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Alcohol and Drug Abuse Program, College of Veterinary Medicine, ..... Research Career Development Award no. DA-00l58 ... Academic Press, Orlando, Florida.

Journal of Neurochemistry Lippincott—Raven Publishers, Philadelphia © 1998 International Society for Neurochemistry

Repeated Cocaine Administration Alters Extracellular Glutamate in the Ventral Tegmental Area Peter W. Kalivas and Patricia Duffy Alcohol and Drug Abuse Program, College of Veterinary Medicine, Washington State University, Pullman, Washington, U.S.A.

Abstract: The present study determined if repeated cocaine injections alter the effect of cocaine on extracellular glutamate in the ventral tegmental area (VTA). All rats were treated with daily cocaine (15 mg/kg i.p. x 2 days, 30 mg/kg i.p. x 5 days) or saline for 7 days. At 21 days after discontinuing the daily injections, a dialysis probe was placed into the VTA and the extracellular levels of glutamate were estimated. A systemic injection of cocaine (15 mg/kg i.p.) elevated extracellular glutamate in the VTA of rats pretreated with daily cocaine but not in the daily saline-pretreated subjects. No significant change in glutamate was produced by a saline injection in either pretreatment group. In a group of rats pretreated with daily cocaine, the D1 antagonist SCH-23390 (30 /2M) was infused through the dialysis probe prior to the acute injections of saline and cocaine. SCH-23390 prevented the increase in extracellular glutamate associated with the acute administration of cocaine. Behavioral data were collected simultaneously with the measures of extracelluar glutamate. The behavioral stimulant effect of cocaine was greater in cocaine-pretreated than saline-pretreated subjects, and the behavioral augmentation in cocainepretreated rats was partly blocked by SCH-23390. These data support the hypotheses that repeated cocaine administration produces an increase in the capacity of D1 receptor stimulation to release glutamate in the VTA and that this mechanism partly mediates behavioral sensitization produced in rats treated with daily cocaine injections. Key Words: Cocaine— Dopamine—Ventral tegmental area—Glutamate—D1 receptor. J. Neurochem. 70, 1497—1502 (1998).

The repeated administration of amphetamine-like psychostimulants results in sensitization of the behavioral response to a subsequent drug injection (Robinson and Becker, 1986; Kalivas and Stewart, 1991). In experimental animals this is characterized by an increase in locomotor activity and motor stereotypies (Segal and Mandell, 1974; Post and Rose, 1976) as well as a facilitated acquisition of drug self-administration (Horger et al., 1990, 1992; Piazza et al., 1990; Schenk et al., 1993). In a subpopulation of psychostimulant addicts, behavioral sensitization is characterized by an increase in psychopathologies such as paranoia and


panic (Sato et al., 1983; Post and Weiss, 1988; Satel and Edell, 1991) and perhaps facilitated relapse (Robinson and Berridge, 1993; Bartlett et al., 1997). The stimulant effect elicited by an acute injection of psychostimulant arises primarily from increased dopamine transmission due to binding to the dopamine transporter (Reith et al., 1986; Ritz et al., 1990), and many studies have revealed that the long-lasting sensitized behaviors are associated with increased dopamine transmission in dopamine axon terminal fields (Robinson et al., 1988; Pettit et al., 1990; Kalivas and Duffy, 1993; Wolf et al., 1993; Paulson and Robinson, 1995; Heidbreder et al., 1996). One mechanism whereby dopamine transmission is augmented is via increased releasability of dopamine from axon terminals. This clearly occurs since dopamine release from striatal tissue slices or synaptosomes is enhanced in rats pretreated with repeated injections of amphetamine or cocaine (Robinson and Becker, 1982; Kolta et al., 1985; Pens et al., 1990). Alternatively, there may exist changes in the excitability of dopamine cells in the ventral mesencephalon of sensitized animals (Tong et al., 1995; White et al., 1995), and the resulting changes in firing frequency or pattern can facilitate dopamine release in the axon terminals (Suaud-Chagny et al., 1992). In simple terms, facilitation of the activity of dopamine cells may arise from augmented excitatory or decreased inhibitory input. There is evidence for alterations in both excitatory and inhibitory neurotransmission in the dopamine cell group in the medial ventral mesencephalon, specifically in the ventral tegmental area (VTA). The iontophoretic application of excitatory amino acids (EAAs) or the stimulation of excitatory afferents from the prefrontal cortex (PFC) more readily activates dopamine neurons in rats pretreated with daily cocaine or amReceived September 19, 1997; revised manuscript received November 14, 1997; accepted November 14, 1997. Address correspondence and reprint requests to Dr. P. W. Kalivas at Department of VCAPP, washington State University, Pullman, WA 99164-6520, U.S.A. Abbreviations used: EAA, excitatory amino acid; PFC, prefrontal cortex; VTA, ventral tegmental area.



