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NEUROPHARMACOLOGY
Institute for Neuroscience, Northwestern University, Evanston, Illinois (T.K.A.) and Research Institute on Addictions, State University of New York at Buffalo, Buffalo, New York (R.-Y.S., S.H.-D.)
Received for publication
July 28, 2006
Accepted
September 26, 2006.
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Abstract |
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S. Examination of the downstream signaling mechanisms reveals that activation of the nonselective cation current requires the stimulation of phospholipase C but not an increase in intracellular calcium. Thus, pharmacological inhibition of phospholipase C reduces the amplitude of IDA. In contrast, buffering intracellular calcium has no effect on the amplitude of IDA. Bath application of transient receptor potential (TRP) channels blockers, 2-aminoethoxydiphenyl borate and SKF96365 [1-(-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole], strongly inhibits IDA amplitude, suggesting the involvement of TRP-like conductance. These results reveal previously unsuspected mechanism by which D2-like DA receptors induce membrane depolarization and enhance the excitability of DR 5-HT neurons.
). In addition, a dysfunction of the 5-HT system is thought to underlie a variety of psychiatric disorders, including anxiety and depression (Arango et al., 2002). Drugs that increase serotonin tone in the brain are the major substances used clinically for the treatment of anxiety and depression disorders (Nemeroff, 2003).
5-HT neurons of the DR receive a dense dopaminergic innervation from midbrain dopamine (DA) neurons (Peyron et al., 1995; Kitahama et al., 2000) and express D2-like (D2, D3) DA receptors (Mansour et al., 1990; Suzuki et al., 1998). These anatomical results suggest that DA input to the DR may play a critical role in the regulation of the function of DR 5-HT neurons. Consistent with this notion, in vivo neurochemical studies have reported that administration of DA receptor agonists increases the synthesis and release of 5-HT in the DR (Ferré et al., 1994; Matsumoto et al., 1996). Direct evidence for a role of DA in the regulation of 5-HT neurons function comes from recent in vitro electrophysiological study showing that activation of DA receptors induces a membrane depolarization and thereby increases the excitability of DR 5-HT neurons (Haj-Dahmane, 2001). This excitatory effect is mediated via the activation of D2-like DA receptors presumably located on DR 5-HT neurons.
D2-like DA receptors, comprising D2, D3, and D4 receptors, regulate neuronal excitability by modulating diverse ionic conductances and synaptic transmission (Greengard, 2001). These receptors are mainly coupled to the G-protein of the Go/Gi family to inhibit adenylyl cyclase (Missale et al., 1998). Generally, activation of these receptors decreases neuronal excitability by inducing a membrane hyperpolarization (Lacey et al., 1987). The ionic mechanism underlying this inhibitory response is best characterized in midbrain DA neurons, where it is widely accepted that the D2-like DA receptor-induced membrane hyperpolarization is mediated by an activation of an inward rectifier potassium current (Lacey et al., 1987). In contrast to midbrain DA neurons, activation of D2-like DA receptors in the DR strongly depolarizes and enhances the excitability of 5-HT neurons (Haj-Dahmane, 2001). Similar membrane depolarization induced by these receptors has also been reported in several other brain regions, such as the nucleus accumbens (Uchimura et al., 1986), the thalamus (Munsch et al., 2005), and hypothalamus (Yang et al., 1991). However, the ionic and signaling mechanisms underlying this excitatory response remain unknown. In the present study, we show that activation of D2-like DA receptors in the DR depolarizes 5-HT neurons via activation of nonselective cation current. The D2-like DA receptor-induced cationic conductance is signaled through phospholipase C pathway and exhibits pharmacological and electrical properties similar to transient receptor potential canonical (TRPC) channels. As such, these results outline a novel mechanism by which D2-like DA receptors increase the intrinsic excitability of DR 5-HT neurons.
