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Stimulation of D2 receptors with quinpirole suppressed spon- taneous firing similarly among all neurons expressing mRNA solely for D2S, D2L, or D3 receptors.
JOURNAL OF NEUROCHEMISTRY

| 2011 | 116 | 966–974

doi: 10.1111/j.1471-4159.2010.07107.x

*Department of Physiology, Sungkyunkwan University School of Medicine and Center For Molecular Medicine, Samsung Biomedical Research Institute, Suwon, Korea  Department of Pathology, Inje University Seoul Paik Hospital, Jeo-dong, Jung-gu, Seoul, Korea

Abstract Dopamine (DA) receptors generate many cellular signals and play various roles in locomotion, motivation, hormone production, and drug abuse. According to the location and expression types of the receptors in the brain, DA signals act in either stimulatory or inhibitory manners. Although DA autoreceptors in the substantia nigra pars compacta are known to regulate firing activity, the exact expression patterns and roles of DA autoreceptor types on the firing activity are highly debated. Therefore, we performed individual correlation studies between firing activity and receptor expression patterns using acutely isolated rat substantia nigra pars compacta DA neurons. When we performed single-cell RT-PCR experiments, D1, D2S, D2L, D3, and D5 receptor mRNA were heteroge-

neously expressed in the order of D2L > D2S > D3 > D5 > D1. Stimulation of D2 receptors with quinpirole suppressed spontaneous firing similarly among all neurons expressing mRNA solely for D2S, D2L, or D3 receptors. However, quinpirole most strongly suppressed spontaneous firing in the neurons expressing mRNA for both D2 and D3 receptors. These data suggest that D2S, D2L, and D3 receptors are able to equally suppress firing activity, but that D2 and D3 receptors synergistically suppress firing. This diversity in DA autoreceptors could explain the various actions of DA in the brain. Keywords: dopamine, dopamine autoreceptor, dopaminergic neuron, spontaneous firing, substantia nigra. J. Neurochem. (2011) 116, 966–974.

Activations of the midbrain dopamine (DA) systems facilitate locomotion, reward-related behaviors, and some types of memory formation (Meltzer et al. 1997; Spanagel and Weiss 1999; Berk and Hyman 2000; Schultz 2004). Impairment of DA function is involved in many neurodegenerative and/or neuropsychiatric disorders, such as Parkinson’s disease (Olanow and Tatton 1999), schizophrenia (Egan and Weinberger 1997), Tourette’s syndrome, and drug addiction (Missale et al. 1998). Dopamine neurons in the midbrain, such as the ventral tegmental area and the substantia nigra pars compacta (SNc), exhibit spontaneous rhythmic firing activities. However, in response to various sets of input information, these neurons are able to generate diverse firing patterns including weak or strong bursts (Grace and Bunney 1984; Hyland et al. 2002). The rate and pattern of spontaneous firing determine DA releases from axon termini as well as from somatodendritic compartments and also establishes phasic and tonic concentrations of DA in target areas and extracellular spaces in the brain (Meltzer et al. 1997; Adell and Artigas 2004).

Five distinct DA receptors which are members of the seven transmembrane domain G-protein coupled receptor family have been isolated and characterized (Daniela et al. 2000). On the basis of biochemical and pharmacological properties, DA receptors are grouped into two subfamilies as follows: the D1like receptor subfamily comprising the D1 and D5 receptors, and the D2-like receptor subfamily comprising the D2, D3, and D4 receptors. Through alternative splicing, the D2 receptor gene encodes two molecularly distinct short and long isoforms, designated D2S and D2L. The D2L receptors have an additional 29 amino acids within the third intracellular loop (Daniela

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Received August 19, 2010; revised manuscript received November 5, 2010; accepted November 8, 2010. Address correspondence and reprint requests to Professor Myoung Kyu Park, Department of Physiology, Sungkyunkwan University School of Medicine, 300 Chunchun-dong, Jangan-ku, Suwon 440-746, Korea. E-mail: [email protected] Abbreviations used: DA, dopamine; GIRK, G-protein-gated inwardly rectifying potassium; SNc, substantia nigra pars compacta; TH, tyrosine hydroxylase.