phetamine injections, respectively (Tong et al., 1995; White et al., 1995). Conversely, Bonci and Williams (1996) revealed a reduction in D 1-mediated GABA input to dopamine cells in rats pretreated with daily cocaine injections. Additionally, there is a reduction in the capacity of D2 autoreceptor agonists to inhibit dopamine neurons in rats pretreated with daily cocaine injections (Wolf et al., 1993). In addition to GABA afferents to the VTA, D1 receptors may be located on BAA afferents. Neurons in the PFC that have excitatory projections to the VTA express D1 receptor mRNA (Lu et al., 1997), and application of a D1 agonist into the VTA or substantia nigra with a microdialysis probe increases the extracellular content of glutamate (Kalivas and Duffy, 1995; Arbaca et al., 1995; Rosales et al., 1997). Thus, by increasing extracellular dopamine in the VTA (Parsons and Justice, 1993), cocaine may indirectly stimulate D1 receptors and thereby enhance excitatory transmission in the VTA. The present experiments explore this possibility by examining cocaine-stimulated elevation of extracellular glutamate in the VTA of rats sensitized to repeated injections of cocaine.

MATERIALS AND METHODS Surgery and animal housing Male Sprague—Dawley rats (Simonsen Laboratories, Gilroy, CA, U.S.A.) were individually housed with food and water available ad libitum in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the NIH. A 12/12-h lightldark cycle was used with the lights on at 6:30 h. One week after discontinuing daily cocaine or saline treatments (see below), rats weighing 270—330 g were anesthetized with Equithesin (3.0 ml/kg; 9.7 g sodium pentobarbital, 42.5 g chloral hydrate, and 21.3 g MgSO4 dissolved in 1 L of 11% ethanol and 42% propylene glycol, vollvol) and mounted in a stereotaxic apparatus. Unilateral dialysis guide cannulae (14-mm, 20gauge stainless steel) were implanted 3 mm dorsal to the VTA [2.5 mm anteroposterior, 0.5 mm mediolateral, —2.5 mm dorsoventral relative to the interaural line according to Pellegrino et al. (1979)] and cemented in place by affixing dental acrylic to three stainless-steel screws tapped into the skull.

lowed by an injection of saline or cocaine (15 mg/kg i.p.), and motor activity was quantified for an additional 120 min. All of the daily injections of cocaine were made between 10:00 and 15:00 h. The photocell apparatus employed consisted of 16 photocell cages (Omnitech, Columbus, OH, U.S.A.) each located in a separate sound-attenuated chamber with individual light and air sources. Each apparatus contained 16 infrared photocells located 2 cm off the cage floor to measure horizontal activity (e.g., a combination of locomotion and stereotypy) and 8 photocells located 6 cm off the floor to estimate vertical activity (e.g., rearing).

