Brain neurotransmitter turnover rates during rat intravenous cocaine self-administration

Neuroscience 117 (2003) 461– 475

BRAIN NEUROTRANSMITTER TURNOVER RATES INTRAVENOUS COCAINE SELF-ADMINISTRATION J. E. SMITH,* T. R. KOVES AND C. CO

DURING

RAT

identify heretofore unrecognized targets by characterizing the neuronal systems involved in cocaine self-administration through assessment of the turnover rates of brain biogenic monoamine and amino acid neurotransmitters. Turnover rates were investigated because they represent the rates of utilization of a neurotransmitter which are thought to directly reflect the activity of the subset of neurons that release it. In addition, the procedures developed by the authors allow the assessment of the turnover rates of multiple neurotransmitters in the same small brain region which has permitted the development of testable hypothesis about underlying neuronal circuitry (Smith et al., 1982, 1984; Goeders and Smith, 1993). It has been widely thought that drugs of abuse produce addictive effects by activating brain systems mediating the hedonic substrates for natural stimuli (i.e. food, water and sexual behavior). The demonstration that electrical brain stimulation of distinct brain regions could serve as a reinforcer (Olds and Milner, 1954) indicated that hedonic processes could be mediated by discrete neuronal pathways. Drugs of abuse are thought to produce positive hedonic effects by chemically activating these neuronal systems, a hypothesis supported by data showing abused drugs to lower thresholds for brain-stimulation reinforcement (Olds and Travis, 1960; Kornetsky et al., 1979). The brain systems responsible for drug self-administration have been studied using animal i.v. self-administration methodologies. These studies have been significantly influenced the last two decades by the dopamine (DA) hypothesis of reinforcement (Wise, 1978) which has focused research efforts on a single neuronal system. Although data from pharmacological blockade (Pozuelo and Kerr, 1972), neurotoxin-induced lesion (Roberts et al., 1977, 1980; Lyness et al., 1979) and microdialysis experiments (Pettit and Justice, 1989, 1991; Weiss et al., 1992; Meil et al., 1995) generally support the involvement of DA neurons in the self-administration of drugs that have direct effects on DA systems, particularly DA innervations of the nucleus accumbens (Roberts et al., 1977, 1980; Roberts and Koob, 1982), these data do not support a ubiquitous role for DA in these processes for all abused drugs. For example, neurotoxin-induced lesions of DA innervations of the nucleus accumbens that disrupt amphetamine (Lyness et al., 1979) and cocaine self-administration (Roberts et al., 1977, 1980; Roberts and Koob, 1982), do not alter opiate self-administration (Pettit et al., 1984; Dworkin et al., 1988a). In addition, DA levels in the extracellular fluid from this brain region that are increased during cocaine selfadministration (Pettit and Justice, 1989; Hemby et al., 1997; Bradberry et al., 2000) have been reported to both

Center for the Neurobiological Investigation of Drug Abuse, Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Wake Forest University, Medical Center Boulevard, Winston-Salem, NC 27157-1083, USA

Abstract—The turnover rates of dopamine, norepinephrine, serotonin, aspartate, glutamate and GABA were measured in 27 brain regions of rats self-administering cocaine and in yoked cocaine- and yoked vehicle-infused controls using radioactive pulse-labeling procedures to identify brain neuronal systems underlying self-administration. Changes in the activity of heretofore unrecognized dopamine, norepinephrine, serotonin, glutamate and GABA innervations of the forebrain specific to cocaine self-administration were found. This included innervations of the nucleus accumbens, ventral pallidum, lateral hypothalamus and the anterior and posterior cingulate, entorhinal–subicular and visual cortices. Turnover rates also were calculated using metabolite/neurotransmitter ratios which were inconsistent with the pulse-label technologies indicating that ratio procedures are not accurate measures of neurotransmitter utilization. Results with the pulselabel technique provide evidence of the involvement of neuronal systems in cocaine self-administration not previously known, some of which may have a broader role in brain reinforcement processes for natural reinforcers (i.e. food, water, etc.) since drugs of abuse are thought to produce reinforcing effects by modulating activity in these endogenous systems. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: dopamine, serotonin, norepinephrine, glutamate, GABA, drug reinforcement.

Drug abuse continues to be a major public health problem and, despite increased scientific investigation, the basic biological processes underlying these disorders are still not understood, often limiting the availability of pharmacological adjuncts for treatment programs. Effective pharmacotherapy for drug abuse requires a knowledge of the critical targets responsible for the abusive action that permits development of selective modulators of these key sites. Cocaine is one of the major drugs of abuse without effective pharmacological adjuncts because of a dearth of such effective selective targets. This study was initiated to *Corresponding author. Tel: ⫹1-336-716-8506; fax: ⫹1-336-7168501. E-mail address: [email protected] (J. E. Smith). Abbreviations: Asp, aspartate; DA, dopamine; DOPAC, dihydroxyphenyl acetic acid; EDTA, ethylenediaminetetraacetic acid; Glu, glutamate; HPLC, high-pressure liquid chromatography; HVA, homovanillic acid; NA, noradrenaline; NMDA, N-methyl-D-aspartate; 5-HT, serotonin; 5-HIAA, 5-hydroxyindole acetic acid; v/v, volume/volume.

0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(02)00819-9

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increase (Wise et al., 1995) and not change (Hemby et al., 1995, 1999) during heroin self-administration. The authors propose that the brain processes underlying compulsive drug use, even in this rodent model, involve complex neuronal circuits that include multiple neurotransmitter systems. The assessments of turnover rates of biogenic monoamine and amino acid neurotransmitters in small brain regions of rats intravenously self-administering cocaine and in controls receiving response-independent infusions of cocaine or the vehicle are expected to identify neuronal subsystems and putative circuitry not previously recognized and potentially permit the identification of new targets for the development of pharmacotherapies for the treatment of cocaine abuse.

EXPERIMENTAL PROCEDURES Animals Thirty-nine adult male Fischer strain F-344 90 –150-day-old rats (Harlan, Indianapolis, IN, USA) were used in groups of three littermates with one allowed to intravenously self-administer cocaine and the other two receiving either identical infusions of cocaine or the vehicle on a schedule yoked to the self-administering rat. Littermates from this inbred strain were used to minimize genetic variation that could affect the experimental measures. Each litter was housed together in group cages in a temperature-controlled environment with unlimited access to food and water on a reversed 12-h light/dark cycle (lights on 1700 – 0500 h). Experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication no. 80-23) revised in 1996. Procedures were reviewed and approved by the Wake Forest University School of Medicine animal care and use committee.

