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Cocaine and dopaminergic actions in rat neostriatum
NeuropharmacologyVol. 32, No. 8, pp. 807-817, 1993 Printed in Great Britain
0028-3908193 $6.00+ 0.00 Pergamon Press Ltd
COCAINE AND DOPAMINERGIC ACTIONS IN RAT NEOSTRIATUM K. D. NANTWI and E. P. SCHOENER Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield, Detroit, MI 48201, U.S.A.
(Accepted 11 January 1993) Summary--Electrophysiologicalexperiments were conducted /n vivo to characterize the involvement of dopamine in effects of cocaine at the cellular level. L-DOPA (preceeded by carbidopa) evoked excitation and depression equally at 0.25 mg/kg; however, at 0.5 and 1.0 mg/kg depression in the basal rate of discharge predominated. Cocaine evoked similar responses. At 0.25 and 0.5mg/kg excitation and depression were elicited in about equal proportion, while at 1.0 mg/kg depression was predominant. Cocaine-induced neuronal changes were reversible by haloperidol. In experiments with specific dopamine antagonists, cocaine-induced neuronal depression was blocked with the D~ antagonist, SCH 23390 at a dose of 0.25 mg/kg. The D e antagonist, eticlopride, did not alter cocaine-induced depression in any specific manner. Excitation, following cocaine, was not altered by intervention with SCH 23390, yet it was blocked with the D 2 antagonist eticlopride at a dose of 0.25 mg/kg. The duration of these changes was consistent with the observed duration of the psychotropic actions of cocaine.
Key words--neostriatum, cocaine, dopamine, D t receptors, D 2 receptors.
Abuse of cocaine is a major health and social problem today, having grown steadily over the past 15 years (Adams and Durell, 1984). Chronic use of cocaine can lead to behavioral pathology, with toxic schizoid manifestations developing in severe cases. The latter is characterized by paranoia, hallucinations and delusions (Siegel, 1978; Jaffe, 1985) purportedly due to long-lasting alterations in the levels of uptake of dopamine (Roy, Bhattacharyga, Pradham and Pradham, 1978). In laboratory animals, acute administration of cocaine increases exploration, locomotion and grooming behavior at small doses (Scheel-Kruger et al., 1977). Larger doses lead to a decrease in stereotyped behavior (Randrup and Munkvad, 1967). Generally, repeated administrations of cocaine stimulate locomotor activity (Schuster and Balster, 1977), increase the intensity of stereotypies (Post and Rose, 1976), induce or exaggerate rotational behavior in animals with 6-OHDA-induced lesions in the .substantia nigra (Lin-Chu, Robinson and Becker, 1985) and enhance susceptibility to drug-induced convulsions with subthreshold doses (Stripling and Ellinwood, 1977). The critical element of dependence on cocaine may lie in its reinforcing euphorigenic effects. Physical dependence develops with repeated administration and termination of use of cocaine can lead to a predictable sequence of behavioral and physiological changes in humans and animals (Carroll, Lac and Nygaard, 1989; Woolverton and Kleeven, 1988). The neurochemical basis for the reinforcing effect of cocaine has not been fully elucidated, but inhibition of reuptake of catecholamines at the presynaptic NP32/s--F
membrane seems to play an important role (Ross and Renyi, 1966; Heikkila, Orlansky, Mytilineau and Coher, 1979; Gawin and Ellingwood, 1988). Studies have indicated that modulation of the sensitivity of dopamine receptors after blockade of monoamine reuptake is an important determinant in long-term effects of cocaine (Roy et aL, 1978). Although there is a current debate on the precise localization and mechanism of this effect, involvement of mesolimbic and mcsocortical dopaminergic systems appears unequivocal (Goeders and Smith, 1983; Goeders, Dworkin and Smith, 1986; Wise, 1987; Dworkin and Smith, 1987; Koob and Bloom, 1988). The dopaminergic system in the neostriatum plays a role in the motor effects of cocaine and may also be involved in reinforcement (Robbins and Sahakian, 1983). The goal of the present investigation was to characterize the manner in which specific dopaminergic mechanism(s) may be implicated in the cellular effects of cocaine. The study sought to determine the doserelated effects of cocaine on spontaneously-active striatal neurons and whether these are mediated by specific dopamine receptors. METHODS
Male Sprague-Dawley rats (200-275 g) were used in these experiments. They were housed 2 in a cage, with food and water available ad libitum, in an environmentally controlled room on a 12/12hr light/dark cycle. Prior to surgery, at 0800 hr the animals were anesthesized with urethane (ethyl carbamate, I g/kg, i.p.). A femoral vein and artery were cannulated to administer fluids and measure arterial
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K.D. NANTWland E. P. SCHOENER
blood pressure, respectively. A tracheotomy was performed to allow for ventilatory assistance, if necessary and to detect respiratory activity with a thermocouple. Body temperature was maintained at 37 + I°C. The subjects were mounted in a cranial stereotaxic apparatus, according to the atlas of Pellegrino, Pelligrino and Cushman (1981). A fronto-temporal craniotomy was performed and the meninges were removed to expose the cortex overlying the striatum. A glass-coated, platinum-iridium electrode (Wohlbarsht, MacNichol and Wagner, 1960), electrolytically etched to a tip diameter of 2-3/~m, was inserted at I mm anterior to Bregrna, 2-3 mm lateral of the midline and 3.0-3.8 mm below the surface for extracellular, spontaneous single unit recording. Signals were fed through a preamplifier and into a bandpass amplifier, conditioned and displayed on a dual-beam storage oscilloscope. The output from the amplifier was monitored continuously and the standard pulse generated by a Schmidt trigger was passed to a physiograph integrator to produce a record of average firing frequency (count-rate histograph over 10 sec periods).
All drugs were dissolved in 0.9% saline, except SCH 23390 and haloperidol, which were first solubilized in a few drops of glacial acetic or tartaric acid, then diluted with 0.9% saline and pH adjusted to 7.2-7.3 with NaOH. All of the test agents were made up in solution each day, except cocaine and eticlopride, which were stored for up to 1 week as a stock solution in sealed, light-tight glass bottles stored in a refrigerator.
General protocol
Histology
One neuron was examined per animal in this study. For each experiment neuronal activity was monitored continuously before, during and after administration of drugs. Average neuronal discharge rate was monitored for 10-15 min to establish a pattern that served as control. Thereafter, a test drug was injected. Changes in discharge rate following treatment with drug were examined and expressed on a normalized basis as a percentage of control. This allowed for the comparison of group data and accounted for individual differences in basal discharge rates. The duration of the effect of drug was defined as the time from onset of change in discharge rate/pattern, until recovery to control level. The time to achieve maximum response was characterized as the period from onset to the time of maximum response. Recovery was determined from vehicle control studies as the baseline discharge rate + 15%, the maximum extent of spontaneous fluctuations. Changes in neuronal firing after administration of drug, which were less than 15%, were considered insignificant. The EDs0 values for L-DOPA and cocaine were estimated from the respective dose-response curves. Biphasic responses were characterized by two distinguishable phases of excitation and depression. In studies on dopamine receptor antagonism, a test/retest paradigm was employed. Specifically, after establishment of a stable discharge rate, the response of a standard dose of cocaine was monitored before and after administration of a specific D, or De dopamine receptor antagonist. The magnitude of cocaineelicited response, before and after the antagonist, was then expressed on a normalized basis as a percentage of control activity. Procaine was administered in
At the end of each experiment, the location of the recording electrode was marked by passing a small current (2 hA) for 15 sec at each polarity. An intravenous overdose of urethane was followed by surgical opening of the thoracic cavity. The vascular system was then perfused by cardiac injection with normal saline (0.9%) and 10% buffered (phosphate) formalin. The brain was dissected from the cranium and fixed in formalin (10%) and butyraldehyde. Frozen serial sections were cut with a cryostat at 40tJm. Every third section was mounted and stained (cresyl violet) for later examination.
separate experiments to control for the possibility that local anesthetic properties of cocaine could be responsible for effects of the drug. Initially, to evaluate the effects of dopamine per se, the precursor Lfl-3,4-dihydroxyphenylethylamine (LDOPA), in conjunction with carbidopa (a specific inhibitor of peripheral aromatic amino acid decarboxylase) were investigated in the same manner as cocaine. The specific dopamine antagonists (R)-(+)8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3benzazepin-7-ol (SCG23390, D~) and eticlopride (D2) were utilized to assess the involvement of discrete dopamine receptors.