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Materials and Methods |
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). All experiments were conducted in compliance with the National Institutes of Health and American Association for Accreditation of Laboratory Animal Care guidelines and were approved by the University at Buffalo Institutional Animal Care and Use Committee. In brief, animals were anesthetized with halothane and killed by decapitation. The brain was quickly removed and cooled in ice-cold standard artificial cerebrospinal fluid (aCSF) solution of the following composition: 119 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl2, 1.3 mM MgSO4, 1 mM NaH2PO4, 26.2 mM NaHCO3, and 11 mM glucose and saturated with 95% O2/5% CO2. A block of brain tissue containing the DR was dissected, and coronal slices (300–400 µm) were cut with a vibratome (Lancer series 1000; Ted Pella Inc., Irvine, CA). Slices were stored for at least 1 h at room temperature in a holding chamber filled with the aCSF solution and continuously bubbled with 95% O2/5% CO2. Slices containing the DR were then transferred one at a time to a recording chamber, in which the slice was held submerged between two nylon nets and continuously perfused with the standard aCSF solution (1–2 ml/min) saturated with 95% O2/5% CO2 at 30 ± 1°C.
Whole-Cell Recordings. Whole-cell recordings were performed from DR neurons using the "blind" patch-clamp technique. Low resistance path electrodes (3–5 M
) were made from borosilicate glass tubing (Warner Instruments Inc., Hamden, CT) using a P97 horizontal puller (Sutter Instruments, Novato, CA). Recording pipettes were filled with an internal solution containing 120 mM potassium gluconate, 10 mM KCl, 10 mM Na2-phosphocreatine, 10 mM HEPES, 1 mM MgCl2, 1 mM EGTA, 2 mM Na2-ATP, and 0.25 mM Na-GTP (pH 7.3, adjusted with KOH and osmolarity, 280 mosM). In some experiments aimed at examining the ionic mechanism of the DA-induced current, recordings were performed using a cesium gluconate-based intracellular solution of the following composition: 120 mM D-gluconic acid, 10 mM CsCl, 20 mM Na2-phosphocreatine, 10 mM HEPES, 1 mM MgCl2, 1 mM EGTA, 2 mM Na2-ATP, and 0.25 mM Na-GTP (pH 7.3, adjusted with CsOH; osmolarity, 280 mosM). Intracellular electric signals were recorded using an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA) in the bridge mode or in the continuous, single-electrode voltage-clamp mode. Membrane voltages and currents were filtered at 3 to 10 kHz and recorded online using a paper chart recorder (model TA 240; Gould Instrument, Valley View, OH). Fast electric events such as voltage ramps were digitized at 10 KHz using the Digidata 1200 interface board (Axon Instruments) and stored on a hard disk using Pclamp 9.0 software (Axon Instruments). DR 5-HT neurons were identified by their electrophysiological properties as described previously (Haj-Dahmane, 2001). Access resistance (10–20 M) was monitored online during the course of the experiments with hyperpolarizing steps (–50 to –100 pA, 200–500 ms). Experiments were discarded when series resistance changed by more than 10%.
Voltage-clamp recordings were performed using the continuous voltage-clamp mode. The access resistance was compensated by up to 80% using the bridge circuit of the amplifier, and the settling time of the membrane current in response to 10 mV hyperpolarization was optimized to <5 ms. The steady-state current-voltage (I-V) plots were obtained using a slow voltage ramps (6 mV/s) from –120 to 10 mV before and during DA application. These experiments were conducted in the presence of tetrodotoxin (TTX) (1 µM) and CdCl2 (200 µM) to block voltage-dependent sodium and calcium current, respectively. The net current-voltage plot of the DA-induced inward current was obtained by digital subtraction of the control I-V curve from that obtained in the presence of DA. The values of membrane potential were corrected for the liquid junction potential that was present between the pipette and the bath solutions (approximately +6 and +12 mV for the potassium- and cesium gluconate-based intracellular solutions, respectively).