Surgical techniques Rats were anesthetized with pentobarbital (50 mg/kg, i.p.; Abbott Laboratories, North Chicago, IL, USA) after pretreatment with atropine methyl nitrate (10 mg/kg, i.p.; Sigma, St Louis, MO, USA) and implanted with venous catheters placed in the right jugular vein using previously described methods (Weeks, 1962, 1972; Pickens and Dougherty, 1972). The catheter (a small piece of polyvinyl-chloride tubing) was inserted into the right posterior facial vein, guided into the right jugular vein until it terminated just outside the right atrium and anchored to muscle in the area of the vein. The other end of the catheter continued subcutaneously to the back where it exited between the scapulae through a polyethylene shoulder harness. Penicillin G procaine (75,000 units, i.m.; Wyeth Laboratories, Philadelphia, PA, USA) was administered in a volume of 0.25 ml before catheterization of the animals. The harness provided a point of attachment for the catheter to a needle-tubing leash that passed out the top of the animal chamber. A leak-proof swivel (Brown et al., 1976) was used to attach the leash to the tubing leading to the infusion pump so that the animals had almost complete freedom of movement. The rats were placed in infusion chambers and allowed 4 days to recover before initiation of experimental procedures. Automatically programmed infusions (0.2 ml delivered over 5.8 s) of heparinized saline (1.7 units/ml) were administered at hourly intervals to maintain functional catheters when animals were not in the daily selfadministration sessions. Patency of catheters was checked periodically by delivering an i.v. infusion of methohexital (10 mg/kg; Eli Lilly, Indianapolis, IN, USA) and determining latency for loss of stability or consciousness which occurs within 1 to 2 s.

Cocaine self-administration Triads of rats with chronic jugular catheters were exposed to three different treatment conditions: self-administration or simultaneous response-independent yoked infusions of either cocaine or vehicle (heparinized-saline) in operant conditioning chambers (Med Associates, St. Albans, VT, USA). One rat in each triad was trained to intravenously self-administer cocaine (0.33 mg/infusion delivered over 5.8 s) on a fixed ratio 2 schedule during daily sessions in operant chambers containing a house light, session light, a stimulus light, response lever, tone source, glass food-pellet dispenser tube and a drinking tube providing unlimited access to water. Another littermate in each triad received simultaneous identical response-independent infusions of cocaine and a third littermate received simultaneous infusions of the vehicle both on a schedule yoked to infusions taken by the self-administering rat. Triads were accommodated to the conditioning chambers by being placed inside for half-hour periods the 3 days preceding catheter implantation. The operant chambers for each triad were housed in the same sound-attenuated cubicle. Daily self-administration sessions that terminated after 6 h or 60 infusions were conducted 7 days per week with the response requirement initially being one lever press which was rapidly increased to two over the first several sessions. Infusions were paired with the onset of a 20-s tone and light compound stimulus and by retraction of the lever for a 20-s timeout period. The self-administering rats were provided 24 one-gram food pellets (Bioserve, Frenchtown, NJ, USA) each 24-h period (an average amount of food consumed during unrestricted access) and the littermates receiving yoked infusions were restricted to the number of food pellets consumed by the self-administering rat to which they were yoked. To control for the anorectic effects of cocaine, the number of food pellets consumed by the self-administering rat was recorded at 0800 and 1700 h each day and that number of pellets was placed in the tubes located in the chambers of the rats receiving yoked infusions.

Pulse-label procedure After 30 days of stable self-administration, each triad was pulse labeled with radioactive precursors for the biogenic monoamine and amino acid putative neurotransmitters 1 h into the self-administration session. Fifty microliters of saline containing 0.4 mCi of 14 D-glucose [U- C] (specific activity 273 mCi per mmol; I.C.N, Irvine, CA, USA), 1.0 mCi of L-[G-3H] tryptophan (specific activity 9.9 Ci per mmol; Amersham, Arlington Heights) and 2.0 mCi L-[ 2,6-3H-tyrosine (specific activity 53 Ci per mmol; Amersham) were administered 60 (N⫽7 triads) or 90 (N⫽6 triads) min prior to killing through the jugular catheters. These time points were chosen because they have been demonstrated to be on the log-linear portion of the decay in radioactivity curve for DA, noradrenaline (NA), serotonin (5-HT) (Co et al., 1982), aspartate (Asp), glutamate (Glu) and GABA (Freeman et al., 1983).

Tissue preparation Animals were killed by immersion in liquid nitrogen until totally frozen (5 min). The heads were separated and allowed to warm to ⫺20 °C and the brains removed. The brains were sectioned in the coronal plane in a cryostat at ⫺18 °C into 750-␮m serial sections. The brain areas of interest (prefrontal, pyriform, motor, somatosensory, anterior cingulate, posterior cingulate, entorhinal-subicular, visual and temporal-auditory cortices, olfactory tubercle, nucleus accumbens, caudate nucleus- putamen, diagonal band preoptic region, ventral pallidum-stria terminalis, amygdala, septum, globus pallidus, hippocampus, substantia nigra, ventral tegmental area, medial hypothalamus, lateral hypothalamus, medial thalamus, lateral thalamus, raphe nuclei, superior and inferior colliculus and brain stem) were microdissected with the aid of a stereomi-

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croscope and the dissected tissue samples stored at ⫺80 °C until analyzed. The frozen tissue samples were individually pulverized at ⫺20 °C in liquid nitrogen with a stainless steel mortar cooled on dry ice. Frozen tissue powder from each sample was transferred to two tubes, one for extraction of the biogenic monoamines and the other for amino acids. Samples that were less than 20 mg were used for measurement of either the amino acids or biogenic amines only.

Freeman et al., 1983; Smith et al., 1982; Dworkin et al., 1995) with the assumption that radiolabel was disappearing from a single open pool (Zilversmit, 1960) since there is no acceptable method for determining CNS intraneuronal compartmentation in vivo. Turnover rates⫽k⫻content where the apparent fractional rate constant (k) was calculated as:

Neurochemical procedures

and t ⁄ was extrapolated from a semilogarithmic plot of the specific radioactivities (d.p.m./pmol or d.p.m./nmol) obtained at the two pulse times on the linear portion of the decay in the radioactivity curve for each neurotransmitter. The apparent fractional rate constants (k) and turnover rates were determined by comparing each animal at the 60-min pulse time with all six animals at the 90-min pulse time. A mean of these measures was calculated and used to represent one turnover measure. Thus, for each of the seven animals in each of the three treatment conditions at the short pulse time, up to seven turnover rates were calculated and these values for each of the three treatment conditions were then used to determine significance of differences in turnover rates. This procedure was used to obtain an accurate error estimate that would represent both the variation in content measurements as well as in specific radioactivities. The turnover rate was expressed as pmol mg protein⫺1 h⫺1 (DA, NA and 5-HT) or nmol mg protein⫺1 h⫺1 (Asp, Glu and GABA) and was the product of each rate constant (h-1) and each content value (pmol mg protein-1 or nmol mg protein-1). Negative values that resulted when the specific radioactivity at the short pulse point was lower than the long pulse point were not included in the calculations.

