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Dopaminergic and GABAergic modulation of glutamate release from rat subthalamic nucleus efferents to the substantia nigra
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Research Report
Dopaminergic and GABAergic modulation of glutamate release from rat subthalamic nucleus efferents to the substantia nigra Theo Hatzipetros, Bryan K. Yamamoto ⁎ Laboratory of Neurochemistry, Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, MA 02118, USA
A R T I C LE I N FO
AB S T R A C T
Article history:
The regulation of the glutamatergic projection from the subthalamic nucleus (STN) to the
Accepted 6 January 2006
substantia nigra (SN) was investigated using dual-probe microdialysis in the awake behaving rat. Reverse dialysis of the cholinergic receptor agonist carbachol (1 mM) into the STN caused an increase in the extracellular concentrations of glutamate and
Keywords:
dopamine in the SN. The increase in glutamate was transient and returned toward
Subthalamic nucleus
basal values despite the continued perfusions of the STN with carbachol. Carbachol-
Substantia nigra
stimulated glutamate release was prolonged by perfusion of the selective D2 dopamine
Glutamate
receptor antagonist raclopride (100 μM) into the SN and was attenuated by the perfusion
Dopamine
of the selective D2-like receptor agonist quinpirole (10 μM). In contrast, perfusion of the D1
GABA
dopamine receptor antagonist SCH-23390 (100 μM) did not alter the carbachol-stimulated
Parkinson's disease
glutamate release even though it increased basal glutamate concentrations. Perfusion of the GABAA receptor antagonist bicuculline (10 μM) into the SN prolonged the carbacholstimulated glutamate release in similar fashion as raclopride. The present findings suggest that somatodendritically released dopamine in the SN regulates glutamate release from subthalamic axon terminals by differentially activating dopamine D2 and D1 receptors. Activation of D2 heteroreceptors, located on STN axon terminals, provides a negative feedback control on stimulated subthalamic glutamate release, while D1 receptor activation preferentially regulates basal glutamate concentrations. The findings of the present study also indicate that GABA exerts an inhibitory control on glutamate release in the SN through GABAA receptors. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
The subthalamic nucleus (STN) is the primary glutamatergic nucleus in the basal ganglia (Smith and Parent, 1988) and provides the main excitatory efferent projections to the substantia nigra pars reticulata (SNr) and the medial globus pallidus (Kita and Kitai, 1987). The STN plays an important role in movement control, and dysregulation of STN activity has
been associated with the pathophysiology of Parkinson's disease (Hamada and DeLong, 1992; Rodriguez et al., 1998). The activity of STN neurons is regulated primarily by an inhibitory GABAergic projection from the globus pallidus (Shink et al., 1996) and by excitatory glutamatergic projections from multiple cortical areas (Fujimoto and Kita, 1993; Maurice et al., 1998). Moreover, the STN neuronal activity is influenced by a cholinergic projection from the pedunculopontine
⁎ Corresponding author. Fax: +1 617 638 5668. E-mail address: [email protected] (B.K. Yamamoto). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.01.015
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nucleus (PPN) (Lavoie and Parent, 1994; Mouroux et al., 1995) and by a direct dopaminergic projection from the substantia nigra pars compacta (SNc) (Hassani et al., 1997). Cholinergic afferents are believed to exert an excitatory effect on STN neuronal activity and are mediated via cholinergic muscarinic M3 receptors (Flores et al., 1996). However, there is no consensus on the nature of the dopaminergic modulation of glutamatergic projections from the STN to the SN (Johnson et al., 1994). The glutamatergic projection from the STN to the SN, referred to as the subthalamonigral pathway, plays a pivotal role in the firing activity and bursting behavior of SNc DA cells (Smith and Grace, 1992) and SNr GABA neurons (Maurice et al., 2003). Glutamate, presumably from subthalamic afferents, stimulates SNc DA cells through an activation of ionotropic (Overton and Clark, 1991; Chergui et al., 1994) and metabotropic (Meltzer et al., 1997) glutamate receptors. The majority of these receptors are located on the long dendritic processes of DA cells, which enter into and branch within the SNr (Chatha et al., 2000). The GABAergic neurons of the SNr also express ionotropic glutamate receptors (Tse and Yung, 2000). It has been demonstrated that stimulation of the STN increases DA cell firing (Benazzouz et al., 2000a) and results in somatodendritic DA release (Rosales et al., 1994; Mintz et al., 1986) as well as GABA release in the SN (Windels et al., 2005; Rosales et al., 1997). The functional role of somatodendritically released DA in the SN is not as well characterized as DA released from nigrostriatal terminals. Previous studies have shown that DA released in the SN functions in the feedback regulation of DA cell activity via D2 autoreceptor activation (Pucak and Grace, 1994) and also in regulating the activity of GABAergic neurons in the SNr through its actions on D1 receptors (Abercrombie and DeBoer, 1997; Waszczak, 1990). In the present study, we investigated the possibility that somatodendritically released DA may play the additional role in regulating glutamate release from subthalamic terminals in the SN. This dopaminergic regulation could be mediated via an activation of D2 heteroreceptors located on asymmetric terminals in the SN that presumably are glutamatergic (Pickel et al., 2002) and/or through the activation of D1 heteroreceptors on glutamate terminals (Rosales et al., 1997). To investigate the regulation of the glutamatergic projection from the STN to the SN, dual-probe microdialysis was used in the awake behaving rat. The cholinergic receptor agonist carbachol was reverse dialyzed into the STN, and extracellular glutamate and dopamine concentrations were simultaneously monitored in the SN. Reverse dialysis of selective dopaminergic receptor agonists and antagonists into the SN during carbachol stimulation of the STN was used to assess the differential contributions of D1 and D2 receptor subtypes on glutamate release in the SN. Furthermore, the contribution of GABA on glutamate release in the SN was assessed by perfusing the selective GABAA antagonist bicuculline into the SN during carbachol stimulation of the STN. It was posited that the administration of the cholinergic receptor agonist carbachol into the STN will stimulate glutamate and, consequently, dopamine release in the SN. It was further hypothesized that the increase in extracellular dopamine within the SN
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negatively modulates the stimulated release of glutamate from the subthalamic terminals via the activation of D2 heteroreceptors.
2.
Results
The placements of the dialysis probes in SN and STN are shown in Fig. 1. The basal concentrations of glutamate and dopamine in the SN were 1524 ± 324 pg/20 μl and 1.4 ± 0.2 pg/20 μl respectively.
2.1. Effect of carbachol perfusions into the STN on extracellular glutamate concentrations in the SN Local perfusions of the cholinergic receptor agonist carbachol (1 mM) into the STN significantly increased extracellular glutamate in the SN to 171 ± 37% of basal concentrations (Fig. 2A). The increase in glutamate was transient and was only observed in the first 20-min dialysate sample after the beginning of carbachol perfusions. Glutamate concentrations then decreased toward basal values despite the continued perfusion of carbachol. No effects of carbachol on glutamate release in the SN were observed when it was perfused into regions adjacent to the STN.