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et al. 2000). D1-like receptors activate adenylyl cyclase through Gs/Golf, but D2-like receptors are linked to Gi/o, which liberates Gai/o and Gbc (Bonci and Hopf 2005). Gai/o subunits inhibit adenylyl cyclase, and Gbc subunits trigger many other signals including activation of K+ channels. In general, D1-like receptors are known to increase the production of cAMP, whereas D2-like receptors are known to suppress cAMP production. Therefore, the primary action of DA in the brain is to activate neurons through D1-like receptors, whereas this activation is antagonized by D2-like receptors (Bonci and Hopf 2005). These actions are well documented in the striatum, where the direct striatonigral pathway is positively activated by D1 receptors, and the indirect striatopallidal pathway is inhibited by D2 receptors (Alexander and Crutcher 1990; Mink and Thach 1993; Kaji 2001; Fisone et al. 2007; Surmeier et al. 2007). Thus, the final outcome of DA functions may be determined by the combinational outcome of all DA signals within the relevant neuronal circuits. However, this model is too simplistic and does not entirely explain the DA system. In fact, D2-like receptors are also found in midbrain DA neurons where they act as autoreceptors. Activation of DA D2-like autoreceptors in the somatodendritic compartment in the midbrain suppresses spontaneous firing mainly by activating G-protein-gated inwardly rectifying potassium (GIRK) channels (Mercuri et al. 1997; Kuzhikandathil et al. 1998; Centonze et al. 2002), and DA autoreceptors expressed at dopaminergic nerve terminals regulate DA synthesis and release (Koeltzow et al. 1998; Dickinson et al. 1999; Fisone et al. 2007), thus acting as a negative feedback controller. However, although it is clear that D2-like autoreceptors regulate DA neuron excitability in the midbrain, the exact roles and types of autoreceptors are highly debated. According to previous reports (Weiner et al. 1991; Diaz et al. 1995; Usiello et al. 2000), D2L, D2S, and D3 receptors are mostly found in the midbrain. Regarding their functional roles, some in vitro biochemical data suggest that D2L and D2S have similar functions, although they possess different coupling affinities for G-proteins (Montmayeur et al. 1993; Guiramand et al. 1995; Jomphe et al. 2006). However, some papers have reported distinct functions of the above two receptors (Koeltzow et al. 1998; Usiello et al. 2000; Lindgren et al. 2003). Furthermore, it has been reported that D3 autoreceptors, despite the presence of D3 autoreceptors in the SNc, do not appear to activate GIRK currents in SNc DA neurons (Davila et al. 2003). At the moment, therefore, the exact types of autoreceptors expressed in SNc DA neurons and their roles on the firing activity in the SNc are still unclear. Especially, the expression patterns of DA autoreceptors in native neurons at the single neuron level have not been thoroughly studied. Therefore, we measured firing activity using the patch clamp technique in acutely dissociated rat SNc DA neurons and directly examined expression patterns of DA autoreceptor types from

the same neurons employing single-cell RT–PCR in order to correlate single-cell gene expression patterns and firing activities. As a result, we found that D1, D2L, D2S, D3, and D5 DA receptors are heterogeneously expressed in SNc DA neurons, and that an individual neuron possesses a various combination of the above receptor types. Through the analysis of correlation between firing activities and particular receptor expression patterns, we could suggest that all of the D2S, D2L, and D3 receptors are able to equally suppress firing activity, and that D2 and D3 receptors appear to synergistically suppress firing in SNc DA neurons.

Materials and methods Preparation of SNc dopamine neurons Postnatal days 9–16 Sprague–Dawley rats regardless of sex were used for this study. Food and water were available ad libitum. All animal procedures were approved by the Sungkyunkwan University Animal Care and Use Committee. After decapitation, the whole brain was removed and immediately transferred to ice-cold oxygenated HEPES-buffered saline containing (in mM) 135 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 25 D-glucose, and 10 HEPES. The pH was adjusted to 7.3 by titration with 10 M NaOH. The brain was cut into coronal slices of 400 lm thickness with a vibratome (Series 1000, St. Louis, MO, USA). The SNc region within each slice was then dissected out with a scalpel blade and digested with papain (4– 8 U/mL; Worthington, Lakewood, NJ, USA) for 30–50 min at 36– 37C in oxygenated HEPES-buffered saline. Next, the digested tissue segments were rinsed with enzyme-free saline and gently triturated with a graded series of fire-polished Pasteur pipettes. The isolated cells were placed onto poly-D-lysine-coated small glass covers. All dissociate neurons were used for physiological experiments within 2 h of removal. Single-cell RT-PCR After measuring the firing activity, neurons were aspirated into a microelectrode pipette via the application of negative pressure. The pipette was filled with a sterile recording solution containing diethylene pyrocarbonate-water, ribonuclease inhibitor, and dithiothreitol (RNAguardTM; Amersham Biosciences, Piscataway, NJ, USA). After aspiration, the microelectrode pipette was broken, and the contents were ejected into a 1.5 mL Eppendorf tube. RNA was isolated using RNEasy kits (Qiagen, Hilden, Germany). Purified RNA was heated to 68C for 5 min and then incubated on ice for 5–10 min. cDNA was synthesized from cellular mRNA through the addition of SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) (1 lL, 200 U/lL), pd(N6) random hexamer (1 lL), RNA-Guard (GE Healthcare, Piscataway, NJ, USA) (1 lL), dithiothreitol (2 lL, 10 mM), dNTP mixture (1 lL, 4 mM each), and 5· reverse transcription buffer (Invitrogen) (4 lL). The reaction mixture (20 lL) was incubated at 36C for 1 h. The reaction was terminated by heating the mixture to 95C for 5 min and then cooling on ice. A cDNA fraction from the single cell was used as a template for conventional PCR amplification. Reaction mixtures contained dNTPs (0.5 lL, 10 mM each), primers (1–1.5 lL, 125 ng/lL), Taq DNA polymerase (0.5 lL,) and 10· buffer (2.5 lL). The

