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Electrical stimulation of reward sites in the ventral tegmental area of the rat increases dopamine transmission… Fiorino, Dennis Frank 1993

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ELECTRICAL STIMULATION OF REWARD SITES IN THE VENTRALTEGMENTAL AREA OF THE RAT INCREASES DOPAMINE TRANSMISSION IN THENUCLEUS ACCUMBENS AS MEASURED BY IN VIVO MICRODIALYSISByDENNIS FRANK FIORINOB.Sc., University of Lethbridge, 1990A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESNeuroscienceWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAApril 1993©Dennis Frank Fiorino, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(SignatureDepartment of  N Eut.OSC NC The University of British ColumbiaVancouver, CanadaDate OQ vs , \''f\1DE-6 (2/88)AbstractIn vivo microdialysis with HPLC-ED was used to measure dopamine (DA),3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in the nucleus accumbens of the rat, prior to,during, and after 15-min periods of electrical brain-stimulation at sites in the ventraltegmental area (VIA) that supported intracranial self-stimulation (ICSS). In the firstexperiment, both ICSS and yoked-stimulation of the VTA evoked significantincreases in extracellular concentrations of DA, its metabolites, and 5-HIAA.Comparable results from ICSS and yoked groups were interpreted as evidence thatthe rewarding properties of VIA stimulation were a causal factor in the elevated DAtransmission in the nucleus accumbens, rather than intense operant behavior.Further evidence for this hypothesis came from a second set of data in whichchanges in extracellular DA levels during the measurement of rate/intensityfunctions for ICSS were positively correlated. 5-HIAA concentrations alsoincreased during ICSS but these changes were not correlated with either ICSS rateor current intensity, suggesting that changes in serotonin metabolism were unlikelyto subserve brain-stimulation reward in the VTA. These results provide furtherevidence that stimulated mesolimbic DA release is a sufficient condition for reward.iiTABLE OF CONTENTSAbstract^  iiList of Tables  vList of Figures ^  viAcknowledgements  viiiINTRODUCTIONHistorical Background ^1GENERAL METHODSSubjects^  16Surgery  16Apparatus ^  16Microdialysis Probe Characteristics ^  17High Performance Liquid Chromatography and ElectrochemicalDetection ^20Intracranial Self-Stimulation Training ....^ 20Instrumentation Overview^  20Statistical Analysis  23Histology ^  23iiiEXPERIMENT 1Effects of ICSS or yoked-stimulation of the VIA on DA, DOPAC, HVA,and 5-HIAA in the ipsilateral nucleus accumbens ^ 24Methods ^  24Results  ^26Discussion  ^44EXPERIMENT 2Effect of varying current intensity during VTA-ICSS on DA, DOPAC,HVA, and 5-HIAA in the ipsilateral nucleus accumbens ^ 46Methods ^  46Results  ^47Discussion  ^59GENERAL DISCUSSION ^  61REFERENCES ^  66ivList of TablesTable 1^Mean basal values for DA, DOPAC, HVA, and 5-HIAA inExperiment 1 and 2 ^28VList of FiguresFigure 1^Diagram of the microdialysis probe assembly  ^19Figure 2^Schematic illustration of the experimental set-up  ^22Figure 3^Effect of self-stimulation or yoked-stimulation of the ventraltegmental area on extracellular dopamine in the nucleusaccumbens  ^31Figure 4 Effect of self-stimulation or yoked-stimulation of the ventraltegmental area on extracellular DOPAC and HVA in thenucleus accumbens . . 33Figure 5 Effect of self-stimulation yoked-stimulation of the ventraltegmental area on extracellular 5-HIAA in the nucleusaccumbens ^ 36Figure 6^Representative placement of a microdialysis probe in theright nucleus accumbens  ^39Figure 7^Location of the tips of bipolar stimulating electrodes for theICSS and Yoked groups in Experiment . .^ 41Figure 8^Location of the tips of bipolar stimulating electrodes for theunstimulated Control animals in Experiment 1 ^43Figure 9^Effect of self-stimulation at low (18pA), medium (22pA), andhigh (27pA) current intensities on extracellular dopamine. .^50viviiFigure 10^Effect of self-stimulation at low (18pA), medium (22pA), andhigh (27pA) current intensities on extracellular DOPAC andHVA ^52Figure 11^Effect of self-stimulation at low (18pA), medium (22pA), andhigh (27pA) current intensities on extracellular 5-HIAA . .^55Figure 12^Location of the tips of bipolar stimulating electrodes for ICSSanimals in Experiment 2 ^58AcknowledgementsMany thanks to my family and friends for their enthusiatic support. Special thanks toChuck Blaha, Ariane Coury, Chris Fibiger, Fred LePiane, and Tony Phillips for theirgenerous assistance in preparing this thesis.1INTRODUCTIONThe discovery that rats would work to stimulate electrically discrete brainareas led to the idea that intracranial self-stimulation (ICSS) could be used touncover the neural mechanisms of reward (Olds & Milner, 1954). Rewardingstimuli have two main organizing effects on behavior (Carr, Fibiger, and Phillips,1989). First, rewarding stimuli can elicit approach responses. Second, rewardscan produce reinforcement. Based upon empirical evidence, Schneirla (1959)proposed two basic motivated behaviors in all animals: approach and withdrawal.Similarly, Young (1959) polarized behaviors into either approach-maintaining oravoidance-terminating patterns. Rewards elicit these approach patterns ofbehavior. An initial observation that led to the discovery of self-stimluation was thatrats would return to the area of the testing box where they received brainstimulation. By this definition, the brain stimulation the rats received is rewarding.In operant terms, reinforcement describes the ability of rewards to increase theprobability that the responses that preceed it will be repeated (Skinner, 1938). Inthe context of this thesis, reinforcement describes how animals perform a well-learned self-stimulation response. According to this perspective, reinforcement canalso be used as a measure of reward. Increased rates of operant respondingreflect the the rewarding quality of the stimulation. These two organizing effects ofreward on behavior are interdependent and appear to share a common underlyingphysiological mechanism. The perception of a rewarding stimulus results in boththe appreciation that the stimulus is a reward (eliciting approach behaviors andreinforcement) and a modulation of response depending upon the attractiveness ofthe reward.A number of scientific findings led to the catecholamine (CA) theory ofreward first proposed by Stein (1964, 1968). Early mapping studies showed thatICSS could be obtained from a wide variety of structures (Crow, 1972a). The2ICSS could be obtained from a wide variety of structures (Crow, 1972a). Themedial forebrain bundle (MFB) supported notably high rates of 1CSS (Fibiger,1978; Crow, 1972a). Newly developed histochemical techniques allowed thetopographical mapping of monoaminergic neurons in the nervous system andascending catecholaminergic fibers were found to travel within the MFB to manyforebrain structures (Fuxe, 1965). It became apparent that there was a goodcorrelation between the location of ascending CA-containing neurons and areasthat supported ICSS (Dresse, 1966). Given that CA neurons, especially thosecontaining noradrenaline (NA), project widely throughout the brain, it is notsurprising that a correlation with ICSS was found (Fibiger, 1978). Althoughcorrelational findings cannot imply causality, it was an important starting point forsubsequent research.More convincing evidence came from pharmacological experiments on1CSS. Drugs that enhance central CA transmission, such as amphetamine (Stein,1964), cocaine (Crow, 1970), and monoamine oxidase inhibitors (Poschel, 1969),were found to facilitate ICSS rates of responding. Drugs that impair central CAtransmission, such as reserpine (Olds, Killam, & Bach-y-Rita, 1956), tetrabenazine(Crow, 1972a), alpha-methyl-p-tyrosine (Cooper, Black, & Paolino, 1971), andchlorpromazine (Crow, 1972a) attenuated ICSS rates.Stein (1964) offered arguments for both NA and for dopamine (DA) as theprimary CA mediating ICSS but settled on a noradrenergic hypothesis of brain-stimulation reward based primarily on pharmacological and anatomical evidence.First, drugs which increased and decreased NA transmission facilitated andattenuated ICSS, respectively. Second, the site of origin of ascendingnoradrenergic fibers corresponded to the main ascending limb of a limbic midbraincircuit which Nauta had identified as an important part of a reward-punishmentsystem in the central nervous system (Stein, 1968). Moreover, the ascending3"noradrenergic" ventral tegmental area (VTA) cells in this circuit were identified byStein as playing an important role in mediating the effects of drugs on ICSS.Dresse (1966) proposed that it was primarily ascending NA, not DA, neurons thatmediated ICSS. This conclusion was due, in part, to an early and erroneous reportthat most neurons of the VTA, an area Dresse found to support high rates of ICSS,were noradrenergic (Fuxe, 1965). Third, ICSS could be obtained from the locuscoeruleus (LC, a noradrenergic nucleus in the brainstem) at points along theascending course of its axons in the dorsal noradrenergic bundle (DNB) prior to itmerging