge,26the voltammetry observed is influenced primarily by doublelayer effects obtained previously with surface-modified electrodes,26resulting in accelerated electron-transfer rates. In particular, the observed behavior might be explained by an electrostatic interaction between the anionic (stearate) groups on the electrode surface and the protonated amine group on the DA side chain a t physiological pH, which would also produce more rapid charge transfer. Such an "adsorption" process is advantageous, since it would lead to increased sensitivity and selectivity to DA.22.28-30
Lane and Blaha
LESIONED
INTACT
0 . 1 0 . 2 -0.1
0
-0.1
0
0.1
0.2
Ag/AgCl ) Figure 3. Representative semidifferentiated voltammograms obtained at stearate-modified graphite paste electrodes in the nucleus accumbens of six freely moving rats after unilateral degeneration of DA nerve terminals with 6-hydroxydopamine (6OHDA). Voltammograms were recorded alternately in both intact (right) and lesioned (left) accumbens of each individual animal. The lesioned side corresponds to each accumbens of these six animals in which DA innervation was selectively destroyed by 6-OHDA injections into the ipsilateral ventral tegmentum 3 weeks prior to voltammetric recordings. Other experimental conditions as described in text. E
(
VOLTS v s .
Alternatively, radioactive tracer35 and electrochemical s t ~ d i e shave ~ ~ shown , ~ ~ that cations can penetrate to an appreciable extent below the surface of graphite paste electrodes. Thus, electrostatic or more specific chemical interactions between the carboxylic groups and the protonated amine side chain embedded into the graphite paste might provide a mechanism for inducing more Nernstian behavior (increased charge transfer) without involving "adsorption" on the electrode surface. This phenomenon would also lead to increased sensitivity and selectivity to DA. Consequently, we propose that a mixed mechanism could result in the voltammetry observed. Given that the surfaces of graphite paste electrodes are extremely complex and obviously do not have welldefined surfaces such as Pt(ll1) and Pt(100), it is unlikely that there exists a single mechanism that explains all. Further studies are in progress to examine the above and other possibilities. Cellular Origin of the Presumed DA Voltammogram in Vivo. When recorded from the nucleus accumbens of freely moving rats, semidifferentiated voltammograms recorded from -0.1 to 4-0.25 V exhibited one distinct peak potential centered a t +0.1 V (Figure 3). To demonstrate that this voltammogram was related to DA nerve terminals, these terminals were unilaterally destroyed as previously r e p ~ r t e d ~ , ~ by , ' ~injection ,~' of 6-OHDA (8 pg/2 pL) into the left ventral tegmentum of six animals. This procedure causes massive (96-99%) depletion of DA and permanent degeneration of DA nerve terminals in the accumbens on the injected ~ide.~,'~'~*'' Each rat was first treated with a 25 mg/kg dose of desipramine to afford protection against damage to the small number of NE terminals innervating this brain region. Three weeks after 6-OHDA injection, voltammograms were recorded alternately as described above with stearate-modified electrodes implanted in both the left and right nucleus accumbens. (35) Chambers, C. A,; Lee, J. K.
J. Electround. Chem.
1967, 14,
309.
(34) Verbiese-Genard, N.; Kauffmann, J. M.; Hanocq, M.; Molle, L. J . Electround. Chem. 1984, 170, 243.
(36) Wang, J.; Freiha, B. A. Anal. Chem. 1984,56, 849. (37) Wang, J.; Deshmukh, B. K.; Bonakdar, M. J . Electround. Chem. 1985,194, 339.
