Apomorphine and dopamine D1 receptor agonists increase the firing rates of subthalamic nucleus neurons

Neuroscience Vol. 72, No. 3, pp. 863-876, 1996

Pergamon

0306-4522(95)00583-8

ElsevierScienceLtd IBRO Printedin Great Britain

APOMORPHINE A N D D O P A M I N E DI RECEPTOR AGONISTS INCREASE THE FIRING RATES OF SUBTHALAMIC N U C L E U S N E U R O N S D. S. KREISS,* L. A. A N D E R S O N and J. R. WALTERSI" Experimental Therapeutics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 10, Room 5C-103, 9000 Rockville Pike, Bethesda, MD 20892-1406, U.S.A. Abstract--The present study investigated the regulation of spontaneous neuronal activity in the subthalamic nucleus by dopamine receptors using in vivo extracellular single unit recording techniques. Subthalamic nucleus neuronal firing rates were doubled by systemic administration of the nonselective dopamine receptor agonist apomorphine. The response to apomorphine was attenuated in animals anesthetized with chloral hydrate or ketamine. The dopamine D2/D3 receptor agonist quinpirole did not alter subthalamic nucleus neuronal firing rates. Firing rates were increased by the D~ receptor agonists SKF 38393 and SKF 82958 two- to three-fold; these increases were reversed by the D~ receptor antagonist, SCH 23390. Autoradiographic studies using [125I]SCH 23982 indicated that D I family receptors were located along the ventral edge of the subthalamic nucleus and the dorsal aspect of the cerebral peduncle. Local administration of SKF 82958 into the subthalamic nucleus doubled neuronal firing rates; these increases were reversed by systemic administration of SCH 23390. Infusion of SCH 23390 into the subthalamic nucleus prevented systemic SKF 38393 from increasing the firing rates of subthalamic nucleus neurons. These results indicate that apomorphine and D~ receptor agonists exert an excitatory influence on subthalamic nucleus neuronal activity. In addition, the excitation induced by D~ receptor agonists appears to be mediated, at least in part, by D~ receptors located in the vicinity of the subthalamic nucleus. The data suggest that basal ganglia output under conditions of increased dopamine receptor stimulation is influenced by the activation of excitatory subthalamic efferent pathways, as opposed to suppression of these pathways as predicted by current models of basal ganglia function. Key words: SKF 38393, SKF 82958, basal ganglia, electrophysiology, autoradiography.

Models of basal ganglia organization typically describe the subthalamic nucleus (STN) as a link from the striatum and external globus pallidus to the entopeduncular nucleus (the rodent homologue of the primate internal globus pallidus) and substantia nigra pars reticulata. 3,27 However, anatomical considerations and a variety of clinical and preclinical studies indicate that this view underestimates the potential role of the STN in the integration and transmission of information throughout the basal ganglia and affiliated structures. For instance, the STN makes reciprocal connections with the external globus pallidus, substantia nigra pars reticulata, *Pharmacology Research Associate Fellow, National Institute of General Medical Sciences, Bethesda, MD 20892, U.S.A. tTo whom correspondence should be addressed. Abbreviations: ANOVA, analysis of variance; LY-171555, quinpirole; MPTP, I-methyl-4-phenyl-1,2,3,6tetrahydropyridine; NMDA, N-methyl-o-aspartate; • SCH 23390, (R)-(+)-7-chloro-8-hydroxy-3-methyl-1phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; SCH 23982, (R)-( + )-8-[125]iodo-7-hydroy-2,3,4,5-tetrahydro-3-methyl-3-3-phenyl-1H-3-benzazepine;SKF 38393, ( + )- I -phenyl-2,3,4,5-tetrahydro-( I H)-3-benzazepine7,8-diol; SKF 82958, (+)-6-chloro-N-allyl-SKF 38393; STN, subthalamic nucleus.

substantia nigra pars compacta, peduncuiopontine tegmental nucleus, entopeduncular nucleus, and the cerebral cortex. In addition, the STN sends efferents to the striatum, nucleus accumbens, and substantia innominata, and receives afferents from the parafascicular nucleus of the thalamus, locus coeruleus, and dorsal raphe nucleus.2'17'18"20"32'43'49.58'72Through these connections the STN has the potential to have a marked influence on many aspects of basal ganglia function. Clinical interest in the STN arises from its role in movement disorders. ~ For more than a half a century it has been known that lesions of the STN produce ballism in humans. 57"93 Similar types of involuntary movements are produced in primates following either electrolytic or excitotoxic lesions of the S T N . 21'34'36"94 Ballism in primates has also been observed following inhibition of STN neuronal activity, produced either by increasing the activity of the inhibitory pallidosubthalamic pathway ~5 or by the putative depolarization block of STN neuronal activity24"34 following local administration of the GABA antagonist bicuculline into the STN. 25 In combination, these studies have led to the hypothesis that hypoactivity of the STN may underlie some types of dyskinesia, such as that occurring in patients with Huntington's chorea 3'24"57'93

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or that occurring in patients with Parkinson's disease following chronic dopamine replacement therapy. 63 Conversely, hyperactivity of the STN is thought to be associated with the akinesia characteristic of Parkinson's disease. 3'27,64This hypothesis is supported by electrophysiological studies reporting increased spontaneous activity of STN neurons in animal models of Parkinson's disease, s'9'61'76 Furthermore, lesions of the STN or high-frequency stimulationinduced depolarization block of the STN 6"7have been shown to alleviate the parkinsonian motor dysfunction in l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP)-treated primates. 5,7'8'33Very recently, symptoms in human Parkinson's patients have also been successfully ameliorated after high-frequency stimulation of the STN. 6"53 Given the association of motor abnormalities and STN dysfunction, a better understanding of the regulation of neuronal activity in the STN could serve to elucidate further the role of the STN in the basal ganglia. The neurotransmitter dopamine plays a critical role in movement, clearly evidenced by the profound motor deficits produced by the loss of dopamine neurons in Parkinson's disease. The influence of dopamine o n the STN is described as indirect by current models of basal ganglia organization.3'27In these models, release of dopamine in the striatum results in a net decrease in STN activity due to disinhibition of the globus pallidus. However, it is likely that additional sites of action contribute to the impact of dopamine on this area. For example, the STN receives a significant input from cortical neurons, which in turn are innervated by midbrain dopaminergic neurons.37'54 Furthermore, the STN itself receives direct input from the dopaminergic cell bodies of the substantia nigra tT'~s'58 and has been shown to contain dopamine86 and dopaminergic varicose terminals, ts,Ss Moreover, both dopamine D l and D 2 receptors have been reported to be localized in the STN. 13.26.30,56 A role for dopamine in the regulation of the neuronal activity of the STN is suggested by studies observing altered firing of STN neurons following dopamine depletion in rodents treated with either reserpine and alpha-methyl-para-tyrosine76 or 6-hydroxydopamine38 and following dopamine depletion in primates treated with MPTP. s'o'6t Other studies indicating an influence of dopamine on the STN have shown that glucose utilization was decreased in the STN following systemic administration of the dopamine D~ receptor antagonist SCH 23390 s4 and was increased following systemic administration of either the dopamine receptor agonist apomorphine t6 or the dopamine releasing agent and uptake inhibitor amphetamine.~.9~ Several studies provide evidence for a local action of dopamine within the STN itself. Two electrophysiology studies have reported that iontophoretic application of dopamine exerts an excitatory effect on STN neurons. 62'7s In another study, iontophoretic

application of dopamine produced mixed changes in the firing rates of both spontaneously firing and glutamate-activated STN neurons. 17 Also, a role for dopamine DI receptor stimulation in the STN in the production of vacuous mouth movements has been identified.73 The present study examined the dopamine receptor-mediated regulation of spontaneous neuronal activity in the STN of rats using in vivo extracellular single-unit recording techniques. To characterize the effects of systemic dopamine receptor stimulation on this area, STN neuronal firing rates were measured following administration of the dopamine DI/D2 receptor agonist apomorphine. The individual effects of stimulating dopamine D~ and D2 receptor subtype families were then investigated. These families include the D l and D 2 receptor subtypes and the D2, D3, and D 4 receptor subtypes, respectively,82 and are referred to as D~ and D 2 receptors hereafter. The response of STN neurons to the dopamine D~ receptor agonists, SKF 3839369'75'81and SKF 829584'68and the D 2 receptor agonist quinpirole (LY 171555)23'67 were measured. In the second stage of the study the presence of D~ receptors in the locale of the STN was determined and the effects following local infusion of dopamine D~ receptor agonists into the STN were examined. EXPERIMENTAL PROCEDURES

