Introduction

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Several lines of evidence suggest that activation of mu and kappa opioid receptors leads to functionally opposite effects. For example, central kappa receptor activation can increase morphine analgesia and antagonize both morphine tolerance and physical dependence in morphine-tolerant animals, as well as antagonize withdrawal responses in human heroin addicts (Aceto et al., 1982; Friedman et al., 1981; Green and Lee, 1988; Lee and Smith, 1984; Tulunay et al., 1981; Wen and Ho, 1982). Electrophysiological studies have demonstrated that intravenous or iontophoretic administration of morphine excites DA cells in the rat VTA and in the substantia nigra pars compacta (Gysling and Wang, 1983; Matthews and German, 1984; Yim and Mogenson, 1980), whereas U50, a kappa receptor agonist, inhibits DA cells (Walker et al., 1987). Microdialysis studies have also consistently demonstrated that i.c.v. or VTA microinjections of morphine, -endorphin, DAMGO or [D-Pen²,D-Pen⁵]enkephalin result in significant increases in extracellular DA and DA metabolite concentrations in the NAcc (Devine et al., 1993; Di Chiara and Imperato, 1988; Leone et al., 1991; Mulder et al., 1984; Narita et al., 1992; Spanagel et al., 1990a, 1990b, 1992; Werling et al., 1988). In contrast, systemic or i.c.v. administration of two kappa agonists, E-2078 (a stable dynorphin analog) or U50, significantly decreases extracellular DA and DA metabolite concentration in the mesolimbic ventral striatum (Devine et al., 1993; Di Chiara and Imperato, 1988; Narita et al., 1992; Spanagel et al., 1990a, 1992; Werling et al., 1988).

A similar dichotomy appears to exist with respect to DA-dependent locomotor behavior. Microinjections of the selective mu agonist DAMGO or the selective delta agonist [D-Pen²,D-Pen⁵]enkephalin i.c.v. (Mickley et al., 1990) or bilaterally into the VTA (Latimer et al., 1987) produce forward locomotion or, after unilateral administration, contraversive circling (Jenck et al., 1988). In contrast, VTA kappa receptors appear not to be involved in opioid-induced locomotion, although both i.c.v. DynA and systemic U50 produce significant decreases in locomotion (Jenck et al., 1988).

Finally, conditioned place preference experiments have further demonstrated that mu and kappa opioid receptor agonists produce positive reinforcing or reward (Mucha and Herz, 1986; Suzuki et al., 1991, 1993) and negative reinforcing or aversive (Bals-Kubik et al., 1989; Barr et al., 1994; Tang and Collins, 1985) effects, respectively. Furthermore, systemic administration of kappa agonists U50 and E-2078 (a stable dynorphin analog) also suppress the morphine-induced place preference (Funada et al., 1993).

Based on the above data, we hypothesize that kappa agonists may antagonize heroin (mu agonist)-reinforced SA behavior, one of the most reliable animal models of the reinforcing effects of addictive drugs. To help explain the possible mechanisms of this hypothesis, the interaction of mu and kappa receptor activation on DA release in the NAcc during heroin SA was investigated simultaneously in freely moving, behaving animals. In addition, although a large body of evidence supports the hypothesis that the mesolimbic DA system plays an essential role in the development and maintenance of heroin SA, the exact relationship between this behavior and mesolimbic DA activity remains incomplete. Whether neurochemical modulation of this system can subsequently alter an animal's drug-taking behavior is also poorly understood. Such data would provide further evidence to support the DA hypothesis of drug abuse and serve as a potential bioassay to explore new agents in the treatment of opiate abuse. As such, in the present study, the high temporal and spatial resolution of in vivo FCV was used to evaluate fluctuations in extracellular DA concentration associated with heroin SA and the role of kappa receptor activation on both heroin SA behavior and DA release in the NAcc.

