Metabotropic glutamate receptors (mGluRs) are a major class of excitatory amino acid receptors. Eight mGluR subtypes, coupled to a variety of effector systems, have been cloned. These receptors have been classified into three groups based on amino acid sequence homology, effector systems, and pharmacological profile. Group I mGluRs increase phosphoinositide turnover, whereas groups II and III mGluRs are negatively coupled to adenylyl cyclase. The striatum possesses a high density of mGluR binding sites, and several mGluR mRNAs and proteins are expressed by striatal neurons. In rats, unilateral striatal injection of the nonsubtype selective mGluR agonist 1-aminocyclopentane-1S,3R-dicarboxylic acid (1S,3R-ACPD) results in contralateral rotation with delayed onset, thought to be secondary to an increase in dopamine release. We sought to determine the mGluR subtype(s) involved, the modulation of the rotation by other basal ganglia neurotransmitter systems, and the functional anatomy underlying the rotational behavior. The group I mGluR agonist 3,5-dihydroxyphenylglycine (DHPG) induced contralateral rotation in a dose-dependent manner, whereas group II and group III agonists were ineffective. Rotation induced by DHPG or 1S,3R-ACPD was attenuated by group I antagonists, but not by group II or group III antagonists. This suggests that the rotation is mediated by group I mGluRs. Rotation induced by DHPG or 1S,3R-ACPD was attenuated by pretreatment with antagonists at muscarinic cholinergic, adenosine A2, dopamine D2, or dopamine D1 receptors. Examination of FOS-like immunoreactivity after group I and group II mGluR agonist administration suggests increased activity in the striatopallidal pathway. However, [14C]-2-deoxyglucose uptake studies indicate increased activity in nuclei of the striatopallidal (indirect) pathway, particularly in the subthalamic nucleus, only after group I mGluR activation.
- metabotropic glutamate receptor
- basal ganglia
- subthalamic nucleus
- adenosine A2 receptors
- muscarinic receptors
Excitatory amino acids (EAAs) are important neurotransmitters in the basal ganglia, and EAAergic agents are being investigated actively as possible pharmacotherapies for basal ganglia disorders. A major class of EAA receptors are the metabotropic glutamate receptors (mGluRs), coupled to second messenger systems via G-proteins. Eight mGluR subtypes have been cloned, several of which possess splice variants. These mGluR subtypes have been categorized into three groups based on their amino acid sequence homology, effector systems, and pharmacological profile. When expressed and activated in transfection systems, group I receptors (mGluRs 1 and 5) stimulate phosphoinositide hydrolysis. Group II (mGluRs 2 and 3) and group III (mGluRs 4–8) receptors inhibit adenylyl cyclase, although with different pharmacological profiles (for review, see Pin and Duvosin, 1995; Roberts, 1995). Ligand-binding studies have shown the striatum to possess a high density of mGluR binding sites (Albin et al., 1992), and several mGluR mRNAs and proteins are expressed by striatal neurons, including members of all three mGluR groups (Abe et al., 1992; Martin et al., 1992; Shigemoto et al., 1992, 1993; Fotuhi et al., 1993; Ohishi et al., 1993a,b; Saugstead et al., 1994; Testa et al., 1994; Joly et al., 1995; Romano et al., 1995).
