Group I mGluRs Coupled to G Proteins Are Regulated by Tyrosine Kinase in Dopamine Neurons of the Rat Midbrain

Alessandro Tozzi,1 Ezia Guatteo,1 Luigi Caputi,2 Giorgio Bernardi,1,2 and Nicola B. Mercuri1,2

 1Istituto di Ricovero e Cura a Carattere Scientifico Fondazione S. Lucia, 00179 Rome; and  2Clinica Neurologica, Università di Tor Vergata, 00133 Rome, Italy


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tozzi, Alessandro, Ezia Guatteo, Luigi Caputi, Giorgio Bernardi, and Nicola B. Mercuri. Group I mGluRs Coupled to G Proteins Are Regulated by Tyrosine Kinase in Dopamine Neurons of the Rat Midbrain. J. Neurophysiol. 85: 2490-2497, 2001. Metabotropic glutamate receptors (mGluRs) modulate neuronal function via different transduction mechanisms that are either dependent or independent on G-protein function. Here we investigated, using whole cell patch-clamp recordings in combination with fluorimetric measurements of intracellular calcium concentration ([Ca2+]i), the metabolic pathways involved in the responses induced by group I mGluRs in dopamine neurons of the rat midbrain. The inward current and the [Ca2+]i increase caused by the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG, 100 µM) were permanently activated and subsequently abolished in cells loaded with the nonhydrolizable GTP-analogue GTP-gamma -S (600 µM). In addition, when GDP-beta -S (600 µM) was dialyzed into the cells to produce the blockade of the G proteins, the DHPG-dependent responses were reduced. When the tissue was bathed with the phospholipase C inhibitor 1-[6[[(17beta )-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]exyl]-1H-pyrrole-2,5-dione (10 µM), the DHPG-induced calcium transients slightly diminished but the associated inward currents were not affected. Interestingly, a substantial depression of the DHPG-induced inward current and transient increase of [Ca2+]i was caused by the protein tyrosine kinase inhibitors tyrphostin B52 (40 µM) and 4',5,7-trihydroxyisoflavone (genistein; 40 µM), whereas genistein's inactive analogue 4',5,7-trihydroxyisoflavone-7-glucoside (40 µM) was ineffective. The blockade of the Src family of tyrosine kinase by 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (20 µM), mitogen-activated protein kinase by 2'-amino-3' methoxyflavone (50 µM), and protein kinase C by staurosporine (1 µM) had no effect on the cellular responses caused by DHPG. The mGluR5-selective antagonist 2-methyl-6-(phenylethynyl)-pyridine (10-100 µM) did not affect the actions of DHPG. Thus our results indicate that the responses, mainly mediated by mGluRs1 in dopamine neurons, are activated by intracellular mechanisms coupled to G proteins and regulated by tyrosine kinases.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The activation of metabotropic glutamate receptors (mGluRs) in the CNS modulates second-messenger pathways and, in turn, influences cellular excitability (Anwyl 1999; Conn and Pin 1997; Pin and Douvoisin 1995; Schoepp and Conn 1993). Eight mGluRs have been identified so far. These have been subdivided into three groups. The group I (mGluR1 and mGluR5) activates phospholipase C (PLC), while groups II (mGluR2 and mGluR3) and III (mGluR4, mGluR6, mGluR7, and mGluR8) are negatively coupled to cyclic AMP (Gomeza et al. 1996; Pin and Douvoisin 1995; Watkins and Collingridge 1994). With regard to the group I mGluRs, they are believed to couple to GTP-binding proteins (G proteins) and, by stimulating phosphoinositide hydrolysis, to mobilize calcium from intracellular stores and depolarize neurons (Bockaert et al. 1993; Congar et al. 1997; Lee and Boden 1997; Pin et al. 1994). Moreover, it has been suggested that the group I mGluRs could use other intracellular systems, which do not require G proteins, to excite different types of central neurons (Guerineau et al. 1995; Heuss et al. 1999; Tempia et al. 1998; Zheng et al. 1995).