To examine the effect of low extracellular sodium ([Na+]o = 27 mM) on the amplitude and I-V curve of DA-induced inward current, aCSF containing low sodium was prepared by substituting NaCl with equimolar concentration of choline-chloride. Because a high concentration of choline can activate nicotinic and muscarinic receptors, these experiments were conducted in the presence of atropine (3 µM) to block any potential muscarinic receptor-induced responses.
In some experiments, the calcium-buffering capacity of the internal solution was increased by replacing 25 mM potassium gluconate with 25 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). In experiments examining the role of postsynaptic G-proteins in the signaling of DA-induced membrane depolarization/inward current, GTP was replaced with either GDPS (200 µM) or GTP
S (200 µM).
Drugs and Drug Application. DA and others drugs were administered by bath application in the aCSF solution at known concentrations. Sodium metabusulphite (100 µM), which had no effect on the excitability of DR 5-HT neurons, was routinely added to the aCSF solution to prevent DA oxidization. To examine the involvement of phospholipase C (PLC) in the signaling of DA-induced inward current, slices were pretreated with a PLC inhibitor, U-73122 (10 µM), or its inactive analog, U-73343 (10 µM), for at least 1 h before recording. U-73122 and U-73343 were first reconstituted in chloroform into single-use aliquots. The aliquots were evaporated to dryness, dissolved in DMSO, and then added to the aCSF solution at the desired concentration. The final dilution of DMSO was 0.001%, which had no significant effect on the excitability of DR 5-HT neurons. Most chemicals and drugs, including forskolin, 8-Br-cAMP, and 2-aminoethoxydiphenyl borate (2-APB), were purchased from Sigma-Aldrich (St. Louis, MO). Quinpirole and sulpiride were obtained from Research Biochemical Incorporated (Natick, MA). TTX was purchased from Alomome Labs (Jerusalem, Israel). U-73122, U-73343, and SKF-93635 were obtained from Biomol (Plymouth Meeting, PA). Ro-32-0432 and chelerythrine chloride were obtained from Calbiochem (San Diego, CA). Results in the text and figures are presented as mean ± S.E.M. Statistical analysis were performed using the Student's paired t test for within-group comparison and the Student's unpaired t test for comparison between groups.
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Results |
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; Haj-Dahmane, 2001). Consistent with earlier findings (Haj-Dahmane, 2001), bath application of DA (3 to 100 µM) elicited a reversible slow membrane depolarization. Generally, the membrane depolarization induced by DA (30 µM) was sufficient to activate voltage-dependent sodium current and initiate a train of action potentials (Fig. 1A1, n = 30). The ability of DA to depolarize DR 5-HT neurons persisted in the presence of TTX (1 µM) and cadmium chloride (200 µM), a manipulation that profoundly reduces neurotransmitter release. Under these conditions, DA (30 µM) depolarized 5-HT neurons by 10.5 ± 3.8 mV (Fig. 1A2, n = 6), indicating that the DA-induced membrane depolarization is mediated by activation of postsynaptic DA receptors located on 5-HT neurons.
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), respectively, elicited a slow reversible membrane depolarization (Fig. 1B1, n = 8). Similar to the effect of DA, the ability of (–) quinpirole to depolarize DR 5-HT neurons persisted in the presence of TTX and cadmium chloride (Fig. 1B2, control = 9.5 ± 4.6 mV; in TTX + CdCl2 = 8 ± 2.5 mV, n = 6, p > 0.05), suggesting that the membrane depolarization is mediated by the stimulation of postsynaptic D2-like DA receptors.
