Cooperative Activation of Dopamine D₁ and D₂ Receptors Increases Spike Firing of Nucleus Accumbens Neurons via G-Protein βγ Subunits

Introduction

Dopamine (DA) in the nucleus accumbens (NAcb) has long been considered an important modulator of addiction and goal-directed behaviors (Spanagel and Weiss, 1999). The shell region of the NAcb in particular is implicated in a number of cellular and behavioral phenomena, especially in relation to addictive drugs (Zahm, 1999). Although a diversity of functional effects and ionic targets can be modulated by DA, the exact role of DA receptor activation in the NAcb is only partially understood (Greengard et al., 1999; Nicola et al., 2000). DA receptors are generally grouped into two subfamilies, the D₁-like receptors and the D₂-like receptors (Missale et al., 1998). Opposing influences of D₁ and D₂ receptor activation on cAMP-dependent signaling have been reported in many studies (Stoof and Kebabian, 1981; Missale et al., 1998), with D₁ receptors acting through the stimulatory G_s-like G_olf, and D₂ receptors acting through the inhibitory G_i/o proteins.

In contrast, results from a number of behavioral studies suggest a cooperative interaction of D₁ and D₂ receptors in the NAcb (Plaznik et al., 1989; Chu and Kelley, 1992; Wolterink et al., 1993; Phillips et al., 1994; Hodge et al., 1997; Ikemoto et al., 1997; Gong et al., 1999; Koch et al., 2000; Nowend et al., 2001). For example, animals will self-administer D₁ and D₂ agonists directly into the NAcb in combination but not alone (Ikemoto et al., 1997). Furthermore, self-administration of amphetamine (Phillips et al., 1994) and ethanol (Hodge et al., 1997), lever pressing for a conditioned reinforcer (Chu and Kelley, 1992; Wolterink et al., 1993), and evaluating the relative cost of obtaining a reward (Koch et al., 2000; Nowend et al., 2001) all may involve coactivation of D₁ and D₂ receptors in the NAcb. Dose-dependent modulation of firing rate and TTX-independent Fos induction by cooperative D₁ and D₂ receptor activation have also been reported (Chiodo and Berger, 1986; Wachtel et al., 1989; Williams and Millar, 1990; Hu and White, 1997; LaHoste et al., 2000).

Despite the implication of D₁and D₂ receptor cooperativity in several behaviors, the specific cellular and biochemical pathways that mediate the interaction between D₁ and D₂ receptors are uncertain. The present study was designed to examine the effects of DAergic agonists on spike firing in medium spiny neurons (MSNs) in the NAcb shell and the cellular mechanisms that might mediate these effects.

Materials and Methods

Slice preparation and electrophysiology. Coronal slices (300 μm) were prepared from male postnatal day 22–28 Sprague Dawley rats (50–80 gm). After cutting, slices recovered at 32°C in carbogen-bubbled artificial CSF (ACSF (in mm: 126 NaCl, 1.6 KCl, 1.2 NaH₂PO₄, 1.2 MgCl₂, 2.4 CaCl₂, 18 NaHCO₃, and 11 glucose, pH 7.2–7.4, 301–305 Osm) for 30 min to 5 hr. During experiments, slices were submerged and continuously perfused (using a peristaltic pump, ∼2 ml/min) with carbogen-bubbled ACSF warmed to 31–32°C and supplemented with CNQX (10 μm, to block AMPA-type glutamate miniature EPSPs), picrotoxin (50 μm, to block GABA_A receptors), and sodium metabisulfite (50 μm), an antioxidant to preserve DAergic reagents (Nicola and Malenka, 1997). CNQX and picrotoxin were added to isolate the cell from several major sources of neurotransmitter input whose release is known to be inhibited by dopamine (Pennartz et al., 1992a; Nicola and Malenka, 1997). In preliminary experiments, DA-induced increases in spike firing were observed in the absence of these three reagents (data not shown). All reagents were bath-applied.