Langmuir, Vol. 6, No. 1, 1990 61
Surface-Modified Electrode Investigations
MODIFIED ELECTRODE As shown in Figure 3, the voltammogram was com0.8 rpletely abolished when recorded from the lesioned side. These results demonstrate directly that the presumed DA irjl voltammogram originates from intact DA nerve termial 0.4 nals in the accumbens, a result not previously reported. Similar results have been obtained from the striatum fold 2 0.2 cnlowing 6-OHDA lesioning pro~edures.'~'~~'~ It is notem Q 0.0 z 4 e e I O 12 14 le 18 20 22 24 2e 2s 30 eo worthy that voltammograms recorded with unmodified electrodes over the potential range examined show AA DAYS AFTER IMPLANTATION and DOPAC to be the major contributors to the signals Figure 4. Base-line voltammetric responses obtained at stearobserved in both striatum and ac~umbens.~**l~ With these ate-modified graphite paste electrodes in the nucleus accumelectrodes, 6-OHDA lesioning fails to significantly affect bens of freely moving rats over a 2-month period following electhe voltammograms obtained from either brain region, trode implantation. Values shown are the means (*SEM) of because they are due predominantly to AA o ~ i d a t i o n . ~ - ~ ~ ~the ' DA peak current measured daily ( N = 8-10 animals/ measurement per day). For clarity, measurements take on every Long-Term Stability of Modified Electrodes in other day are shown from day 2 to day 30. Variations to day Brain Tissue. Whereas our previous work has investi60 were similar to those shown for the first month. Experimengated changes in DA release for periods of up to 1 2 h or tal conditions as described in text. more in anesthetized or freely moving animals, little attention has been paid to the long-term stability of the stearatemodified electrode in brain tissue. Such long-term stability is of primary importance to understanding the role of central DA function as it relates to a variety of anaw I tomical, pharmacological,and behavioral conditions. StudGEL 750 mg/kg ies investigating regeneration of damaged DA neurons, viability of tissue explants, adaptive changes in DA neurotransmission, effects of repeated pharmacological treatments, and various behavioral paradigms are just a few examples which may require continuous monitoring for periods of weeks to months or longer. Accordingly, we examined the voltammetric characteristics of modified graphite paste electrodes in the striatum and nucleus 5 0 accumbens of freely moving rats prior to any imposed a 0 stimulus over an extended period of time following elec0 1 2 3 4 5 trode implantation. TIME AFTER GEL INJECTION (HOURS) Mean base-line values (ASEM) for each brain region Figure 5. Time courses obtained with stearate-modified graphwere determined by averaging the peak current of the ite paste electrodes in the nucleus accumbens of freely moving DA voltammogram from different animals implanted with rats following injection of y-butyrolactone (GBL, 750 mg/kg ip). The top panel shows actual semidifferentiated voltammoone electrode on a given day after surgery. These valgrams obtained consecutively prior to, during, and after GBL ues were obtained a t 1300-1400 h over a 2-month recordadministration to one animal. Data shown in the bottom panel ing period. An overall mean value (ASEM) for each brain correspond to the times shown in the top panel and are expressed region was determined by averaging all base-line means as the mean (hSEM) percentage change from the mean preinfrom postoperative day 2 to day 60. jection base-line peak currents obtained from six animals. For clarity, every second measurement taken is shown. Other experBase-line responses of semidifferentiated voltammoimental conditions as described in text. grams obtained from individual animals on each test day did not vary by more than 5% (fSEM) within a 3-5-h recording session. This within-animal variation remained preliminary evidence has shown that, in some animals, relatively stable and constant over the entire 2-month these electrodes give stable and reproducible voltammorecording period (data not shown). Modified electrodes grams for as long as 1 year after implantation. implanted in the striatum and nucleus accumbens yielded Effects of Dopamine Cell Activity on Dopamine overall mean peak currents of 0.23 f 0.04 nA s-l/' (100% Efflux. A necessary prerequisite for meaningful interf 17%) and 0.22 f 0.03 nA s-'' (100% f 15%), respecpretations of in vivo measurements is that the observed tively ( n = 14 electrodes/brain region per 58 days). The voltammogram corresponds to DA which is dependent variations about the mean (15-17 %) were comparable on the conduction of impulses (action potentials) along to those deduced in a similar manner in anesthetized rats the axons of DA neurons. To investigate this possibil(8-10%).2s,3s-40 The amplitudes of the voltammetric sigity, rats were injected with y-butyrolactone (GBL), an nals measured in the nucleus accumbens of unanestheagent that selectively suppresses or abolishes the spontized, freely moving rats are shown in Figure 4 as a functaneous firing of DA neurons depending on the dose tion of time after implantation. The voltammetric sige m p l ~ y e d . ~The ' effect of GBL on representative voltanal becomes stable in a m p l i t u d e 2-4 days after mmograms recorded from a modified electrode placed in implantation of the electrode and remains stable for a the nucleus accumbens of a conscious, unrestrained anigiven animal and for a given electrode over the entire mal and the time courses of this effect are shown in Figpostimplantation test period. Comparable results were ure 5. obtained from the striatum (data not shown). Although Administration of GBL (750 mg/kg ip) rapidly decreased the test period was restricted to a period of 2 months, the voltammetric peak current to undetectable levels. This loss of signal was observed a t 1 h following injection, (J.
