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Dopamine activates inward rectifier K+ channel in acutely dissociated rat substantia nigra neurones
Neuropharmacology 39 (2000) 191–201 www.elsevier.com/locate/neuropharm
Dopamine activates inward rectifier K + channel in acutely dissociated rat substantia nigra neurones Soko Uchida, Norio Akaike, Junichi Nabekura
*
Department of Physiology, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi Higashi-ku, Fukuoka 812-8582, Japan Accepted 18 May 1999
Abstract The effect of dopamine (DA) was investigated on acutely dissociated rat substantia nigra pars compacta (SNc) neurones by using patch clamp recording. The SNc neurones could be classified into two groups. About 75% of large neurones (⬎30 µm in diameter) were tyrosine hydroxylase (TH) positive while almost all small neurones (⬍20 µm) were TH negative. In the large neurones, DA hyperpolarized the membrane, resulting in a reduction of the frequency of spontaneous action potentials in current-clamp mode and induced an inward rectifier K + current in voltage-clamp mode. Quinpirole, a D2 receptor agonist, mimicked the DA action. S(⫺)-sulpiride, a D2 receptor antagonist, inhibited the DA-induced current (IDA) more effectively than SKF83566, a D1 receptor antagonist. Intracellular application of either guanosine 5’-O-(2-thiodiphosphate) (GDP-βS) or pertussis toxin (IAP) suppressed IDA. Guanosine 5’-O-(3-thiotriphosphate) (GTP-γS) sustained the DA response. Modulators for cAMP such as forskolin and isobutylmethylxathine, H-89, a protein kinase A inhibitor, and chelerythrine, a protein kinase C inhibitor, had no effect on IDA. The frequency of DA-induced single channel currents in the inside-out patch configuration, for which the unitary conductance was 56.6pS, was greatly reduced by the replacement of GTP with GDP perfused at the cytosolic side. These results suggest that DA acts on a D2like receptor and activates directly an IAP-sensitive G protein coupled with inward rectifier K + channels, resulting in a decrease in the spontaneous firing activities of rat SNc dopaminergic neurones. 2000 Elsevier Science Ltd. All rights reserved. Keywords: Dopamine; K + channel; Substantia nigra pars compacta
1. Introduction The substantia nigra (SN) as well as the ventral tegmental area possesses the densest area of dopamine (DA)-containing neurones in the central nervous system. The dopaminergic neurones of the substantia nigra pars compacta (SNc) send their axons to the striatum (Nauta and Cole, 1974), and to themselves through recurrent fibers (Cheramy et al., 1981). SNc dopaminergic neurones have the so-called ‘autoreceptor’ for DA (Bunney et al., 1973; Cheramy et al., 1981). The application of DA to SNc neurones hyperpolarizes the membrane and inhibits the firing rate of the neurones (Bunney et al., 1973; Groves et al., 1975; Lacey et al., 1988; Mercuri et al., 1992). However, DA does not cause either a hyp* Corresponding author. Tel.: +81-92-642-6090; fax: +81-92-6426094. E-mail address: [email protected] (J. Nabekura)
erpolarization of the cell membrane or an inhibition of spontaneous firing of the SN dopaminergic neurones in D2 receptor-deficient mice (Mercuri et al., 1997). These reports suggest that SN dopaminergic neurones have a functional negative feedback system for controlling their own activity through a D2 type receptor. Recent molecular biological approaches have classified the DA receptor into five groups (D1, D2, D3, D4, and D5) (Bunzow et al., 1988; Sokoloff et al., 1990; Zhou et al., 1990; Sunahara et al., 1991; Van Tol et al., 1991). For the D2 receptor, an activation of the D2 dopamine receptor opens K + channels and inhibits Ca2+ channels (Vallar and Meldolesi, 1989). It is also well known that DA receptors are coupled with a GTP binding protein (G protein) (Castro and Strange, 1993) and regulate an intracellular second messenger system. DA inhibits forskolin-stimulated cyclic adenosine 3’-5’ monophosphate (cAMP) production (Bates et al., 1991; Johansson and Westlind-Danielsson, 1994) and induces a phosphatidyl inositol-linked mobilization of intracellular Ca2+
0028-3908/00/$ - see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 9 9 ) 0 0 1 1 1 - 2
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(Liu et al., 1992) in mouse L-cells thymidine kinase deficient (Ltk -) fibroblast cells, stably expressing the rat D2S or human D2L receptor, respectively. Stimulation of either the D2L or D2S receptor expressed in C6 glia and human enbryonic kidney (HEK) 293 cells sensitizes cAMP accumulation (Watts and Neve, 1996). However, cAMP does not mediate the DA-induced current (IDA) response in cultured SN neurones (Kim et al., 1995). The detailed mechanisms for DA action on native SNc neurones have not been well understood. In the present study, we thus investigated the modulatory effects of DA on neuronal activity and its possible intracellular signalling pathways of acutely dissociated rat SNc neurones using a patch clamp technique.
2. Methods 2.1. Cell preparation Neurones were acutely dissociated from the rat SNc as described previously (Inomata et al., 1993; Nabekura et al., 1996). Briefly, 2-week-old Wistar rats of both sexes were decapitated under pentobarbital intraperitoneal anaesthesia. The brain was quickly removed from the skull and serial coronal slices of mesencephalon were obtained at a thickness of 400 µm with a microslicer (DTK-1000, Dosaka, Kyoto, Japan). Following 30–60 min maintenance in an incubation medium equilibrated with 95% O2 and 5% CO2 at room temperature (21– 23°C), the slices were treated with dispase (2000 Protease Unit/ml) at 31°C for 60 min. After the enzyme treatment, the slices were stored in the incubation medium for 1 h. Thereafter, the region of the SNc was carefully punched out with an electrically polished injection needle under a binocular microscope (SM 2–1, Nikon, Tokyo, Japan). The punched-out pieces were mechanically triturated with fine glass pipettes in a 35 mm culture dish (Primaria Falcon, Lincoln Park, New Jersey, USA) filled with the standard external solution under a phase-contrast microscope (TMS-1, Nikon). The dissociated neurones which adhered to the dish within 10 min were used for electrophysiological recordings. 2.2. Electrical measurements Nystatin-perforated whole cell recordings (Akaike and Harata, 1994) were employed for almost all electrical measurements. In some experiments conventional whole-cell recording and inside-out patch recording (Hamill et al., 1981) were used. Patch pipettes were made from barosilicate glass tube (1.5 mm o.d., 0.9 mm i.d.; G-1.5, Narishige, Tokyo, Japan) in a two-stage vertical pipette puller (PP 83, Narishige). The resistance between the recording electrode filled with internal solution and the reference electrode in the standard external
solution was 5–7 M⍀. In single channel experiments, the pipettes were coated with silicone (Shin-Etsu, Tokyo, Japan) near their tips to reduce the electrical capacitance. The neurones were visualized with phase-contrast equipment on an inverted microscope (IMT-2, Olympus, Tokyo, Japan). The current and voltage were measured with a patch-clamp amplifier (EPC-7, List Medical, Darmstadt, Germany), monitored on both a storage oscilloscope (MS-5100 A, Iwatsu Electric, Tokyo, Japan) and a pen recorder (Recti-Hoiz-8K, Nippondenki San-ei, Tokyo, Japan), and stored on videotapes after digitization with a pulse-coded modulation processor (PCM-501 ESN, Nihon Koden, Tokyo, Japan). The membrane currents were filtered at 1 kHz (E-3201A, NF Electronic Instruments, Tokyo, Japan). For single channel currents, data were filtered using a four-pole lowpass Bessel-type ⫺3 dB corner frequency of 1–2 kHz and sampled every 0.1–0.5 ms onto the hard disk of an IBM 386 computer (PS/V Entry, Nippon IBM, Tokyo, Japan) using pCLAMP software (version 6.0, Axon Instruments, USA). Unitary current amplitudes were measured by forming a histogram of baseline and openlevel datum points, and by fitting these histograms with Gaussian curves using a least-square algorithm to find the area under each curve. In single channel current recordings, the inward currents are shown as downward deflections. The currentvoltage (I–V) relationship was plotted against the inverted pipette potential (membrane potential, Vm). Neither the number of channels in the patch (N) nor the open probability of the individual channel (Po) can be determined. However, a variable NPo (the open probability of the patch) was determined by NPo=⌺[NP(n)] in which P(n) is the probability of the record to stay at a level at which n channels open simultaneously (Friedrich et al., 1988). 2.3. Solution The ionic composition of the incubation medium was (mM): 124 NaCl, 5 KCl, 1.2 KH2PO4, 23 NaHCO3, 2.4 CaCl2, 1.3 MgSO4 and 10 glucose. The pH of the incubation medium was adjusted to 7.4 with 95% O2 and 5% CO2. The ionic composition of the standard solution was (mM): 150 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2,10 glucose, and 10 HEPES. External solution containing 20 mM K + was prepared by replacing NaCl with equimolar KCl. The pH of these external solutions was adjusted to 7.4 with tris (hydroxymethyl) aminomethane (Tris-OH). The ionic composition of the internal (patch pipette) solution for the nystatin-perforated patch recording was (mM): 50 KCl, 100 K-methansulfonate and 10 HEPES. Nystatin was dissolved in methanol at 10 mg/ml, then the stock solution was diluted with the internal solution just before use at a final concentration of 200 µg/ml. The internal solution for the conventional whole-cell patch
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recording had the following composition of (mM): 114 K-methansulfonate, 40 NaCl, 0.25 CaCl2, 3 MgCl2, 0.5 EGTA, 2 Na2-ATP, 0.3 GTP (or GDP-βs or GTP-γs), and 5 HEPES. The pH of these internal solutions were adjusted to 7.2 with Tris-OH. The ionic composition of the intracellular solution for the inside-out patch recordings was (mM): 106 K-methansulfonate, 40 NaCl, 2 Na2-ATP, 0.1 GTP (or GDP), 1 MgCl2, 5 EGTA, 5 HEPES, pH 7.2 with Tris-OH.
national Inc, USA), and 0.2% normal bovine serum at 4°C overnight. Thereafter the neurones were treated with FITC-conjugated goat anti-rabbit IgG (1: 225, Biomedical Technologies Inc., USA) for 30 min at room temperature. Images of the neurones with TH staining were collected with a CCD camera (Hamamatsu Photonics, Hamamatsu, Japan) attached to a fluorescent microscope (Axolal, Zeiss, Switzerland) and stored in a computer using Image Shot (Alpha, Fukuoka, Japan).