An alteration of the functional interbalance between glutamate and dopamine in the basal ganglia has been implicated in motor and psychiatric disorders (Albin et al. 1989; Carlsson and Carlsson 1990; Davis et al. 1991). Therefore the activity of mGluRs in the ventral midbrain are critically involved in the expression of motor behaviors and sensitization to amphetamine by influencing the dopaminergic output in the terminal fields (Kim and Vezina 1998; Swanson and Kalivas 2000; Vezina and Kim 1999). Metabotropic glutamate receptors are found on dopamine neurons (Shigemoto et al. 1992; Testa et al. 1994) where they provide vital important support for growth (Plenz and Kitaj 1998), mediate presynaptic and postsynaptic effects (Bonci et al. 1997), and control neuronal discharge (Meltzer et al. 1997; Mercuri et al. 1993). In addition, the stimulation of excitatory fibers within the ventral midbrain induces a slow metabotropic depolarizing and/or hyperpolarizing potential in dopamine cells that is mediated by group I mGluRs (Fiorillo and Williams 1998; Shen and Johnson 1997). In line with these results, we have previously demonstrated that mGluR I agonists produce a sodium-dependent inward current and promote release of calcium from intracellular stores in these neurons (Guatteo et al. 1999; Mercuri et al. 1993). Although we have hypothesized that these two effects are not interdependent (Guatteo et al. 1999), their molecular mechanisms are not completely known yet. Therefore the purpose of the present study is to investigate the transduction processes mediating the cellular responses caused by the activation of group I mGluRs in rat dopamine neurons maintained in vitro in a slice preparation, using the whole cell patch-clamp recordings in combination with fluorimetric measurements of [Ca2+]i. Thus our principal goals are to assess if G proteins are involved in the effects of the activation of mGluRs I, if PLC activation is required for the membrane current production and calcium release from the internal stores, and if protein tyrosine kinase (PTK) activation is an important factor in the intracellular pathways leading to the expression of the (S)-3,5-dihydroxyphenylglycine (DHPG)-induced responses.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue preparation

The preparation of the midbrain tissue was performed as described previously (Mercuri et al. 1995). Albino Wistar rats of either sex (150-200 g) were anesthetized with ketamine and killed. All experiments were carried out according to guidelines of the Comitato Etico of the Tor Vergata University on the use of animals in research. The brain was rapidly removed, and horizontal slices (thickness, 250 µm) were cut in 10-14°C physiological saline using a vibratome and starting from the ventral surface of the midbrain. The artificial cerebrospinal fluid solution (ACSF) contained (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, 10 glucose, and 18 NaHCO3; gassed with 95% O2-5% CO2. After incubation in a reservoir (1-2 h, 34°C), a single slice was transferred into a recording chamber and completely submerged in ACSF with continuously flowing (2.5 ml/min) solution at 33-34°C (pH 7.4). The chamber was mounted on the stage of an upright microscope Axioskop (Carl Zeiss, Oberkochen, Germany) equipped for infrared video microscopy (Hamamatsu, Hamamatsu City, Japan) and video microfluorimetry (ImproVision, Coventry, UK).

Electrophysiology

Whole cell patch-clamp recordings were obtained with an amplifier (Axopatch 1D, Axon Instruments, Foster City, CA) from visually identified dopamine neurons (Guatteo et al. 1999) using pipettes made from 1.5-mm borosilicate glass (WPI, Sarasota, FL) and pulled with a PP 83 Narishige puller (Tokyo). The resistance of the patch pipette was around 4 MOmega when filled with a standard solution containing (in mM) 145 K+-gluconate, 0.1 CaCl2, 2 MgCl2, 10 HEPES, 0.75 EGTA, 2 Mg-ATP, and 0.3 Na3GTP (pH 7.35). In a subset of experiments, Na3GTP (GTP) was substituted with a nonhydrolizable analogue GTP-gamma -S trilithium salt (GTP-gamma -S) or with GDP-beta -S trilithium salt (GDP-beta -S, both 0.6 mM). For microfluorimetry (see following text), 125 µM calcium green-1 exapotassium salt (Molecular Probes, Leiden, The Netherlands) was added to the pipette solution. Series resistance was partially compensated using the amplifier. The membrane voltage and current were digitized at 5 kHz through a Digidata 1200B A/D converter acquired and analyzed using pClamp and Axoscope software (Axon Instruments).

