Nruropharmacoloyy Vol. Il. pp. 559-564 Q Pergamon PressLtd 1978. Printedin GreatBritain

BEHAVIOURAL AND NEUROCHEMICAL EFFECTS OF REPEATED ADMINISTRATION OF COCAINE IN RATS S. N. ROY, A. K. BHAITACHARYYA,

Department

of Pharmacology,

SIKTA PRADHAN

and S. N.

PRADHAN

Howard University College of Medicine, Washington, D.C. 20059, U.S.A. (Accepted 9 February 1978)

Summary-The effects of repeated administration of cocaine (15 mg/kg, i.p. twice daily at 8-hr intervals) were investigated on the spontaneous motor activity (SMA) and stereotypy (ST) as well as on the various neurotransmitters (e.g. norepinephrine, NE; dopamine, DA; serotonin, S-HT; acetylcholine, ACh) in different brain areas (e.g. diencephalon-midbrain, DM; pons-medulla, PM; caudate nucleus, CN)

in rats. Following repeated injections of cocaine, both SMA and ST gradually increased, reaching a peak in each case on about the 9th day, then gradually decreased up to the 18th or 20th day, after which the activities were maintained at minimum level which was slightly higher than normal levels. Concomitantly, the DA level in the CN and DM increased and 5-HT in the DM and PM decreased reaching their maximum or minimum levels following cocaine injections on the 9th day; these changes were gra@ally minimized by the 18th day and remained so up to the 30th day. There were also slight changes in NE and ACh levels. It thus appears that, following repeated cocaine administrations, the changes in the drug-induced behavioural effects can be correlated roughly with the changes in the DA level in the CN and the S-HT levels in the DM and PM.

20 and 4Omg/kg, (i.p.) doses (Downs and Eddy, Repeated administrations of cocaine have been 1932b). Tolerance was also shown to develop to the reported to cause increased sensitivity (or reversed discriminative stimulus properties of 2.5 and 5 mg/kg tolerance) to the drug as demonstrated by a variety of behavioural effects in different species of animals: (i.p.) doses of cocaine (McKenna and Ho, 1977). Cocaine is known to produce its effects on various excitement and hyperactivity in monkeys and dogs such as norepinephrine (Downs and Eddy, 1932a; Tatum and Seevers, 1929); central neurotransmitters abnormal visual searching (Post, Kopanda and Black, (NE), dopamine (DA), serotonin (5-HT) and acetylcholine (ACh) (for review see Scheel-Kriiger et aI., 1976; Post, 1977); hyperactivity and stereotypy in rats (Ho, Taylor, Estevez, Englert and McKenna, 1977; 1977). However, the effects of repeated administration of cocaine with respect to these neurotransmitters Post, 1977; Stripling and Ellinwood, 1977; Scheelhave not been adequately studied. Such a study was Kruger, Braestrup, Nielson, Golembiowska and undertaken in the present investigation in an attempt Mogilnicka, 1977); dyskynesia in monkeys (Post, 1977); catalepsy in dogs (Gutierrez-Noriega, 1950) to correlate the induced neurochemical changes with the chronic behavioural effects of cocaine, and the and monkeys (Post, 1977); hyperthermia in dogs (Gutierrez-Noriega and Zapata-Ortiz, 1944) and seiz- results are presented in this report. ures and death in monkeys (Post, 1977), dogs (Gutierrez-Noriega and Zapata-Ortiz, 1944) and rats (Downs METHODS and Eddy, 1932b; Stripling and Ellinwood, 1977). Male albino rats (Wistar-derived Walter Reed Reported administration of doses in man of up to strain) 200-25Og body weight were used throughout 10 g a day (Jaffee, 1975) indicates a great deal of tolerance considering 1.2 g to be the toxic dose, and even ‘the experiment. The animals were given food and 20mg to have been fatal (Caldwell and Sever, 1974). water ad Iibitum. For behavioural studies the rats were housed in individual cages. Acute tolerance was shown with respect to cocaineinduced increase in heart rate and respiratory rate Behaoioural methods and EEG arousal in monkeys (Matsuzaki, Misra and (a) Spontaneous motor activity (SMA). The SMA of Mule, 1975). Tolerance to cocaine-induced anorexia and weight loss was demonstrated in some species, individual rats was recorded by Selective Activity especially at low doses. Thus, repeated injections of Meters (Model S, Columbus Instruments, Ohio). A cocaine (15 mg/‘kg, s.c.) failed to alter weight gain in rat was kept in a plastic cage that was placed over an Activity Meter and covered at the top with a dogs (Colpaert, Kuyps, Niemegeers and Janssen, wooden board with several holes. During 40-min 1976). In rats, a depression of weight gain occurred following repeated injections of 75 mg/kg, but not of daily sessions, SMA was recorded every 10min; the activity during the initial 10&n period was conKey words: cocaine, repeated injections, behavioural sidered as exploratory activity. After 5-7 days of effects, neurochemical effects, rats. adaptation to the experimental situation during which