To further confirm the involvement of D2-like DA receptors, we tested the effect of sulpiride, a nonselective D2-like DA receptor antagonist that binds to both D2 and D3 receptors with equal affinity (Seeman and Van Tol, 1994), on the ability of DA and (–) quinpirole to induce a slow membrane depolarization. As illustrated in Fig. 1C, pretreatment of slices with sulpiride (3 µM) totally abolished the membrane depolarization induced by DA (30 µM). The average amplitude of the membrane depolarization induced by DA (30 µM) obtained in control condition and in the presence of sulpiride was 10.5 ± 3.8 and 0.25 ± 0.34 mV (Fig. 1D, n = 8, p < 0.001). Likewise, in the presence of sulpiride, the selective D2-like agonist, quinpirole (30 µM), failed to depolarize DR 5-HT neurons (Fig. 1D, control = 9.5 ± 4.6 mV, in sulpiride = 0.35 ± 0.16, n = 7, p < 0.01). Taken together, these results strongly indicate that DA depolarizes DR 5-HT neurons via activation of postsynaptic D2-like DA receptors.
DA Activates a Nonselective Cationic Conductance. Previous studies in other neuronal preparations have suggested that activation of D2-like DA receptors can induce a membrane depolarization through a reduction in potassium conductance (Uchimura et al., 1986; Higashi et al., 1989). To test whether a similar ionic mechanism also mediated the D2-like receptor-induced membrane depolarization in DR 5-HT neurons, we first assessed the impact of DA on the holding current and membrane conductance of DR 5-HT neurons in voltage-clamp mode. As illustrated in Fig. 2A1, in DR 5-HT neurons voltage clamped at –70 mV, bath application of DA (30 µM) elicited a reversible inward shift of the holding current. The average amplitude of the DA-induced inward current (IDA) was 65 ± 6.5 pA (n = 15). Further examination of the changes in membrane conductance using a hyperpolarizing voltage step (10 mV, 500 ms) applied before and during DA application revealed that the IDA was consistently associated with a significant increase in membrane conductance (Fig. 2, A2 and A3; Gm control = 1.89 ± 0.32 nS, Gm DA = 2.75 ± 0.56 nS, n = 6, p < 0.05). We next examined the ability of DA to induce an inward current using cesium gluconate-based intracellular solution and in the presence of extracellular cesium (2 mM), a manipulation that blocks most potassium channels. We found that bath application of DA (30 µM) still induced a robust inward current in all of the DR 5-HT neurons tested (Fig. 2B, n = 7). The average amplitude of IDA recorded under these conditions was essentially comparable with that obtained using potassium gluconate-based intracellular solution (Fig. 2C, IDA control = 65 ± 6.9 pA, IDA cesium = 68 ± 7.5 pA, p > 0.05, n = 7). These results suggest that IDA in the DR 5-HT neuron is unlikely to be mediated by a blockade of potassium current.
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To characterize the ionic mechanism of IDA, we assessed its current-voltage (I-V) relationship using a slow voltage ramps (6 mV/s) from –120 to 10 mV applied before DA (30 µM) application and at the peak of the inward current. As illustrated in Fig. 3B, the I-V relationship of IDA recorded with a cesium gluconate-based intracellular solution and in the presence of TTX (1 µM) was linear and exhibited a positive slope conductance at potential between –120 and 0 mV. The average reversal potential of IDA was –15 ± 8 mV (Fig. 3B, n = 8), suggesting the involvement of mixed cationic conductance. To further confirm that IDA is mainly mediated by a nonselective cation current, we next examined the impact of lowering the extracellular concentration of sodium on the amplitude and the reversal potential of IDA. As illustrated in Fig. 3C, replacing sodium chloride in the extracellular solution with choline chloride profoundly reduced the amplitude of IDA. The average amplitudes of IDA recorded in control condition and in low extracellular sodium were 65 ± 10 and 15 ± 8 pA, respectively (Fig. 3E, p < 0.05, n = 8). Lowering the extracellular concentration of sodium also resulted in a negative shift of the reversal potential of the IDA (Fig. 3D, Erev control = –15 ± 8 mV; Erev in low sodium = –45 ± 10 mV, n = 8, p < 0.05). Because sodium substitution per se had no effect on the cell input resistance, the reduction in IDA amplitude and the shift of its reversal potential was unlikely to be attributed to a loss of voltage control of the dendritic arbor. Taken together, these results indicate that IDA is mainly mediated by an activation of nonselective cationic conductance permeable to sodium ions.