with the MFB, and in a number of terminal regions of this pathway (e.g.dorsal hippocampus) (Phillips & Fibiger, 1989). A neurochemical study using thepush-pull cannula technique provided further support for the NA hypothesis. Stein& Wise (1969) found that in unanesthetized rats given an intraventricular injectionof tritiated-NA, MFB stimulation caused an increase in radioactivity in perfusatesfrom the lateral hypothalamus or amygdala. Non-rewarding brain stimulation didnot.Subsequently, an overwhelming amount of evidence was collected thatrefuted the noradrenergic hypothesis (see Fibiger, 1978). First, a histofluorescencestudy determined that the cell bodies of the VTA contain DA (Ander), DahlstrOm,Fuxe, Larsson, Olson, Ungerstedt, 1966), and not NA terminals as previouslyclaimed (Fuxe, 1965). Second, because early pharmacological studies used drugsthat act on both noradrenergic and dopaminergic systems, it was impossible todiscern whether one or both neurotransmitters mediated ICSS reward (Fibiger,1978). When specific noradrenergic and dopaminergic drugs were used, initialstudies failed to use methods that made a clear distinction between reward andmotor/performance factors (Stellar & Rice, 1989). One study that used the specificNA receptor antagonist phenoxybenzamine and extinction as a rate-free measureof reward showed that DA, but not NA, blockade, impaired MFB self-stimulation4(Fouriezos, Hanson, & Wise, 1978). The extinction method assumes that reducingreward with drugs will produce an extinction curve similar to one produced whenstimulation is turned off in a self-stimulating animal. Further, it was found that therelative potencies of neuroleptics to decrease ICSS reward was better correlated toDA, rather than NA, receptor binding properties (Gallistel & Davis, 1983). Third, aseries of lesion experiments provided compelling evidence that NA was notnecessary for brain-stimulation reward. Bilateral lesions of the DNB using 6-hydroxydopamine (6-0HDA), a selective catecholaminergic neurotoxin, failed todisrupt LC self-stimulation despite reducing hippocampal and cortical NA by 96.7%(Clavier, Fibiger, & Phillips, 1976). lpsilateral or bilateral electrolytic lesions of theLC did not effect self-stimulation of the DNB despite reducing cortical NA by over80% (Clavier & Routtenberg, 1976). Finally, 6-0HDA lesions of the DNB, whichreduced hippocampal NA by 97%, had no effect on hippocampus self-stimulationor its facilitation by amphetamine (Phillips, van der Kooy, & Fibiger, 1977; van derKooy, Fibiger, & Phillips, 1977). Given these data, it was clear that the NAhypothesis of brain-stimulation reward was not tenable.Just as anatomical experiments correlating self-stimulation sites withnoradrenergic neurons provided support for the NA theory of reward, the samestrategy provided the first evidence for a dopaminergic substrate in brain-stimulation reward (Phillips & Fibiger, 1989). Although the hypothesis that DAneurons exclusively mediate ICSS reward can be rejected on purely anatomicalgrounds, the possibility remains that stimulation of these neurons is sufficient forreward (Fibiger, 1978). Early studies had shown that ICSS could be obtained inventral mesencephalic areas corresponding to the VTA and SN (Routtenberg &Malsbury, 1969; Olds & Olds, 1963). Crow (1972b) conducted a detailed mappingstudy of the ventral mesencephalon confirming positive ICSS sites in the VTA andSN and, noting that these were DA rather than NA-containing cell bodies,5concluded that DA neurons support ICSS. Further evidence came from amoveable stimulating electrode study which found that positive ICSS sites in themesencephalon were confined to an area of DA-containing cell bodies and thatICSS thresholds and response rates were proportional to the density ofdopaminergic neurons at the electrode tip (Corbett & Wise, 1980). The majorterminal fields of the mesotelencephalic DA systems have also been found tosupport ICSS, including the nucleus accumbens (Phillips, Brooke, & Fibiger, 1975),striatum (Phillips, Carter, & Fibiger, 1976), prefrontal cortex (Rolls & Cooper, 1973),central amygdala (Wurtz & Olds, 1963), and septum (Olds & Milner, 1954).Pharmacological experiments have shown that systemically-administeredneuroleptics attenuate MFB-stimulation reward and, in some cases, produceoperant motor/performance deficits (Stellar & Rice, 1989; Fibiger, 1978). Thisillustrates a pervasive problem in research investigating the pharmacological basisof ICSS. Since DA systems are known to serve important motor functions (Fibiger,1978) and also appear to play a role in brain-stimulation reward, care must betaken to distinguish between the attenuating actions of neuroleptics on ICSSreward and their impairment of general operant motor performance. It was notsurprising that early studies investigating neuroleptic-induced attenuation of ICSSprovided equivical results (Fibiger, 1978).The introduction of novel rate-free and reward-specific behavioral measuresled to more convincing evidence for the involvement of DA in ICSS reward.Fouriezos et al. (1978) had shown that certain doses of pimozide attenuatedreward independent from its impairing effects on motor performance. Franklin(1978) used a rate-frequency curve-shift method to further confirm that pimozidecould selectively reduce reward. In this method, lateral and vertical curve shiftsoccured independently and reflected changes in reward and performance,respectively. The reward-attenuating effect of pimozide was also shown to be6counteracted by amphetamine treatment (Gallistel & Karras, 1984). Similarly,direct injections of the neuroleptic cis-flupenthixol into the nucleus accumbenswere found to reduce MFB-stimulation reward while producing only mild motorimpairments, as assessed by a rate-frequency curve shift (Stellar & Rice, 1989).Another paradigm where animals could regulate electrical stimulation usingon and off responding, provided further evidence that neuroleptic administrationproduced both reward and motor effects (Liebman, Hall, & Prowse, 1982).Neuroleptics also were found to raise ICSS stimulation thresholds using anautotitration-of-threshold method (Neill, Garr, Clark, & Britt, 1982), a self-adjustingprocedure where the stimulation current is decreased after a fixed number ofresponses. In this case, animals could adjust the level of stimulating current to avalue that was perceivably rewarding by pressing an adjacent lever. It is worthnoting, however, that in contrast to systemic injections, direct injections ofneuroleptics into the nucleus accumbens lowered ICSS stimulation thresholds(Neill et al., 1982).The conditioned place preference technique is another rate-free behavioralparadigm which can test undrugged animals after a training period where theyhave learned to either avoid or approach a distinctive environment that had beenrepeatedly paired with aversive or rewarding stimuli, respectively. Using thisparadigm, Ettenberg & Duvauchelle (1988) found that rats developed a preferenceto places paired with ICSS, an effect which could be blocked by systemically-administered haloperidol in a dose-dependent manner.Studies which have examined the effects of direct and indirect DA agonistson ICSS behavior have also provided convincing evidence of DA involvement.Amphetamine and cocaine have been shown to increase reward by loweringstimulation thresholds without altering stimulation detection thresholds (Kornetsky& Esposito, 1981). Using the self-regulation of duration paradigm, amphetamine7(Liebman, Gerhardt, & Prowse, 1982b) and nomifensine (Gerhardt & Liebman,1985) were found to increase ICSS reward. Amphetamine injection into thenucleus accumbens also enhanced reward (Stellar & Rice, 1989). ICSS was alsofacilitated by the specific DA uptake blocker GBR-12909 (Phillips, Blaha, & Fibiger,1989). Apomorphine, a direct DA receptor agonist, can produce increases in self-stimulation behavior, if administered in small doses, but generally has been foundto disrupt ICSS (Wauquier & Niemegeers, 1973). The latter effect may haveresulted from a non-contingent reward signal competing with ICSS (Stellar & Rice,1989). Collectively, the application of rate-free behavioral paradigms andpharmacological data have supported a role for DA in brain-stimulation reward.!psilateral versus contralateral DA pathway lesion experiments have alsocontributed to understanding neurochemical substrates of brain-stimulation rewardindependent of performance effects. Phillips & Fibiger (1978) found that ICSS ofthe VTA was attenuated severely by selective ipsilateral destruction of DApathways at the level of the hypothalamus using 6-0HDA. Further, self-stimulationat terminal sites of this pathway (i.e. nucleus accumbens and medial prefrontalcortex) in these lesioned animals was only marginally affected which suggestedthat DA does not exclusively mediate ICSS in these two regions (Phillips & Fibiger,1989). A subsequent study using ipsilateral and contralateral 6-0HDA lesions, incombination with rate-intensity measures, found that only lesions ipsilateral to theVTA electrode reduced reward (Fibiger, LePiane Jakubovic, & Phillips, 1987). Adrastic attenuation of