Microfluorimetry

The fluorescent ion indicator calcium green-1 was loaded into the cell via the patch pipette and excited via a ×40 water-immersion objective (Olympus, Hamburg, Germany) by epi-illumination with light provided by a 75-W Xenon lamp. Excitation light was band-pass filtered at 480 nm, and emission light passed a barrier filter of 500 nm and was detected by a CCD camera (Photonic Science, Millham, UK). A set of images was acquired at 6- or 12-s interval. Time courses of fluorescence values were calculated over regions of the neuron that included the cell body ("regions of interest," defined as those pixels that exhibit at least 20-30% of maximal specific fluorescence) and were corrected for background fluorescence measured from image regions free of dye fluorescence (IonVision software, ImproVision, Coventry, UK). The fluorescent signal was relatively homogenous over the cell body and initial portion of the proximal dendrites; thus the analysis was restricted to this part of the neuron. Recordings of changes of [Ca2+]i are expressed as percentage of (Ft - F0)/F0 values (Delta F/F%), where Ft is the emitted fluorescence at time t, and F0 is the emitted fluorescence at time 0 of the cell excited at 480-nm wavelength. Data analysis was done using Origin 4.1 software (Microcal, Northampton, MA).

Statistical analysis

Values in the text are expressed as means ± SE. Student's t-test or one-way/two-way ANOVA test for repeated measures, followed by the Tukey test for post hoc analysis when differences were significant were used. Significance levels of 0.01-0.05 (*) or 0.001 (**) were considered. Traces show the most representative example within a set of experiments. Bars in the plots represent averaged responses (calcium transients and currents) obtained from each single experiment and under each condition.

Drug application

DHPG, 7(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate (CPCCOEt), 2-methyl-6-(phenylethynyl)-pyridine (MPEP), 4',5,7-trihydroxyisoflavone (Genistein), and N-(2-phenylethyl)-3,4-dihydroxybenzyldenecyanoacetamide (Tyrphostin B52) were obtained from Tocris Cookson (Bristol, UK); 2'-amino-3' methoxyflavone (PD 98059) was obtained from Calbiochem (Milan, Italy). 1-[6[[(17beta )-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]exyl]-1H-pyrrole-2,5-dione (U73122) was from RBI (Milan, Italy). 4',5,7-Trihydroxyisoflavone-7-glucoside (Genistin), staurosporine and dopamine were from Sigma (Milan, Italy). 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]- pyrimidine (PP1) was from Alexis (Postfach, Switzerland).

CPCCOEt, PD98052, genistein, genistin, tyrphostin B52, U73122, PP1, and staurosporine were dissolved in DMSO just before a set of experiments, and the final concentration of each compound included DMSO 1:1000 diluted. DHPG was applied via a glass pipette, which was positioned close to the cell body of the patched neuron and connected to a pneumatic pico-pump set at 10-20 psi, 2 s (Picospritzer, WPI, Sarasota, FL). The other drugs were bath-applied by switching the solution to one containing known concentrations of drugs. An exchange of the solution in the recording chamber occurred in about 1 min.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Properties of the dopamine cells

Patch-clamp whole cell recordings were performed on 76 visually and electrophysiologically identified "principal" dopamine neurons in the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA). These cells were identified by their fusiform cell body and long unbranched proximal dendrites extending in the plane of the slice (Grace and Onn 1989). In current-clamp mode they fired spontaneously at 1.6 ± 0.2 Hz (n = 5). They showed a hyperpolarizing/outward response to dopamine (30 µM) and a pronounced hyperpolarization-activated inward current, Ih (Grace and Onn 1989; Johnson and North 1992; Lacey et al. 1989; Mercuri et al. 1995; Yung et al. 1991). When held at -60 mV, the average holding current was -58 ± 12 pA, n = 8. In general, no electrophysiological differences were found in neurons of the SNc (n = 46) and VTA (n = 30), and those cells responded to the mGluR agonist in the same way.

DHPG-induced responses on dopaminergic neurons

The specific mGluR group-I agonist DHPG (Conn and Pin 1997; Schoepp et al. 1994) was locally applied via a glass pipette placed close to the soma of neurons voltage-clamped at -60 mV (Guatteo et al. 1999). Figure 1B represents a dopamine neuron in the whole-cell configuration. Repeated puffs of DHPG (100 µM, 20 psi, 2 s) induced a transient inward current associated with a rapid increase in [Ca2+]i (Fig. 1C). Mean amplitudes were 249 ± 17 pA (n = 37) for inward currents (Fig. 2B, bottom) and 70 ± 3% Delta F/F (n = 37) for calcium transients (Fig. 2B, top).