Intrastriatal injection of the nonsubtype selective mGluR agonist 1-aminocyclopentane-1S,3R-dicarboxylic acid (1S,3R-ACPD) results in vigorous contralateral rotation in rats (Sacaan et al., 1991, 1992;Kaatz and Albin, 1995). The basis for this behavioral change appears to be increased output of dopaminergic nigrostriatal neurons on the injected side, because there is an increase in dopamine and dopamine metabolites after in vivo intrastriatal injection of 1S,3R-ACPD (Sacaan et al., 1992). This does not appear to be a direct effect on dopamine release, because 1S,3R-ACPD does not increase dopamine release from striatal slices in vitro (Sacaan et al., 1992). Examination of Fos-like immunoreactivity (FLIR), a presumed indicator of neuronal activity changes, suggests that dopamine release occurs secondary to activation of the subthalamic nucleus (STN), and lesions of the STN result in blockade of 1S,3R-ACPD-induced contralateral rotation (Kaatz and Albin, 1995). We hypothesize that activation of striatal mGluRs results in increased activity of striatopallidal projection neurons with consequent disinhibition of the STN. The STN sends a major excitatory projection to the substantia nigra pars compacta (SNc) (Smith and Grace, 1992), and we believe that it is via this projection that increased dopamine release occurs after stimulation of striatal mGluRs. We performed a series of experiments with FLIR and mapping of local cerebral glucose metabolism (lCMRglu) with the [14C]-2-deoxyglucose autoradiographic method (Sokoloff, 1977).
A major feature of Parkinson’s Disease (PD) is overactivity of the STN (Albin et al., 1989). The possible STN overactivity seen after 1S,3R-ACPD suggests that mGluR antagonists may be useful for pharmacotherapy in PD. However, a clearer understanding of the pharmacology of mGluR-induced rotation is needed before this knowledge can be applied to pharmacotherapy of PD. In addition to testing our hypothesis about the functional anatomy of the rotational behavior, we addressed two additional questions. First, which mGluR subtype(s) is involved in the rotational behavior? Second, how do other neurotransmitter systems of the basal ganglia modulate mGluR-induced rotation?
MATERIALS AND METHODS
Drugs. 1-Aminocyclopentane-1S,3R-dicarboxylic acid (1S,3R-ACPD), (R,S)-3,5-dihydroxyphenylglycine (DHPG), (2S,3S,4S)-CCG (L-CCG-I), L(+)-2-amino-4-phosphonobutyric acid (L-AP4), α-methyl-4-phosphono-phenylglycine (MPPG), α-methyl-4-tetrazolylphenylglycine (MTPG), 1-aminoindan-1,5-dicarboxylic acid (UPF523), and α-methyl-4-carboxy-phenylglycine (MCPG) (Tocris Cookson, St. Louis, MO) were dissolved in 0.1 m phosphate buffer (PB), and pH was adjusted with 4N NaOH. 8-(3-Chlorostyryl) caffeine (CSC) and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (RBI, Natick, MA) were dissolved in 40% DMSO and diluted to volume with PB. 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethyl-carboxamido adenosine HCl (CGS 21680) (RBI) was dissolved in PB. SKF38393, SCH23390, eticlopride, quinpirole, and scopolamine (RBI) were dissolved in ddH2O. Haloperidol, USP (RBI) was dissolved in glacial acetic acid and diluted to volume with PB.
Intrastriatal injections. Male Sprague Dawley rats (Harlan, Indianapolis, IN) weighing 200–300 gm were used in all experiments. Rats were anesthetized with ether and mounted in a small animal stereotaxic apparatus (Kopf, Tujunga, CA). Through a burr hole in the skull, a 25 μl Hamilton syringe was introduced in the striatum, and drug was injected in a 2 μl volume over a 2 min period. Coordinates for striatal injection relative to bregma with the incisor bar at 0 mm were anterior–posterior, +1.0 mm; medial–lateral, 2.6 mm; dorsal–ventral, −5.7 mm (Paxinos and Watson, 1986). After injection, the syringe was left in place for 5 min and then slowly withdrawn. Animals were allowed to recover for 4 hr in bedded cages before rotational behavior was measured.
Rotational behavior. Four hours after intrastriatal injection, rats were placed in a clear hemispherical container and frequency of rotations ipsilateral and contralateral to the side of injection was recorded for a 5 min period. One rotation was defined as a 360° turn without a change of direction. Previous work from our laboratory (Kaatz and Albin, 1995) and others (Sacaan et al., 1991,1992) has determined this to be a reliable measure of rotational behavior, because contralateral rotations plateau at 3–4 hr after injection and remain constant for several hours. Significant differences in rotational behavior between groups were established by ANOVA in conjunction with Fisher’s protected least-significant difference post hoc comparisons (p < 0.05; Statview 4.01, Abacus Concepts, Berkeley, CA).