asymptotic ICSS response rates on only the side of the lesionled to an important observation. Specifically, that this aspect of the curve-shiftmethod, traditionally used as an index of performance, does not work withmanipulations which block all reward (Phillips & Fibiger, 1989).Various lesion experiments also suggested a dissociation between the twomajor ascending dopaminergic pathways in the brain: the mesocorticolimbic and8nigrostriatal systems. Unilateral kainic acid lesions of the dorsal striatumpreferentially reduced brain-stimulation reward in the SN but not the 'TEA (Phillips,LePiane, & Fibiger, 1982). lpsilateral 6-0HDA lesions of the mesencephalic DApathways were found to produce only a transient attenuation in ICSS rates from theSN (Clavier & Fibiger, 1977). Since contralateral lesions produced similarimpairments, ICSS attenuation was thought to be due to motor deficits. Also,amphetamine facilitated ICSS in the SN contralateral and not ipsilateral to thelesion. Collectively, these results suggested that although dopaminergic neuronsmay not exclusively mediate SN-stimulation reward, they are involved (Clavier &Fibiger, 1977).There is still much debate over whether stimulated release of DA is sufficientfor reward (Phillips et al., 1989; Fibiger & Phillips, 1987; Gallistel, 1986). Ex vivoand in vivo biochemical analyses provide a more direct approach in addressingthis issue.Ex Vivo ExperimentsQuantitative 2-deoxyglucose (2-DG) autoradiography has been used to mapfunctional neural pathways based on altered rates of glucose utilization, a markerof metabolic activity (Kennedy, Des Rosiers, Jehle, Reivich, Sharpe, & Sokoloff,1975). A selective pattern of metabolic activation in dopaminergic terminal fieldsfollowing self-stimulation of the VTA and SN has been shown using the 2-DGtechnique (Porrino, 1987; Porrino, Esposito, Seeger, & Crane, 1985; Porrino,Esposito, Seeger, Crane, Pert, & Sokoloff, 1984). In contrast, other 2-DG studies(Gallistel, Gomita, Yadin, & Campbell, 1985; Gallistel, Karreman, & Reivich, 1977;Yadin, Guarini, & Gallistel, 1983) found little evidence for activation ofdopaminergic projections by self-stimulation. Methodological differences betweenthese studies may account for these seemingly equivocal results. First, whereas9Porrino et al. directly stimulated dopaminergic neurons in the ventralmesencephalon, Gallistel and Yadin stimulated portions of the MFB. The MFB is aheterogenous bundle with widespread ascending and descending components(see Nauta & Haymaker, 1969) whereas the mesencephalic dopamine system iswell-defined in terms of its projections and topography (Fallon, 1988; Domesick,1988; Swanson, 1976). Second, Gallistel and Yadin used brief pulses (0.1 msec)of square wave current. Porrino et al. used longer pulses corresponding to a 60 Hzsine wave current (about 0.8 msec, peak to peak). It has been suggested thatcurrent parameters commonly used in self-stimulation experiments (i.e. squarewave, brief pulses), are inadequate to excite the thin, unmyelinated DA fibers(Yeomans, Maidment, & Bunney, 1988). Further, square wave stimulation of theMFB is only effective in releasing striatal DA when pulses longer than 0.5 msec areused (Millar, Stamford, Kruk, & Wightman, 1985).Another technique that has been used to map active brain areas in responseto chronic MFB self-stimulation is cytochrome oxidase (CO) histochemistry(Bielajew, 1991). Active areas are denoted by an increase in CO, a key enzyme incellular metabolism. Bielajew found that only self-stimulation with longer pulsedurations (2.0 msec) increased metabolic activity in dopaminergic projection areassuch as the frontal cortex and the olfactory tubercle. It is interesting that increasedCO activity was observed in the nucleus accumbens with only brief pulse durations(0.1 msec).Ex vivo measurements of DA and its metabolites 3,4-dihydroxyphenylaceticacid (DOPAC) and homovanillic acid (HVA) in the nucleus accumbens immediatelyfollowing self-stimulation have provided some more direct evidence for increasedDA transmission during ICSS (Phillips et al., 1989). Increased DOPAC/DA ratios inthe nucleus accumbens and prefrontal cortex after self-stimulation of the VTA havealso been reported (Simon, Stinus, Tassin, Lavielle, Blanc, Thierry, Glowindki, & Le10Moal, 1979). In contrast to the above studies, increases in DOPAC were observedin the olfactory tubercle, but not in the nucleus accumbens or striatum followingVIA self-stimulation (Mitchell, Nicoleau, Arbuthnott, & Yates, 1982). This study alsofailed to see changes in DOPAC in the nucleus accumbens or striatum afterposterior hypothalamic self-stimulation. In another study, however, increased DAturnover was observed in the nucleus accumbens and striatum after self-stimulation of the hypothalamus in mice (Garrigues & Cazala, 1983).The finding that some types of operant behavior may increase the rate oftransport of DOPAC in some brain regions (Heffner, Vosmer, & Seiden, 1984)raised an important cautionary point. That is, ICSS-related increases in DAmetabolism may be due to the motoric act of lever pressing. These problems havebeen partially addressed in two ex vivo studies. In the first, increases in tyrosinehydroxylase (TH) activity, as measured by accumulated 3,4-dihydroxyphenylalanine levels, were observed in dopaminergic terminal fieldssuch as the nucleus accumbens, striatum, and olfactory tubercle after leverpressing for stimulation of the VIA (Phillips, Jakubovic, & Fibiger, 1987). Nochanges in TH activity were seen after lever pressing for food on a fixed-ratioschedule that produced operant rates comparable to ICSS of the VIA. Resultsindicated a role for DA in VTA-ICSS independent of operant behavior. A secondstudy incorporated a group of "yoked" animals which received non-contingentelectrical stimulation to the VIA to control for increases in dopaminergic acitivtydue to operant responding. Fibiger, Jakubovic, LePiane, & Phillips (1987) foundcomparable DOPAC and HVA to DA ratios in the ipsilateral nucleus accumbens,striatum, and olfactory tubercle in both self-stimulating and yoked animals.It has been suggested that DOPAC may not be related to neuronal activityand is an unreliable index of DA release (Westerink, Hofsteede, Damsma, and deVries, 1988; ZetterstrOm, Sharp, Collin, & Ungerstedt, 1988). DOPAC, lacking an11active uptake mechanism, is able to diffuse more readily through extracellularspace and may be partly derived from distal tissue. Microdialysis (MD)experiments investigating voltage-dependent release of neurochemicals suggestthat only 50% of basal DOPAC formation is related to neuronal activity (Westerink,Tuntler, Damsma, Rollema, and de Vries, 1987). Further, a major source ofDOPAC comes from intracellular catabolism of newly synthesized or cytoplasmicDA and, therefore, may reflect metabolism of DA prior to release (Butcher,Fairbrother, Kelly, & Arbuthnott, 1988).An obvious problem with ex vivo analyses is relating behavior withneurochemical levels in post-mortem tissue. Also, tissue assays may not be anaccurate reflection of what is happening in the extracellular space, the critical siteof action of transmitters. The recent development of techniques such aselectrochemistry and MD provide a way in which the neurochemistry in theextracellular space can be monitored in vivo.In Vivo ExperimentsMany studies have examined the efflux of DA after MFB stimulation using invivo electrochemistry. This technique measures changes in oxidation currentproportional to the concentration of electroactive species, such as DA, in the vicinityof an electrochemical probe (Blaha & Phillips, 1990). Millar et al. (1985) reportedthat both sine and square wave stimuli were equally effective in releasing DA in thestriatum but only when pulse widths greater than 0.5 msec were used. Manyelectrochemistry studies have used 50 to 75 Hz sine wave stimuli successfully toinduce DA efflux in the dorsal and ventral striatum (Williams & Millar, 1990;Stamford, Kruk, & Millar, 1988, 1986a, b; May & Wightman, 1989; Kuhr, Wightman,& Rebec, 1987; Kuhr, Bigelow, & Wightman, 1986; Stamford, Kruk, Millar, &Wightman, 1984).12Gratton, Hoffer, & Gerhardt (1988) found that stimulation (200-800 pA, 20-100 Hz, 0.1 msec cathodal square wave, 500 msec trains) of the VTA or MFB inanesthetized rats with a previous history of ICSS resulted in increases in theelectrochemical measurement of DA. Further, the ICSS-induced increase in theelectrochemical signal was potentiated by nomifensine. A series ofelectrochemical experiments examining the effect of experimenter-administeredstimulation and self-stimulation of the VTA on DA efflux in the nucleus accumbensin unanesthetized rats provide more convincing support for the role of DA in VTA-stimulation reward (Blaha & Phillips, 1990). Stimulation (10-30 pA, 60 Hz sinewave, 0.2 sec trains) of the VTA caused increases in the DA oxidation current.Blaha & Phillips (1990) emphasized some important correlations. First, there was aclose relationship between the threshold current for ICSS and the threshold forproducing a change in the electrochemical signal in the nucleus accumbens.Second, there was a positve correlation