G
G
H
(38) Lane, R. F.; Blaha, C. D. Brain Res. Bull. 1987,18,135. (39) Blaha, C.D.;Lane, R. F. Neurosci. Lett. 1987,78,199. (40) Lane, R. F.; Blaha, C. D.; Phillips, A. G. Prog. NeuroPsychopharmacol. & Biol. Psychiatry 1987,12,297.
(41) Roth, R. H.In Neurophysiology of Dopaminergic Systems; Chiodo, L. A., Freeman, A. S., Eds.; Lakeshore Publishing Co.: New York, 1987;p 187.
62 Langmuir, Vol. 6, No. 1, 1990
;;120
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APOMORPHINE
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Lane and Blaha ASCORBATE
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Figure 6. Time courses obtained with stearate-modified graphite paste electrodes in the nucleus accumbens of conscious,unrestrained rats following injection of apomorphine (APO, 100 p g / kg sc). Results are expressed as the mean (ISEM) percentage change from the mean preinjection base-line peak currents obtained from six individual animals administered APO once every 2 weeks. Note that ascorbic acid (1 g/kg ip) given after APO did not alter the base-line responses. Other experimental conditions as described in text.
remained absent for 2 h and 40 min, and then slowly returned to preinjection values in approximately 4 h. Similar time courses were observed when GBL was injected into six different animals on six different occasions (Figure 5). Our finding that the voltammogram is completely abolished by GBL shows that the changes are not caused by passive diffusion or other modes of mass transfer from cellular elements to the electrode surface, caused by damage to nerve terminals due to electrode implantation. Rather, it directly demonstrates that the voltammogram depends on the maintenance of normal DA neuronal activity and thus reflects nerve impulse mediated release of endogenous DA. In a series of experiments distinct from measurements of the long-term variations in the base-line DA voltammogram, responses to injections of apomorphine were determined in separate groups of freely moving rats by using the daily protocol described above. Since the effects of apomorphine on DA efflux have previously been reported in anesthetized animals, the motivation behind this experiment was to demonstrate the reproducibility of the voltammogram to a receptor-mediated drug stimulus that decreases DA release with modified electrodes exhibiting long survival periods in awake animals. Apomorphine has been shown to inhibit the activity of DA neurons, an effect attributed to its direct stimulation of DA receptors?2 Administered in appropriately high doses, stimulation of DA receptors by apomorphine leads to an activation of neuronal feedback mechanisms capable of completely inhibiting DA release. Figure 6 shows that injection of apomorphine (100 pg/kg sc) into six different animals once every 2 weeks produced an immediate decrease in DA release that reached undetectable levels 30 min after injection and returned to preinjection values in about 110 min. During recording sessions, the variations in the changes in the magnitudes of the responses recorded every 10 min for at least 5 h never exceeded 10% of the mean value of the peak currents at each time point during the response to the drug. Combined with the long-term experiments in unperturbed animals (Fig(42) Grace, A. A.; Bunney, B. 1092.