	Materials and Methods

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Surgical preparation. Twenty-six male Long-Evans rats (Sasco, Madison, WI) weighing 300 to 450 g at the time of surgery were housed individually and maintained on a 12-hr light/dark cycle with free access to food and water (lights on from 8:00 p.m. to 8:00 a.m.). With the animals under sodium pentobarbital anesthesia (60 mg/kg i.p.), rats were implanted with a chronic Silastic jugular catheter that was passed subcutaneously to exit between the shoulders. After a 3-day recovery, rats were trained to self-administer heroin (0.06 mg/kg/injection i.v.) for 5 days. Rats demonstrating stable SA behavior (10-min interinjection intervals, 20% change in total SAs per session) were again anesthetized with sodium pentobarbital, and a carbon fiber electrode was stereotaxically implanted into the NAcc (coordinates: 1.7 mm anterior to bregma, 1.6 mm lateral to midline and 7.2-7.4 mm ventral to the surface of the cortex). In some animals, a 30-gauge stainless-steel guide cannulae was also implanted into the anterior portion of the ipsilateral lateral ventricle (coordinates: 0.8 mm posterior to bregma, 1.6 mm lateral to midline and 3.2-3.4 mm ventral to the surface of the cortex). To minimize coating of the carbon fiber by tissue fragments during implantation, the dura and pia mater were prepunctured with a 23-gauge syringe needle. Ag/AgCl reference and stainless-steel ground electrodes were implanted into the ipsilateral and contralateral parietal cortex, respectively. Miniature pin connectors soldered to the three electrodes were inserted into a plastic strip connector and secured with acrylic dental cement to four stainless steel screws threaded into the skull. The jugular catheter was passed subcutaneously to terminate on the head assembly.

Microelectrode fabrication and in vitro calibration. Electrodes were fabricated from a single 8-µm-diameter carbon fiber that extended 250 µm beyond the tip of a pulled glass capillary and fixed in the capillary by a drop of Epon Resin mixed with O-phenylendiamine (1 g of Epon Resin: 0.14 g of O-phenylendiamine). The electrode assembly was baked at 300°C for 3 hr until the melted Epon Resin reached the capillary tip. Electrodes were coated with a 5% Nafion solution (Aldrich Chemical, Milwaukee, WI), an ion-selective polymer that promotes the passage of cations such as DA and impedes the passage of anions, primarily AA and the DA metabolite, DOPAC. Electrodes were dipped into 5% Nafion, air-dried and baked at 85°C for 5 min; the entire procedure was repeated five or six times. Before implantation, electrodes were calibrated for their DA sensitivity and their selectivity to DA against AA and DOPAC in a 0.1 M phosphate-buffered saline solution consisting of 154 mM NaCl, 78 mM Na₂HPO₄·7H₂O and 18 mM NaH₂PO₄ ·H₂O, pH 7.2 to 7.4.

FCV. Electrochemical measurements were performed with a microcomputer-based voltammetric instrument (IVEC10; Medical Systems, Greenvale, NY). The FCV waveform consisted of one cycle of a triangle wave initiated at 0 mV vs. Ag/AgCl and swept between 1.0 and 0.5 mV with a scan rate of 50 V/sec and a repetition rate of 1.0 Hz. For quantification, the electrode oxidation current was integrated between 400 and 900 mV and converted to dopamine concentration using the in vitro calibration data obtained before electrode implantation according to the working hypothesis that signal changes were due entirely to changes in DA concentration. To estimate electrode DA selectivity in vivo, several rats received i.p. injections of apomorphine, a D₁/D₂ receptor agonist, or nomifensin, a DA reuptake inhibitor, to modulate endogenous extracellular DA concentration changes while the NAcc electrochemical signal was recorded.

Heroin SA Procedure. Electrochemical recordings were performed during daily 5-hr heroin SA sessions starting on the third day after electrode implantation. Rats were placed in a light-attenuated chamber (30 × 40 × 60 cm) equipped with a lever mounted 5 cm above the cage floor on one side wall. To minimize extraneous electrical interference, a low-current bias preamplifier was attached directly onto the animal's head assembly and connected to the recording apparatus by a shielded cable through a low-impedance electrical commutator (Airflyte, Bayonne, NJ). The intravenous catheter was connected to a syringe pump (Razel, Stamford, CT) through polyethylene tubing, and a liquid swivel was attached to the commutator. Each depression of the lever delivered an infusion of heroin (100 µl) over a 10-sec period. Depending on the experiment, heroin doses were 0.06, 0.1 and/or 0.2 mg/kg/injection dissolved in sterile saline. A 60-W white light situated above the chamber was simultaneously illuminated with each drug infusion. Usually, the first 30 to 60 min of each recording session consisted of base-line electrochemical measurements with the lever removed.