All experiments were performed using whole-cell recording, except where specifically indicated that an amphotericin perforated patch was used. Patch clamping was performed using visualized infrared differential interference contrast with 2.5–3.5 MΩ electrodes. Current pulses were applied using Clampex 8.0 and an Axo-1D patch amplifier in current-clamp mode (Axon Instruments, Foster City, CA). On breaking into neurons, the resting membrane potentials were between -95 and -80 mV. In most experiments, the membrane potential for each neuron was set to approximately -85 mV using the patch amplifier ∼5 min after breaking into a cell, except for experiments involving intracellular perfusion with okadaic acid, norokadaone, or G_βγ subunits, in which the resting membrane potential of cells was maintained at given potential (either -90 or -80 mV) throughout experiments to compensate for small drifts in membrane potential that can occur after break-in. Series resistance correction was 15–20 MΩ. To record EPSPs, a bipolar stimulating electrode was placed ∼100 μm lateral to the recording electrode. Afferents were stimulated with 10 pulses at 20 Hz (20 μsec) every 30 sec using a Master 8 (AMPI, Jerusalem, Israel), and EPSPs were recorded using Clampex in current-clamp mode.

For voltage-clamp experiments, ACSF was modified to contain 3.6 mm MgCl₂, 0.2 mm CaCl₂, 0.1 mm CdCl₂, and 1 μm TTX to block calcium and sodium current. Neurons were held at -80 mV with the patch amplifier. Our voltage protocol for determining effects of DA and 4-aminopyridine (4-AP) on potassium currents involved a series of 11 steps. In each step, a neuron was hyperpolarized to -100 mV for 700 msec and then depolarized for 300 msec. The depolarization ranged from -80 to +20 mV across the 11 steps. This protocol was applied every 30 sec. For analysis, magnitude of the total evoked potassium current was determined 280 msec into the current response after depolarization to 0 mV.

All data are shown as mean ± SEM. Unless otherwise indicated, all statistics were performed using a two-tailed, unpaired t test.

Analysis of spike firing. All such data were analyzed using Axograph (Axon Instruments, Foster City, CA). To calculate percent change in spiking, a current pulse was selected that exhibited approximately seven or eight spikes at baseline. The same current pulse was used for all time points of a given cell. Spike firing rates during the 3 min before addition of the reagent were averaged, and this value was normalized to 100%. Statistical significance was determined for the average spike firing change during the last 2 min of exposure to reagents. In most cases, statistical significance of changes in spike rate for a particular experimental condition was determined in comparison with an appropriate control condition. For experiments in which we systematically varied resting membrane voltage (V_m-rest) and determined the relationship between change in V_m-rest and change in spike firing, we first determined, for each cell, the change in number of spikes per millivolt change in V_m-rest. Then, to make these data comparable with the percent change in spike firing measured with DAergic agonists, we chose a baseline number of spikes of 7.2 (the mean number of spikes before addition of DAergic agonists in the cells shown in Fig. 5C) and determined the percent increase in spike firing per millivolt change in V_m-rest for each cell.

We attempted to estimate the proportion of neurons that responded to a given treatment, and, as is commonly observed (Uchimura et al., 1986; Surmeier et al., 1995), we found that most but not all MSNs responded to DAergic agonists. Approximately 70% of cells responded under whole-cell conditions, and ∼80% of cells responded under perforated patch conditions (with a threshold of 15% increase in spiking to be considered responding), suggesting that a small washout of some signaling molecules might have occurred under whole-cell conditions. However, to avoid the arbitrary designation required to delineate cells as responders or not, we included all neurons exposed to a given condition in all of our analyses.