Biogenic monoamines. Biogenic monoamines and metabolites were extracted from 15 to 50 mg of pulverized tissue powder with 0.4-ml ice-cold 1-N formic acid/acetone (volume/volume [v/v]: 15/85) and lipids removed with a heptane/chloroform wash (v/v: 8/1). 3,4-Dihydroxybenzylamine was added to the tissue powder/ extraction buffer slurry for each sample as an internal standard to correct for recovery. The aqueous layer was taken to dryness under N2 and stored at ⫺20 °C until analyzed. The content and specific radioactivity of DA, NA and 5-HT were concurrently measured with high-pressure liquid chromatography (HPLC) and electrochemical detection and the peaks collected and radioactivity determined with liquid scintillation spectrometry (Co et al., 1982; Dworkin et al., 1995). The content of metabolites (dihydroxyphenyl acetic acid [DOPAC], 5-hydroxyindole acetic acid [5-HIAA] and homovanillic acid [HVA]) were also measured. The samples were reconstituted in the mobile phase (0.05-M citrate-phosphate, pH 3.7, containing 0.4-mM sodium octylsulfate [Fisher Scientific] and 10% methanol) and injected into a C18 reverse-phase column (0.46⫻25 cm, 5 ␮m). The biogenic monoamines and metabolites were eluted over 20 min with the isocratic mobile phase at a 1.0 ml/min flow rate. Retention times in minutes were: NA, 4.7; 3,4dihydroxybenzylamine, 6.6; DOPAC, 7.0; DA, 9.3; 5-HIAA, 10.0; HVA, 16.7; and 5-HT, 20.2. Individual peaks were collected and radioactivity was determined using liquid scintillation spectrometer. Proteins were measured in the pellets (Lowry et al., 1951), content (pmol/mg protein) determined from the internal standards after correction for recovery and the specific radioactivity (d.p.m./ pmole) calculated for each sample. Amino acids. The amino acid neurotransmitters were assayed for content and specific radioactivity using a modification (Dworkin et al., 1995) of a previously reported procedure (Jones and Gilligan, 1983) with HPLC and fluorescence detection. Amino acids were extracted from 10 to 15 mg of frozen tissue powder with 0.4 ml 4 °C methanol and homoserine added as an internal standard. The extracts were dried at 37 °C under a stream of dry nitrogen and stored at ⫺20 °C until assay. The samples were reconstituted in methanol and reacted with O-pthaldialdehyde reagent (100 mg O-pthaldialdehyde in 0.5 ml methanol, 100 ␮l 2-mercaptoethanol in 1.9 ml of 0.4-M borate, pH 9.5) for 2 min and injected into a gradient HPLC system (Gilson Model 201) using an autoinjector (Gilson 401 and 231). A C18 reverse-phase column (0.46⫻15 cm, 5 ␮m) was used with fluorometric detection (Gilson 121) (excitation 305–395 nm, emission 430 – 470 nm). The mobile phase was 0.1-M sodium acetate, pH 6.2, containing 0.1-mM EDTA and increasing concentrations of methanol (15–50%) with a 1.3 ml/min flow rate. Retention times in minutes were: Asp, 2.4; Glu, 4.0; GABA, 20.4. Individual amino acid peaks were collected with a fraction collector (Gilson Model 202 with controller) and the radioactivity in each was determined with liquid scintillation spectrometry. Proteins were measured in the pellets (Lowry et al., 1951), content (nmol/mg protein) was determined from the internal standards after correction for recovery and the specific radioactivity (d.p.m./nmol) calculated for each sample.

Turnover rate calculation Radioactive pulse-label procedure. Turnover rates were determined with previously reported methods (Co et al., 1982;

k ⫽ ln2/t1/2 12

Metabolite: neurotransmitter content ratios for DA and 5-HT. Turnover rates for DA and 5-HT were also calculated from DOPAC/DA, (DOPAC⫹HVA)/DA and 5-HIAA/5-HT ratios for direct comparisons with the pulse-label technique since these are often used as a measure of turnover (Bailey et al., 2000; Frink et al., 1996; Takeo et al., 1997).

Statistical analysis The individual turnover rates calculated as outlined above were then assessed for significant differences by comparing the cocaine self-administering animals with the yoked cocaine-infused controls (designated as the cocaine self-administration effect) and the cocaine-yoked infused controls with the yoked vehicle-infused controls (designated as the cocaine effect). Student’s t-tests were used to test these two differences between three means without correction for multiple comparisons within a given brain region because of the assumption that turnover rates of each neurotransmitter are an independent measure and because there is a dearth of evidence on the interdependence of multiple neuronal systems within a given brain region.

RESULTS I.v. cocaine self-administration Animals acquired i.v. cocaine self-administration within the first five sessions with the response requirement raised to the terminal fixed ratio 2 value within the first several days and stable baselines and patterns of drug intake were obtained by the tenth session. The number of sessions of exposure prior to the pulse-labeling session was 36.0⫾2.5 days for all groups (all values presented are means⫾ standard deviations unless otherwise specified). The total drug intake for the 13 triads was 51.6⫾6.7 infusions per session that corresponds to 17.0⫾2.2 mg/day. The interinfusion intervals did not differ for the two pulse-label time

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Fig. 1. Significant changes in the turnover rates of dopamine in brain regions of i.v. cocaine self-administering, yoked infused-cocaine and yoked infused-vehicle groups. Turnover rates in the nucleus accumbens were 10 times higher (left y axis) than other brain regions (right y axis). Significant differences found between the self-administering rat and the yoked cocaine-infused animals were considered to be a “self-administration effect” (significance symbol positioned over the self-administration group data) while significant differences between the yoked vehicle-infused group and the yoked cocaine group (when the self-administering group was of the same magnitude and not significantly different from the yoked cocaine-infused group data) were considered to be a “cocaine effect” (significance symbol positioned over the yoked cocaine-infused group data). The significance of differences between means assessed with Student’s t-test were: *⫽P⬍0.05; †⫽P⬍0.01; ‡⫽P⬍0.001 for an n⫽13 (seven at the 60-min pulse time and six at the 90-min pulse time per treatment group).

groups (60 min [6.6⫾1.2 min] or 90 min [6.1⫾1.6 min]) even though the total number of infusions differed during the pulse-label interval (60 min [10.7⫾2.3] and 90 min [15.5⫾4.0]) since the 90-min pulse interval had an additional 30 min of access to self-administration. Food intake Nineteen to twenty-two 1-g food pellets were generally consumed each 24-h period. The average daily intake for the three treatment conditions during the 36-day self-administration period were: cocaine self-administration, 18.5⫾0.9; yoked cocaine infused 19.7⫾2.2; yoked vehicle infused, 22.0⫾1.2. Yoking food available to the cocaine self-administering rat resulted in similar daily caloric intake and body weights of the littermates within each triad without restricting the intake of the vehicle-infused animals. Neurotransmitter turnover rates: pulse-label method The changes in neurotransmitter turnover rates observed (Figs. 1–7) were generally of three types: (1) those that were the result of cocaine and seen in both the selfadministering and the yoked cocaine-infused groups (cocaine effect); (2) those that were the result of the ability to self-administer and seen only in the self-administering group (self-administration effect); and (3) those that were affected by the cocaine and seen in the yoked cocaine group but were reversed by the ability to self-administer (reversal effect).