2.2. Effect of carbachol perfusions into the STN on extracellular dopamine concentrations in the SN Local perfusions of carbachol (1 mM) into the STN significantly increased extracellular dopamine in the SN (Fig. 2B). The maximum DA concentrations occurred at 40 min after the beginning of carbachol perfusions and after the rise in glutamate in the STN. The peak extracellular DA concentrations were 124 ± 7% of basal concentrations. No effects of carbachol on dopamine release in the SN were observed when it was perfused into regions adjacent to the STN (data not shown).
2.3. Effect of raclopride and SCH 23390 perfusions into the SN on glutamate release in the SN during carbachol stimulation of the STN Local perfusions of the selective D2 dopamine receptor antagonist raclopride (100 μM) into the SN slightly enhanced the carbachol-stimulated glutamate release to 197 ± 26% of baseline concentrations. Notably, however, raclopride also prolonged the increase in carbachol-stimulated glutamate release throughout the perfusion period (Fig. 3). In contrast, local perfusions of the selective D1-like receptor antagonist SCH-23390 (100 μM) into the SN had no effect on carbacholstimulated subthalamic glutamate release in the SN at any time point (Fig. 3). In the absence of subthalamic stimulation by carbachol, raclopride perfusions did not change extracellular glutamate concentrations in the SN as compared to baseline concentrations (data not shown). In contrast, in the absence of subthalamic stimulation by carbachol, SCH perfusions in the SN caused significant increases in glutamate
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Fig. 1 – Location of the microdialysis probes in the subthalamic nucleus (A) and the substantia nigra (B). The positions of the probes were determined by postmortem histological analysis and are shown onto diagrams taken from the Paxinos and Watson (1986) rat brain atlas. STN probes that were considered a “hit” are represented in dark grey color (n = 8), and STN probes that were considered a “miss” are represented in light grey color (n = 7). SN probes were all “hits” and are represented in dark grey color (n = 15).
throughout the perfusion period which reached maximum levels of 164 ± 23% of baseline values.
2.4. Effect of quinpirole perfusions into the SN on carbachol-stimulated subthalamic glutamate release in the SN Local intranigral perfusions of the selective D2-like receptor agonist quinpirole (10 μM) into the SN attenuated the carbachol-stimulated glutamate release at the 20-min time point (Fig. 4). In the absence of subthalamic stimulation by carbachol, quinpirole perfusions did not change extracellular glutamate concentrations in the SN as compared to baseline concentrations (data not shown).
2.5. Effect of bicuculline perfusions into the SN on carbachol-stimulated subthalamic glutamate release in the SN Local perfusions of the selective GABAA receptor antagonist bicuculline (10 μM) into the SN prolonged the carbacholstimulated glutamate release throughout the perfusion period. The maximum increase was 178 ± 36% of baseline concentrations and occurred 40 min after the beginning of perfusions (Fig. 5). In the absence of subthalamic stimulation by carbachol, bicuculline perfusions did not change extracellular glutamate concentrations in the SN as compared to baseline concentrations (data not shown).
3.
Discussion
The overall objective of these studies was to investigate the regulation of the glutamatergic projection from the subthalamic nucleus to the substantia nigra. Carbachol perfusions into the STN caused an increase in the extracellular concentrations of glutamate and dopamine in the SN. The increase in extracellular glutamate was transient and returned toward basal values despite the continued perfusions of the STN with carbachol. Carbachol-stimulated glutamate release was prolonged by perfusion of the selective D2 dopamine receptor antagonist raclopride into the SN and was attenuated by the perfusion of the selective D2-like receptor agonist quinpirole. In contrast, the D1 dopamine receptor antagonist SCH-23390 did not alter the carbachol-stimulated glutamate release even though it increased basal glutamate concentrations. The GABAA receptor antagonist bicuculline prolonged the carbachol-stimulated glutamate release in similar fashion as raclopride. Consistent with previous studies using carbachol microinjections into the STN (Flores et al., 1996; Rosales et al., 1997), reverse dialysis of carbachol into the STN increased extracellular glutamate concentrations in the SN. These effects of carbachol are most likely mediated via the activation of muscarinic M3 cholinergic receptors present in the STN (Flores
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Fig. 3 – Effect of SCH and raclopride perfusions into the SN on carbachol-stimulated subthalamic glutamate release in the SN. Local perfusions of raclopride (100 μM) into the SN are represented by the open circles (n = 6). Local perfusions of SCH-23390 (100 μM) into the SN are represented by the closed triangles (n = 5). Control values for this figure (closed circles) are Dulbecco's perfusions into the SN and are reproduced from Fig. 1A (n = 8). Glutamate data are presented as percent of baseline. All data are mean ± SEM. *P b 0.05 vs. control, #P b 0.05 vs. baseline.
Fig. 2 – Effect of carbachol perfusions into the subthalamic nucleus on extracellular glutamate (A) and dopamine (B) concentrations in the SN. For part (A), local carbachol (1 mM) perfusions into the STN are represented by the closed circles (n = 8). Controls (open circles) are carbachol perfusions into regions adjacent to the STN (n = 7). For part (B), local perfusions of carbachol into the STN are represented by the closed circles (n = 7). Controls (open circles) are carbachol perfusions into regions adjacent to the STN (n = 5). Glutamate and dopamine data are presented as percent of baseline. All data are mean ± SEM. *P b 0.05 vs. control, #P b 0.05 vs. baseline.
et al., 1996) and not nicotinic cholinergic receptors since the latter do not contribute significantly to the activity of subthalamic neurons (Feger et al., 1979). Carbachol stimulation of the STN also increased extracellular dopamine within the SN, as previously demonstrated (Rosales et al., 1994; Mintz et al., 1986). This effect is probably mediated by subthalamic glutamatergic afferents that increase the activity of SNc DA cells and cause the release of somatodendritic DA mainly by activating ionotropic NMDA receptors (Overton and Clark, 1991; Chergui et al., 1994) present on the somatodendritic processes of SNc DA cells (Smith et al., 1996). Moreover, the observed sequence of events in the current study demonstrating that the maximal increase in DA concentrations occurs after the rise in glutamate and provides further evidence to suggest that the increase in dopamine was a result of the carbachol-induced STN stimulation of glutamate release in the SN.
Although reverse dialysis of carbachol into the STN increased the extracellular concentrations of glutamate in the SN, the increase was transient and returned to basal values despite the continued perfusion of carbachol. One possible explanation is that cholinergic receptor desensitization in the STN may have contributed to the lack of a sustained increase in extracellular glutamate within the SN. However, this possibility is unlikely since local pressure application of high concentrations (10 mM) of carbachol did not reduce the
Fig. 4 – Effect of quinpirole perfusions into the SN on carbachol-stimulated subthalamic glutamate release in the SN. Local perfusions of quinpirole (10 μM) into the SN are represented by the open circles (n = 7). Control values for this figure (closed circles) are Dulbecco's perfusions into the SN and are reproduced from Fig. 1A (n = 8). All data are mean ± SEM. *P b 0.05 vs. quinpirole, #P b 0.05 vs. baseline.