Reagents. Whole-cell experiments were performed with potassium methanesulfonate- or potassium chloride-based solutions. KMeS/KOH, 0.95% (v/v); methanesulfonic acid, 0.76% (v/v); 20 mm HEPES; 0.2 mm EGTA; 2.8 mm NaCl; 2.5 mg/ml Mg-ATP; 0.25 mg/ml GTP, pH 7.2–7.4, 275–285 Osm; and KCl/KCl, 144 mm replacing KOH and methanesulfonic acid. Amphotericin experiments used the methanesulfonate-based solution, except without ATP and GTP. Amphotericin was made fresh as a 30 mg/ml stock in DMSO, sonicated, added at 0.2% (v/v) to a pipette solution containing 0.25 mg/ml pluronic acid, and sonicated again. Peptide sequences were as follows: FVIII, YEDSYEDISAYLLSKNNAIPR; and SPβγ, DALRIQMEERFMASNPSKVSYEPIT (Ma et al., 1997; synthesized by Synpep, Dublin, CA). Peptides were prepared as a 500× stock in DMSO and kept at -80°C. Peptides were used within 2 weeks of dilution in DMSO. In half of the peptide experiments, the experimenter did not know the identity of the peptide. Purified bovine whole-brain G_βγ subunits (Calbiochem, Lucerne, Switzerland; Huang et al., 1998) were aliquoted and kept at–80°C. Maltose-binding protein (MBP; a generous gift from Dr. Dorit Ron and Alicia Vagts, Ernest Gallo Clinic and Research Center), which is approximately the same molecular weight as G_βγ (∼50 kDa for MBP and ∼46 kDa for G_βγ), was prepared in the same vehicle as G_βγ subunits, with a final concentration of 20 μm DTT, 20 μm EGTA, and 0.002% Lubrol.

Most reagents were prepared fresh each day, including 4-aminopyridine, DA, sodium metabisulfite (all in Ringer's solution), ω-conotoxin-GVIA and ω-agatoxin (in water), eticlopride, propylnorapomorphine (NPA), okadaic acid, quinpirole, SCH23390, and U0126 (all in DMSO), and nifedipine (in 95% ethanol; Bargas et al., 1994). Dendrotoxin (Alomone Labs, Jerusalem, Israel), CNQX, Rp isomer of cAMPS (Rp-cAMPs) (Biolog, La Jolla, CA), and amphetamine were dissolved in water and kept at -20°C. SKF81297, SKF82957, forskolin, and dideoxy-forskolin were dissolved in DMSO and kept at -20°C. More than 85% of D₁ agonist experiments used SKF81297, and, when tested, we saw similar results with both. Picrotoxin was dissolved in water and kept as a room temperature stock. Unless otherwise indicated, all reagents were made at 1:1000 stock and were purchased from Sigma (St. Louis, MO) or Research Biochemicals (Natick, MA).

Results

Discussion

The present study shows that DA increases spike firing in MSNs from the NAcb shell. This enhancement of spike firing requires coactivation of D₁ and D₂ receptors, because neither agonist alone modifies spike firing, and the effect of DA is inhibited by either a D₁ oraD₂ receptor antagonist. The increased spike firing after coactivation of D₁ and D₂ receptors is mediated intracellularly by G_βγ and cAMP-dependent processes. Finally, our biophysical and pharmacological studies suggest that enhancement of spike firing occurs primarily through inhibition of a slow A-type potassium current (I_As).

Our results may provide a cellular mechanism to explain observations from behavioral studies suggesting a cooperative action of D₁ and D₂ receptors in the NAcb. For example, rats will self-administer D₁ and D₂ agonists into the NAcb in combination but will not self-administer either alone (Ikemoto et al., 1997). Both synergistic and additive effects of D₁ and D₂ receptor activation in the NAcb have been reported by studies of conditioned reinforcement (Chu and Kelley, 1992; Wolterink et al., 1993). Also, results from studies of amphetamine self-administration (Phillips et al., 1994) and evaluating the relative cost of obtaining a reward (Koch et al., 2000; Nowend et al., 2001) are suggestive of a cooperative role for D₁ and D₂ receptors in the NAcb in behavioral expression. Although D₁–D₂ interaction is not observed for all behaviors involving NAcb DA (Coccurello et al., 2000), these studies suggest that D₁ and D₂ receptors interact cooperatively in the expression of a number of reward- and motivation-related behaviors mediated by the NAcb.