238,
s. J . Pharmacol. E x p . Ther. 1986,
ure 4), these results demonstrate that changes in the DA voltammogram can be reproducibly monitored over prolonged periods of time and indicate the suitability of the modified electrodes for continuous monitoring of the effects of chronic administration of a variety of pharmacological agents on changes in DA efflux in the CNS of conscious, behaving animals. As shown in Figure 6, injection of AA (1g/kg ip) after the effects of apomorphine had subsided produced no increases in oxidation current, consistent with previous observations that the modified electrodes do not respond to increases in AA concentration in the ECF, an effect opposite to that observed with unmodified electrodes.9,22-2428 New Insights into the Pharmacology of Antipsychotic Drugs. Biochemical and anatomical evidence supports the existence of two major ascending dopaminergic pathways in the mammalian brain.43 The nigrostriatal and mesolimbic-cortical dopaminergic systems originate from two subpopulations of DA-containing cells located in the substantia nigra zona compacta and ventral tegmental area of the midbrain (mesencephalon).These two nuclei give rise to neuronal projections which innervate, to a large extent, the striatum and the nucleus accumbens of the forebrain, respectively. In the rat, the nigrostriatal and mesolimbic DA systems are believed to mediate separate modes of behavior (e.g., repetitive stereotyped movements and cognitive function and motivation, r e ~ p e c t i v e l y ) .In ~ ~man, these neuronal pathways also appear to be differentially involved with the expression of certain neurological diseases and pathological disorders (e.g., Parkinson's disease and s ~ h i z o p h r e n i a ) . ~ ~ . ~ ~ In recent years, theories concerning the pathogenesis of schizophrenia have focused on brain DA systems, in part due to the linear correlation between neuroleptic drug affinity for central DA (D, subtype) receptors in animals and antipsychotic potency in humans, while no such correlation has been demonstrated for any other central receptor.47 The drugs used to treat schizophrenia, the so-called antipsychotic (neuroleptic) drugs, have proven invaluable as pharmacologicaltools in delineating the function of these two brain DA systems. Acute treatment with antipsychotic drugs causes a prompt increase in the synthesis, metabolism, and efflux of DA in those forebrain regions that contain the terminals of the major ascending DA neurons.23,28729,48 These neurochemical responses are believed to reflect a compensatory increase in the activity of DA neurons in response to acute blockade of pre- and postsynaptic DA receptors. That the therapeutic potency of different neuroleptic drugs correlates well with their affinity for the DA 0, receptor and that DA receptor blockade occurs almost immediately after neuroleptic administration are at variance with the observation that, in man, chronic treatment is required for their therapeutic actions and, in some cases, the development of involuntary motor responses, including Parkinson-like (neurological) side effects and tardive (43) Lindvall, 0. In The Neurobiology of Dopamine; Horn, A. S., Korf, J., Westerink, B. H. C., Eds.; Academic Press: New York, 1979; p 319. (44) Iversen, S. D.; Koob, G. F. In Nonstriatal Dopaminergic Neurons; Costa, E., Gessa, G. L., Eds.; Raven Press: New York, 1977; p 133. (45) Hornykiewicz, 0. Neuroscience 1978, 3, 773. (46) Crow, T. J. In Neuroleptics and Schizophrenia; Simister, J. M., Ed.; Plenum Press: Lundbeck, 1979; p 29. (47) Peroutka, S. J.; Snyder, S. H. Am. J. Psychiatry 1980, 137, 1618. (48) Roth, R. H. In Neuroleptics: Neurochemical, Behauioral and Clinical Perspectiues; Coyle, J. T., Enna, S. J., Eds.; Raven Press: New York, 1983; p 119.
