A role for non-NMDA excitatory amino acid receptors in regulating the basal activity of rat globus pallidus neurons and their activation by the subthalamic nucleus

BRAIN RESEARCH ELSEVIER

Brain Research 666 (1994) 21-30

Research report

A role for non-NMDA excitatory amino acid receptors in regulating the basal activity of rat globus pallidus neurons and their activation by the subthalamic nucleus Robert P. Soltis 1, Lisa A. Anderson, Judith R. Waiters *, Mark D. Kelland 2 Experimental Therapeutics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bldg. 10, Room 5C103 10, Center Dr. MSC 1406, 9000 Rockuille Pike, Bethesda, MD 20892-1406, USA Accepted 13 September 1994

Abstract

We have investigated the hypothesis that excitatory amino acid (EAA) receptors in the globus pallidus (GP) play a significant role in maintaining the firing rates of GP neurons under basal conditions and following activation of the subthalamic nucleus (STN). Drugs were infused directly into the GP and/or STN while the extracellular single unit activity of Type II GP neurons was recorded in ketamine-anesthetized rats. Local infusions of the EAA agonists NMDA (30-300 pmol/200 nl) or AMPA (0.1-1 pmol/200 nl) elicited increases in the firing rate of GP neurons in a dose-dependent fashion. Infusion of the GABA A receptor antagonist bicuculline methiodide (1-10 pmol/100 nl) into the STN also elicited dose-related increases in the firing rate of GP neurons. Intrapallidal infusion of the non-NMDA (AMPA/kainate) receptor antagonist NBQX (0.1-1.0 nmol) reduced the basal firing rate of GP neurons by 40%. In contrast, the NMDA antagonist MK-801 (0.01-0.1 nmol) produced no significant effect on basal firing rate. Intrapallidal infusion of the non-selective EAA receptor antagonist kynurenic acid or NBQX reversed or blocked the increase in firing rate of GP neurons following bicuculline-induced activation of the STN. Similar treatment with MK-801, however, had no significant effect on this response. These results indicate that tonic stimulation of non-NMDA receptors plays an important role in maintaining the basal activity of GP neurons and in mediating the effects of increased excitatory input from subthalamic afferent neurons.

Keywords: Globus pallidus; Subthalamic nucleus; Extracellular recording; NBQX; MK-801; AMPA; Kainate; NMDA 1. Introduction

One of the two major output pathways of the basal ganglia involves the pallidal complex (in rodents, the globus pallidus (GP) and entopeduncular nucleus) and the subthalamic nucleus (STN) (for reviews see [3,24,59]). The GP receives a major input from the neostriatum, and projects primarily to the STN. The STN relays pallidal output to the entopeduncular nucleus and substantia nigra pars reticulata, the primary

* Corresponding author. Fax: (1) (301) 496-6609. 1 Present address: Department of Pharmacology, Drake University, College of Pharmacy and Health Sciences, Des Moines, IA 50311, USA. 2 Present address: Department of Psychology, St. Anselm College, 100 St. Anselm Drive, Manchester, NH 03102-1310, USA. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 1 1 3 6 - 2

output nuclei of the basal ganglia, and also projects back to the GP. Both striatopallidal and pallidosubthalamic neurons are thought to exert inhibitory effects, utilizing G A B A as their neurotransmitter [22,39,62,68], whereas STN efferents are believed to be excitatory, using an excitatory amino acid (EAA; e.g. glutamate) as their neurotransm~tter [1,11,40,41,60,61,69]. Pallidal neurons are tonically active in vivo, and fire at relatively high rates [4,9,18]. Intracellular recording studies have indicated that excitatory drive from the STN to the GP may be an important factor in regulating the basal activity of G P neurons [40,41,57,64]. STN neurons are also tonically active, and may relay cortical, thalamic and pedunculopontine tegmental input to the GP; they also have the potential to provide pallidosubthalamic feedback [13,41,45]. The role of the STN and its putative utilization of E A A neurotransmitters have recently become of interest in the area of Parkinson's disease research. In