between the current intensity, the ICSSrate, and the DA oxidation signal. Finally, the DA uptake blockers, nomifensine,cocaine, and GBR-12909, facilitated both the ICSS rate and the DA oxidationsignal in a correlative manner.In vivo MD is a widely-used technique that permits the monitoring ofneurotransmitters and their metabolites in the extracellular space of discrete brainregions of freely-moving animals (Ungerstedt, 1991; Benveniste & HOttemeier,1990; DiChiara, 1990; Westerink, Damsma, Rollema, De Vries, & Horn, 1987). Inthis method, a hollow dialysis fiber tube is implanted stereotaxically in a specificbrain area. Low molecular weight compounds diffuse down their concentrationgradients from the extracellular fluid across the dialysis membrane and into aphysiological salt solution that pert uses through the dialysis fiber at a constant rate.The fluid (dialysate) is then collected and assayed using standard analyticalprocedures. The most popular methods of analysis combine compound separation13via high performance liquid chromatography (HPLC) and compound quantificationvia electrochemical (ED) or fluorometric detection.MD has been applied to the study of DA efflux and metabolism afterelectrical brain stimulation (Cenci, Kalen, Mandel, & BjOrklund, 1992; Manley,Kuczenski, Segal, Young, & Groves, 1992; Tepper, Creese, & Schwartz, 1991).Tepper et al. (1991) reported that MFB stimulation (3-100 Hz) activatingantidromically-identified SN neurons could produce changes in striatalextracellular DA as measured by MD. Similarly, Manley et al. (1992) foundincreases in extracellular DA levels in response to stimulation of the MFB usingfrequencies in the range of normal dopaminergic cell firing (4-10 Hz). Both thesestudies observed DA augmentation using MD perfusates containing high calciumconcentrations (2.3-2.4 mM). However, when a lower calcium concentration wasused (1.2 mM), no increases in extracellular DA were observed (Tepper et al.,1991).Using MD, Nakahara, Ozaki, Miura, Mieura, & Nagatsu (1989a) found thatICSS (100 Hz; biphasic square pulses, 0.3 msec duration) of the MFB resulted inincreases in nucleus accumbens levels of the DA metabolites, DOPAC and HVA,and the serotonin metabolite, 5-HIAA, but no significant increases in DA. Thisgroup suggested that the small increases in DA due to ICSS were masked by anICSS-induced facilitation of reuptake of released DA (Nakahara, 1991). In asubsequent experiment, ICSS caused a markedly increased efflux of DA in thenucleus accumbens in rats pretreated with the DA uptake blocker, nomifensine(Nakahara, Ozaki, Vapoor, & Nagatsu, 1989b). These rats were re-implanted witha MD probe three days later and, after nomifensine pretreatment, experimenter-administered stimulation caused a similar increase in extracellular DA (Nakaharaet al., 1989b). These results suggested that elevations in nucleus accumbens DAwere due to the rewarding effects of brain stimulation and not to the motoric act of14lever pressing. Additionally, a recent experiment has shown a preferentialactivation of the mesolimbic DA system by MFB ICSS that resulted in increased DAlevels in the medial frontal cortex and nucleus accumbens, but not the striatum, innomifensine-pretreated rats (Nakahara, Fuchikami, Ozaki, Iwasaki, & Nagatsu,1992). Moreover, Miliaressis, Emond, & Merali (1991) found increases inextracellular DA in the nucleus accumbens as a result of MFB or VTA ICSS whenlonger pulse durations were used. When shorter pulse durations were employed,ICSS failed to produce detectable increases in dialysate DA. To date, MD studieshave yielded equivocal results concerning the role of DA in ICSS.The present series of experiments were designed to investigate whetherrewarding electrical stimulation of the VTA is accompanied by increases inextracellular DA in the nucleus accumbens as measured by MD. Importantmethodological differences from previous ICSS-MD studies included: 1)stimulating electrodes in close proximity to the DA perikarya in the VTA rather thanthe MFB, 2) allowing a sufficient amount of time to elapse after implantation of theMD probe (18-24 hrs) to maximize release-dependent DA, 3) the use ofphysiological levels of calcium (1.3 mM) in the MD perfusate, and 4) the testing ofdrug-free animals (i.e. no DA uptake blockers).Experiment 1 incorporated two important control groups. The inclusion of a"yoked" group, which received stimulation non-contingently, controlled for thepossibility that increases in DA transmission in the nucleus accumbens could bedue to intense operant activity accompanying VTA-ICSS. Both self-stimulating andyoked animals were compared to unstimulated control animals which had a similarhistory of self-stimulation.Experiment 2 further tested the hypothesis of a dopaminergic substrate forbrain-stimulation reward by examining the effect of varying current intensities onVTA-ICSS and DA efflux in the ipsilateral nucleus accumbens as measured by MD.15A positive correlation would be expected if extracellular DA in the nucleusaccumbens mirrors the rewarding value of current intensity and self-stimulationresponse.16GENERAL METHODSSubjectsAdult male Long Evans rats (300-400 g, Charles River Canada, Inc., St.Constance, Québec) were housed individually in Plexiglas boxes (dried corn cobbedding) on a 12:12 h light-dark cycle with free access to food and water.SurgeryAnimals were anesthetized with ketamine hydrochloride (100 mg/kg, i.p.)and xylazine (10 mg/kg, i.p.) prior to stereotaxic surgery. A bipolar stimulatingelectrode (Plastic Products Co.) was inserted into the VTA (coordinates frominteraural zero, flat skull: anterior 3.5 mm, dorsal 1.8 mm, lateral 0.5 mm frommidline) and a MD probe guide cannula (15 mm, 19 g) was implanted above theipsilateral nucleus accumbens (coordinates from Bregma, flat skull: anterior 1.8mm, lateral 1.0 mm, ventral 1.0 mm from dura). Both implants were securedchronically to the skull with six set screws and dental acrylic.ApparatusICSS testing was conducted in a Plexiglas box (24 cm x 25 cm x 30 cm) witha wire mesh floor. A single removeable lever (4 cm x 8 cm) was mounted on onewall 3 cm from the floor. The Plexiglass box was housed in a modified ventilated17and insulated plastic chamber (Colbourn Instruments) which served to shield theanimal from distracting stimuli. A small hole on top of the chamber allowed thepassage of MD tubing and electrical cables from the animal to a dualelectrical/double channel liquid swivel (modified lnstech 375D) located outside thebox.Microdialysis Probe CharacteristicsMD probes (Fig. 1A) used in Experiment 1 were of a concentric designconsisting of a semi-permeable hollow fiber (340 pm o.d., 65 000 M.W. cut-off,Filtral 12, Hospal), a PE50 inlet tubing, a fused silica outlet tubing (75 pm i.d x 150pm o.d.) and a 24 g stainless steel cannula (34 mm). Epoxy (Devcon 2-Ton) wasused to seal joints and plug the dialysis fiber tip. The inlet tubing was connected toa syringe pump (Harvard, model 22). MD probes used in Experiment 2 wereidentical to those used in Experiment 1 except that a long PE10 outlet tubing wasglued over a shortened fused silica tubing (10 cm). The outlet tubing was thenconnected to the second liquid channel of the swivel which led to an automaticinjector (Valco Instruments). Typical in vitro recovery at 21°C and a 1.5 pL/min flowrate was 15% for DA and 11°/0 for the metabolites.A MD probe guide collar (Fig. 1B) secured the MD probe inside the guidecannula (Fig. 1C).Figure 1. Diagram of the microdialysis probe assembly. The microdialysis probe(A) is secured in the microdialysis probe collar (B) by a top-end set screw. Theprobe is then inserted into the guide cannula (C) which has been implanted overthe area of interest. The probe-collar assembly is then secured over the guidecannula by a bottom-end set screw.18Thread for4— Protective CoilAttachmentSet Screws4—i4—BrassC. Guide Cannula4—Stainless SteelTubing (19 ga.)19A. Microdialysis^B. MicrodialysisProbe^ Probe CollarHigh Performance Liquid Chromatography and Electrochemical DetectionMicrodialysate analytes were separated by reverse phase chromatography(Beckman ultrasphere column, ODS 3 pm, 7.5 cm, 4.6 mm i.d.) using a 0.083 Msodium acetate buffer (pH 3.5, 3% methanol). The glassy carbon workingelectrode was set at +0.650 V. The apparatus consisted of a Spectra Physics 8810HPLC pump, a Rheodyne 7125 injector (Experiment 1) or a Valco Instruments 2-Position autoinjector (EC1OW) and Digital Valve Sequence Programmer(Experiment 2), an EGG 400 electrochemical detector, and a Shimadzu CR3Aintegrator.Intracranial Self-Stimulation TrainingOne week after surgery, animals were screened for ICSS behavior in theoperant box connected via leads to the swivel. The stimulation current ranged from6-26 pA (60 Hz sine wave; 200 ms trains). Responsive animals were then testedon an ascending current intensity program (5 min stimulation periods; 2 pA steps)for 3-5 days. Rats (n=23) that maintained stable rates of ICSS (>750 presses/15min) were selected for the MD experiments.Instrumentation Overview20Fig. 2 is a schematic illustration of instruments used in Experiments 1 and 2.21Figure 2. A schematic illustration of