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The tissues were homogenized (100 mg/ml) in icecold 0.4 N perchloric acid, centrifuged in a Sorvall (RCZ-B model) at 4” for 20min at lO,Wrpm. Norepinephrine, DA and 5-HT were extracted simultaneously from the supernatant of the tissue homogenate according to the method of Cox and Perhach (1973). Norepinephrine and DA were assayed by the method of Chang (1964) and Spano and Neff (1971). Serotonin was extracted and determined according to the procedure of Ansell and Beeson (1968) and Maickel, Cox, Saillant and Miller (1968). Acetylcholine was assayed according to the procedure of Shea and Aprison (1973) with slight modification (Pradhan, Roy and Pradhan 1978).

saline (1 ml/kg) was injected intraperitoneally daily before the session, SMA was more or less stabilized. Following this control period, treatment with cocaine WS started. Cocaine HCI dissolved in saline in 1.5% solution was injected intraperitoneally in 15 mg/kg dose daily before the session and again after 8 hr. (b) Nor& and stereotyped hehaviour. The rats were observed for their exploratory (e.g. sniffing, rearing, jumping. biting) and other normal spontaneous (e.g. licking and grooming) behaviour. After cocaine treatment, some of these behaviours became stereotyped. They were subjectively scored and recorded every 10 min by an experienced observer. The responses were grouped into three score categories: (i) “licking” their bodies or the cage walls--(1 score); (ii) “sniffing and searching” (Sn-Sc) inside the cage-(2 scores); (iii) “rearing and biting” (RB) the hole edges of the wooden board covering the cage-(3 scores). Although the categories were scored differently, they were not weighted while measuring the total stereotypic responses (ST) that represent only the sum of unweighted categories.

Data analysis

In both behavioural and neurochemical experiments, the data of each parameter from several rats at a particular experimental condition were used to calculate the mean and the standard error of the mean (fS.E.). Student’s t-test was performed to evaluate the statistical significance of the change from the control. Drug effects were calculated in terms of percentage change from the controls in neurochemical and some behavioural experiments.

Neurochemical procedures

At specified intervals following injection of cocaine (15 mg/kg, i.p.), naive rats were decapitated after being subjected to microwave radiation focused on the skull for 2 set in a Litton Microwave Oven (Model No. 70/50; 3.5 kw; 2450 MHz; 1300 W output). The brain was quickly removed and different areas, such as the caudate nucleus (CN), the diencephalon-midbrain (DM) and the pons-medulla (PM) were dissected out at 4”; DM and PM were assayed for NE and 5-HT; DM and one CN were assayed for DA, and the other CN was used for ACh assay.

RESULTS

Behavioural eficts

During the pre-drug (control) period, the rats showed high activity counts during the first 10min due to initial exploratory activity that decreased during the subsequent 10 min. Stereotyped behaviour was minimal and represented only some normal exploratory or other spontaneous behaviour. Levels of these

15uo-

1400-

12LYJSMA

-2

1

5

10

DAYS OF COCAINE TREATMENT

15

20

(15 mg/kg, Lp.. twice

25 daily1

Fig. 1. Effects of repeated injections (15 mgfig, i.p., twice daily at 8-hr intervals) of cocaine on spontaneous motor activity (SMA) and stereotypy (ST) with its components (rearing-biting, RB; sniffingsearching, Sn-Sc; “ticking” is not shown here). Data represent mean 4 SE. of responses from 4 rats. Control levels of behavioural activities are given for day -2 and day 0 (not marked; between day 1 and day -2).