22

R.P. So#is el al. / Brain Research 666 (1994) 21-30

monkeys r e n d e r e d p a r k i n s o n i a n by a d m i n i s t r a t i o n of the n e u r o t o x i n 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP), the activity of n e u r o n s in the G P a n d STN is altered [52], and the p a r k i n s o n i a n symptoms can be alleviated by i n d u c i n g lesions of the STN [5,7,25] or by high-frequency electrical stimulation of the STN [6]. Systemic a d m i n i s t r a t i o n of the n o n - N M D A ( A M P A / k a i n a t e ) receptor a n t a g o n i s t 2,3-dihydroxy-6nitro-7-sulfamoyl-benzo(F)quinoxaline(NBQX) potentiates the a n t i p a r k i n s o n i a n actions of L - D O P A a n d d o p a m i n e agonists in some animal models of Parkinson's disease [43,47,48] (but see also [20,49]). N M D A antagonists also modify the effects of L - D O P A a n d d o p a m i n e agonists in animal models of P a r k i n s o n ' s disease, although the n e t effect of this i n t e r a c t i o n varies with species, b r a i n region a n d agonist p r i m i n g [10,14,17,19,31,42,53,63]. T h e s e data suggest that the basal ganglia pathways utilizing E A A s play an important part in basal ganglia f u n c t i o n a n d dysfunction. A b e t t e r u n d e r s t a n d i n g of the roles of the individual E A A receptor subtypes in m e d i a t i n g the effects of specific pathways within the basal ganglia should promote a more rational t h e r a p e u t i c approach to the t r e a t m e n t of basal ganglia disorders, in a d d i t i o n to providing insight into the relationships b e t w e e n the different basal ganglia nuclei. In the p r e s e n t study, we have e x a m i n e d the relative roles of the N M D A and n o n - N M D A ( A M P A / k a i n a t e ) subtypes of E A A receptors in the G P in r e g u l a t i n g tonic activity a n d s u b t h a l a m i c - i n d u c e d increases in activity of G P n e u r o n s of the rat. W e have focused on Type I1 n e u r o n s of the GP, one of two n e u r o n a l cell types that may be distinguished based on extracellular waveforms [34,54]. Type II pallidal cells constitute approximately half the tonically active cells e n c o u n t e r e d in the G P in k e t a m i n e - a n e s t h e t i z e d rats a n d exhibit a biphasic waveform with an initial postive c o m p o n e n t , followed by a negative c o m p o n e n t [34]. T h e second cell type in the globus pallidus (Type I n e u r o n s ) displays a smaller, biphasic waveform with an initial negative c o m p o n e n t [34]. R e c o r d i n g s in this study were limited to Type II n e u r o n s primarily because their signal is generally larger than that of Type I n e u r o n s a n d therefore more readily m a i n t a i n e d d u r i n g m a n i p u l a t i o n s r e q u i r e d for the m i c r o i n f u s i o n p a r a d i g m s used here. I n the first series of experiments, we d e t e r m i n e d w h e t h e r Type II G P n e u r o n s could be activated by either local infusion of the E A A agonists N-methyl-Daspartate ( N M D A ) or alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid ( A M P A ) or by infusion of the G A B A A receptor a n t a g o n i s t bicuculline m e t h i o dide (bicuculline) into the STN. Secondly, we investigated the role of E A A receptor subtypes in m e d i a t i n g the tonic n e u r o n a l activity of Type II G P n e u r o n s by infusing the n o n - c o m p e t i t i v e N M D A a n t a g o n i s t dizocilpine (MK-801) or the competitive n o n - N M D A re-

ceptor a n t a g o n i s t N B Q X into the GP. Finally, we e x a m i n e d the extent to which these E A A receptor subtypes are involved in m e d i a t i n g increases in pallidal activity b r o u g h t about by b i c u c u l l i n e - i n d u c e d activation of the STN.

2. Materials and methods 2.1. Surgical procedures

Male Sprague-Dawley rats (225-325 g, Taconic Farms, Germantown, NY) were housed under environmentally-controlled conditions with continuous access to laboratory chow and water. Rats were anesthetized with ketamine (150 mg/kg, ip; supplemented as needed) and positioned in a stereotaxic instrument with the incisor bar 3.2 mm below the horizontal plane [58]. Once the overlying skin and connective tissue were cleared from the skull, the portion of the skull overlying the GP and the STN was removed. Body temperature was maintained at 37-38°C with a heating pad. Procedures were carried out in strict compliance with the NIH Guide for the Care and Use of Laboratory Animals, 2.2. Extracellular recording