the experimental set-up. A perfusion solutionwas pumped through a swivel to an implanted microdialysis probe. In Experiment1, the microdialysis samples were collected in tubes and manually injected into anHPLC-ED system (1). In Experiment 2, samples continued through the swivel intoan autoinjector (2) and were injected into an HPLC-ED system every 15 min. TheHPLC-ED system consisted of an HPLC pump, an injector, a column, anelectrochemical detector, and an integrator. Animals received electrical stimulationeither non-contingently or by pressing a lever mounted in a Plexiglas box. Currentpassed from the stimulator through a swivel to an implanted stimulating electrode.23Statistical AnalysisNeurochemical data, expressed as a percentage of the average of the firstthree baseline samples, were analyzed using analyses of variance (ANOVA) withrepeated measures. A significant interaction in the ANOVA's was followed by post-hoc comparisons using Student t-tests. All tests were performed using a statisticalsoftware package (BMDP Inc.).HistologyAfter the MD experiment, animals were sacrificed with an overdose of chloralhydrate and transcardially perfused with physiological saline (0.9% NaCI indistilled water) and phosphate buffered formalin (4% paraformaldehyde). Brainswere quick-frozen with CO2 and sliced (40 pm sections) using a cryostatmicrotome. Sections were mounted on glass slides (coated with 2% gelatin) andstained with cresyl violet for subsequent examination under a light microscope.EXPERIMENT 1Effects of ICSS or yoked-stimulation of the WA on DA, DOPAC, HVA, and 5-HIAAin the ipsilateral nucleus accumbensExperiment 1 examines whether rewarding electrical stimulation of the VTAproduces any changes in DA transmission in the ipsilateral nucleus accumbens asmeasured by MD. This experiment includes a "yoked" stimulation group as acontrol for the possibility that lever-pressing, rather than brain-stimulation reward,increases DA efflux during ICSS. An unstimulated control group with comparablesurgical and behavioral experience is also included. Important methodologicalconsiderations include 1) stimulating electrodes in close proximity to the DAperikarya in the VTA, 2) MD probes implanted 18-24 hrs prior to behavioral testingto maximize release-dependent DA, 3) the use physiological levels of calcium (1.3mM) in the MD perfusate, and 4) testing of drug-free animals.MethodsAnimals were assigned randomly to three groups: 1) ICSS (n=5), 2) yoked(n=5), and 3) unstimulated controls (n=5). ICSS animals performed a bar pressresponse to receive VTA stimulation. Each yoked animal was paired with a self-stimulating animal exhibiting a similar rate-intensity profile and the two animalswere placed in separate test boxes at the same time. Only the yoked animal of thepair was implanted with a MD probe. No lever was available to yoked animalsduring testing. Although current intensity was the same for both animals, yokedanimals received stimulation at a rate determined by their ICSS partner. Rats in2425the unstimulated control group were connected to a stimulation lead but did notreceive stimulation during testing. Control subjects had a comparable experienceof ICSS prior to the MD experiment.MD probes were inserted 18-24 h prior to the experiment (ventral 7.8 mmfrom dura). Each probe was then connected to the swivel and the animal remainedin the test box overnight with free access to food and water. No lever was presentin the test box overnight. The probe was flushed overnight at 0.15 plimin withperfusate (10 mM sodium phosphate buffer, pH 7.4, 1.3 mM CaCl2, 3.0 mM KCI, 1.0mM MgCl2, and 147 mM NaCI). On the day of the experiment, the flow rate of theperfusate was increased to 1.5 pL/min and dialysate samples were collected atleast one hour later. If an ICSS animal was being tested, the lever was remountedin the test box. Dialysate samples were collected every 15 min in Eppendorfmicrotubes positioned just below the swivel. The samples were injected manuallyonto an HPLC-ED system.Combined neurochemical and stimulation protocols for the ICSS and yokedgroups consisted of: 1) baseline measures of neurotransmitter and metabolites(minimum of 60 min); 2) the first stimulation period (15 min) during which eachanimal either engaged in bar pressing for ICSS or received yoked brain-stimulation (current = 22 pA); 3) a post-stimulation recovery period with no brain-stimulation available for 90 min; and 4) a second stimulation period conducted withidentical stimulation parameters to the first test phase. Dialysate samples werecollected from unstimulated control animals for at least 4.5 h.ResultsVTA-StimulationThe ICSS group averaged 1281 trains of brain-stimulation (range = 1058 to1560) in the first stimulation period and 1568 (range = 999 to 2153) in the secondstimulation period 90 min later. The yoked group received an average of 1821stimulations (range = 1720 to 1977) in the first period and 1987 stimulations (range= 1685 to 2240) in the second. There was no significant difference in the averagenumber of stimulations received between the ICSS and the yoked groups orbetween stimulation periods for each group.Both ICSS and yoked animals exhibited approach and exploratorybehaviors such as forward locomotion, rearing, and sniffing during stimulationperiods.Neurochemical AnalysesMean baseline concentrations of DA, DOPAC, HVA, and 5-HIAA are given inTable 1A.DopamineThere were no significant differences in basal values between the threegroups. The ANOVA revealed that during and following each of the stimulationperiods, DA in both the ICSS and yoked-stimulation groups differed significantlyfrom the unstimulated control group (F1,8 = 10.44; p < 0.01 and F1,8 = 26.43; p <0.01). There were no significant differences in dialysate DA concentrationsbetween the ICSS and yoked control groups over each phase of the experiment.Post-hoc comparisons revealed significant increases in DA relative to unstimulated2627Table 1. Mean basal values for DA, DOPAC, HVA, and 5-HIAA in (A) Experiment1 and (B) Experiment 2. Values are represented as the mean of the averages ofthree baseline samples from each animal. Range values are means of eachbaseline sample across animals. Values are uncorrected for probe recovery.A. EXPERIMENT 1.Analyte Group Mean (nM) Range (nM)Dopamine Control 0.50 ± 0.03 0.49 ± 0.03 to 0.52 ± 0.04ICSS 0.85 ± 0.19 0.81 ± 0.17 to 0.87 ± 0.20Yoked 0.84 ±0.19 0.83 ±0.05 to 0.87 ±0.09DOPAC Control 302.7 ± 74.6 292.1 ± 71.2^to 313.4 ± 77.0ICSS 346.5 ± 66.7 343.8 ± 63.6^to 350.4 ±68.7Yoked 499.0 ± 126.4 486.4 ± 126.8 to 506.0 ± 127.8HVA Control 147.9 ± 25.2 145.9± 23.2 to 149.0 ±25.3ICSS 107.9 ±5.5 105.2 ±5.8^to 110.3 ±7.2Yoked 134.2 ± 28.4 132.0 ± 25.9 to 136.3 ± 29.75-HIAA Control 110.5 ± 18.0 110.0 ± 18.4 to^111.1 ± 18.7ICSS 100.1 ± 15.6 98.2 ± 13.9^to 103.0 ± 18.5Yoked 104.9 ± 11.2 104.2± 11.8 to^106.3± 11.1B. EXPERIMENT 2.Analyte Mean (nM) Range (nM)Dopamine 0.72 ± 0.12 0.71 ± 0.11^to 0.73 ± 0.13DOPAC 675.1 ± 117.2 659.4 ± 108.2 to 686.8 ± 120.6HVA 212.2 ± 23.1 209.5 ±21.1^to 214.3 ±23.15-HIAA 143.9 ± 10.2 142.9 ± 10.0^to 144.8 ± 10.22829controls that peaked during each ICSS period. Fig. 3A shows that in the first ICSSperiod, stimulation of the VTA was accompanied by a 96.5 ± 28.2 % increase inextracellular DA above baseline control values. DA remained elevated above pre-stimulation values for 30 min post-stimulation. The second ICSS period againproduced an increase in extracellular DA of 178.3 ± 48.1 %. In this instance, DAremained significantly elevated for 60 min post-stimulation.Stimulation of the VIA in yoked animals (Fig. 3B) was also associated withincreases in extracellular DA of 93.6 ± 16.7% and 114.8 ± 14.1 % in the first andsecond stimulation periods, respectively. DA remained significantly elevated for upto 75 min after each stimulation period.Dopamine MetabolitesDOPACSelf-stimulation produced maximal increases in extracellular DOPAC of 63.3± 5.0 % and 54.3 ± 14.4% after the first and second stimulation periods,respectively (Fig. 4A). Maximal increases were obtained in the first sample afterthe stimulation period and these values differed significantly from those of theunstimulated control group (F1,8 = 130.78; p < 0.0005). Significant increases wereobserved during the stimulation periods and DOPAC remained elevated for 60 min(first stimulation period) and 30 min (second stimulation period) post-stimulation.Stimulation in the yoked group also resulted in extracellular increases inDOPAC that also reached maximal values (144.5 ± 8.5%) in the first post-stimulation samples (Fig. 4B). The first stimulation period produced a significantincrease in DOPAC lasting 60 min post-stimulation. The second stimulation periodalso produced a significant increase in DOPAC which remained significantlyelevated for 15 min post-stimulation. The maximum30Figure 3. Effect of (A) self-stimulation (ICSS) or (B) yoked-stimulation (Yoked) ofthe ventral tegmental area on extracellular dopamine (DA) in the nucleusaccumbens. Stimulation-induced effects are expressed as percent change relativeto the average of four pre-stimulation baseline measures (100%). Bar graphs(bottom panels) show the number of stimulations received in each 15-minstimulation period. +p<0.10, *p<0.05, "p<0.01, ***p<0.001.B• ICSS--*--- Control._ .