Materials

( + )SKF 38393 [( ± )- l-phenyl-2,3,4,5-tetra-hydro-(lH)-3benzazepine-7,8-diol] hydrochloride, SKF 82958 [(+)-6chloro-N-allyl-SKF 38393] hydrobromide, (+) SCH 23390 [(R)-( + )-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4, 5tetrahydro-lH-3-benzazepine] hydrochloride, ( - ) quinpirole hydrochloride, and ketanserin tartrate were obtained from Research Biochemicals International (Natick, MA, U.S.A.). Apomorphine hydrochloride was obtained from Sigma Chemical Co. (St Louis, MO, U.S.A.). Haloperidol was obtained from McNeil Pharmaceutical (Spring House, PA, U.S.A.). Gallamine triethiodide was obtained from American Cyanamid Co. (Pearl River, NY, U.S.A,). Mepivaeaine hydrochloride was obtained from Winthrop Pharmaceuticals (New York, NY, U.S.A.). SCH 23982 [(R)-(+)-8-[125]iodo-7-hydroy-2,3,4,5-tetrahydro-3-methyl3-3-phenyl-lH-3-benzazepine] was obtained from Dupont NEN Research Products (Boston, MA, U.S.A.). Drug doses refer to the weight of the salts. Surgical procedures

Male Sprague-Dawley rats (250-350 g, Taconic Farms, Germantown, NY, U.S.A.) were housed under environmentally-controlled conditions with free access to laboratory chow and water. All experiments were conducted in accordance with National Institutes of Health's Guide for Care and Use o f Laboratory Animals. In addition, experimental procedures used were designed to minimize animal discomfort. At the beginning of an experiment, rats were anesthetized with halothane, a tracheotomy was performed, and the trachea was intubated with a cannula. Rats were maintained under halothane anesthesia during all surgical procedures) ° Incision sites and pressure points were thoroughly infiltrated with the long acting local anesthetic, mepivacaine hydrochloride. Rats were then placed into a stereotaxic instrument and, using a hydraulic microdrive, a

Dopamine receptor agonists increase firing rates recording electrode was lowered through a small burr hole drilled in the skull to the STN at the following Paxinos and Watson TM coordinates: 4.9 mm anterior to the lambdoid suture, 2.2 mm lateral to lambda, and 6.8-8.0 mm ventral to the dura. For experiments involving local administration into the STN, the following additional surgical procedures were performed at the beginning of the experiment. A 22 gauge guide cannula was lowered to the STN at an angle of 30° from the vertical through a second small burr hole made in the skull at the following Paxinos and Watson 74 coordinates: 5.0 mm anterior to the lambdoid suture, 2.0 mm lateral to lambda, and 7.7 mm ventral to the dura. A 28-gauge injection cannula, extending 1 mm beyond the end of the guide cannula, connected to polyethylene tubing (PE 20) filled with drug solution, was then lowered through the guide cannula for eventual delivery of drug or vehicle (occurring at least 20 min later). For experiments involving local administration of SCH 23390 into the STN, fused silica tubing (Polymicro Technologies; Phoenix, AZ, U.S.A.) extending 1.3 mm beyond the tip of the guide cannula, connected to PE 20 tubing filled with drug solution, was used for drug delivery. Once all surgical procedures were completed, the animal was immobilized with 16mg/kg gallamine triethiodide (Davis and Geck; Pearl River, NY, U.S.A.) administered through a lateral tail vein and artificially respired via the intubated cannula on room air at a rate adjusted to maintain an expired CO2 of 3.5-4.2% as measured by a CO2 analyser. Eye-drops of a tetrahydrozoline HC1 solution were applied to prevent corneal drying. Body temperature was maintained at 37-38°C using a heating pad and a rectal thermometer. In a separate set of experiments, instead of the above procedure in which rats were locally anesthetized and artificially respired, animals were systemically anesthetized with chloral hydrate (400 mg/kg) or ketamine (150 mg/kg), administered intraperitoneally (i.p.). Animals were maintained under anesthesia throughout these experiments by administration of supplemental injections of the anesthetics.

Extracellular single unit recordings Extracelhilar single unit activity of spontaneously firing STN neurons was recorded with single barrel glass microelectrodes filled with 2% Pontamine Sky Blue dye in 2 M NaC1) ° The electrode tips were broken back to a diameter of 1-2 #m. Electrode in vitro impedances ranged between 3.0-6.0 Mr] (at 135 Hz). Extracellularly recorded action potentials were passed through a high input-impedance amplifier and monitored on an oscilloscope and audiomonitor. All STN neurons recorded exhibited a biphasie ( + / - ) waveform. Discriminated signals were stored both on computer disk and chart paper and analysed using the Rate/Interspike Interval Data Acquisition and Analysis Program for personal computers (Symbolic Logic; Dallas, TX, U.S.A.). Only one cell per animal was studied. Neurons were identified by their stereotaxic location and by histological location of the electrode tip following iontophoresis of Pontamine Sky Blue at the completion of an experiment.

Drug administration To investigate the effects of systemic administration of drugs, a basal firing rate was established over a 4-5 min period, and then drugs or saline were administered through the tail intravenously (i.v.) as a bolus, unless otherwise indicated. Firing rates were monitored for at least 10 min following drug administration, and in some cases, a dopamine receptor antagonist was then administered to investigate the potential for reversal of the drug effect. In indicated experiments, SKF 38393 was administered in 2-4 sequential doses at 1 min intervals. Drugs were dissolved in deionized water, except for apomorphine which was dissolved in saline (0.9% NaCI), at varying concentrations such that a volume of I ml was injected per kg rat body weight. SCH 23390 was dissolved in a small volume (50-80 #1) of 0.001 M HC1 and

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then brought up to a final volume of 0.5 mg/ml with deionized water. To investigate the effects of local administration of drugs into the STN, a basal firing rate was established over a 4-5 min period, and then drugs or vehicle were slowly infused into the STN at a rate of 0.1 #l/min over 3 min using a microinfusion syringe pump (Harvard Apparatus, Model 22; South Natick, MA, U.S.A.). Firing rates were monitored for at least 10 min following the infusion. Locally infused drugs were dissolved in saline at a volume of either 1.0 or 10.0/lg/#1. Occasionally, gentle heating was necessary to dissolve the drug.

Receptor autoradiography Naive rats were killed by decapitation, and their brains removed and frozen at - 7 0 ° C until further use. Twenty micrometer sections were cut using a refrigerated cryostat and thaw-mounted on to gelatin-coated slides and stored at -70°C. On the day of a binding experiment, consecutive slide-mounted sections were preincubated for 10rain in 50 mM Tris-HCl buffer (120raM NaCI, 5 mM KC1, 2 mM CaCI2, and 1 mM MgCI2; pH 7.4) at 4°C and then incubated at 25°C for 45 min in 50 mM Tris-HCl buffer containing 0.5 nM of the D 1 receptor antagonist [t2q]SCH 23982 (2200 Ci/mmol, Dupont NEN Research Products; Boston, MA, U.S.A.) and 50 nM ketanserin to block ligand binding to serotonin 5-hydroxytryptamine2 receptors, s2 Non-specific binding was determined in the presence of I # M SCH 23390. Sections were exposed to LKB 3H-Ultro-film (LKB Instruments, Gaithersburg, MD, U.S.A.) for 18 h at 4°C. The film was developed in Kodak D-19. The optical density of the STN region of the autoradiogram was quantified using the Image program, Version 1.42, for Macintosh computers (NIH; Betbesda, MD, U.S.A.). Nonspecific binding was subtracted and optical density values were converted into fmoles [t2q]SCH 23982 specifically bound per nag of tissue using calibrated [125I]microscales (Amersham; Bucks, U.K.). To compare the location of radiolabeled ligand with specific brain structures, the tissue slides used for autoradiography were stained with Cresyl Violet and cover slips attached.

Data analysis Firing rates were averaged over 4-10min following administration of drug or saline, unless otherwise indicated, and were expressed as a percentage of basal firing. To determine differences between experimental groups, the means -I- S.E.M. were calculated and analysed using analysis of variance (ANOVA) at P < 0.05 followed by Dunnett's post hoc test or by using Student's t-test (GraphPad InStat, Version 2.04; San Diego, CA, U.S.A.). RESULTS

U n d e r basal conditions, S T N n e u r o n s recorded in these experiments exhibited s p o n t a n e o u s firing, characterized by a n irregular p a t t e r n . Firing rates o f S T N n e u r o n s recorded in locally anesthetized rats r a n g e d f r o m 1.6 to 26.0 spikes per second. T h e overall m e a n value o f basal firing rate for S T N n e u r o n s was 8.5 + 0 . 5 spikes per second ( m e a n + S . E . M . , n = 122). M e a n basal firing rates o f experimental g r o u p s did n o t differ f r o m one a n o t h e r , as indicated by A N O V A (F[14,121] = 1.11).