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Fig. 1. Metabotropic glutamate receptor (mGluR)1-mediated intracellular calcium and current responses in dopamine neurons. A: hyperpolarizing voltage commands from a VH of -60 to -120 mV (bottom) activated a voltage- and time-dependent Ih (top) in the dopaminergic neuron shown in B. B: infrared image of a patch-clamped dopamine neuron loaded with the indicator calcium green-1. RE, recording electrode; PP, puff pipette (- - -); ROI, region of interest in which the fluorescence intensity was measured. C: simultaneous [Ca2+]i (top) and current (bottom) recordings from the cell shown in A and B. The repeated pressure ejections of (S)-3,5-dihydroxyphenylglycine (DHPG; 100 µM, 2 s, 2- to 4-min interval), elicited inward currents associated with transient elevations of [Ca2+]i.



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Fig. 2. Effects of intracellular dialysis with the GTP-analogue, GTP-gamma -S, on the DHPG-induced responses. A: the 1st application of DHPG evokes an inward current (bottom) and increases in [Ca2+]i (top), but both responses do not fully recover. The following applications of DHPG progressively reduce membrane currents but do not produce elevations in [Ca2+]i. In spite of this, an intracellular calcium increase is still observed when the cell is depolarized by a voltage step from -60 to -30 mV (inset). B: the plots show mean values and SEs of the DHPG-induced [Ca2+]i (top) and current (bottom) signals at different minutes from the beginning of the whole cell recording, in control condition (intracellular solution containing GTP, n = 37) and during the intracellular dialysis with GTP-gamma -S (600 µM). Note that the DHPG-induced calcium rise in GTP-gamma -S is already reduced at 2 min (**P < 0.001, n = 11) compared with controls. The DHPG-induced currents at 2 min are not significantly different from controls (P = 0.99, n = 12). Both calcium and current transients at 4 and 6 min are significantly reduced compared with the 2-min application (**P < 0.001).

The current reached a peak in 1.9 ± 0.2 s and decay time constant (tau ) was 2.4 ± 0.5 s (n = 3). The [Ca2+]i reached a peak in 5.7 ± 0.3 s and recovered with a decay time constant of 12.4 ± 0.8 s (n = 3). Both decay time constants (tau ) were obtained by fitting the traces with a monoexponential function. To avoid desensitization (Guatteo et al. 1999), the cellular responses to the agonist were usually evoked every 2 min and remained constant up to 30 min from the onset of the recording.

Cellular responses to DHPG are G-protein dependent

To examine whether signaling pathways linked to activation of group I mGluR involve G proteins (Conn and Pin 1997), we exchanged the intracellular solution containing GTP with a solution containing GTP-gamma -S (600 µM), a nonhydrolysable analogue of GTP that keeps G proteins in a long-term activated state (Lacey et al. 1988). At the first DHPG puff, 2-6 min after the establishment of whole cell configuration, a transient increase in [Ca2+]i coupled to an inward current occurred. However, at the following puffs, the corresponding current and calcium rise did not fully return to the baseline, leading to a progressive increase in the holding current and [Ca2+]i. Figure 2A shows that 6 min after the onset of recordings (3rd DHPG puff) was sufficient to completely abolish both DHPG-induced signals. Indeed, mean values for calcium transients at 4 and 6 min (2nd and 3rd puff) were reduced to 3 ± 1% Delta F/F and to 1 ± 0.1% Delta F/F, n = 11, **P < 0.001. Mean values for inward currents at 4 and 6 min were reduced to 48 ± 8 pA and to 18 ± 7 pA, n = 12, **P < 0.001.

Under these conditions, a depolarizing pulse (30 mV, 20 s) applied to the membrane of the recorded cell was still able to cause a [Ca2+]i increase, suggesting that the calcium influx from the extracellular space was not affected. When GTP-gamma -S was dialyzed, the first DHPG puff application (at 2 min) evoked a smaller calcium transient (% Delta F/F = 30 ± 10, n = 11) respect to the puff at 2 min in control cells (% Delta F/F = 70 ± 3, n = 37; ANOVA, Tukey test, **P < 0.001), whereas no significant changes of the current amplitude were measured at this time point (Fig. 2B, bottom, 249 ± 17 pA, n = 37 for controls and 262 ± 33 pA, n = 12 in GTP-gamma -S, ANOVA, Tukey test, P = 0.99).