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IDA Is Signaled via G-Protein and Phospholipase C Pathway. To determine whether the signaling of IDA required G-protein activation, we first tested the impact of intracellular application of GDPS, a membrane impermeable inhibitor of G-proteins, on the ability of DA to induce IDA. To insure that this manipulation was effective in inhibiting the function of G-proteins, we also monitored the 5-HT-induced outward potassium current, a response known to be mediated by activation of G-proteins (Innis et al., 1988). As illustrated in Fig. 4A, in 5-HT neurons recorded with a pipette solution containing GTP (250 µM) bath application of 5-HT (30 µM) induced an outward current, whereas administration of DA (30 µM) elicited a prominent inward current. Replacing GTP with GDPS (250 µM) in the pipette solution completely blocked the 5-HT-induced potassium current (Fig. 4B, n = 6), indicating an effective blockade of G-proteins. Under these conditions, bath application of DA (30 µM) failed to induce an inward current (Fig. 4B, n = 6). Overall, the amplitude of IDA recorded with GTP and GDPS was 63 ± 8 and 6 ± 5 pA, respectively (Fig. 4D, n = 6, p < 0.01). We next examined the effect of GTPS, an irreversible activator of G-protein, on the DA-induced inward current. As expected for a G-protein-mediated response, in 5-HT neurons loaded with GTPS, bath application of DA (30 µM) induced a persistent inward current (Fig. 4C, n = 4). Combined, these results demonstrate that activation of G-proteins is required for the signaling of IDA in DR 5-HT neurons.
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). As such, it is conceivable that this pathway may be involved in the signaling of IDA. If inhibition of adenylyl cyclase is required for the signaling of IDA, activation of this enzyme should at least reduce the amplitude IDA. To test this possibility, we examined the effect of forskolin (10 µM), a lipophilic adenylyl cyclase activator, on the amplitude of IDA. We found that in slices treated with forskolin, bath application of DA (30 µM) still induced a robust inward current with a comparable amplitude with that obtained in control slices (IDA control = 62 ± 4.5 pA; IDA forskolin = 68 ± 6.5 pA, n = 5, p > 0.05, data not shown). Likewise, treatment of slices with the membrane-permeable cAMP analog, 8-Br-cAMP, which presumably can increase the intracellular cAMP level, failed to significantly affect the amplitude of IDA (IDA control = 62 ± 4.5 pA; IDA in 8-Br-cAMP = 65 ± 6.8; p > 0.05; n = 5). Together, these results suggest that inhibition of adenylyl cyclase is unlikely to contribute to the signaling of IDA in DR 5-HT neurons.
Several reports have provided evidence that D2-like DA receptors can also activate PLC and increase the hydrolysis of phosphatidylinositol-4,5 biphosphate (Vallar et al., 1990; MacKenzie et al., 1994). Thus, we investigated whether pharmacological inhibition of PLC/phosphatidylinositol-4,5 biphosphate pathway could affect IDA. As shown in Fig. 5A2, pretreatment of slices with U-73122 (10 µM), an inhibitor of PLC, profoundly reduced the amplitude of IDA (IDA control = 61.66 ± 4.2 pA; IDA in U-73122 = 10.1 ± 4.1, p < 0.05; n = 8, Fig. 5B). In contrast, treatment of slices with U-73433 (10 µM), an inactive analog of U-73122, had no significant effect on IDA amplitude (IDA control = 61.66 ± 4.2 pA; IDA in U 73433 = 59 ± 9.1 pA, n = 4, p > 0.05, Fig. 4, A3 and B). These results suggest that activation of PLC is required for the signaling of IDA.