Dose–effect curves for the mGluR agonists 1S,3R-ACPD, DHPG, L-CCG-I, and L-AP4 were obtained by intrastriatal injection of these agents. Likewise, dose–effect curves were obtained for the mGluR antagonists MCPG, UPF523, MTPG, and MPPG by intrastriatal co-injection of each agent at various concentrations with a constant concentration of DHPG (0.5 μmol) or 1S,3R-ACPD (0.75 μmol).
CSC, DPCPX, CGS 21680, SKF 38393, SCH 23390, haloperidol, eticlopride, quinpirole, or scopolamine were administered intraperitoneally 20 min before intrastriatal injection of 1S,3R-ACPD or DHPG.
STN lesions. Animals were anesthetized with ketamine/xylazine (10:3, 1 mg/kg, i.p.) and mounted in a small animal stereotaxic apparatus. Ibotenic acid (6 μg in 0.5 μl) was injected unilaterally in the STN with a 25 μl Hamilton syringe. Coordinates for STN injection relative to bregma with the incisor bar at −3.4 mm were anterior–posterior, −3.7 mm; medial–lateral, 2.3 mm; dorsal–ventral, −8.4 mm (Paxinos and Watson, 1986). After injection, the syringe was left in place for 5 min and then slowly withdrawn. Animals were allowed to recover for 8 d. To select animals with appropriate lesions, animals were challenged with apomorphine (0.5 mg/kg, s.c.) on day 9 to test for ipsilateral rotational behavior (Delfs et al., 1995). Animals demonstrating >10 ipsilateral rotations/5 min were used in this experiment. On day 12, animals underwent the [14C]-2-deoxyglucose procedure as described below. At the conclusion of the experiment, the extent of STN lesions was determined from Nissl-stained sections by computer-assisted morphometry (Imaging Research Systems, Ontario, Canada), comparing the volume of the STN on the lesioned side versus the nonlesioned side.
Immunohistochemistry. Immediately after measurement of rotational behavior, rats were anesthetized deeply with pentobarbital (100 mg/kg, i.p.) and perfused transcardially with 100 ml PB followed by 250 ml 4% paraformaldehyde in PB. Brains were removed, post-fixed overnight in 4% paraformaldehyde in PB, and cryoprotected in 20% sucrose in PB for 24 hr. Sections (40 μm) were cut on a sliding microtome, saving sections from the forebrain through the midpons in 0.02% sodium azide in PB. Sections were processed for FLIR with an affinity-purified polyclonal antiserum directed against a conserved portion of the fos protein (Cambridge Biomedical Research, Wilmington, DE). Dilution of the antibody was 1:8000, and FLIR was visualized by the avidin–biotin conjugate technique using a Vectastain kit (Vector, Burlingame, CA) with NiCl2 enhancement. Sections were mounted on gelatin-coated slides, coverslipped, and viewed under light microscopy. Adjacent sections were stained with cresyl violet to confirm injection sites.
Histology. After measurement of rotational behavior, animals were anesthetized deeply with 0.4 ml ketamine/xylazine (10:3) and brains were removed rapidly and frozen over dry ice. Brains were sliced in 20 μm sections and thaw-mounted on gelatin-coated slides. Tissue was fixed over paraformaldehyde vapor for several days, stained with cresyl violet, and examined under light microscopy to confirm injection sites.