To better establish the linkage between the mGluR I activation and G proteins, six neurons were also patched with a solution in which GTP was substituted with GDP-beta -S, an inactive analogue of GDP (Eckstein et al. 1979). Figure 3A shows the DHPG-induced calcium increase (top) and inward current (bottom) after 8 and 30 min from the onset of the recording of a cell dialyzed with GDP-beta -S. Figure 3B reports mean values of the inward current and [Ca2+]i at 8 min (I = 86 ± 14 pA, bottom, % Delta F/F = 60 ± 9, n = 6, top) and after 30 min from the onset of the recording (I = 24 ± 3 pA, bottom, % Delta F/F = 21 ± 6, n = 6, top) compared with control values reported in Fig. 2B. Note that calcium transients at 8 min were comparable in amplitudes to controls (ANOVA, P = 0.1), whereas inward currents at the same time point were already significantly reduced (ANOVA, Tukey test, **P < 0.001). Both signals were almost abolished within 30 min of GDP-beta -S dialysis with respect to the signals measured at 8 min (ANOVA, Tukey test, *P < 0.05). This clearly suggests that G proteins are involved in the effects induced by DHPG.



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Fig. 3. Effects of intracellular dialysis with the GDP-analogue GDP-beta -S on the DHPG-induced responses. A, top: [Ca2+]i increases caused by 2 puff applications of DHPG in a GDP-beta -S (600 µM)-loaded neuron at 8 and 30 min after breakthrough into whole cell configuration. Bottom: the corresponding inward currents are shown. Note the progressive depression of the responses caused by the blockade of G proteins. B: mean amplitudes of the mGluR-induced [Ca2+]i increase (top) and inward currents (bottom). Mean values for calcium transients at 8 min of recording are not significantly different compared with controls (P = 0.1), but after 30 min, both responses are strongly affected (*P < 0.05, n = 6) by GDP-beta -S.

Metabotropic glutamate responses are partially dependent on phospholipase C but not on protein kinase C activation

mGluRs I are known to be coupled to the hydrolysis of membrane phosphoinositides by PLC and to the subsequent production of intracellular inositol-(1,4,5)-triphosphate (IP3) that enhances the release of calcium from intracellular stores. However, bath application of the membrane permeable PLC inhibitor U73122 (10 µM) (Chen et al. 1994; Lee and Boden 1997; Netzeband et al. 1997) for 15-20 min had a small reducing effect on the DHPG-induced calcium rise but did not change the DHPG-induced current (Fig. 4). In fact, in the presence of U73122 the amplitude of calcium transients was reduced to 82 ± 7% of control, n = 9 (t-test, **P < 0.001) (Fig. 4B, top), but the amplitude of the inward currents was not affected by the drug (102 ± 3% of control, n = 9, t-test, P = 0.8, bottom). The hydrolysis of phosphatidil inositol leads to diacylglycerol (DAG) production that in turn stimulates PKC. To investigate whether PKC activation is involved in the DHPG-induced calcium release and/or membrane currents, we bath applied staurosporine (1 µM) (Bonci and Williams 1997), a potent inhibitor of PKC, and evaluated DHPG-induced calcium and current amplitudes at 6-8 and 14-16 min after the onset of staurosporine application. Staurosporine had no effect on both DHPG-induced inward current (93 ± 11% of control, at 6-8 min and 96 ± 10% of control at 14-16 min, ANOVA, P = 0.99) and calcium increase (% Delta F/F = 108 ± 9, at 6-8 min and 96 ± 10, at 14-16 min, n = 3, ANOVA, P = 0.86, data not shown).



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Fig. 4. Phospholipase C (PLC) is not important in the generation of the DHPG-induced responses. A: bath application of the PLC inhibitor U-73122 (10 µM) slightly reduces the amplitude of the DHPG-induced increase in [Ca2+]i (top), while it does not affect the inward current (bottom, holding potential at -60 mV). B: mean values of the [Ca2+]i increase (top) in the presence of U-73122 (n = 9, **P < 0.001) and after wash out of the drug. Values are expressed as percentage of control (· · ·). Note that calcium signal fully recovered. Bottom plots illustrate the lack of effects of the PLC blocker on the DHPG-induced currents (n = 9, P = 0.8). · · ·, control amplitudes.