2.4. Drugs
2.6. Statistics
The drugs used in the present study included; dispase [Godo Shuei, Tokyo, Japan], dopamine, l-ascorbic acid, BaCl2 [Ishizu pharmaceutional Co, LTD, Osaka, Japan], nystatin, guanosine 5’-O-(3-thiotriphosphate) (GTP-γS), islet-activating protein (IAP, pertussis toxin), isobutylmethylxathine (IBMX), forskolin, chelerythrine [Sigma, St. Louis, MO, USA], quinpirole, (±)-1-phenyl-2,3,4,5tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride (SKF38393), s(⫺)-sulpiride, (±)-7-bromo-8-hydroxy3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SKF83566), R(+)-2-dipropylamino-7hydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (R(+)-7-OH-DPAT) [Reseach Biocemicals International, MA,USA], guanosine 5’-triphosphate (GTP), guanosine 5’-diphosphate (GDP) [Yamasa, Chiba, Japan], guanosine 5’-O-(2-thiodiphosphate) (GDP-βS) [BIOMOL Research Laboratories, Inc, PA, USA], N-[2-(bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide 2HCl (H-89) [Seikagaku corporation, Tokyo, Japan], tetraethyl ammonium-Cl (TEA) [Tokyo Kasei, Tokyo, Japan]. The drugs insoluble in water were first dissolved in dimethylsulfoxide (DMSO) and then further diluted in the external solution. The final concentrations of DMSO were always less than 0.1%, in which DMSO alone had no effect on the membrane potential or electrical activity. l-ascorbic acid was added to the dopamine solution to prevent oxidative degradation of dopamine. The final concentration of l-ascorbic acid was less than 100 µM, in which l-ascorbic acid alone did not produce any effects. The drugs were applied using a rapid application system termed the ‘Y-tube method’ (Murase et al., 1990), which enables the external solution surrounding a neurone to exchange within 20 ms.
Data were presented as the mean±standard error of the mean (S.E.M.). For constructing the concentrationresponse curves, the data were fitted to a modified Michaelis-Menten equation by using least-squares fitting: I=(Imax×Cn)/(Cn+EC50n), where I was the druginduced current, Imax was the maximum response, C was the concentration of an agonist, EC50 was the concentration that evokes a half-maximum response and n was the Hill’s coefficient. For constructing concentrationinhibition curves, the data were fitted to the following equation by using least-squares fitting: I=1⫺{Imax×Cn/(Cn+IC50n)}, where I was the current amplitude normalized to the control response without an antagonist, C was the concentration of an antagonist, and IC50 was the concentration for half-inhibition.
2.5. Immunocytochemistry Following electrical recordings, the dishes were filled with a solution of 4% paraformaldehyde dissolved in 0.1 M phosphate-buffered saline (PBS), pH 7.4 for 1 h. Then the neurones were incubated in PBS containing 30% sucrose at 4°C overnight. After being incubated in a solution containing 0.25% Triton X-100 for 30 min, the neurones were placed into PBS containing rabbit antityrosine hydroxylase (TH; 1:2000, Chemicon Inter-
3. Results 3.1. DA responses The SNc neurones could be divided into two groups according to their soma size. One group of neurones exhibits a large soma ranging between 30 µm and 40 µm in diameter. The other group is composed of small neurones (15–20 µm in diameter). Eighteen out of 24 large SNc neurones (75.0%) were shown to be immunopositive for TH staining, while 23 out of 24 small neurones (95.8%) were TH negative. This result indicates that DA neurones are a large size (Fig. 1A and B). Under current-clamp conditions, the large SNc neurones showed spontaneous action potentials at a regular rate ranging from 0.4 to 2.6 Hz (1.2±0.27 Hz, mean±S.E.M., n=7) in the standard extracellular solution ([K +]o; 5 mM). The application of 10⫺6 M DA immediately hyperpolarized the cell membrane associated with a reduction in firing rate (Fig. 1C). However, the action potentials gradually reappeared even in the presence of 10⫺6 M DA (Fig. 1C). After washing out DA, both the membrane potential and firing rates recovered to those before DA application. Under voltage-clamp conditions, 10⫺6 M DA induced a peak followed by a gradual decrease of inward current in 22 out of 24 large neurones examined in a 20 mM K + external solution at a holding poten-
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Fig. 1. Acutely dissociated SNc neurones of the rat. (A) Translucent images of large (a) and small (b) neurones acutely dissociated from the SNc of the rat. (B) Epifluorescent images of neurones presented in A with immunofluorescent staining for tyrosine hydroxylase (TH). Only the large neurone (a), but not the small neurone (b) is TH positive. Scale bar, 50 µm. (C) DA (10⫺6 M) reduced the frequency of spontaneous action potentials recorded using the nystatin-perforated patch clamp recording configuration under current-clamp. Action potentials gradually reappeared in the continuous presence of DA.
tial (VH) of ⫺60 mV (Fig. 2Aa), while only 1 out of 24 small neurones responded to 10⫺6 M DA. For the DA response, there was a statistical difference between the two groups (P⬍0.01, χ2 test). Thus, we chose large SNc neurones (DA neurones) for further experiments.
3.2. DA receptor subtype Extracellular solution containing 20 mM K + and a VH of ⫺60 mV were employed in almost all experiments for convenience to analyze data because of the marked
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Fig. 2. Responses of DA receptor agonists and antagonists. (Aa) Peak amplitude of the inward responses to DA (top) or quinpirole (bottom) increased in a concentration-dependent manner. (Ab) The concentration-response relationships for DA receptor agonists. SKF38393 (up to 10⫺5 M) did not induce any current. (Ba) s(⫺)-sulpiride inhibited the 10⫺6 M DA-induced current in a concentration-dependent manner. The neurones were pretreated with the DA receptor antagonist for 2 min before 10⫺6 M DA. (Bb) The concentration-inhibition relationships for DA antagonists on the IDA. All responses were normalized to the peak current induced by 10⫺6 M DA without antagonists in each neurone. All recordings were performed in external solution containing 20 mM K + at a VH of ⫺60 mV in voltage-clamp conditions. Each point and vertical line shows the mean±S.E.M. of 4–9 neurones.
inward rectification of IDA (Fig. 3). The peak amplitude of the DA response increased in a concentration-dependent manner (Fig. 2A). Quinpirole (LY171555), a D2 receptor agonist, mimicked DA action (Fig. 2Aa). The respective values for the threshold concentration, the EC50 and Hill’s coefficient were 10⫺8 M, 1.55×10⫺7 M and 1.36 for DA (n=9), and 10⫺8 M, 2.35×10⫺7 M and 0.91 for quinpirole (n=9). On the other hand, SKF38393, a D1 receptor agonist, induced no current at a concentration of up to 10⫺5 M in the DA neurones (n=4, Fig. 2Ab). The maximum currents normalized to the peak amplitude of the 10⫺6 M DA response were 1.08 (n=9) for DA and 0.92 (n=9) for quinpirole. S(⫺)-sulpiride, a D2 receptor antagonist, reversibly inhibited 10⫺6 M IDA in a concentration-dependent manner with a IC50 of 4.52×10⫺9 M (n=5, Fig. 2B). SKF83566, a potent D1 receptor antagonist, inhibited 10⫺6 M IDA with an IC50 of 3.53×10⫺6 M (n=5, Fig. 2Bb). These results indicate that IDA is activated mainly by D2 receptors.
3.3. Ionic mechanism of IDA The I–V relationship for IDA showed an inward rectification (Fig. 3). The reversal potentials of the IDA (EDA) estimated from the I–V relationships were ⫺80 mV (n=4) and ⫺43 mV (n=4) in the standard external solution containing 5 mM and 20 mM K +, respectively (Fig. 3B). These values were close to the respective theoretical K + equilibrium potentials (⫺82.7 mV for 5 mM K + and ⫺49.0 mV for 20 mM K +) calculated by the Nernst equation at the given K + concentrations of the external and pipette solutions. These findings indicate that activation of the D2 receptor opens the inward rectifier K + channel in SNc DA neurones. 3.4. Contribution of GTP binding protein to IDA Intracellular perfusion with 0.3 mM GTP using a conventional whole-cell recording mode maintained a constant amplitude of IDA over 30 min (Fig. 4Aa and B). On the other hand, the intracellular application of 0.3
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Fig. 3. Current-voltage (I–V) relationship for the DA response. (A) DA (10⫺6 M) -induced currents at various VHs. All current traces were obtained from the same neurone in external solution containing 20 mM K + under voltage-clamp mode. (B) I–V relationships for DA-induced responses in the external solution with two different K + concentrations (open circle; 5 mM, close circle; 20 mM). All responses were normalized to the peak current induced by 10⫺6 M DA in the external solution containing 20 mM K + at a VH of ⫺40 mV (*). Each point and vertical bar shows the mean±S.E.M. of four neurones. Intracellular K + concentration ([K +]i) was 150 mM.
Fig. 4. Involvement of G protein in DA response. (A) Representative recordings of the IDA after intracellular perfusion with 0.3 mM GTP (a), 0.3 mM GDP-βS (b) or 0.3 mM GTP-γS (c) by using the conventional whole-cell patch recording mode. The horizontal bars indicate the periods of 10⫺6 M DA application. The numbers above the horizontal bars show the time after the rupture of the patch membrane. All recordings were carried out in external solution containing 20 mM K + at a VH of ⫺60 mV. (B) Relative peak amplitudes of 10⫺6 M DA responses with GTP (open circles), GDP-βs (close circles) and IAP (close triangles) as a function of time after rupture of membrane. All responses were normalized to the peak amplitude of the IDA obtained at 1 min after rupture of the membrane in each cell. Each point and vertical bar shows the mean±S.E.M. of five to eight neurones.
mM GDP-βS, a non-hydrolyzable GDP analogue, caused a rapid suppression of the IDA (Fig. 4Ab and B). Intracellular application of 0.3 mM GTP-γS, a nonhydrolyzable GTP analogue, sustained the inward current induced by the first application of 10⫺6 M DA. During this sustaining period, a second application of DA
failed to induce an IDA of maximum amplitude (n=3, Fig. 4Ac). IAP (1 µg/ml) applied intracellularly gradually decreased the peak amplitude of IDA (Fig. 4B). These results suggest that an IAP-sensitive GTP binding protein (G protein) may be involved in the IDA in SNc DA neurones.