[14C]-2-deoxyglucose autoradiography. For 4 d before surgery, animals were habituated to rodent restrainers. On the day of the experiment, animals were implanted with a femoral vein catheter and then underwent intrastriatal injection of DHPG (1 μmol), L-CCG-I (1 μmol), or PB vehicle, as described above. After intrastriatal injection, animals were placed in rodent restrainers. At 195 min after intrastriatal injection, animals received an intravenous injection of [14C]-2-deoxyglucose (100 μCi/kg) (ARC, St. Louis, MO). At 240 min, animals were killed by intravenous injection of pentobarbital (50 mg/kg) followed by 0.1 m KCl (0.5 ml) and brains were removed rapidly and frozen over dry ice. Brains were sliced in 20 μm sections on a Lipshaw cryostat. Sections were thaw-mounted on gelatin-coated slides and exposed along with calibrated [14C] plastic standards (ARC) on β-max Hyperfilm (Amersham, Arlington Heights, IL) for 7 d. Sections were then fixed over paraformaldehyde vapor and stained with cresyl violet to confirm injection sites and aid in image analysis. Autoradiograms were analyzed by quantitative densitometry using an MCID-M1 image analysis system (Imaging Research Systems). Optical density measurements for each region were taken bilaterally in a minimum of five brain sections. Measurements of each structure were made in each section in which the structure was visible, and as large an area as possible was sampled. Tissue 14C concentrations were determined from the optical densities and a calibration curve obtained from co-exposed [14C] standards. For each animal, ratios of injected/uninjected side were made for each region (Patel et al., 1985). For each condition (vehicle, DHPG, STN lesion + DHPG, or L-CCG-I), the mean ratio for each region was determined. Mean ratios were compared by repeated-measures ANOVA (region, within-subjects factor) in conjunction with Bonferroni–Dunn post hoc comparisons (Statview 4.01, Abacus Concepts). Significant differences between drug treatments within each region were determined with 95% confidence intervals. Additional comparisons were made by determining the relative glucose utilization in each region as a ratio of the 14C concentration of the region to the mean 14C concentration of whole brain uptake for that animal (relative 2-deoxyglucose uptake) (Mitchell and Crossman, 1984; Sharp et al., 1993; Trugman and James, 1993). The ratio of region/whole brain uptake could then be used for comparisons with Student’s t tests. Statistical significance was taken as p < 0.05.
Ipsilateral rotations were infrequent, and there were no significant differences between groups in all the rotational behavior experiments. Additionally, administration of the mGluR antagonists UPF523, MCPG, MTPG, or MPPG alone produced no significant level of rotation. None of the mGluR agonists or antagonists produced histological evidence of toxicity at 24–48 hr after acute intrastriatal injection at any of the doses administered.
Intrastriatal administration of the selective type I mGluR agonist DHPG induces contralateral rotation in a dose-dependent manner. In addition, it appears to be more potent than the nonselective mGluR agonist 1S,3R-ACPD (Fig. 1). L-CCG-I (group II ≫ group I) does not elicit any significant level of rotation at any of the doses administered, nor does the group III agonist L-AP4 (Fig. 1). The group I mGluR antagonists UPF523 and MCPG (group I ≥ group II) attenuate contralateral rotation induced by DHPG or 1S,3R-ACPD, whereas the group II/III antagonists MTPG and MPPG have no effect at any of the doses administered (Fig. 2).
Purinergic, cholinergic, and dopaminergic modulation
Pretreatment with the adenosine A2 receptor antagonist CSC (3 mg/kg, i.p.) significantly reduced contralateral rotation induced by intrastriatal DHPG, whereas pretreatment with the adenosine A1 receptor antagonist DPCPX (5 mg/kg, i.p.) had no effect. Additionally, pretreatment with the adenosine A2 receptor agonist CGS 21680 (0.5 mg/kg, i.p.) significantly potentiated DHPG-induced contralateral rotation (Fig. 3). These results are similar to those obtained previously in our lab with 1S,3R-ACPD-induced contralateral rotation (Kearney and Albin, 1995).
Pretreatment with the muscarinic cholinergic antagonist scopolamine (5 mg/kg, i.p.) significantly reduced contralateral rotation induced by striatal 1S,3R-ACPD (1 μmol) or DHPG (1 μmol) (Fig.4) by 35 and 42%, respectively.