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We next examined whether a rise in intracellular calcium was required for the activation of IDA. To this end, DR 5-HT neurons were recorded with a potassium gluconate-based intracellular solution containing a high concentration of BAPTA (25 mM), a fast calcium chelator. The ability of BAPTA to buffer the transient increase in intracellular calcium was monitored by the amplitude of the current underlying the afterhyperpolarizing potential (IAHP), a response known to be mediated by calcium-activated potassium current (Pan et al., 1994). We found that dialysis of 5-HT neurons with BAPTA (25 mM) for at least 10 min strongly reduced the amplitude of the IAHP (IAHP at 1 min = 155 ± 15 pA; IAHP at 10 min = 25 ± 12 pA; n = 6) but failed to affect the ability of DA (30 µM) to induce an inward current. Overall, the amplitude of IDA recorded with an internal solution containing BAPTA (25 mM) was similar to that obtained using a pipette solution containing a low concentration of calcium chelator EGTA (1 mM) (Fig. 5D; IDA control = 58.33 ± 9.21 pA; IDA in BAPTA = 56.66 ± 11.8 pA, p > 0.05, n = 6). These findings indicate that a rise in cytosolic calcium is not required for the activation of IDA in DR 5-HT neurons.
TRP Channel Blockers Profoundly Reduce IDA. TRPC is a family of proteins that forms a diverse group of nonselective cation currents (Harteneck et al., 2000; Clapham et al., 2001). The TRPC family consists of seven members (TRPC1–TRPC7), which are widely distributed within the mammalian brain (Strübing et al., 2001; Riccio et al., 2002). One prominent feature of TRPC channels is that they are activated in response to receptor-mediated stimulation of different PLC isoforms (Clapham et al., 2001). Our findings that the IDA requires PLC activation and exhibits an I-V relationship comparable with some TRPC currents raised the possibility that the cation channels activated by D2-like DA receptors may belong to the TRPC channel family. To test this possibility, we examined the effect of 2-APB and SKF-93635, which are known to block TRPC channels (Li et al., 1999; Clapham et al., 2001), on the amplitude of IDA. As illustrated in Fig. 6A, treatment of brain slices with either 2-APB or SKF96365 (100 µM) strongly reduced the amplitude of IDA, albeit the blockade of IDA by 2-APB was less pronounced than that obtained with SKF-93635 (IDA control = 54.5 ± 6.9 pA; IDA in 2-APB = 15.6 ± 4.7 pA, IDA in SKF96365 = 5.45 ± 5.2 pA, n < 0.05, n = 8, Fig. 6B). Because 2-APB is also a blocker of IP3 receptors, it is conceivable that the reduction of IDA amplitude simply reflects a blockade of calcium release from an IP3-sensitive store. However, this possibility is highly unlikely since an increase in intracellular calcium is not required for the activation of IDA. Taken together, these pharmacological results suggest that IDA may be mediated by TRPC channels.
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Discussion |
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Ionic Mechanism of the D2-Like DA Receptor-Induced Membrane Depolarization. Stimulation of D2-like DA receptors has been shown to induce a slow membrane depolarization in several other brain regions (Uchimura et al., 1986; Yang et al., 1991; Munsch et al., 2005). Results from a previous study in accumbens neurons have suggested that this excitatory response is mediated by a decrease in potassium conductance (Uchimura et al., 1986). This conclusion, however, was based on the finding that the membrane depolarization was associated with a small or no increase in membrane resistance. Unlike these neurons, we found that in DR 5-HT neurons, the D2-like DA receptor-induced membrane depolarization is predominantly mediated by an increase in nonselective cationic conductance.
Several lines of evidence support this conclusion. First, the D2-like DA receptor-induced membrane depolarization and inward current were consistently associated with a significant increase in membrane conductance. In addition, blockade of potassium currents with cesium had no effect on the amplitude of IDA. These observations exclude the possibility that IDA is mediated by a reduction in potassium conductance. Second, the amplitude of IDA always increased linearly with membrane hyperpolarization and displayed a reversal potential at –15 mV. The reversal potential determined within the technical limitation inherent to clamping neurons in brain slices exhibited an intermediate value between the equilibrium potentials for sodium (ENa = +56 mV) and potassium ions (Ek =–104 mV), suggesting the involvement of a mixed cationic conductance. Consistent with this notion, lowering the extracellular concentration of sodium reduced the amplitude of IDA and induced a negative shift of it reversal potential, thus indicating that the sodium ion is a main charge carrier.