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Fig. 5 – Effect of bicuculline perfusions into the SN on carbachol-stimulated subthalamic glutamate release in the SN. Local perfusions of bicuculline (10 μM) into the SN are represented by the open circles (n = 4). Control values for this figure (closed circles) are Dulbecco's perfusions into the SN and are reproduced from Fig. 1A (n = 8). Glutamate data are presented as percent of baseline. All data are mean ± SEM. *P b 0.05 vs. control.
firing of subthalamic neurons (Falkenburger et al., 2001). It is also possible that subsequent to carbachol-induced STN stimulation, DA is released from striatal terminals, which in turn activates the polysynaptic indirect pathway of the basal ganglia to inhibit STN activity. This scenario is also unlikely since polysynaptic-induced changes in extracellular glutamate do not happen within the time-frame of our studies but are more likely to take place over the span of several hours (Stephans and Yamamoto, 1994). Another possibility that was considered and addressed in the current study is that the subthalamonigral glutamatergic projection is tightly regulated by an inhibitory dopaminergic feedback mechanism initiated within the substantia nigra at the glutamatergic terminals. Several studies have shown that somatodendritically released DA within the SN plays a role in the regulation of SNc DA and SNr GABA cell activity. However, very few studies to date have suggested that dopamine can regulate basal glutamate release from STN axon terminals (Rosales et al., 1997; Abarca et al., 1995). The current study extended these findings to show that antagonism of D2 receptors within the SN enhances and prolongs the increase in glutamate produced by carbachol stimulation of the STN (Fig. 3). Moreover, D2 receptor agonism attenuated the carbachol-induced increase in glutamate. Therefore, the increase in dopamine observed within the SN during carbachol stimulation of the STN (Fig. 2B) may limit the increase in glutamate via activation of D2 receptors located on asymmetric excitatory terminals (Pickel et al., 2002). The D2 mediated inhibition of glutamate release, however, appears to be dependent on STN activity since D2 dopamine receptor blockade did not affect the basal concentrations of extracellular glutamate in the SN in the absence of STN stimulation. In contrast, perfusion of the D1 receptor antagonist SCH23390 into the SN did not enhance nor prolong the transient
increase in extracellular glutamate produced by carbachol (Fig. 3). These findings do not suggest a role for D1 receptors in the regulation of stimulated glutamate release from STN terminals. SCH-23390 did increase though extracellular glutamate in the SN in the absence of carbachol stimulation of the STN, a finding that is consistent with a study by Rosales et al. (1997) which suggested a role for D1 receptors on the regulation of basal glutamate concentrations. Therefore, it appears that D1 receptors are preferentially activated to maintain tonic glutamate release. Collectively, these results provide the first evidence to suggest that somatodendritically released dopamine in the SN through an activation of D2 dopamine heteroreceptors provides a negative feedback control on stimulated or phasic glutamate release from STN terminals within the SN. In addition, D1 dopamine heteroreceptor activation regulates basal glutamate concentrations in the SN in the absence of STN stimulation or during tonic STN activity. Another possibility that was considered in this study is that the regulation of subthalamonigral glutamate transmission can be mediated by the inhibitory neurotransmitter GABA that also is increased in response to subthalamic stimulation (Rosales et al., 1997). GABA in the SN is critically involved in the regulation of DA cell activity by tonically suppressing the excitatory inputs (Kitai et al., 1999) via GABAA receptor activation localized on DA cell bodies (Tepper et al., 1995; Paladini et al., 1999). In the present study, perfusion of the GABAA receptor antagonist bicuculline into the SN during carbachol stimulation of the STN effectively enhanced the increase in glutamate release produced by STN stimulation (Fig. 5). This indicates that GABA, in addition to DA, negatively regulates glutamate release from STN axon terminals either through a direct activation of GABAA heteroreceptors on the STN axon terminals or indirectly by decreasing SNc DA cell activity. A recent study has shown that DA depolarizes STN neuron via a direct nigrosubthalamic pathway (Cragg et al., 2004). Therefore, GABA-induced inhibition of the nigrosubthalamic dopaminergic projection could inhibit the release of glutamate in the SN from subthalamic neurons. Conversely, inhibition of GABA cells in the SN with bicuculline could disinhibit the nigrosubthalamic DA path to increase subthalamic glutamatergic efferent activity to the SN. The observed changes in extracellular glutamate in the SN were designed to be selectively reflective of stimulation of the STN. Although additional glutamatergic projections to the SN that originate from the frontal cortex (Carter, 1982) and the PPN (Tokuno et al., 1988) may also contribute to the basal concentrations of extracellular glutamate in the SN, it is unknown if these or other glutamatergic projections are under the same dopaminergic control as those originating from the STN. Although areas adjacent to the STN may have unknown glutamatergic projections to the SN or may affect STN activity, our findings illustrate that when regions adjacent to the STN were perfused with carbachol (Fig. 2A), no changes in glutamate concentrations were observed in the SN. Therefore, the increases in glutamate within the SN observed during carbachol perfusions most likely originate from the STN. The current findings have implications for the pathophysiology and pharmacotherapy of Parkinson's disease (PD). It
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has been hypothesized that the progressive loss of SNc DA cells in PD results from an increased glutamate transmission in the SN and a subsequent excitotoxic cell death (Rodriguez et al., 1998). Moreover, lesions or high-frequency stimulation of the STN reduce most of the motor impairments associated with PD (Parkin et al., 2001; Benazzouz et al., 2000b), presumably by reducing the activity of the subthalamonigral pathway. Based on these past findings and the current study, modulation of the excitatory cholinergic stimulation of the subthalamonigral glutamatergic pathway at the level of the STN may be therapeutically beneficial. Perhaps more important, however, is that modulation of the glutamate release by the D2 receptor within the SN occurs only under conditions of STN stimulation and suggests that the subthalamonigral glutamatergic pathway can be tightly regulated primarily at the axon terminal region by presynaptic inhibitory D2 heteroreceptors. Thus, these findings further support the use of D2 agonists that target not only the striatum but also target the SN for the modulation of aberrant or augmented subthalamonigral glutamate release in relation to the symptomatic treatment of PD and possibly for the prevention of the progressive excitotoxic degeneration of dopamine cell bodies in the substantia nigra.
4.
Experimental procedures
4.1.
Animals
Adult male Sprague–Dawley rats (200–300 g; Harlan Sprague Dawley, IN, USA) were used in all experiments. Rats were housed four per cage prior to surgery/dialysis and were maintained on a 12:12-h light/dark cycle in a temperature- and humidity-controlled environment. Food and water were available ad libitum. Animal care was in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals which was approved and monitored by the Institutional Animal Care and Use Committee of Boston University. 4.2.