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It is well known that activation of the D2 receptor regulates intracellular cAMP production (Bates et al., 1991; Liu et al., 1992; Johansson and Westlind-Danielsson, 1994; Kim et al., 1995; Watts and Neve, 1996). In the present study, however, the DA response persisted in the presence of forskolin (10⫺5 M), an adenylyl cyclase activator, and IBMX (10⫺6 M), a phosphodiesterase inhibitor (105.9±4.9% peak amplitude of IDA compared with that without these modulators, P⬎0.1; paired t-test, n=5). H-89 (10⫺6 M), a protein kinase A (PKA) inhibitor, did not block the DA response (87.5±11.1% of control, P⬎0.1, n=4). Pretreatment with chelerythrine (3×10⫺7 M), a protein kinase C (PKC) inhibitor, for 3 min before DA application did not block the IDA (99.7±6.2% of control, P⬎0.1, n=4). These results indicate that IDA is mediated by an IAP-sensitive G protein, but is independent of PKA and PKC pathways. 3.5. DA-induced single channel current In the inside-out patch recording configuration, 10⫺7 M DA in the patch pipette and 0.3 mM GTP applied to the cytoplasmic side increased single channel activities at a membrane potential of ⫺70 mV (Fig. 5Aa, n=7).
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Replacement of GTP with 0.3 mM GDP on the cytoplasmic side markedly decreased the DA-induced single channel activities (Fig. 5Ab, n=7). The open probabilities of the patch (Npo) were 0.0021±0.0004 and 0.0004±0.0002 with the bath solution containing 0.3 mM GTP and 0.3 mM GDP, respectively. The respective values for open time and close time were 0.372 ms±0.063 ms and 78.7 ms±7.2 ms for GTP, and 0.285 ms±0.02 ms and 218 ms±36 ms for GDP. There is a statistically significant difference in the close time between the two groups (P⬍0.01, paired t-test). Respective mean amplitudes for the first and second peaks were 2.93 pA and 6.98 pA for GTP and 3.02 pA and 7.18 pA for GDP, indicating no differences in the mean amplitude of DA-induced single channel current between GTP and GDP treatment (Fig. 5Ba and b). In addition, I–V relationships revealed that there was a remarkable DAinduced inward rectification even at the single channel current level and that the maximal single channel conductance of the DA-induced inward current was 56.6 pS at a VH between ⫺100 and +10 mV (Fig. 6).
Fig. 5. Effects of GDP and GTP on DA-operated single channel activity. The inside-out patch recording mode was employed to record the DAinduced single channel currents. (A) Current traces with 0.3 mM GTP (a) and 0.3mM GDP (b) applied to the cytosolic side solution. The horizontal thin and broken lines show the close (c) and open (o) levels, respectively. The patch pipette contained 10⫺7 M DA. The membrane potential (Vm) was ⫺70 mV. (B) The amplitude histograms of the currents induced by 10⫺7 M DA with 0.3 mM GTP (a) or 0.3mM GDP (b). The amplitude histograms were fitted by a Gaussian curve with a peak value of 2.93 pA (a), and 3.02 pA (b). Sampling time of single channel events was two minutes.
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Fig. 6. I–V relationship for DA-induced single channel current. (A) The DA-induced single channel current obtained at various Vms. The patch pipette contained 10⫺7 M DA. The horizontal thin and broken lines indicate the close and the open levels, respectively. (B) The I–V relationship for the DA-induced single channel currents. Each point and vertical bar shows the mean±S.E.M. of three neurones. Almost all vertical bars are small enough to be masked within the solid circles.
4. Discussion
4.2. DA receptor subtype
4.1. Classification of SNc neurones
Recent molecular biological techniques have revealed five cloned subtypes of dopamine receptors; D1, D2, D3, D4 and D5 (Bunzow et al., 1988; Zhou et al., 1990; Sokoloff et al., 1990; Van Tol et al., 1991; Sunahara et al., 1991; Sibley and Monsma, 1992). Structural and pharmacological analyses led to the division of these receptors into two classes: the D1- and D2-like receptors. The D1-like subfamily consists of D1 and D5 receptors, while the D2-like family consists of D2L and D2S isoforms, as well as D3 and D4 receptors (Sibley and Monsma, 1992). In both slices and culture, the hyperpolarization produced by DA was mimicked by a D2 receptor agonist quinpirole (Lacey et al., 1987; Kim et al., 1995). Also in this study using acutely dissociated neurones, quinpirole mimicked DA, but the maximal amplitude of the quinpirole-induced current in the DA neurones was only 85% of that induced by DA (Fig. 2Ab). This result suggests two possibilities; 1) a partial, not full, agonistic action of quinpirole on the D2 receptor in SNc neurones, 2) an additional involvement of a D3 type receptor in the DA response. However, clear discrimination between the D2like receptors is difficult because selective agonists and antagonists for D2 and D3 receptors are not yet available. Moreover, the differences in Kd and Ki among most
The present study demonstrated that DA acted on a D2-like receptor and activated an inward rectifier K + channel in large neurones acutely dissociated from the rat SNc. On the other hand, DA did not induce any current in small SNc neurones. In addition, large neurones showed spontaneous action potentials at a regular rate ranging from 0.4 to 2.6 Hz. This is compatible with a previous report that DA neurones of the SNc fired spontaneous action potentials in the range of 1–8 Hz in a brain slice preparation (Lacey et al., 1989). Of cultured (Kim et al., 1997) and acutely dissociated SNc neurones (present study), 57% and 75% stained positive for TH, respectively. Both DA neurones and non-DA neurones have been reported to be of a similar size in cultured SNc neurones (Kim et al., 1997). On the other hand, DA neurones are estimated to be larger in size than non-DA neurones with a mean conductance of 22 nS and 4 nS, respectively, in the slice preparation (Lacey et al., 1989). This is in accord with the present result showing that DA neurones were larger in soma size than non-DA neurones (Fig. 1 and Results). Some morphological characteristics such as size might change in culture.
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agonists and antagonists of D2-like receptors are within one order of magnitude between D2 and D3 receptors (Burris et al., 1995). On the other hand, SKF38393, a D1 agonist, did not induce any current in the DA neurones (Fig. 2Ab). In addition, a D2 antagonist was more effective on the DA-induced current than the D1 antagonist (Fig. 2Bb). These pharmacological studies indicate that IDA is mediated by a D2-like receptor but not D1like receptor activation in SNc DA neurones. 4.3. DA receptor coupled with K+ channel K + channels which are characterized by an inward rectification of the current can be functionally classified into two groups. One is the inward rectifier K + (IRK) channel family. The activity of IRK channels is not modulated by G proteins, and the open time of the channel is 10–200 ms (Kubo et al., 1993a). The other is the G protein-gated inward rectifier K + (GIRK) channel family. The channel activities of this family are modulated by G proteins, and the channel open time is 0.5– 2 ms (Kubo et al., 1993b; Grigg et al., 1996). The present study showed that the DA-induced inward rectifier K + current in the DA neurones had characteristics similar to those of the GIRK family, because of the involvement of a G protein in the channel activation and of the similar open time, approximately 0.3 ms. The GIRK channels have been reported to conduct K + at or near the resting membrane potential and to play an important role in controlling cell excitability (Wickman and Clapham, 1995). The mRNAs of GIRK1, GIRK2, GIRK3 and GIRK4 (mGIRK1, mGIRK2, mGIRK3 and mGIRK4, respectively) have been cloned (Kubo et al., 1993b; Lesage et al., 1994; Duprat et al., 1995). Northern blot analysis has revealed their distributions: mGIRK4 is restricted to the heart; mGIRK2 and mGIRK3 transcripts are specifically present in the brain; mGIRK1 is expressed in both heart and brain (Lesage et al., 1995). Single channel analyses of K + currents mediated by cloned GIRK1, GIRK2, GIRK1 and 2, and GIRK2 and 4 in an expression system demonstrate that there is only one population of channels with very similar properties to each other, characterized by a unitary conductance of about 40 pS and a mean open time duration of less than 1 ms. On the other hand, native GIRK channels recorded in different neuronal cell types have been reported to possess other properties of unitary conductance varying from 38 to 55 pS and an open time of the order of 2 ms (Lesage et al., 1995). Our results indicating that the unitary conductance of DA-operated channels was 56.6 pS in SNc neurones corresponded with Lesage’s report (1995). An immunohistochemical approach has revealed that mGIRK1, mGIRK2 and their expressed proteins exist in both soma and dendrites of dopaminergic neurones in the SNc (Liao et al., 1996). However it should be a target for further study to elucidate which subunits
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of the GIRK channels form functional channels in the DA neurones of rat SNc. 4.4. Possible pathway between DA receptor and K+ channel In freshly dissociated SNc neurones, the intracellular application of GDP-βS, the non-hydrolyzable analogue of GDP which competitively inhibits the binding of GTP to the α-subunit of G protein, inhibited the DA-induced K + current (Fig. 4Ab and B). On the other hand, an intracellular application of GTP-γS, the non-hydrolyzable analogue of GTP, which activates the G protein persistently, induced an irreversible modulation of IDA (Fig. 4Ac). These results indicate that the DA response in SNc neurones is mediated by a G protein. Additional support for the involvement of a G protein in IDA was revealed by the open probabilities in the inside-out patch recording combined with cytoplasmic GDP-GTP exchange (Fig. 5). It is controversial whether the G protein participating in the DA action on SNc neurones is IAP-sensitive or insensitive. In rats treated with an injection of IAP into the SN regions, the reduction of spike frequency by DA agonists disappears (Innis and Aghajanian, 1987). In cultured SNc neurones of rats, the DA agonist-induced response is completely abolished by IAP pretreatment (Kim et al., 1995). On the other hand, the hyperpolarization induced by DA in SN neurones is not affected by the intracerebroventricular injection of IAP (Lacey et al., 1988). In the present study, the intracellular application of IAP eliminated DA-induced K + currents in DA neurones (Fig. 4B). This result indicates that IDA is mediated by IAP-sensitive G proteins, i.e., Gi and Go, in the DA neurones of the rat SNc. It is well known that G proteins are coupled to effective ion channels through the activation of various intracellular second messengers. DA reduces the spontaneous spike frequency of DA neurones in the SNc by altering cAMP production (Shi and Bunney, 1992). Kim et al. (1995) reported that the quinpirole-induced response was larger in cAMP-loaded neurones compared with controls, but that cAMP did not have any effect on the K + conductance. The present study showed that the application of forskolin, a direct activator of adenylyl cyclase, and IBMX, an inhibitor of phosphodiesterase, had no effect on the IDA. H-89, an inhibitor of PKA, and chelerythrine, an inhibitor of PKC, did not affect the IDA. In addition, DA could elicit currents even in the inside-out patch recording mode, in which soluble second messengers and other cytoplasmic factors are presumably lost and/or diluted. These results suggest that the activation of the D2-like receptor opens K + channels through an IAP-sensitive G protein and that the coupling between the G protein and K + channel seems to be ‘direct’, rather than mediated by intracellular soluble second messengers in the SNc DA neurones.