Effects of repeated cocaine injections

561

Table 1. Neurotransmitter concentrations in different brain areas in rats Brain areas Diencephalonmidbrain Pons-medulla Caudate nucleus

Concentrations* (pg/g) 5-HT DA

NE 0.48 + 0.02

0.27 f 0.01

0.67 f 0.02 0.66 * 0.03

0.63 f 0.01 2.75 + 0.1

7.59 + 0.01

* Mean k SE. from 4 rats in each group. NE-norepinephrine; DA-dopamine; SHT-serotonin; choline.

behavioural activities during 40-min sessions on day -2 and day 0 are shown in Figure 1. Following administration of a dose of cocaine (15 mg/kg, i.p.), SMA began to increase, reaching its peak within 10min and then gradually declined during the next 30 min. Various components of stereotyped behaviour began to develop and gradually increased until the end of 4Omin sessions. Although all the three components of this abnormal behaviour increased, the increase of RB was comparatively most pronounced. Following repeated administration of cocaine (15 mg/kg i.p. twice daily at 8-hr intervals), there were gradual increases in both SMA and total ST with its components reaching their peaks on about the 9th day. The RB responses were maximally increased compared to two other components of stereotypy. From the 10th day onwards, there were gradual decreases of SMA and the ST components up to the 18th to 20th day, after which the rate of decrease of these behaviours was very low, although all the activities were maintained at levels slightly higher than normal. Figure 1 illustrates the changes in SMA and ST with its components RB and Sn-Sc (licking is not shown) following repeated administration of cocaine. Neurochemical effects The levels of various

neurotransmitters e.g. NE, DA, 5-HT and ACh in different brain areas in the control rats are given in Table 1. ‘The effects of a dose (15 mg/kg, i.p., 20 and 60 min after injection) of cocaine on the levels of these neurotransmitters in the respective brain areas were investigated at intervals of several days following the repeated administration of the same dose (15 mg/kg, twice daily at 8-hr intervals) up to 30 days, as shown in Figure 2 and Table 2. At 20min post-drug on day 0 a significant (P < 0.01) increase of the DA level in the CN and significant (P < 0.001) decreases of the 5-HT levels in the PM and DM were observed. There were also slight increases of the DA level in the DM, and of ACh level in the CN, and decreases of the NE levels in the DM and PM, On day 9 following repeated administration, the changes of the DA level in the CM and of the 5-HT levels in the DM and PM at

ACh

ACh-acetyl-

20min post-drug were found to be maximum; NE levels in the DM and PM and the ACh level in the CN also showed slight increases. On day 18 and day 30 at 20min post-drug, levels of all the transmitters (except those of ACh in CN on day 18 and of NE in PM on day 30) were decreased. Compared to the changes in the neurotransmitter levels at 20min following cocaine injection, those at 60min post-drug on various days after its repeated injections were usually reduced or even reversed and generally in the opposite direction (Table 2). Some changes were further enhanced; of those, five (marked $ in Table 2) were +4% of the control or smaller and appeared to be insignificant, and only two (marked (j) were more pronounced. DISCUSSION The present study shows that repeated administration of cocaine in rats caused a gradual increase of SMA and stereotypy up to the 9th day after which their increases became less and less up to the 18th to 20th day, although still remaining above the conCOCAINE

(15 mg/kg, i.p.. twice daily) t-* -

DA-CN DA-DM ACh-CN

O---O -

NE-DM NE-PM

Fig. 2. Alterations in the effects of cocaine on various neurotransmitter (NE, norepinephrine; DA, dopamine; 5-HT, serotonin; ACh, acetylcholine) levels in discrete brain areas (DM, diencephalon-midbrain; PM, ponsmedulla; CN, caudate nucleus) following its repeated administration (15 mg/kg, i.p., twice daily at 8-hr intervals) in rats.

S. N. ROY

562

et al.

Table 2. Relative changes in the neurotransmitter levels at 60 min with respect to that at 20min following injection of cocaine (15 mg/kg, i.p.) on different days after its repeated administration (15 mg/kg, i.p.. twice daily at 8-hr intervals) in rats Neurotransmitter and brain area* NE DA 5-HT ACh

Day 0 DM PM CN DM DM PM CN

Change? in the transmitter level Day 9 Day 30 Day I8

+5 +8 -19 +I$ +29 +38 +41

-9 +4$ -10 -I +3 +12 -4

-It. -11 +22 +9 +4 -11 + 48s

+31 +240 +1 +I +I +14 +34

* NE, norepinephrine; DA, dopamine; 5-HT. serotonin; ACh, acetylcholine; DM, diencephalon-midbrain; PM, pons-medulla; CN, caudate nucleus. t The change (increase +; decrease -) in the neurotransmitter levels at 60min (or 40min for day 30) is with reference to the respective change at 20min post-drug from the same experiment, the quantitative data for which is presented in Figure 2. The change at either time is calculated in terms of percentage change from the control. The change at 6Omin is usually in the opposite direction with respect to the corresponding change at 20 min, except