Microdialysis probe construction
Dialysis probes were of a concentric flow design and were constructed as previously described (Yamamoto and Pehek, 1990; Yamamoto and Davy, 1992). The length of the dialysis membrane was 1 mm for the STN probes and 1.5 mm for the SN probes. The dead volume of all probes was calculated so as to synchronize the timing and the initiation of drug perfusion with sample collection. 4.3.
Surgical probe implantation
Rats were anesthetized with a combination of xylazine (6 mg/kg, i. p.) and ketamine hydrochloride (70 mg/kg, i.p.) and were mounted into a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA) for the implantation of the microdialysis probes into the SN and the ipsilateral STN. The skull was exposed, holes were drilled, and probes were slowly lowered to the following co-ordinates aimed at STN: AP −3.8 mm, ML ±2.4 mm as referenced from bregma, DV −7.3 mm as referenced from dura, and at SN (at a 15° lateral angle): AP −5.6 mm, ML ±4.2 mm as referenced from bregma, DV −7.5 mm as referenced from dura (Paxinos and Watson, 1986). The probes were secured to the skull with three stainless steel machine screws and cranioplastic cement. Rats were transferred into the dialysis cages and were allowed to recover over night. Dialysis was conducted on the following morning.
4.4.
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In vivo microdialysis
After a 2-h prebaseline perfusion period, the microdialysis probes were perfused with modified Dulbecco's phosphate-buffered saline (138 mM NaCl, 2.7 mM KCl, 0.5 mM MgCl2, 1.5 mM KH2PO4, 1.2 mM CaCl2, and 5.0 mM d-glucose, pH 7.4) until stable baseline concentrations of DA or glutamate were achieved in the dialysate. The perfusion medium was then switched to one containing the treatment drug and was reverse dialyzed into the brain for 1 h. The perfusion medium was then switched back to Dulbecco's for 1 h of post-drug collection. The perfusion was conducted at a constant rate of 1.5 μl/min using a microinfusion pump (Harvard Apparatus, Holliston, MA, USA). Dialysate samples (20 μl) were collected from the outflow of the probe every 20 min. At the end of each experiment, the rats were killed by decapitation, and the probes were manually perfused with green dye before they were extracted. The brains were removed and frozen to be sectioned later in 40-μm-thick coronal slices for probe placement verification. The green dye was used to determine the location of the dialysis membrane, and the exact area that was perfused. 4.5.
Measurement of extracellular dopamine and glutamate
Separate groups of rats were used for the measurement of dopamine and glutamate in microdialysates. Dialysate aliquots (20 μl) were assayed for dopamine by high-performance liquid chromatography (HPLC) coupled to electrochemical detection. Separation was achieved with a 3-μm C18 column (100 × 2.0 mm; Phenomenex, Torrance, CA, USA) and a mobile phase consisting of 25 mM citric acid, 20 mM sodium acetate, 0.1 mM EDTA, 0.215 mM octyl sodium sulfate, and 5% methanol (pH 3.90). Electrochemical detection was performed with a BAS Model LC-4B electrochemical detector (BAS Instruments, West Lafayette, IN, USA) and a glassy carbon electrode maintained at a potential of 0.6 V. Data were collected using EZChrom Elite application software (Scientific Software Inc., Pleasanton, CA, USA). Glutamate was assayed from 20 μl aliquots by HPLC coupled to fluorescence detection following precolumn derivatization with opthaldialdehyde as described by (Donzanti and Yamamoto, 1988) with slight modifications. A 10 μl aliquot of the derivatization reagent was automatically added to the 20 μl dialysate sample by an ESA Model 542 autosampler (ESA Inc, Chelmsford, MA, USA), mixed, and allowed to react for exactly 90 s before injection onto the column. Separation was achieved with a 3-μm C18 column and a mobile phase consisting of 0.1 M sodium phosphate, 0.1 mM EDTA, and 10% methanol (pH 6.65). Fluorescence detection was performed with a Waters 474 Fluorescence Detector (Waters Corp, Milford, MA, USA). The excitation and emission wavelengths were set at 340 and 440 nm respectively. Data were collected using EZChrom Elite application software. 4.6.
Drugs
Carbamylcholine chloride (carbachol), s-(−)-raclopride, r-(+)-SCH23390, (+)-bicuculline, (−)-quinpirole, and Dulbecco's phosphatebuffered saline were all purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Optimal and pharmacologically active drug concentrations were chosen based on published reports in which carbachol (Moor et al., 1995), raclopride (Rahman et al., 2001), SCH-23390 (Rossi et al., 2005), bicuculline (Zackheim and Abercrombie, 2005), and quinpirole (Tanganelli et al., 2004) were intracerebrally reverse dialyzed. 4.7.
Data analysis
Glutamate and dopamine data are presented as a percent of baseline to standardize across groups and allow for effective comparisons between the different treatments. Dialysis data are presented as means ± SEM. Statistical significance was determined
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by a two-way ANOVA with repeated measures coupled with Bonferroni's post hoc test (P b 0.05).
Acknowledgments This work was supported by DA07606 and DAMD 17-99-1-9479.