These behavioral observations are quite intriguing, given that D₁ and D₂ receptors are traditionally thought to oppositely couple to the G-protein–PKA system (Missale et al., 1998). G_βγ provides a mechanism by which G_s- and G_i/o-coupled receptors, such as D₁ and D₂, respectively, can act cooperatively to activate PKA (Sunahara et al., 1996; Watts and Neve, 1997), especially perhaps for behaviors involving NAcb PKA signaling (Self et al., 1998; Sutton et al., 2000). Interestingly, a recent paper demonstrated that self-administration of ethanol is significantly reduced after inhibition of G_βγ function in the NAcb (Yao et al., 2002), in agreement with decreased ethanol consumption observed after block of D₁ or D₂ receptors within the NAcb (Hodge et al., 1997). Here, enhancement of spike firing after coactivation of D₁ and D₂ receptors required both G_βγ- and cAMP-dependent processes. In particular, intracellular perfusion with G_βγ enabled D₁ but not D₂ enhancement of spike firing, indicating that G_βγ derived from D2 was required for a spike firing increase. These data raise the interesting possibility that the DAergic signaling pathway we have identified mediates self-administration of ethanol and perhaps other behaviors.

Our results are also consistent with studies suggesting that PKA plays a major role in DA signaling in MSNs (Greengard et al., 1999). We should note that there are likely to be multiple forms of DA receptor and G_βγ signaling, including presynaptic modulation (McGinty, 1999) and interaction with signaling pathways other than PKA (Seiden et al., 1993; Sunahara et al., 1996; Missale et al., 1998; Hernandez-Lopez et al., 2000). Also, although we used relatively high concentrations of DA (Pennartz et al., 1992a; Nicola and Malenka, 1997), it is likely that the high density of dopamine transporters and strong dopamine transporter activity around MSNs (Uchimura and North, 1990; Jones et al., 1995; Hersch et al., 1997) greatly reduce the extracellular concentration of DA. Finally, the relatively slow onset of response after application of agonists, which has been observed in other studies (Nicola et al., 1996), likely reflects both the time required to superfuse the slice with a reagent and the time for intracellular signaling events to occur. For example, enhancement of spike firing after bath application of direct I_As antagonists (e.g., 4-AP) still takes several minutes, suggesting that some delay in onset of drug action is to be expected in brain slice experiments.

Our data also indicate that DA might increase the firing rate of MSNs in the NAcb in vivo. However, in vivo studies of DAergic modulation of MSN spike firing have produced mixed results, with observations of both excitation and inhibition (for review, see Siggins, 1978; Nicola et al., 2000). Several factors might contribute to these discrepancies. First, DAergic reduction of firing might be attributable to inhibition of glutamate release (Pennartz et al., 1992a; Nicola et al., 1996; Nicola and Malenka, 1997). Second, several studies have observed dose-dependent effects of DA, with lower doses activating and higher doses inhibiting firing (Chiodo and Berger, 1986; Wachtel et al., 1989; Williams and Millar, 1990; Hu and White, 1997). In this regard, DA release after stimulation of the ventral tegmental area or the median forebrain bundle, which might result in more moderate DA levels compared with direct application, can strongly enhance spike firing in MSNs (Chiodo and Berger, 1986; Gonon and Sundstrom, 1996). Thus, DA likely has multiple effects, perhaps depending on dose or signaling context, but there is a strong precedent for DAergic activation of MSNs. Of particular interest are recent studies of NAcb firing in response to cues that indicate food reward (S. M. Nicola, I. A. Yun, K. T. Wakabayashi, and H. L. Fields, unpublished observations). In some NAcb cells, firing rates increase during presentation of the cue, and this enhancement of firing is greatly reduced by VTA inactivation. VTA inactivation or infusion of dopamine receptor antagonists into the NAcb also greatly inhibits behavioral responding to the cue. Taken together, these data suggest that DA enhances firing in a set of NAcb neurons, and that this change in firing may be important for proper task performance after the cue is observed.