Systemic administration o f apomorphine T h e d o p a m i n e D I / D 2 receptor agonist a p o m o r p h i n e at a dose o f 0 . 3 2 m g / k g i.v. (equivalent to 1/zmol/kg) consistently increased S T N n e u r o n a l firing rates, as s h o w n in Fig. 1 (top panel) a n d Fig. 2.

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Peak increases in firing rate averaged over a 2-min interval occurred between 1 and 8 min following intravenous injection of apomorphine. The effects of a p o m o r p h i n e were measured for 4-10 rain after drug injection in many cells (n = 8); however, in a number of cells (n = 6) the effects o f apomorphine were measured for only 0 - 2 m i n after drug injection (indicated by triangular symbols in Fig. 2). The data from these two groups were combined since there was no difference (as determined by Student's t-test) between the effects of apomorphine measured at the

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Fig. I. Representative histograms illustrating the effects of intravenous administration of apomorphine, quinpirole, and SKF 38393 on STN neuronal firing rates. Arrows indicate the time at which the indicated drug was administered. The top panel illustrates a 4-fold increase in firing rate produced by 0.32 mg/kg apomorphine, which was reversed with haloperidol (0.2 mg/kg). The middle panel illustrates that 0.26 mg/kg quinpirole did not alter the firing rates of STN neurons. The bottom panel illustrates an approximately 2-fold increase in firing rate produced by 10 mg/kg SKF 38393, which was reversed with SCH 23390 (0.5 mg/kg).

TREATMENT

Fig. 2. The effects on STN neuronal firing rates produced by a series of dopaminergic agents. The following drugs were intravenously administered (ordered left to right on graph): SAL (saline, n =7), APO (apomorphine, 0.32 mg/kg, n = 14), QUIN (quinpirole, 0.26 mg/kg, n = 6), SKF 38393 (20mg/kg, n = 16), SKF 82958 (0.43 mg/kg, n = 7), SCH 23390 (0.5 mg/kg, n = 8), and HAL (haloperidol, 0.2 mg/kg, n = 9). Bar height indicates mean changes (+ 1 S.E.M.) in firing rate, expressed as percentage of basal values. Open symbols illustrate the individual responses to drug observed among STN neurons. Circles indicate cases in which drug was given as a single bolus and squares indicate cases in which SKF 38393 was given in 2--4 sequential doses. Drug effects were measured 4-10 rain post injection, except for cases indicated by triangles in which firing rate was measured 0-2 min following apomorphine injection. For reference, the dashed line indicates 100% basal firing rate. Asterisks indicate a significant increase in firing rate as compared to values of saline-treated controls, using ANOVA followed by Dunnett's post hoc comparisons (*P < 0.05; **P < 0.01). two time intervals either in the same cells or in different groups o f cells. A p o m o r p h i n e increased firing rates to a m e a n of 200 + 16% (n = 14) of basal firing rates. This increase was significant compared with rates of saline-treated controls (n = 7), measured 4 - 1 0 m i n post-injection, as indicated by A N O V A (F[6,66] = 10.16, P < 0.01) and Dunnett's post hoc test (P < 0.05). Apomorphine-induced increases in firing rates were reversed to or below basal firing rates by administration of the dopamine D2 receptor antagonist haloperidol (0.2 mg/kg i.v.) in eight out of 11 cells (for example see Fig. 1, top panel). As shown in Fig. 2, when administered alone, haloperidol (0.2 m g / k g i.v., n = 9) did not alter firing rates in comparison to rates of saline-treated controls, as indicated by D u n n e t t ' s post hoc test. T o investigate the effect of anesthesia on the ability of a p o m o r p h i n e to increase firing rates, animals were anesthetized with either chloral hydrate (400 m g / k g i.p.) or the N-methyl-D-aspartate ( N M D A ) receptor antagonist ketamine (150mg/kg i.p.). M e a n basal firing rates of STN neurons in rats under ketamine anesthesia (18.5 + 3.2 spikes/s, n = 8)

Dopamine receptor agonists increase firing rates were significantly higher than m e a n basal firing rates in locally paralysed rats (8.5 + 1.2 spikes/see, n = 14), as indicated by A N O V A (F[2,29] = 6.62, P < 0.01), followed by Dunnett's post hoc test (P < 0.01). In contrast, mean basal firing rates o f rats under chloral hydrate anesthesia (8.6 _ 2.6 spikes/s, n = 8) did not differ from those in locally paralysed rats. As shown in Fig. 3, the effects o f a p o m o r p h i n e were significantly attenuated by the presence of chloral hydrate or ketamine, as indicated by A N O V A (F[2,29] = 10.85, P <0.01), followed by D u n n e t t ' s post hoc tests (P < 0.01).

Systemic administration of quinpirole The dopamine D2 receptor agonist quinpirole did not alter the mean firing rate o f S T N neurons, as shown in Fig. 1 (middle panel) and Fig. 2. The averaged effects of quinpirole at a dose o f 0.26 m g / k g i.v. (equivalent to 1 #mol/kg, n = 6) did not differ from the effects of saline administration, as indicated by Dunnett's post hoc test.

Systemic administration of SKF 38393 The dopamine Dl receptor agonist S K F 38393 increased the firing rates of S T N neurons, as shown in Fig. 1 (bottom panel) and Fig. 2. Peak increases in o 25O

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Fig. 4. The effects on STN neuronal firing rates following intravenous administration of SKF 38393 at doses of 2.5 mg/kg (n = 6), 5 mg/kg (n = 6), l0 mg/kg (n = 8), and 20 mg/kg (n = 16, data identical to those presented in Fig. 2). Control rats were treated with saline (n = 7, data identical to those presented in Fig. 2). Bar height indicates mean changes ( + 1 S.E.M.) in firing rate expressed as a percentage of basal values measured 4-10 min following either a bolus administration or the last of a sequential dose administration. Open symbols illustrate the individual responses to drug observed among STN neurons. Circles indicate cases in which the drug was given as a single bolus; squares indicate cases in which the drug was given in 2-4 sequential doses. For reference, the dashed line indicates 100% basal firing rate. Asterisks indicate a significant increase in firing rate as compared to control values, indicated by ANOVA followed by Dunnett's post hoc comparisons (P < 0.01).

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ANESTHETIC PREPARATION Fig. 3. The effects of anesthesia on the ability of apomorphine to increase STN neuronal firing rates. Animals were treated with apomorphine (0.32 mg/kg i.v.) in the presence of local anesthetic (n = 14, data identical to that in Fig. 2)

or in the presence of either chloral hydrate (400 mg/kg i.p., n = 8 ) or ketamine (150mg/kg i.p., n = 8 ) . Bar height indicates mean changes (+ l S.E.M.) in firing rate, expressed as percentage of basal values. Open symbols illustrate the individual responses to drug observed among STN neurons. Circles indicate cases in which firing rate was measured 4-10 rain following apomorphine injection; triangles indicate cases in which firing rate was measured 0-2 rain following apomorphine injection. For reference, the dashed line indicates 100% basal firing rate. Asterisks indicate the group mean is significantly different from the locally anesthetiz~ group, using ANOVA followed by Dunnett's post hoc comparisons (P < 0.01).

firing rates occurred between 4 and 8 min following bolus or the last o f a series o f injections of S K F 38393. Figure 4 shows the dose dependence of the increase in firing rates produced by S K F 38393 at doses o f 2.5, 5, l0 and 20 m g / k g i.v (equivalent to 8.6, 17.1, 34.2 and 6 8 . 5 # m o l / k g , respectively). In some cases the S K F 38393 dose was administered as a single bolus (n = 9), indicated by the circular symbols in Figs 2 and 4, and in other cases the S K F 38393 was administered in a series o f 2 - 4 sequential doses given at 1-min intervals (n = 7), indicated by the square symbols in Figs 2 and 4. F o r a particular dose, there was no difference (as indicated by Student's t-test) between the increase in firing rates 4-10 min following a bolus administration or 4-10 min following the last o f a sequential dose administration, thus the results were pooled. Administration of S K F 38393 at 2 . 5 m g / k g increased firing rates to a mean of 140 + 18% (n = 6) of basal firing, 5 mg/kg increased firing rates to 165 + 22% (n = 6) of basal firing, 10 m g / k g increased firing rates to 165 + 23% (n = 8) o f basal firing, and 20 mg/kg increased firing rates to 257 + 28% (n = 16) of basal firing (Fig. 4). A N O V A indicated a significant effect of dose (F[4,42] = 5.66, P < 0.01), and post hoc comparisons using Dunnett's test indicated that the m e a n increase in firing rate produced by 20 mg/kg S K F 38393 was significantly

D.S. Kreiss et al.