Pretreatment with the nonsubtype selective dopamine antagonist haloperidol (0.3 mg/kg, i.p.) reduced contralateral rotation induced by DHPG (1 μmol) by 40% (Fig. 5 A). This is similar to a previous result from Sacaan et al. (1992) that showed a similar reduction in 1S,3R-ACPD-induced rotation by haloperidol. Pretreatment with the dopamine D2 antagonist eticlopride (0.5 mg/kg, i.p.) significantly attenuated contralateral rotation induced by DHPG (0.5 μmol) or 1S,3R-ACPD (0.75 μmol), whereas pretreatment with the dopamine D2 agonist quinpirole (1 mg/kg, i.p.) had no effect on DHPG- or 1S,3R-ACPD-induced contralateral rotation (Fig. 5 C). Pretreatment with the dopamine D1 antagonist SCH 23390 (0.1 mg/kg, i.p.) attenuated rotation induced by DHPG, whereas it completely blocked rotation induced by 1S,3R-ACPD. The dopamine D1 agonist SKF 38393 (5 mg/kg) significantly attenuated rotation induced by 1S,3R-ACPD, but only reduced moderately DHPG-induced rotation (Fig. 5 B).
Intrastriatal injection of vehicle produces virtually no FLIR in the basal ganglia, although there is some FLIR in the parafascicular nucleus of the thalamus and hypothalamus (data not shown). Intrastriatal injection of DHPG produced a pattern of FLIR identical to that seen after 1S,3R-ACPD administration (Kaatz and Albin, 1995). Although there was virtually no FLIR in the striatum and SNc, there was a marked increase in FLIR in the globus pallidus (GP), entopeduncular nucleus (EP), STN, and substantia nigra pars reticulata (SNr) on the injected side (Fig. 6), as well as in the ventrobasal thalamus (data not shown). Intrastriatal injection of L-CCG-I produced a pattern of FLIR similar to that seen after 1S,3R-ACPD and DHPG (Fig. 6), although the magnitude of FLIR in the STN is not as great as that seen after DHPG (Fig. 6 C). After intrastriatal DHPG, virtually every neuron in the STN is labeled. However, after intrastriatal L-CCG-I, there are STN neurons that do not show FLIR.
Intrastriatal injection of L-AP4 produced a distinctive pattern of FLIR. There was a marked increase in FLIR in the striatum and nucleus accumbens. However, there was little FLIR in the GP, EP, STN, SNr, and SNc (Fig. 7).
Local cerebral glucose metabolism
Animals receiving a unilateral, striatal injection of DHPG demonstrated asymmetric increased lCMRglu in the following basal ganglia structures: GP (+12%), STN (+44%), SNr (+24%), SNc (+49%), and EP (+42%). In addition, increased lCMRglu was seen in the following basal ganglia projection regions: ventroanterior nuclei (VA, +25%), intralaminar nuclei (IL, +12%), and ventrolateral nuclei (VL, +16%) of the thalamus; lateral habenula (+15%); and the intermediate (SCint, 35%) and deep layers (SCdp, +26%) of the superior colliculus (Figs. 8, 9). Vehicle-injected controls showed no significant side-to-side differences. There was a small decrease in lCMRglu in the striatum on the injected side in both DHPG (−8%) and vehicle (−10%) injected animals, which is most likely the result of mechanical damage caused by injection.
Ibotenic acid lesion of the STN resulted in 65% loss of STN volume (Fig. 10). STN lesions decreased the mGluR stimulation effects on basal ganglia and projection area lCMRglu. The only areas that still showed significant increased lCMRglu after DHPG administration are the LH (+12%), SNc (+30%), and the EP (+23%) (Figs. 8, 9). After vehicle administration in animals with STN lesions, there were no significant differences versus intact, vehicle-injected controls (data not shown).
L-CCG-I-injected animals showed significant increased lCMRglu in the SNc (+11%). However, they show decreased lCMRglu in the striatum (−30%), VA thalamus (−23%), IL thalamus (−13%), VL thalamus (−19%), and the SCint (−7%) and SCdp (−4%) (Figs. 8, 9). Comparison of the relative 2-deoxyglucose uptake of the uninjected side in STN-lesioned, DHPG-injected rats versus intact vehicle-injected controls revealed that these changes represent actual decreases in lCMRglu on the injected side, and not increased lCMRglu on the uninjected side (Student’s t test, p < 0.05).
