Journal (tt NeurochemistryLippincolt—Raven Publishers, Philadelphia© 1996 International Society for Neurochemistry

Nicotine Effects on Dopamine Clearancein Rat Nucleus Accumbens

Carl Hart and Charles Ksir

Department of Psychology and Neuroscience Program, University ot Wyoming, Laramie, Wyoming, U.S.A.

Abstract: In vivo voltammetry was used to measure theclearance of exogenously applied dopamine (DA) in thenucleus accumbens following acute systemic nicotineadministration in urethane-anesthetized rats. The IVEC-5system was used for continuous in vivo electrochemicalmeasurements. A finite amount of DA was pressure-ejected (25—100 nI, 200 ,uM barrel concentration) at 5-mm intervals from micropipettes (tip diameter, 10—15 1km)positioned 250 ± 50 ~smfrom the recording electrode.The peak DA concentration after each DA ejection wassignificantly decreased in rats following nicotine, but notin rats given saline. In addition, when mecamylamine wasadministered 20 mm before nicotine it clearly antagonizednicotine effects. These results suggest that nicotine mayactually facilitate DA transporter systems within the nu-cleus accumbens. Key Words: Nicotine—Dopaminetransporter—Nucleus accumbens—In vivo voltammetry.J. Neurochem. 66, 216—221 (1996).

Self-administration studies suggest that dopamine(DA) is an important component in the reinforcingeffects of nicotine. Corrigall and Coen (1991) reportedthat nicotine self-administration is significantly re-duced after administration of DA antagonists. Theyfurther demonstrated that microinfusions of the nico-tinic antagonist dihydro-~3-erythroidineinto the ventraltegmental area (VTA) reduced nicotine self-adminis-tration (Corrigall et al., 1994). Autoradiographic stud-ies have revealed that nicotinic cholinergic receptorsare located on the cell bodies of dopaminergic neurons(Clarke and Pert, 1985). in addition, nicotinic agonistscan cause direct release of DA from nerve terminals(Rowell et al., 1987). Systemic or microiontophoreticadministration of nicotine causes an increase in thefiring rate of dopaminergic neurons of the substantianigra and VTA (Lichtensteiger et al., 1982; Clarkeet al., 1985; Grenhoff et al., 1986). Presumably thisincrease in rate of firing leads to increased DA releasein the striatum and nucleus accumbens (NAS). Fur-thermore, this release of DA by nicotine has beenshown to be greater in the NAS when compared withother brain regions, including the caudate-putamenarea (Rowell et al., 1987; Lapin et al., 1989). In vivo

microdialysis studies have also demonstrated that nico-tine stimulates DA release preferentially in the NAScompared with other regions (Imperato et al., 1986).

In vivo voltammetry may also be used to measureDA levels in brain tissue. A major advantage of thisapproach is the high sampling rate, as the DA concen-tration can be measured on a second-by-second basis.The use of in vivo voltammetry seems well suited forexamination of DA concentrations after drug adminis-tration. Brazell et al. (1990) combined invivo microdi-alysis and voltammetry to measure endogenous DAlevels in the NAS compared with those levels foundin the caudate-putamen following acute nicotine ad-ministration. Their findings revealed that nicotine does,in fact, preferentially augment NAS DA release. Theseresults are in agreement with the dialysis study of Tm-perato et a!. (1986). However, the procedure used byBrazell et al. (1990) was unable to address possiblemechanisms through which this effect is mediated. Forexample, the observed increase in NAS extracellularDA levels could have been due to increased DA releasein the NAS or to nicotine’s ability to inhibit the DAtransporter system. Cass et al. (1992) were interestedin a similar question with systemic cocaine. In theirexperiment in vivo voltammetry was used to measurethe clearance and diffusion of exogenously applied DAin the striatum and NAS to elucidate the possible mech-anism(s) responsible for the higher DA levels ob-served in the NAS following cocaine administration.They concluded that inhibition of the DA transporter(reuptake) system was responsible for the higher DAlevels and that this effect was greater in the NAS thanin the striatum.

Although in vivo voltammetry has been used to mea-sure the clearance and diffusion of exogenously ap-

Received March 22, 1995; revised manuscript received July 24,1995; accepted August 8, 1995.

Address correspondence and reprint requests to Dr. C. Ksir atDepartment of Psychology, Box 3415, University Station, Universityof Wyoming, Laramie, WY 82071, U.S.A.

Abbreviations used: DA, dopamine; MPTP, l-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NAS, nucleus accumbens; VTA, ventraltegmental area.