Drugs
Analysis of data All data were statistically analyzed using a 1-way analysis of variance (ANOVA) with repeated measures on one variable (dose). Neuronal activity was analyzed as a function of pretreatment and post hoc discharge rate. Individual comparisons between respective means were made with the Scheffe's F-test.
RESULTS A total of 173 striatal neurons was studied. The site of activity was histologically verified (Fig. 1). The average frequency of basal spikes, determined from physiographic count-rate records, ranged from 0.1 to 9 impulses per sec, with a mean of 2.9 + 0.6 impulses per sec for neurons that were depressed and 2.16+0.36 impulses per sec for those that were excited after eocene. Changes in the average firing rate, following administration of test drugs, did not appear to be related to the basal activity on either an absolute or normalized basis.
Fig. 1. Photomicrograph of a coronal section from the brain of a typical subject. Arrow indicates the site of neuronal recording produced by current passed directly (2 nA for 15 set) through the electrode. Abbreviations: cpu, caudate putamen; cc, corpus callosum; Is, lateral septal nucleus.
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Cocaine and dopaminergic actions Table 2. Effectsof cocaine on single unit dischargerate
Experiments with L-DOPA Administration of L-DOPA was followed by obvious changes in neuronal activity including excitatory, depressant and biphasic modulation of discharge rate (Table 1). All of these changes were temporally dissociated from transient changes in blood pressure and heart rate that occurred immediately after the injection and no variation of action potential shape or amplitude was observed. The predominant effect on neuronal discharge rate was a dose-related depression. Neuronal responses were qualitatively related to dose and evident at all doses within 6 min. It is noteworthy that at 0.25 mg/kg, L-DOPA produced depression and excitation in about equal proportions. Biphasic/bimodal responses were observed at 0.25 and 0.5 mg/kg L-DOPA and, in each of these cases, excitation preceded depression. Latency to onset and duration of depression were inversely and directly related to dose, respectively. Unlike depression, excitatory responses were random in magnitude and duration. In 6 experiments testing the involvement of dopamine receptors, the antagonist haloperidol (0.10.5 mg/kg) completely blocked all neuronal changes evoked by L-DOPA in a test/retest paradigm and when administered during the peak effect of L-DOPA. In the latter instance, recovery was accelerated.
Experiments with procaine Procaine, a local anesthetic like cocaine, was administered at 0.25, 0.5, l, 2 and 4 mg/kg, according to the same protocol as L-DOPA and cocaine. Selection of the doses of procaine was based on prior experience in similar experiments with neurons in the nucleus accumbens, dorsal raphe nucleus and the locus coeruleus in the rat (Pitts and Marwah, 1986). Even at the largest dose tested (4 mg/kg), procaine did not appreciably alter the discharge frequency of neurons. However, in some experiments, the action potential spike amplitude was decreased substantially at the largest dose. In all of these experiments, administration of procaine was followed by a (20-25%) decrease in blood pressure. The duration of this effect was random and neuronal activity did not vary reliably with it.
Experiments with cocaine Intravenous (i.v.) infusion of cocaine was followed consistently by significant modulation in discharge
0.25 0.50 1.0 Saline
9 7 7 4
Percentage Percentage Percentage Percentage depressed excited biphasic no response 33.3 57.1 85.7 0
33.3 28.6 14.3 0
11.1 14.3 0 0
Dose of drug Percentage Percentage Percentage Percentage (mg/kg) N depressed excited biphasic no response
0.25 o.5o l.OO
Saline
44 36 44 22
47.7 44.4 54.5 0
43.2 44.4 36.3 0
6.8 5.6 4.6 0
2.3 5.6 4.6 100
Cocaine-induced changes in neuronal behavior were related to dose of drug. At the smaller doses, depression and excitation were evenly distributed while, depression predominated at largest dose. Neurons were classified as nonresponsive if the they manifested <15% change in discharge rates following treatment.
rate (Table 2). A few neurons (5.6%) exhibited a biphasic response (excitation preceeding depression) similar to the behavior of striatal neurons following administration of dopamine via push-pull perfusion (Schoener, 1984) or the administration of L-DOPA. The onset of measurable change was evident within three min. Administration was frequently accompanied by a transient peripheral response (Fig. 2), manifested as an increase in blood pressure and a reflex decrease in heart rate. Neither the nature nor the magnitude of neuronal modulation was related to this. The pressor change was most likely due to activation of ~-adrenoceptors in peripheral blood vessels, as reported by Pitts and Marwah (1986). No modulation of the amplitude of action potentials occurred in these experiments, as they did following procaine. At small doses, approximately equal numbers of neurons were excited and depressed, while at the largest dose tested depression clearly predominated. Intervention with haloperidol (0.25 mg/kg) blocked or significantly attenuated all subsequent responses to cocaine.
Inhibition A significant proportion of the neurons tested was depressed (n = 61, r = 0.817) in a dose-related manner, following administration of cocaine (Fig. 3). While this inhibition was not related to the baseline mean discharge rate, slower units (0.1-0.25 ips) did not always recover fully to the original firing rate. The mean latency to onset and the time to achieve maximum depression for cocaine did not vary as a function of dose. However, the duration of effect of the drug was significantly related to the dose of cocaine (P < 0.05), with a maximum at 0.5 mg/kg. Certain aspects of cocaine-induced depression, the magnitude and duration of action in particular, were similar to L-DOPA-induced depression.
Excitation
Table 1. Effects of L-DOPA
Dose of drug (mg/kg) N
811
22.2 0 0 I00
Administration of L-DOPA selectively altered neuronal behavior. At a small dose, the responses were variable but depression and excitation were more prevalent than biphasic and nonresponsive cases. With larger doses, depression predominated.
The magnitude of neuronal facilitation was less robust than the inhibition (36--45% elevation, n = 51, r = 0.546). Typically, this increase was evident within 3 min and was more commonly observed at the small dose (0.25 mg/kg) than at larger doses. Latency to onset of excitation (2.18-2.89rain) was not significantly different from that for depression (2.26-2.36 min). The magnitude of excitation was not
K. D. NANTWl and E. P. SCHOENER
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Fig. 2. Depression of neuronal discharge rate after intravenous administration of cocaine in many experiments. Cardiovascular changes after injection of drug were always transient and temporally unrelated to the duration of neuronal depression. related to dose of drug. The most reliable dose--effect relationship was manifested for duration of action and paralleled that for depression, with an apparent m a x i m u m response occurring at 0.5 mg/kg.
D 1 antagonist S C H 23390
At a standard dose of 0.25mg/kg, SCH 23390 blocked or attenuated depression induced by cocaine
DEPRESSION OF NEOSTRIATAL NEURONS
FOLLOWING COCAINE 100'
**,* ! **,t
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0.25
0.5
1
DOSE OF COCAINE ( m g / k g ) Fig. 3. Cocaine depressed neuronal activity in a dose-related manner. Changes in mean firing rate were highly significant (P ~<0.0001)** when compared to vehicle (saline) controls. Percentage inhibition was significantly greater at 0.5* and 1" mg/kg than at 0.25 mg/kg (P <.0.05).