for those marked with $ (+4x or smaller) and 5 (higher percentages). trol levels. Thus, while, initially, cocaine produced sensitization or reverse tolerance, subsequently a gradual “relative tolerance” could be observed in its effect. The initial reverse tolerance in the present study is similar to that observed by other investigators (lot. cit.). However, the present observations agree more closely with some findings of Post (1977) and Ho, et al. (1977). Post (1977) observed prominent hyperactivity and stereotypy during the first 446 weeks followed by a decrease in stereotypy from the second month of cocaine administration in monkeys, although behaviours in rats as well as some inhibitory behaviours in monkeys were gradually increased with repeated injections over a period of 20-28 weeks. Ho et al. (1977) reported stimulation of SMA in rats, that gradually increased for about 7 days and then decreased during the subsequent period up to 45 days following repeated cocaine injections (10 mg/kg, i.p. daily). Several hypotheses proposed by different investigators regarding the mechanism of functional or behavioural supersensitivity following repetitive stimulant administration have been discussed by Post (1977). These include alterations in the dopamine receptor (such as supersensitivity, activation, imbalance of two types of receptors, alterations in the agonist-antagonist conformation), positive nigra-neostriatal feedback loop and self-inhibition by dopaminergic neurones, and increased tyrosine hydroxylase activity. Increase of tyrosine hydroxylase activity has been demonstrated in the striatium and hypothalamusthalamus following repeated administration of cocaine by Ho er al. (1977). Ho et al. (1977) also showed that half-life of unchanged cocaine in the brain of rats repeatedly injected with the drug was shorter than that in saline-treated animals. They suggested that although cocaine is metabolized faster in

rats repeatedly injected with cocaine compared to single dose-treated animals, the continuous accumulation of the unchanged drug in tissues and fluids may account for the linear increase of motor activity during the initial 7 days. However, Post (1977) reported that cocaine levels in blood and cerebrospinal fluid (CSF) reach approximately similar peaks and have similar half-lives during both initial and chronic cocaine injections and the alterations of their cocaine levels with chronic administration are not sufficient to account for the progressive effects on behaviour or seizures. The present neurochemical data showed that increase in the DA levels in the CN and decrease in the 5-HT levels in the DM and PM, as observed after a single injection of cocaine reported previously from this laboratory (Pradhan et al., 1978) are further accentuated following its daily injections up to the 9th day. Thus, concomitant drug-induced changes in stereotypy and the DA level in the CN with repeated cocaine injection appear to be correlated. However. Post et al. (1976) failed to show any difference in the levels of the dopamine metabolite (homovanillic acid, HVA) after acute (first injection) or chronic (twice daily for 5 days per week for 13 weeks) cocaine administration in the cisternal CSF of monkeys. 5-Hydroxyindoleacetic acid levels in the cisternal CSF were not significantly elevated. Spontaneous motor activity has been shown to reflect the changes in NE level (Dolphin, Jenner and Marsden. 1976a, b; Maj, Grabowska and Mogilnicka, 1971) with or without the change in the DA level (Herman, 1970; Broitman and Donoso, 1971); however, the NE level does not show any significant change in this experiment. One of the reasons could be that the NE level usually increases at about 10min after cocaine injection and then decreases below the normal as shown in a previous study (Pradhan et al., 1978). Such an increase in the

563

Effects of repeated cocaine injections

NE level could not be seen in these experiments except on day 9, since they were conducted 20min after the drug injection; however, following repeated injections the decrease in the NE levels in the DM and PM was reversed, although only slight decreases were found on the 9th day. On day 18 and day 30, the levels of NE in the brain area after cocaine injection were below the normal and the increases in SMA were minimum. Thus a rough correlation could be Seen in the changes in NE levels and SMA following acute and chronic cocaine injections. The initial increases of NE and DA levels and their subsequent decrease in certain brain areas, particularly on day 0 and day 9 during chronic cocaine administration as shown in the present as well as in previous experiments (Pradhan et a[., 1978), probably indicate release of these transmitters that may account for the effects on SMA and stereotypy. On day 18 and day 30 such changes were minimal; the levels of NE and DA were below the normal. Concomitantly the behavioural changes were also minimal. A comparison of changes in the neurotransmitter levels at 20 min and 60 min post-drug on various days (Table 2) indicate that the later data were indicative of recovery processes in action. Because of some variability in the data, it was difficult to establish any trend in day-to-day changes in such a recovery process following repeated injections of cocaine. These experiments thus show that repeated daily administration of cocaine tends to show sensitization (or reverse tolerance) to SMA and stereotypy up to the 9th day following which a “relative tolerance” begins to develop although behavioural stimulation still persists to a certain extent. These drug-induced behavioural changes at various phases during chronic adminis~ation can be correlated to the changes in the levels of neurotransmitters (particularly NE and DA) in certain brain areas.

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