216

NICOTINE EFFECTS ON DOPAMINE CLEARANCE 217

plied DA in the NAS after cocaine administration, thistechnique has not been used to study nicotine’s effects.Nicotine, like cocaine, has been shown to have rein-forcing properties, and reinforcement seems to be de-pendent on the ability to increase NAS DA release(Corrigall et al., 1992; Corrigall and Coen, 1994).Moreover, the recent demonstration by Izenwasser etal. (1991) that nicotine inhibits DA uptake in vitroprovides further support for the notion that nicotineactions on DA neurons may be analogous to cocaine.Thus, we originally hypothesized that in vivo volt-ammetric findings from systemically administered nic-otine should yield similar results as reported by Casset al. (1992): inhibition of DA uptake processes. How-ever, recent preliminary findings from our laboratoryindicated that nicotine may have the opposite effect,that of enhancing DA c!earance (Ksir et a!., 1995).The current experiments are a more controlled andextensive effort to characterize the effect of nicotineon the clearance of exogenous DA.

MATERIALS AND METHODS

AnimalsTwenty-seven adu!t male Sprague—Dawley rats, weighing

250—350 g, were used in these experiments. Animals werehoused three or four per cage and allowed free access tofood and water. Lights were maintained on a 12-h light—dark cycle.

Electrodes and recording apparatusRecordings were made using Nafion-coated (5% solution;

Aldrich Chemical Co., Milwaukee, WI, U.S.A.) single ormultiple carbon fiber [three fibers (fiber diameter, 30 tim;exposed length of 100 ,um) sea!ed in a glass capillary]working electrodes. These electrodes have been shown to beselective to themonoamine neurotransmitterscompared withascorbic acid (Gerhardt et al., 1984).

Electrochemical measurements were performed with a mi-crocomputer-controlled apparatus (IVEC-5; Medical Sys-teins Corp., Greenvale, NY U.S.A.). A square-wave oxida-tion potential of 0.55 V, with respect to the reference elec-trode, was applied to the working e!ectrode for 100 ms ata rate of 5 Hz. This potential allows for the oxidation ofelectroactive molecules, such as DA, at the surface of theelectrode tip. The resulting oxidation current was digitallyintegrated during the last 80 ms of the pulse. The reversecurrent flow generatedby the reduction ofthe oxidized mole-cules was digitized in the same manner when the potentialwas dropped back to its resting level (0.0 V for 100 ms).

Immediately before each recording session, each electrodewas calibrated for its sensitivity to DA against ascorbic acid.Calibrations were performed in vitro at room temperatureusing DA solutions prepared with 0.1 M phosphate-bufferedsaline (pH 7.4). Electrodes showed excellent linearity overa DA concentration range from 2 to 8

1~M;calibrations wereperformed in solutions containing a fixed concentration of250 ,uM ascorbic acid to mimic brain extracellular levels.All electrodes used exhibited a DA to ascorbic acid selectiv-ity ratio of >500:1 (Gerhardt et al., 1984, 1987). Electrodesensitivity to DA averaged 22,000 ±2,000 digital countsper 1 jiM change in DA concentration. An Ag/AgC1 wire

served as a reference electrode. A micropipette (tip diameter,10—15 jim) containing 200 pM DA (pH 7.2) was attachedto the working electrode at a distance of 250 ± 50 jim toallow pressure ejection of DA into the NAS.

SurgeryRats were anesthetized with urethane (1.25 g/kg of body

weight) 1.5 h before surgery. Body temperature wasmain-tained at ~—37°Cwith an isothermal pad. A tracheotomy wasperformed, and the animal was mounted in a Kopf stereo-taxic device with the incisor bar positioned 3.3 mm below theinteraural line. A 3.0-mm-diameter hole was drilled slightlyanterior to bregma. The dura was removed to allowinsertionof the micropipette/Nafion-coated carbon fiberelectrode as-sembly into the NAS [1.2 mm anterior, 2.5 mm lateral tobregma, and 7.0—7.3 mm below brain surface (Paxinos andWatson, 1986)1. The reference electrode was placed in therostral part of the brain away from the recording electrodeand cemented in place with dental acrylic.

ProcedureTest ejections were conducted in the overlying cerebral

cortex (not a DA-rich region) to ensure that signals weresatisfactory. The electrode assembly was then lowered intothe NAS. After a delay of 5 mi DA pressure ejections(25—100 nI) were begun at 5-mm intervals into the NAS.The amount of DA ejected was monitored by measuring themovement of the fluid meniscus in the micropipette aftereach ejection using a dissection microscope with an eyepiecereticule. Pressure and time parameters were adjusted follow-ing each of the first several releases to obtain peak DA signalamplitudes of 1 jiM. The pressure and time parametersthen remained constant throughout the remainder of the ex-periment. Before drug administration at least three sequentialDA signals were obtained that varied by no more than 10%.When this requirement was met subjects received saline (n= 8), 1 mg/kg of mecamylamine (n = 3), or 0.4 mg/kgof nicotine (n = 10). In six animals mecamylamine wasadministered 20 mm before nicotine. All drugs were admin-istered subcutaneously. DA was pressure-ejected every 5mm for an additional minimum of 60 mm after the test drugadministration.