Cocaine and dopaminergic actions
813
ATTENUATION OF COCAINE-INDUCED DEPRESSlONBY D 1 ANTAGONIST, SCH 23390
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0.5
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DOSE OF DRUG (mg/kg)
Fig. 4. At a standard effective dose, the D 1 antagonist, SCH 23390 blocked and attenuated depression elicited by cocaine at 0.25 and 0.50 mg/kg, respectively; however, at the largest dose of cocaine, the antagonist was not effective. **Indicate P < 0.05 determined by Scheffe F-test. (Figs 4 and 5) in a test/retest paradigm. In control experiments, SCH 23390 itself evoked depression with a mean + SEM of 39.0 _ 5.9% and 41.0 + 5.3% at 0.1 (n = 6) and 0.25 (n = 7) mg/kg, respectively. Two neurons were nonresponsive and another manifested a biphasic response after the antagonist challenge at 0.1 and 0.25 mg/kg, respectively. When employed in the same test/retest paradigm for experiments in which cocaine initially provoked facilitation, SCH 23390 did not yield reliable results. In 6 experiments, cocaine-elicited excitation was blocked in one, unaltered in two, increased in two and reversed in one.
of control experiments elicited a modest excitation of 41.8 + 10.8% and 30.8 + 3.3% above control discharge rate at 0.1 and 0.25mg/kg, respectively. Three neurons were nonresponsive to the antagonist at 0.1 mg/kg (n = 1) and 0.25mg/kg (n = 2 ) . One neuron was depressed following eticlopride (0.1 mg/kg). When the same protocol was utilized in experiments with cocaine that initially revealed depression, the effects of cocaine following eticlopride produced wide fluctuations in neuronal behavior, precluding any definite inference (Table 3).
Cocaine, SCH 23390 and eticlopride Eticlopride (1)2 antagonist) At a standard dose of 0.25 mg/kg, eticlopride significantly and reliably attenuated excitatory changes that followed administration of cocaine (Fig. 6). A typical record of antagonism in a test/retest paradigm clearly illustrates this interaction (Fig. 7). Administration of the antagonist alone in a series (n = 5)
0.25 mg/kg 0.25 mg/kg Coc SCH 23390
0.25 mg/kg Coc
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In a small number of additional experiments (4), it was possible to confirm the dopamine receptor selectivity using both D~ and D2 receptor-specific antagonists with cocaine. This demonstrated the effect of both dopamine receptor types on a single striatal neuron and their selective involvement in the specific neuronal responses elicited by cocaine (Fig. 8).
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Fig. 5. In a test/retest paradigm, neuronal depression elicited by 0.25 mg/kg cocaine was attenuated, following intervention with a challenge dose of SCH 23390 (0.25 mg/kg), as illustrated in this physiographic record.
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K.D. NAN'rWIand E. P. SCHOENER COCAINE AND DOPAMINE2 RECEPTOR ANTAGONIST, ETICLOPRIDE
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Fig. 6. At a challenge dose of 0.25 mg/kg, the D2 antagonist, eticlopride reversed facilitation after cocaine (0.25 and 0.5 mg/kg). Eticlopride itself modestly enhanced the neuronal discharge in control experiments. DISCUSSION
Several lines of evidence have indicated that behavioral manifestations of cocaine may be mediated by dopamine in the mesocorticolimbic system (Roberts, Koob, Klonoff and Fibiger, 1980; Goeders and Smith, 1986). Cellular actions of cocaine may be mediated similarly. To test this hypothesis, electrophysiological experiments were conducted on single, spontaneously-active neurons in the neostriatum of the rat. This area has been implicated in conditioned reinforcement (Robbins, Watson, Gaskin and Ennis, 1983) and is well-endowed with high affinity dopamine binding sites (Marshall, O'Dell, Navarrette and Rosenstein, 1990). Effects o f cocaine on single spontaneously-active units
In addition to its well-known action as a local anesthetic, cocaine is known to inhibit the reuptake of monoamines (Heikkila et al., 1975; Ross and 0.25 mg/kg Coc
Renyi, 1966). In the present experiments, effects of cocaine on neostriatal neurons were due either directly or indirectly to alteration(s) in activity of dopamine receptors. This was first indicated by antagonism of cocaine-induced changes with haloperidol, consistent with observations of Einhorn, Johansen and White (1988) in the nucleus accumbens of the rat. Increased concentration of dopamine in striatal interstitium has been observed by in vivo microdialysis following administration of cocaine (Bradberry and Roth, 1988; Nicolaysen, Pan and Justice, 1988; Hurd, Kehr and Ungerstedt 1988), supporting the idea that increased extracellular striatal dopamine, following administration of cocaine may be related to the neuronal behavior observed here. The effects of L-DOPA, observed in the current study, were consistent with those of cocaine and indirectly support a dopaminergic role in the cellular actions of cocaine. The possibility that the local anesthetic properties of cocaine were responsible for changes in neuronal activity was investigated by examining the effects of procaine. As in studies of the locus coeruleus of the rat (Pitts and Marwah, 1986) and the nucleus accumbens (Einhorn et al., 1988), procaine had no effect on the discharge rate of striatal neurons, short of local anesthesia at the larger doses (Taylor, 1959). Systemic administration of cocaine provoked a variety of changes in the discharge rate of neostriatal neurons. Depression, excitation and biphasic responses (excitation preceding depression) were observed. These effects were temporally distinct from brief pressor changes that occurred immediately after administration of drug. Similar findings were reported in the previous studies of Pitts and Marwah (1986) and Einhorn et al. (1988), under experimental conditions like those employed here. Cardiovascular effects of the drug were not likely to have been responsible for these neuronal changes. The majority of neurons tested showed a dosedependent depression in discharge rate and duration of effect after cocaine. This depression was unrelated 0.25 mg/kg Eric
0.25 mg/kg Coc
2.0
0
w
w o. 0
I
5 rain
I
Fig. 7. Excitation elicited by cocaine was susceptible to attenuation by the D2 antagonist, eticlopride. As illustrated in this physiograph of a test/retest paradigm, the initial neuronal excitation after cocaine was blocked after intervention with the antagonist.
Cocaine and dopaminergic actions Table 3. Effectsof eticlopride* on cocaine-elicitedinhibition Percentage inhibition pre-
Dose of cocaine
mg/kg 0.25 0.50
N 3 4
antagonist 47.67-[- 15.5 39.75_+7.3
i.00
7
87.14 -t- 7.3*
Percentage inhibition post-
antagonist 41.33 + 30.14 36.4_ 15.7 61.43+ 8.12
*Standard dose of 0.25mg/kg. The D2antagonist,eticlopridedid not alter cocaine-elicitedneuronal depressionreliably.The magnitudeof depressionwas significant (P < 0.05) at 1.0mg/kg. Interventionwith the antagonist in the test/retest paradigm provokedwide fluctuations. to basal activity, since comparable effects were observed with neurons of diverse firing rates and patterns, following administration of cocaine. Qiao, Daugherty, Wiggins and Dafny (1990) and Lacey Mercuri and North (1990) recently reported that locally administered cocaine induced depression in a majority of neostriatal, substantia nigra and ventral tegmental neurons tested. Latency to onset of depression was evident within 3 min here, as it was in studies of the nucleus accumbens (Einhorn et al., 1988). Neuronal recovery from depression was achieved usually within 20 rain. By 30 min, discharge rate had returned to pretreatment levels. This time--course paralleled the reportedly short duration of psychoactive effects of the drug (Fischman and Schuster, 1982). Depression of the rate of neuronal discharge after administration of cocaine was blocked by the nonselective dopamine receptor antagonist haloperidol. In subsequent experiments with selective dopamine antagonists, the D I ligand SCH 23390 blocked cocaine-induced depression in a dose-related manner, implying receptor selectivity for this cocaine-induced effect. In vitro studies on cells of the nucleus accumbens have shown similar selectivity (Uchimura and North, 1990). In the current investigation, the D2 receptor antagonist eticlopride did not produce a consistent reversal of cocaine-induced neuronal depression. In fact, following intervention with eticlopride, marked variations in neuronal discharge rates ensued. Therefore, it may be argued that D 2 receptors
0.5mg/kgCoc
815
are not directly involved in cocaine-induced inhibition of neuronal discharge. While cocaine-induced depression has been observed in other in vivo studies (Einhorn et al., 1988; Qiao et ai., 1990), the implication of selective DIreceptor involvement is novel. This action could be mediated postsynaptically, given the concentration of postsynaptic D~ receptors in the caudate-putamen (Altar and Marien, 1987). However, in light of the complex neuronal circuitry in the striatum, involvement of other neurotransmitters would seem plausible. The principal neurotransmitter at the terminals of the nigrostriatal pathway is dopamine (Anden, Carlsson, Dahlstrom, Fuxe, Hiltarp and Larsson, 1964). Both Dm and D 2 receptors are localized postsynaptically (Altar and Marien, 1987) and on intrinsic neurons (Girault, Spampinato, Glowinski and Bcsson, 1986). Since a large portion of striatal neurons utilize 7-aminobutyric acid (GABA, Ottersen and Storm-Mathisen, 1984), this may be of functional importance (Scheel-Kruger, 1986). Release of GABA in the striatum is modulated by dopamine (van der Heyden, Venema and Korf, 1980) and stimulation of D] and D2 receptors can facilitate (Girault et al., 1986) or inhibit (Tossman and Ungerstedt, 1986) release of endogenous GABA in the dorsal striatum, respectively. Depression of neuronal activity by cocaine could have been mediated through direct postsynaptic action; however, it is also possible that it occurred indirectly through enhanced release of GABA on neurons that interact with dopamine-responsive neurons. Striatal dopaminergic terminals make synaptic contact with intrinsic GABA-containing neurons (Kubota, Inagaki, Kito and Wu, 1987) and may influence their function. The preferential activation of striatal D] receptors is consistent with the predominant neuronal depression after cocaine. Facilitation of neuronal activity by cocaine occurred at all doses but most frequently at the smallest dose (0.25 mg/kg). Its magnitude was not related to
0.5mg/kgCoe
0.25mg/kgEllc
2"0I Q, 0
0.25 mg/kg SCH 23390
5 rain
Fig. 8. Selective blockade of both depression and excitation elicited by cocaine in single neurons was induced by specific dopamine receptor antagonists. In the physiograph recording above, SCH 23390 blocked depression a n d apparently unmasked excitation. Facilitation, following a second administration of cocaine, was reversed by eticlopride.