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thermal cycling program for primer sets was 40 cycles of 94C for 30 s, 58–61C for 30 s, and 72C for 45 s. Previously reported DA receptor primers (Surmeier et al. 1996) that had been developed from GenBank sequences for tyrosine hydroxylase (TH) with commercially available software (DNAstar) were used. For the TH cDNA, the primers were TH-sense 5¢-AAC TCT CCA CGG TGT ACT GGT T-3¢ and antisense 5¢-TGA AGC TCT CGG ACA CAA AGT A-3¢. After identification, neurons were tested for the presence of DA receptor mRNA. The primer sequences were as follows: D1 receptor-sense 5¢-GAC AAC TGT GAC ACA AGG TTG AGC-3¢ and antisense 5¢-AAT ACA GTC CTT GGA GAT GGA GCC-3¢; D2 receptor-sense 5¢-GCA GTC GAG CTT TCA GAG CC-3¢ and antisense 5¢-TCT GCG GCT CAT CGT CTT AAG-3¢; D3 receptor-sense 5¢-CAT CCC ATT CGG CAG TTT TCA A-3¢ and antisense 5¢-TGG GTG TCT CAA GGC AGT GTC T-3¢; D4 receptor-sense 5¢-TCA TGC TAC TGC TTT ACT GGG CCA-3¢ and antisense 5¢-TCT GAG AGA GGT CTG ACT CTG GTC-3¢; D5 receptor-sense 5¢-AGT CGT GGA GCC TAT GAA CCT GAC-3¢ and antisense 5¢-GCG TCG TTG GAG AGA TTT GAG ACA-3¢ (Surmeier et al. 1996; Vysokanov et al. 1998). PCR products were visualized with ethidium bromide stain and separated using electrophoresis in 1.5–2% agarose gels. Measuring cytosolic Ca2+ levels The isolated SNc cells were incubated with 2 lM fura-2AM at room temperature (20–24C) for 20–30 min. The cells were then washed twice with normal physiological salt solution. All cells were used within 2 h of isolation. Single-cell fluorescence intensity was measured using an Olympus IX70 inverted microscope (40· objective or 60· water immersion objective) attached to a chargecoupled device image intensifier camera (Quantix, Roper Scientific Inc., Tucson, AZ, USA) and Metafluor software (Molecular Devices, Sunnyvale, CA, USA). Dual excitation at 340/380 was used with a 400-nm dichroic mirror and emitted light was collected with a 450-nm long pass filter as described previously (Choi et al. 2003). Measuring electrical activities The patch clamp system (EPC-9, HEKA) was used to measure spontaneous firing activities. Patch pipettes were made with a micropipette puller (MODEL P-97; Sutter Instrument, Novato, CA, USA), and pipette tips were polished with Narishige Forge (Micro Forge MF-830, Narishige, Japan). The resistances of the patch pipettes were 2–3 MW. Cell-attached configurations were established in the current clamp mode. In the cell-attached patch experiments, the electrical signals were continuously sampled at 2 kHz (1 kHz filter) and stored in an IBM-compatible computer for further analysis. In this case, the patch pipettes were filled with the bath solution. The electrical signals were very similar according to the extracellular recordings (Grace and Bunney 1983a,b). Frequency conversion of spontaneous action potentials was performed with Igor ver. 4 (WaveMetrics Inc., Nimbus, Portland, USA), and some of the data were analyzed with Origin ver. 7.0 (OriginLab corporation, Northampton, MA, USA). Statistics The one-way ANOVA test was used, and p-values less than 0.05 were regarded as significant.