Limbic regions. Eight changes in turnover specific to self-administration were seen in limbic regions that included four increases and four decreases. The nucleus accumbens which has been implicated in drug self-administration had three changes that included increases in DA (Fig. 1) and decreases in Glu (Fig. 5) and GABA (Fig. 4) turnover. The ventral pallidum also showed an increase in DA and a decrease in Glu turnover while the medial hypothalamus had a decrease in 5-HT (Fig. 2) and the lateral hypothalamus an increase in DA. DA turnover also was increased in the septum. Five changes in turnover were seen in limbic regions resulting from cocaine alone (seen in both groups receiving cocaine) that included three increases and two decreases. Increases in DA turnover were seen in the amygdala and in NA (Fig. 3) in the lateral and medial hypothalamus, while decreases were seen in 5-HT in the ventral pallidum, and NA in the septum. One reversal effect was observed in 5-HT in the hippocampus (Fig. 7). Diencephalon. One turnover rate change was found in the diencephalon that was specific to self-administration: a decrease in NA in the lateral thalamus. Two changes were a direct result of cocaine alone that included increases in 5-HT in the lateral thalamus and in GABA in the globus pallidus. Two reversals were observed in the diencephalon that included DA in the diagonal band-preoptic nucleus region and 5-HT in the caudate nucleus putamen.

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Fig. 2. Significant changes in the turnover rates of serotonin in brain regions of i.v. cocaine self-administering, yoked infused-cocaine and yoked infused-vehicle groups. The turnover rates of serotonin were approximately four times higher in the lateral thalamus (right y axis) than in the medial thalamus, somatosensory cortex, substantia nigra and ventral pallidum (left y axis). Significant differences found between the self-administering rat and the yoked cocaine-infused animals were considered to be a “self-administration effect” (significance symbol positioned over the self-administration group data) while significant differences between the yoked vehicle-infused group and the yoked cocaine group (when the self-administering group was of the same magnitude and not significantly different from the yoked cocaine-infused group) were considered to be a “cocaine effect” (significance symbol positioned over the yoked cocaine-infused group data). The significance of differences between means assessed with Student’s t-test were: *⫽P⬍0.05; †⫽P⬍0.01 for an n⫽13 (seven at the 60-min pulse time and six at the 90-min pulse time per treatment group).

Cerebral cortex. Seven turnover rate changes were seen in the cerebral cortex specific to the self-administering rats (all decreases). In limbic cortical regions these included decreases in Glu in the posterior cingulate cortex, GABA in the anterior cingulate, in Glu and Asp (Fig. 6) in the entorhinal subicular cortex. In addition, there were decreases in NA and GABA turnover in the somatosensory cortex and in Glu in the visual cortex. Two decreases in turnover resulted from exposure to cocaine alone that included DA and 5-HT in the somatosensory cortex. Four changes in turnover resulted from reversals that included DA in the motor, visual and entorhinal subicular cortices and GABA in the posterior cingulate cortex.

lateral hypothalamus and increases in the caudate nucleus–putamen and somatosensory cortex) (Table 1) and 15 decreases in 5-HT turnover rates (prefrontal, pyriform, motor, somatosensory, entorhinal-subicular and visual cortices, olfactory tubercle, diagonal band–preoptic area, septum, hippocampus, medial and lateral hypothalamus, medial and lateral thalamus and the brain stem) were identified with this technique (Table 2). All were drug-effect changes seen in both groups receiving cocaine except for one that would be classified as a reversal (DA in the medial hypothalamus).

Brain stem nuclei. Five decreases in turnover were seen in the brain stem specific to self-administration that included decreases in 5-HT in the substantia nigra, Glu in the raphe nuclei and DA, Glu and Asp in the remaining brain stem. One change in the brain stem occurred that was the result of exposure to cocaine, a decrease in DA turnover in the substantia nigra.

Neurotransmitter turnover-rate measurements in cocaine self-administering rats identified sub-populations of DA-, NA-, 5-HT-, GABA- and Glu-releasing neurons that have not been previously recognized as involved in the brain processes that underlie these complex behaviors. These include DA innervations of the lateral hypothalamus, ventral pallidum–stria terminalis and septum, Glu innervations of the nucleus accumbens and ventral pallidum–stria terminalis likely originating in the prefrontal and frontal cortex and 5-HT innervations of the medial hypothalamus (Fig. 8). Selective changes were found also in the cerebral cortex

Neurotransmitter turnover rates: DOPAC/DA and 5-HIAA/5-HT ratio. Five changes in DA turnover rates (decreases in the diagonal band–preoptic area, medial and

DISCUSSION

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Fig. 3. Significant changes in the turnover rates of noradrenaline in brain regions of i.v. cocaine self-administering, yoked infused-cocaine and yoked infused-vehicle groups. Significant differences found between the self-administering rat and the yoked cocaine-infused animals were considered to be a “self-administration effect” (significance symbol positioned over the self-administration group ddata) while significant differences between the yoked vehicle-infused group and the yoked cocaine group (when the self-administering group was of the same magnitude and not significantly different from the yoked cocaine-infused group) were considered to be a “cocaine effect” (significance symbol positioned over the yoked cocaine-infused group data). The significance of differences between means assessed with Student’s t-test were: *⫽P⬍0.05; †⫽P⬍0.01 for an n⫽13 (seven at the 60-min pulse time and six at the 90-min pulse time per treatment group).

that have not been recognized previously that include noradrenergic and GABA innervations of the somatosensory and anterior cingulate cortices and Glu innervations of the posterior cingulate, entorhinal and visual cortices.

Biogenic monoamine neurotransmitter involvement The significant changes in the turnover rates of DA included increases in DA turnover in the nucleus accum-

Fig. 4. Significant changes in the turnover rates of GABA in brain regions of i.v. cocaine self-administering, yoked infused-cocaine and yoked infused-vehicle groups. Significant differences found between the self-administering rat and the yoked cocaine-infused animals were considered to be a “self-administration effect” (significance symbol positioned over the self-administration group data) while significant differences between the yoked vehicle-infused group and the yoked cocaine group (when the self-administering group was of the same magnitude and not significantly different from the yoked cocaine-infused group) were considered to be a “cocaine effect” (significance symbol positioned over the yoked cocaine-infused group data). The significance of differences between means assessed with Student’s t-test were: *⫽P⬍0.05; †⫽P⬍0.01; ‡⫽P⬍0.001 for an n⫽13 (seven at the 60-min pulse time and six at the 90-min pulse time per treatment group).