All animal procedures were in strict accordance with theNIH Guidefor the Care and Use ofLaboratory Animals andwere approved by the institutional Animal Care Committee.

HistologyAt the end of each experiment, each animal was perfused

with 10% formalin. The brain was removed and later sec-tioned and stained with cresyl violet. Recording sites wereidentified by localizing the electrode track to its most ventralextent. Electrode tracks that were not confined to the NASwere not included in the data analysis.

RESULTS

DA clearanceFollowing each pressure ejection of DA into the

NAS, a transient and reproducible electrochemical sig-nal was observed. These peaks were characterized bythree parameters: rise time (measure of the time ittakes the pressure-ejected DA from the micropipetteto reach its peak amplitude), peak amplitude, and halftime, which is defined as the time it takes each signal

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218 C. HART AND C. KSIR

FIG. 1. Measured concentration of DA for 5 mm following atypical pressure ejection of DA in the NAS before and after sys-temic nicotine (Nic). The rise time for the higher peak was 32s. The half-decay time was 37 s.

to decrease to 50% of the peak amplitude. The valuesof these parameters at 15, 10, and 5 mm before drugtest were averaged to obtain a baseline value for eachanimal.

The groups’ baseline values for these parameterswere not significantly different before administrationof the test drug. Mean ±SEM baseline peak amplitudefor the saline group was 1.9 ±0.95 jiM, for the nicotinegroup was 1.7 ±0.72 jiM, for the mecamylamine!nicotine group was 1.4 ±0.31 jiM, and for the meca-mylamine only group was 1.0 ±0.37 jiM. For risetime the respective groups’ baseline values were 17.7±10.5, 32.6 ±20.1, 21.9 ±9.4, and 21.6 ± 15.4 s.For the half time the baseline values were 57.6 ±43.2,101.2 ±56.7, 68.6 ±30.6, and 63.9 ±47.1 s. Althougheach of the measures varied from one animal to an-other, based on the volume of DA ejected and distancebetween the ejection pipette and recording electrode,each animal’s measures were consistent within an ex-periment. Therefore, postinjection drug effects werecalculated as a percentage of that animal’s baseline foreach measure.

Drug effectsFollowing the injection of nicotine, DA peak heights

were consistently lower than baseline (Fig. 1). To de-termine whether a significant change occurred follow-ing drug administration, the percent baseline values forpeak amplitudes, rise time, and half time of the DAsignal from rats that received nicotine alone, salineonly, mecamylamine alone, and mecamylamine 20 mmbefore nicotine were compared. A two-factor ANOVAwith repeated measures found a significant differencein peak amplitude change between the groups (F= 7.60, df= 3, 23,p <0.01). In addition, there wasa significant effect of postinjection time (F = 5.29, df= 12, p < 0.01) and a significant group by time inter-action (F = 2.42, df = 36, 276, p < 0.01). Figure2 shows change in peak amplitudes as a function ofpostmnjection time for each group. Further analysisshowed that the nicotine group was significantly differ-ent from the other three groups in that it produced amuch lower peak amplitude in the signal (p < 0.01).The mecamylamine/nicotine group was not differentfrom the saline group, implying ablockade of the nico-tine effect.

No significant differences in rise times were ob-served among the groups (Fig. 3). However, there wasa significant effect of time (F = 2.55, df = 12, p<0.01). It appears that the significant time effect mayhave resulted from an increase in the rise times overthe session in the mecamylamine/nicotine and meca-mylamine groups. However, when each of these groupswas analyzed separately, this trend was not significant.

When ANOVA was used to analyze half decay timesfor each group, no significant differences were ob-served, On inspection of the half-time data (Fig. 4),it appears that there was an increase in the mecamyl-amine/nicotine and mecamylamine groups. However,when these groups were analyzed separately this in-crease was not significant.

DISCUSSIONNicotine is thought to exert effects on DA neurons

in both the nigrostriatal and VTA-NAS pathways. The

FIG. 2. Mean ±SEM (bars) peak amplitude of DAsignals, expressed as a percentage of baseline val-ues. The arrow indicates the time of drug injection.