Tyrosine kinase mediates the DHPG-induced responses

It has been recently reported that group I mGluRs can modulate either a nonselective cationic current in CA3 hippocampal cells or Ca2+ mobilization in Chinese hamster ovary (CHO) cells by the phosphorylation of tyrosine substrates (Heuss et al. 1999; Umemori et al. 1997). To test for this possibility in dopaminergic neurons, we investigated the effects of the nonspecific tyrosine kinase inhibitor genistein (Wang and Salter 1994) on the DHPG-induced responses. Bath applications of genistein for 4-16 min (40 µM) reversibly reduced the inward current and elevation in [Ca2+]i (Fig. 5A) to 39 ± 11% (n = 10, t-test, *P < 0.01) and 47 ± 12% (n = 10, t-test, *P < 0.01) of controls, respectively (Fig. 5B). The effect of genistein partially washed out within 5 min. To further clarify the involvement of PTKs, we bath applied tyrphostin B52 (40 µM), a compound that is selective for epidermal growth factor receptor (EGFR)-kinase and other PTKs (Gazit et al. 1991). Tyrphostin B52 significantly reduced, in a poorly reversible manner, both DHPG-induced inward current (I = 25 ± 12%, n = 6, t-test, *P < 0.05) and [Ca2+]i elevation (% Delta F/F = 32 ± 3%, n = 6, t-test, *P < 0.01).



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Fig. 5. Tyrosine kinase is involved in the intracellular pathways activated by group I mGluRs. A: a repeated pressure ejection of DHPG in the close vicinity of the patched neuron induces transient increases of intracellular calcium (top) and inward currents (bottom). The responses to the agonist are reversibly depressed by the bath application of the tyrosine kinase inhibitor genistein (40 µM, horizontal bar). B, top: mean values as percentage of control (dotted line) of calcium increases in the presence of genistein (dark gray bars, n = 10, *P < 0.01), tyrphostin B52 (black bars, n = 6, *P < 0.01) and genistein's inactive analogue, genistin (light gray bars, 40 µM, n = 4, P = 0.8). Bottom: the mean amplitude as percentage of control of the DHPG-induced currents in the presence of genistein (left, n = 10, *P < 0.01), of tyrphostin B52 (middle, n = 6, *P < 0.05), and of the inactive analogue genistin (right, n = 4, P = 0.4).

Figure 5B shows mean amplitudes of calcium (top) and current (bottom) signals evoked by DHPG application, expressed as percentage of control, in the presence of genistein (40 µM), tyrphostin B52 (40 µM), and the genistein's inactive analogue genistin (40 µM). Note that genistin did affect neither the current nor the calcium amplitudes (I = 84 ± 5% of control, n = 4, t-test, P = 0.4; Delta F/F = 98 ± 4% of control, n = 4, t-test, P = 0.8), and this was tested in two of the experiments in which genistein was used.

To assess the possible involvement of the Src-family of tyrosine kinase (Boxall et al. 1996) in modulating the DHPG-induced responses, we performed experiments in the presence of the specific Src-kinase inhibitor PP1 (20 µM) (Hanke et al. 1996; Heuss et al. 1999). In the presence of this compound, DHPG produced an inward current and calcium signal that were not significantly different from control (not shown). Indeed, intracellular calcium transients were 110 ± 13% of control at 6-8 min and 105 ± 16% of control at 14-16 min (n = 3, ANOVA, P = 0.92), and the inward currents were 95 ± 5% of control at 6-8 min and 90.5 ± 5% of control at 14-16 min (n = 3, ANOVA, P = 0.93).

Another possible target of the tyrosine kinase-dependent and mGluR I-dependent intracellular pathway is the activation of the MAPK cascade. For this reason, we investigated whether the inhibition of the extracellular signal regulated kinase (ERK) (Ferraguti et al. 1999; Fiore et al. 1993) might be involved in the induction of the effects of mGluRs. PD 98059 (50 µM), a specific inhibitor of the two best characterized members of ERK, ERK1/ERK2 (Alessi et al. 1995) did not change the inward current and increase of [Ca2+]i caused by locally applied DHPG. In the presence of PD 98059, calcium transients were 106 ± 13% of control at 6-8 min and 86 ± 6% of control at 14-16 min (n = 4, ANOVA, P = 0.42, not shown) and the inward currents were 74 ± 13% of control at 6-8 min and 89 ± 7% of control at 14-16 min (n = 4, ANOVA, P = 0.76, not shown).