Cation-nonselective currents have been shown to mediate slow membrane depolarization in a several other brain regions (Shen and North, 1992; Haj-Dahmane and Andrade, 1996). In some neuronal preparations, these cationic current are voltage-dependent (Haj-Dahmane and Andrade, 1996; Faber et al., 2006), whereas in others, they seem to be voltage-independent (Shen and North, 1992; Crépel et al., 1994). In the present study, we found that IDA in DR 5-HT neurons is not voltage-dependent because its amplitude increases with membrane hyperpolarization and exhibits a linear I-V relationship. A similar voltage-independent nonselective cation current has recently been shown to mediate the membrane depolarization of DR 5-HT neurons induced by orexin receptors (Liu et al., 2002). Thus, our results further support the notion that nonselective cation currents play a critical role in the regulation of the excitability of DR 5-HT neurons.
Most cation-nonselective currents known to mediate slow membrane depolarization, with the exception of cyclic nucleotide-gated cation channels and hyperpolarization-activated cation current (IH), are thought to be activated in response to the stimulation of Gq/G11-coupled receptors, such as M1 muscarinic receptor (Haj-Dahmane and Andrade, 1996), group I metabotropic glutamate receptors (Kim et al., 2003), and orexin receptors (Liu et al., 2002). In the present study, we found that the DA-induced cationic current (IDA) in DR 5-HT neurons is signaled by D2-like DA receptors, which are mainly coupled to Go/Gi type G-proteins (Missale et al., 1998), indicating that cation-nonselective conductances can also be activated by Go/Gi coupled receptors. Such ionic mechanism may also mediate the membrane depolarization induced by D2-like DA receptors in other brain areas (Yang et al., 1991; Munsch et al., 2005).
Previous studies have shown that an increase in intracellular calcium is required for the activation of some nonselective cationic conductances (Swandulla and Lux, 1985; Crépel et al., 1994). These calcium-activated cationic currents, commonly called CAN currents (for review, see Partridge et al., 1994), have been shown to mediate the membrane depolarization induced by the group I metabotropic receptor in CA1 pyramidal neurons (Crépel et al., 1994). In the present study, however, we found that activation of IDA does not require an increase in intracellular calcium because loading 5-HT neurons with a high concentration of calcium chelator BAPTA (25 mM) had no effect on the amplitude of the DA-induced cationic current. This finding indicates that IDA is unlikely to be mediated by the calcium-activated nonselective cation current (CAN).
Signaling Mechanisms of IDA. Previous studies have shown that D2-like DA receptors are primarily coupled to Go/Gi-type G-proteins (Missale et al., 1998). As expected for a G-protein-mediated response, we found that intracellular application of GDPS, which blocks G-protein function, totally abolishes IDA, whereas in the presence of GTPS, which irreversibly activates G-protein, stimulation of D2-like DA receptors induces a persistent IDA. These results demonstrate that IDA is signaled via G-protein-dependent mechanisms.
We have shown that activation of adenylyl cyclase with forskolin has no significant effect on the amplitude of IDA. In addition, the ability of D2-like DA receptors to activate IDA is also preserved in the presence of 8-Br-cAMP, a membrane-permeable analog of cAMP. Hence, although stimulation of D2-like DA receptor is known to inhibit adenylyl cyclase (Missale et al., 1998), our data suggest that this pathway is unlikely to be involved in the signaling of IDA. Instead, we found that pharmacological inhibition of PLC with U-73122 almost abolished IDA, indicating that activation of PLC is required for the signaling of IDA. Interestingly, activation of PLC pathway has also been shown to mediate the D2-like receptor-induced inhibition of calcium current in striatal neurons (Hernández-Lopez et al., 2000). Thus, our results are consistent with the notion that D2-like DA receptors can activate the PLC pathway (Vallar et al., 1990; MacKenzie et al., 1994), and through this pathway, they modulate a variety of membrane conductances, including the nonselective cation current described in the present study.