Surface-Modified Electrode Investigations
Langmuir, Vol. 6, No. 1, 1990 63
alterations in extracellular DA concentrations predicted d y ~ k i n e s i a . ~ Thus, ” ~ ~ some other, time-dependent proto occur following chronic antipsychotic administration. cess appears to be responsible for their therapeutic and Therefore, the ability to directly detect changes in DA adverse side effects. release is required to adequately address this hypotheIt is of particular significance that so-called “classical” sis. antipsychotic drugs (e.g., haloperidol and chlorpromazine) often induce these motor side effects during conAlthough the time-dependent therapeutic and adverse tinuous treatment, whereas other antipsychotic agents effects of repeated antipsychotic treatment have been termed “atypical” (e.g., clozapine and thioridazine) are attributed, a t least in part, to depolarization block in DA relatively free from such side effect^.^^,^' Although based cells, electrophysiological assessments of DA cell activon somewhat conflicting biochemical and electrophysioity following repeated drug treatment do not yield direct logical evidence, the induction of neurological side effects information concerning the functional consequences of by classical antipsychotics is thought to be related to conthis condition on DA release from DA nerve terminal fields tinuous blockade of DA function in the nigrostriatal DA in the CNS. Given the data presented herein and system, whereas the therapeutic effectiveness of both elsewhere,” our in vivo electrochemical techniques appear classes of drugs may be related to their antagonistic action particularly well suited to study changes in extracellular on mesolimbic/mesocortical DA ~ystems.~’Recent elecDA concentrations as they relate to the differences between trophysiological studies have shown that after 3-4 weeks the two classes of antipsychotic drugs with regard to their of daily administration of classical neuroleptics the numsite and time-dependent mechanisms of action on DA ber of spontaneously active DA neurons within the subsystems in the brain. We have recently reported that stantia nigra zona compacta and the adjacent ventral repeated (21-day) treatment with haloperidol produces tegmental area are substantially In cona significant decrease in basal DA release in the striatrast, similar treatment with atypical antipsychotic drugs tum and nucleus a c c u m b e n ~ , ~sites ~ , ~of~ nigrostriatal -~~ inactivates only DA neurons in the ventral t e g m e n t ~ m . ~ l - ~ ~and mesolimbic DA nerve terminals, respectively. EviThese effects are thought to model both the clinical effidence was also presented suggesting that these effects cacy and neurological side effect potential of these comwere due to induction of depolarization block in DA neupounds in man.42’51,53 rons. We have now extended these findings by examinDirect intracellular recordings from identified DA cells ing the effects of similar treatments with both classical inactivated after repeated haloperidol treatment have (haloperidol, chlorpromazine, and (-)-sulpiride) and atypestablished that the reduction in DA cell activity is due ical (clozapine and thioridazine) antipsychotics on basal to the development of a rather unusual phenomenon desDA release in the striatum and nucleus accumbens. For ignated as depolarization block and that apomorphine comparative purposes, metoclopramide was examined administration restores spontaneous activity in these since, at low doses, it exerts only weak antipsychotic effects neurons.42 Although the ionic mechanisms underlying but readily induces adverse side effects.55 Desipramine this phenomenon remain poorly understood, the inhibiand promethazine were selected since they fail to induce tion of DA cell firing following continuous neuroleptic depolarization block in midbrain DA neurons after repeated treatment is thought to produce overexcitation of DA neutreatmen~~l’~~ rons, an effect which eventually elevates the DA cell memConsistent with previous observation^,^^,^^-^^ repeated brane potential above its normal resting state. This