——--+--- Yoked.^A^--•--- Control350c 300S 250ca.0e 2004 15001001500I^I^I^I I IIIIIII^IIIII^IIII^1111-11^llllll^0 60^120^180^240^0^60^120^180^240Time (min)32Figure 4. Effect of self-stimulation or yoked-stimulation of the ventral tegmentalarea on extracellular DOPAC and HVA in the nucleus accumbens. Stimulation-induced effects are expressed as percent change relative to the average of fourpre-stimulation baseline measures (100%). The top panels show DOPAC changesin the (A) ICSS and (B) Yoked groups as compared to the unstimulated Controlgroup. The middle panels show HVA changes in the (C) ICSS and (D) Yokedgroups as compared to the unstimulated Control group. Bar graphs (bottompanels) show the number of stimulations received in each 15-min stimulationperiod. +p<0.10, *p<0.05, "p<0.01, ***p<0.001.B—6-- YokedA^--•--- Control•^ICSS--*--- ControlInt.*AM200175150125100 -r^1^1^1 II^1^1^1^1^1^1^1^1^1^1^1^1^1^1^1 I 1^1^1^1^10 60^120^180^240^0 60^120Time (min)DIIIII180^24034increase was 38.1 ± 11.4%. All of these values differed significantly from those ofthe unstimulated control group (F1,8 = 35.25; p < 0.0005).HVASelf-stimulation produced significant increases above baseline (Fig. 4C) inextracellular HVA (63.3 ± 13.5%, first stimulation period and 69.7 ± 23.2%, secondstimulation period). The maximum increases occured 30 min post-stimulation.Significant increases were seen during ICSS in the first period and levelsremained significantly elevated for 60 min post-stimulation. Following the secondstimulation period, a significant increase in HVA was seen only after the stimulationsession and levels remained significantly elevated for 30 min.Yoked-stimulation also resulted in significant increases (F1,8 = 126.89; p <0.0005) in extracellular HVA (Fig. 4D). The first stimulation produced an immediateand significant increase in HVA that persisted for 60 min. The maximal increasewas 60.1 ± 5.6%. The second stimulation period produced an increase in HVA butonly reached significance (p < 0.05) in two samples: 15 and 60 min post-stimulation.Serotonin Metabolite5-HIAAICSS was accompanied by long-lasting increases in extracellular 5-HIAAconcentrations (F1,8 = 38.00; p < 0.001). Significant increases in 5-HIAA were seenduring ICSS periods and these had not returned to pre-stimulation baseline valuesafter 75 min (Fig.5A). Maximal increases were obtained in the samples obtainedimmediately following the stimulation periods. Increases of 32.3 ± 6.0% (firststimulation period) and 42.6 ± 12.5% (second stimulation period) were observed.Figure 5. Effect of (A) self-stimulation (ICSS) or (B) yoked-stimulation (Yoked) ofthe ventral tegmental area on extracellular 5-HIAA in the nucleus accumbens.Stimulation-induced effects are expressed as percent change relative to theaverage of four pre-stimulation baseline measures (100%). Bar graphs (bottompanels) show the number of stimulations received in each 15-min stimulationperiod. -Fp<0.10, *p<0.05, "p<0.01, ***p<0.001.35• ICSS----4,-- Control--+-- Yoked--*--- ControlA BTc 200750co 175.01500......ex 125<-± 1006l 101111^I^I^11111120^180^2401.I ^1ill^1120^180,,,240^0Time (min)37As with ICSS, yoked-stimulation (Fig. 5B) resulted in increases in 5-HIAAthat were persistent and relatively stable (F1,8 = 32.20; p < 0.001). The firststimulation caused an immediate increase in 5-HIAA which lasted for 90 min post-stimulation. This elevated level of 5-HIAA was stable and increases ranged from18.6 ± 8.6% (first stimulation period) to 25.3 ± 4.8% (fourth post-stimulationsample). The second stimulation period caused a further increase in 5-HIAA to amaximum of 37.2 ± 5.2% (first sample post-stimulation). Extracellular 5-HIAAremained significantly different from baseline control values for 90 min followingthe second period of VTA-stimulation, at which point the experiment wasterminated.HistologyHistological examination of brain sections confirmed that MD probes were inthe nucleus accumbens. Placements extended from about 1.2 to 2.2 mm anteriorto Bregma (mode = 1.7 mm) and, based on the deepest point of insertion, theexposed membrane of the MD probe was fully in the nucleus accumbens. Fig. 6shows a representative placement of a MD probe and extent of damage in thenucleus accumbens.The tips of the bipolar electrodes were in the VTA in all animals used in themicrodialysis experiments. Fig. 7 shows electrode tip placements for ICSS andyoked animals in Experiment 1. Control animal electrode tips (Fig. 8) were in thesame areas.38Figure 6. Representative placement of a microdialysis probe (2 mm exposeddialysis membrane) and extent of damage in the right nucleus accumbens.Approximately 1.7 mm anterior to Bregma (flat skull). Coronal section redrawn fromPaxinos & Watson (1982).mmcoco40Figure 7. Location of the tips of bipolar stimulating electrodes for the ICSS(squares) and Yoked (triangles) groups in Experiment 1. All electrodes wereimplanted in the right hemisphere. Yoked placements are drawn on the left side forclarity. Serial coronal sections redrawn from Paxinos and Watson (1982). Thenumbers on the right of each section refer to their position relative to Bregma (mm).ww 8'9-ww E°9-;T.42Figure 8. Location of the tips of bipolar stimulating electrodes for the unstimulatedControl animals in Experiment 1. All electrodes were implanted in the righthemisphere. Coronal sections redrawn from Paxinos and Watson (1982). Thenumbers on the right of each section refer to their position relative to Bregma (mm).ww E"9-WW 817-44DiscussionSelf- or yoked-stimulation of the VTA in drug-free rats was accompanied bysignificant increases in extracellular DA concentrations in the ipsilateral nucleusaccumbens as assessed by MD. Increased concentrations of the DA metabolites,DOPAC and HVA, also were associated with VTA-stimulation. The pattern ofchange of DOPAC and HVA relative to the 15-min stimulation session differs fromDA in that the maximum increases were not observed during this period, but in thefirst and second post-stimulation periods, respectively. Significant increases in theserotonin metabolite, 5-HIAA also were observed following VTA-stimulation.Although the increases in 5-HIAA concentrations coincided with stimulationperiods, the sustained elevation of the metabolite, unlike DA or DOPAC, suggeststhat serotonin activity in the nucleus accumbens may not be associated directlywith brain-stimulation reward.The inclusion of two important control groups in this experiment aided indiscerning neurochemical changes due to rewarding brain-stimulation from thosethat might be attributed to non-specific environmental stimuli and handling (i.e. theunstimulated control group) or from operant responding (i.e. the yoked controlgroup). Aside from an early influential report that non-contingent electricalstimulation of ICSS sites was aversive (Steiner, Beer, & Shaffer, 1969), manystudies have demonstrated that brain-stimulation can be rewarding if administerednon-contingently to animals with a previous history of ICSS (Ettenberg &Duvauchelle, 1988; Tsang & Stutz, 1984; Ettenberg, Laferriere, Milner, & White,1981). In an elegant and convincing experiment, Ettenberg & Duvauchelle (1988)showed that rats developed a conditioned place preference to a locationassociated with non-contingent MFB stimulation at self-produced rates. Since theplace preference technique has been shown to be sensitive to both rewarding and45aversive treatments, it would be difficult to defend the suggestion that non-contingent brain-stimulation is aversive.Therefore, it is reasonable to attribute increased DA efflux in yoked animalsduring VIA-stimulation to the rewarding effects of brain-stimulation. It follows, then,that increased DA efflux in ICSS animals during self-stimulation is due to brain-stimulation reward rather than operant behavior. Both ICSS and yoked animalsengaged in locomotion, rearing, and sniffing during VTA-stimulation. Thepossibility that these behaviors may increase DA release in the nucleusaccumbens still exists.46EXPERIMENT 2Effect of varying current intensity during VTA-1CSS on DA, DOPAC, HVA, AND 5-HIAA in the ipsilateral nucleus accumbensExperiment 2 examined the effects of varying current intensities on DA effluxin the ipsilateral nucleus accumbens as measured by microdialysis. The currentintensities ranged from low intensities, just above ICSS threshold, to highintensities producing optimal ICSS rates. The order of intensities used duringtraining and on a given test day was randomized to minimize any contrast effects.A positive correlation between ICSS responses and the magnitude of theincrease in extracellular DA concentrations in the nucleus accumbens wouldprovide further support for a dopaminergic substrate for VTA-ICSS.MethodsOnce stable rate-intensity profiles were obtained, animals (n=7) were testedfor 6 days on a three-intensity program which would be used in the microdialysisphase of the experiment. The program consisted of three, 15-min stimulationperiods, each at a different intensity (i.e. high, H, medium, M, or low, L, current),spaced 90 min