These electrophysiological studies, taken together with the behavioral results described above, suggest that the D₁ and D₂ cooperative interaction we have characterized in brain slices may have particular relevance in vivo. However, we should note that, as in vivo, in vitro studies of DAergic effects on MSN function have produced mixed results (Nicola et al., 2000). Some studies from NAcb slices found no DA-related changes in postsynaptic properties (Pennartz et al., 1992a; Nicola et al., 1996; Nicola and Malenka, 1997), whereas others observed significant DA-dependent changes in input resistance or resting membrane potential (Uchimura et al., 1986; Uchimura and North, 1990; O'Donnell and Grace, 1996). The latter may reflect action of DA on cell types other than MSNs (Yasumoto et al., 2002), and such influences were negated here by studying DAergic signaling in relative pharmacological isolation. Some discrepancies may also relate to differences among MSNs from the dorsal striatum, NAcb core, and NAcb shell (Kelly and Nahorski, 1987; Calabresi et al., 1992; Pennartz et al., 1992b; O'Donnell and Grace, 1993; Paxinos, 1995; Thomas et al., 2000). In particular, D₁ and D₂ receptors in the dorsal striatum are more clearly segregated in the so-called “patch” and “matrix” compartments, whereas such distinction is much less clear in the NAcb shell (Paxinos, 1995). Because slice studies from the dorsal striatum have generally not addressed the compartmental localization of the neurons under investigation, differential signaling among compartments could contribute to the variety of DAergic effects observed in vitro (for review, see Nicola et al., 2000).

Pharmacological and biophysical analyses suggest that the DA-mediated enhancement of spike firing observed here was mediated by inhibition of I_As and was not associated with a change in function of several other channel types, including sodium and L-, N-, and P/Q-type calcium channels, although we should note that other groups have observed modulation of sodium channels by DA in the NAcb (Zhang et al., 1998). Pharmacological inhibitors of I_As, such as α-dendrotoxin and 4-AP (5–60 μm), enhanced spike firing and occluded the effects of DA on spike firing. Also, using voltage-clamp methods, we observed a small but significant inhibition of potassium currents by 4-AP and DA (Surmeier and Kitai, 1993). In this regard, Bekkers and Delaney (2001) found that inhibition of I_As produced only a small decrease in total potassium current but led to a significant enhancement in spike firing, consistent with the critical role I_As contributes to action potential firing (Nisenbaum et al., 1994; Wickens and Wilson, 1998; Mahon et al., 2000). Although these results, particularly occlusion of DAergic effects after inhibition of I_As function, suggest that DA enhances spike firing by inhibiting I_As, we should acknowledge that additional effects beyond those on I_As cannot be definitively excluded, and that changes in V_m-rest in particular can account for the changes in spike firing in a small percentage of cells.

On the basis of analyses of the up and down state transitions and biophysical properties, it has been suggested that potassium channels are key regulators of excitability of MSNs (Wickens and Wilson, 1998). I_As activates at voltages around spike threshold, and inhibition of I_As may allow previously subthreshold synaptic input to elicit action potential firing (Nisenbaum et al., 1994; Wickens and Wilson, 1998; Mahon et al., 2000). In agreement, we found that DAergic activation increased the number of spikes fired during synaptic stimulation. Our data predict that DA will enhance the number of spikes fired in vivo during an up-state transition or during any other coherent glutamate excitation, with little effect on the hyperpolarized down state. This is consistent with the idea that DA is modulatory and normally requires glutamate receptor activation for DAergic effects to be observed (Chiodo and Berger, 1986; Nicola et al., 2000). Thus, by inhibition of I_As, coactivation of D₁ and D₂ receptors in the NAcb shell could enhance glutamate-mediated cellular excitation and thereby contribute to the expression of goal-directed behaviors.