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Kita, H., 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. Kitai, S.T., Shepard, P.D., Callaway, J.C., Scroggs, R., 1999. Afferent modulation of dopamine neuron firing patterns. Curr. Opin. Neurobiol. 9, 690–697. Lavoie, B., Parent, A., 1994. Pedunculopontine nucleus in the squirrel monkey: projections to the basal ganglia as revealed by anterograde tract-tracing methods. J. Comp. Neurol. 344, 210–231. Maurice, N., Deniau, J.M., Glowinski, J., Thierry, A.M., 1998. Relationships between the prefrontal cortex and the basal ganglia in the rat: physiology of the corticosubthalamic circuits. J. Neurosci. 18, 9539–9546. Maurice, N., Thierry, A.M., Glowinski, J., Deniau, J.M., 2003. Spontaneous and evoked activity of substantia nigra pars reticulata neurons during high-frequency stimulation of the subthalamic nucleus. J. Neurosci. 23, 9929–9936. Meltzer, L.T., Serpa, K.A., Christoffersen, C.L., 1997. Metabotropic glutamate receptor-mediated inhibition and excitation of substantia nigra dopamine neurons. Synapse 26, 184–193. Mintz, I., Hammond, C., Guibert, B., Leviel, V., 1986. Stimulation of the subthalamic nucleus enhances the release of dopamine in the rat substantia nigra. Brain Res. 376, 406–408. Moor, E., DeBoer, P., Auth, F., Westerink, B.H., 1995. Characterisation of muscarinic autoreceptors in the septohippocampal system of the rat: a microdialysis study. Eur. J. Pharmacol. 294, 155–161. Mouroux, M., Hassani, O.K., Feger, J., 1995. Electrophysiological study of the excitatory parafascicular projection to the subthalamic nucleus and evidence for ipsi- and contralateral controls. Neuroscience 67, 399–407. Overton, P., Clark, D., 1991. N-methyl-D-aspartate increases the excitability of nigrostriatal dopamine terminals. Eur. J. Pharmacol. 201, 117–120. Paladini, C.A., Celada, P., Tepper, J.M., 1999. Striatal, pallidal, and pars reticulata evoked inhibition of nigrostriatal dopaminergic neurons is mediated by GABA(A) receptors in vivo. Neuroscience 89, 799–812. Parkin, S., Nandi, D., Giladi, N., Joint, C., Gregory, R., Bain, P., Scott, R., Aziz, T.Z., 2001. Lesioning the subthalamic nucleus in the treatment of Parkinson's disease. Stereotact. Funct. Neurosurg. 77, 68–72. Paxinos, G., Watson, C., 1986. The Rat Brain in Stereotaxic Coordinates. Academic Press, Sydney. Pickel, V.M., Garzon, M., Mengual, E., 2002. Electron microscopic immunolabeling of transporters and receptors identifies transmitter-specific functional sites envisioned in Cajal's neuron. Prog. Brain Res. 136, 145–155. Pucak, M.L., Grace, A.A., 1994. Evidence that systemically administered dopamine antagonists activate dopamine neuron firing primarily by blockade of somatodendritic autoreceptors. J. Pharmacol. Exp. Ther. 271, 1181–1192. Rahman, S., Engleman, E., Simon, J., McBride, W.J., 2001. Negative interaction of dopamine D2 receptor antagonists and GBR 12909 and GBR 12935 dopamine uptake inhibitors in the nucleus accumbens. Eur. J. Pharmacol. 414, 37–44. Rodriguez, M.C., Obeso, J.A., Olanow, C.W., 1998. Subthalamic nucleus-mediated excitotoxicity in Parkinson's disease: a target for neuroprotection. Ann. Neurol. 44, S175–S188. Rosales, M.G., Flores, G., Hernandez, S., Martinez-Fong, D., Aceves, J., 1994. Activation of subthalamic neurons produces NMDA receptor-mediated dendritic dopamine release in substantia nigra pars reticulata: a microdialysis study in the rat. Brain Res. 645, 335–337. Rosales, M.G., Martinez-Fong, D., Morales, R., Nunez, A., Flores, G., Gongora-Alfaro, J.L., Floran, B., Aceves, J., 1997. Reciprocal interaction between glutamate and dopamine in the pars
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Dopaminergic and GABAergic modulation of glutamate release from rat subthalamic nucleus efferents to the substantia nigra Theo Hatzipetros, Bryan K. Yamamoto ⁎ Laboratory of Neurochemistry, Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, MA 02118, USA
A R T I C LE I N FO
AB S T R A C T
Article history:
The regulation of the glutamatergic projection from the subthalamic nucleus (STN) to the
Accepted 6 January 2006
substantia nigra (SN) was investigated using dual-probe microdialysis in the awake behaving rat. Reverse dialysis of the cholinergic receptor agonist carbachol (1 mM) into the STN caused an increase in the extracellular concentrations of glutamate and
Keywords:
dopamine in the SN. The increase in glutamate was transient and returned toward
Subthalamic nucleus
basal values despite the continued perfusions of the STN with carbachol. Carbachol-
Substantia nigra
stimulated glutamate release was prolonged by perfusion of the selective D2 dopamine
Glutamate
receptor antagonist raclopride (100 μM) into the SN and was attenuated by the perfusion
Dopamine
of the selective D2-like receptor agonist quinpirole (10 μM). In contrast, perfusion of the D1
GABA
dopamine receptor antagonist SCH-23390 (100 μM) did not alter the carbachol-stimulated
Parkinson's disease
glutamate release even though it increased basal glutamate concentrations. Perfusion of the GABAA receptor antagonist bicuculline (10 μM) into the SN prolonged the carbacholstimulated glutamate release in similar fashion as raclopride. The present findings suggest that somatodendritically released dopamine in the SN regulates glutamate release from subthalamic axon terminals by differentially activating dopamine D2 and D1 receptors. Activation of D2 heteroreceptors, located on STN axon terminals, provides a negative feedback control on stimulated subthalamic glutamate release, while D1 receptor activation preferentially regulates basal glutamate concentrations. The findings of the present study also indicate that GABA exerts an inhibitory control on glutamate release in the SN through GABAA receptors. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
The subthalamic nucleus (STN) is the primary glutamatergic nucleus in the basal ganglia (Smith and Parent, 1988) and provides the main excitatory efferent projections to the substantia nigra pars reticulata (SNr) and the medial globus pallidus (Kita and Kitai, 1987). The STN plays an important role in movement control, and dysregulation of STN activity has
been associated with the pathophysiology of Parkinson's disease (Hamada and DeLong, 1992; Rodriguez et al., 1998). The activity of STN neurons is regulated primarily by an inhibitory GABAergic projection from the globus pallidus (Shink et al., 1996) and by excitatory glutamatergic projections from multiple cortical areas (Fujimoto and Kita, 1993; Maurice et al., 1998). Moreover, the STN neuronal activity is influenced by a cholinergic projection from the pedunculopontine
⁎ Corresponding author. Fax: +1 617 638 5668. E-mail address: [email protected] (B.K. Yamamoto). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.01.015
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nucleus (PPN) (Lavoie and Parent, 1994; Mouroux et al., 1995) and by a direct dopaminergic projection from the substantia nigra pars compacta (SNc) (Hassani et al., 1997). Cholinergic afferents are believed to exert an excitatory effect on STN neuronal activity and are mediated via cholinergic muscarinic M3 receptors (Flores et al., 1996). However, there is no consensus on the nature of the dopaminergic modulation of glutamatergic projections from the STN to the SN (Johnson et al., 1994). The glutamatergic projection from the STN to the SN, referred to as the subthalamonigral pathway, plays a pivotal role in the firing activity and bursting behavior of SNc DA cells (Smith and Grace, 1992) and SNr GABA neurons (Maurice et al., 2003). Glutamate, presumably from subthalamic afferents, stimulates SNc DA cells through an activation of ionotropic (Overton and Clark, 1991; Chergui et al., 1994) and metabotropic (Meltzer et al., 1997) glutamate receptors. The majority of these receptors are located on the long dendritic processes of DA cells, which enter into and branch within the SNr (Chatha et al., 2000). The GABAergic neurons of the SNr also express ionotropic glutamate receptors (Tse and Yung, 2000). It has been demonstrated that stimulation of the STN increases DA cell firing (Benazzouz et al., 2000a) and results in somatodendritic DA release (Rosales et al., 1994; Mintz et al., 1986) as well as GABA release in the SN (Windels et al., 2005; Rosales et al., 1997). The functional role of somatodendritically released DA in the SN is not as well characterized as DA released from nigrostriatal terminals. Previous studies have shown that DA released in the SN functions in the feedback regulation of DA cell activity via D2 autoreceptor activation (Pucak and Grace, 1994) and also in regulating the activity of GABAergic neurons in the SNr through its actions on D1 receptors (Abercrombie and DeBoer, 1997; Waszczak, 1990). In the present study, we investigated the possibility that somatodendritically released DA may play the additional role in regulating glutamate release from subthalamic terminals in the SN. This dopaminergic regulation could be mediated via an activation of D2 heteroreceptors located on asymmetric terminals in the SN that presumably are glutamatergic (Pickel et al., 2002) and/or through the activation of D1 heteroreceptors on glutamate terminals (Rosales et al., 1997). To investigate the regulation of the glutamatergic projection from the STN to the SN, dual-probe microdialysis was used in the awake behaving rat. The cholinergic receptor agonist carbachol was reverse dialyzed into the STN, and extracellular glutamate and dopamine concentrations were simultaneously monitored in the SN. Reverse dialysis of selective dopaminergic receptor agonists and antagonists into the SN during carbachol stimulation of the STN was used to assess the differential contributions of D1 and D2 receptor subtypes on glutamate release in the SN. Furthermore, the contribution of GABA on glutamate release in the SN was assessed by perfusing the selective GABAA antagonist bicuculline into the SN during carbachol stimulation of the STN. It was posited that the administration of the cholinergic receptor agonist carbachol into the STN will stimulate glutamate and, consequently, dopamine release in the SN. It was further hypothesized that the increase in extracellular dopamine within the SN
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negatively modulates the stimulated release of glutamate from the subthalamic terminals via the activation of D2 heteroreceptors.