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4.5. DA receptor and neuronal function The dopaminergic neurones in the SNc exhibit DA receptors in the somatodendritic region and axon terminal (Cheramy et al., 1981). Activation of autoreceptors in the somatodendritic region inhibits the spontaneous activities of dopaminergic neurones (Groves et al., 1975). Somatodendritic DA release evoked in the SNc is reduced by D2 receptor agonists and enhanced significantly by D2 receptor antagonists during appropriate electrical stimulation (Cragg and Greenfield, 1997). Activation of the autoreceptors located at the axon terminals inhibits DA synthesis and DA release (Starke et al., 1989). Because we employed dissociated SNc neurones which retained only their soma and proximal dendrites in this study, somatodendritic autoreceptors might be involved in the IDA. In addition, our results that the inhibition of spontaneous activity by DA is probably mediated by a direct coupling between G proteins and K + channels suggest a possible role of IDA in the rapid onset-offset negative feedback regulation of neuronal excitability in SNc DA neurones. Acknowledgements We would like to give our appreciation to Ms. Min Li for critical reading of the manuscript. This work was supported by Grant-in-Aids for Scientific Research (1167044 to J.N. and 10044301 and 10470009 to N.A.) and by Grant-in-Aids for Scientific Research on priority area (11170240) to J.N. from the Ministry of Education, Science and Culture, Japan. References Akaike, N., Harata, N., 1994. Nystatin perforated patch recording and its applications to analyses of intracellular mechanisms. Japanese Journal of Physiology 44, 433–473. Bates, M.D., Senogles, S.E., Bunzow, J.R., Liggett, S.B., Civelli, O., Caron, M.G., 1991. Regulation of responsiveness at D2 dopamine receptors by receptor desensitization and adenylyl cyclase sensitization. Molecular Pharmacology 39, 55–63. Bunney, B.S., Walters, J.R., Roth, R.H., Aghajanian, G.K., 1973. Dopaminergic neurons: effect of antipsychotic drugs and amphetamine on single cell activity. Journal of Pharmacology and Experimental Therapeutics 185, 560–571. Bunzow, J.R., Van Tol, H.H., Grandy, D.K., Albert, P., Salon, J., Christie, M., Machida, C.A., Neve, K.A., Civelli, O., 1988. Cloning and expression of a rat D2 dopamine receptor cDNA. Nature 336, 783–787. Burris, K.D., Pacheco, M.A., Filtz, T.M., Kung, M.P., Kung, H.F., Molinoff, P.B., 1995. Lack of discrimination by agonists for D2 and D3 dopamine receptors. Neuropsychopharmacology 12, 335–345. Castro, S.W., Strange, P.G., 1993. Coupling of D2 and D3 dopamine receptors to G-proteins. Federation of European Biochemical Societies 315, 223–226. Cheramy, A., Leviel, V., Glowinski, J., 1981. Dendritic release of dopamine in the substantia nigra. Nature 289, 537–542.
Cragg, S.J., Greenfield, S.A., 1997. Differential autoreceptor control of somatodendritic and axon terminal dopamine release in substantia nigra, ventral tegmental area, and striatum. Journal of Neuroscience 17, 5738–5746. Duprat, F., Lesage, F., Guillemare, E., Fink, M., Hugnot, J.P., Bigay, J., Lazdunski, M., Romey, G., Barhanin, J., 1995. Heterologous multimeric assembly is essential for K + channel activity of neuronal and cardiac G-protein-activated inward rectifiers. Biochemical and Biophysical Research Communications 212, 657–663. Friedrich, F., Paulmichl, M., Kolb, H.A., Lang, F., 1988. Inward rectifier K channels in renal epithelioid cells (MDCK) activated by serotonin. Journal of Membrane Biology 106, 149–155. Grigg, J.J., Kozasa, T., Nakajima, Y., Nakajima, S., 1996. Single-channel properties of a G-protein-coupled inward rectifier potassium channel in brain neurons. Journal of Neurophysiology 75, 318–328. Groves, P.M., Wilson, C.J., Young, S.J., Rebec, G.V., 1975. Self-inhibition by dopaminergic neurons. Science 190, 522–529. Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J., 1981. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflu¨gers Archives 391, 85–100. Innis, R.B., Aghajanian, G.K., 1987. Pertussis toxin blocks autoreceptor-mediated inhibition of dopaminergic neurons in rat substantia nigra. Brain Research 411, 139–143. Inomata, H., Nabekura, J., Akaike, N., 1993. Suppression of taurine response in acutely dissociated substantia nigra neurons by intracellular cyclic AMP. Brain Research 615, 347–350. Johansson, M.H., Westlind-Danielsson, A., 1994. Forskolin-induced up-regulation and functional supersensitivity of dopamine D2 long receptors expressed by Ltk- cells. European Journal of Pharmacology 14 (269), 149–155. Kim, K.M., Nakajima, Y., Nakajima, S., 1995. G protein-coupled inward rectifier modulated by dopamine agonists in cultured substantia nigra neurons. Neuroscience 69, 1145–1158. Kim, K.M., Nakajima, S., Nakajima, Y., 1997. Dopamine and GABA receptors in cultured substantia nigra neurons: correlation of electrophysiology and immunocytochemistry. Neuroscience 78, 759– 769. Kubo, Y., Baldwin, T.J., Jan, Y.N., Jan, L.Y., 1993a. Primary structure and functional expression of a mouse inward rectifier potassium channel. Nature 362, 127–133. Kubo, Y., Reuveny, E., Slesinger, P.A., Jan, Y.N., Jan, L.Y., 1993b. Primary structure and functional expression of a rat G-proteincoupled muscarinic potassium channel. Nature 364, 802–806. Lacey, M.G., Mercuri, N.B., North, R.A., 1987. Dopamine acts on D2 receptors to increase potassium conductance in neurones of the rat substantia nigra zona compacta. Journal of Physiology 392, 397– 416. Lacey, M.G., Mercuri, N.B., North, R.A., 1988. On the potassium conductance increase activated by GABAB and dopamine D2 receptors in rat substantia nigra neurones. Journal of Physiology 401, 437– 453. Lacey, M.G., Mercuri, N.B., North, R.A., 1989. Two cell types in rat substantia nigra zona compacta distinguished by membrane properties and the actions of dopamine and opioids. Journal of Neuroscience 9, 1233–1241. Lesage, F., Duprat, F., Fink, M., Guillemare, E., Coppola, T., Lazdunski, M., Hugnot, J.P., 1994. Cloning provides evidence for a family of inward rectifier and G-protein coupled K + channels in the brain. Federation of European Biochemical Societies Letters 353, 37–42. Lesage, F., Guillemare, E., Fink, M., Duprat, F., Heurteaux, C., Fosset, M., Romey, G., Barhanin, J., Lazdunski, M., 1995. Molecular properties of neuronal G-protein-activated inwardly rectifying K + channels. Journal of Biological Chemistry 270, 28660–28667. Liao, Y.J., Jan, Y.N., Jan, L.Y., 1996. Heteromultimerization of Gprotein-gated inwardly rectifying K + channel proteins GIRK1 and
S. Uchida et al. / Neuropharmacology 39 (2000) 191–201
GIRK2 and their altered expression in weaver brain. Journal of Neuroscience 16, 7137–7150. Liu, Y.F., Civelli, O., Zhou, Q.Y., Albert, P.R., 1992. Cholera toxinsensitive 3’,5’-cyclic adenosine monophosphate and calcium signals of the human dopamine-D1 receptor: selective potentiation by protein kinase A. Molecular Endocrinology 6, 1815–1824. Mercuri, N.B., Calabresi, P., Bernardi, G., 1992. The electrophysiological actions of dopamine and dopaminergic drugs on neurons of the substantia nigra pars compacta and ventral tegmental area. Life Sciences 51, 711–718. Mercuri, N.B., Saiardi, A., Bonci, A., Picetti, R., Calabresi, P., Bernardi, G., Borrelli, E., 1997. Loss of autoreceptor function in dopaminergic neurons from dopamine D2 receptor deficient mice. Neuroscience 79, 323–327. Murase, K., Randic, M., Shirasaki, T., Nakagawa, T., Akaike, N., 1990. Serotonin suppresses N-methyl-d-asparatate responses in acutely isolated spinal dorsal horn neurons of the rat. Brain Research 525, 84–91. Nabekura, J., Omura, T., Akaike, N., 1996. Alpha 2 adrenoceptor potentiates glycine receptor-mediated taurine response through protein kinase A in rat substantia nigra neurons. Journal of Neurophysiology 76, 2447–2454. Nauta, H.J., Cole, M., 1974. Efferent projections of the subthalamic nucleus. Transactions of the American Neurological Association 99, 170–173. Shi, W.X., Bunney, B.S., 1992. Roles of intracellular cAMP and protein kinase A in the actions of dopamine and neurotensin on midbrain dopamine neurons. Journal of Neuroscience 12, 2433–2438.
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Sibley, D.R., Monsma, F.J. Jr., 1992. Molecular biology of dopamine receptors. Trends in Pharmacological Science 13, 61–69. Sokoloff, P., Giros, B., Martres, M.P., Bouthenet, M.L., Schwartz, J.C., 1990. Molecular cloning and characterization of a novel dopamine receptor (D3) as a target for neuroleptics. Nature 347, 146–151. Starke, K., Gothert, M., Kilbinger, H., 1989. Modulation of neurotransmitter release by presynaptic autoreceptors. Physiological Reviews 69, 864–989. Sunahara, R.K., Guan, H.C., O’Dowd, B.F., Seeman, P., Laurier, L.G., Ng, G., George, S.R., Torchia, J., Van Tol, H.H., Niznik, H.B., 1991. Cloning of the gene for a human dopamine D5 receptor with higher affinity for dopamine than D1. Nature 350, 614–619. Vallar, L., Meldolesi, J., 1989. Mechanisms of signal transduction at the dopamine D2 receptor. Trends in Pharmacological Science 10, 74–77. Van Tol, H.H., Bunzow, J.R., Guan, H.C., Sunahara, R.K., Seeman, P., Niznik, H.B., Civelli, O., 1991. Cloning of the gene for a human dopamine D4 receptor with high affinity for the antipsychotic clozapine. Nature 350, 610–614. Watts, V.J., Neve, K.A., 1996. Sensitization of endogenous and recombinant adenylate cyclase by activation of D2 dopamine receptors. Molecular of Pharmacology 50, 966–976. Wickman, K., Clapham, D.E., 1995. Ion channel regulation by G proteins. Physiological Reviews 75, 865–885. Zhou, Q.Y., Grandy, D.K., Thambi, L., Kushner, J.A., Van Tol, H.H., Cone, R., Pribnow, D., Salon, J., Bunzow, J.R., Civelli, O., 1990. Cloning and expression of human and rat D1 dopamine receptors. Nature 347, 76–80.