Our results suggest that contralateral rotation induced by unilateral striatal mGluR activation is mediated by group I mGluRs. First, the group I agonist DHPG (Ito et al., 1992; Schoepp et al., 1995) elicits dose-dependent rotation and is more potent than the nonselective agonist 1S,3R-ACPD, whereas group II and group III agonists induced no rotation. Second, group I antagonists UPF523 (Pellicciari et al., 1995; Sacaan et al., 1996) and MCPG (Hayashi et al., 1992) attenuate rotation induced by 1S,3R-ACPD or DHPG. The Group II/III antagonists MTPG and MPPG (Jane et al., 1995; Beddingfield et al., 1996) have no effect, even at higher doses, although they are more potent at their respective receptors than UPF523 and MCPG are at group I receptors (Jane et al., 1995; Pellicciari et al., 1995; Beddingfield et al., 1996). Additionally, chronic lithium treatment, which impairs mGluR-mediated PI signaling, suppresses 1S,3R-ACPD-induced rotational behavior (K. Kaatz and R. Albin, unpublished observations).
Rotation induced by intrastriatal DHPG or 1S,3R-ACPD is modulated similarly by agents that interact with other neurotransmitter systems of the basal ganglia. Pretreatment with a low dose of haloperidol (0.3 mg/kg) resulted in an ∼50% reduction of contralateral rotation induced by 1S,3R-ACPD (Sacaan et al., 1992) or DHPG, supporting the role of dopamine in mediating this behavior. To investigate further mGluR/dopamine receptor interactions, we examined the role of the D1 and D2 dopamine receptor subtypes. The D2 antagonist eticlopride decreased contralateral rotation induced by both 1S,3R-ACPD and DHPG to approximately the same extent, whereas the D2 agonist quinpirole had no effect. The D1 antagonist SCH23390 decreased both 1S,3R-ACPD- and DHPG-induced rotation. However, whereas SCH23390 reduced DHPG-induced rotation by ∼65%, it blocked 1S,3R-ACPD-induced rotation completely. The D1 agonist SKF38393 moderately reduced DHPG-induced rotation, whereas it reduced 1S,3R-ACPD-induced rotation by ∼50%. We propose that this difference is the result of an interaction between dopamine D1 and group II mGluRs. Preliminary results from our laboratory show that when rats are pretreated with SCH23390 or SKF 38393, intrastriatal L-CCG-I induces modest ipsilateral rotation (L. Darling, J. Kearney, and R. Albin, unpublished observations).
We reported previously on adenosine A2 receptor-mediated modulation of 1S,3R-ACPD-induced contralateral rotation (Kearney and Albin, 1995). We found similar adenosine A2receptor-mediated modulation of DHPG-induced contralateral rotation. Specifically, the A2 antagonist CSC attenuates DHPG-induced contralateral rotation, whereas the A2 agonist CGS 21680 potentiates rotation. The adenosine A1 antagonist DPCPX had no effect on rotation induced by either DHPG or 1S,3R-ACPD. Our results suggest that an interaction occurs between mGluRs and adenosine A2 receptors. The distribution of adenosine A2receptors in the striatum is restricted primarily to striatopallidal neurons (Schiffman et al., 1991). A potential site of action of mGluR–adenosine interaction is striatopallidal neurons, which also express mGluRs 1a and 5 (S. Tallaksen-Greene, K. Kaatz, C. Romano, and R. Albin, unpublished observations).