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Fig. 5. Significant changes in the turnover rates of glutamate in brain regions of i.v. cocaine self-administering, yoked infused-cocaine and yoked infused-vehicle groups. Significant differences found between the self-administering rat and the yoked cocaine-infused animals were considered to be a “self-administration effect” (significance symbol positioned over the self-administration group data). The significance of differences between means assessed with Student’s t-test were: *⫽P⬍0.05; †⫽P⬍0.01 for an n⫽13 (seven at the 60-min pulse time and six at the 90-min pulse time per treatment group).

bens, ventral pallidum, septum, lateral hypothalamus and the brain stem. It is not surprising that increases in DA turnover were seen in the nucleus accumbens since it has long been thought that these DA innervations have a major role in cocaine self-administration (Roberts et al., 1977,

1980; Roberts and Koob, 1982; Pettit et al., 1984). The elevated extracellular fluid levels of DA in the nucleus accumbens shown in rats during cocaine self-administration are consistent with the increased turnover observed here (Pettit and Justice, 1989; Hemby et al., 1997). The

Fig. 6. Significant changes in the turnover rates of aspartate in brain regions of i.v. cocaine self-administering, yoked infused-cocaine and yoked infused-vehicle groups. Significant differences found between the self-administering rat and the yoked cocaine-infused animals were considered to be a “self-administration effect” (significance symbol positioned over the self-administration group data). The significance of differences between means assessed with Student’s t-test were: †⫽P⬍0.01 for an n⫽13 (seven at the 60-min pulse time and six at the 90-min pulse time per treatment group).

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Fig. 7. Significant reversals in the turnover rates of dopamine, GABA, and serotonin in brain regions of i.v. cocaine self-administering, yoked infused-cocaine and yoked infused-vehicle groups. Cocaine alone (yoked cocaine-infused versus yoked vehicle-infused groups) produced a significant increase or decrease in turnover that was reversed by the opportunity to self-administer the drug (yoked cocaine-infused versus the cocaine self-administering groups). Significance symbol for a self-administration effect are positioned over the self-administration group data and for the cocaine effect over the yoked cocaine-infused group data. The significance of differences between means assessed with Student’s t-test were: *⫽P⬍0.05; †⫽P⬍0.01; ‡⫽P⬍0.001 for an n⫽13 (seven at the 60-min pulse time and six at the 90-min pulse time per treatment group).

actions of cocaine in the nucleus accumbens could be through a DA inhibition of Glu inputs from the cortex which would be consistent with the increased turnover rates of DA and decreased turnover rates of Glu seen here. This is supported by data showing a decrease in Glu-evoked firing in this region in rats with a history of self-administration (White et al., 1995). DA innervations of the lateral hypothalamus and ventral pallidum have not been heretofore identified as important in this behavior. The increase in DA in the ventral pallidum is consistent with the effects of 6-hydroxydopamine lesions of this region which disrupt cocaine place preference conditioning (Gong et al., 1997) and with recent data from this laboratory showing increased extracellular fluid levels of DA during cocaine self-administration (Sizemore et al., 2000). In addition, elevated extracellular fluid levels of DA in the lateral hypothalamus have also been observed during cocaine selfadministration (unpublished observations from this laboratory). The increases in DA turnover during self-administration in the lateral hypothalamus may represent a neuronal interface for reinforcement systems with behaviorally relevant hypothalamic nuclei, while the increase in the ventral pallidum–stria terminalis indicates the involvement of the behaviorally relevant extended amygdala. The decreases in 5-HT turnover in the medial hypothalamus and substantia nigra specific to self-administration suggest a release from tonic serotonergic inhibition of behaviorally relevant hypothalamic nuclei and striatal motor systems. However, pharmacological blockade experiments have shown systemic administration of antagonists for 5-HT receptor subtypes (5-HT1A, 5-HT1C, 5-HT2 and

5-HT3) either do not alter i.v. cocaine self-administration (Peltier and Schenk, 1991; Lacosta and Roberts, 1993; Peltier et al., 1994) or do so in a non-specific manner (Peltier and Schenk, 1993). The non-specific effect would be consistent with a modulation of the release of inhibition of the substantia nigra ascending DA fibers that innervate the caudate-putamen. However, enhancement of serotonergic tone by antagonism of 5-HT reuptake with fluoxetine (Richardson and Roberts, 1991) or with administration of the precursor L-tryptophan (McGregor et al., 1993) reduced break points in rats on progressive-ratio schedules of self-administration. It has recently been shown that administration of a 5-HT1B/1A receptor agonist facilitated the reinforcing properties of cocaine (Parsons et al., 1998) and potentiated the cocaine-induced increases in DA in the nucleus accumbens (Parsons et al., 1999). Thus, the elevation of DA in the nucleus accumbens and increased turnover could partially result from increased synaptic levels of 5-HT that potentiate the actions of cocaine on DA. Furthermore, 5,7-dihydroxytryptamine-induced lesions of 5-HT innervations of the nucleus accumbens appear to either increase the activating effects of cocaine on lever pressing and/or increase the reinforcing efficacy of i.v. cocaine administration since higher break points were seen in rats responding on a progressive-ratio schedule (Loh and Roberts, 1990). These apparent differences in the role of 5-HT in cocaine self-administration clearly warrant further investigation. Decreases in NA turnover seen in the lateral thalamus and somatosensory cortex also await further characterization.

Table 1. Turnover rates calculated from metabolite/neurotransmitter ratios for dopamine in brain regions of triads of rats either intravenously self-administering cocaine or receiving yoked infusions of cocaine or vehicle Content (pmol/mg protein) DOPAC

Somatosensory cortex YS 3.7⫾0.2 YCa 3.7⫾0.2 SA 3.8⫾0.2 Caudate putamen YR 118.1⫾7.6 YC 131.6⫾6.2 SA 120.0⫾4.2 Medical hypothalamus YS 4.2⫾0.2 YC 4.6⫾0.2 SA 4.8⫾0.2 Lateral hypothalamus YS 4.9⫾0.3 YC 4.3⫾0.2 SAa 4.2⫾0.2 Diagonal band pre-optic YS 15.1⫾1.3 YCa 13.1⫾1.2 SAa 12.2⫾1.1 Nucleus accumbens YS 116.0⫾6.4 YC 113.1⫾6.9 SAa 105.0⫾3.1 Ventral pallidum, stria terminalis YS 27.6⫾1.8 YC 24.0⫾2.3 SAa 21.7⫾1.3 Septum YS 14.1⫾0.9 YC 12.7⫾0.9 SAa 12.4⫾0.7 Brain stem YS 3.3⫾0.2 YC 2.6⫾0.2* SAa 2.9⫾0.2 Amygdala YS Outside YCa Measurement SA Limitation Substantia nigra YS Outside YCa Measurement SA Limitation Motor cortex YS 3.8⫾0.2 YCa 4.0⫾0.2 SAa 4.1⫾0.2 Entorhinal subicular cortex YS Outside YCa Measurement SAa Limitation Visual cortex YS Outside YCa Measurement SAa Limitation