Extracellular neuronal signals were sampled using a single barrel glass micropipette (tip diameter less than 2/~m) filled with 2 M NaC1 containing 1% Pontamine sky blue. Electrode impedances were 2-6 megaohms measured in vitro at 135 Hz. The electrode was lowered into the GP (0.9 mm posterior, 2.9 mm lateral to bregma; 5-7 mm ventral to brain surface) and advanced with a hydraulic microdrive. Neuronal signals recorded by the electrode were passed through an amplifier, filtered and monitored on an oscilloscope and audiomonitor. The signals were relayed to a window discriminator with the digital output representing single action potentials from a spontaneously active neuron. Only Type II pallidal neurons (those with a biphasic, initially positive waveform) were recorded [34]. The output was recorded on a strip chart recorder and by computer with the aid of the Rate/Interspike Interval Data Acquisition and Analysis Program (Symbolic Logic, Dallas, TX), which stored all data for future statistical analysis. 2.3. Drug administration

NBQX was dissolved in distilled water; all other drugs were dissolved in saline. Drugs were infused into the GP and/or the STN while simultaneously recording neuronal activity in the GP. Prior to recording baseline neuronal activity, a stainless steel guide cannula (28-gauge, Plastics One, Roanoke, VA) was stereotaxically placed into the GP (1.0 mm posterior, 3.0 mm lateral to bregma; 5.7 mm ventral to brain surface) and/or into the STN (3.5 mm posterior, 2.4 mm lateral to bregma; 8.1 mm ventral to brain surface). The guide cannulae were positioned at a 15° angle (oriented laterally for the GP and caudally for the STN) relative to the vertically-oriented recording electrode. Once a stable baseline was attained (4-6 min of sampling), a stainless steel injector cannula (33-gauge, Plastics One, Roanoke, VA) connected to a 1 /xl Hamilton syringe with polyethylene tubing was carefully inserted into the guide cannula. The tip of the injector cannula extended 0.5 mm beyond the tip of the guide cannula. Drugs or saline vehicle were infused into the GP at a rate of 200 nl over 2 min and into the STN at a rate of 100 nl over 30 s with the use of an infusion pump (Harvard model 22). At the end of the infusion, the injector cannula was left in place for 1 min and then carefully removed. In the five experiments examining the dose-re-

R.P. Soltis et al. / Brain Research 666 (1994) 21-30 sponse relationship to infusion of bicuculline into the STN, rats received 3 injections spaced 5 min apart. This interval was chosen in consideration of the time course of maximal effect and recovery, based on observations from pilot experiments. In the remaining experiments, rats received no more than two injections per site. In another set of experiments, drugs were infused into the striatum at sites 1.0-1.5 mm dorsal to the GP while recording from neurons in the GP. Drugs were infused at a rate of 200 nl over 2 min. At the end of each experiment, rats were deeply anesthetized with ketamine and the infusion and recording sites were marked by infusion (100-200 nl) or iontophoresis (25 /zA for 15 min) of 1% Pontamine sky blue. The infusion and recording sites were then verified by histological examination. Only cases in which recording sites were localized to the GP and in which infusion sites were correctly placed in the GP, STN, and/or striatum were used in data analyses.

2.4. Statistical analysis Results are expressed as means _+S.E. Values for the changes in firing rate were determined at the plateaus occurring during the 10 min following the end of the infusion. Dose-response relationships and group differences were analyzed using analysis of variance (ANOVA), repeated measures ANOVA, Dunnett's test a n d / o r contrast analysis (BMDP Statistical Software). The 5% limits of probability were accepted as significant.

23

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3. Results

3.1. Infusion of EA4 agonists into the GP 400

Intrapallidal infusion of the excitatory amino acid agonists AMPA or NMDA increased the firing rate of Type II GP neurons in ketamine-anesthetized rats (Fig. 1). Both AMPA and NMDA elicited increases in firing rate in a dose-dependent manner and to similar magnitudes (Fig. 2). At the highest dose tested for each agent, AMPA and NMDA produced significant increases in firing rate (> 125% of baseline) in 5 out of 5 ceils. The onset of these changes occurred within 1 min after the end of the infusion period. Infusion of saline vehicle into the GP had no significant effect on the basal firing rate of this cell type.

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300 pmol Fig. 1. Histogram tracings taken from three separate experiments depicting the effects of local infusion of saline vehicle (200 nl, top), AMPA (1 pmol, middle) and NMDA (300 pmol, bottom) on the firing rate of Type II GP neurons in ketamine-anesthetized rats.