Dopamine activates inward rectifier K + channel in acutely dissociated rat substantia nigra neurones Soko Uchida, Norio Akaike, Junichi Nabekura
*
Department of Physiology, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi Higashi-ku, Fukuoka 812-8582, Japan Accepted 18 May 1999
Abstract The effect of dopamine (DA) was investigated on acutely dissociated rat substantia nigra pars compacta (SNc) neurones by using patch clamp recording. The SNc neurones could be classified into two groups. About 75% of large neurones (⬎30 µm in diameter) were tyrosine hydroxylase (TH) positive while almost all small neurones (⬍20 µm) were TH negative. In the large neurones, DA hyperpolarized the membrane, resulting in a reduction of the frequency of spontaneous action potentials in current-clamp mode and induced an inward rectifier K + current in voltage-clamp mode. Quinpirole, a D2 receptor agonist, mimicked the DA action. S(⫺)-sulpiride, a D2 receptor antagonist, inhibited the DA-induced current (IDA) more effectively than SKF83566, a D1 receptor antagonist. Intracellular application of either guanosine 5’-O-(2-thiodiphosphate) (GDP-βS) or pertussis toxin (IAP) suppressed IDA. Guanosine 5’-O-(3-thiotriphosphate) (GTP-γS) sustained the DA response. Modulators for cAMP such as forskolin and isobutylmethylxathine, H-89, a protein kinase A inhibitor, and chelerythrine, a protein kinase C inhibitor, had no effect on IDA. The frequency of DA-induced single channel currents in the inside-out patch configuration, for which the unitary conductance was 56.6pS, was greatly reduced by the replacement of GTP with GDP perfused at the cytosolic side. These results suggest that DA acts on a D2like receptor and activates directly an IAP-sensitive G protein coupled with inward rectifier K + channels, resulting in a decrease in the spontaneous firing activities of rat SNc dopaminergic neurones. 2000 Elsevier Science Ltd. All rights reserved. Keywords: Dopamine; K + channel; Substantia nigra pars compacta
1. Introduction The substantia nigra (SN) as well as the ventral tegmental area possesses the densest area of dopamine (DA)-containing neurones in the central nervous system. The dopaminergic neurones of the substantia nigra pars compacta (SNc) send their axons to the striatum (Nauta and Cole, 1974), and to themselves through recurrent fibers (Cheramy et al., 1981). SNc dopaminergic neurones have the so-called ‘autoreceptor’ for DA (Bunney et al., 1973; Cheramy et al., 1981). The application of DA to SNc neurones hyperpolarizes the membrane and inhibits the firing rate of the neurones (Bunney et al., 1973; Groves et al., 1975; Lacey et al., 1988; Mercuri et al., 1992). However, DA does not cause either a hyp* Corresponding author. Tel.: +81-92-642-6090; fax: +81-92-6426094. E-mail address: [email protected] (J. Nabekura)
erpolarization of the cell membrane or an inhibition of spontaneous firing of the SN dopaminergic neurones in D2 receptor-deficient mice (Mercuri et al., 1997). These reports suggest that SN dopaminergic neurones have a functional negative feedback system for controlling their own activity through a D2 type receptor. Recent molecular biological approaches have classified the DA receptor into five groups (D1, D2, D3, D4, and D5) (Bunzow et al., 1988; Sokoloff et al., 1990; Zhou et al., 1990; Sunahara et al., 1991; Van Tol et al., 1991). For the D2 receptor, an activation of the D2 dopamine receptor opens K + channels and inhibits Ca2+ channels (Vallar and Meldolesi, 1989). It is also well known that DA receptors are coupled with a GTP binding protein (G protein) (Castro and Strange, 1993) and regulate an intracellular second messenger system. DA inhibits forskolin-stimulated cyclic adenosine 3’-5’ monophosphate (cAMP) production (Bates et al., 1991; Johansson and Westlind-Danielsson, 1994) and induces a phosphatidyl inositol-linked mobilization of intracellular Ca2+
0028-3908/00/$ - see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 9 9 ) 0 0 1 1 1 - 2
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(Liu et al., 1992) in mouse L-cells thymidine kinase deficient (Ltk -) fibroblast cells, stably expressing the rat D2S or human D2L receptor, respectively. Stimulation of either the D2L or D2S receptor expressed in C6 glia and human enbryonic kidney (HEK) 293 cells sensitizes cAMP accumulation (Watts and Neve, 1996). However, cAMP does not mediate the DA-induced current (IDA) response in cultured SN neurones (Kim et al., 1995). The detailed mechanisms for DA action on native SNc neurones have not been well understood. In the present study, we thus investigated the modulatory effects of DA on neuronal activity and its possible intracellular signalling pathways of acutely dissociated rat SNc neurones using a patch clamp technique.
2. Methods 2.1. Cell preparation Neurones were acutely dissociated from the rat SNc as described previously (Inomata et al., 1993; Nabekura et al., 1996). Briefly, 2-week-old Wistar rats of both sexes were decapitated under pentobarbital intraperitoneal anaesthesia. The brain was quickly removed from the skull and serial coronal slices of mesencephalon were obtained at a thickness of 400 µm with a microslicer (DTK-1000, Dosaka, Kyoto, Japan). Following 30–60 min maintenance in an incubation medium equilibrated with 95% O2 and 5% CO2 at room temperature (21– 23°C), the slices were treated with dispase (2000 Protease Unit/ml) at 31°C for 60 min. After the enzyme treatment, the slices were stored in the incubation medium for 1 h. Thereafter, the region of the SNc was carefully punched out with an electrically polished injection needle under a binocular microscope (SM 2–1, Nikon, Tokyo, Japan). The punched-out pieces were mechanically triturated with fine glass pipettes in a 35 mm culture dish (Primaria Falcon, Lincoln Park, New Jersey, USA) filled with the standard external solution under a phase-contrast microscope (TMS-1, Nikon). The dissociated neurones which adhered to the dish within 10 min were used for electrophysiological recordings. 2.2. Electrical measurements Nystatin-perforated whole cell recordings (Akaike and Harata, 1994) were employed for almost all electrical measurements. In some experiments conventional whole-cell recording and inside-out patch recording (Hamill et al., 1981) were used. Patch pipettes were made from barosilicate glass tube (1.5 mm o.d., 0.9 mm i.d.; G-1.5, Narishige, Tokyo, Japan) in a two-stage vertical pipette puller (PP 83, Narishige). The resistance between the recording electrode filled with internal solution and the reference electrode in the standard external
solution was 5–7 M⍀. In single channel experiments, the pipettes were coated with silicone (Shin-Etsu, Tokyo, Japan) near their tips to reduce the electrical capacitance. The neurones were visualized with phase-contrast equipment on an inverted microscope (IMT-2, Olympus, Tokyo, Japan). The current and voltage were measured with a patch-clamp amplifier (EPC-7, List Medical, Darmstadt, Germany), monitored on both a storage oscilloscope (MS-5100 A, Iwatsu Electric, Tokyo, Japan) and a pen recorder (Recti-Hoiz-8K, Nippondenki San-ei, Tokyo, Japan), and stored on videotapes after digitization with a pulse-coded modulation processor (PCM-501 ESN, Nihon Koden, Tokyo, Japan). The membrane currents were filtered at 1 kHz (E-3201A, NF Electronic Instruments, Tokyo, Japan). For single channel currents, data were filtered using a four-pole lowpass Bessel-type ⫺3 dB corner frequency of 1–2 kHz and sampled every 0.1–0.5 ms onto the hard disk of an IBM 386 computer (PS/V Entry, Nippon IBM, Tokyo, Japan) using pCLAMP software (version 6.0, Axon Instruments, USA). Unitary current amplitudes were measured by forming a histogram of baseline and openlevel datum points, and by fitting these histograms with Gaussian curves using a least-square algorithm to find the area under each curve. In single channel current recordings, the inward currents are shown as downward deflections. The currentvoltage (I–V) relationship was plotted against the inverted pipette potential (membrane potential, Vm). Neither the number of channels in the patch (N) nor the open probability of the individual channel (Po) can be determined. However, a variable NPo (the open probability of the patch) was determined by NPo=⌺[NP(n)] in which P(n) is the probability of the record to stay at a level at which n channels open simultaneously (Friedrich et al., 1988). 2.3. Solution The ionic composition of the incubation medium was (mM): 124 NaCl, 5 KCl, 1.2 KH2PO4, 23 NaHCO3, 2.4 CaCl2, 1.3 MgSO4 and 10 glucose. The pH of the incubation medium was adjusted to 7.4 with 95% O2 and 5% CO2. The ionic composition of the standard solution was (mM): 150 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2,10 glucose, and 10 HEPES. External solution containing 20 mM K + was prepared by replacing NaCl with equimolar KCl. The pH of these external solutions was adjusted to 7.4 with tris (hydroxymethyl) aminomethane (Tris-OH). The ionic composition of the internal (patch pipette) solution for the nystatin-perforated patch recording was (mM): 50 KCl, 100 K-methansulfonate and 10 HEPES. Nystatin was dissolved in methanol at 10 mg/ml, then the stock solution was diluted with the internal solution just before use at a final concentration of 200 µg/ml. The internal solution for the conventional whole-cell patch
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recording had the following composition of (mM): 114 K-methansulfonate, 40 NaCl, 0.25 CaCl2, 3 MgCl2, 0.5 EGTA, 2 Na2-ATP, 0.3 GTP (or GDP-βs or GTP-γs), and 5 HEPES. The pH of these internal solutions were adjusted to 7.2 with Tris-OH. The ionic composition of the intracellular solution for the inside-out patch recordings was (mM): 106 K-methansulfonate, 40 NaCl, 2 Na2-ATP, 0.1 GTP (or GDP), 1 MgCl2, 5 EGTA, 5 HEPES, pH 7.2 with Tris-OH.
national Inc, USA), and 0.2% normal bovine serum at 4°C overnight. Thereafter the neurones were treated with FITC-conjugated goat anti-rabbit IgG (1: 225, Biomedical Technologies Inc., USA) for 30 min at room temperature. Images of the neurones with TH staining were collected with a CCD camera (Hamamatsu Photonics, Hamamatsu, Japan) attached to a fluorescent microscope (Axolal, Zeiss, Switzerland) and stored in a computer using Image Shot (Alpha, Fukuoka, Japan).
