Adaptation to Repeated Cocaine Administration in Rats

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Ann. N.Y. Acad. Sci. 965: 172–179 (2002). © 2002 New York Academy of Sciences.

Adaptation to Repeated Cocaine Administration in Rats

ZBIGNIEW K. BINIENDA,a FREDERICO PEREIRA,a,c

KENNETH ALPER,b WILLIAM SLIKKER, JR.,a ANDSYED F. ALIa

aDivision of Neurotoxicology, NCTR/FDA, Jefferson,Arkansas 72029, USAbBrain Research Laboratories, New York UniversityMedical Center, New York, New York 10016, USAcInstituto de Farmacologia e Terapeutica Experimental,University of Coimbra Medical School, Coimbra, Portugal

ABSTRACT: Quantitative electroencephalogram (EEG) studies in cocaine-dependent human patients show deficits in slow-wave brain activity, reflectedin diminished EEG power in the delta and theta frequency bands. In thepresent study, electrophysiological measures were monitored in 10 nonanesthe-tized, adult male Sprague-Dawley rats via bipolar, epidural electrodes implant-ed over the somatosensory cortex. Control electrocorticograms (ECoG) wererecorded twice within a two-week interval to establish a baseline. Rats weresubsequently injected daily with cocaine HCl at 15 mg/kg, i.p., for two weeks.The ECoG was recorded during a 1-h session one day after the last injection.Total concentrations of dopamine (DA) and its metabolites were assayed in cau-date nucleus (CN) and frontal cortex (FC) using HPLC/EC. Compared withcontrols, marked increases in DA concentrations were observed in both re-gions. The DA turnover decreased significantly. The power spectra, obtainedby use of a fast Fourier transformation, revealed a significant decrease in slow-wave delta frequency bands following repeated exposure to cocaine. These dataare consistent with reported findings in humans that repeated exposures to co-caine result in a decrease in slow-wave brain activity. Further studies are nec-essary to establish whether regional alterations in blood flow and metabolicactivity may underlie such observations.

KEYWORDS: rat; brain; cocaine; EEG; slow wave; delta frequency

INTRODUCTION

Repeated exposure to cocaine (COC) has been shown to sensitize animals to thelocomotor-activating effects of this agent, leading to hyperactivity and stereotypy.1,2

Behavioral sensitization to psychostimulants including COC is thought to involve

Address for correspondence: Dr. Zbigniew K. Binienda, Division of Neurotoxicology, HFT-132, FDA/NCTR, Jefferson, AR 72079-9502. Voice: 870-543-7920; fax: 870-543-7745.

[email protected]

173BINIENDA et al.: COCAINE ADMINISTRATION

increased dopamine (DA) neurotransmission in nigrostriatal and mesolimbic path-ways.3 Alterations of the gamma-aminobutyric acid (GABA) receptor system maycontribute to those behavioral changes as well, since repeated injections of COC de-crease GABA receptor in the striatum.4 In addition, it was shown that the increasedresponsiveness of dopaminergic neurons to glutamate in the ventral tegmental area,after chronic exposure to COC, selectively involved ionotropic glutamate receptorsAMPA.5 Recently, an increase in glutamate receptor subunit levels (GluR2/3) wasobserved in rats that developed behavioral sensitization to repeated COC exposure.6

Quantitative electroencephalogram (EEG) studies in COC-dependent human pa-tients show deficits in slow-wave brain activity, reflected by diminished EEG powerin the delta and theta bands.7,8 A decrease in low-frequency electrocorticograms(ECoG) after chronic or repeated COC exposure may be associated with reducedmetabolic activity in the frontal cortex,9 and could be related to alterations in corticalprocessing modulated by DA.10 Experimental induction of addiction to COC in hu-man subjects is impossible due to ethical constraints, necessitating a need for a rel-evant animal model. The aim of this study was to evaluate alterations indopaminergic neurotransmission and ECoG following repeated COC administrationin rats.

MATERIAL AND METHODS

Animals

Adult, male CD strain, Sprague-Dawley rats from the FDA/NCTR breeding col-ony were used for this study. Animals were kept under controlled environmentalconditions (temperature 22°C, relative humidity 45–55%, 12-h light/dark cycle) andhoused individually with food and water supplied ad libitum. Animal care and useprocedures were in accordance with the American Association for Accreditation ofLaboratory Animal Care (AAALAC) guidelines and approved by the InstitutionalAnimal Care and Use Committee (IACUC).