3.2. Infusion of a GABA antagonist into the STN Infusion of the GABA A receptor antagonist bicuculline into the STN increased the firing rate of all Type II GP neurons tested. In the dose-response studies (Fig. 3; line graph), infusion of 1, 2 and 7 pmol/100 nl of bicuculline resulted in a dose-dependent activation of the firing rates of the GP neurons recorded (each infusion separated by 5 min; cumulative doses of 1, 3 and 10 pmol, n = 5). Single injections of bicuculline (10 pmol/100 nl; Fig. 3, bar graph; n = 5) caused an increase in firing rate similar in magnitude to that induced by the cumulative dosing protocol. The increase began within 90 s after the end of the infusion and reached a plateau within 5-10 min, The neuronal

activity of 7 out of 10 cells was monitored until firing rate returned to baseline values, over a period of 10 to 20 min. The remaining 3 cells of this group went into apparent depolarization block, characterized by a decrease in amplitude and an increase in duration of the action potential. Infusion of saline vehicle into the STN produced no significant changes in the firing rate of GP cells over a period of 10 min (n = 5).

3.3. Infusion of F__MAantagonists into the GP Intrapallidal infusion of the non-NMDA EAA receptor antagonist NBQX reduced the firing rate of Type II GP neurons (Fig. 4, main graph). At doses of

R.f: Soltis et al. / Brain Research 666 (I994) 21-30

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Fig. 2. Percent changes in firing rate of Type 11 GP neurons caused by intrapallidal infusion of A M P A (open bars) or N M D A (crossed bars). For each bar, n = 5. Asterisks denote significant difference from saline vehicle controL.

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0.3 and 1.0 nmol, N B Q X significantly decreased firing rate in 4 out of 5 and 5 out of 5 cells, respectively, over a 20 min time period. However, only 1 out of 5 cells was significantly inhibited by local infusion of 3.0 nmol NBQX. To examine the possibility that the 3.0 nmol dose of N B Q X might be exerting an effect outside of the GP (and counteracting the local effect), N B Q X was infused into the striatum at sites 1.0-1.5 mm dorsal to the GP while neuronal activity in the GP was monitored. Infusion of 1.0 nmol of NBQX, a dose that consistently decreased the firing rate of GP neurons by approximately 40% when infused locally, significantly increased the firing rate of 2 out of 5 GP cells and had no effect on the remaining 3 cells, when infused at sites in the striatum (Fig. 4, inset). In contrast to NBQX, intrapallidal infusion of the N M D A receptor antagonist MK-801 had no effect on

15

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Fig. 4. Time course of effects on basal firing rate of Type II GP neurons caused by intrapallidal infusion of saline vehicle or 0.1-3.0 nmol NBQX. Inset: time course of effects on basal firing rate of Type II GP neurons caused by infusion of 1.0 nmol N B Q X into globus pallidus or striatum. Asterisks denote significant difference from saline control.

the firing rate of Type II GP neurons (Fig. 5). At doses of 0.01 and 0.1 nmol, MK-801 produced no significant changes in basal firing rate over the course of 20 min, as compared to saline vehicle controls. However, at the highest dose tested, 1.0 nmol, MK-801 significantly increased the firing rates of 3 out of 5 cells. To assess the possibility that this latter effect was the result of diffusion, MK-801 was infused into the striatum at sites

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Fig. 5. Time course of effects on basal firing rate of Type II GP neurons caused by intrapallidal infusion of saline vehicle or 0.01-1.0 nmol MK-801. Inset: time course of effects on basal firing rate of Type II GP neurons caused by infusion of 0.1 nmol MK-801 into globus pallidus or striatum. Asterisks denote significant difference from saline control.

R.P. Soltis et al. / Brain Research 666 (1994) 21-30

25

EAA receptors were playing a role in this process. To examine the role of specific subtypes of EAA receptors in mediating this response, either the non-NMDA receptor antagonist NBQX or the NMDA receptor antagonist MK-801 were infused into the GP 5 min prior to infusion of bicuculline into the STN. Infusion of NBQX (1.0 nmol) into the GP attenuated the maximal bicuculline-induced increase in firing rate of Type II GP neurons. In contrast, similar local pretreatment with MK-801 (0.1 nmol) failed to alter the increase in firing rate of GP neurons following activation of the STN (Figs. 8 and 9).