2.4. Drugs
2.6. Statistics
The drugs used in the present study included; dispase [Godo Shuei, Tokyo, Japan], dopamine, l-ascorbic acid, BaCl2 [Ishizu pharmaceutional Co, LTD, Osaka, Japan], nystatin, guanosine 5’-O-(3-thiotriphosphate) (GTP-γS), islet-activating protein (IAP, pertussis toxin), isobutylmethylxathine (IBMX), forskolin, chelerythrine [Sigma, St. Louis, MO, USA], quinpirole, (±)-1-phenyl-2,3,4,5tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride (SKF38393), s(⫺)-sulpiride, (±)-7-bromo-8-hydroxy3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SKF83566), R(+)-2-dipropylamino-7hydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (R(+)-7-OH-DPAT) [Reseach Biocemicals International, MA,USA], guanosine 5’-triphosphate (GTP), guanosine 5’-diphosphate (GDP) [Yamasa, Chiba, Japan], guanosine 5’-O-(2-thiodiphosphate) (GDP-βS) [BIOMOL Research Laboratories, Inc, PA, USA], N-[2-(bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide 2HCl (H-89) [Seikagaku corporation, Tokyo, Japan], tetraethyl ammonium-Cl (TEA) [Tokyo Kasei, Tokyo, Japan]. The drugs insoluble in water were first dissolved in dimethylsulfoxide (DMSO) and then further diluted in the external solution. The final concentrations of DMSO were always less than 0.1%, in which DMSO alone had no effect on the membrane potential or electrical activity. l-ascorbic acid was added to the dopamine solution to prevent oxidative degradation of dopamine. The final concentration of l-ascorbic acid was less than 100 µM, in which l-ascorbic acid alone did not produce any effects. The drugs were applied using a rapid application system termed the ‘Y-tube method’ (Murase et al., 1990), which enables the external solution surrounding a neurone to exchange within 20 ms.
Data were presented as the mean±standard error of the mean (S.E.M.). For constructing the concentrationresponse curves, the data were fitted to a modified Michaelis-Menten equation by using least-squares fitting: I=(Imax×Cn)/(Cn+EC50n), where I was the druginduced current, Imax was the maximum response, C was the concentration of an agonist, EC50 was the concentration that evokes a half-maximum response and n was the Hill’s coefficient. For constructing concentrationinhibition curves, the data were fitted to the following equation by using least-squares fitting: I=1⫺{Imax×Cn/(Cn+IC50n)}, where I was the current amplitude normalized to the control response without an antagonist, C was the concentration of an antagonist, and IC50 was the concentration for half-inhibition.
2.5. Immunocytochemistry Following electrical recordings, the dishes were filled with a solution of 4% paraformaldehyde dissolved in 0.1 M phosphate-buffered saline (PBS), pH 7.4 for 1 h. Then the neurones were incubated in PBS containing 30% sucrose at 4°C overnight. After being incubated in a solution containing 0.25% Triton X-100 for 30 min, the neurones were placed into PBS containing rabbit antityrosine hydroxylase (TH; 1:2000, Chemicon Inter-
3. Results 3.1. DA responses The SNc neurones could be divided into two groups according to their soma size. One group of neurones exhibits a large soma ranging between 30 µm and 40 µm in diameter. The other group is composed of small neurones (15–20 µm in diameter). Eighteen out of 24 large SNc neurones (75.0%) were shown to be immunopositive for TH staining, while 23 out of 24 small neurones (95.8%) were TH negative. This result indicates that DA neurones are a large size (Fig. 1A and B). Under current-clamp conditions, the large SNc neurones showed spontaneous action potentials at a regular rate ranging from 0.4 to 2.6 Hz (1.2±0.27 Hz, mean±S.E.M., n=7) in the standard extracellular solution ([K +]o; 5 mM). The application of 10⫺6 M DA immediately hyperpolarized the cell membrane associated with a reduction in firing rate (Fig. 1C). However, the action potentials gradually reappeared even in the presence of 10⫺6 M DA (Fig. 1C). After washing out DA, both the membrane potential and firing rates recovered to those before DA application. Under voltage-clamp conditions, 10⫺6 M DA induced a peak followed by a gradual decrease of inward current in 22 out of 24 large neurones examined in a 20 mM K + external solution at a holding poten-
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Fig. 1. Acutely dissociated SNc neurones of the rat. (A) Translucent images of large (a) and small (b) neurones acutely dissociated from the SNc of the rat. (B) Epifluorescent images of neurones presented in A with immunofluorescent staining for tyrosine hydroxylase (TH). Only the large neurone (a), but not the small neurone (b) is TH positive. Scale bar, 50 µm. (C) DA (10⫺6 M) reduced the frequency of spontaneous action potentials recorded using the nystatin-perforated patch clamp recording configuration under current-clamp. Action potentials gradually reappeared in the continuous presence of DA.
tial (VH) of ⫺60 mV (Fig. 2Aa), while only 1 out of 24 small neurones responded to 10⫺6 M DA. For the DA response, there was a statistical difference between the two groups (P⬍0.01, χ2 test). Thus, we chose large SNc neurones (DA neurones) for further experiments.
3.2. DA receptor subtype Extracellular solution containing 20 mM K + and a VH of ⫺60 mV were employed in almost all experiments for convenience to analyze data because of the marked
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Fig. 2. Responses of DA receptor agonists and antagonists. (Aa) Peak amplitude of the inward responses to DA (top) or quinpirole (bottom) increased in a concentration-dependent manner. (Ab) The concentration-response relationships for DA receptor agonists. SKF38393 (up to 10⫺5 M) did not induce any current. (Ba) s(⫺)-sulpiride inhibited the 10⫺6 M DA-induced current in a concentration-dependent manner. The neurones were pretreated with the DA receptor antagonist for 2 min before 10⫺6 M DA. (Bb) The concentration-inhibition relationships for DA antagonists on the IDA. All responses were normalized to the peak current induced by 10⫺6 M DA without antagonists in each neurone. All recordings were performed in external solution containing 20 mM K + at a VH of ⫺60 mV in voltage-clamp conditions. Each point and vertical line shows the mean±S.E.M. of 4–9 neurones.
inward rectification of IDA (Fig. 3). The peak amplitude of the DA response increased in a concentration-dependent manner (Fig. 2A). Quinpirole (LY171555), a D2 receptor agonist, mimicked DA action (Fig. 2Aa). The respective values for the threshold concentration, the EC50 and Hill’s coefficient were 10⫺8 M, 1.55×10⫺7 M and 1.36 for DA (n=9), and 10⫺8 M, 2.35×10⫺7 M and 0.91 for quinpirole (n=9). On the other hand, SKF38393, a D1 receptor agonist, induced no current at a concentration of up to 10⫺5 M in the DA neurones (n=4, Fig. 2Ab). The maximum currents normalized to the peak amplitude of the 10⫺6 M DA response were 1.08 (n=9) for DA and 0.92 (n=9) for quinpirole. S(⫺)-sulpiride, a D2 receptor antagonist, reversibly inhibited 10⫺6 M IDA in a concentration-dependent manner with a IC50 of 4.52×10⫺9 M (n=5, Fig. 2B). SKF83566, a potent D1 receptor antagonist, inhibited 10⫺6 M IDA with an IC50 of 3.53×10⫺6 M (n=5, Fig. 2Bb). These results indicate that IDA is activated mainly by D2 receptors.
3.3. Ionic mechanism of IDA The I–V relationship for IDA showed an inward rectification (Fig. 3). The reversal potentials of the IDA (EDA) estimated from the I–V relationships were ⫺80 mV (n=4) and ⫺43 mV (n=4) in the standard external solution containing 5 mM and 20 mM K +, respectively (Fig. 3B). These values were close to the respective theoretical K + equilibrium potentials (⫺82.7 mV for 5 mM K + and ⫺49.0 mV for 20 mM K +) calculated by the Nernst equation at the given K + concentrations of the external and pipette solutions. These findings indicate that activation of the D2 receptor opens the inward rectifier K + channel in SNc DA neurones. 3.4. Contribution of GTP binding protein to IDA Intracellular perfusion with 0.3 mM GTP using a conventional whole-cell recording mode maintained a constant amplitude of IDA over 30 min (Fig. 4Aa and B). On the other hand, the intracellular application of 0.3
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Fig. 3. Current-voltage (I–V) relationship for the DA response. (A) DA (10⫺6 M) -induced currents at various VHs. All current traces were obtained from the same neurone in external solution containing 20 mM K + under voltage-clamp mode. (B) I–V relationships for DA-induced responses in the external solution with two different K + concentrations (open circle; 5 mM, close circle; 20 mM). All responses were normalized to the peak current induced by 10⫺6 M DA in the external solution containing 20 mM K + at a VH of ⫺40 mV (*). Each point and vertical bar shows the mean±S.E.M. of four neurones. Intracellular K + concentration ([K +]i) was 150 mM.
Fig. 4. Involvement of G protein in DA response. (A) Representative recordings of the IDA after intracellular perfusion with 0.3 mM GTP (a), 0.3 mM GDP-βS (b) or 0.3 mM GTP-γS (c) by using the conventional whole-cell patch recording mode. The horizontal bars indicate the periods of 10⫺6 M DA application. The numbers above the horizontal bars show the time after the rupture of the patch membrane. All recordings were carried out in external solution containing 20 mM K + at a VH of ⫺60 mV. (B) Relative peak amplitudes of 10⫺6 M DA responses with GTP (open circles), GDP-βs (close circles) and IAP (close triangles) as a function of time after rupture of membrane. All responses were normalized to the peak amplitude of the IDA obtained at 1 min after rupture of the membrane in each cell. Each point and vertical bar shows the mean±S.E.M. of five to eight neurones.
mM GDP-βS, a non-hydrolyzable GDP analogue, caused a rapid suppression of the IDA (Fig. 4Ab and B). Intracellular application of 0.3 mM GTP-γS, a nonhydrolyzable GTP analogue, sustained the inward current induced by the first application of 10⫺6 M DA. During this sustaining period, a second application of DA
failed to induce an IDA of maximum amplitude (n=3, Fig. 4Ac). IAP (1 µg/ml) applied intracellularly gradually decreased the peak amplitude of IDA (Fig. 4B). These results suggest that an IAP-sensitive GTP binding protein (G protein) may be involved in the IDA in SNc DA neurones.