conadministration of haloperidol reduced the basal release dition leads to an increase in the firing threshold of these of DA in both the st,riatum and nucleus accumbens as DA neurons and an impairment of the action potential evidenced by a significant reduction in generating mechanism. In this particular case, apomorthe voltammetric peak currents for DA (38% and 58% vs phine’s effectiveness in reversing the neurolepticvehicle controls, respectively: Figure 7C,D). In sharp coninduced state of inactivation of DA neurons has been attribtrast, clozapine decreased basal DA release in the nucleus uted to its ability to repolarize the DA neuronal memaccumbens (30% vs vehicle controls) but had no effect brane to such an extent that normal spontaneous activity on the release of DA in the striatum (Figure 7E,F). is r e i n ~ t a t e d . ~ ~ Recent studies have shown that systemic administraAn important consequence of these findings is that DA tion of the direct-acting DA agonist apomorphine to drugrelease from DA nerve terminal regions should be marknaive animals results in hyperpolarization of midbrain edly attenuated. However, ex vivo biochemical assessDA neurons with inhibition of spontaneous discharge42 ments have shown that chronic administration of antipand a decrease in DA release in DA nerve terminal regions sychotics results in tolerance to, and in some cases comas depicted here (cf. Figure 6) and el~ewhere.~’Consisplete reversal of, the increase in forebrain DA turnover tent with these effects, administration of apomorphine (see ref 48). Thus, the tolerance to the chronic effects (100 Fg/kg iv) to vehicle-treated control animals reduced of these drugs on DA metabolism may be directly related the DA voltammetric signal to undetectable values in both to their ability to induce depolarization block in central brain regions (Figure 7A,B). The time courses of these DA systems. In contrast, recent in vivo electrochemical effects were comparable to those shown in Figure 6. studies have shown a dissociation between the mechaIn marked contrast, after repeated treatment with halonisms responsible for the induction of depolarization block peridol, apomorphine administration reversed the decreases and the effects of atypical antipsychotics on extracelluin DA release in both brain regions to values that did lar DOPAC levels following repeated treatment.54 Connot differ significantly from vehicle-treated controls. Repsequently, DA metabolism may not accurately reflect the resentative examples of voltammograms recorded from (49) Crowley, T. J.; Hydinger-Macdonald, M. Arch. Gen. Psychiaanimals treated repeatedly with vehicle or haloperidol try 1981, 38, 668. before and at the peak effect of apomorphine are shown (50) Gerlach, J . ; Simmelsgard, H. Psychopharmacology 1978, 59, in Figure 7C,D. Actual time courses of these effects are 1 nk shown in Figure 8. As shown in A and B of Figure 8, the (51) Chiodo, L. A.; Bunney, B. S. J . Neurosci. 1983, 3, 1607. (52) Chiodo, L. A.; Bunney, B. S. J . Neurosci. 1985,5, 2539. apomorphine-induced increase in DA release began at (53) White, F. J.; Wang, R. Y . Science 1983, 221, 1054. the decreased basal level observed after repeated treat(54) Maidment, N. T.; Marsden, C. A. Eur. J . Pharmacol. 1987, 136, 141. ment with haloperidol and increased to peak values within
64
Langmuir, Vol. 6, No. 1, 1990
Lane and Blaha
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Figure 8. Time courses of the effects of a challenge injection of apomorphine (APO, 100 pg/kg iv) on basal DA release in the striatum and nucleus accumbens of animals treated repeatedly for 21 days with drug vehicle (A-D), haloperidol (A,B),and clozapine (C) ( 0 ) .Note that in the case where repeated drug treatment had no effect on DA release (C), injection of APO completely inhibited DA release, whereas in every case showing a decrease in DA release (A,B,D) APO administration reversed these decreases to values that did not differ significantly from vehicle-treated controls (0or - -). All results are means *SEM from groups of six animals and are expressed as a percentage of the mean base-line DA release value measured in control rats. This value is designated as 100% and is shown as the first data point of the vehicle-treated time courses "(0or - -)". 