Drug treatment. During the first 2 to 3 days of recording, all 26 rats received heroin (0.06 mg/kg/infusion) on a continuous reinforcement (FR1) schedule. Rats were then divided into three groups: (1) heroin SA dose-response rats received 0.06, 0.1 and 0.2 mg/kg/injection for an additional 3 to 5 days (n = 14); some rats received naloxone (10 mg/kg i.p. 5-10 min before SA) on the last day of recording; (2) rats receiving heroin SA (0.06 mg/kg/injection) plus U50 (0.1 or 0.5 mg/kg), a selective kappa agonist, coadministered for 3 to 5 days (n = 6) with subsequent U50 (0.5 mg/kg/injection) substituted for heroin during SA behavior, followed by experimentor delivered (passive) U50; and (3) rats receiving heroin SA (0.06 mg/kg/injection) plus pretreatment with nor-BNI, a selective kappa antagonist (1 µg i.c.v.) or saline at 10 min before testing for 2 days (n = 6) with subsequent DynA (1 µg i.c.v.; 1 injection/hr) pretreatment administered during heroin (0.06 mg/kg) SA for an additional 2 to 3 days (n = 4).

Histology. On completion of each experiment, rats were deeply anesthetized with pentobarbital and transcardially perfused with phosphate-buffered saline followed by 10% formalin. Brains were sectioned at 40 µm, and electrode and i.c.v. guide cannulae tips were verified histologically.

Data reduction and statistical analyses. Only data obtained during regularly spaced self-injections (10-min interinjection intervals) were analyzed. To increase signal detection threshold, the relative FCV signal changes were averaged across trials with the lever press set to zero (0 nM DA, 0 min) for both time and amplitude in a manner similar to that used to analyze electrical evoked potentials. Signal values were first averaged across the 60 consecutive 1-sec oxidation cycles each minute during each SA trial and then averaged across all trials within each session. Finally, all repeated sessions for each rat and all rats in each group were combined, thereby significantly increasing the signal detection threshold. Data are expressed as mean amplitude ± S.E.M. and expressed in nanomolar concentration of DA based on the in vitro calibration factor of each electrode. Two-way analyses of variance were used to analyze DA release alterations before and after heroin SA and drug treatment effects. Student's t tests were applied to analyze the effects of drug treatment on number of heroin SAs in each 5-hr session. Significance was set at P  .05.

	Results

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Heroin SA behavior. Typically, all rats rapidly learned the operant task and reliably self-administered heroin within the first week of training. The few rats with unstable SA and poor electrochemical responses were eliminated from the study. Stereotypy developed over the 5 to 10 daily SA sessions, resulting in decreased general locomotor behavior, whereas total SAs increased slightly. Although SA behavior varied across different rats and sessions, there were no significant differences in either the pattern of responses or mean SA rate across sessions (data not shown). The SA rate was maximal in the first hour (4.35 ± 0.20) of a session, followed by a reduced rate of 2.6 to 3.2 injections/hr with a mean interinjection interval of 15 to 25 min at 0.06 mg/kg/injection. Total heroin intake decreased in a dose-dependent manner with increasing heroin doses (see fig. 2B).

NAcc electrochemical signal changes during heroin SA. When data from all heroin SA trials in a given rat were averaged, three major electrochemical signal response patterns were seen: a monophasic response increase (8 of 14 rats), a biphasic initial signal increase followed by a decrease (3 of 14) and an initial signal decrease followed by an increase (3 of 14). All three patterns were seen after SA of 0.06 and 0.1 mg/kg/injection of heroin, although only monophasic response increases were seen after high (0.2 mg/kg/injection) heroin dose SA. These three electrochemical response patterns are depicted in figure 1A (from several representative rats during individual heroin SA trials).