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It is well known that activation of the D2 receptor regulates intracellular cAMP production (Bates et al., 1991; Liu et al., 1992; Johansson and Westlind-Danielsson, 1994; Kim et al., 1995; Watts and Neve, 1996). In the present study, however, the DA response persisted in the presence of forskolin (10⫺5 M), an adenylyl cyclase activator, and IBMX (10⫺6 M), a phosphodiesterase inhibitor (105.9±4.9% peak amplitude of IDA compared with that without these modulators, P⬎0.1; paired t-test, n=5). H-89 (10⫺6 M), a protein kinase A (PKA) inhibitor, did not block the DA response (87.5±11.1% of control, P⬎0.1, n=4). Pretreatment with chelerythrine (3×10⫺7 M), a protein kinase C (PKC) inhibitor, for 3 min before DA application did not block the IDA (99.7±6.2% of control, P⬎0.1, n=4). These results indicate that IDA is mediated by an IAP-sensitive G protein, but is independent of PKA and PKC pathways. 3.5. DA-induced single channel current In the inside-out patch recording configuration, 10⫺7 M DA in the patch pipette and 0.3 mM GTP applied to the cytoplasmic side increased single channel activities at a membrane potential of ⫺70 mV (Fig. 5Aa, n=7).
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Replacement of GTP with 0.3 mM GDP on the cytoplasmic side markedly decreased the DA-induced single channel activities (Fig. 5Ab, n=7). The open probabilities of the patch (Npo) were 0.0021±0.0004 and 0.0004±0.0002 with the bath solution containing 0.3 mM GTP and 0.3 mM GDP, respectively. The respective values for open time and close time were 0.372 ms±0.063 ms and 78.7 ms±7.2 ms for GTP, and 0.285 ms±0.02 ms and 218 ms±36 ms for GDP. There is a statistically significant difference in the close time between the two groups (P⬍0.01, paired t-test). Respective mean amplitudes for the first and second peaks were 2.93 pA and 6.98 pA for GTP and 3.02 pA and 7.18 pA for GDP, indicating no differences in the mean amplitude of DA-induced single channel current between GTP and GDP treatment (Fig. 5Ba and b). In addition, I–V relationships revealed that there was a remarkable DAinduced inward rectification even at the single channel current level and that the maximal single channel conductance of the DA-induced inward current was 56.6 pS at a VH between ⫺100 and +10 mV (Fig. 6).
Fig. 5. Effects of GDP and GTP on DA-operated single channel activity. The inside-out patch recording mode was employed to record the DAinduced single channel currents. (A) Current traces with 0.3 mM GTP (a) and 0.3mM GDP (b) applied to the cytosolic side solution. The horizontal thin and broken lines show the close (c) and open (o) levels, respectively. The patch pipette contained 10⫺7 M DA. The membrane potential (Vm) was ⫺70 mV. (B) The amplitude histograms of the currents induced by 10⫺7 M DA with 0.3 mM GTP (a) or 0.3mM GDP (b). The amplitude histograms were fitted by a Gaussian curve with a peak value of 2.93 pA (a), and 3.02 pA (b). Sampling time of single channel events was two minutes.
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Fig. 6. I–V relationship for DA-induced single channel current. (A) The DA-induced single channel current obtained at various Vms. The patch pipette contained 10⫺7 M DA. The horizontal thin and broken lines indicate the close and the open levels, respectively. (B) The I–V relationship for the DA-induced single channel currents. Each point and vertical bar shows the mean±S.E.M. of three neurones. Almost all vertical bars are small enough to be masked within the solid circles.
4. Discussion
4.2. DA receptor subtype
4.1. Classification of SNc neurones
Recent molecular biological techniques have revealed five cloned subtypes of dopamine receptors; D1, D2, D3, D4 and D5 (Bunzow et al., 1988; Zhou et al., 1990; Sokoloff et al., 1990; Van Tol et al., 1991; Sunahara et al., 1991; Sibley and Monsma, 1992). Structural and pharmacological analyses led to the division of these receptors into two classes: the D1- and D2-like receptors. The D1-like subfamily consists of D1 and D5 receptors, while the D2-like family consists of D2L and D2S isoforms, as well as D3 and D4 receptors (Sibley and Monsma, 1992). In both slices and culture, the hyperpolarization produced by DA was mimicked by a D2 receptor agonist quinpirole (Lacey et al., 1987; Kim et al., 1995). Also in this study using acutely dissociated neurones, quinpirole mimicked DA, but the maximal amplitude of the quinpirole-induced current in the DA neurones was only 85% of that induced by DA (Fig. 2Ab). This result suggests two possibilities; 1) a partial, not full, agonistic action of quinpirole on the D2 receptor in SNc neurones, 2) an additional involvement of a D3 type receptor in the DA response. However, clear discrimination between the D2like receptors is difficult because selective agonists and antagonists for D2 and D3 receptors are not yet available. Moreover, the differences in Kd and Ki among most
The present study demonstrated that DA acted on a D2-like receptor and activated an inward rectifier K + channel in large neurones acutely dissociated from the rat SNc. On the other hand, DA did not induce any current in small SNc neurones. In addition, large neurones showed spontaneous action potentials at a regular rate ranging from 0.4 to 2.6 Hz. This is compatible with a previous report that DA neurones of the SNc fired spontaneous action potentials in the range of 1–8 Hz in a brain slice preparation (Lacey et al., 1989). Of cultured (Kim et al., 1997) and acutely dissociated SNc neurones (present study), 57% and 75% stained positive for TH, respectively. Both DA neurones and non-DA neurones have been reported to be of a similar size in cultured SNc neurones (Kim et al., 1997). On the other hand, DA neurones are estimated to be larger in size than non-DA neurones with a mean conductance of 22 nS and 4 nS, respectively, in the slice preparation (Lacey et al., 1989). This is in accord with the present result showing that DA neurones were larger in soma size than non-DA neurones (Fig. 1 and Results). Some morphological characteristics such as size might change in culture.
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agonists and antagonists of D2-like receptors are within one order of magnitude between D2 and D3 receptors (Burris et al., 1995). On the other hand, SKF38393, a D1 agonist, did not induce any current in the DA neurones (Fig. 2Ab). In addition, a D2 antagonist was more effective on the DA-induced current than the D1 antagonist (Fig. 2Bb). These pharmacological studies indicate that IDA is mediated by a D2-like receptor but not D1like receptor activation in SNc DA neurones. 4.3. DA receptor coupled with K+ channel K + channels which are characterized by an inward rectification of the current can be functionally classified into two groups. One is the inward rectifier K + (IRK) channel family. The activity of IRK channels is not modulated by G proteins, and the open time of the channel is 10–200 ms (Kubo et al., 1993a). The other is the G protein-gated inward rectifier K + (GIRK) channel family. The channel activities of this family are modulated by G proteins, and the channel open time is 0.5– 2 ms (Kubo et al., 1993b; Grigg et al., 1996). The present study showed that the DA-induced inward rectifier K + current in the DA neurones had characteristics similar to those of the GIRK family, because of the involvement of a G protein in the channel activation and of the similar open time, approximately 0.3 ms. The GIRK channels have been reported to conduct K + at or near the resting membrane potential and to play an important role in controlling cell excitability (Wickman and Clapham, 1995). The mRNAs of GIRK1, GIRK2, GIRK3 and GIRK4 (mGIRK1, mGIRK2, mGIRK3 and mGIRK4, respectively) have been cloned (Kubo et al., 1993b; Lesage et al., 1994; Duprat et al., 1995). Northern blot analysis has revealed their distributions: mGIRK4 is restricted to the heart; mGIRK2 and mGIRK3 transcripts are specifically present in the brain; mGIRK1 is expressed in both heart and brain (Lesage et al., 1995). Single channel analyses of K + currents mediated by cloned GIRK1, GIRK2, GIRK1 and 2, and GIRK2 and 4 in an expression system demonstrate that there is only one population of channels with very similar properties to each other, characterized by a unitary conductance of about 40 pS and a mean open time duration of less than 1 ms. On the other hand, native GIRK channels recorded in different neuronal cell types have been reported to possess other properties of unitary conductance varying from 38 to 55 pS and an open time of the order of 2 ms (Lesage et al., 1995). Our results indicating that the unitary conductance of DA-operated channels was 56.6 pS in SNc neurones corresponded with Lesage’s report (1995). An immunohistochemical approach has revealed that mGIRK1, mGIRK2 and their expressed proteins exist in both soma and dendrites of dopaminergic neurones in the SNc (Liao et al., 1996). However it should be a target for further study to elucidate which subunits
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of the GIRK channels form functional channels in the DA neurones of rat SNc. 4.4. Possible pathway between DA receptor and K+ channel In freshly dissociated SNc neurones, the intracellular application of GDP-βS, the non-hydrolyzable analogue of GDP which competitively inhibits the binding of GTP to the α-subunit of G protein, inhibited the DA-induced K + current (Fig. 4Ab and B). On the other hand, an intracellular application of GTP-γS, the non-hydrolyzable analogue of GTP, which activates the G protein persistently, induced an irreversible modulation of IDA (Fig. 4Ac). These results indicate that the DA response in SNc neurones is mediated by a G protein. Additional support for the involvement of a G protein in IDA was revealed by the open probabilities in the inside-out patch recording combined with cytoplasmic GDP-GTP exchange (Fig. 5). It is controversial whether the G protein participating in the DA action on SNc neurones is IAP-sensitive or insensitive. In rats treated with an injection of IAP into the SN regions, the reduction of spike frequency by DA agonists disappears (Innis and Aghajanian, 1987). In cultured SNc neurones of rats, the DA agonist-induced response is completely abolished by IAP pretreatment (Kim et al., 1995). On the other hand, the hyperpolarization induced by DA in SN neurones is not affected by the intracerebroventricular injection of IAP (Lacey et al., 1988). In the present study, the intracellular application of IAP eliminated DA-induced K + currents in DA neurones (Fig. 4B). This result indicates that IDA is mediated by IAP-sensitive G proteins, i.e., Gi and Go, in the DA neurones of the rat SNc. It is well known that G proteins are coupled to effective ion channels through the activation of various intracellular second messengers. DA reduces the spontaneous spike frequency of DA neurones in the SNc by altering cAMP production (Shi and Bunney, 1992). Kim et al. (1995) reported that the quinpirole-induced response was larger in cAMP-loaded neurones compared with controls, but that cAMP did not have any effect on the K + conductance. The present study showed that the application of forskolin, a direct activator of adenylyl cyclase, and IBMX, an inhibitor of phosphodiesterase, had no effect on the IDA. H-89, an inhibitor of PKA, and chelerythrine, an inhibitor of PKC, did not affect the IDA. In addition, DA could elicit currents even in the inside-out patch recording mode, in which soluble second messengers and other cytoplasmic factors are presumably lost and/or diluted. These results suggest that the activation of the D2-like receptor opens K + channels through an IAP-sensitive G protein and that the coupling between the G protein and K + channel seems to be ‘direct’, rather than mediated by intracellular soluble second messengers in the SNc DA neurones.