30-35 min. The duration of these effects (120-125 min) was identical with those observed in vehicle-treated control animals following apomorphine administration. Of particular significance, after prolonged treatment with clozapine, apomorphine administration temporarily increased DA release to control values only in the nucleus accumbens, while maximally decreasing the release of DA in the striatum (Figure 7E and 7F; Figure 8C and 8D). The effects of repeated treatment with the various antipsychotic and control drugs tested on basal DA release levels and the maximal effects of a subsequent challenge
Figure 9. Effects of repeated (21-day) treatment with antipsychotic and non-antipsychotic drugs on basal DA release in the striatum and nucleus accumbens. Note that in every case where repeated drug treatment had no effect on DA release injection of apormorphine (APO,100 kg/kg iv) completely inhibited DA release, whereas in every case showing a decrease in DA release APO administration reversed these decreases to values that did not differ significantly from vehicle-treated controls. Bars show mean values kSEM from six rats per drug group and are expressed as a percentage of the mean basal DA release value measured in vehicle-treated controls. This value is designated as 100% (open bars). Numbers beneath abscissa represent drug doses. Asterisks indicate a significant difference (p < 0.001) from vehicle controls. Abbreviations: HAL, haloperidol;CPZ, chlorpromazine;(-)SUL, (-)-sulpiride; (+)SUL, (+)-sulphide;CLOZ, clozapine;THIO, thioridazine; MET, metoclopramide; PRO, promethazine; DMI, desipramine. injection of apomorphine are summarized in histogram form in Figure 9. Similar to the effects of haloperidol, chlorpromazine and (-)-sulpiride significantly reduced the basal release of DA in both the striatum and nucleus accumbens (37% and 39%; 54% and 38% vs vehicle controls, respectively). Consistent with the effects of clozapine, thioridazine reduced the level of basal DA release only in the nucleus accumbens (41% vs vehicle controls). In contrast, metoclopramide treatment produced effects that were opposite to those of clozapine or thioridazine in that DA release was decreased only in the striatum (32% vs vehicle controls). In all cases where repeated drug treatment induced a reduction in DA release, administration of apomorphine temporarily restored DA release to levels comparable to vehicle-treated control animals (Figure 9). The effects of these neuroleptic agents appeared to be due to selective DA receptor blockade since repeated administration of (+)-sulphide, promethazine, or desipramine, drugs lacking in antipsychotic efficacy or adverse side effects, failed to alter basal DA release or apomorphine-induced decreases in DA release in either the striatum or the nucleus accumbens (Figure 9). The present results provide evidence that in vivo voltammetric methods can be applied to investigate the sites and mechanisms of action of neuroleptic agents on central dopaminergic function. Combined with electrophysiological data, the observed reductions in DA release following repeated neuroleptic treatment appear to be related to the ability of these drugs to inactivate DA neurons in the substantia nigra zona compacta and ventral tegmentum. The ability of apomorphine to reverse the decreases in DA release strongly implicates depolarization block as the mechanism responsible for these effects. These results further suggest that the inability of atypical antip-
Langmuir, Vol. 6, No. 1, 1990 65
Surface-Modified Electrode Investigations