816
K.D. NA~rrwl and E. P. SCHOE~dt
dose and was significantly less than the magnitude of depression at all doses. Based in part on reversal by haloperidol o f L-DOPA-induced facilitation, and the differential affinity of D~ and De receptors for dopamine (Creese, Sibley, Hamblin and Left, 1982) it was tempting to speculate that specific activation of dopamine receptors of the D~ type mediated this neuronal change. This was confirmed by the consistent reversal of facilitation after intervention with the selective D2 receptor antagonist, eticlopride. While precise localization of the receptors was not undertaken in the present study, it is conceivable that postsynaptic (Altar and Marien, 1987) and intrinsic striatal (Girault et al., 1986) D2 receptors were implicated. The random effect of SCH 23390 on cocaineinduced facilitation, strongly suggests that activation of D1 receptors was not involved with excitation. Substantial evidence for specificity in the effects of cocaine came from the biphasic responses of some neurons and the observation that depression and facilitation could be blocked selectively by SCH 23390 and eticlopride, respectively. These findings indicate that D~ and D2 receptors may be present and functional on the same neuron as reported by Akaike, Ohno, Sasa and Takaori (1987) and inferred from earlier studies of dopaminergic actions on formation of cyclic adenosine monophosphate (cAMP) in slices o f brain (Stoof and Kebabian, 1981) and primary cell cultures (Chneiweiss, Glowinski and Premont, 1988). Acknowledgements--These studies were supported in part
by grants from the National Institute on Health (GM 08167) and Alcohol, Drug Abuse, and Mental Health Administration (MH 471 81). The authors are indebted to A. Barnett of the Schering-Plough Corporation for kindly providing the SCH 23390 used in these studies. The assistance of Michael Parizon and David Bradley Jr in the histological preparations is most appreciated. We are grateful to Dr Robin Barraco for his helpful discussions.
REFERENCES
Adams E. H. and Durrell J. (1984) A growing public health problem. In: Cocaine: Pharmacology, Effects, and Treatment of Abuse (Grabowski J., Ed.). National Institute on Drug Abuse Research Monograph 50, Rockville, Maryland. Akaike A., Ohno Y., Sasa M. and Takaori S. (1987) Excitatory and inhibitory effects of dopamine on neuronal activity of the caudate nucleus neurons in vitro. Brain Res. 418: 262-272. Altar C. A. and Marien M. R. (1987) Picomolar affinity of [12sI]SCH 23982 for D~ receptors in brain demonstrated with digital subtraction autoradiography. J. Neurosci. 7: 213-222. Anden N. E., Carlsson A., Dahlstrom A., Fuxe K. L., Hillarp N. A. and Larsson, K. (1964) Demonstration and mapping out of neostriatal dopamine neurons. Life Sci. 3: 523-530. Carroll M. E., Lac S. T. and Nygaard S. T. (1989) A currently available nondrug reinforcer prevents the acquisition or decreases the maintenance of cocaine-reinforced behavior. Psychopharmacology 97: 23-29. Chneiweiss H., Glowinski J. and Premont J. (1988) Mu and delta opiate receptors coupled negatively to adenylate
cyclase on embryonic neurons from the mouse striatum in primary cultures. J. Neurosci. 8: 3376-3382. Creese I., Sibley D. R., Hamblin M. W. and LeftS. E. (1983) The classification of dopamine receptors: relationship to radioligand binding. A. Rev. Neurosci. 6: 43-71. Einhorn L. C., Johansen P. A. and White F. J. (1988) Electrophysiological effects of cocaine in the mesoaccumhens dopamine system: studies in the ventral tegmental area. J. Neurosci. 8: 100-112. Fischman M. W. and Schuster C. R. (1982) Cocaine selfadministration in humans. Fedn Proc. 41: 241-246. Gawin F. H., and Ellinwood E. H. (1988) Cocaine and ct.her stimulants: actions, abuse and treatment. New Engl. J. Med. 318:1173-1182. Girault J. A., Spampinato U., Glowinski J. and Besson M. J. (1986) In vivo release of3H ~,-Aminobutyric acid in the rat neostriatum--I 1. Opposing effects of D~ and D 2 dopamine receptor stimulation in the dorsal caudate putamen. Neuroscience 19: i 109-1117. Goeders N. E. and Smith J. E. (1983) Cortical dopaminergic involvement in cocaine reinforcement. Science 221: 773-775. Goeders N. E., Dworkin S. I. and Smith J. E. (1986) Neuropharmacological assessment of cocaine selfadministration into medial prefrontal cortex. Pharmac. Biochem. Behav. 24: 1429-1440. Heikkila R. E., Orlansky H., Mytilineau C. and Cohen G. (1975) Amphetamine: evaluation of D- and L-isomers as releasing agents and uptake inhibitors for [3H]dopamine and norepinephrine in slices of rat neostriatum and cerebral cortex. J. Pharmac. Exp. Ther. 194: 47-56. van der Heyden J. A. M., Venema K. and Korf J. (1980) In vivo release of endogenous GABA from rat striatum: inhibition by dopamine. J. Neurochem. 34: 1338-1341. Hurd Y. L., Kehr J. and Ungerstedt U. (1988) In vivo microdialysis as a technique to monitor drug transport: Correlation of extracellular cocaine levels and dopamine overflow in rat brain. J. Neurochem. 51: 1314-1316. 129: 379-385. Jaffe J. H. (1985) Forward. In: Cocaine Use in America: Epidemiologic and Clinical Perspectives. (Kozel N. J. and Adams E. H,, Ed.), p. v. National Institute on Drug Abuse Research Monograph 61, U.S. Government Printing Office, Washington, District of Columbia. Koob G. F. and Bloom F. E. (1988) Cellular and molecular mechanisms of drug dependence. Science, Wash. D.C. 242:715-723. Kubota Y., Inagaki S., Kito S. and Wu J.-Y. (1987) Dopaminergic axons directly make synapses with GABAergic neurons in the rat neostriatum. Brain. Res. 406: 147-156. Lacey M. G., Mercuri N. B. and North R. A. (1990) Actions of cocaine on rat dopaminergic neurones in vitro. Br. J. Pharmac. 99: 731-735. Lin-Chu G., Robinson T. E. and Becker J. B. (1985) Sensitization of rotational behavior produced by a single exposure to cocaine. Pharmac. Biochem. Behav. 22: 901-903. Marshall J. F., O'Dell S. J., Navarrete R. and Rosenstein A. J. (1990) Dopamine high-affinity transport in rat brain: major differences between dorsal and ventral striatum. Neuroscience 37:11-21. Nicolaysen L. C., Pan H. and Justice J. B. (1988) Extracellular cocaine and dopamine concentrations are linearly related in rat neostriatum. Brain Res. 456: 317-323. Ottersen O. P. and Storm-Mathisen J. (1984) Glutamateand GABA-containing neurons in the mouse and rat brain, as demonstrated with a new immunocytochemical technique. J. Comp. Neurol. 229: 374-392. Pellegrino L. J., Pellegrino A. S. and Cushman A. J. (1981) A Stereotaxic Atlas of the Rat Brain, 2 edn. Plenum Press, New York.
