Application of semidifferential electroanalysis to studies of neurotransmitters in the central nervous system

J. Electroanal. Chem., 95 (1979) 117--122

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© Elsevier Sequoia S.A., Lausanne -- Printed in The Netherlands

Preliminary note APPLICATION OF SEMIDIFFERENTIAL ELECTROANALYSIS TO STUDIES OF NEUROTRANSMITTERS IN THE CENTRAL NERVOUS SYSTEM

ROSS F. LANE*, A R T H U R T. HUBBARD and CHARLES D. BLAHA

Department of Chemistry, University of California, Santa Barbara, Calif. 93106 (U.S.A.) (Received l l t h September 1978)

INTRODUCTION

The development of electroanalytical methods for the study of monoamine neurotransmitters in the central nervous system (CNS) is an exciting research area of recent interest [1--8]. The basis of this interest is the possibility of performing direct and, preferably, continuous in vivo measurements of the extracellular concentrations of these transmitters. This offers particular promise for obtaining information regarding the synaptic events extant at the neuronal membrane [9, 10]. For example, measurements of drug and electricallystimulated release of dopamine [2--5] and serotonin [5, 6], and efflux of their metabolites [3, 7, 8] have now been described. Determination of the endogenous (natural) concentrations of the catecholamines, serotonin and their respective metabolites is complicated since many of these compounds oxidize too similarly to allow their individual detection by corn ventional electroanalytical methods such as cyclic voltammetry or chronoamperometry [3, 9, 10]. Consequently, an important objective of this work was to develop methods for achieving maximum electrochemical discrimination, while permitting examination of large and small signals simultaneously. Simplicity and low cost of the instrumentation are also of primary importance. We have previously shown that differential pulse voltammetry [11] displays favorable resolution of the current-potential profiles observed from brain tissue [2, 3]. The nature of the electrode surface was also demonstrated to be important. This preliminary note explores the potential of semidifferential electroanalysis, introduced by Goto and Ishii [12]. The theory of such curves for both reversible and irreversible electrode processes at planar and spherical electrodes has been described [12, 13]. Experimental confirmation of the theory has been reported by Oldham et al. [14], who also described analog instrumentation capable of semidifferentiating the current directly. We report here for the first time results of experiments which indicate the advantages of semidifferential electroanalysis for studies of neurotransmitters in the CNS.

*To whom correspondence should be addressed.

118 EXPERIMENTAL Apparatus Carbon paste (graphite/Nujol) microelectrodes having tip diameters between 125 and 250 #m were used as the working electrodes. The larger electrodes were prepared by press-fitting 30-gauge Teflon tubing over 29 or 30 gauge stainless steel wire. Smaller electrodes were prepared from stainless steel Teflon-coated wire of 0.13--0.18 mm o.d. Complete descriptions of electrode fabrication have been given elsewhere [3,6,8,9]. All potentials were measured and are reported with respect to a miniature Ag/AgC1 electrode (1M NaC1) constructed as previously described [2]. This electrode was inserted into a 22-gauge stainless steel guide cannula which served as the auxiliary electrode and facilitated its placement in brain tissue. Operational amplifiers (Teledyne/Philbrick, Model 1026 with BQ-100 booster) were utilized in a conventional multipurpose electrochemical circuit [1]. Analog semidifferentiation of the d.c. current was accomplished by means of circuit B shown in Fig.4 of ref.14, modified only by employing a feedback resistance of 107 ~2. The ladder n e t w o r k used in this circuit [15] was constructed from components having±l% tolerances (Century Components, Canyon Country, Calif.). Cyclic and semiderivative voltammograms (also termed derivative neopolarograms [13,14] ) were recorded simultaneously on separate X-Y recorders. Chemicals and routes of administration D-amphetamine sulfate (Sigma) and L-5-hydroxytryptophan hydrochloride (Calbiochem) were used as received. DL-seryl-2-(2,3,4-trihydroxybenzyl) hydrazine hydrochloride (Serazide, RO4-4602, Hoffman-LaRoche) was generously supplied by Dr. L.D. Lytle. The drugs, dissolved in isotonic saline, were administered either intraperitoneally (i.p.) or intravenously (i.v.) via a tail vein. Dosages are specified in the text. All solutions were prepared immediately prior to use. Procedure Male Sprague-Dawley rats weighing 300--400 g were anesthetized with chloral hydrate (400 mg/kg, i.p.) and placed in a stereotaxic instrument (Model 1204, David Kopf, Tujunga, Calif.). After appropriate incisions and burr holes had been made the carbon paste electrode was implanted into the caudate nucleus [16] at a rate of approximately 200 pm per minute, thereby minimizing damage to brain tissue during placement of the electrode [3]. The reference-auxiliary electrode combination was placed in contralateral frontal cortex, a b o u t 5 mm from the working electrode. Because of the low current ranges encountered, placement of the reference electrode was not critical. Cyclic and semiderivative voltammograms, were then recorded every ten minutes until reproducible current-potential curves were obtained. Stability was evidenced by reproducibility of voltammetric peak currents to +3% and was achieved after five or six potential scans. These procedures are described in greater detail elsewhere [2, 3].