Results Expression patterns of dopamine autoreceptors in the dissociated single dopamine neurons from SNc Among many dissociated neurons, some of neurons had a very large soma attached to multiple long dendrites, whose lengths were often greater than 200 lm, as shown in Fig. 1a. When the electrical phenomena and tyrosine hydroxylase (TH, a marker of dopaminergic neurons) expression were examined in these characteristic neurons, more than 90% of these large neurons were confirmed to be dopaminergic in our experimental conditions (Fig. 1a, and see Choi et al. 2003; Kim et al. 2004, 2007). Most of these neurons showed regular spontaneous firings at 2–4 Hz and responded to DA application. To examine the types of DA receptors expressed in the SNc, RT-PCR was performed in brain tissues containing only the SNc, the same areas used for the isolation of dissociated neurons. In this brain area, all types of mRNA for DA receptors except D4 receptors were found based on positive amplification of TH mRNA (Fig. 1b). In Fig. 1b, two bands for the D2 receptors indicated the presence of two different sizes of this receptor. Primers for D4 receptors were also tested in the whole entire brain, and positive amplification of D4 receptors (Fig. 1b, right) was observed. Therefore, it seems that D1, D2, D3, and D5 receptors but not D4 receptors were expressed in the SNc. Next, to verify the expressions of the above DA receptors in the SNc DA neurons, single-cell RT-PCR was performed in the dissociated single neurons. When a neuron was identified as dopaminergic according to PCR amplification with primers for TH, further amplification was performed with primers that specifically amplified mRNA for specific DA receptors (D1–D5 receptors). As shown in Fig. 1c and d, among the TH-expressing neurons (total number of neurons = 47), mRNA for D2L, D2S, and D3 receptors were most frequently found. D2L, D2S, and D3 receptor mRNA were expressed in 66.0% (31/47), 44.7% (21/47), and 55.3% (26/47) of the TH-expressing SNc neurons, respectively. D1 receptor mRNA was only expressed in 4.6% (2/47) and D5 receptor mRNA was expressed in 8.5% (4/47) of the THexpressing SNc neurons (Fig. 1c and d). In 36.2% (17/47) of the neurons examined, both D2S and D2L receptors were found together, and 25.5% (12/47) of the neurons expressed all three D2S, D2L and D3 receptors. The neurons purely expressing both D2S and D2L except any other receptors represented just 8.5% (4/47, Fig. 1c and d) of the total neurons. Dose-dependent inhibition of spontaneous firing by dopamine in acutely dissociated SNc dopamine neurons Under cell-attached current-clamp conditions, tentative DA neurons showing large soma with multiple large dendrites

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(c) Fig. 2 Dose dependence of dopamine-induced inhibition of spontaneous firing. (a) Spontaneous firing activity was recorded from acutely isolated dopamine neurons using cell-attached current–clamp mode. A transmitted image of one of the acutely isolated dopamine neurons is seen with a recording patch pipette. Bath applications of dopamine (10 nM to 100 lM) inhibited firing activity in a dose-dependent manner. (b) Dose–response curve (n = 50 cells). The duration of the first strong inhibition of spontaneous firing was analyzed. (c) By counting firing numbers during the first and second 50 s periods, the inhibitions of the first strong inhibition (during the first 50 s immediately after dopamine application) and the later sustained inhibition (the second steady-state inhibition, between 50 and 100 s) were analyzed.

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Fig. 1 Identification of SNc dopaminergic neurons and expression patterns of dopamine receptor subtypes. (a) Transmitted and immunofluorescence images of an acutely isolated dopaminergic neuron. Expression of tyrosine hydroxylase (TH) was confirmed by staining with TH antibody and FITC-conjugated secondary antibody. (b) RTPCR of mRNAs for dopamine receptor subtypes in brain slices containing the SNc. In the SNc, all subtypes of dopamine receptor mRNA except D4 were expressed together with TH mRNA. D4 receptor mRNA was detected in whole brain tissues. (c) Single-cell RT-PCR. Expression patterns of mRNAs for dopamine receptor subtypes in 47 neurons. (d) Fraction of each dopamine receptor mRNA expression in 47 neurons; D1R = 4.3%; D2S = 44.7%, D2L = 66.0%, D3R = 55.3%, and D5R = 8.5%. *Statistical difference (p < 0.05).