2.
Results
The placements of the dialysis probes in SN and STN are shown in Fig. 1. The basal concentrations of glutamate and dopamine in the SN were 1524 ± 324 pg/20 μl and 1.4 ± 0.2 pg/20 μl respectively.
2.1. Effect of carbachol perfusions into the STN on extracellular glutamate concentrations in the SN Local perfusions of the cholinergic receptor agonist carbachol (1 mM) into the STN significantly increased extracellular glutamate in the SN to 171 ± 37% of basal concentrations (Fig. 2A). The increase in glutamate was transient and was only observed in the first 20-min dialysate sample after the beginning of carbachol perfusions. Glutamate concentrations then decreased toward basal values despite the continued perfusion of carbachol. No effects of carbachol on glutamate release in the SN were observed when it was perfused into regions adjacent to the STN.
2.2. Effect of carbachol perfusions into the STN on extracellular dopamine concentrations in the SN Local perfusions of carbachol (1 mM) into the STN significantly increased extracellular dopamine in the SN (Fig. 2B). The maximum DA concentrations occurred at 40 min after the beginning of carbachol perfusions and after the rise in glutamate in the STN. The peak extracellular DA concentrations were 124 ± 7% of basal concentrations. No effects of carbachol on dopamine release in the SN were observed when it was perfused into regions adjacent to the STN (data not shown).
2.3. Effect of raclopride and SCH 23390 perfusions into the SN on glutamate release in the SN during carbachol stimulation of the STN Local perfusions of the selective D2 dopamine receptor antagonist raclopride (100 μM) into the SN slightly enhanced the carbachol-stimulated glutamate release to 197 ± 26% of baseline concentrations. Notably, however, raclopride also prolonged the increase in carbachol-stimulated glutamate release throughout the perfusion period (Fig. 3). In contrast, local perfusions of the selective D1-like receptor antagonist SCH-23390 (100 μM) into the SN had no effect on carbacholstimulated subthalamic glutamate release in the SN at any time point (Fig. 3). In the absence of subthalamic stimulation by carbachol, raclopride perfusions did not change extracellular glutamate concentrations in the SN as compared to baseline concentrations (data not shown). In contrast, in the absence of subthalamic stimulation by carbachol, SCH perfusions in the SN caused significant increases in glutamate
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Fig. 1 – Location of the microdialysis probes in the subthalamic nucleus (A) and the substantia nigra (B). The positions of the probes were determined by postmortem histological analysis and are shown onto diagrams taken from the Paxinos and Watson (1986) rat brain atlas. STN probes that were considered a “hit” are represented in dark grey color (n = 8), and STN probes that were considered a “miss” are represented in light grey color (n = 7). SN probes were all “hits” and are represented in dark grey color (n = 15).
throughout the perfusion period which reached maximum levels of 164 ± 23% of baseline values.
2.4. Effect of quinpirole perfusions into the SN on carbachol-stimulated subthalamic glutamate release in the SN Local intranigral perfusions of the selective D2-like receptor agonist quinpirole (10 μM) into the SN attenuated the carbachol-stimulated glutamate release at the 20-min time point (Fig. 4). In the absence of subthalamic stimulation by carbachol, quinpirole perfusions did not change extracellular glutamate concentrations in the SN as compared to baseline concentrations (data not shown).
2.5. Effect of bicuculline perfusions into the SN on carbachol-stimulated subthalamic glutamate release in the SN Local perfusions of the selective GABAA receptor antagonist bicuculline (10 μM) into the SN prolonged the carbacholstimulated glutamate release throughout the perfusion period. The maximum increase was 178 ± 36% of baseline concentrations and occurred 40 min after the beginning of perfusions (Fig. 5). In the absence of subthalamic stimulation by carbachol, bicuculline perfusions did not change extracellular glutamate concentrations in the SN as compared to baseline concentrations (data not shown).
3.
Discussion
The overall objective of these studies was to investigate the regulation of the glutamatergic projection from the subthalamic nucleus to the substantia nigra. Carbachol perfusions into the STN caused an increase in the extracellular concentrations of glutamate and dopamine in the SN. The increase in extracellular glutamate was transient and returned toward basal values despite the continued perfusions of the STN with carbachol. Carbachol-stimulated glutamate release was prolonged by perfusion of the selective D2 dopamine receptor antagonist raclopride into the SN and was attenuated by the perfusion of the selective D2-like receptor agonist quinpirole. In contrast, the D1 dopamine receptor antagonist SCH-23390 did not alter the carbachol-stimulated glutamate release even though it increased basal glutamate concentrations. The GABAA receptor antagonist bicuculline prolonged the carbachol-stimulated glutamate release in similar fashion as raclopride. Consistent with previous studies using carbachol microinjections into the STN (Flores et al., 1996; Rosales et al., 1997), reverse dialysis of carbachol into the STN increased extracellular glutamate concentrations in the SN. These effects of carbachol are most likely mediated via the activation of muscarinic M3 cholinergic receptors present in the STN (Flores
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Fig. 3 – Effect of SCH and raclopride perfusions into the SN on carbachol-stimulated subthalamic glutamate release in the SN. Local perfusions of raclopride (100 μM) into the SN are represented by the open circles (n = 6). Local perfusions of SCH-23390 (100 μM) into the SN are represented by the closed triangles (n = 5). Control values for this figure (closed circles) are Dulbecco's perfusions into the SN and are reproduced from Fig. 1A (n = 8). Glutamate data are presented as percent of baseline. All data are mean ± SEM. *P b 0.05 vs. control, #P b 0.05 vs. baseline.