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RESULTS AND DISCUSSION

Cyclic and semiderivative voltammograms recorded from the caudate nucleus are shown in Fig.1. Within the potential region examined four oxidation peaks can be distinguished as the potential is scanned to more positive potentials (Fig.lA). No reduction waves appear upon scan reversal over the same potential region [3,9]. It is to be noted that the appearance of four discernible faradaic peaks are only observed with scan rates at or below 10 mV s -1 . At higher scan rates the individual oxidation processes merge and only two broad signals are observed in the voltammogram. This deterioration in the voltammetric response has been related to unequal shifts in peak potentials with scan rate among the various reactants, resulting in peak overlap [ 3]. However, even with slow linear sweep voltammetry considerable overlap of the oxidation peaks persists and the faradaic currents appear to contain a significant contribution from background currents. Consequently, measurement of peak heights and accurate location of peak potentials is difficult. The corresponding semiderival~ve voltammogram is shown in Fig.lB. The semiA

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120 derivative voltammogram exhibits a noticeably better baseline and the more symmetrical peak shapes predicted by previous reports [13,14]. In addition, the relative improvement in the ratio o f peak currents to background current is apparent. A more detailed examination of this aspect of the technique is deferred to alater communication. It can also be seen from Fig.lB that the discrimination between peaks is better in the semiderivative mode than with slow sweep cyclic voltammerry. In particular, note t h a t the small wave that can just be discerned in F i g . l A at potentials just positive of the first peak is now well defined and well separated from the larger preceding peak. The separation in peak potentials (Ep = 0.18V and 0.34V) is 160mV and both signals can be measured simultaneously. The third Signal in the voltammogram (Ep = 0.51V) is also completely separated from the preceding oxidation process, although the current does not decay to baseline at potentials on the positive side of this peak due to some overlap with the much larger signal at the most positive potential (Ep = 0.69V). That semidifferentiating the current represents a distinct improvement in readout format is clearly evident. Studies presented elsewhere have now identified most of the species responsible for the faradaic currents shown in Fig.1. Briefly, the peak at +0.18 V is due to the summed response of ascorbic acid (AA) and the neurotransmitter dopamine (DA) present in extracellular fluid [3,9]. The peak at +0.34V is due to the transmitter 5-hydroxytryptamine (5-HT, i.e., serotonin) with perhaps a minor contribution from its metabolite 5-hydroxyindoleacetic acid [6]. The electrochemical detection of endogenous serotonin in brain tissue has not been previously accomplished. Oxidation of the major metabolite homovanillic acid (HVA) is responsible for the signal observed at +0.51V [3]. The origin of the peak at +0.69V has n o t been definitely established as of the present time. The measurement of 5-HT in the presence of DA (and AA) is a matter of practical importance [17]. The effect of amphetamine administration illustrates h o w the present instrumentation can be used to advantage for simultaneous measurement of 5-HT in the presence of large concentrations of the more easily oxidized DA. Amphetamine-stimulated release of DA from caudate tissue has n o w been demonstrated by several electrochemical methods [2,4,5]. Conversely, this drug does n o t increase 5-HT efflux [6,18]. Fig.2A shows a series of semiderivative 0.3

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121

voltammograms resulting from the injection of amphetamine. With time the peak current at +0.18V increases in magnitude as more and more DA is released into extracellular fluid. At the same time there is essentially no change in the 5-HT signal. At a time corresponding to maximal DA release (curve d in Fig.2A) the 5-HT peak current is approximately an order of magnitude less than the peak height at +0.18V but is still clearly resolved. In contrast, the determination of 5-HT at this time is not possible by linear sweep voltammetry because the large DA wave completely masks the oxidation peak for 5-HT (Fig.2B). As an additional example Fig.3 shows a semiderivative voltammogram in which the 5-HT response was selectively increased by i.p. administration of 5-hydroxytryptophan (5-HTP). 5-HTP is the biological precursor of 5-HT, which, when injected systemically, enters the brain and is rapidly decarboxylated to 5-HT. Pretreatment with the compound R04-4602 (in appropriate doses) prevents decarboxylation of 5-HTP in the periphery, thus allowing more of this amino acid to enter the brain at low doses [19]. Approximately two hours after the injection the 5-HT signal was increased ten-fold with respect to the peak height correspond. ing to the endogenous 5-HT concentration. It is evident from Fig.3 that the readout with this technique still permits straightforward measurement of all four signals under these conditions. 0.7

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In summary, semidifferential electroanalysis should prove extremely valuable in studies of neuronal processes in which participation of DA, 5-HT (and, presumably, norepinephrine) is implicated. The method has the advantage of monitoring these transmitters and/or their metabolites simultaneously. This additional information is of particular importance in view of recent evidence suggesting various functional interrelations between catecholamine and serotonin-containing systems in the brain [17]. Due to the heterogeneity of brain tissue, precise measurements of their concentrations remains a problem to be solved [3]. To this end, the en-

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hanced sensitivity and favorable readout form, coupled with the simplicity and low cost of the instrumentation [14], make the semiderivative approach highly attractive. ACKNOWLEDGEMENTS The support of this work by the National Institute of Neurological and Communicative Disorders and Stroke is gratefully acknowledged.

REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

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