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Systemic administration of SKF 82958 A second dopamine D l receptor agonist, S K F 82958, also consistently increased the firing rates of STN neurons, as shown in Fig. 2. Peak increases in firing rates occurred between 2 and 8 min following injection of S K F 82958. S K F 82958 at a dose of 0.43 mg/kg i.v. (equivalent to 1/~mol/kg) increased firing to a mean of 294 ± 47% (n = 7) of basal firing rates. A N O V A (F[6,63]=10.96, P < 0 . 0 1 ) and D u n n e t t ' s post hoc test indicated that the increase in firing rates was significant compared with rates of saline-treated controls ( P < 0 . 0 1 ) . S K F 82958induced increases in firing rates were reversed in five out of seven cells by administration of SCH 23390 (0.5 mg/kg i.v.). The reversals ranged between 34 and 68% of the firing rates measured 4-10 min following the administration of S K F 82958.

different from the mean rate of saline-treated controls (n = 7, P < 0.01). S K F 38393-induced increases in firing rates were reversed to or below basal firing rates by administration of the dopamine D~ receptor antagonist SCH 23390 (0.5mg/kg i.v.) in 12 out of 14 cells (for example see Fig. 1, bottom panel). When administered alone, SCH 23390 (0.5 mg/kg i.v., n = 8) did n o t alter firing rates in comparison to rates of salinetreated controls using D u n n e t t ' s post hoc test, as shown in Fig. 2. Histological localization of the electrode tip at the end of an experiment indicated that occasionally the recorded cell did not lie within the boundaries of the STN, but rather within the zona incerta. It is interesting to note that in contrast to STN cells, the zona incerta cells did not respond consistently to administration of S K F 38393. F o r example, the m e a n firing rate of zona ineerta cells following administration of 2 0 m g / k g S K F 38393 was 1 1 1 + 2 1 % (n = 7) of basal values (data not shown). These observations support the anatomical specificity of the STN neuronal response to the dopamine D~ receptor agonist.

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Fig. 5. The effects on STN neuronal firing rates following local administration of SKF 82958 into the STN. The top left panel is a representative histogram illustrating a 1.5-fold increase in firing rate produced by 0.3 pg SKF 82958, which was reversed with intravenous SCH 23390. The bottom left panel illustrates an approximately 2-fold increase in firing rate produced by 3.0 pg SKF 82958, which was also reversed with SCH 23390. The horizontal bars indicate the time during which SKF 82958 was locally infused into the STN and the arrows indicate the time at which SCH 23390 (0.5 mg/kg) was administered through the tail vein. The right panel illustrates effects of local administration of 0.3/~g SKF 82958 (n = 7), 3.0/~g SKF 82958 (n = 8), or saline (n = 6) into the STN. Bar height indicates mean changes (+ 1 S.E.M.) in firing rates measured 4-10 rain following infusion of drug, expressed as a percentage of basal values. Open symbols illustrate the individual responses to drug observed among STN neurons. For reference, the dashed line indicates 100% basal firing rate. Asterisk indicates a significant increase in firing rate compared with values of saline-treated controls, using Student's t-test (P < 0.05).

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Fig. 6. The effects of local administration of SCH 23390 into the STN on the ability of intravenous administration of SKF 38393 to increase STN neuronal firing rates. The left panels are representative histograms illustrating the response to intravenous SKF 38393 alone (top left panel) and in the presence of local infusion of SCH 23390 into the STN (bottom left panel). The arrows indicate the time at which SKF 38393 (20 mg/kg) was administered through the tail vein, and the horizontal bar in the bottom panel indicates the time during which SCH 23390 (0.3 #g) was locally infused into the STN. The fight panel illustrates the averaged drug effects on STN neuronal firing rates. Animals were treated with either intravenous SKF 38393 alone (20 mg/kg, n = 16, data identical to those presented in Fig. 2), intravenous SKF 38393 (20 mg/kg) 4 min following local administration of SCH 23390 into the STN (0.3 #g, n -- 10), local administration of SCH 23390 into the STN alone (0.3 #g, n = 7), or local administration of saline into the STN (n = 6, data identical to those presented in Fig. 5). Bar height indicates mean changes (+ 1 S.E.M.) in firing rate measured 4-10 min following intravenous SKF 38393 injection or local saline infusion or 0-4 min following SCH 23390 local infusion, expressed as percentage of basal values. Open symbols illustrate the individual responses to drug observed among STN neurons. Circles indicate cases in which SKF 38393 was given as a single bolus; squares indicate cases in which SKF 38393 was given in 2-4 sequential doses. For reference, the dashed line indicates 100% basal firing rate. Asterisks indicate that the group mean was significantly different from the group treated with intravenous SKF 38393 in combination with local SCH 23390, using ANOVA followed by Dunnett's post hoc comparisons (P < 0.01). 140 + 23% (n = 7) of basal firing and infusion of 3.0 g g (equivalent to 2 4 m M ) S K F 82958 increased firing rates to 199 + 33% (n = 8) o f basal firing, as shown in the fight panel of Fig. 5. A N O V A indicated a trend toward a significant effect of dose (F[2,20] = 3.12, P = 0.07), and Student's t-test indicated that the mean increase in firing rate produced by the higher dose of S K F 82958 was significant (P < 0.05) as compared to saline infusion (n = 6). The S K F 82958-induced firing rate increases were reversed to or below basal firing rates by systemic administration of S C H 23390 (0.5 m g / k g i.e.) in two out of three cells at the lower dose and in three out of three cells at the higher dose (for examples see Fig. 5, left panels).

this effect may have been nonspecific. Local infusion of 3.0 # g (equivalent to 34 m M ) S K F 38393 increased S T N neuronal firing rates to 292 + 54% (n = 7) of basal firing rates (data not shown). Unlike the immediate increase in firing rates following local infusion of S K F 82958, peak increases in firing rates following the local infusion o f S K F 38393 were delayed, occurring at 6 - 8 min post-infusion. Moreover, the increases in firing rates induced by local infusion of S K F 38393 were not reversed by systemic administration of S C H 23390 (0.5 m g / k g i.v.) in four out o f four cells (data not shown). These results suggest that local infusion o f S K F 38393 may induce nonspecific excitatory effects.

Local administration of SKF 38393

Local administration of SCH 23390 in combination with systemic administration of SKF 38393

Local administration of S K F 38393 into the S T N also increased S T N neuronal firing rates; however,

The ability of systemically administered S K F 38393 to increase firing rates o f S T N neurons was

D.S. Kreiss et al.

870

completely blocked by local administration of SCH 23390 into the STN, as shown in Fig. 6. A dose of 0.3 ltg (equivalent to 3.1 mM) SCH 23390 was locally infused into the STN 4 min before the systemic administration of 20 mg/kg S K F 38393. As described previously, the mean STN neuronal firing rate following systemic administration of S K F 38393 was 257 + 28% (n = 16) of basal firing. Following combined systemic administration of S K F 38393 and local administration of SCH 23390, the mean firing rate ( 1 2 4 + 2 1 % of basal firing, n = 10) was not significantly different from the mean rate of salinetreated controls. The blockade of the effects of systemically administered agonist by local administration of the antagonist was statistically significant, as indicated by A N O V A (F[3,38] = 9.89, P < 0.01) and Dunnett's post hoc test (P < 0.01). Local administration of SCH 23390 into the STN alone did not alter subthalamic neuronal firing rates, measured 0 - 4 m i n following the infusion, as indicated by Dunnett's post hoc test (n = 7, Fig. 6, right panel).