usually showed spontaneous action potentials with a regular rhythm (2.9 ± 0.4 Hz, n = 76). In these neurons, DA application suppressed spontaneous firing, as previously

reported, mainly by activating GIRK currents (Uchida et al. 2000). Low concentrations of DA (10–100 nM) suppressed spontaneous action potentials, thereby decreasing the spontaneous firing rate (Fig. 2a, first trace) but often failed to completely inhibit spontaneous firings (failure was observed in two out of four neurons at 10 nM and two out of six neurons at 100 nM). The firing rate (Hz) was decreased from 2.02 ± 0.30 to 1.45 ± 0.35 (n = 4) with the application of 10 nM DA and from 2.48 ± 0.82 to 2.24 ± 0.64 (n = 6) with 100 nM DA. However, high concentrations of DA (‡ 1 lM) usually caused two phases of firing inhibition, the initial complete firing inhibition and the subsequent incomplete suppression of firings (n = 15, Fig. 2a). After the first phase of firing inhibition (complete suppression of spontaneous firing), the firing activity gradually reappeared even in the presence of DA (the second sustained phase). During this period, the firing rate was not fully recovered to the rate that was observed before DA application. When cells were stimulated with higher doses of DA (‡ 1 lM), the duration of the first phase of firing inhibition was increased; nevertheless, spontaneous firing activity reappeared after some time regardless of dose. Even when exposed to DA at concentrations greater than 100 lM, spontaneous firing activity was not completely suppressed (Fig. 2a). Interestingly, first-phase inhibition was dosedependent, but second phase sustained inhibition was not

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(Fig. 2c). The initial complete inhibition by DA application was always seen at 1 lM DA, and the strongest inhibition was observed at 100 lM (IC50 = 7.8 lM, n = 30; Fig. 2b). However, the second phase sustained suppression was always observed when DA suppressed firing and did not appear to be dose-dependent (Fig. 2a and c). These results may suggest that DA-induced inhibition of spontaneous firing may not be mediated via only one mechanism. As it has been reported that direct stimulation of the DA somatodendritic autoreceptor activates not only GIRK current but also at least three additional potassium currents including the delayed rectifier (IK), an anomalous rectifier current (IANOM), and a transient A current (IA) (Liu et al. 1994; White 1996), it may be reasonable to conclude that DA may activate several signaling pathways resulting in the inhibition of firing activity with different sensitivities, including low sensitivity and high sensitivity pathways (Lane et al. 2008). As another alternative explanation, the constant fraction of incomplete inactivation of GIRK currents may be responsible for the dose-independent second-phase partial inhibition. Dopamine-induced firing inhibition and cytosolic calcium changes in spontaneously firing dopamine neurons Previously, we reported that a proper cytosolic Ca2+ level ([Ca2+]c) is essential for the generation and maintenance of spontaneous firing in SNc DA neurons (Kim et al. 2007). In addition, it has been reported that the activation of DA receptors causes an increase in [Ca2+]c via Ca2+ release from intracellular stores in some cell types (Lin et al. 1995; Wong et al. 2001; So et al. 2009). Therefore, to investigate whether DA autoreceptors are involved in Ca2+ signaling in SNc DA neurons, spontaneous firing and [Ca2+]c were simultaneously measured in acutely dissociated DA neurons. As shown in Fig. 3a, application of 10 lM DA induced two phases of firing inhibition, as previously described. At the same time, changes in [Ca2+]c were also observed during the period of DA application. In this case, [Ca2+]c was not increased but rather decreased (n = 13; Fig. 3a and d). Therefore, there seems to be no DA -induced Ca2+ release mechanism in SNc DA neurons. The reason for the decrease in [Ca2+]c caused by DA seems to be related to suppression of spontaneous firing activity. As we have previously reported (Choi et al. 2003; Kim et al. 2007), spontaneous firing itself causes Ca2+ influx by activating voltage-operated Ca2+ channels and thereby maintaining basal [Ca2+]c at a level slightly greater than that of silent state neurons. Thus, the decrease of [Ca2+]c caused by DA may be explained by decreased basal Ca2+ influx because DA suppressed spontaneous firing. When the changes of [Ca2+]c were plotted against DA concentration, the response range for [Ca2+]c changes as shown in Fig. 3b was very similar to the range for firing inhibition (Fig. 2b). This result supports the idea that DA decreases basal [Ca2+]c levels by suppressing spontaneous firing activity.

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Fig. 3 Dopamine decreased cytosolic Ca2+ concentration ([Ca2+]C) with suppression of spontaneous firing rate. (a) Application of dopamine (10 lM) inhibited spontaneous firing with a decrease in cytosolic Ca2+ level. (b) The dose–response curve of the dopamine-induced decreases of [Ca2+]C (basal ratio = 0.6). (c) Blocking of spontaneous firing by TTX decreased [Ca2+]C levels. In this condition, further application of dopamine did not affect [Ca2+]C levels. In the presence of TTX (0.5 lM), Ca2+-free solutions of dopamine did not affect [Ca2+]C levels. (d) Comparison of Ca2+ changes among the above groups.