Sacaan et al. (1992) reported an increase in striatal acetylcholine release, which occurs after striatal 1S,3R-ACPD, approximately coincident with the onset of contralateral rotation. This suggests that striatal acetylcholine may be important for induction of rotation. The density of presynaptic striatal cholinergic markers is among the highest of any brain region, and cholinergic effects within the striatum are thought to be mediated primarily by muscarinic receptors. Additionally, M1, M3, and M5muscarinic cholinergic receptors share a major signal transduction pathway with group I mGluRs, PLC activation. The muscarinic cholinergic antagonist scopolamine attenuated contralateral rotation induced by 1S,3R-ACPD or DHPG. This could be the result of activation of group I mGluRs on cholinergic interneurons. Takeshita et al. (1996) reported mGluRs 1 and 5 on striatal cholinergic neurons suppress leak K+ conductance, thereby playing a role in controlling the membrane potential of the cholinergic neurons. Immunocolocalization studies have shown co-localization of cholinergic neurons with mGluR1a and mGluR5 in the striatum (Tallaksen-Greene, Kaatz, Romano, and Albin, unpublished observations).
Functional anatomical correlates
To examine the functional anatomical correlates of mGluR agonist-induced rotation, we examined FLIR as well as lCMRglu mapping. Although FLIR is often used as a marker of neuronal activation, some caution must be used in the interpretation of FLIR results. FLIR changes may be described more accurately as reflecting achange in the basal level of neuronal activity at the level of gene transcription (Morgan and Curran, 1991). There is evidence that some mismatch may occur for FLIR and 2-deoxyglucose in some brain regions, and neurons in some areas do not show FLIR under any stimulus conditions (Dragunow and Faull, 1989). For example, in the kindling model of epilepsy, the SNc showed no increase in c-fos mRNA, although it has an increased firing rate (Labiner et al., 1993). Several characteristics of the [14C]-2-deoxyglucose technique complement FLIR results and their interpretation. First, it is quantitative, which allowed us to make comparisons between different conditions such as intact versus STN-lesioned conditions. Second, it allowed us to determine the direction of regional neuronal activity changes (i.e., inhibition vs excitation).
The pattern of FLIR seen after 1S,3R-ACPD (Kaatz and Albin, 1995) and DHPG administration is identical. There is increased FLIR in the GP, EP, STN, SNr, and ventrobasal thalamus. The same pattern of FLIR is also seen after administration of L-CCG-I (group II ≫ group I). However, L-CCG-I does not produce rotational behavior. This suggests that there may be a lower threshold for generation of FLIR than for rotational behavior. Labiner et al. (1993) showed that there is little correlation between the magnitude of increase in FOS expression and change in neuronal activity, suggesting that there is a threshold for induction of FOS, beyond which there is little relationship between expression and neuronal activity.
Although L-CCG-I and DHPG produced the same pattern of FLIR, they produced very different patterns of lCMRglu. L-CCG-I administration resulted in decreased lCMRglu in basal ganglia projection areas, including the VA, VL and IL thalamus, SCint, and SCdp. The small increases in lCMRglu in some basal ganglia regions after intrastriatal L-CCG-I in the basal ganglia are most likely attributable to weak stimulation of group I mGluRs by L-CCG-I.
After intrastriatal DHPG administration, there was increased lCMRglu in the GP, STN, EP, SNr, and SNc. Additionally, we saw increases in lCMRglu in downstream projection areas, including VA, VL and IL thalamus, LH, SCint, and SCdp, suggesting increased basal ganglia output on the injected side. Conventionally, lCMRglu reflects increased glucose utilization in synaptic terminals and dendrites. However, in some cases in which neurons are extremely active, somal metabolism may also be evident (Di Rocco et al., 1989). We propose that this is the case within the STN. STN integrity is essential for rotational behavior after intrastriatal injection of mGluR agonists, and virtually every neuron in the STN shows FLIR (Kaatz and Albin, 1995). Additionally, regions receiving STN afferents such as the GP, EP, SNr, and SNc show increased lCMRglu, suggesting that the STN terminals are more active after stimulation of striatal group I mGluRs. In particular, the SNc, which receives an excitatory input from the STN, shows a 50% increase in lCMRglu. This suggests that the SNc is being activated, which may result in increased dopamine release in the striatum. One would also predict that the SNr/EP, which receives an excitatory input from the STN, would be activated as well. This prediction is borne out in that we observed increased lCMRglu in the SNr/EP as well as in their projection regions, including the LH, VA, VL and IL thalamus, SCint, and SCdp.