Dopamine D t receptor autoradiography Dopamine D~ receptor autoradiography confirmed the presence of dopamine D~ receptors (30-40 fmoles/mg tissue) in the vicinity of the STN, as shown in Fig. 7. Direct comparison of the location of autoradiographic label (['2sI]SCH 23982) in individual tissue slices (Fig. 7, bottom panel) with the structural boundaries of the STN visualized in the same tissue slice by Cresyl Violet stain (Fig. 7, top panel) indicated that Dj receptors were located along the ventral edge of the STN which borders the cerebral peduncle. DI receptors were also found throughout the cerebral peduncle, with the highest densities occurring along the dorsal aspect, bordering the STN. D~ receptors were not observed in the dorsal regions of the STN. DISCUSSION

Effects of systemic administration of apomorphine The present study examined the regulation of neuronal activity in the STN of the rat by dopamine receptor stimulation. STN neuronal firing rates were increased by the dopamine DJD2 receptor agonist apomorphine approximately 2-fold relative to basal firing rates. This apomorphine-induced increase in STN firing rates was not predicted by the working model of the basal ganglia that has been discussed in the literature over the past several years, s'27 The model's prediction arises from evidence that dopamine receptor stimulation in the striatum has an inhibitory effect on GABAergic striatopallidal activity, resulting in disinhibition of pallidal neuronal activity.~°.12a°m This increase in the activity of pallidal neurons is predicted to cause a reduction of STN neuronal activity as a consequence of the increased acti¢ity of the GABAergic pallidosubthalamic path-

way. 77's3 In fact, electrophysiological studies of the globus pallidus from this laboratory have found that one of two major subpopulations of pallidal neurons--termed Type II cells on the basis of the positive/negative pattern of the extracellularly recorded action potentialS---responds as the model predicts to systemically administered apomorphine, by showing an increase in firing rateJ °'ll's7 However, recently, a second major subpopulation of pallidal neurons--termed Type I cells, displaying a negative/positive extracellular action potential pattern-has been studied and found to show a consistent decrease in firing rate following apomorphine. 46 Thus, this latter observation has made it difficult to predict how the apomorphine-mediated changes in globus pallidus output will affect STN activity. As yet, the role of pallidal efferents to the STN in mediating the unexpected response of STN neurons to the dopamine nonselective receptor agonist remains unclear. Because anesthesia has been shown to attenuate the effects of dopamine receptor stimulation in other areas of the basal ganglia, the effect of anesthesia on the ability of apomorphine to increase the firing rates of STN neurons was explored in the present study. Anesthesia produced by either chloral hydrate or the noncompetitive N M D A receptor antagonist ketamine 95 significantly attenuated apomorphineinduced increases in firing rates. The attenuation of apomorphine's effects by chloral hydrate is probably not related to the effects of the anesthetic on basal firing, as basal firing rates of STN neurons in the chloral hydrate preparation did not differ from those in the locally anesthetized preparation (which were comparable to basal rates reported in previous studies31'38'76"79). However, the mean basal firing rate of STN neurons in the ketamine preparation was significantly higher than that in the locally anesthetized preparation. Hence, one explanation for the attenuation of apomorphine's effects under ketamine anesthesia is the elevated frequency of basal firing. Alternatively, it is possible that ketamine and chloral hydrate are reducing/blocking activity in pathways critical to the expression of the apomorphine-mediated effect. The ability of ketamine to attenuate the effects of apomorphine on STN neuronal activity suggests that tonic N M D A receptor stimulation plays a role in the pathways mediating this effect. In the globus pallidus, ketamine anesthesia has been shown to decrease the basal firing rates of both subpopulations of pallidal neurons. ~ Perhaps the decreased firing rates of globus pallidus neurons contribute to the increased basal firing rates of STN neurons. In contrast to observations in the STN, ketamine anesthesia did not attenuate apomorphine's effects on either subpopulation of pallidal neurons, '~ adding support to the idea that sites other than the globus pallidus play a major role in mediating the effects of apomorphine on STN neurons.

Dopamine receptor agonists increase firing rates

Effects o f systemic administration o f a D z receptor agonist Evidence from this study indicates that both dopamine D : and D~ receptor subtypes contribute to the response o f S T N neurons to dopaminergic stimulation. A role for the D2 receptor subtype in mediating the apomorphine-induced increase in S T N

871

neuronal firing rates is implicated by the ability o f the D z receptor antagonist haloperidoP °,4' to reverse a p o m o r p h i n e ' s effects on STN neuronal activity. However, the m e a n firing rate of STN neurons was not altered by administration of the selective D2 receptor agonist, quinpirole, at a dose of 0.26 mg/kg. Thus, while blockade o f D 2 receptor stimulation

Fig. 7. Location of dopamine D I receptors in the vicinity of the STN as indicated by autoradiography. The top panel portrays the ventral half of a coronal section through a rat midbrain stained with Cresyl Violet, corresponding to Paxinos and Watson ~3 coordinate: 4.9 mm anterior to the lambdoid suture. The STN, labeled by an arrow, is a small lens-shaped structure lying along the dorsal aspect of the cerebral peduncle (cp). The bottom panel illustrates the same tissue slice, labeled with [~2sI]SCH 23982. Areas of darker labeling indicate the presence of D I ~-ceptors. Comparison of the autoradiographic label (bottom panel) with the location of the STN (top panel) shows that D~ receptors are located in the vicinity of the STN, primarily along the ventral edge of the nucleus.

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appears to counter the effects of a mixed dopamine receptor agonist, increasing the level of D 2 receptor stimulation alone is not sufficient to produce significant alterations in the firing rates of STN neurons. Effects o f systemic administration o f Dl receptor agonists

In contrast to the lack of effect following administration of a D 2 receptor agonist, STN neuronal firing rates were markedly elevated by systemic administration of two dopamine Dj receptor agonists. The selective D t receptor agonist SKF 38393 dose-dependently increased the firing rates of STN neurons. A 2-3-fold increase over basal rates was produced by 20mg/kg SKF 38393. A similar increase in STN firing rates was produced by a second D~ receptor agonist, SKF 82958, at a dose of 0.43 mg/kg. The approximately 50-fold higher in vivo potency of SKF 82958 in comparison with the potency of SKF 38393 is in concordance with the higher in vivo potency of SKF 82958 for inducing rat stereotypic behavior 59 and with the higher in vitro affinity of SKF 82958 for the D t receptor. 4 The pharmacological specificity of these D~ receptor agonist-mediated effects was confirmed by the ability of systemic administration of the selective D~ receptor antagonist SCH 2339041"42"69 to reverse the D 1 receptor agonist-induced increases in firing rates.

Effects o f local administration of D 1 receptor agonists and antagonist into the subthalamic nucleus

Evidence that D~ receptors located in the vicinity of the STN could mediate the D t receptor agonistinduced increases in neuronal activity was obtained from experiments in which dopaminergic agents were directly infused into the STN. Previous studies concerning the relationship between infusion volume, rate of delivery, and extent of diffusion in brain tissue suggest that the local drug infusions in the present study remained relatively confined to the STNfl "6°,~ Local administration of SKF 82958 at a dose of 3.0/tg, but not 0.3/tg, increased STN neuronal firing rates to a magnitude similar to that produced by systemic administration of D~ receptor agonists. The pharmacological selectivity of this effect was further substantiated by the ability of a subsequent systemic administration of SCH 23390 to reverse fully the effects of the locally infused D~ receptor agonist. The excitation of STN neurons following localized stimulation of D~ receptors in the vicinity of the STN is in concordance with previous observations of the excitatory effects of iontophoretic application of dopamine in the STN.62, 78

The effects of stimulation of D~ receptors in the vicinity of the STN by local administration of SKF 38393 were also investigated. Like locally administered SKF 82958,oSKF 38393 increased STN neuronal firing rates. However, in contrast to locally Location o f D t receptors in the vicinity o f the subtha- administered SKF 82958, the local SKF 38393lamic nucleus induced increases appeared to have been a result of The present study demonstrates that D~ family nonselective effects specifically associated with local receptors located within the vicinity of the STN play administration of SKF 38393. First, the local SKF an important role in the electrophysiologicai response 38393-induced increases in firing rates could not be to systemic administration of D~ receptor agonists. reversed by systemic administration of the dopamine Autoradiographic results confirmed that dopamine D~ receptor antagonist SCH 23390. Secondly, the D~ receptors are located within the vicinity of the increase induced by local administration of SKF STN. The highest density of D~ receptor binding 38393 had a delayed onset of several minutes, inconoccurred along the ventral edge of the STN and the sistent with the typical time course of local adminisdorsal aspect of the cerebral peduncle. The occur- tration studies. Additionally, toxic or nonselective fence of D~ receptors in the STN has been reported excitatory effects of SKF 38393 have been previously in a number of histological studies. 14.lS"26,2s'3°'55,73's°'s9 documented following administration locally in vivo 47 In contrast, a recent study by Johnson and colleagues or via bath application in vitro, s5 although in the reported that the D~ receptors are not located within present experiments no obvious tissue damage was the borders of the STN, but are located nearby in the evident following the local SKF 38393 infusions. In cerebral peduncle. ~ The autoradiographic results of summary, the experiments involving local administhe current study are in general agreement with both tration of SKF 38393 were inconclusive. Additional evidence for the role of Dt receptors in viewpoints. Side-by-side comparison of the location of radiolabeled D t receptor ligand with the structural the vicinity of the STN in mediating the effects of boundaries of the STN as visualized by Cresyl Violet systemically administered Dt receptor agonists on stain (see Fig. 7) revealed that the location of D~ STN neurons was obtained from studies in which receptors is along the STN-cerebral peduncle border. SCH 23390 was locally administered into the STN As morphological studies have shown that the den- prior to systemic administration of SKF 38393. Lodrites of STN neurons extend across the ventral STN calized antagonism of the D~ receptors in the vicinity boundary into the cerebral peduncles, ~5'4s it seems of the STN completely blocked the effects of sublikely that at least some of the D~ receptors visualized sequent ubiquitous activation of D~ receptors followalong the STN-cerebral peduncle border have the ing systemic administration of the D1 receptor potential to modulate locally the STN neuronal agonist. This result suggests that D~ receptor agonistinduced increases in STN neuronal firing rates are activity.