Fig. 2 – Effect of carbachol perfusions into the subthalamic nucleus on extracellular glutamate (A) and dopamine (B) concentrations in the SN. For part (A), local carbachol (1 mM) perfusions into the STN are represented by the closed circles (n = 8). Controls (open circles) are carbachol perfusions into regions adjacent to the STN (n = 7). For part (B), local perfusions of carbachol into the STN are represented by the closed circles (n = 7). Controls (open circles) are carbachol perfusions into regions adjacent to the STN (n = 5). Glutamate and dopamine data are presented as percent of baseline. All data are mean ± SEM. *P b 0.05 vs. control, #P b 0.05 vs. baseline.
et al., 1996) and not nicotinic cholinergic receptors since the latter do not contribute significantly to the activity of subthalamic neurons (Feger et al., 1979). Carbachol stimulation of the STN also increased extracellular dopamine within the SN, as previously demonstrated (Rosales et al., 1994; Mintz et al., 1986). This effect is probably mediated by subthalamic glutamatergic afferents that increase the activity of SNc DA cells and cause the release of somatodendritic DA mainly by activating ionotropic NMDA receptors (Overton and Clark, 1991; Chergui et al., 1994) present on the somatodendritic processes of SNc DA cells (Smith et al., 1996). Moreover, the observed sequence of events in the current study demonstrating that the maximal increase in DA concentrations occurs after the rise in glutamate and provides further evidence to suggest that the increase in dopamine was a result of the carbachol-induced STN stimulation of glutamate release in the SN.
Although reverse dialysis of carbachol into the STN increased the extracellular concentrations of glutamate in the SN, the increase was transient and returned to basal values despite the continued perfusion of carbachol. One possible explanation is that cholinergic receptor desensitization in the STN may have contributed to the lack of a sustained increase in extracellular glutamate within the SN. However, this possibility is unlikely since local pressure application of high concentrations (10 mM) of carbachol did not reduce the
Fig. 4 – Effect of quinpirole perfusions into the SN on carbachol-stimulated subthalamic glutamate release in the SN. Local perfusions of quinpirole (10 μM) into the SN are represented by the open circles (n = 7). Control values for this figure (closed circles) are Dulbecco's perfusions into the SN and are reproduced from Fig. 1A (n = 8). All data are mean ± SEM. *P b 0.05 vs. quinpirole, #P b 0.05 vs. baseline.
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Fig. 5 – Effect of bicuculline perfusions into the SN on carbachol-stimulated subthalamic glutamate release in the SN. Local perfusions of bicuculline (10 μM) into the SN are represented by the open circles (n = 4). Control values for this figure (closed circles) are Dulbecco's perfusions into the SN and are reproduced from Fig. 1A (n = 8). Glutamate data are presented as percent of baseline. All data are mean ± SEM. *P b 0.05 vs. control.
firing of subthalamic neurons (Falkenburger et al., 2001). It is also possible that subsequent to carbachol-induced STN stimulation, DA is released from striatal terminals, which in turn activates the polysynaptic indirect pathway of the basal ganglia to inhibit STN activity. This scenario is also unlikely since polysynaptic-induced changes in extracellular glutamate do not happen within the time-frame of our studies but are more likely to take place over the span of several hours (Stephans and Yamamoto, 1994). Another possibility that was considered and addressed in the current study is that the subthalamonigral glutamatergic projection is tightly regulated by an inhibitory dopaminergic feedback mechanism initiated within the substantia nigra at the glutamatergic terminals. Several studies have shown that somatodendritically released DA within the SN plays a role in the regulation of SNc DA and SNr GABA cell activity. However, very few studies to date have suggested that dopamine can regulate basal glutamate release from STN axon terminals (Rosales et al., 1997; Abarca et al., 1995). The current study extended these findings to show that antagonism of D2 receptors within the SN enhances and prolongs the increase in glutamate produced by carbachol stimulation of the STN (Fig. 3). Moreover, D2 receptor agonism attenuated the carbachol-induced increase in glutamate. Therefore, the increase in dopamine observed within the SN during carbachol stimulation of the STN (Fig. 2B) may limit the increase in glutamate via activation of D2 receptors located on asymmetric excitatory terminals (Pickel et al., 2002). The D2 mediated inhibition of glutamate release, however, appears to be dependent on STN activity since D2 dopamine receptor blockade did not affect the basal concentrations of extracellular glutamate in the SN in the absence of STN stimulation. In contrast, perfusion of the D1 receptor antagonist SCH23390 into the SN did not enhance nor prolong the transient
increase in extracellular glutamate produced by carbachol (Fig. 3). These findings do not suggest a role for D1 receptors in the regulation of stimulated glutamate release from STN terminals. SCH-23390 did increase though extracellular glutamate in the SN in the absence of carbachol stimulation of the STN, a finding that is consistent with a study by Rosales et al. (1997) which suggested a role for D1 receptors on the regulation of basal glutamate concentrations. Therefore, it appears that D1 receptors are preferentially activated to maintain tonic glutamate release. Collectively, these results provide the first evidence to suggest that somatodendritically released dopamine in the SN through an activation of D2 dopamine heteroreceptors provides a negative feedback control on stimulated or phasic glutamate release from STN terminals within the SN. In addition, D1 dopamine heteroreceptor activation regulates basal glutamate concentrations in the SN in the absence of STN stimulation or during tonic STN activity. Another possibility that was considered in this study is that the regulation of subthalamonigral glutamate transmission can be mediated by the inhibitory neurotransmitter GABA that also is increased in response to subthalamic stimulation (Rosales et al., 1997). GABA in the SN is critically involved in the regulation of DA cell activity by tonically suppressing the excitatory inputs (Kitai et al., 1999) via GABAA receptor activation localized on DA cell bodies (Tepper et al., 1995; Paladini et al., 1999). In the present study, perfusion of the GABAA receptor antagonist bicuculline into the SN during carbachol stimulation of the STN effectively enhanced the increase in glutamate release produced by STN stimulation (Fig. 5). This indicates that GABA, in addition to DA, negatively regulates glutamate release from STN axon terminals either through a direct activation of GABAA heteroreceptors on the STN axon terminals or indirectly by decreasing SNc DA cell activity. A recent study has shown that DA depolarizes STN neuron via a direct nigrosubthalamic pathway (Cragg et al., 2004). Therefore, GABA-induced inhibition of the nigrosubthalamic dopaminergic projection could inhibit the release of glutamate in the SN from subthalamic neurons. Conversely, inhibition of GABA cells in the SN with bicuculline could disinhibit the nigrosubthalamic DA path to increase subthalamic glutamatergic efferent activity to the SN. The observed changes in extracellular glutamate in the SN were designed to be selectively reflective of stimulation of the STN. Although additional glutamatergic projections to the SN that originate from the frontal cortex (Carter, 1982) and the PPN (Tokuno et al., 1988) may also contribute to the basal concentrations of extracellular glutamate in the SN, it is unknown if these or other glutamatergic projections are under the same dopaminergic control as those originating from the STN. Although areas adjacent to the STN may have unknown glutamatergic projections to the SN or may affect STN activity, our findings illustrate that when regions adjacent to the STN were perfused with carbachol (Fig. 2A), no changes in glutamate concentrations were observed in the SN. Therefore, the increases in glutamate within the SN observed during carbachol perfusions most likely originate from the STN. The current findings have implications for the pathophysiology and pharmacotherapy of Parkinson's disease (PD). It
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has been hypothesized that the progressive loss of SNc DA cells in PD results from an increased glutamate transmission in the SN and a subsequent excitotoxic cell death (Rodriguez et al., 1998). Moreover, lesions or high-frequency stimulation of the STN reduce most of the motor impairments associated with PD (Parkin et al., 2001; Benazzouz et al., 2000b), presumably by reducing the activity of the subthalamonigral pathway. Based on these past findings and the current study, modulation of the excitatory cholinergic stimulation of the subthalamonigral glutamatergic pathway at the level of the STN may be therapeutically beneficial. Perhaps more important, however, is that modulation of the glutamate release by the D2 receptor within the SN occurs only under conditions of STN stimulation and suggests that the subthalamonigral glutamatergic pathway can be tightly regulated primarily at the axon terminal region by presynaptic inhibitory D2 heteroreceptors. Thus, these findings further support the use of D2 agonists that target not only the striatum but also target the SN for the modulation of aberrant or augmented subthalamonigral glutamate release in relation to the symptomatic treatment of PD and possibly for the prevention of the progressive excitotoxic degeneration of dopamine cell bodies in the substantia nigra.