To further verify that the decrease in spontaneous firing rate was responsible for the DA -induced [Ca2+]c decrease, firings were completely suppressed with 500 nM TTX, and 10 lM DA was reapplied. As shown in Fig. 3c, there were no changes in [Ca2+]c in this condition (n = 13), suggesting that the decrease of [Ca2+]c was caused by the suppression of spontaneous firing. Although the application of DA decreased [Ca2+]c in a dose-dependent manner (Fig. 3b), to verify whether DA triggers any Ca2+ release from intracellular stores, which may be mitigated by reduced Ca2+ influxes, DA was applied immediately after removing extracellular Ca2+. In the Ca2+free medium, DA did not affect [Ca2+]c (n = 7; Fig. 3c and d). Thus, in SNc DA neurons, DA autoreceptors do not appear to directly activate Ca2+ signals but rather passively decrease [Ca2+]c levels by suppressing spontaneous firing activity. Dopamine suppresses firing activity by activation of D2-like receptors Which types of autoreceptors were responsible for suppression of spontaneous firing and the decrease of [Ca2+]c was investigated. As SNc DA neurons express many types of DA receptors, as shown in Fig. 1, a D1-like receptor agonist, SKF38393, and a D2-like receptor agonist, quinpriole, were used. As shown in Fig. 4a, SKF38393 did not evoke any changes in the firing rate or [Ca2+]c (n = 9; Fig. 4a and b), but quinpirole produced the same responses as those seen in

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(b) Fig. 4 Suppression of spontaneous firing and decrease in [Ca2+]c by D2 dopamine receptors. (a) A specific D1-like dopamine receptor agonist, SKF38393 (10 lM), did not inhibit spontaneous firing or alter [Ca2+]c levels, but a specific D2-like dopamine receptor agonist, quinpirole (10 lM), lowered spontaneous firing rates and [Ca2+]c levels. (b) The durations in which spontaneous firing was completely inhibited; changes in [Ca2+]c caused by SKF38393 or quinpirole are summarized. (c) Dopamine application in the presence of D1- or D2like receptor antagonists. Although sulpiride (10 lM) blocked the effect of dopamine, SCH39166 (10 lM) did not alter the effect of dopamine. Inset image shows a cell-attached recording neuron.

DA application, in that the firing rate and [Ca2+]c were decreased (n = 18; Fig. 4a and b). Next, specific antagonists for DA receptors were used. When 10 lM DA was applied in the presence of S())-sulpiride, a specific D2-like receptor antagonist, the responses to DA were completely abolished. As shown in Fig. 4c, in the acutely isolated DA neurons, sulpiride itself did not affect the basal firing rate, but it completely blocked the effect of DA if applied prior to DA. However, SCH39166, a specific D1-like receptor antagonist, did not block the DA-induced firing inhibition (n = 10; Fig. 4c). All of these data indicate that DA suppresses firing activity by activating D2-like receptors in SNc DA neurons. Correlation between dopamine autoreceptor expression pattern and firing activity Although D2-like receptors were dominantly expressed in SNc DA neurons (D2S, D2L and D3), in which they suppressed firing activity, many DA receptors were heterogeneously expressed in each SNc neuron (Fig. 1c). As the exact DA autoreceptor types responsible for firing inhibition are still uncertain, the DA autoreceptors responsible for firing inhibition were sought. First, firing activity was recorded and quinpirole was added, then single-cell RT-PCR was performed in the same cell. Consequently, various responses to quinpirole were observed in neurons expressing various sets of DA autoreceptors. As shown in Fig. 5, among the

Fig. 5 Correlation study between quinpirole-induced firing inhibition and receptor expression pattern. (a) D2 receptor expression and quinpirole-induced suppression of spontaneous firing. The expressions of D2S, D2L, D3 and D2/D3 receptors were examined using single-cell RT-PCR. A D2-like receptor agonist, quinpirole 10 lM, inhibited spontaneous firing in all cases. Representative photos of neurons expressing each type of DA receptor are seen with a recording patch pipette. In cell-attached mode, spontaneous firing was suppressed by application of 10 lM quinpirole. (b) Comparison of relative inhibitions of spontaneous firings by quinpirole in the neurons expressing only D2S, D2L, D3, or both D2 and D3 receptors, respectively. *Statistical difference (p < 0.05, one-way ANOVA).

recorded neurons, some expressed mRNA for only D2L (n = 10), D2S (n = 3), and D3 receptors (n = 6), respectively. Some neurons expressed mRNA for both D2S and D2L receptors (n = 4) and others expressed mRNA for all three D2S, D2L, and D3 receptors. Interestingly, 10 lM quinpirole suppressed firing in all of the recorded neurons. The firing inhibitions were very similar among the neurons expressing mRNA for only D2L (n = 10), D2S (n = 3), or D3 receptors (n = 6), respectively (Fig. 5a and b). Basal firing rates were not significantly different between subgroups (ANOVA, p > 0.05). When the firing inhibition was analyzed as shown in Fig. 5b, there were no statistical differences among the above three groups. Representative data are presented in Fig. 5a, together with mRNA expression patterns. In the upper two traces of Fig. 5a, the expression of only one type of D2 receptor mRNA, either D2S or D2L, was confirmed. These data suggest that all of the D2L, D2S, and D3 receptors are able to suppress firing activity at an equal potency in DA neurons.