Dopamine receptor agonists increase firing rates mediated, at least in part, by D~ receptors located in the vicinity of the STN. Possible origins o f D 1 receptor-bearing afferents

The D~ receptors in the vicinity of the STN are located on afferent terminals and not on STN cell dendrites or bodies, as in situ hybridization studies report an absence of D~ receptor m R N A in the STN and cerebral peduncles? °,55 The origin of these D~ receptor-bearing afferents is unclear. However, the afferents probably do not arise from the globus pallidus, as D~ receptor m R N A is undetectable in this structure) ° Another possible source of the afferents is the thalamus, a structure known to contain D~ receptor m R N A ) ° However, it is most likely that the Dt receptor-bearing pathways are STN excitatory afferents from the cerebral cortex, 2'17A8"2°'32 which also contains D~ receptor m R N A ) ° Interestingly, morphological studies of cortical efferents to the STN describe a projection pathway that overlies the anatomical location of dopamine D~ receptors observed in the vicinity of the STN, i.e. the cortical efferents pass through the dorsal aspect of the cerebral peduncles before terminating in the STN? The glutamatergic nature of cortical and thalamic efferents to the STN 2'29'35"5°'78enables one to speculate that D~ receptors located on STN afferent terminals may act to increase glutamate release in the vicinity of STN dendrites. Potentiation of glutamate release by local D t receptor stimulation has, in fact, been demonstrated in other brain regions, such as the substantia nigra pars reticulata ~ and ventral tegmental area. 45 CONCLUSIONS

In conclusion, this study demonstrates that apomorphine, as well as D~ receptor agonists, increase the firing rates of STN neurons. Moreover, these results suggest that the excitatory effects of D~ receptor agonists on STN neuronal activity are mediated, at least in part, by D~ receptors located within the vicinity of the STN. An important implication of these findings is that basal ganglia output under conditions of increased dopaminergic stimulation is influenced by the activation of the excitatory subthalamic efferent pathways.

873

The idea that the subthalamic efferent pathways would be activated by dopamine receptor stimulation counters predictions of current models of basal ganglia organization. Current models propose that enhanced dopamine receptor stimulation would (i) increase the activity of the inhibitory striatonigral pathway, (ii) increase the activity of the inhibitory pallidonigral pathway, and (iii) decrease the activity of the excitatory subthalamonigral pathway. Thus, the predicted individual effects, as well as the summed net effect, of these three convergent input pathways is an attenuation of substantia nigra pars reticulata and entopeduncular nucleus neuronal activity. From such predictions arose the hypothesis that dopamine receptor stimulation reduces inhibitory efferent output from the substantia nigra pars reticulata and entopeduncular nucleus, thereby disinhibiting the thalamus (i.e. opening the "thalamic filter"), activating cortical areas, and in the end, augmenting arousal and behavioral output. 19"22 However, electrophysiological studies examining the response of substantia nigra pars reticulata neurons to dopamine receptor agonists have not found results consistent with these predictions in normal animals. Systemically administered apomorphine induces a mixture of responses in the substantia nigra pars reticulata, with a substantial percentage of cells responding with increases in firing rate. 9° In addition , D~ receptor agonists have been reported to produce increases in substantia nigra pars reticulata neuronal activity. 39'88"92The results of the present study suggest that increased excitatory efferent input from the subthalamic nucleus contributes to these observed firing rate changes in the substantia nigra pars reticulata. They further suggest that the net effect of enhanced dopamine receptor stimulation on basal ganglia output is more complex than current models predict and reflects a dynamic interplay of excitatory and inhibitory influences arising from the actions of dopamine receptor agonists at both striatal and extrastriatal sites. Acknowledgements--We would like to most kindly thank

Ms Anne Kask for her assistance with the receptor autoradiography and Dr Debra Bergstrom for her comments on earlier drafts of this manuscript.

REFERENCES

1. Abarca J., Gysling K., Roth R. H. and Bustos G. (1995) Changes in extracellular levels of glutamate and aspartate in rat substantia nigra induced by dopamine receptor ligands: in vivo microdialysisstudies. Neurochem. Res. 20, 159-169. 2. Afsharpour S. (1985) Topographical projections of the cerebral cortex to the subthalamie nucleus. J. comp. Neurol. 236, 14-28. 3. Albin R. L., Young A. B. and Penney J. B. (1989) The functional anatomy of basal ganglia disorders. Trends Neurosci. 12, 366-375. 4. Andersen P. H. and Jansen J. A. (1990) Dopamine receptor agonists: selectivity and dopamine DL receptor efficacy. Eur. J. Pharmac. 188, 335-347. 5. Aziz T. Z., Peggs D., Sambrook M. A. and Crossman A. R. (1991) Lesion of the subthalamic nucleus for the alleviation of l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP)-induced Parkinsonism in the primate. Moo. Disord. 6, 288-292.

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D . S . Kreiss et al.