Instrumentation

Rats in the COC group were instrumented under general isoflurane anesthesia.Upon placing the animal’s head in the stereotaxic apparatus, the skin and muscle tis-sue on the upper cranium were incised and tissues reflected to expose the skull. Fivestainless-steel screws (size No. 0-80 × 1/8-in. length) were installed as epidural elec-trodes in the undersized holes drilled in the scull above the somatosensory cortex(3 mm laterally from the sagittal fissure and 1 and 4 mm posterior to bregma). Thescrews were soldered to the connector via stainless-steel wires. The connector wasattached to the skull using dental cement, and a ground wire was implanted underthe skin in the dorsal neck, as previously described.11

ECoG Recording and Cocaine Administration

The baseline ECoG was recorded twice at a seven-day interval during a 1-h ses-sion (reference run) after a seven-day postsurgical recovery. The ECoG was recordedvia a tether and swivel system. During recording, animals remained in a microdial-

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ysis bowl placed inside a Faraday cage. The signals were amplified using BioAmp100 differential amplifiers and a CyberAmp 380 signal conditioner (Axon Instru-ments, Inc., Union City, CA) to pass frequencies of 1–40 Hz and processed withLabView software (National Instruments, Austin, TX). The power spectra obtainedby use of fast Fourier transform (FFT) were divided into 1.25–4.50 Hz (delta), 4.75–6.75 Hz (theta), 7.00–9.50 Hz (alpha-1), 9.75–12.50 Hz (alpha-2), 12.75–18.50 Hz(beta-1), and 18.75–35.00 Hz (beta-2) frequency bands. Following the baselineECoG recording, rats were injected (i.p.) daily with cocaine hydrochloride obtainedfrom the Research Triangle Institute (Research Triangle Park, NC) at 15 mg/kg fortwo weeks. The ECoG signals were recorded again during a 1-h session approxi-mately 24 h following the last COC injection.

Neurochemical Analysis

Neurochemical assessments were performed in the COC-treated rats and a sepa-rate group of rats (n = 5) used as control for this analysis. All rats were sacrificed bydecapitation upon completion of the EEG recording. Their brains were removed, fro-zen on dry ice, and stored at −80°C until dissection of the caudate nucleus (CN) andfrontal cortex (FC) according to Glowinski and Iversen.12 Concentrations of DA andits metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid(HVA), were assayed using a high-performance liquid chromatography method cou-pled with electrochemical detection (HPLC/EC) according to Ali et al.13

Statistical Analysis

After scanning for noise (time points where all signals were outside the first or99th percentiles of the entire run), data for each animal at a given frequency bandwere adjusted to the reference run in that band. Each power measurement duringtreatment was divided by the median power in the reference run (baseline or refer-ence level). In this way, all animals were placed on a similar scale despite factors thatmade the absolute power different between animals. Once all the animals were ad-justed to similar scales, a log-transform was used to normalize the data. Power dif-ference between a given interval (5 or 30 min) and the reference-level period wastested against zero using the Student’s t-test at the significance level p < 0.05.

The two-sample Student’s t-test was applied to compare monoamine levels be-tween control and treated rats at p < 0.05.

RESULTS AND DISCUSSION

Repeated administration of COC produced enhancement of locomotor activityand stereotyped behavior (pronounced sniffing) observed by the first week of treat-ment within less than 20 min after the injection. The COC was administered to ratsusing a paradigm that produces sensitization, as reported in other studies.1,6,14 Nosignificant changes in power spectra were observed when comparing the baselineECoG recordings during the two sessions recorded at a seven-day interval. However,compared with the baseline obtained from the reference-run recording, the powerspectra analyzed in rats treated with COC for two weeks revealed a significant de-


crease in slow-wave frequency bands, lasting until 30 min after the beginning of re-cording (FIG. 1). There was also a decrease of power observed in alpha-1, alpha-2,and beta-1 frequency bands, but much less pronounced and lasting only about 15 min(FIG. 2). The values recovered near baseline after approximately 30 min of recording(FIGS. 1 and 2). The recovery of the EEG signals observed during recording may berelated to the resting-state animals assumed with time, compared to the initial alert-ness related to the initiation of the recording procedure.