Lesions of the STN completely block rotational behavior after unilateral striatal 1S,3R-ACPD administration (Kaatz and Albin, 1995). STN lesions abolished lCMRglu increases in the STN, SNr, and basal ganglia projection areas including the thalamus, SCint, and SCdp. This supports the crucial role that the STN plays in the induction of rotational behavior. The only regions that still had significant increased lCMRglu were the EP, SNc, and LH. However, the magnitude of these increases was half that seen in intact rats. The lesions of the STN were not complete, and ∼35% of STN neurons remained. The incompleteness of the lesions would also account for some of the small, but nonsignificant, increases seen in the VL thalamus, SCint, and SCdp as well.
Previous results suggested that mGluR agonist-induced rotation occurs via a complex, multisynaptic mechanism that ultimately results in increased dopamine release on the injected side (Sacaan et al., 1992). Studies of FLIR suggested that the increase in dopamine release occurs secondary to activation of the STN (Kaatz and Albin, 1995), and lesions of the STN block 1S,3R-ACPD-induced rotational behavior (Kaatz and Albin, 1995), suggesting that the STN plays a crucial role in the induction of mGluR-induced rotation. The SNc receives an excitatory afferent from the STN (Smith and Grace, 1993), and we hypothesize that the STN induces the SNc to increase dopamine release through this projection. The level of STN activity is regulated indirectly by the striatum by modulation of GP neuron activity via the striatopallidal projection. Our functional anatomy studies suggest that activation of group I mGluRs in the striatum results in increased activity of the striatopallidal projection, which results in disinhibition of the STN with consequent excitation of the SNc as well as of the SNr/EP (Fig. 11).
We report evidence to support the role of group I mGluRs in mediating rotational behavior after intrastriatal administration of mGluR agonists. However, at this point, additional elucidation of the group I subtype involved is impossible, because there are no suitable pharmacological agents to differentiate between mGluRs 1 and 5. Recently, data from genetic knock-out experiments have distinguished between mGluRs 1 and 5 in cerebellar function. Mice deficient in mGluR1 show severe motor coordination problems and ataxia (Aiba et al., 1994;Conquet et al., 1994), whereas motor coordination appears normal in mice lacking mGluR5 (Wojtowicz et al., 1996). Basal ganglia function in these knock-out mice has not been examined as yet. Intrastriatal administration of DHPG in these knock-out mice may provide definitive evidence for involvement of mGluR1 and/or mGluR5 in mGluR-mediated rotational behavior.
mGluRs have been suggested as useful targets for pharmacotherapy in PD (Schoepp and Conn, 1993; Kaatz and Albin, 1995; Nicoletti et al., 1996). A hallmark feature of PD is overactivity of the STN, which may lead to progressive excitotoxic death of the remaining dopamine-containing neurons of the SNc (Saji et al., 1996) and hyperstimulation of the GPi/SNr (GPi = EP in rodents) resulting in a reduction in motor activity (Albin et al., 1989). We have shown that stimulation of striatal group I mGluRs results in probable STN hyperactivity. This suggests that group I mGluR antagonists may alleviate STN hyperactivity and may be useful for pharmacotherapy of PD. However, a clearer understanding of the physiological role of mGluRs within the basal ganglia is needed before this knowledge can be applied to PD pharmacotherapy.
This work was supported by grants from the Lucille P. Markey Trust, the Michigan Parkinson Foundation, and the Geriatrics Research, Education and Clinical Center of the Ann Arbor VAMC, and US Public Health Service Grants AG08671 and NS0722. We thank Tim Desmond and Laura Darling for technical assistance and Kenneth Guire at the Center for Statistical Consultation and Research, University of Michigan. We thank the anonymous reviewers for their constructive criticism.
Correspondence should be addressed to Dr. Roger L. Albin, 1014 Neuroscience Lab Building, 1103 E. Huron Street, Ann Arbor, MI 48104.