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Among the recorded 28 neurons, there were five neurons that expressed mRNA for both D3 and D2 receptors. Surprisingly, 10 lM quinpirole strongly suppressed firing activity in these neurons (Fig. 5a, bottom), despite the basal firing rate was not different from the neurons only expressing mRNA for either D2 or D3. Along with mRNA for both D2S and D2L receptors, these neurons also had D3 receptor mRNA (Fig. 5b). These data suggest that D3 receptors in the presence of D2 receptors synergistically suppress firing activity, although this combination was found in a small number of recorded SNc DA neuron (5/28). These data are summarized in Fig. 5b.

Discussion Dopamine actions in the brain depend purely on DA receptor types and their locations within neuronal circuits. Many past studies using various pharmacological and genetic approaches have suggested that pre-synaptic and post-synaptic DA receptors have distinct functions (Usiello et al. 2000; Lindgren et al. 2003), and that DA autoreceptors act in the feedback regulation of firing as well as in DA synthesis and release (Diaz et al. 1995; Mercuri et al. 1997; Koeltzow et al. 1998; Adell and Artigas 2004). However, because of the lack of specific agonists and antagonists for each DA receptor type and the complex actions and expressions of DA receptors in neuronal networks, the exact functions and locations of DA receptors remain unclear. Although D2S, D2L, and D3 receptors are believed to be expressed in the dopaminergic neurons of the SNc (Weiner et al. 1991; Usiello et al. 2000; Davila et al. 2003), there is little agreement regarding the participation of these autoreceptor types in firing regulation. One of the major reasons for this would be ascribed to the lack of direct confirmation of receptor types and functional correlations in actual dopaminergic neurons at the single cell level. Most experiments have been performed in mice in which a specific type of DA receptor was deleted or inserted and in which agonists and antagonists for D2-like or D1-like DA receptors were used. In addition, the interpretations of these in vivo experiments have usually been based on the assumption of homogeneous expression patterns of DA autoreceptor types in midbrain DA neurons. If all DA neurons express the same patterns of DA autoreceptors, the expected in vivo responses to DA receptor agonists/antagonists would be the same. However, although mRNA for D2S, D2L, and D3 receptors were detected in whole tissues containing the ventral tegmental area or SNc (Weiner et al. 1991; Usiello et al. 2000; Davila et al. 2003), there are varying reports on the functional roles of the autoreceptors in regard to the firing activities of dopaminergic neurons according to the experimental conditions. Therefore, in this study we directly investigated the expression patterns of mRNAs for DA autoreceptors at the single-cell level, as well as their

regulatory roles on firing activity using acutely dissociated DA neurons from the rat SNc. Single-cell RT-PCR experiments showed that D1, D2, D3, and D5 receptor mRNA were expressed in 4.3, 74.5, 55.3, and 8.5% in the TH-expressing DA neurons, respectively (Fig. 1), suggesting heterogeneous expression of DA autoreceptors in the SNc. The acutely dissociated DA neurons showed spontaneous action potentials at 2.9 ± 0.4 Hz (n = 93) with a regular rhythm and possessing multiple long dendrites (Fig. 1), presumably representing good somatodendritic response to DA receptor stimulation. However, the DA neurons recorded did not exhibit a full response to DA receptor stimulation since distal dendritic compartments were usually lost in our experimental conditions. In the acutely isolated DA neurons, DA decreased spontaneous firing activity (IC50 = 7.8 lM, n = 13) with a decrease in cytosolic Ca2+ level (IC50 = 1 lM, Figs 2 and 3). This decrease was blocked by the DA D2/D3 receptorspecific antagonist sulpiride but not by the D1/D5 receptorspecific antagonist SCH39166. Quinpirole, a specific D2/D3 receptor agonist, mimicked DA (Fig. 4), and the inhibitory effects of quinpirole on the neurons expressing mRNA for D2S receptors, D2L receptors, and D3 receptors were similar (Fig. 5). However, quinpirole exerted a greater suppression on firings in the neurons expressing mRNA for both the D2 and D3 receptors. From these results, it was concluded that (i) both short and long forms of D2 DA autoreceptors inhibit spontaneous firings; (ii) D3 autoreceptors suppress spontaneous firing activity; (iii) the potencies of firing inhibition by D2L, D2S, or D3 autoreceptors seem to be similar; (iv) DA receptors lower cytosolic Ca2+ levels by suppressing spontaneous firings; and (v) D2 and D3 receptors appear to synergistically suppress firing in the DA neurons of the rat SNc. There is not much difference of the molecular structures between D2S and D2L receptors, except that D2L receptors have an additional 29 amino acids in the third intracellular loop (Daniela et al. 2000). However, it has been reported that they have distinct roles in pre- and post-synaptic sites (Usiello et al. 2000). D2S receptors appear to regulate TH enzyme activity in pre-synaptic DA neurons (Lindgren et al. 2003), and the activation of D2S receptors appears to regulate the electrical activities of dopaminergic neurons by acting at pre-synaptic sites, as DA agonists modulate firing activity in D2L knockout mice (Centonze et al. 2002). However, D2L receptors seem to work mainly at the post-synaptic site and involve the activity of D1 receptor-mediated locomotion (Usiello et al. 2000; Lindgren et al. 2003). Another study reported that D2S and D2L receptors appear to be equipotent in the regulation of neuronal excitability and neurotransmitter release using Enhanced Green Fluorescent Protein (EGFP)tagged DA D2 receptor isoforms in primary cultured neurons (Jomphe et al. 2006). In addition, many biochemical data suggest that the midbrain DA neurons express many types of