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Fig. 6. Histogram tracing depicting the effects of intrapallidal infusion of kynurenic acid (30 nmol; KYN) on the increase in firing rate of Type II GP neurons induced by subthalamic infusion of bicuculline (10 pmol).

1.0-1.5 mm dorsal to the GP while GP neurons were recorded. Infusion of 0.1 nmol MK-801, a dose that produced no significant effect on basal firing rate when infused into the GP, significantly increased firing rate in 4 out of 5 GP cells when infused at sites in the striatum (Fig. 5, inset). To determine if local EAA receptors were involved in mediating the increase in firing rate of GP neurons resulting from activation of the STN, EAA receptor antagonists were infused into the GP before or after infusion of bicuculline into the STN. Local infusion of the non-selective EAA antagonist kynurenic acid (30 nmol/200 nl; n = 7) reversed the increase in firing rate of GP neurons elicited by blockade of GABA A receptors in the STN (Fig. 6 and 7), indicating that pallidal 300

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Fig. 7. Time course of effects on firing rate of Type 1I GP neurons caused by intrapallidal infusion of kynurenic acid (30 nmol; KYN, n = 7) following infusion of bicuculline (10 pmol) into the subthalamic nucleus. Asterisk denotes significant difference from control.

The data presented in this study provide evidence that local EAA receptors play an important role in mediating the basal activity of Type II GP neurons, as well as the increased neuronal activity in the GP associated with activation of the STN. Intrapallidal infusion of EAA agonists and subthalamic infusion of the GABA g receptor antagonist bicuculline both resulted in dose-related increases in GP neuronal firing rates. These effects occurred within 30 s following intrapallidal infusion and within 90 s following subthalamic infusion, and demonstrate that GP neurons may be excited either by direct activation of NMDA or AMPA receptors in the GP or by activation of an excitatory afferent pathway from the STN. These results confirm and extend previous studies which have found that infusion of bicuculline into the STN (doses of 80 pmol in 200 nl) increases the firing rate of STN neurons, as well as the majority of GP neurons [21,61]. These changes are thought to result from activation of STN neurons following blockade of the tonic inhibitory GABAergic input to the STN from the GP. Microinjection of higher concentrations of bicuculline decreases subthalamic activity due to depolarization block [61]. In the present study, the concentration of bicuculline used was four times lower and the injection volume half that used in the above studies. Therefore, the excitatory responses observed in the Type II GP neurons here are likely to be the result of excitation, and not depolarization blockade, of STN projection neurons. A role for EAA receptors in mediating the increase in the firing rate of GP neurons induced by activation of the STN was suggested by the ability of the nonselective EAA antagonist kynurenic acid to reverse this response when infused into the GP. Moreover, the results indicate that the specific EAA receptor subtypes mediating this effect are non-NMDA receptors. The non-NMDA (AMPA/kainate) receptor antagonist, NBQX, reduced the firing rate of GP neurons when locally infused into the GP and also attenuated

R.P. Soltis et al. / Brain Research 666 (1004) 21-30

3)

the increase in firing rate induced by activation of the STN. In contrast, N M D A receptors do not appear to play a significant role in the modulation of the basal activity of Type II GP neurons or in mediating excitatory input from the STN. Infusion of MK-801 into the GP failed to reduce the firing rate of Type II GP neurons and also failed to affect the increase in GP cell firing rate following bicuculline-induced activation of the STN. NMDA receptors are present in the GP, however, and Type II GP neurons were responsive to local infusions of NMDA. Thus, pallidal N M D A receptors that influence Type II GP neurons may mediate responses to EAAs from inputs from other sources,

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Fig. 8. Histogram tracings taken from three separate experiments depicting the effects of subthalamic infusion of bicuculline on the firing rate of Type II GP neurons following intrapallidal pretreatment with saline vehicle (200 nl, top), NBQX (1.0 nmol, middle), or MK-801 (0.1 nmol, bottom).