Microdialysis The dialysis probes were constructed as described by Robinson and Wishaw (1988), with 1.0—1.5 mm of active dialysis membrane exposed at the tip. The probes were inserted through the guide cannula into the VTA the night prior to the dialysis experiment, which was conducted 3 weeks after the last daily injection of cocaine or saline. The next day, dialysis buffer (2.7 mM KC1, 140 mIl‘! NaC1, 1.4 mM CaC12, 1.2 mM MgCl2, 5.0 mM D-glucose, plus 0.2 mM phosphatebuffered saline to give a pH value of 7.4) was advanced through the probe at a rate of 1.9 ‚el/min via a syringe pump (Harvard Instruments, Boston, MA, U.S.A.) for 2 h. Twentyminute baseline samples were collected for 100 mm, and then the rats received a systemic injection of saline (1 ml! kg i.p.) followed 80 min later by cocaine (15 mg/kg i.p.; donated by the National Institute of Drug Abuse). After cocaine, dialysis samples were collected for an additional 180 min. Thus, dialysis samples were collected for a total of 360 min. In seven of the rats pretreated with daily cocaine, the D1 antagonist SCH-23390 (30 ‚eM; Research Biochemicals International, Natick, MA, U.S.A.) was introduced into the VTA via the dialysis probe. After collecting the baseline dialysis samples and 40 min before injecting saline, dialysis buffer containing SCH-23390 was perfused through the VTA for the remainder of the experiment. Thus, in the animals receiving SCH-23390, the experiment was an additional 40 min in duration (i.e., 400 mm). SCH-23390 was dissolved in the dialysis perfusion buffer, and cocaine was dissolved in saline. During both baseline and drug administration, samples were taken every 20 min. The dialysis experiment was conducted in a photocell cage to permit the simultaneous monitoring of motor activity (Omnitech). At the end of the experiment, the dialysis probe was removed and the animal was administered an overdose of pentobarbital (>100 mg!

kg i.p.).

Daily cocaine treatment and behavioral measures

Quantification of glutamate content

Rats were pretreated with daily cocaine or saline injections for 1 week. All rats were placed in the photocell apparatus 24 h prior to beginning daily cocaine or saline injections. After a 1-h adaptation, rats were injected with saline (1.0 mI/kg i.p.) and returned to the photocell cage where photocell beam breaks were quantified for 60 min. The next day, the subjects were divided into daily cocaine (n = 22) and daily saline (n = 10) treatment groups. On the first day, all rats were adapted to the photocell box for 60 min. Following the adaptation period, the subjects were administered either saline or cocaine (15 mg/kg i.p.), and motor activity was monitored for an additional 2 h. Over the next 5 days, rats were given either daily saline or cocaine (30 mg/kg i.p.) in their home cage. On the last day of daily injections, the rats were again adapted to the photocell boxes for 60 mm, fol-

The concentration of glutamate was determined using HPLC with fluorometric detection. The dialysis samples were collected into 10 ‚eI of 0.1 M HC1 containing 2.0 pmol of homoserine as the internal standard. The mobile phase for glutamate is described by Moghaddam (1993). A reversed-phase column (10 cm, 3 ~imODS; Bioanalytical Systems, West Lafayette, IN, U.S.A.) was used to separate the amino acids, and precolumn derivatization of amino acids with o-phthalaldehyde was performed using an autosampler (Gilson Medical Supplies, Middleton, WI, U.S.A.). Gluta-

J. Neurochem., Vol. 70, No. 4, 1998

mate was detected by a fluorescence spectrophotometer (Shi-

madzu, Columbia, MD, U.S.A.) using an excitation wavelength of 300 nm and an emission wavelength of 400 nm. Peak heights were measured, normalized to the internal standard, and compared with an external standard curve for quan-



TABLE 1. Development of behavioral sensitization to cocaine Treatment group


Saline Cocaine

10 15 7


First daily injection

Last daily injection

Paired t


4,407 ±683 15,587 ± 1,629

6,042 ±825 33,777 ±2,623

1.93 5.61


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