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4.5. DA receptor and neuronal function The dopaminergic neurones in the SNc exhibit DA receptors in the somatodendritic region and axon terminal (Cheramy et al., 1981). Activation of autoreceptors in the somatodendritic region inhibits the spontaneous activities of dopaminergic neurones (Groves et al., 1975). Somatodendritic DA release evoked in the SNc is reduced by D2 receptor agonists and enhanced significantly by D2 receptor antagonists during appropriate electrical stimulation (Cragg and Greenfield, 1997). Activation of the autoreceptors located at the axon terminals inhibits DA synthesis and DA release (Starke et al., 1989). Because we employed dissociated SNc neurones which retained only their soma and proximal dendrites in this study, somatodendritic autoreceptors might be involved in the IDA. In addition, our results that the inhibition of spontaneous activity by DA is probably mediated by a direct coupling between G proteins and K + channels suggest a possible role of IDA in the rapid onset-offset negative feedback regulation of neuronal excitability in SNc DA neurones. Acknowledgements We would like to give our appreciation to Ms. Min Li for critical reading of the manuscript. This work was supported by Grant-in-Aids for Scientific Research (1167044 to J.N. and 10044301 and 10470009 to N.A.) and by Grant-in-Aids for Scientific Research on priority area (11170240) to J.N. from the Ministry of Education, Science and Culture, Japan. References Akaike, N., Harata, N., 1994. Nystatin perforated patch recording and its applications to analyses of intracellular mechanisms. Japanese Journal of Physiology 44, 433–473. Bates, M.D., Senogles, S.E., Bunzow, J.R., Liggett, S.B., Civelli, O., Caron, M.G., 1991. Regulation of responsiveness at D2 dopamine receptors by receptor desensitization and adenylyl cyclase sensitization. Molecular Pharmacology 39, 55–63. Bunney, B.S., Walters, J.R., Roth, R.H., Aghajanian, G.K., 1973. Dopaminergic neurons: effect of antipsychotic drugs and amphetamine on single cell activity. Journal of Pharmacology and Experimental Therapeutics 185, 560–571. Bunzow, J.R., Van Tol, H.H., Grandy, D.K., Albert, P., Salon, J., Christie, M., Machida, C.A., Neve, K.A., Civelli, O., 1988. Cloning and expression of a rat D2 dopamine receptor cDNA. Nature 336, 783–787. Burris, K.D., Pacheco, M.A., Filtz, T.M., Kung, M.P., Kung, H.F., Molinoff, P.B., 1995. Lack of discrimination by agonists for D2 and D3 dopamine receptors. Neuropsychopharmacology 12, 335–345. Castro, S.W., Strange, P.G., 1993. Coupling of D2 and D3 dopamine receptors to G-proteins. Federation of European Biochemical Societies 315, 223–226. Cheramy, A., Leviel, V., Glowinski, J., 1981. Dendritic release of dopamine in the substantia nigra. Nature 289, 537–542.
Cragg, S.J., Greenfield, S.A., 1997. Differential autoreceptor control of somatodendritic and axon terminal dopamine release in substantia nigra, ventral tegmental area, and striatum. Journal of Neuroscience 17, 5738–5746. Duprat, F., Lesage, F., Guillemare, E., Fink, M., Hugnot, J.P., Bigay, J., Lazdunski, M., Romey, G., Barhanin, J., 1995. Heterologous multimeric assembly is essential for K + channel activity of neuronal and cardiac G-protein-activated inward rectifiers. Biochemical and Biophysical Research Communications 212, 657–663. Friedrich, F., Paulmichl, M., Kolb, H.A., Lang, F., 1988. Inward rectifier K channels in renal epithelioid cells (MDCK) activated by serotonin. Journal of Membrane Biology 106, 149–155. Grigg, J.J., Kozasa, T., Nakajima, Y., Nakajima, S., 1996. Single-channel properties of a G-protein-coupled inward rectifier potassium channel in brain neurons. Journal of Neurophysiology 75, 318–328. Groves, P.M., Wilson, C.J., Young, S.J., Rebec, G.V., 1975. Self-inhibition by dopaminergic neurons. Science 190, 522–529. Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J., 1981. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflu¨gers Archives 391, 85–100. Innis, R.B., Aghajanian, G.K., 1987. Pertussis toxin blocks autoreceptor-mediated inhibition of dopaminergic neurons in rat substantia nigra. Brain Research 411, 139–143. Inomata, H., Nabekura, J., Akaike, N., 1993. Suppression of taurine response in acutely dissociated substantia nigra neurons by intracellular cyclic AMP. Brain Research 615, 347–350. Johansson, M.H., Westlind-Danielsson, A., 1994. Forskolin-induced up-regulation and functional supersensitivity of dopamine D2 long receptors expressed by Ltk- cells. European Journal of Pharmacology 14 (269), 149–155. Kim, K.M., Nakajima, Y., Nakajima, S., 1995. G protein-coupled inward rectifier modulated by dopamine agonists in cultured substantia nigra neurons. Neuroscience 69, 1145–1158. Kim, K.M., Nakajima, S., Nakajima, Y., 1997. Dopamine and GABA receptors in cultured substantia nigra neurons: correlation of electrophysiology and immunocytochemistry. Neuroscience 78, 759– 769. Kubo, Y., Baldwin, T.J., Jan, Y.N., Jan, L.Y., 1993a. Primary structure and functional expression of a mouse inward rectifier potassium channel. Nature 362, 127–133. Kubo, Y., Reuveny, E., Slesinger, P.A., Jan, Y.N., Jan, L.Y., 1993b. Primary structure and functional expression of a rat G-proteincoupled muscarinic potassium channel. Nature 364, 802–806. Lacey, M.G., Mercuri, N.B., North, R.A., 1987. Dopamine acts on D2 receptors to increase potassium conductance in neurones of the rat substantia nigra zona compacta. Journal of Physiology 392, 397– 416. Lacey, M.G., Mercuri, N.B., North, R.A., 1988. On the potassium conductance increase activated by GABAB and dopamine D2 receptors in rat substantia nigra neurones. Journal of Physiology 401, 437– 453. Lacey, M.G., Mercuri, N.B., North, R.A., 1989. Two cell types in rat substantia nigra zona compacta distinguished by membrane properties and the actions of dopamine and opioids. Journal of Neuroscience 9, 1233–1241. Lesage, F., Duprat, F., Fink, M., Guillemare, E., Coppola, T., Lazdunski, M., Hugnot, J.P., 1994. Cloning provides evidence for a family of inward rectifier and G-protein coupled K + channels in the brain. Federation of European Biochemical Societies Letters 353, 37–42. Lesage, F., Guillemare, E., Fink, M., Duprat, F., Heurteaux, C., Fosset, M., Romey, G., Barhanin, J., Lazdunski, M., 1995. Molecular properties of neuronal G-protein-activated inwardly rectifying K + channels. Journal of Biological Chemistry 270, 28660–28667. Liao, Y.J., Jan, Y.N., Jan, L.Y., 1996. Heteromultimerization of Gprotein-gated inwardly rectifying K + channel proteins GIRK1 and
S. Uchida et al. / Neuropharmacology 39 (2000) 191–201
GIRK2 and their altered expression in weaver brain. Journal of Neuroscience 16, 7137–7150. Liu, Y.F., Civelli, O., Zhou, Q.Y., Albert, P.R., 1992. Cholera toxinsensitive 3’,5’-cyclic adenosine monophosphate and calcium signals of the human dopamine-D1 receptor: selective potentiation by protein kinase A. Molecular Endocrinology 6, 1815–1824. Mercuri, N.B., Calabresi, P., Bernardi, G., 1992. The electrophysiological actions of dopamine and dopaminergic drugs on neurons of the substantia nigra pars compacta and ventral tegmental area. Life Sciences 51, 711–718. Mercuri, N.B., Saiardi, A., Bonci, A., Picetti, R., Calabresi, P., Bernardi, G., Borrelli, E., 1997. Loss of autoreceptor function in dopaminergic neurons from dopamine D2 receptor deficient mice. Neuroscience 79, 323–327. Murase, K., Randic, M., Shirasaki, T., Nakagawa, T., Akaike, N., 1990. Serotonin suppresses N-methyl-d-asparatate responses in acutely isolated spinal dorsal horn neurons of the rat. Brain Research 525, 84–91. Nabekura, J., Omura, T., Akaike, N., 1996. Alpha 2 adrenoceptor potentiates glycine receptor-mediated taurine response through protein kinase A in rat substantia nigra neurons. Journal of Neurophysiology 76, 2447–2454. Nauta, H.J., Cole, M., 1974. Efferent projections of the subthalamic nucleus. Transactions of the American Neurological Association 99, 170–173. Shi, W.X., Bunney, B.S., 1992. Roles of intracellular cAMP and protein kinase A in the actions of dopamine and neurotensin on midbrain dopamine neurons. Journal of Neuroscience 12, 2433–2438.
201
Sibley, D.R., Monsma, F.J. Jr., 1992. Molecular biology of dopamine receptors. Trends in Pharmacological Science 13, 61–69. Sokoloff, P., Giros, B., Martres, M.P., Bouthenet, M.L., Schwartz, J.C., 1990. Molecular cloning and characterization of a novel dopamine receptor (D3) as a target for neuroleptics. Nature 347, 146–151. Starke, K., Gothert, M., Kilbinger, H., 1989. Modulation of neurotransmitter release by presynaptic autoreceptors. Physiological Reviews 69, 864–989. Sunahara, R.K., Guan, H.C., O’Dowd, B.F., Seeman, P., Laurier, L.G., Ng, G., George, S.R., Torchia, J., Van Tol, H.H., Niznik, H.B., 1991. Cloning of the gene for a human dopamine D5 receptor with higher affinity for dopamine than D1. Nature 350, 614–619. Vallar, L., Meldolesi, J., 1989. Mechanisms of signal transduction at the dopamine D2 receptor. Trends in Pharmacological Science 10, 74–77. Van Tol, H.H., Bunzow, J.R., Guan, H.C., Sunahara, R.K., Seeman, P., Niznik, H.B., Civelli, O., 1991. Cloning of the gene for a human dopamine D4 receptor with high affinity for the antipsychotic clozapine. Nature 350, 610–614. Watts, V.J., Neve, K.A., 1996. Sensitization of endogenous and recombinant adenylate cyclase by activation of D2 dopamine receptors. Molecular of Pharmacology 50, 966–976. Wickman, K., Clapham, D.E., 1995. Ion channel regulation by G proteins. Physiological Reviews 75, 865–885. Zhou, Q.Y., Grandy, D.K., Thambi, L., Kushner, J.A., Van Tol, H.H., Cone, R., Pribnow, D., Salon, J., Bunzow, J.R., Civelli, O., 1990. Cloning and expression of human and rat D1 dopamine receptors. Nature 347, 76–80.