Cocaine and dopaminerglc actions Pitts D. K. and Marwah J. (1986) Electrophysiologlcal actions of cocaine on noradrenergic neurons in the rat locus coeruleus. J. Pharmac. Exp. Ther. 240: 345-351. Post R. M. and Rose H. (1976) Increasing effects of repetitive cocaine administration in the rat. Nature, Lond. 260: 731-732. Qiao, J.-T., Dougherty P. M., Wiggins R. C. and Dafny N. (1990) Effects of microiontophoretic application of cocaine, alone and with receptor antagonists, upon the neurons of the medial prefrontal cortex, nucleus accumhens and caudate nucleus of rats. Neuropharmacology 29: 379-385. Randrup A. and Munkvad I. (1967) Stereotyped activity produced by amphetamine in several species and man. Psychopharmacology 11: 300-311. Robbins T. W., Watson B. A., Gaskin M. and Ennis C. (1983) Contrasting interactions of pipradrol, d-amphetamine, cocaine, cocaine analogues, apomorphine and other drugs with conditioned reinforcement. Psychopharmacology 80:113-119. Roberts D. C. S., Koob G. F., Klonoff P. and Fibiger, H. C. (1980) Extinction and recovery of cocaine selfadministration following 6-hydroxydopamine lesions of the nucleus accumbens. Pharmac. Biochem. Behav. 12: 781-787. Ross S. B. and Renyi A. L. (1966) Uptake of some tritiated sympathomimetic amines by mouse brain cortex in vitro. Acta Pharmac. Tox. 24: 297-309. Roy S. N., Bhattacharyya A. K., Pradhan S. and Pradhan S. N. (1978) Behavioral and neurochemical effects of repeated administration of cocaine in rats. Neuropharmacology 17: 559-564. Scheei-Kruger J. (1986) Dopamine-GABA interactions: evidence that GABA transmits, modulates and mediates
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dopaminerglc functions in the basal ganglia and the limbic system. J. Acta Neurol. Scand. 73: Suppl. 107, 1-49. Schoener E. P. and Elkins D. P. (1984) Neuronal response to dopamine in rat neostriatum. A push-pull perfusion study. Neuropharmacology 23: 611-616. Schuster C. R. and Balster R. L. (1977) The discriminative stimulus properties of drugs. In: Advances in Behavioral Pharmacology (Thompson T. Y. and Dews P. B., Eds), Voi. 1, pp. 85-138. Academic Press, New York. Siegel R. K. (1978) Cocaine hallucinations. Am. J. Psychiat. 135: 309-314. Stoof J. C. and Kebabian J. W. (1981) Opposing roles for Dz and D 2 dopamine receptors in efflux of cyclic AMP from rat neostriatum. Nature 294: 366-368. Stripling J. S. and Ellinwood E. H. (1977) Sensitization to cocaine following chronic administration in the rat. In: Cocaine and other Stimulants (Ellinwood E. H. Jr and Kilbey M. M., Eds), pp. 327-351. Plenum Press, New York. Taylor R. E. (1959) Effect of procaine on electrical properties of squid axon membrane. Am. J. Physiol. 196: 1071-1078. Tossman U. and Ungerstedt U. (1986) The effect of apomorphine on the potassium-evoked overflow of GABA in rat striatum studied by microdialysis. Eur. 3". Pharmac. 123: 295-298. Uchimura N. and North R. A. (1990) Actions of cocaine on rat nucleus aceumbens neurones/n vitro. Br. J. Pharmac. 99:. 736-740. Wohlbarsht M. L., MacNichol E. F. Jr and Wagner H. G. (1960) Glasscoated platinum microelectrode. Science 132: 1309-1310. Woolverton W. L. and Kleven M. S. (1988) Evidence for cocaine dependence in monkeys, following prolonged exposure. Psychopharmacology 94: 288-291.
0028-3908193 $6.00+ 0.00 Pergamon Press Ltd
COCAINE AND DOPAMINERGIC ACTIONS IN RAT NEOSTRIATUM K. D. NANTWI and E. P. SCHOENER Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield, Detroit, MI 48201, U.S.A.
(Accepted 11 January 1993) Summary--Electrophysiologicalexperiments were conducted /n vivo to characterize the involvement of dopamine in effects of cocaine at the cellular level. L-DOPA (preceeded by carbidopa) evoked excitation and depression equally at 0.25 mg/kg; however, at 0.5 and 1.0 mg/kg depression in the basal rate of discharge predominated. Cocaine evoked similar responses. At 0.25 and 0.5mg/kg excitation and depression were elicited in about equal proportion, while at 1.0 mg/kg depression was predominant. Cocaine-induced neuronal changes were reversible by haloperidol. In experiments with specific dopamine antagonists, cocaine-induced neuronal depression was blocked with the D~ antagonist, SCH 23390 at a dose of 0.25 mg/kg. The D e antagonist, eticlopride, did not alter cocaine-induced depression in any specific manner. Excitation, following cocaine, was not altered by intervention with SCH 23390, yet it was blocked with the D 2 antagonist eticlopride at a dose of 0.25 mg/kg. The duration of these changes was consistent with the observed duration of the psychotropic actions of cocaine.
Key words--neostriatum, cocaine, dopamine, D t receptors, D 2 receptors.