apart. The order of intensities used in the program was randomizedand a specific combination was not repeated over the 6 days. The L current wasthe intensity at which an animal responded at approximately 100-150 presses/ 5min. The H current was the intensity at which the animal's pressing rate reachedan asymptote. The M current was the midpoint value between the L and H current.47As in Experiment 1, each animal was implanted with a microdialysis probeand connected to the swivel and left in the test box overnight with free access tofood and water. No lever was present in the test box overnight. The probe wasflushed overnight at 1.5 pUmin with perfusate.The day of the experiment, the lever was remounted in the test box.Dialysate samples were injected automatically into the HPLC-ED system every 15min. Testing consisted of: 1) baseline measures of neurotransmitter andmetabolites (minimum of 60 min); 2) an ICSS period (15 min) where each animalengaged in bar pressing for VIA stimulation; 3) a post-ICSS recovery period withno brain-stimulation available for 90 min; and 4) a second and third 15 min ICSSperiod, each followed by a 90 min recovery period. A different intensity was usedfor each ICSS period. Again, the order of intensities used on a given test day wasrandomized across animals with at least one subject in each of the following sixsequences: LMH, LHM, MLH, MHL, HLM, HML.ResultsVTA-StimulationThe mean current intensities for the three stimulation conditions were:L=18.0 ±1.7 pA; M=22.0 ± 1.7 pA; and H=26.7 ± 2.0 pA. During ICSS, the meannumber of lever presses per 15 min at each intensity were: L intensity, 502 ± 61; Mintensity, 1133 ± 109; and H intensity, 1629 ± 80. There was a positive correlationbetween the number of lever presses and the current intensity (Pearson'sP=0.0048; r=0.5781).48Neurochemical AnalysesMean baseline levels of DA, DOPAC, HVA, and 5-HIAA are presented inTable 1B.DopamineFig. 9. shows baseline DA levels and increases associated with ICSS at thethree current intensities. The DA level at time = 0 min for each current intensitycorresponded to the fourth baseline sample. Statistical comparisons were madebetween this baseline sample and changes in DA following ICSS. Furthercomparisons made between samples following ICSS and the sample precedingstimulation, although not shown, yielded almost identical results. Self-stimulationat the L intensity resulted in an increase in extracellular DA of 22.0 ± 3.6%(p<0.05). After the stimulation period, there was an immediate return to values thatwere not statistically different from baseline. A greater increase of 48.0 ± 6.3% wasobserved following ICSS at the M intensity and these levels remained significantlyelevated for 15 min following the stimulation period. Self-stimulation at the Hintensity was accompanied by an increase to 185.2 ± 13.7% of pre-stimulationbaseline. Again, the augmentation outlasted the ICSS bout by 15 min (p<0.10). Bythe second post-stimulation sample, DA concentrations were still elevated but wereapproaching baseline values.Dopamine MetabolitesDOPACThe top panel of Fig.10 shows DOPAC concentrations before, during and afterICSS at each of the three stimulation intensities. Although small increases wereseen after ICSS at both the L and M intensities, the only statistically significantchanges were obtained after ICSS at the H intensity. In this case,49Figure 9. Effect of self-stimulation at low (18pA), medium (22pA), and high (27pA)current intensities on extracellular dopamine (DA). Stimulation-induced effects areexpressed as percent change relative to the average of four pre-stimulationbaseline measures (100%). Bar graphs (bottom panels) show the number ofstimulations received in each 15-min stimulation period. +p<0.10, *p<0.05,"p<0.01.Mean Current Intensity22 pA,-,27 pA,-,.18 pA,-,-1-1-r0 01 ^-2000c1 1 1 1 1 1^1^1 1 1 1 1 1^11 1 1 1 1 1 1^0 3- 1500 n.500 5=0.....1^- 1000 -acri45 90 0 45 90 0 45 90Time (min)200175150,4.)t_ 1254c100 II-we51Figure 10. Effect of self-stimulation at low (18pA), medium (22pA), and high (27pA)current intensities on extracellular DOPAC (top panels) and HVA (middle panels).Stimulation-induced effects are expressed as percent change relative to theaverage of four pre-stimulation baseline measures (100%). Bar graphs (bottompanels) show the number of stimulations received in each 15-min stimulationperiod. +p<0.10, *p<0.05, "p<0.01.Mean Current Intensity18 pA^22 pA^27 pAr's ru52175 -150 -125 -100 - Way .?Irkkiii,175 -150 -125 -100 - woe*ft- 2000- 1500I — 1000- 5001^1 1 1 1 1^1 I^1 1 1 1 1^1 I III ..^00 45 90 0 45 90 0 45 90Time (min)-r-r-r053significant increases in DOPAC were seen during the ICSS period and maximumincreases (146.9 ± 12.7%) were obtained in the first post-stimulation sample.Levels remained elevated for 30 min post-stimulation.HVASelf-stimulation resulted in significant increases in HVA at each of the threecurrent intensities (lower panel, Fig. 10). After an ICSS period at the L current,HVA increased by 19.0 ± 2.6% (first post-stimulation sample) and remainedelevated for 45 min post-stimulation. Self-stimulation at the M current elevatedHVA which persisted for 60 min post-stimulation. The maximal increase at thisintensity was 29.1 ± 5.0%. At both L and M intensities, changes in HVA did notreach statistical significance until the sample following the ICSS period. ICSS atthe high current intensity resulted in an immediate increase in HVA which reacheda maximum (154.5 ± 8.6%) in the second sample post-stimulation. After H intensitystimulation, HVA remained significantly elevated for 90 min following the ICSSperiod.Serotonin Metabolite5-HIAAAlthough increased 5-HIAA concentrations were observed following Lintensity ICSS, they were not statistically significant. Significant elevations inextracellular 5-HIAA were observed with both M and H intensities (Fig. 11). Self-stimulation at M intensity was accompanied by a significant increase during theICSS period and in the first and third post-stimulation samples. The maximumincrease in 5-HIAA at M intensity was 31.2 ± 10.2%. Self-stimulation at the Hintensity was followed by a significant increase in 5-HIAA in the first sample post-stimulation (34.9 ± 8.9%) and they remained elevated for 30 min post-stimulation.54Figure 11. Effect of self-stimulation at low (18pA), medium (22pA), and high (27pA)current intensities on extracellular 5-H IAA. Stimulation-induced effects areexpressed as percent change relative to the average of four pre-stimulationbaseline measures (100%). Bar graphs (lower panels) show the number ofstimulations received in each 15-min stimulation period. +p<0.10, *p<0.05,"p<0.01, ***p<0.001.22 pA^27 pA18 pAri. Pilo'TT 1/111111^111111110^0 45 90 0 45 90Cg.—2000 3C—150051000 =co—500 —L(A30 45 90^B.Mean Current IntensitycMEIN• ^175 —coC 13.0 150 -0.... 125 -4`Z. 100ZlbTime (min)56HistologyHistological examination of brain sections confirmed that microdialysisprobes were in the nucleus accumbens. Placements were in the same range ofsections as those in Experiment 1 and extended from about 1.2 to 2.0 mm anteriorto Bregma (mode = 1.7 mm). Based on the deepest point of insertion, the exposedmembrane of the microdialysis probe was fully in the nucleus accumbens.The location of the electrode tips from animals in Experiment 2 are shown in Fig.12.57Figure 12. Location of the tips of bipolar stimulating electrodes for ICSS animals inExperiment 2. All electrodes were implanted in the right hemisphere andplacements were distributed within 0.3 mm of this section. Coronal sectionredrawn from Paxinos and Watson (1982). The number on the right of the sectionrefers to its position relative to Bregma (mm).-4.8 mmcria)59DiscussionAs in Experiment 1, self-stimulation of the VTA was accompanied bysignificant increases in extracellular DA concentrations in the ipsilateral nucleusaccumbens. There was a positive correlation between the magnitude ofextracellular DA and the number of lever presses. Augmented elevations ofextracellular DA at higher intensities is likely due to an increased recruitment ofdopaminergic fibers carrying the reward signal.DA concentrations associated with ICSS remained elevated above pre-stimulation baseline values for extended periods. A comparison of the temporalpatterns of extracellular DA concentrations at the three current intensities suggeststhat the more intense the brain-stimulation, the longer DA values remain elevated.Such sustained increases in DA have also been observed in dialysates from thenucleus accumbens of male rats following a 30-min session of copulation(Damsma, Pfaus, Wenkstern, Phillips, and Fibiger, 1992; Pfaus, Damsma,Nomikos, Wenkstern, Blaha, Phillips, and Fibiger, 1990). DA returned to pre-copulation values within 20-30 min after receptive females were removed from thetest chamber. The hysteresis of nucleus accumbens DA following strongstimulation by either natural stimuli or electrical brain-stimulation may haveimportant implications for the neurochemical functioning of this system and fortheories of the functional correlates of dopaminergic transmission. First, theprolonged increases in extracellular DA may indicate temporary saturation