Ferger and his collaborators reported that rats injected daily for 10 days withCOC at 10 mg/kg, but not 20 mg/kg, led to a decrease in all spectral EEG frequencybands.14 The effect lasted until 45 min after COC administration. In contrast, an in-crease in the alpha-1 power was observed following repeated COC injections at 20mg/kg. In the present study, the reduction in slow-wave activity was observed 24 hafter the last COC injection. A decrease in low-frequency brain activity has been as-sociated with reduced metabolic activity in the frontal cortex, as shown by positronemission tomography and EEG correlation analyses.9

TABLE 1 shows the effect of repeated COC administration on the levels of DA andits metabolites in CN and FC. The concentration of DA increased significantly inboth regions, suggesting that this COC treatment regimen increased the tyrosine hy-droxylase (TH) activity. Compared with controls, the levels of DA metabolites de-creased concomitantly with a significant decrease in DA turnover, calculated asDOPAC + HVA/DA ratio. Attenuation of DA levels in medial prefrontal cortex ob-served with increasing DA in the nucleus accumbens has been reported in rats treatedwith COC at 15 mg/kg for five days. Those observations were made using microdi-alysis techniques that determine extracellular DA concentrations.16 An increase inthe extracellular DA concentration in the axon terminals of striatum and nucleus ac-cumbens has been reported in association with sensitization to amphetamine and co-caine,17,18 and is consistent with sensitization to cocaine, inducing an increase intotal content of striatal DA. The ECoG findings in the present study resemble theoutcome of studies of COC-dependent human subjects. In human studies, a markeddeficit in slow-wave brain activity, along with an enhancement of activity in betapower band, was frequently observed (see Ref. 10). The animal model presentedhere may prove to be useful in the study of human COC dependence.

TABLE 1. Concentrations of dopamine (DA) and its metabolites DOPAC and HVA(ng/100-mg wet weight of tissue) in the caudate nucleus and frontal cortex of ratsinjected i.p. with cocaine daily at 15 mg/kg, i.p., for a 14-day period

Region DA DOPAC HVA Turnover Region

Caudate Nucleus Control 1085.8 ± 27.7 137.3 ± 8.1 81.5 ± 7.1 0.20 ± 0.01

Cocaine 1696.8* ± 174.5 112.0 ± 26.7 71.0 ± 10.5 0.12* ± 0.02

Frontal Cortex Control 78.3 ± 8.3 60.3 ± 3.9 62.0 ± 4.6 1.57 ± 0.12

Cocaine 197.0* ± 9.5 37.7 ± 19.5 76.8 ± 23.6 0.57* ± 0.20

NOTE: Turnover calculated as DOPAC + HVA/DA. Data are expressed as mean ± SEM; n = 5rats; *p < 0.05 significantly different from control.


FIGURE 1. Brain activity in rats injected with cocaine daily at 15 kg/day, i.p., for a 14-day period. Power values calculated in 30-min time intervals as percent of the 1-h reference-level power assigned as a value of 100% in each band. Mean ± SEM; n = 5. *p < 0.05 sig-nificantly different from reference level (baseline). Frequency waves: delta (1.25–4.50 Hz);theta (4.75–6.75 Hz); alpha-1 (7.00–9.50 Hz); alpha-2 (9.75–12.50 Hz); beta-1 (12.75–18.50 Hz); beta-2 (18.75–35.00 Hz).


FIGURE 2. Brain activity in rats injected with cocaine daily at 15 kg/day, i.p., for a 14-day period. Power values calculated in 5-min time intervals as percent of the 1-h reference-level power assigned as a value of 100% in each band. Mean ± SEM; n = 5. *p < 0.05 sig-nificantly different from reference level (baseline). Frequency waves: delta (1.25–4.50 Hz);theta (4.75–6.75 Hz); alpha-1 (7.00–9.50 Hz); alpha-2 (9.75–12.50 Hz); beta-1 (12.75–18.50 Hz); beta-2 (18.75–35.00 Hz).


CONCLUSIONS

Data gathered in the present study are consistent with reported findings in hu-mans that repeated exposures to COC result in a decrease in slow-wave brain activ-ity. A decrease in DA metabolism and increase in total DA concentration in CN andFC was also observed. Further studies are necessary to establish whether regional al-terations in blood flow and metabolic activity may underlie such observations.

ACKNOWLEDGMENTS

The authors thank Brett Thorn (R.O.W. Sciences) for the statistical analysis of theEEG data.

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Adaptation to Repeated Cocaine Administration in Rats

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