Abuse of cocaine is a major health and social problem today, having grown steadily over the past 15 years (Adams and Durell, 1984). Chronic use of cocaine can lead to behavioral pathology, with toxic schizoid manifestations developing in severe cases. The latter is characterized by paranoia, hallucinations and delusions (Siegel, 1978; Jaffe, 1985) purportedly due to long-lasting alterations in the levels of uptake of dopamine (Roy, Bhattacharyga, Pradham and Pradham, 1978). In laboratory animals, acute administration of cocaine increases exploration, locomotion and grooming behavior at small doses (Scheel-Kruger et al., 1977). Larger doses lead to a decrease in stereotyped behavior (Randrup and Munkvad, 1967). Generally, repeated administrations of cocaine stimulate locomotor activity (Schuster and Balster, 1977), increase the intensity of stereotypies (Post and Rose, 1976), induce or exaggerate rotational behavior in animals with 6-OHDA-induced lesions in the .substantia nigra (Lin-Chu, Robinson and Becker, 1985) and enhance susceptibility to drug-induced convulsions with subthreshold doses (Stripling and Ellinwood, 1977). The critical element of dependence on cocaine may lie in its reinforcing euphorigenic effects. Physical dependence develops with repeated administration and termination of use of cocaine can lead to a predictable sequence of behavioral and physiological changes in humans and animals (Carroll, Lac and Nygaard, 1989; Woolverton and Kleeven, 1988). The neurochemical basis for the reinforcing effect of cocaine has not been fully elucidated, but inhibition of reuptake of catecholamines at the presynaptic NP32/s--F
membrane seems to play an important role (Ross and Renyi, 1966; Heikkila, Orlansky, Mytilineau and Coher, 1979; Gawin and Ellingwood, 1988). Studies have indicated that modulation of the sensitivity of dopamine receptors after blockade of monoamine reuptake is an important determinant in long-term effects of cocaine (Roy et aL, 1978). Although there is a current debate on the precise localization and mechanism of this effect, involvement of mesolimbic and mcsocortical dopaminergic systems appears unequivocal (Goeders and Smith, 1983; Goeders, Dworkin and Smith, 1986; Wise, 1987; Dworkin and Smith, 1987; Koob and Bloom, 1988). The dopaminergic system in the neostriatum plays a role in the motor effects of cocaine and may also be involved in reinforcement (Robbins and Sahakian, 1983). The goal of the present investigation was to characterize the manner in which specific dopaminergic mechanism(s) may be implicated in the cellular effects of cocaine. The study sought to determine the doserelated effects of cocaine on spontaneously-active striatal neurons and whether these are mediated by specific dopamine receptors. METHODS
Male Sprague-Dawley rats (200-275 g) were used in these experiments. They were housed 2 in a cage, with food and water available ad libitum, in an environmentally controlled room on a 12/12hr light/dark cycle. Prior to surgery, at 0800 hr the animals were anesthesized with urethane (ethyl carbamate, I g/kg, i.p.). A femoral vein and artery were cannulated to administer fluids and measure arterial
807
808
K.D. NANTWland E. P. SCHOENER
blood pressure, respectively. A tracheotomy was performed to allow for ventilatory assistance, if necessary and to detect respiratory activity with a thermocouple. Body temperature was maintained at 37 + I°C. The subjects were mounted in a cranial stereotaxic apparatus, according to the atlas of Pellegrino, Pelligrino and Cushman (1981). A fronto-temporal craniotomy was performed and the meninges were removed to expose the cortex overlying the striatum. A glass-coated, platinum-iridium electrode (Wohlbarsht, MacNichol and Wagner, 1960), electrolytically etched to a tip diameter of 2-3/~m, was inserted at I mm anterior to Bregrna, 2-3 mm lateral of the midline and 3.0-3.8 mm below the surface for extracellular, spontaneous single unit recording. Signals were fed through a preamplifier and into a bandpass amplifier, conditioned and displayed on a dual-beam storage oscilloscope. The output from the amplifier was monitored continuously and the standard pulse generated by a Schmidt trigger was passed to a physiograph integrator to produce a record of average firing frequency (count-rate histograph over 10 sec periods).
All drugs were dissolved in 0.9% saline, except SCH 23390 and haloperidol, which were first solubilized in a few drops of glacial acetic or tartaric acid, then diluted with 0.9% saline and pH adjusted to 7.2-7.3 with NaOH. All of the test agents were made up in solution each day, except cocaine and eticlopride, which were stored for up to 1 week as a stock solution in sealed, light-tight glass bottles stored in a refrigerator.
General protocol
Histology
One neuron was examined per animal in this study. For each experiment neuronal activity was monitored continuously before, during and after administration of drugs. Average neuronal discharge rate was monitored for 10-15 min to establish a pattern that served as control. Thereafter, a test drug was injected. Changes in discharge rate following treatment with drug were examined and expressed on a normalized basis as a percentage of control. This allowed for the comparison of group data and accounted for individual differences in basal discharge rates. The duration of the effect of drug was defined as the time from onset of change in discharge rate/pattern, until recovery to control level. The time to achieve maximum response was characterized as the period from onset to the time of maximum response. Recovery was determined from vehicle control studies as the baseline discharge rate + 15%, the maximum extent of spontaneous fluctuations. Changes in neuronal firing after administration of drug, which were less than 15%, were considered insignificant. The EDs0 values for L-DOPA and cocaine were estimated from the respective dose-response curves. Biphasic responses were characterized by two distinguishable phases of excitation and depression. In studies on dopamine receptor antagonism, a test/retest paradigm was employed. Specifically, after establishment of a stable discharge rate, the response of a standard dose of cocaine was monitored before and after administration of a specific D, or De dopamine receptor antagonist. The magnitude of cocaineelicited response, before and after the antagonist, was then expressed on a normalized basis as a percentage of control activity. Procaine was administered in
At the end of each experiment, the location of the recording electrode was marked by passing a small current (2 hA) for 15 sec at each polarity. An intravenous overdose of urethane was followed by surgical opening of the thoracic cavity. The vascular system was then perfused by cardiac injection with normal saline (0.9%) and 10% buffered (phosphate) formalin. The brain was dissected from the cranium and fixed in formalin (10%) and butyraldehyde. Frozen serial sections were cut with a cryostat at 40tJm. Every third section was mounted and stained (cresyl violet) for later examination.
separate experiments to control for the possibility that local anesthetic properties of cocaine could be responsible for effects of the drug. Initially, to evaluate the effects of dopamine per se, the precursor Lfl-3,4-dihydroxyphenylethylamine (LDOPA), in conjunction with carbidopa (a specific inhibitor of peripheral aromatic amino acid decarboxylase) were investigated in the same manner as cocaine. The specific dopamine antagonists (R)-(+)8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3benzazepin-7-ol (SCG23390, D~) and eticlopride (D2) were utilized to assess the involvement of discrete dopamine receptors.
Drugs
Analysis of data All data were statistically analyzed using a 1-way analysis of variance (ANOVA) with repeated measures on one variable (dose). Neuronal activity was analyzed as a function of pretreatment and post hoc discharge rate. Individual comparisons between respective means were made with the Scheffe's F-test.
RESULTS A total of 173 striatal neurons was studied. The site of activity was histologically verified (Fig. 1). The average frequency of basal spikes, determined from physiographic count-rate records, ranged from 0.1 to 9 impulses per sec, with a mean of 2.9 + 0.6 impulses per sec for neurons that were depressed and 2.16+0.36 impulses per sec for those that were excited after eocene. Changes in the average firing rate, following administration of test drugs, did not appear to be related to the basal activity on either an absolute or normalized basis.
Fig. 1. Photomicrograph of a coronal section from the brain of a typical subject. Arrow indicates the site of neuronal recording produced by current passed directly (2 nA for 15 set) through the electrode. Abbreviations: cpu, caudate putamen; cc, corpus callosum; Is, lateral septal nucleus.
809
Cocaine and dopaminergic actions Table 2. Effectsof cocaine on single unit dischargerate
Experiments with L-DOPA Administration of L-DOPA was followed by obvious changes in neuronal activity including excitatory, depressant and biphasic modulation of discharge rate (Table 1). All of these changes were temporally dissociated from transient changes in blood pressure and heart rate that occurred immediately after the injection and no variation of action potential shape or amplitude was observed. The predominant effect on neuronal discharge rate was a dose-related depression. Neuronal responses were qualitatively related to dose and evident at all doses within 6 min. It is noteworthy that at 0.25 mg/kg, L-DOPA produced depression and excitation in about equal proportions. Biphasic/bimodal responses were observed at 0.25 and 0.5 mg/kg L-DOPA and, in each of these cases, excitation preceded depression. Latency to onset and duration of depression were inversely and directly related to dose, respectively. Unlike depression, excitatory responses were random in magnitude and duration. In 6 experiments testing the involvement of dopamine receptors, the antagonist haloperidol (0.10.5 mg/kg) completely blocked all neuronal changes evoked by L-DOPA in a test/retest paradigm and when administered during the peak effect of L-DOPA. In the latter instance, recovery was accelerated.
Experiments with procaine Procaine, a local anesthetic like cocaine, was administered at 0.25, 0.5, l, 2 and 4 mg/kg, according to the same protocol as L-DOPA and cocaine. Selection of the doses of procaine was based on prior experience in similar experiments with neurons in the nucleus accumbens, dorsal raphe nucleus and the locus coeruleus in the rat (Pitts and Marwah, 1986). Even at the largest dose tested (4 mg/kg), procaine did not appreciably alter the discharge frequency of neurons. However, in some experiments, the action potential spike amplitude was decreased substantially at the largest dose. In all of these experiments, administration of procaine was followed by a (20-25%) decrease in blood pressure. The duration of this effect was random and neuronal activity did not vary reliably with it.