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Regulation of spontaneous firing by dopamine autoreceptors | 973

DA autoreceptors including both D2S and D2L receptors (Usiello et al. 2000; Davila et al. 2003; Lindgren et al. 2003; Jomphe et al. 2006). As studies discriminating between D2S and D2L receptor actions in the brain are usually hampered by the lack of specific agonists and antagonists, experiments using genetically modified mice such as D2L or D2 knockout mice do not guarantee the exact localization and function of each receptor (Lin 2008). Uncertainties with regard to the location of each receptor type within neuronal networks add further complexities to the interpretation of in vivo studies. Therefore, to determine the exact roles and expression types of autoreceptors in DA neurons, direct confirmation of each receptor and its role in firing regulation should be examined in actual neurons at the single-cell level. To this end, we directly investigated DA autoreceptormediated regulation of firings using acutely isolated DA neurons from normal SD rats. Although DA receptors were heterogeneously expressed in an individual dopaminergic neuron, we found that some of the DA neurons expressed only one type DA receptor mRNA, either D2S or D2L. In these dopaminergic neurons, quinpirole similarly inhibited spontaneous firings, confirming that D2S (0.4 ± 0.2 Hz) and D2L (0.5 ± 0.1 Hz) receptors are able to similarly suppress firing in dopaminergic neurons (Fig. 5). However, in our experimental conditions, we could not rule out the possibility that the protein levels of the receptors were different. Even though mRNA for a specific subtype of DA receptor was not detected in single-cell RT-PCR, there is a very rare possibility that mRNA at an undetectable level may express functional amount of receptors. Interestingly, in normal conditions, the number of neurons expressing only D2L receptor mRNA (12/47 cells) was larger than the number of neurons expressing only D2S receptor mRNA (3/47 cells) (Fig. 1c). In addition, the inhibition of spontaneous firings by quinpirole in the neurons expressing both D2L and D2S receptor mRNA (0.4 ± 0.1 Hz) was not much different from that in neurons expressing either D2S or D2L receptor mRNA. However, most of the neurons expressing D3 receptor mRNA were found to co-express D2 receptor mRNA (12/47 cells, Fig. 1c), and these neurons showed the strongest inhibition of firings by quinpirole (Fig. 5). These heterogeneous expression patterns and functional differences may be important for dopaminergic functions in the brain and should be considered in the interpretation of in vivo experimental data. In recent studies using D2-KO mice, DA neurons from mice lacking D2 receptors but still expressing D3 receptors were shown to be insensitive to DA and quinpirole based on in vitro electrophysiological recordings (Mercuri et al. 1992, 1997; Davila et al. 2003). However, in those studies, the investigators did not confirm DA receptor expression patterns for all of the neurons recorded. Therefore, their results may be different from those of our recordings. In addition, they

recorded neurons from D2-KO mice, while we recorded neurons from SD rats. Nevertheless, we suggest that not all of the dopaminergic neurons in the SNc are homogeneous, and that they express various combinations of receptors. Therefore, interpretation of in vivo data of DA receptor agonists and antagonists should be conducted carefully.

Acknowledgements This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (no. 20090080712) and by a grant of the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (no. A090371). The authors declare no competing interests.

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