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FIG. 3. Mean ±SEM (bars) rise time of DA sig-nals, expressed as a percentage of baseline val-ues. The arrow indicates the time of drug injec-tion.

results from the present study support this view, atleast with reference to the NAS. However, when invivo voltammetry was used to measure the clearanceof exogenously applied DA, nicotine, unlike cocaine(Cass et al., 1992), caused a significant decrease inthe peak DA signals. This effect was blocked by pre-treatment with systemic mecamylammne, which sug-gests that nicotine’s actions are in part mediatedthrough nicotinic receptors. As previously reported byothers (Schwartz et al., 1984; Clarke et al., 1985),nicotinic receptors are located on the dendrites, cellbodies, and axons of DA-containing neurons. Thus, atthis time it is not possible for us to determine themechanism by which nicotine enhances DA clearance.There exist at least three possible explanations for ourfindings. First, nicotine might exert effects directly onDA uptake processes. Nicotinic receptors on DA termi-nals might somehow interact with the DA transportmechanism to stimulate clearance. This, however, isin conflict with the reports that nicotine inhibits DAreuptake in the striatum (Izenwasser et al., 1991) anda later experiment conducted by Izenwasser and Cox

(1992) that found nicotine to have no effect on theDA transporter in the NAS. A possible explanation forthese differences is that Izenwasser et a!. (1991) andIzenwasser and Cox (1992) used in vitro brain slicetechniques, whereas our experiments were conductedwith intact animals.

A second possible mechanism is that nicotine couldinteract with DA terminals to enhance DA release.Increased release of DA might indirectly lead to anenhancement of the reuptake process as a means toclear the increased amounts of DA in the synapse.Evidence that nicotinic receptors on DA nerve termi-nals modulate DA release provides further support forthis explanation (Rapier et al., 1990; Grady et al.,1992). Once DA is released, it may modulate the DAtransporter through activation of presynaptic D2 recep-tors (Meiergerd et al., 1993; Parsons et al., 1993; Cassand Gerhardt, 1994).

Finally, nicotine may bind to nicotinic receptors onthe cell bodies of DA neurons, causing an increase infiring rate of the cell, resulting in enhanced release andreuptake of DA. This hypothesis is consistent with

FIG. 4. Mean ±SEM (bars) half-time of DA sig-nals, expressed as a percentage of baselineval-ues. The arrow indicates the time of drug injec-tion.

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220 C. HART AND C. KSIR

findings by other laboratories, which have reported in-creases in firing rates and DA turnover rates in re-sponse to nicotine (Lichtensteiger et al., 1982; Clarkeet a!., 1985; Grenhoff et al., 1986). In fact, it wasrecently reported by Nisell et al. (1994) that localmicroinfusion of mecamylamine into the VTA, but notinto the NAS, blocked the effect of systemic nicotinein releasing DA in the NAS. Those results make itappear that the most likely explanation of the currentresults is the third one: Nicotine acts on the VTA toenhance DA release in the NAS, which then enhancesDA uptake in the NAS, perhaps by blocking D2 recep-tors.

The recent work by Behmand and Hank (1992),which found that nicotine enhances I -methyl-4-phe-nyl- 1 ,2,3,6-tetrahydropyridine (MPTP) neurotoxicity,provides additional support for the hypothesis that nic-otine facilitates the DA transporter. Although themechanism by which nicotine enhances MPTP neuro-toxicity remains unknown, presumably nicotine,through its actions on the DA transporter, causes agreater portion of 1 -methyl-4-phenylpyridinium(MPP~),the eventual neurotoxin, to be taken up byDA neurons, therefore resulting in an enhanced neuro-toxic effect. Taken together, these data provide strongsupport for nicotine’s role in potentiating DA reuptakemechanisms.

An additional interesting finding from the currentexperiment is that the time of onset of nicotine’s effect(20 mm) is consistent with the maximal behavioraleffects produced by nicotine (Ksir et al., 1985), whichimplies that the behavioral effects seen with nicotinemay be mediated through its actions on the NAS. Ina recent study, Benwell and Balfour (1992) combinedin vivo microdialysis and locomotor activity monitor-ing to assess nicotine’s effects on NAS DA release andlocomotor activity. They concluded that the enhancedlocomotor response observed in rats is associated withnicotine’s ability to increase DA secretion in the NAS.This conclusion is supported by many other studiesthat have assessed nicotine’s effects on locomotion(Clarke et a!., 1988; Museo and Wise, 1990). How-ever, the correlation between the current results andprevious behavioral findings is not perfect, in that thebehavioral effects of 0.4 mg/kg of nicotine in a nico-tine-naive rat would be limited to a duration of ~=30mm. The effect on DA clearance had not returned tobaseline 60 mm after injection in the current experi-ment. One possible explanation for this difference inthe apparent duration of the drug’s effects may bethat the current experiments were carried out underurethane anesthesia, which might alter the pharmacoki-netics of nicotine or the responses of the dopaminergicneurons themselves.

Acknowledgment: The authors would like to thank theRocky Mountain Center for Sensor Technology at the Uni-versity of Colorado Health Sciences Center, Denver, CU,

for technical assistance. This work was supported in part bygrant BNS 9110308 from the National Science Foundation.

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