Turnover rates HVA

DA

DOPAC

DOPAC⫹HVA

DA

DA

3.8⫾0.2 5.8⫾0.5* 5.9⫾0.4

9.1⫾0.6 7.7⫾0.5 7.8⫾0.6

0.42⫾0.03 0.50⫾0.03 0.52⫾0.04

0.86⫾0.06 1.25⫾0.07* 1.32⫾0.10

32.0⫾2.1 46.9⫾2.0* 47.3⫾2.1

816.3⫾32.1 713.6⫾21.7† 731.1⫾32.21

0.15⫾0.01 0.20⫾0.02 0.17⫾0.01

0.20⫾0.01 0.26⫾0.02* 0.24⫾0.01

17.2⫾1.0 21.9⫾0.9‡ 20.0⫾0.8

0.25⫾0.01 0.21⫾0.01* 0.24⫾0.01*

19.0⫾1.0 22.6⫾1.8 19.6⫾0.9

0.26⫾0.01 0.20⫾0.01* 0.22⫾0.01

93.7⫾10.8 99.0⫾11.3 100.2⫾11.1

0.18⫾0.01 0.15⫾0.01* 0.13⫾0.01

845.3⫾42.9 801.3⫾17.2 849.0⫾42.4

0.14⫾0.01 0.14⫾0.01 0.13⫾0.01

149.1⫾16.2 151.4⫾16.7 164.0⫾17.8

0.20⫾0.01 0.17⫾0.01 0.15⫾0.02

5.1⫾0.8 5.5⫾0.6 6.3⫾0.3

68.2⫾4.7 71.9⫾4.7 78.4⫾5.4

0.21⫾0.01 0.18⫾0.02 0.16⫾0.01

0.29⫾0.02 0.28⫾0.03 0.26⫾0.02

2.1⫾0.1 2.1⫾0.2 2.7⫾0.2

12.4⫾0.7 9.7⫾0.8 9.9⫾0.8

0.26⫾0.01 0.28⫾0.02 0.31⫾0.03

0.44⫾0.02 0.51⫾0.05 0.59⫾0.05

0.35⫾0.02 0.40⫾0.03 0.37⫾0.05

0.72⫾0.05 0.87⫾0.07 0.83⫾0.12

36.4⫾1.9 43.9⫾2.7 43.3⫾2.0

0.18⫾0.01 0.20⫾0.01 0.18⫾0.01

25.1⫾1.1 29.7⫾1.5† 26.9⫾1.0 49.3⫾5.3 49.4⫾4.1 58.3⫾4.3 3.6⫾0.2 4.7⫾0.4* 4.8⫾0.2

11.6⫾1.1 10.6⫾0.8 13.0⫾1.7 3.3⫾0.3 3.7⫾0.2 3.6⫾0.3 15.5⫾1.3 19.3⫾1.9 15.5⫾1.0

DOPAC indicates dihydroxyphenyl acetic acid; HVA, homovanillic acid; DA, dopamine; YS, yoked saline infused; YC, yoked cocaine infused; and SA, self-administration. a Indicates changes observed in the turnover rates using the radioactive pulse labeling procedure. * Significance difference measured by one-way analysis of variance followed by Student-Newman-Keuls method of analysis. Values are mean⫾S.E.M.

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Table 2. Turnover rates calculated from metabolite/neurotransmitter ratios for serotonin in brain regions of triads of rats either intravenously self-administering cocaine or receiving voked infusions of cocaine or vehicle Content (pmol/mg protein)

Turnover rates

5-HIAA

5-HIAA

5-HT

5-HT Prefrontal cortex YS 18.0⫾1.0 YC 13.2⫾0.9 SA 13.1⫾0.8 Pyriform cortex YS 11.3⫾0.5 YC 8.3⫾0.8* SA 9.1⫾0.4 Motor cortex YS 15.7⫾1.0 YC 11.3⫾0.8* SA 10.8⫾0.6 Somatosensory cortex YS 15.3⫾0.6 YCa 12.2⫾0.7* SA 11.4⫾0.6 Entorhinal subicular cortex YS 15.9⫾0.9 YC 10.8⫾0.5* SA 9.7⫾0.8 Visual cortex YS 18.5⫾1.2 YC 13.6⫾1.2* SA 11.5⫾0.7 Olfactory tubercle YS 21.1⫾1.2 YC 16.7⫾1.0* SA 17.3⫾1.0 Diagonal band, pre-optic YS 19.3⫾0.9 YC 15.6⫾1.1* SA 15.4⫾1.0 Septum YS 16.2⫾0.7 YC 12.9⫾0.6* SA 12.6⫾0.8 Hippocampus YS 28.4⫾1.6 YCa 20.1⫾1.3* SAa 21.6⫾1.2 Medical hypothalamus YS 23.5⫾1.4 YC 18.3⫾1.1* SAa 17.9⫾1.0 Lateral hypothalamus YS 31.6⫾1.5 YCa 25.5⫾1.4* SA 24.7⫾1.3 Medial thalamus YS 31.1⫾2.0 YC 25.4⫾1.5* SA 24.8⫾1.2

60.2⫾3.5 55.4⫾2.4 55.0⫾2.4

0.31⫾0.03 0.24⫾0.01* 0.23⫾0.01

29.7⫾1.0 30.8⫾1.4 30.6⫾0.8

0.39⫾0.02 0.30⫾0.02* 0.30⫾0.01

30.6⫾1.6 26.8⫾1.2 27.3⫾1.0

0.52⫾0.03 0.42⫾0.03* 0.40⫾0.02

29.0⫾1.8 27.9⫾1.3 29.3⫾1.1

0.57⫾0.03 0.44⫾0.03* 0.40⫾0.02

34.4⫾0.9 35.5⫾1.2 33.3⫾1.4

0.46⫾0.02 0.30⫾0.01* 0.29⫾0.02

40.5⫾1.4 43.1⫾2.2 38.9⫾1.2

0.46⫾0.03 0.31⫾0.02* 0.30⫾0.02

64.4⫾3.7 59.0⫾3.4 66.9⫾4.3

0.34⫾0.03 0.30⫾0.03* 0.27⫾0.02

50.3⫾2.0 50.5⫾1.8 53.1⫾1.4

0.38⫾0.02 0.31⫾0.02* 0.29⫾0.02

36.0⫾1.0 40.5⫾0.9‡ 43.0⫾2.3

0.44⫾0.02 0.32⫾0.02* 0.29⫾0.01

37.4⫾1.2 35.5⫾1.6 37.8⫾1.4

0.76⫾0.04 0.57⫾0.02* 0.57⫾0.03

51.9⫾2.4 58.5⫾2.9 58.8⫾2.6

0.46⫾0.02 0.31⫾0.01* 0.30⫾0.01

57.1⫾1.5 62.4⫾2.2 62.7⫾2.3

0.56⫾0.02 0.41⫾0.02* 0.39⫾0.02

56.5⫾2.2 61.0⫾2.1 60.9⫾2.4

0.55⫾0.03 0.41⫾0.03* 0.41⫾0.02

Table 2. (Continued) Content (pmol/mg protein)