released from DA nerve terminals (see ref 58), can, upon sychotic drugs to decrease striatal DA release may be acute systemic or central administration, induce a conrelated to their lower potential for causing neurological dition resembling depolarization block in the nucleus side effects and that a decrease in limbic DA release fola ~ c u m b e n s . Moreover, ~ ~ ~ ~ , ~electrophysiological ~~ studlowing prolonged drug treatment may be involved in the ies have shown that acute injection of the CCK antagodelayed onset of therapeutic efficacy in man. nist proglumide reverses depolarization block following A t present, it is uncertain why repeated treatment with repeated haloperidol a d m i n i ~ t r a t i o n .Additionally, ~~ preclozapine and thioridazine does not lead to depolarizaliminary voltammetric experiments have shown that the tion block of substantia nigra DA cells and a decrease in neuropeptide neurotension (NT) exhibits pharmacologDA release in the striatum, although there are several ical actions resembling those of ~ l o z a p i n e .Combined, ~~ possible explanations. First, it has been proposed that these data suggest that CCK and NT may be involved these drugs do not cause neurological side effects becauses in the effects of neuroleptics and perhaps may be essenthey have, in addition to antidopaminergic actions, antitial in the time-dependent expression of depolarization cholinergic properties in the CNS.52 Second, these agents block in midbrain DA neurons. exhibit a,-noradrenergic receptor blocking properties that also may in part underlie their differential pharmacologAcknowledgment. We thank McNeil Laboratories ical actions.52 Finally, it is possible that clozapine and (haloperidol), Smith, Kline & French Laboratories (chlothioridazine affect postsynaptic DA receptors differrpromazine hydrochloride), Sandoz Pharmaceuticals (clozently in striatal and limbic regions.56 apine and thioridazine hydrochloride), Ravizza ((-)In accord with these alternatives, recent electrophysisulpiride and (+)-sulpiride), Wyeth Laboratories (prometological and in vivo voltammetric studies have shown that h a z i n e ) , a n d USV L a b o r a t o r i e s ( d e s i p r a m i n e repeated coadministration of either an anticholinergic drug hydrochloride) for generously supplying the drugs used or an a,-noradrenergic antagonist with a typical antipin this study. We also thank Siva P. Hari, Richard K. sychotic (haloperidol) prevents both the development of Fancher, and Jean-Michel Rivet for laboratory assisdepolarization block in nigrostriatal DA neurons and the tance. This work was supported in part by USPHS Grants reduction in basal DA release in the s t r i a t ~ m . ~ ~In? ~ ~ ~NS " 13556 and MH 17148 and by the McKnight Research this context, it is of interest to note that several clinical Foundation. studies have shown that coadministration of a neurolepRegistry No. DA, 51-61-6; NE, 51-41-2; HAL, 52-86-8; CPZ, tic, such as haloperidol, with anticholinergic drugs results 50-53-3; (-)SUL, 23672-07-3; CLOZ, 5786-21-0; THIO, 50-52-2; in a reduction in neurological side effect^.^' MET, 364-62-5; stearic acid, 57-11-4. These findings, together with a growing body of evidence, suggest that several different neural systems and (58) Kalivas, P. W. Neurosci. Biobehau. Reu. 1985, 9, 573. (59) Lane, R. F.; Blaha, C. D.; Phillips, A. G. Brain Res. 1986,397, perhaps several neuromodulators also may be important 200. for the induction and maintenance of depolarization block (60) Blaha, C. D.; Phillips, A. G.; Lane, R. F. Regulat. Peptides in DA neurons. For example, we have recently shown 1987.,~ 17. 301. (61) Phillips, A. G.; Lane, R. F.; Blaha, C. D. Trends Pharmacol. that cholecystokinin (CCK),a neuropeptide co-stored and ~~
~~
~
Sci. 1986, 7, 126.
Stanley, M.; Lautin, A.; Rotrosen, J.; Gershon, S.; Kleinberg, D.Psychopharmacology 1981, 71, 219. (56) Altar, C. A.; Wasley, A. M.; Neale, R. F.; Stone, G. A. Brain (55)
Res. Bull. 1986, 16, 517. (57) Lane, R. F.; Blaha, C. D.; Rivet, J. M. Brain Res. 1988, 460,
398.
(62) Phillips, A. G.; Blaha, C. D.; Fibiger, H. C.; Lane, R. F. Ann. N. Y.Acad. Sci. 1988, 537, 347. (63) Chiodo, L. A.; Bunney, B. S. J. Neurosci. 1987, 7, 629. (64) Blaha. C. D.: P h i h s . A. G.: Fibiner. H. C.: Lane. R. F. Ann. N . Y . Acad. Sc'i. 1988.537. i78. (65) Miner, D. J.;'Rice, J. R.; Riggin, R. M.; Kissinger, P. T. Anal. Chem. 1981,53, 2258. 1
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