Experiments with cocaine Intravenous (i.v.) infusion of cocaine was followed consistently by significant modulation in discharge
0.25 0.50 1.0 Saline
9 7 7 4
Percentage Percentage Percentage Percentage depressed excited biphasic no response 33.3 57.1 85.7 0
33.3 28.6 14.3 0
11.1 14.3 0 0
Dose of drug Percentage Percentage Percentage Percentage (mg/kg) N depressed excited biphasic no response
0.25 o.5o l.OO
Saline
44 36 44 22
47.7 44.4 54.5 0
43.2 44.4 36.3 0
6.8 5.6 4.6 0
2.3 5.6 4.6 100
Cocaine-induced changes in neuronal behavior were related to dose of drug. At the smaller doses, depression and excitation were evenly distributed while, depression predominated at largest dose. Neurons were classified as nonresponsive if the they manifested <15% change in discharge rates following treatment.
rate (Table 2). A few neurons (5.6%) exhibited a biphasic response (excitation preceeding depression) similar to the behavior of striatal neurons following administration of dopamine via push-pull perfusion (Schoener, 1984) or the administration of L-DOPA. The onset of measurable change was evident within three min. Administration was frequently accompanied by a transient peripheral response (Fig. 2), manifested as an increase in blood pressure and a reflex decrease in heart rate. Neither the nature nor the magnitude of neuronal modulation was related to this. The pressor change was most likely due to activation of ~-adrenoceptors in peripheral blood vessels, as reported by Pitts and Marwah (1986). No modulation of the amplitude of action potentials occurred in these experiments, as they did following procaine. At small doses, approximately equal numbers of neurons were excited and depressed, while at the largest dose tested depression clearly predominated. Intervention with haloperidol (0.25 mg/kg) blocked or significantly attenuated all subsequent responses to cocaine.
Inhibition A significant proportion of the neurons tested was depressed (n = 61, r = 0.817) in a dose-related manner, following administration of cocaine (Fig. 3). While this inhibition was not related to the baseline mean discharge rate, slower units (0.1-0.25 ips) did not always recover fully to the original firing rate. The mean latency to onset and the time to achieve maximum depression for cocaine did not vary as a function of dose. However, the duration of effect of the drug was significantly related to the dose of cocaine (P < 0.05), with a maximum at 0.5 mg/kg. Certain aspects of cocaine-induced depression, the magnitude and duration of action in particular, were similar to L-DOPA-induced depression.
Excitation
Table 1. Effects of L-DOPA
Dose of drug (mg/kg) N
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22.2 0 0 I00
Administration of L-DOPA selectively altered neuronal behavior. At a small dose, the responses were variable but depression and excitation were more prevalent than biphasic and nonresponsive cases. With larger doses, depression predominated.
The magnitude of neuronal facilitation was less robust than the inhibition (36--45% elevation, n = 51, r = 0.546). Typically, this increase was evident within 3 min and was more commonly observed at the small dose (0.25 mg/kg) than at larger doses. Latency to onset of excitation (2.18-2.89rain) was not significantly different from that for depression (2.26-2.36 min). The magnitude of excitation was not
K. D. NANTWl and E. P. SCHOENER
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Fig. 2. Depression of neuronal discharge rate after intravenous administration of cocaine in many experiments. Cardiovascular changes after injection of drug were always transient and temporally unrelated to the duration of neuronal depression. related to dose of drug. The most reliable dose--effect relationship was manifested for duration of action and paralleled that for depression, with an apparent m a x i m u m response occurring at 0.5 mg/kg.
D 1 antagonist S C H 23390
At a standard dose of 0.25mg/kg, SCH 23390 blocked or attenuated depression induced by cocaine
DEPRESSION OF NEOSTRIATAL NEURONS
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DOSE OF COCAINE ( m g / k g ) Fig. 3. Cocaine depressed neuronal activity in a dose-related manner. Changes in mean firing rate were highly significant (P ~<0.0001)** when compared to vehicle (saline) controls. Percentage inhibition was significantly greater at 0.5* and 1" mg/kg than at 0.25 mg/kg (P <.0.05).
Cocaine and dopaminergic actions
813
ATTENUATION OF COCAINE-INDUCED DEPRESSlONBY D 1 ANTAGONIST, SCH 23390
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Fig. 4. At a standard effective dose, the D 1 antagonist, SCH 23390 blocked and attenuated depression elicited by cocaine at 0.25 and 0.50 mg/kg, respectively; however, at the largest dose of cocaine, the antagonist was not effective. **Indicate P < 0.05 determined by Scheffe F-test. (Figs 4 and 5) in a test/retest paradigm. In control experiments, SCH 23390 itself evoked depression with a mean + SEM of 39.0 _ 5.9% and 41.0 + 5.3% at 0.1 (n = 6) and 0.25 (n = 7) mg/kg, respectively. Two neurons were nonresponsive and another manifested a biphasic response after the antagonist challenge at 0.1 and 0.25 mg/kg, respectively. When employed in the same test/retest paradigm for experiments in which cocaine initially provoked facilitation, SCH 23390 did not yield reliable results. In 6 experiments, cocaine-elicited excitation was blocked in one, unaltered in two, increased in two and reversed in one.
of control experiments elicited a modest excitation of 41.8 + 10.8% and 30.8 + 3.3% above control discharge rate at 0.1 and 0.25mg/kg, respectively. Three neurons were nonresponsive to the antagonist at 0.1 mg/kg (n = 1) and 0.25mg/kg (n = 2 ) . One neuron was depressed following eticlopride (0.1 mg/kg). When the same protocol was utilized in experiments with cocaine that initially revealed depression, the effects of cocaine following eticlopride produced wide fluctuations in neuronal behavior, precluding any definite inference (Table 3).
Cocaine, SCH 23390 and eticlopride Eticlopride (1)2 antagonist) At a standard dose of 0.25 mg/kg, eticlopride significantly and reliably attenuated excitatory changes that followed administration of cocaine (Fig. 6). A typical record of antagonism in a test/retest paradigm clearly illustrates this interaction (Fig. 7). Administration of the antagonist alone in a series (n = 5)
0.25 mg/kg 0.25 mg/kg Coc SCH 23390
0.25 mg/kg Coc
t
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In a small number of additional experiments (4), it was possible to confirm the dopamine receptor selectivity using both D~ and D2 receptor-specific antagonists with cocaine. This demonstrated the effect of both dopamine receptor types on a single striatal neuron and their selective involvement in the specific neuronal responses elicited by cocaine (Fig. 8).
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Fig. 5. In a test/retest paradigm, neuronal depression elicited by 0.25 mg/kg cocaine was attenuated, following intervention with a challenge dose of SCH 23390 (0.25 mg/kg), as illustrated in this physiographic record.