Pharmacology of metabotropic glutamate receptors

DHPG is a specific agonist for group I mGluRs, therefore the mGluR1 or the mGluR5 subtypes, most likely, mediate the responses described in the preceding text. To differentiate between these two receptors, we took advantage of the fact that the compound CPCCOEt preferentially antagonizes responses mediated by mGluR1 (Casabona et al. 1997) while MPEP those mediated by mGluR5 (Gasparini et al. 1999). We previously reported that CPCCOEt (100 µM) depressed the DHPG-induced inward current and the elevation in [Ca2+]i (Guatteo et al. 1999, Fig. 5). Now we found that the specific mGluR5 antagonist MPEP 10 µM (Gasparini et al. 1999; Heuss et al. 1999) did not antagonize the inward current or the DHPG-mediated Ca2+ signals (I = 102 ± 1% of control, t-test, P = 0.13, Fig. 6B, bottom; Delta F/F% = 127 ± 13% of control, n = 4, t-test, P = 0.09). MPEP up to 100 µM did not reduce the DHPG-induced inward currents and the calcium transients (n = 2, data not shown).



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Fig. 6. Pharmacology of the DHPG-induced responses. A: calcium transients (top) and inward currents (bottom) induced by pressure ejection of DHPG on a dopamine neuron. The presence of the mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP; 10 µM) in the bath for 16 min has no effect on the currents amplitudes or on the calcium transients. Conversely, the mGluR1 antagonist 7(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate (CPCCOEt, 100 µM) reversibly reduces currents and [Ca2+]I transients within 3-5 min. B: mean values of calcium (top) and current amplitudes (bottom) in control condition and in the presence of MPEP. Means were not significantly different from control for calcium transients (n = 4, P = 0.09) and for inward currents (n = 4, P = 0.13).

This pharmacological profile identifies the receptor mediating the DHPG-induced responses in dopamine neurons as mGluR1-like.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Intracellular events leading to the DHPG-induced responses

The main finding of the present study is that in midbrain dopaminergic neurons, the inward currents and associated intracellular calcium transients, induced by the activation of group I mGluRs, are G-protein dependent and apparently require a protein tyrosine kinase. Although G-protein-unlinked inward currents are reported to be caused by mGluRs agonists in hippocampal CA3 and dorsolateral septal neurons (Guerineau et al. 1995; Zheng et al. 1995), the fact that the DHPG-induced current and intracellular calcium signal are blocked by the dialysis with GTP-gamma -S and strongly reduced by the intracellular diffusion of GDP-beta -S, confirms the involvement of G proteins when group I mGluRs are activated in dopamine neurons. This evidence is also in agreement with the results previously published by Shen et al. (Shen and Johnson 1997) concerning the G-protein dependency of the mGluR-induced excitatory postsynaptic currents in VTA dopamine neurons. Moreover, as already reported by these authors, we observed that GDP-beta -S has a longer time course than GTP-gamma -S in affecting mGluR-mediated responses. We might speculate that, as shown in other cell types (Gruol et al. 1996; Saugstad et al. 1998; Umemori et al. 1997), the mGluR-activated responses in dopamine cells are possibly mediated by Gq, a class of G-protein insensitive to pertussis toxin (Pin and Douvoisin 1995).

Furthermore, the stimulation of group I mGluRs is usually believed to activate, via G proteins, PLC, which in turn catalyzes IP3 and DAG production from membrane phosphatides. IP3 induces the release of Ca2+ from internal stores and DAG the activation of PKC (Bockaert et al. 1993; Casabona et al. 1997; Irving et al. 1992; Kawabata et al. 1998; Linden et al. 1994; Nakanishi 1994; Netzeband et al. 1997; Pin and Douvoisin 1995). However, the involvement of this classical intracellular pathway in the DHPG-induced responses is not consistent with our experimental evidence. In fact, the insensitivity of the DHPG-evoked inward current to U73122 and to staurosporine, together with the slight effect of U73122 but not staurosporine in reducing [Ca2+]i, demonstrates that the DHPG-induced effects are mainly independent of PLC and PKC activation. This is also supported by the fact that both drugs were used at saturating concentration, as currently reported in the literature (Bonci and Williams 1997; Lee and Boden 1997; Tempia et al. 1998). Accordingly, group I mGluR-mediated responses not coupled to PLC and unlinked to PKC have been already described in Purkinje cells and in heterologous expression systems (Casabona et al. 1997; Hirono et al. 1998; Tempia et al. 1998).