Turnover rates

5-HIAA

5-HIAA

5-HT

5-HT Lateral thalamus YS 349.3⫾22.3 YC 264.7⫾14.7* SA 260.6⫾12.1 Brain stem YS 35.9⫾2.2 YC 27.6⫾1.6* SA 26.2⫾1.6 Ventral pallidum, stria terminalis YS 32.2⫾2.4 YCa 30.9⫾1.9 SA 30.1⫾1.7 Caudate putamen YS 24.0⫾1.7 YCa 20.8⫾1.1 SAa 21.9⫾1.0

413.3⫾17.2 398.8⫾16.9 400.8⫾12.4

0.84⫾0.04 0.67⫾0.02* 0.65⫾0.02

51.0⫾1.7 52.5⫾1.3 52.5⫾1.7

0.70⫾0.03 0.52⫾0.02* 0.50⫾0.02

56.6⫾3.4 58.6⫾2.1 60.8⫾3.2

0.57⫾0.03 0.53⫾0.03 0.49⫾0.03

33.2⫾1.2 30.3⫾0.9 30.7⫾1.3

0.74⫾0.03 0.71⫾0.03 0.74⫾0.04

5-HIAA indicates 5-hydroxyindole acetic acid; 5-HT, serotonin; YS, yoked saline infused; YC, yoked cocaine infused; and SA, self-administration. a Indicates changes observed in the turnover rates using the radioactive pulse-labeling procedure. * Significance difference measured by one-way analysis of variance followed by Student-Newman-Keuls method of analysis. Values are mean⫾S.E.M.

Amino acid neurotransmitter involvement One of the major findings of this study are the significant decreases in Glu turnover rates in the self-administering rats in the nucleus accumbens, ventral pallidum, posterior cingulate cortex, visual cortex, entorhinal-subicular cortex, raphe nuclei and brain stem. Although it can be argued that the Glu turnover rates may not result primarily from neurotransmitter function, the heterogeneous nature and loci where these changes are seen as well as recent experiments using microdialysis (Gray et al., 1999; Wolf and Xue, 1999; Rawls and McGinty, 2000) support a significant role in synaptic processes. If one assumes that these Glu turnover-rate changes reflect primarily metabolic cellular requirements, then the largest impact on cerebral metabolism would likely result from the administration of cocaine (differences between the yoked cocaine-infused and yoked vehicle-infused rats). However, no such differences were seen. The differences in Glu turnover were between the self-administering and the yoked cocaine-infused rats, indicating that subtle behavioral differences were responsible for these changes. Attributing these turnover changes to metabolism differences rather than a neurotransmitter function is not parsimonious. The significant decreases in Glu turnover in the nucleus accumbens are important since Glu has been clearly shown to have a role in the psychomotor stimulant actions of cocaine. The decreased turnover rates of Glu in the nucleus accumbens could result from a direct action of cocaine on Glu outputs from cortical regions. Glu innerva-

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471

Fig. 8. Rat brain neuronal pathways identified with turnover-rate measures to be involved in the brain processes that underlie cocaine selfadministration and the other pharmacological effects of cocaine. The red closed circles represent dopaminergic neuronal pathways; the light-blue closed triangles represent norepinephrine; the dark-blue closed squares represent serotonin; the magenta open triangles represent aspartate; the purple open circles represent GABA; and the green open squares represent glutamate circuitry. Question marks next to these symbols indicated an unknown origin of these innervations. The top panel shows the pathways implicated in cocaine self-administration and bottom panel those resulting from a drug effect. Abbreviated for brain regions are: ACC, anterior cingulate cortex; Amy, amygdala; ESC, entorhinal–subicular cortex; GP, globus pallidus; Hipp, hippocampus; LC, locus coeruleus; LH, lateral hypothalamus; LT, lateral thalamus; MH, medial hypothalamus; NAcc, nucleus accumbens; PCC, posterior cingulate cortex; PFC, prefrontal cortex; RN, raphe nuclei; Sep, septum; SMC, somatosensory cortex; SN, substantia nigra; VC, visual cortex; VP, ventral pallidum; VTA, ventral tegmental area.

tions of the nucleus accumbens represent primarily input from the prefrontal (Gorelova and Yang, 1997), frontal and cingulate cortices (Fuller et al., 1987). This could occur if cocaine enhanced the effects of DA, NA or 5-HT in these cortical regions by blocking re-uptake resulting in an inhibition of these neurons, or if a decrease in Glu tone in these areas was part of the mechanisms underlying posi-

tive hedonic processes. The decrease in Glu turnover during self-administration is consistent with neuropharmacological data showing administration of an N-methyl-Daspartate (NMDA) Glu-receptor antagonist (Pulvirenti et al., 1992) or ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid Glu-receptor agonist (Cornish et al., 1999) directly into the nucleus accumbens to increase i.v. co-

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caine self-administration in a manner consistent with enhanced reinforcing efficacy (Cornish et al., 1999). This could result from a direct action of Glu on DA neurotransmission since injections of Glu, a Glu uptake inhibitor or a low dose of NMDA into the nucleus accumbens decreased extracellular DA concentrations while higher doses of NMDA increased these levels (Taber et al., 1996). In contrast, DA has been shown to inhibit Glu-evoked firing of cells in this structure suggesting a reciprocal inhibitory relationship between these two neuronal systems (White et al., 1995). The turnover rate measures also found increased turnover of DA and decreased turnover of Glu in the nucleus accumbens during cocaine self-administration which is consistent with such a reciprocal inhibitory relationship, but inconsistent with the involvement of Glu in this structure in the locomotor effects of the drug. However, brain neurotransmitter turnover rates in self-administering rats are different than in rats receiving response-independent administration (Smith et al., 1982, 1984; Dworkin et al., 1995). It is not clear that data collected with responseindependent drug delivery is applicable or generalizable to the drug self-administration milieu. GABA turnover rates were decreased in the nucleus accumbens and anterior cingulate and somatosensory cortices during self-administration. This is consistent with the observations that systemic pretreatment with GABAB agonists attenuate the reinforcing effects of cocaine (Brebner et al., 1999; Campbell et al., 1999; Kushner et al., 1999; Roberts et al., 1996; Roberts and Andrews, 1997; Shoaib et al., 1998). The site of action of these agonists could be either the nucleus accumbens or the ventral tegmental area since injections of a GABAB receptor agonist directly into each of these areas attenuated rat cocaine intake (Shoaib et al., 1998). GABA neurons in the shell of the nucleus accumbens receive significant presynaptic DA modulation through D2 receptors (Delle Donne et al., 1997) suggesting one mechanism for the effects of cocaine on GABA tone that is consistent with the decreased turnover rates seen here. The facilitation of GABA release (Tanganelli et al., 1994) or blockade of degradation (Gerasimov et al., 2000) in the nucleus accumbens which decreased DA release appears to be mediated by GABAB receptors as well (Ashby et al., 1999). However, whether these GABA effects are mediated by intrinsic neurons or innervations from the ventral tegmental area is not known. The decreased turnover rates of GABA in the nucleus accumbens during cocaine self-administration and decreased reinforcing effects of cocaine following administration of GABAB agonists (Roberts et al., 1996; Roberts and Andrews, 1997; Brebner et al., 2000), suggest that nucleus accumbens extracellular fluid levels would be decreased during self-administration which is consistent with the observation that the injection of GABAB receptor agonists into this structure decreases cocaine intake (Shoaib et al., 1998). Cortical processing It has long been apparent that reinforcers are recognized as salient stimuli through sensory systems that require cortical processing. Environmental stimuli with hedonic