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Fig. 9. Bar graphs represent the effects on basal firing rate of Type I1 GP neurons caused by intrapallidal infusion of 200 nl saline, 1.0 nmol NBQX and 0.1 nmol MK-801 (open bars) and subsequent infusion of 10 pmol bicuculline into the subthalamic nucleus (closed bars). Single asterisk denotes a significant difference from basal firing rate following intrapallidal infusions. Double asterisk denotes a significant difference from saline/bicuculline following STN infusion of bicuculline. In all cases, the bicuculline percent basal firing rate (closed bars) was calculated based on the firing rate following intrapallidal infusions (saline, NBQX or MK-801).

such as the thalamus or the pedunculopontine nucleus [16,30,37,44,65]. Interestingly, infusion of the highest dose of MK-801 into the GP increased the firing rate of these pallidal neurons. Results from experiments in which MK-801 was infused into the striatum suggested that this increase was caused by diffusion of the drug along the cannula tract into the striatum. MK-801, at doses that had no effect when infused into the GP, elicited increases in the firing rate of GP cells when infused into the striatum. Thus, while N M D A receptor antagonism within the GP does not affect pallidal activity, N M D A antagonism within the striatum indirectly increases pallidal firing rates, presumably by decreasing inhibitory striatal input to the GP. In similar experiments, NBQX was infused into the striatum during pallidal recordings. The dose of NBQX which most effectively reduced the firing rates of GP neurons when infused into the GP, was ineffective when infused into the striatum. This result indicates that the inhibitory effect of NBQX was mediated locally within the GP. However, the highest dose of NBQX infused into the GP did not decrease GP activity, suggesting that this dose, like the highest dose of MK-801 examined, caused a disinhibition of GP activity. This disinhibition is presumably the result of spread of drug to the striatum and a reduction in the activity of inhibitory striatopallidal neurons. One factor worthy of consideration in interpreting the effects of N M D A and MK-801 in these experiments is the use of ketamine as the anesthetic agent. Ketamine, like MK-801, is considered to be a non-competitive, use-dependent N M D A receptor antagonist

R.P. So#is et al. // Brain Research 666 (1994) 21-30

[26,28,50]. However, a number of studies indicate that ketamine, when used as an anesthetic, does not induce a generalized block of NMDA receptors and differs in its effects from MK-801. Ketamine has in fact frequently been used as an anesthetic or anesthetic supplement in studies examining the role of glutamate or EAA-containing pathways in vivo [21,23,38,40,61,66]. Ketamine was considered preferable to other anesthetics in this case because previous investigations have shown that this anesthetic has less of an effect on the tonic activity of GP neurons and their responses to pharmacological agents, such as dopamine agonists [8,33,34]. In the present study, ketamine anesthesia clearly did not prevent local infusion of NMDA from stimulating the activity of GP neurons and did not prevent MK-801 from exerting an effect in the striatum. In agreement with our results, Kessler et al. [36] have demonstrated that in ketamine-anesthetized rats, the ability of NMDA to induce swallowing responses is not blocked. Likewise, Kelland and Chiodo [32] have shown that iontophoresis of NMDA effectively increases the firing rate of accumbens neurons in ketamine-anesthetized rats, although the magnitude of this effect was reduced as compared to urethane-anesthetized rats. The dose a n d / o r method of administration of ketamine appears to play a role in its ability to exert NMDA receptor blockade. When administered at subanesthetic doses, ketamine has been shown to significantly attenuate some responses to NMDA. For example, a low dose of ketamine administered intravenously blocks NMDA-induced depression of antidromic field potentials in the rat abducens nucleus [55] (see also [15]). In a recent study comparing in vivo effects of ketamine and MK-801, we demonstrated that intraperitoneally-administered ketamine at anesthetic doses (150 mg/kg) does not mimic the ability of MK801 to modify the excitatory effects of apomorphine on firing rates of Type II GP neurons or the ability of apomorphine to induce stereotypy in rats [33]. Intravenous administration of much lower doses of ketamine (5 mg/kg) did however, influence these apomorphine-induced effects. Moreover, higher doses of MK-801 do not induce a ketamine-like anesthesia in mice or rats [29, 33]. Taken together with the results from the present study, it appears that ketamine anesthesia is not associated with a generalized efficacious blockade of NMDA receptors in the central nervous system. The possibility, however, that some NMDAmediated responses are dampened in ketamine-treated animals must be considered. The ability of NBQX to reduce, but not completely inhibit, the basal firing rate of Type II GP neurons and the absence of a direct effect of MK-801 infusion in the GP suggest that mechanisms other than tonic EAA input play a role in maintaining the basal activity of