View larger version (37K): [in this window] [in a new window]   Fig. 1.   Representative original oxidation current signal changes (calibrated in nM of DA) during heroin SA. A, Different patterns of electrochemical responses during heroin (0.06 mg/kg) SA in individual rats. B, Dose-dependent responses (saline, 0.06, 0.1 and 0.2 mg/kg/injection heroin) derived from one rat. C, Tonic signal change during a single -hr continuous SA session. Arrows (in A and B) indicate heroin SA response time. Plots illustrate the 2 min before and 10 to 15 min after heroin SA. Arrows (in C) indicate heroin SAs within a whole session. OX and RED in A and C represent oxidation and reduction currents, respectively. Numbers in each panal correspond to date, rat and file; for example,1229-4-1/2/3 means Dec. 29/rat 4/files 1-3.

View larger version (40K): [in this window] [in a new window]   Fig. 2.   A, Dose-dependent increases in DA-dependent electrochemical signal during heroin-reinforced SA behavior averaged across all rats. B, Effects of heroin dose on number of SAs/5-hr session. Note that as dose increased, DA concentration increased and SA behavior decreased. Naloxone (10 mg/kg i.p., 10 min before heroin SA) pretreatment significantly blocked heroin (0.06 mg/kg/injection)-induced DA release. Saline administration did not significantly alter DA release. Values represent mean ± S.E.M. * Significantly different (P < .05) from 0.06 mg/kg heroin group; n = number of rats and SA= total number of SAs at that dose.

Effects of the selective kappa agonists U50 and DynA on heroin SA behavior and NAcc DA release. Coadministration of U50 (0.1 mg/kg) with heroin (0.06 mg/kg) significantly reduced and subsequently reversed the heroin-induced increase in NAcc DA (fig. 3A). With a latency of 3 min after SA, the electrochemical signal rapidly decreased and reached a maximal reduction of 184 ± 75 nM below base line within 8 min and gradually recovered to base-line levels within 15 min. Concurrent with the decrease in DA release, SA behavior significantly increased from 16.3 ± 2.2 to 28.8 ± 5.9 SAs/5-hr session. However, increasing the dose of U50 to 0.5 mg/kg led to a significant decrease in SA behavior over the 5-hr session (fig. 3B), with most SA behavior occurring within the first half hour of the session and virtually ceasing thereafter. At this high dose (0.5 mg/kg) of coadministered U50, the electrochemical signal was irregular and unstable, with an increase in general locomotor behavior (not shown). Consistent with these data, i.c.v. pretreatment with 1 µg DynA produced a similar decrease in heroin-induced DA release and likewise significantly increased SA behavior from 16.3 ± 2.2 to 28.1 ± 7.5 SAs/5-hr session (fig. 3). Similar effects were also observed after a higher dose (2 µg) of DynA was administered.

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Effects of heroin alone (0.06 mg/kg) and heroin coadministered with U50 (0.1 mg/kg) or DynA pretreatment (1 µg i.c.v.) on NAcc DA release (A) and heroin SA behavior (B). In A, both agents similarly antagonized and later reversed the NAcc DA release during heroin SA. In B, low-dose (0.1 mg/kg) U50 increased heroin SA behavior, while high-dose (0.5 mg/kg) U50 reduced SA behavior to saline levels. Data for heroin SA taken from figure 2 for comparison. * P < .05, ** P < .01, different from heroin alone; n = number of rats and SA = total number of SAs at that dose.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Effect of passive U50 (0.5 mg/kg, n = 6, total injections = 20) on basal DA electrochemical signal in the NAcc. Note the similar response pattern as that seen in figure 3 when U50 was coadministered with heroin or the heroin response after DynA pretreatment. Inset, inability of U50 (0.5 mg/kg) to substitute for heroin during SA in rats previously trained to SA heroin. ** P < .01, different from base line.