properties (i.e. food, liquids, sexual cues, etc.) or acquired hedonic properties through association with such stimuli, require cortical processing for recognition before activity in brain-stem circuitry would be recruited and/or modulated. Although drugs could circumvent these processes by direct activation of brain-stem nuclei similar to electrical brain stimulation, the identification of turnover-rate changes in innervations of the entorhinal–subicular, anterior and posterior cingulate and visual cortices demonstrates the importance of cortical processing in drug selfadministration. These cortical systems may have a broader role in the central events that underlie general reinforcement. Potential aversive properties The response-independent presentation of a drug as in the yoked cocaine-infused controls is not a neutral stimulus and may actually have stressful or noxious consequences (Siegel, 1982). Accordingly, some of the changes in neurotransmitter turnover rates in the response-independent cocaine-infused group could represent these aversive properties. Candidate neuronal systems underlying these potential noxious properties include the observed reversal effects such as the decrease in 5-HT turnover rates in the hippocampus and caudate-putamen (Fig. 7). This interpretation is consistent with the hypothesis that the anxiogenic effects of cocaine may be the result of decreased activity of ascending projections from the dorsal raphe nucleus (Costall et al., 1989). Recent data support an anxiogenic action of cocaine which is a component of the self-administration environment (Goeders and Guerin, 1996a,b; Goeders, 1998). The increase in GABA turnover rates in the posterior cingulate cortex and in DA in the diagonal band and entorhinal–subicular and visual cortices in the yoked cocaine-infused group that are reversed in the self-administration group are additional candidates for mediating these anxiogenic actions. Turnover rate calculation differences Large discrepancies were found between turnover rates calculated with the pulse-label technique and metabolite/ neurotransmitter ratios. Although these turnover rates were calculated from the same samples, in no case was the effect obtained with ratios in agreement with data obtained with the pulse-label techniques. Neurotransmitter turnover rates determined with radioactive pulse labeling have been developed to estimate the involvement of the functional pool of neurotransmitter in brain function. The functional pool is defined as that portion that is readily releasable to meet the requirements of the neuron to transmit information. This pool is believed to be the most recently synthesized neurotransmitter, so that a “most recently synthesized, first out” principle is hypothesized to predominate. Radioactive pulse-labeling techniques, selectively label this functional pool so that turnover rates determined by this method are generally thought to reflect activity in this pool. The 19 changes in turnover rates calculated from metabolite/neurotransmitter ratios result directly from changes in the tissue content of DOPAC,

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HVA or 5-HIAA. Fourteen of the 15 changes in 5-HT turnover rates estimated with the ratio technique are the direct result of changes in the content of 5-HIAA, while two of the four changes in DA turnover rates were the result of changes in the content of DOPAC and/or HVA. When changes in the same brain region were identified, the direction of the change was opposite to that seen with the pulse-label technique (septum). The content of DA and 5-HT changed in four of these regions (DA, caudate-putamen, amygdala and medial hypothalamus; 5-HT, septum). Although the dynamics of tissue neurotransmitter-metabolite content may be partially the result of the rate of degradation of the neurotransmitter, other unrelated factors can also be responsible (transport mechanisms, enzymatic processes responsible for removal, blood flow, cerebral spinal fluid dynamics). Tissue content of neurotransmitter metabolites are generally considered questionable measures of neuronal activity. The significant changes in ratios calculated here result only from changes in metabolite content. In addition, in circumstances where a turnover-rate increase would be expected (DA in the nucleus accumbens and ventral pallidum), these were not detected with the ratio method. These discrepant results determined in the same animals with two different procedures raise questions as to the utility of the metabolite/ neurotransmitter ratios as measures of turnover rates and provide support for the use of pulse-label procedures in investigations of neurotransmitter utilization as biological substrates of behavior.

CONCLUSIONS The major finding of this research is the striking differences between the passive cocaine-infused and the cocaine selfadministering rats (referred to as the “self-administration effect”). Although these two groups of animals had identical histories of cocaine exposure, the ability of the selfadministering animals to control such exposure with a simple response had substantial neurochemical consequences (Fig. 8). The brain mechanisms underlying cocaine self-administration clearly involve DA neurons which has been known for some time. However, the involvement of DA innervations of the ventral pallidum, septum and lateral hypothalamus has not been previously demonstrated. A number of other neuronal systems also participate in the circuits that are responsible for this behavior that include 5-HT innervations of the medial hypothalamus and substantia nigra as well as noradrenergic innervations of the lateral thalamus and somatosensory cortex. In addition, GABA-releasing neurons in the nucleus accumbens and anterior cingulate and somatosensory cortices also appear to be involved. The largest number of changes associated with this behavior was seen in Glu-releasing neurons in the nucleus accumbens, ventral pallidum, brain stem, raphe nuclei and the posterior cingulate, entorhinal and visual cortices. These data build on previous findings and suggest a role of discrete sub-populations of DA-, 5-HT-, Glu- and GABA-releasing neurons in the brain processes underlying cocaine self-administration.

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Whether these neuronal systems identified in this turnover rate study are necessary to or participate in the circuits that are responsible for these complex behaviors awaits appropriate verification with such techniques as microdialysis (Hemby et al., 1997, 1999), lesions induced with selective neurotoxins (Dworkin et al., 1988a,b,c), selective receptor alkylating agents (Martin et al., 1995), intracranial administration of receptor agonists and antagonists (Cornish et al., 1999; Dewey et al., 1997; Shoaib et al., 1998) and conditional knockout or knock-in models in self-administering animals (Hikida et al., 2001). Since subsets of these identified systems likely represent neuronal systems mediating reinforcement in general, these data may have broader application to understanding the biological basis of behavior. Acknowledgements—Supported in part by USPHS grants DA03628, DA06634 and DA00114.

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(Accepted 5 October 2002)