27

these cells [40,41]. Interestingly, Robledo and F6ger [61] demonstrated that infusion of the GABA A receptor agonist muscimol into the STN inhibited the firing rate of STN neurons by 100% and decreased the firing rate of GP neurons by only 44%, a reduction similar in magnitude to that observed here following intrapallidal infusions of NBQX. Thus, the tonic effects of STN input to GP Type II neurons appear to be mediated largely through non-NMDA receptors, with the remaining basal tone being mediated by other mechanisms. Systemic administration of EAA antagonists produce similar effects and support these conclusions [70]. On average, intravenous administration of MK-801 failed to affect GP activity (at doses up to 3.3 mg/kg), but intravenous NBQX induced a partial decrease in the firing rate of Type II GP neurons on average of

35% [70]. In these experiments, striatal infusion of the NMDA antagonist, MK-801, resulted in an increase in the firing rates of Type II GP neurons, presumably due to reduced inhibitory striatopallidal input. In contrast, systemic administration of MK-801 on average does not affect the firing rates of either Type II GP neurons or Type I striatal neurons, although some individual neurons are responsive in both areas [70]. This difference is likely to reflect the different methods of drug administration used. In particular, the lack of effect following systemic administration may result from multiple sites of drug action that mask the local effect of MK-801 within the striatum. The known distribution of NMDA and non-NMDA EAA receptors in the basal ganglia may underlie the results of these experiments. The observation that the non-NMDA, AMPA-preferring antagonist, NBQX [67], exerts a more pronounced effect on the activity of Type II GP neurons than does the NMDA receptor antagonist, MK-801, is consistent with the relative concentrations of AMPA and NMDA binding sites in the GP. AMPA and metabotropic receptors are relatively more dense than NMDA and kainate sites in this region [2]. Significantly higher receptor densities, but still relatively more AMPA than NMDA binding, is reported in the striatum [2]. These results contribute to our understanding of the possible sites of action mediating the behavioral effects of MK-801 and NBQX. MK-801 produces ataxia, stereotypies and hyperlocomotion in normals rats and modifies the behavioral effects of dopamine agonists and antagonists in normal and 6-hydroxydopamine-lesioned rats [10,12,14,19,27,42,46,53,56,71,72]. The absence of a direct inhibitory effect of MK-801 on the activity of Type I1 GP neurons and the inability of MK-801 to block increased input from the STN, as shown here, suggest that this compound does not mediate its behavioral effects through actions in the GP. On the other hand, MK-801 does alter the response of

28

R.P. Soltis et al. /Brain Research 666 (1904) 21 30

Type I1 GP neurons to systemically-administered apomorphine without significantly affecting baseline activity in the GP [35]. Thus, an 'upstream' site of action for MK-801, i.e. N M D A receptor blockade-induced modulation of the effects of dopamine agonists on striatal output, would appear likely to be contributing to the behavioral effects of MK-801. In fact, systemically-administered MK-801 has also been shown to alter the effects of apomorphine on the activity of striatal neurons [35]. Conversely, it is possible that the ability of NBQX to directly reduce the tonic activity of Type II GP neurons contributes to the decreased locomotion and catalepsy induced by this drug [51,56]. In addition to the Type II neurons of the GP evaluated in these experiments, a second population of neurons exists within the GP. These cells, referred to as Type I neurons, comprise approximately 50% of the spontaneously active cells in the GP and can be distinguished from Type II neurons on the basis of their extracellular wave form [34]. Type I neurons may differ from Type II neurons in their connections within the basal ganglia, since these neuronal populations show opposite changes in firing rate following systemic apomorphine [34]. The influence of EAAs on Type I neurons of the GP remains to be investigated. In conclusion, the present study demonstrates that non-NMDA EAA receptors mediate, at least in part, the basal firing rate of Type II GP neurons. We have observed that blockade of N M D A receptors produces no significant effect in this population of GP neurons, even though local administration of N M D A robustly excites Type II GP neurons. In addition, non-NMDA EAA receptors, but not N M D A receptors, appear to mediate the excitatory effects of STN projections to the GP. These data indicate a significant role for non-NMDA EAA receptors in the pallidal/STN output pathway of the basal ganglia, and suggest Type II GP neurons as a site of action for the antiparkinsonian effects of NBQX in some animal models (e.g. [43,48]).

Acknowledgements The authors wish to thank Dr. Debra Bergstrom for helpful discussions and advice.

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