Effects of nor-BNI on heroin SA and DA release during heroin SA. In contrast to that seen with kappa receptor agonists, pretreatment with 1 µg i.c.v. nor-BNI (a selective kappa antagonist) 10 min before the heroin SA session resulted in a biphasic action: a modest decrease in heroin-stimulated DA release within 30 to 60 min, followed by a significant enhancement in DA release during heroin SA that lasted for 24 hr. The mean peak signal amplitude increased from 96.9 ± 21.3 nM after heroin alone to 172.8 ± 19.7 nM after heroin plus nor-BNI pretreatment (fig. 5). Likewise, DA signal duration increased from 11 min to 30 min after nor-BNI pretreatment. Nor-BNI (1 µg i.c.v.) alone also elevated the DA signal with a latency of 30 to 40 min. Heroin SA behavior decreased, although nonsignificantly, after nor-BNI from 16.2 ±2.2 to 13.0 ± 5.1 SAs/5 hr.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Effects of pretreatment with the selective kappa receptor antagonist nor-BNI (1 µg i.c.v.) on DA release in the NAcc. Note the significant increase in DA signal in the absence of significant changes in heroin SA behavior (P > .05).

In vivo electrode calibration. To estimate relative electrode selectivity to DA in vivo, two drugs known to act on dopaminergic transmission were administered at the end of several experiments. Apomorphine (1.0 mg/kg i.p., n = 6), a D₁/D₂ receptor agonist (which is assumed to depress impulse-dependent DA release), sharply decreased the electrochemical signal with a maximal (398.9 ± 98.8 nM) effect seen at 4 min and lasting 20 min. In contrast, nomifensine, a DA reuptake inhibitor, augmented basal DA efflux with a maximal signal of 295.1 nM DA observed 5 to 10 min after drug administration and lasting 30 min (fig. 6).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Pharmacological evidence for the DA-dependent nature of the FCV electrochemical signal. Note the significant signal increase after intravenous administration of 7 mg/kg nomifensine (a DA reuptake inhibitor) and signal decrease after 1.0 mg/kg (i.p.) apomorphine. PA, passive administration. * P < .05, ** P < .01 different from base line.

Histology. All electrode tips were located in the NAcc, with 19 located in the medioventral (shell) and 7 located in the laterodorsal (core) part of the nucleus (fig. 7). All the guide cannulae tips were located in the anterior portion of ipsilateral lateral ventricle (not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Reconstruction of electrode tip positions in the NAcc. Note that the majority (19 of 26) of the recording sites were located in the shell region, with only a small portion (7 of 26) in the core region of the NAcc. CPu, caudate putamen.

	Discussion

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The high temporal and spatial resolution of FCV was used in freely behaving rats to measure real-time NAcc DA release and its modulation by opioid kappa receptors during heroin-reinforced SA. The two major findings in this report are (1) heroin SA behavior caused a dose-dependent, naloxone-reversible increase in DA efflux in the NAcc that was inversely proportional to the number of drug SAs (i.e., with increasing heroin dose, DA release increased but SA responses per 5-hr session decreased), and (2) central kappa opioid receptor activation by U50 (a selective kappa agonist) or DynA (an endogenous kappa agonist) significantly decreased basal DA release and antagonized heroin-stimulated DA release during SA. In contrast, blockade of kappa opioid receptors by nor-BNI, a selective kappa receptor antagonist, facilitated SA-induced DA release while nonsignificantly decreasing heroin SA behavior.

Role of the mesolimbic DA system in mediating heroin reinforcing effects. A large body of evidence supports the hypothesis that the mesolimbic DA system plays a major role in heroin-reinforced behaviors. First, the NAcc contains significant intrinsic dynorphinergic and enkephalinergic interneurons and is innervated by -endorphin fibers originating in the medial basal hypothalamus (Johnson et al., 1980; Watson et al., 1982; Weber et al., 1982). Both D₁ and D₂ receptors are also well documented in the mesolimbic system (Boyson et al., 1986). Second, unilateral microinjections of morphine into the VTA induces contralateral rotation, which is indicative of an increase in dopaminergic striatal neurotransmission (Holmes et al., 1983).