814
K.D. NAN'rWIand E. P. SCHOENER COCAINE AND DOPAMINE2 RECEPTOR ANTAGONIST, ETICLOPRIDE
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Fig. 6. At a challenge dose of 0.25 mg/kg, the D2 antagonist, eticlopride reversed facilitation after cocaine (0.25 and 0.5 mg/kg). Eticlopride itself modestly enhanced the neuronal discharge in control experiments. DISCUSSION
Several lines of evidence have indicated that behavioral manifestations of cocaine may be mediated by dopamine in the mesocorticolimbic system (Roberts, Koob, Klonoff and Fibiger, 1980; Goeders and Smith, 1986). Cellular actions of cocaine may be mediated similarly. To test this hypothesis, electrophysiological experiments were conducted on single, spontaneously-active neurons in the neostriatum of the rat. This area has been implicated in conditioned reinforcement (Robbins, Watson, Gaskin and Ennis, 1983) and is well-endowed with high affinity dopamine binding sites (Marshall, O'Dell, Navarrette and Rosenstein, 1990). Effects o f cocaine on single spontaneously-active units
In addition to its well-known action as a local anesthetic, cocaine is known to inhibit the reuptake of monoamines (Heikkila et al., 1975; Ross and 0.25 mg/kg Coc
Renyi, 1966). In the present experiments, effects of cocaine on neostriatal neurons were due either directly or indirectly to alteration(s) in activity of dopamine receptors. This was first indicated by antagonism of cocaine-induced changes with haloperidol, consistent with observations of Einhorn, Johansen and White (1988) in the nucleus accumbens of the rat. Increased concentration of dopamine in striatal interstitium has been observed by in vivo microdialysis following administration of cocaine (Bradberry and Roth, 1988; Nicolaysen, Pan and Justice, 1988; Hurd, Kehr and Ungerstedt 1988), supporting the idea that increased extracellular striatal dopamine, following administration of cocaine may be related to the neuronal behavior observed here. The effects of L-DOPA, observed in the current study, were consistent with those of cocaine and indirectly support a dopaminergic role in the cellular actions of cocaine. The possibility that the local anesthetic properties of cocaine were responsible for changes in neuronal activity was investigated by examining the effects of procaine. As in studies of the locus coeruleus of the rat (Pitts and Marwah, 1986) and the nucleus accumbens (Einhorn et al., 1988), procaine had no effect on the discharge rate of striatal neurons, short of local anesthesia at the larger doses (Taylor, 1959). Systemic administration of cocaine provoked a variety of changes in the discharge rate of neostriatal neurons. Depression, excitation and biphasic responses (excitation preceding depression) were observed. These effects were temporally distinct from brief pressor changes that occurred immediately after administration of drug. Similar findings were reported in the previous studies of Pitts and Marwah (1986) and Einhorn et al. (1988), under experimental conditions like those employed here. Cardiovascular effects of the drug were not likely to have been responsible for these neuronal changes. The majority of neurons tested showed a dosedependent depression in discharge rate and duration of effect after cocaine. This depression was unrelated 0.25 mg/kg Eric
0.25 mg/kg Coc
2.0
0
w
w o. 0
I
5 rain
I
Fig. 7. Excitation elicited by cocaine was susceptible to attenuation by the D2 antagonist, eticlopride. As illustrated in this physiograph of a test/retest paradigm, the initial neuronal excitation after cocaine was blocked after intervention with the antagonist.
Cocaine and dopaminergic actions Table 3. Effectsof eticlopride* on cocaine-elicitedinhibition Percentage inhibition pre-
Dose of cocaine
mg/kg 0.25 0.50
N 3 4
antagonist 47.67-[- 15.5 39.75_+7.3
i.00
7
87.14 -t- 7.3*
Percentage inhibition post-
antagonist 41.33 + 30.14 36.4_ 15.7 61.43+ 8.12
*Standard dose of 0.25mg/kg. The D2antagonist,eticlopridedid not alter cocaine-elicitedneuronal depressionreliably.The magnitudeof depressionwas significant (P < 0.05) at 1.0mg/kg. Interventionwith the antagonist in the test/retest paradigm provokedwide fluctuations. to basal activity, since comparable effects were observed with neurons of diverse firing rates and patterns, following administration of cocaine. Qiao, Daugherty, Wiggins and Dafny (1990) and Lacey Mercuri and North (1990) recently reported that locally administered cocaine induced depression in a majority of neostriatal, substantia nigra and ventral tegmental neurons tested. Latency to onset of depression was evident within 3 min here, as it was in studies of the nucleus accumbens (Einhorn et al., 1988). Neuronal recovery from depression was achieved usually within 20 rain. By 30 min, discharge rate had returned to pretreatment levels. This time--course paralleled the reportedly short duration of psychoactive effects of the drug (Fischman and Schuster, 1982). Depression of the rate of neuronal discharge after administration of cocaine was blocked by the nonselective dopamine receptor antagonist haloperidol. In subsequent experiments with selective dopamine antagonists, the D I ligand SCH 23390 blocked cocaine-induced depression in a dose-related manner, implying receptor selectivity for this cocaine-induced effect. In vitro studies on cells of the nucleus accumbens have shown similar selectivity (Uchimura and North, 1990). In the current investigation, the D2 receptor antagonist eticlopride did not produce a consistent reversal of cocaine-induced neuronal depression. In fact, following intervention with eticlopride, marked variations in neuronal discharge rates ensued. Therefore, it may be argued that D 2 receptors
0.5mg/kgCoc
815
are not directly involved in cocaine-induced inhibition of neuronal discharge. While cocaine-induced depression has been observed in other in vivo studies (Einhorn et al., 1988; Qiao et ai., 1990), the implication of selective DIreceptor involvement is novel. This action could be mediated postsynaptically, given the concentration of postsynaptic D~ receptors in the caudate-putamen (Altar and Marien, 1987). However, in light of the complex neuronal circuitry in the striatum, involvement of other neurotransmitters would seem plausible. The principal neurotransmitter at the terminals of the nigrostriatal pathway is dopamine (Anden, Carlsson, Dahlstrom, Fuxe, Hiltarp and Larsson, 1964). Both Dm and D 2 receptors are localized postsynaptically (Altar and Marien, 1987) and on intrinsic neurons (Girault, Spampinato, Glowinski and Bcsson, 1986). Since a large portion of striatal neurons utilize 7-aminobutyric acid (GABA, Ottersen and Storm-Mathisen, 1984), this may be of functional importance (Scheel-Kruger, 1986). Release of GABA in the striatum is modulated by dopamine (van der Heyden, Venema and Korf, 1980) and stimulation of D] and D2 receptors can facilitate (Girault et al., 1986) or inhibit (Tossman and Ungerstedt, 1986) release of endogenous GABA in the dorsal striatum, respectively. Depression of neuronal activity by cocaine could have been mediated through direct postsynaptic action; however, it is also possible that it occurred indirectly through enhanced release of GABA on neurons that interact with dopamine-responsive neurons. Striatal dopaminergic terminals make synaptic contact with intrinsic GABA-containing neurons (Kubota, Inagaki, Kito and Wu, 1987) and may influence their function. The preferential activation of striatal D] receptors is consistent with the predominant neuronal depression after cocaine. Facilitation of neuronal activity by cocaine occurred at all doses but most frequently at the smallest dose (0.25 mg/kg). Its magnitude was not related to
0.5mg/kgCoe
0.25mg/kgEllc
2"0I Q, 0
0.25 mg/kg SCH 23390
5 rain
Fig. 8. Selective blockade of both depression and excitation elicited by cocaine in single neurons was induced by specific dopamine receptor antagonists. In the physiograph recording above, SCH 23390 blocked depression a n d apparently unmasked excitation. Facilitation, following a second administration of cocaine, was reversed by eticlopride.
816
K.D. NA~rrwl and E. P. SCHOE~dt
dose and was significantly less than the magnitude of depression at all doses. Based in part on reversal by haloperidol o f L-DOPA-induced facilitation, and the differential affinity of D~ and De receptors for dopamine (Creese, Sibley, Hamblin and Left, 1982) it was tempting to speculate that specific activation of dopamine receptors of the D~ type mediated this neuronal change. This was confirmed by the consistent reversal of facilitation after intervention with the selective D2 receptor antagonist, eticlopride. While precise localization of the receptors was not undertaken in the present study, it is conceivable that postsynaptic (Altar and Marien, 1987) and intrinsic striatal (Girault et al., 1986) D2 receptors were implicated. The random effect of SCH 23390 on cocaineinduced facilitation, strongly suggests that activation of D1 receptors was not involved with excitation. Substantial evidence for specificity in the effects of cocaine came from the biphasic responses of some neurons and the observation that depression and facilitation could be blocked selectively by SCH 23390 and eticlopride, respectively. These findings indicate that D~ and D2 receptors may be present and functional on the same neuron as reported by Akaike, Ohno, Sasa and Takaori (1987) and inferred from earlier studies of dopaminergic actions on formation of cyclic adenosine monophosphate (cAMP) in slices o f brain (Stoof and Kebabian, 1981) and primary cell cultures (Chneiweiss, Glowinski and Premont, 1988). Acknowledgements--These studies were supported in part
by grants from the National Institute on Health (GM 08167) and Alcohol, Drug Abuse, and Mental Health Administration (MH 471 81). The authors are indebted to A. Barnett of the Schering-Plough Corporation for kindly providing the SCH 23390 used in these studies. The assistance of Michael Parizon and David Bradley Jr in the histological preparations is most appreciated. We are grateful to Dr Robin Barraco for his helpful discussions.
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