of theDA uptake processes. Second, since this condition occurs during copulation in themale rat, it may have functional significance. For example, it may be a prerequisitefor the activation of immediate-early genes, such as c-fos, whose expression isinduced by both copulation in the male rat (Robertson, Pfaus, Atkinson, Matsumara,Phillips, and Fibiger, 1991) and VTA-ICSS (Fiorino, Robertson, Phillips, Fibiger,60and Swindale, 1992). The large and sustained increase in DA following strongstimuli is also consistent with a parasynaptic (Herkenham, 1987) orneuromodulatory role for extracellular DA.61GENERAL DISCUSSIONSelf-stimulation of VTA was accompanied by significant increases inextracellular DA concentrations in the ipsilateral nucleus accumbens. This result isin general agreement with MD (Nakahara et al., 1992, 1991, 1989a, 1989b;Miliaressis et al., 1991), electrochemistry (Blaha & Phillips, 1990; Phillips et al.,1989), ex vivo analysis of DA/DOPAC ratios (Fibiger et al., 1987), and in vivotyrosine hydroxylase activity experiments (Phillips et al., 1987). Important controlsfor operant responding (i.e. yoked animals) and non-specific environmental stimuli(i.e. unstimulated control animals) strengthen the argument that the observedincreases in extracellular DA after VTA-ICSS were due to rewarding properties ofbrain-stimulation.There was a prolonged elevation of extracellular DA above pre-stimulationbaseline values after VTA-stimulation, sometimes lasting up to 60 min post-stimulation at high current intensities. A sustained elevation in extracellular DAafter VTA-ICSS has also been observed in electrochemistry experiments (Blaha &Phillips, 1990). Whereas the decline in lever press rate occured quickly (i.e. within3 min) after a 5 pA decrement in stimulation current intensity, oxidation currentcorresponding to extracellular DA concentration declined more slowly (i.e. 10 min).As mentioned previously, this phenomenon may have functional significanceincluding a role in parasynaptic transmission which may be facilitated by large andprolonged elevations of DA. Another function may be the regulation of geneexpression via the induction of immediate-early genes such as c-fos. There isevidence that Fos (the protein product of c-fos) and Jun proteins form heterodimerswhich, upon binding, may regulate the expression of selected target genes in thecell (Morgan & Curran, 1990). Cell culture studies suggest that the proenkephalingene may be regulated by Fos and Jun (Sonnenberg, Rauscher, Morgan, and62Curran, 1990). Given that opiate peptides, such as enkephalin, activate DA cells toincrease accumbens-dependent behaviors (Stellar & Rice, 1989), it is possiblethat, following large increases in extracellular DA, a cascade of gene expressionoccurs culminating in the production of substances that can potentiatedopaminergic activity at a different level.The temporal pattern of changes in extracellular DOPAC concentration afterICSS is consistent with the assumption that DOPAC is mainly a product ofintracellular metabolism of recently released DA, recaptured by the presynapticterminal via an uptake mechanism. Regardless, many findings suggest thatDOPAC is a poor index of DA release (Westerink et al., 1988; ZetterstrOm et al.,1988).Evidence for serotonergic involvement in ICSS is equivocal (Stellar & Rice,1989). Experiments 1 and 2 demonstrated that VTA-stimulation was associatedwith increased 5-HIAA concentrations. Unlike DA or DOPAC, however, 5-HIAAconcentrations never returned to pre-stimulation baseline values following thestimulation period. Although neurotransmitter release may not be inferred directlyfrom metabolite concentrations, this finding suggests that if serotonin is involved inVTA ICSS, its role is limited and/or indirect.Results from Experiment 2 concerning the positive correlation betweenextracellular DA concentrations and ICSS responding should be interpreted withcaution. It is assumed that increasing current intensity results in increasedrecruitment of neurons that are activated by stimulation near the tip of the electrode.Increasing extracellular DA concentrations in the nucleus accumbens, such as afteradministration of cocaine or amphetamine, results in increased VTA-ICSS rates.The larger increases in DA concentrations observed after VTA-ICSS at higherintensities may likely reflect a combination of an increased number ofdopaminergic neurons being activated and increased rates of ICSS responding63due to the reinforcing properties of augmented DA concentrations in the nucleusaccumbens. The confound is difficult to avoid. One alternative is to keep thenumber of stimulations constant while varying the current intensity, possibly byemploying a higher fixed-ratio of responding at higher intensities. Anotherapproach is to use varying stimulation frequency while holding current intensityconstant. In this way, the number of neurons activated by stimulation would beheld relatively constant. This approach, however, may yield confounded results forother reasons. The terminals of many VTA neurons contain both DA and theneuropeptides, cholecystokinin (CCK) and neurotensin (Lundberg & HOkfelt,1983). CCK, for example, has excitatory and inhibitory effects on DA release in themedial posterior and anterior nucleus accumbens, respectively (Crawley, 1991). Ithas been suggested that the differential release of classical neurotransmitters,such as DA, and colocalized neuropeptides, such as CCK, may be frequency-coded. That is, low frequency firing may favor the release of classicalneurotransmitters, while high frequency firing may favor peptide release (HOkfelt,Everitt, Holets, Meister, Melander, Schalling, Staines, and Lundberg, 1986). If thisis true, release of peptides during high frequency stimulation may attenuate orfacilitate dopaminergic modulation of reward, depending on the locus of action inthe nucleus accumbens.While other MD studies have provided evidence for and against adopaminergic substrate of brain-stimulation reward, methodological problems inthese experiments raise questions about their results. Although Nakahara et al.(1992, 1991, 1989a, 1989b) observed increased concentrations of DOPAC andHVA in the nucleus accumbens after self-stimulation of the VTA, they foundsignificant increases in DA only in rats pre-treated with the uptake blocker,nomifensine. Some aspects of their methodology may account for this finding.First, their perfusate calcium concentration was 2.3 mM. The physiological64concentration of extracellular calcium of rat striatum has been measured at 1.2 mM(Moghaddam & Bunney, 1989). Perfusate solutions containing high calciumconcentrations have been shown to enhance basal release of DA (Cenci et al.,1992; Moghaddam & Bunney, 1989; Westerink, Tuntler, Damsma, Rollema, & DeVries, 1988) and alter the pharmacological response of DA systems (Timmerman &Westerink, 1991; Moghaddam & Bunney, 1989). One would predict, however, thatthe use of a perfusate high in calcium would facilitate larger increases inneurotransmitter after electrical stimulation. This was not the case. A betterexplanation involves the issue of the latency to begin MD testing after implantationof the probe. Nakahara began dialysing three hours after probe implantation. Ithas been suggested, based upon histological, functional, metabolic, and bloodflow changes after probe insertion, that the optimum time to begin MD is 8 to 48hours post-implantation (Benveniste & I-10ttemeier, 1990). At this time, brain tissueis tetrodotoxin- and calcium-sensitive, both indices of voltage-dependentneurotransmitter release (Benveniste & Hattemeier, 1990). A large, release-independent pool of DA may have masked any increase in extracellular DA due toVTA-ICSS. Further, a recent experiment questioning the validity of using repeatedMD probe insertions (Camp & Robinson, 1992) casts doubt on the integrity of thecomparison made between ICSS rats and rats which received experimenter-administered stimulation.The MD study by Miliaressis et al. (1991) found increases in nucleusaccumbens DA as a result of MFB- or VTA-ICSS only when longer pulse durationsof stimulation were used. First, this group did not mention the calciumconcentration of their MD perfusate. The importance of this element has beenillustrated above. Second, much of their experiment was performed on a smallnumber of animals (four) over two days after the probe was implanted. Dramaticreactions such as edema, hemorrhaging, and gliosis in the tissue adjacent to the65probe have been reported within two days of implantation and the reliability ofmicrodialysis at this point becomes questionable (Benveniste & HOttemeier, 1990).Finally, this group did not include histological verification of electrode placements.Their stereotaxic coordinates from the Paxinos and Watson stereotaxic atlas(1982) appear to place their electrodes somewhere in the cerebellum. Even iftheir electrodes were located in the MFB and VTA, differences between thecharacteristics of brain-stimulation reward at these two sites have been noted andare not trivial (Phillips & Fibiger, 1989). Although Miliaressis et al. 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