6. Benabid A. L., Pollak P., Gross C., Hoffmann D., Benazzouz A., Gao D. M., Laurent A., Gentil M. and Perret J. (1994) Acute and long-term effects of subthalamic nucleus stimulation in Parkinson's Disease. Stereotact. Funct. Neurosurg. 62, 76-84. 7. Benazzouz A., Gross C., Frger J., Boraud T. and Bioulac B. (1993) Reversal of rigidity and improvement in motor performance by subthalamic high-frequency stimulation in MPTP-treated monkeys. Eur. J. Neurosci. 5, 382-389. 8. Bergman H., Wichmann T. and DeLong M. R. (1990) Reversal of experimental parkinsonism by lesions of the subthalamic nucleus. Science 249, 1436-1438. 9. Bergman H., Wichman T., Karmon B. and DeLong M. R. (1994) The primate subthalamic nucleus. II. Neuronal activity in the MPTP model of Parkinsonism. J. NeurophysioL 72, 507-520. 10. Bergstrom D. A., Bromley S. D. and Walters J. R. (1982) Apomorphine increases the activity of rat globus pallidus neurons. Brain Res. 238, 266-271. 11. Bergstrom D. A., Bromley S. D. and Waiters J. R. (1984) Dopamine agonists increase pallidal unit activity: attenuation by agonist pretreatment and anesthesia. Eur. J. Pharmac. 100, 3-12. 12. Bernardi G., Marciani M. G., Morocutti C., Pavone F. and Stanzione P. (1978) The action of dopamine on rat caudate neurons intracellularly recorded. Neurosci. Lett, 8, 235-240. 13. Bouthenet M. L., Martes M. P., Sales N. and Schwartz J. C. (1987) A detailed mapping of dopamine D-2 receptors in rat central nervous system by autoradiography with [~25I] iodosulpride. Neuroscience 20, 117-155. 14. Boyson S. J., McGonigle P. and Molinoff P. B. (1986) Quantitative autoradiographic localization of the Dj and D 2 subtypes of dopamine receptors in rat brain. J. Neurosci. 6, 3177-3188. 15. Brown L. L., Makman M. H., Wolfson L. I., Dvorkin B., Warner C. and Katzman R. (1979) A direct role of dopamine in the rat subthalamic nucleus and an adjacent intrapeduncular area. Science 266, 1416-1418. 16. Brown L. L. and Wolfson L. I. (1978) Apomorphine increases glucose utilization in the substantia nigra, subthalamic nucleus, and corpus striatum of rat. Brain Res. 140, 188-193. 17. Campbell G. A., Eckhardt M. J. and Weight F. F. (1985) Dopaminergic mechanisms in subthalamic nucleus of rat: analysis using horseradish peroxidasc and microiontophoresis. Brain Res. 333, 261-270. 18. Canteras N. S., Shammah-Lagnado S. J., Silva B. A. and Ricardo J. A. (1990) Afferent connections of the subthalamic nucleus: a combined retrograde and anterograde horseradish peroxidase study in the rat. Brain Res. 513, 43-59. 19. Cartsson M. and Carlsson A. (1990) Interactions between glutamatergic and monoaminergic systems within the basal ganglia--implications for schizophrenia and Parkinson's disease. Trends Neurosci. 13, 272-276. 20. Carpenter M. B., Carleton S. C., Keller J. T. and Conte P. (1981) Connections of the subthalamic nucleus in the monkey. Brain Res. 224, 1-29. 21. Carpenter M. B., Whittier J. R. and Mettler F. A. (1950) Analysis of choreoid hyperkinesia in the rhesus monkey. J. comp. NeuroL 92, 293-332. 22. Chevalier G. and Deniau J. M. (1990) Disinhibition as a basic process in the expression of striatal functions. Trends Neurosci. 13, 277-280. 23. Cohen M. L., Shaar C. J. and Colbert W. E. (1984) ct2-Agonist activity of the dopamine DA2-agonist LY171555. J. Cardiovasc. Pharmac. 6, 1245-1248. 24. Crossman A. R. (1987) Primate models of dyskinesia: the experimental approach to the study of basal ganglia-related involuntary movement disorders. Neuroscience 21, 1-40. 25. Crossman A. R., Sambrook M. A. and Jackson A. (1984) Experimental hemichorea/hemiballismus in the monkey. Brain 107, 579-596. 26. Dawson T. M., Barone P., Sidhu A., Wamsley J. K. and Chase T. N. (1988) The D I dopamine receptor in the rat brain: quantitative autoradiographic localization using an iodinated ligand. Neuroscience 26, 83-100. 27. DeLong M. R. (1990) Primate models of movement disorders of basal ganglia origin. Trends Neurosci. 13, 281-285. 28. Dubois A., Savasta M., Curet O. and Scatton B. (1986) Autoradiographic distribution of the D t agonist [3H] SKF38393 in the rat brain and spinal cord. Comparison with the distribution o f D 2 dopamine receptors. Neuroscience 19, 125-137. 29. Frger J., Mouroux M., Benazzouz A., Boraud T., Gross C. and Crossman A. R. (1994) The subthalamic nucleus: a more complex structure than expected. In The Basal Ganglia 1V: N e w Ideas and Data on Structure and Function (ed. Percheron G.), pp. 371-382. Plenum Press, New York. 30. Fremeau R. T., Duncan G. E., Fornaretto M. G., Dearry A., Gingrich J. A., Breese G. R. and Caron M. G. (1991) Localization of D I dopamine receptor mRNA in brain supports a role in cognitive, affective, and neuroendocrine aspects of dopaminergic neurotransmission. Proc. natn Acad. Sci. U.S.A. 88, 3772-3776. 31. Fujimoto K. and Kita H. (1993) Response characteristics of subthalamic neurons to the stimulation of the sensorimotor cortex in the rat. Brain Res. 609, 185-192. 32. Groenewegen H. J. and Berenise H. W. (1990) Connections of the subthalamic nucleus with ventral striatopallidal parts of the basal ganglia in the rat. J. comp. Neurol. 294, 607-622. 33. Guridi J., Luquin M. R., Herrero M. T. and Obeso J. A. (1993) The subthalamic nucleus, a possible target for stereotaxic surgery in Parkinson's Disease. Mov. Disord. 8, 421-429. 34. Hamada I. and DeLong M. R. (1992) Excitotoxic acid lesions of the primate subthalamic nucleus result in transient dyskinesias of the contralateral limbs. J. Neurophysiol. 68, 1850-1858. 35. Hammond C., Deniau J. M., Rouzaire-Dubois B. and Frger J. (1978) Peripheral input to the rat subthalamic nucleus: an electrophysiological study. Neurasci. Lett. 9, 171-176. 36. Hammond C., Frger J., Bioulac B. and Souteyrand J. P. (1979) Experimental hemiballism in the monkey produced by unilateral kainic acid lesion in corpus Luysii. Brain Res. 171, 577-580. 37. H6kfelt T., Ljungdahl A., Fuxe K. and Johansson O. (1974) Dopamine nerve terminals in the rat limbic cortex: aspects of the dopamine hypothesis of schizophrenia. Science 184, 177-179. 38. Hollerman J. R. and Grace A. A. (1992) Subthalamic nucleus cell firing in the 6-hydroxydopamine-treated rat: basal activity and response to haloperidol. Brain Res. 590, 291-298. 39. Huang K. X. and Waiters J. R. (1994) Electrophysiological effects of SKF 38393 in rats with reserpine treatment and 6-hydroxydopamine-induced nigrostriatal lesions reveal two types of plasticity in D~ dopamine receptor modulation of basal ganglia output. J. Pharmac. exp. Ther. 271, 1434-1443. 40. Hyttel J. (1978) Effects of neuroleptics on 3H-haloperidol and 3H-cis(Z)-flupenthixol binding and on adenylate cyclase activity in vitro. Life Sci. 23, 551-556.

Dopamine receptor agonists increase firing rates

875

41. Hyttel J. (1983) SCH 23390---the first selective dopamine D-I antagonist. Eur. J. Pharmac. 91, 153-154. 42. Iorio L. C., Barnett A., Leitz F. H., Houser V. P. and Korduba C. A. (1983) SCH 23390, a potential benzazepine antipsychotic with unique interactions on dopaminergic systems. J. Pharmac. exp. Ther. 226, 462-468. 43. Jackson A. and Crossman A. R. (1981) Subthalamic nucleus efferent projection to the cerebral cortex. Neuroscience 6, 2367-2377. 44. Johnson A. E., Coirini H., Kallstrom L. and Wiesel F. A. (1994) Characterization of dopamine receptor binding sites in the subthalamic nucleus. NeuroReport 5, 1836-1838. 45. Kalivas P. W. and Duffy P. (1995) D~ receptors modulate glutamate transmission in the ventral tegmental area. J. Neurosci. 15, 5379-5388. 46. Kelland M. D., Soltis R. P., Anderson L. A., Bergstrom D. A. and Waiters J. R. (1995) In vivo characterization of two cell types in the rat globus pallidus which have opposite responses to dopamine receptor stimulation: comparison of electrophysiological properties and responses to apomorphine, dizocilpine and ketamine anesthesia. Synapse 20, 338-350. 47. Kelley A. E., Delfs J. M. and Chu B. (1990) Neurotoxicity induced by the D-I agonist SKF 38393 following microinjection into rat brain. Brain Res. 532, 342-346. 48. Kita H., Chang H. T. and Kitai S. T. (1983) The morphology of intraceUularly labeled rat subthalamic neurons: a light microscopic analysis. J. comp. Neurol. 215, 245-257. 49. Kita H. and Kitai S. T. (1987) Efferent projections of the subthalamic nucleus in the rat: light and electron microscopic analysis with the PHA-L method. J. comp. Neurol. 260, 435-452. 50. Kitai S. T. and Deniau J. M. (1981) Cortical inputs to the subthalamus: intracellular analysis. Brain Res. 214, 411-415. 51. Kreiss D. S. and Lucki I. (1994) Differential regulation of serotonin (5-HT) release in the striatum and hippocampus by 5-HTIA autoreceptors of the dorsal and median raphe nuclei. J. Pharmac. exp. Ther. 269, 1268-1279. 52. Leysen J. E., Niemegeers C. J. E., Van Nueten J. M. and Laduron P. M. (1982) [3H]Ketanserin (R 41 468), a selective 3H-ligand for serotonin2 receptor binding sites. Molec. Pharmac. 21, 301-314. 53. Limousin P., PoUak P., Benazzouz A., Hoffmann D., 12 Bas J. F., Broussolle E., Perret J. E. and Benabid A. L. (1995) Effect on parkinsonian signs and symptoms of bilateral subthalamic nucleus stimulation. Lancet 354, 91-95. 54. Lindvall O. and Bj6rklund A. (1974) The organization of the ascending catecholamine neuron systems in the rat brain as revealed by glyoxylic acid fluorescence method. Acta Physiol. scand. SuppL 412, i-48. 55. Mansour A., Meador-Woodruff J. H., Zhou Q., Civelli O., Akil H. and Watson S. J. (1992) A comparison of D~ receptor binding and mRNA in rat brain using receptor autoradiography and in situ hybridization techniques. Neuroscience 46, 959-971. 56. Martes M. P., Bouthenet M. L., Sales N., Sokoloff P. and Schwartz J. C. (1985) Widespread distribution of brain dopamine receptors evidenced with [lz5I]iodosulpride, a highly selective ligand. Science 228, 752-755. 57. Martin J. P. (1927) Hemichorea resulting from a local lesion of the brain. Brain 50, 637-651. 58. Meibach R. C. and Katzman R. (1979) Catecholaminergic innervation of the subthalamic nucleus: evidence for a rostral continuation of the A9 (substantia nigra) dopaminergic cell group. Brain Res. 173, 364-368. 59. Meyer M. E. and Shults J. M. (1993) Dopamine Di receptor family agonists, SK&F 38393, SK&F 77434, and SK&F 82958, differentially affect locomotor activities in rats. Pharmac. Biochem. Behav. 46, 269-274. 60. Millan M. H., Patel S., Mello L. M. and Meldrum B. S. (1986) Focal injection of 2-amino-7-phosphonoheptane acid into prepiriform cortex protects against pilocarpine-induced limbic seizures in rats. Neurosci. Lett. 70, 69-74. 61. Miller W. C. and DeLong M. R. (1987) Altered tonic activity of neurons in the globus pallidus and subthalamic nucleus in the primate MPTP model of Parkinsonism. In The Basal Ganglia 11. Structure and Function (eds Carpenter M. B. and Jayaraman A.), Vol. 32, pp. 415-427. Plenum Press, New York. 62. Mintz I., Hammond C. and Frger J. (1986) Excitatory effect of iontophoretically applied dopamine on identified neurons of the rat subthalamic nucleus. Brain Res. 375, 172-175. 63. Mitchell I. J., Boyce S., Sambrook M. A. and Crossman A. R. (1992) A 2-deoxyglucose study of the effects of dopamine agonists on the Parkinsonian primate brain. Brain 115, 809-824. 64. Mitchell I. J., Brotchie J. M., Graham W. C., Page R. D., Robertson R. G., Sambrook M. A. and Crossman A. R. (I 989) In Basal Ganglia IlL Advances in the Understanding o f Neuronal Mechanisms in Movement Disorders (eds Bernardi G., Carpenter M. B., Di Chiara G., MoreUi M. and Stanzione P.), pp. 607-616. Plenum Press, New York. 65. Mitchell I. J., Jackson A., Sambrook M. A. and Crossman A. R. (1989) The role of the ~subthalamic nucleus in experimental chorea. Brain 112, 1533-1548. 66. Myers R. D. (1966) Injection of solutions into cerebral tissue, relation between volume and diffusion. Physiol. Behav. 1, 171-174. 67. Nagahama S., Chert Y. F., Lindheimer M. D. and Oparil S. (1986) Mechanism of the pressor action of LY171555, a specific dopamine D2 receptor agonist, in the conscious rat. d. Pharmac. exp. Ther. 236, 735-743. 68. O'Boyle K. M., Gaitanopoulos D. E., Brenner M. and Waddington J. L. (1989) Agonist and antagonist properties of benzazepine and thienopyridine derivatives at the D~ dopamine receptor. Neuropharmacology 28, 401-405. 69. O'Boyle K. M. and Waddington J. L. (1984) Selective and stereospecific interaction of R-SK&F 38393 with 3H-piflutixol but not 3H-spiperone binding to striatal D~ and D 2 dopamine receptors. Eur. J. Pharmac. 98, 433-436. 70. Pan H. S., Engber T. M., Chase T. N. and Waiters J. R. (1990) The effects of striatal lesion on turning behavior and globus pallidus single unit response to dopamine agonist administration. Life Sci. 46, 73-80. 71. Pan H. S., Penney J. B. and Young A. B. (1985) y-Aminobutyric acid and benzodiazepine receptor changes induced by unilateral 6-hydroxydopamine lesions of the medial forebrain bundle. J. Neurochem. 45, 1396-1404. 72. Parent A. and Smith Y. (1987) Organization of efferent projections of the subthalamic nucleus in the squirrel monkey as revealed by retrograde labeling methods. Brain Res. 436, 296-310. 73. Parry T. J., Eberle-Wang K., Lucki I. and Chesselet M. F. (1994) Dopaminergic stimulation of subthalamic nucleus elicits oral dyskinesa in rats. Exp. Neurol. 128, 181-190. 74. Paxinos G. and Watson C. (1986) The Rat Brain in Stereotaxic Coordinates. Academic Press, Florida. 75. Pfeiffer F. R., Wilson J. W., Weinstock J., Kuo G. Y., Chambers P. A., Holden K. G., Hahn R. A., Wardell J. R., Tobia A. J., Setler P. E. and Sarau H. M. (1982) Dopaminergic activity of substituted 6-chloro-l-phenyl-2,3,4,5-tetrahydro-lH-3-benzazepines. 3". reed. Chem. 25, 352-358.