Protein kinase-dependent responses

It is known that the brain contains high levels of PTK activity (Hirano et al. 1988). Antibody staining for phosphotyrosine demonstrates that developmental and regional PTK variations within the brain are present (Cudmore and Gurd 1991). It has been already reported that DHPG induces a G-protein-independent cationic current that requires the phosphorylation of tyrosine in CA3 hippocampal neurons (Heuss et al. 1999). A tyrosine kinase-dependent reduction of IAHP is also induced by mGluRs in dentate granule neurons (Abdul-Ghani et al. 1996). In agreement with these studies, we found that the inhibition of tyrosine kinase activity by genistein and by tyrphostin B52 reduced both the mGluR-mediated inward current and the [Ca2+]i signal in dopamine neurons. The lack of effect of the inactive analogue genistin also confirms a specific modification of the tyrosine phosphorylation sites by DHPG. It is not known which tyrosine kinase is involved in the responses caused by mGluR (Boxall and Lancaster 1998). Heuss et al. (1999) have shown that the nonreceptor Src-family PTK is responsible for the synaptic activation of mGluR1 in CA3 pyramidal cell of rat hippocampus. Here, we report that Src-kinase seems not to contribute to the mGluR transduction pathway in dopamine cells because the Src-kinase specific inhibitor PP1, applied at the same concentration reported by these authors (Heuss et al. 1999), does not affect the responses that depend on the activation of mGluR I. MAPKs have been also reported to be regulated by mGluRs or by PKA/PKC in CHO, cortical glial, and hippocampal CA1 cells (Ferraguti et al. 1999; Kurino et al. 1995; Peavy and Conn 1998; Roberson et al. 1999). Conversely, our data obtained using the ERK1/2 inhibitor PD 98059, used at same concentration that was effective in other types of neurons, exclude the possibility that MAPKs are directly implicated in the mechanisms responsible for the generation of the DHPG-induced inward current and release of [Ca2+]i in the dopaminergic cells.

One possibility might be that, in dopamine neurons, the targets of PTK are either the mGlu receptors or the G proteins. In fact, Umemori et al. (1997) demonstrated that PTK inhibitors act before the activation of a Gq/11 protein and that stimulation of Gq/11-protein-coupled receptor induces phosphorylation of a tyrosine residue on the Galpha q/11 subunit, which is essential for the activation of the G protein. Alternatively, PTK could directly phosphorylate the metabotropic glutamate receptor protein (Orlando et al. 1999) as it has been described for the N-methyl-D-aspartate receptors (Lu et al. 1999). Another possibility is that the responses to DHPG might be dependent on Homer proteins, known to be linked to group I mGluRs and to tyrosine phosphatases (Tu et al. 1998). Thus it could be possible that the level of tyrosine phosphorilation regulate these transduction pathways.

Subtype of receptors

The results obtained with subtype specific antagonists suggest that the metabotropic receptor responsible for the induction of the inward current and increase in [Ca2+]i is mGluR1-like. In fact, while CPCCOEt (mGluR1 antagonist) reduced the electrophysiological and Ca2+ responses to DHPG (Guatteo et al. 1999), MPEP (mGluR5 antagonist) was ineffective. These findings are also consistent with a prominent expression of mGluR1 in dopamine cells (Kosinski et al. 1998; Testa et al. 1994).

Conclusions

Taken together, our electrophysiological and fluorimetric observations suggest that tyrosine kinase regulates the G-protein-dependent inward current and intracellular calcium signal caused by the activation of mGluR I in dopamine cells. Although no functional relationship exists between intracellular Ca2+ increase and the inward current response (Guatteo et al. 1999), here we demonstrate that both phenomena share common metabolic pathways that involve G protein and tyrosine kinase activation. The induction of the inward current has obvious enhancing effects on the excitability of the cells, while the release of calcium from intracellular stores can cause a secondary membrane hyperpolarization (Fiorillo and Williams 1998).

It might be also speculated that the phosphorilation of proteins by mGluR-linked PTK are involved in trophic processes and in enduring changes of synaptic signals (Berridge 1998; Boxall et al. 1996; Merlin et al. 1998; Nakanishi 1994; Phenna et al. 1995) on the dopaminergic cells, and this in turn could influence motor behavior (Conquet et al. 1994) and sensitization to drugs of abuse (Vezina and Kim 1999).


    ACKNOWLEDGMENTS

We thank Dr. Marco Capogna for critical reading of the manuscript and M. Federici for technical assistance.


    FOOTNOTES

Address for reprint requests: N. B. Mercuri, Experimental Neurology Laboratory, IRCCS Fondazione S. Lucia, 00179 Rome, Italy (E-mail: mercurin{at}med.uniroma2.it).

Received 4 December 2000; accepted in final form 9 March 2001.


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ABSTRACT
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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society