Although IDA is activated via stimulation of PLC, we found that PKC activators and inhibitors fail to mimic and inhibit DA-induced current, respectively, suggesting that IDA is unlikely to be gated by PKC-dependent protein phosphorylation. Likewise, because elevation of intracellular calcium is not required for the activation of IDA, it is unlikely that IP3-induced calcium release from internal stores contributes to the activation of IDA. As such, it is possible that IDA is directly activated by DAG or by a yet unknown second messenger system downstream from PLC.
Possible Involvement of TRP Channels. Although cation-nonselective currents have been widely described in the mammalian brain, the molecular identities of these channels remain unknown. Recently, convergent lines of evidence indicate that TRPC proteins are primary candidate. Thus, all seven members of the TRPC family are widely expressed in the mammalian brain (Strübing et al., 2001; Riccio et al., 2002). Moreover, depending on the subunit composition, in vitro expression of TRPC proteins leads to diverse membrane conductances that resemble many feature of native nonselective cationic currents recorded in CNS neurons. For instance, coexpression of TRPC1 with TRPC4 or TRPC5 forms heteromultimers that generate a voltage-dependent nonselective cation current (Strübing et al., 2001) with an I-V curve strikingly similar to the native cationic current activated by muscarinic receptors in cortical neurons (Haj-Dahmane and Andrade, 1996). In contrast, expression of homomeric TRPC proteins forms cation-nonselective currents that display linear I-V relationships at negative membrane potentials (Hofmann et al., 2002; Plant and Schaefer, 2003). In the present study, we found that IDA also exhibits a linear I-V curve. In addition, like TRPC channels, IDA is also signaled via a PLC-dependent pathway and is profoundly reduced by 2-APB and SKF96365. These pharmacological results combined with the functional similarities between IDA and TRPC channels suggest that IDA is most likely mediated by TRPC channels. This notion is also supported by a recent study showing that DR 5-HT neurons express at least four members of TRPC family (TRPC1, TRPC3, TRPC5, and TRPC6) (Sergeeva et al., 2003). However, future studies using molecular approaches are required to confirm that IDA is indeed meditated by TRPC channels and to determine which TRPC protein(s) is the target of DA modulation in the DR 5-HT neurons.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: DR, dorsal raphe; IP3, inositol 1,4,5-trisphosphate; 5-HT, serotonin; DA, dopamine; TRPC, transient receptor potential canonical; aCSF, artificial cerebrospinal fluid; TTX, tetrodotoxin; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; PLC, phospholipase C; U-73122, 1-(6-((17b-3-methoxyestra-1,3,5 (10)-trien-17-yl)amino)hexyl)1-H-pyrole-2,5-dione; U-73343, 1-(6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl) 2,5 pyrrolidine-dione; DMSO, dimethyl sulfoxide; 2-APB, 2-aminoethoxydiphenyl borate; SKF-93635, 1-[2-(4-methoxyphenyl)-2-[3-(4methoxyphenyl)propoxy]ethyl-1H-imidazole hydrochloride; AHP, afterhyperpolarizing potential; SKF38393, 2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; DAG, diacylglycerol; PKC, protein kinase C; SKF96365, 1-(-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole; 8-Br-cAMP, 8-bromo-cyclic AMP; Ro-32-0432, 2-{8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyridol [1,2-a]indol-3-yl}-3-(1-methyl-1H-indol-3-yl)maleimide-HCl.
Address correspondence to: Dr. Samir Haj-Dahmane, Research Institute on Addictions, 1021 Main Street, Buffalo, NY 14203. E-mail: dahmane{at}ria.buffalo.edu
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