876

D . S . Kreiss et al.

76. Robledo P. and F6ger J. (1991) Acute monoaminergic depletion in the rat potentiates the excitatory effects of the subthalamic nucleus in the substantia nigra pars reticulata but not in the pallidal complex. J. neural Transm. 86, 115-126. 77. Rouzaire-Dubois B., Hammond C., Hamon B. and F6ger J. (1980) Pharmacological blockade of the globus pallidus-induced inhibitory response of subthalamic cells in the rat. Brain Res. 200, 321-329. 78. Rouzaire-Dubois B. and Scarnati E. (1987) Pharmacological study of the cortical-induced excitation of subthalamic nucleus neurons in the rat: evidence for amino acids as putative neurotransmitters. Neuroscience 21, 429-440. 79. Ryan L. J., Sanders D. J. and Clark K. B. (1992) Auto- and cross-correlation analysis of subthalamic nucleus neuronal activity in neostriatal- and globus pallidal-lesioned rats. Brain Res. 583, 253-261. 80. Savasta M., Dubois A. and Scatton B. (1986) Autoradiographic localization ofD~ dopamine receptors in the rat brain with [3H]SCH 23390. Brain Res. 375, 291-301. 81. Sibley D. R., Left S. E. and Creese I. (1982) Interactions of novel dopaminergic ligands with D-1 and D-2 dopamine receptors. Life Sci. 31, 637-645. 82. Sibley D. R. and Monsma F. J. (1992) Molecular biology of dopamine receptors. Trends Neurosci. 13, 61-69. 83. Smith Y., Bolam J. P. and von Krosigk M. (1990) Topographical and synaptic organization of the GABA-containing pallidosubthalamic projection in the rat. Eur. J. Neurosci. 2, 500-511. 84. Trugrnan J. M. and James C. L. (1993) D t dopamine agonist and antagonist effects on regional cerebral glucose utilization in rats with intact dopaminergic innervation. Brain Res. 607, 270-274. 85. Twery M. J., Thompson L. A. and Waiters J. R. (I 994) Intracellularly recorded response of rat striatal neurons in vitro to fenoldopam and SKF 38393 following lesions of midbrain dopamine ceils. Synapse 18, 67-78. 86. Versteeg D. H. G., VanDer Guten J., De Jong W. and Palkovits M. (1976) Regional concentrations of noradrenaline and dopamine in rat brain. Brain Res. 113, 563-574. 87. Waiters J. R., Bergstrom D. A., Carlson J. H., Chase T. N. and Braun A. R. (1987) D 1 dopamine receptor activation required for postsynaptic expression of D 2 agonist effects. Science 236, 719-722. 88. Waiters J. R., Bergstrom D. A., Carlson J. H., Weick B. G. and Pan H. S. (1987) Stimulation of D-1 and D-2 dopamine receptors: synergistic effects on single unit activity in basal ganglia output nuclei. In Neurophysiology of Dopaminergic Systems--Current Status and Clinical Perspectives (eds Chiodo L. A. and Freeman A. S.), pp. 285-316. Lakeshore, Detroit, MI. 89. Wamsley J. K., Gehlert D. R., Filloux F. M. and Dawson T. M. (1989) Comparison of the distribution of D-I and D-2 dopamine receptors in the rat brain. J. Chem. Neuroanat. 2, 119-137. 90. Waszczak B. L., Lee E. K., Ferraro T., Hare T. A. and Waiters J. R. (1984) Single unit responses of substantia nigra pars reticulata neurons to apomorphine: effects of striatal lesions and anesthesia. Brain Res. 306, 307-318. 91. Wechsler L. R., Savaki H. E. and Sokoloff L. (1979) Effects of D- and L-amphetamine on local cerebral glucose utilization in the conscious rat. J. Neurochem. 32, 15-22. 92. Weick B. G. and Waiters J. R. (1987) Effects of D~ and D 2 dopamine receptor stimulation on the activity of substantia nigra pars reticulata neurons in 6-hydroxydopamine lesioned rats: D~/D 2 coactivation induces potentiated responses. Brain Res. 405, 234-246. 93. Whittier J. R. (1947) Ballism and the subthalamic nucleus (nucleus hypothalamicus; corpus Luysi). Arch. Neurol. Psychiat. 58, 672-692. 94. Whittier J. R. and Mettler F. A. (1949) Studies on the subthalamic nucleus of the rhesus monkey. II. Hyperkinesia and other physiologic effects of subthalamic lesions, with special reference to the subthalamic nucleus of Luys. J. comp. Neurol. 90, 319-372. 95. Yamamura T., Harada K., Okamura A. and Kemmotsu O. (1990) Is the site of action of ketamine anesthesia the N-methyl-D-aspartate receptor? Anesthesiology 72, 704-710. (Accepted 1 December 1995)