Role of the mesolimbic DA system in mediating aversive effects of opioids. Activation of kappa opioid receptors or blockade of endogenous mu opioid receptors by naloxone or naltrexone produces aversive effects in drug-dependent or drug-naive animals (Barr et al., 1994; Spanagel et al., 1994a) and is dysphorigenic in humans (Crowley et al., 1985; Pfeiffer et al., 1986). Mesolimbic D₁ receptors apparently are involved in both the reinforcing and aversive effects of opioids (Leone and Di Chiara, 1987; Shippenberg and Herz, 1987, 1988). The kappa agonist U50 has been demonstrated to inhibit DA cells in the VTA (Walker et al., 1987). Microdialysis studies further demonstrate that kappa agonists can inhibit NAcc basal DA release (Devine et al., 1993; Di Chiara and Imperato, 1988; Narita et al., 1992; Spanagel et al., 1990a, 1992; Werling et al., 1988). A similar inhibition of kappa agonists on calcium-dependent K⁺-stimulated [³H]DA release was also demonstrated in a rat striatal synaptosomal preparation in vitro (Ronken et al., 1993). Together, these data suggest that mu and kappa agonists may act at the same dopaminergic substrate but exert opposite effects. However, it was not known whether similar opposite actions would occur immediately after mu or kappa agonist administration alone in freely behaving rats or whether antagonistic interactions would occur when the agonists were coadministered.

Neurochemical mechanisms of actions of mu and kappa agonists on DA release. The mechanism of mu agonist-induced increases in DA release in the mesolimbic system has been well characterized. Kelley et al. (1980) proposed that opioid agonist-induced increases in A10 DA cell firing rates result from an indirect, disinhibitory action in the VTA. In support of this hypothesis, two types of neurons in the rat VTA have been electrophysiologically identified (Johnson and North, 1992). Ventral mesencephalic 6-hydroxydopamine lesions fail to alter VTA [¹²⁵I]DAMGO binding, but quinolinic acid lesions of this area substantially decrease DAMGO binding, suggesting that a dense population of mu receptors reside on secondary interneurons rather than on the primary VTA dopaminergic cells themselves (Dilts and Kalivas, 1989; Mansour et al., 1988). Met-enkephalin reduces the GABA components of VTA-evoked synaptic potentials, and both DAMGO and enkephalin hyperpolarize nondopaminergic interneurons in the VTA (Johnson and North, 1992) and rat hippocampus (Madison and Nicoll, 1988). Direct evidence has demonstrated that morphine presynaptically inhibits GABA release from GABAergic interneurons in the rat hippocampus (Cohen et al., 1992) and midbrain (Renno et al., 1992), suggesting that opioid-induced actions on DA cells may be mediated by GABAergic interneurons. Furthermore, a direct GABAergic inhibitory modulation on dopaminergic activity has been found. Microiontophoretic application of GABA into the VTA increases DA neuronal spike height while decreasing impulse flow (Matthews and German, 1984), which is thought to be a consequence of DA cell hyperpolarization. Kalivas et al. (1990) found that VTA microinjections of the GABA_B agonist baclofen antagonized increases in NAcc DA after VTA microinjections of DAMGO. Similarly, superfusion of baclofen produces hyperpolarization and increases potassium conductance in rat substantia nigra neurons, suggesting that the effect of GABA on dopaminergic neurons is mediated through GABA_B receptors located on DA cells (Lacey et al., 1988; Pinnock, 1984). In contrast, GABA_A agonists appear to act presynaptically on GABAergic neurons to produce inhibition of these neurons, yielding a net disinhibition of dopaminergic neurons (Churchill et al., 1992; Grace and Bunney, 1979).

Comparision of FCV and chronoamperometry. One of the major findings in this study is the dominant monophasic (DA) signal increase observed immediately after heroin SA. Although initial signal decreases or biphasic signal changes were also observed (occasionally) in several rats (20%), our major observation is not consistent with electrochemical results obtained using chronoamperometry (Kiyatkin, 1994; Kiyatkin et al., 1993). Several methodological variables may help explain this apparent disparity. Perhaps the most important difference between the present report and those of Kiyatkin et al. is that our rats were pretrained for up to 1 week to SA heroin before electrode implantation and FCV recording. Because Kiyatkin was interested in studying the development rather than the maintenance of SA behavior, rats in those studies were initially drug naive when recordings began. Thus, those biphasic chronoamperometric signals were derived from animals during their first exposures to heroin SA, whereas our FCV signals were from animals well experienced with the procedure and drug. It has been speculated that DA responses to heroin in the NAcc may differ with periods of exposure to heroin, suggesting that some adaptive changes may have occurred within the mesolimbic DA system (Self et al., 1995; Tjon et al., 1994), leading to the differences in electrochemical data.