4.
Experimental procedures
4.1.
Animals
Adult male Sprague–Dawley rats (200–300 g; Harlan Sprague Dawley, IN, USA) were used in all experiments. Rats were housed four per cage prior to surgery/dialysis and were maintained on a 12:12-h light/dark cycle in a temperature- and humidity-controlled environment. Food and water were available ad libitum. Animal care was in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals which was approved and monitored by the Institutional Animal Care and Use Committee of Boston University. 4.2.
Microdialysis probe construction
Dialysis probes were of a concentric flow design and were constructed as previously described (Yamamoto and Pehek, 1990; Yamamoto and Davy, 1992). The length of the dialysis membrane was 1 mm for the STN probes and 1.5 mm for the SN probes. The dead volume of all probes was calculated so as to synchronize the timing and the initiation of drug perfusion with sample collection. 4.3.
Surgical probe implantation
Rats were anesthetized with a combination of xylazine (6 mg/kg, i. p.) and ketamine hydrochloride (70 mg/kg, i.p.) and were mounted into a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA) for the implantation of the microdialysis probes into the SN and the ipsilateral STN. The skull was exposed, holes were drilled, and probes were slowly lowered to the following co-ordinates aimed at STN: AP −3.8 mm, ML ±2.4 mm as referenced from bregma, DV −7.3 mm as referenced from dura, and at SN (at a 15° lateral angle): AP −5.6 mm, ML ±4.2 mm as referenced from bregma, DV −7.5 mm as referenced from dura (Paxinos and Watson, 1986). The probes were secured to the skull with three stainless steel machine screws and cranioplastic cement. Rats were transferred into the dialysis cages and were allowed to recover over night. Dialysis was conducted on the following morning.
4.4.
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In vivo microdialysis
After a 2-h prebaseline perfusion period, the microdialysis probes were perfused with modified Dulbecco's phosphate-buffered saline (138 mM NaCl, 2.7 mM KCl, 0.5 mM MgCl2, 1.5 mM KH2PO4, 1.2 mM CaCl2, and 5.0 mM d-glucose, pH 7.4) until stable baseline concentrations of DA or glutamate were achieved in the dialysate. The perfusion medium was then switched to one containing the treatment drug and was reverse dialyzed into the brain for 1 h. The perfusion medium was then switched back to Dulbecco's for 1 h of post-drug collection. The perfusion was conducted at a constant rate of 1.5 μl/min using a microinfusion pump (Harvard Apparatus, Holliston, MA, USA). Dialysate samples (20 μl) were collected from the outflow of the probe every 20 min. At the end of each experiment, the rats were killed by decapitation, and the probes were manually perfused with green dye before they were extracted. The brains were removed and frozen to be sectioned later in 40-μm-thick coronal slices for probe placement verification. The green dye was used to determine the location of the dialysis membrane, and the exact area that was perfused. 4.5.
Measurement of extracellular dopamine and glutamate
Separate groups of rats were used for the measurement of dopamine and glutamate in microdialysates. Dialysate aliquots (20 μl) were assayed for dopamine by high-performance liquid chromatography (HPLC) coupled to electrochemical detection. Separation was achieved with a 3-μm C18 column (100 × 2.0 mm; Phenomenex, Torrance, CA, USA) and a mobile phase consisting of 25 mM citric acid, 20 mM sodium acetate, 0.1 mM EDTA, 0.215 mM octyl sodium sulfate, and 5% methanol (pH 3.90). Electrochemical detection was performed with a BAS Model LC-4B electrochemical detector (BAS Instruments, West Lafayette, IN, USA) and a glassy carbon electrode maintained at a potential of 0.6 V. Data were collected using EZChrom Elite application software (Scientific Software Inc., Pleasanton, CA, USA). Glutamate was assayed from 20 μl aliquots by HPLC coupled to fluorescence detection following precolumn derivatization with opthaldialdehyde as described by (Donzanti and Yamamoto, 1988) with slight modifications. A 10 μl aliquot of the derivatization reagent was automatically added to the 20 μl dialysate sample by an ESA Model 542 autosampler (ESA Inc, Chelmsford, MA, USA), mixed, and allowed to react for exactly 90 s before injection onto the column. Separation was achieved with a 3-μm C18 column and a mobile phase consisting of 0.1 M sodium phosphate, 0.1 mM EDTA, and 10% methanol (pH 6.65). Fluorescence detection was performed with a Waters 474 Fluorescence Detector (Waters Corp, Milford, MA, USA). The excitation and emission wavelengths were set at 340 and 440 nm respectively. Data were collected using EZChrom Elite application software. 4.6.
Drugs
Carbamylcholine chloride (carbachol), s-(−)-raclopride, r-(+)-SCH23390, (+)-bicuculline, (−)-quinpirole, and Dulbecco's phosphatebuffered saline were all purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Optimal and pharmacologically active drug concentrations were chosen based on published reports in which carbachol (Moor et al., 1995), raclopride (Rahman et al., 2001), SCH-23390 (Rossi et al., 2005), bicuculline (Zackheim and Abercrombie, 2005), and quinpirole (Tanganelli et al., 2004) were intracerebrally reverse dialyzed. 4.7.
Data analysis
Glutamate and dopamine data are presented as a percent of baseline to standardize across groups and allow for effective comparisons between the different treatments. Dialysis data are presented as means ± SEM. Statistical significance was determined
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by a two-way ANOVA with repeated measures coupled with Bonferroni's post hoc test (P b 0.05).
Acknowledgments This work was supported by DA07606 and DAMD 17-99-1-9479.
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