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The Journal of Physiology logoLink to The Journal of Physiology
. 2012 Feb 27;590(Pt 10):2273–2284. doi: 10.1113/jphysiol.2011.225672

Regulation of polysynaptic subthalamonigral transmission by D2, D3 and D4 dopamine receptors in rat brain slices

Ke-Zhong Shen 1, Steven W Johnson 1,2
PMCID: PMC3424752  PMID: 22371474

Abstract

Dopamine depletion in experimental models of Parkinson's disease promotes burst firing of neurons in the subthalamic nucleus (STN) and substantia nigra zona reticulata (SNR). A synaptically generated form of burst firing has been shown to arise from complex excitatory postsynaptic currents (EPSCs) that are evoked in SNR neurons by STN stimulation. The present experiments were designed to characterize actions of dopamine on complex EPSCs in slices of rat brain. Using patch pipettes to record whole-cell currents under voltage clamp, dopamine (30 μm) caused a reversible 64% reduction in complex EPSC charge. This effect was partially mimicked by D2, D3 and D4 receptor agonists, and the action of dopamine could be nearly completely blocked by the combined effects of the D2/3 antagonist sulpiride and the D4 antagonist L-745,870. Local application of dopamine to the STN caused a larger inhibition of the complex EPSC (55% reduction) than did dopamine application to the SNR (15% reduction). Simple, monophasic EPSCs, which were evoked in SNR neurons by stimulating the SNR close to the recording pipette, were inhibited to a smaller extent compared to complex EPSCs. Bursts of action potentials evoked in SNR neurons by STN stimulation were inhibited by dopamine to a greater extent than was spontaneous firing. These results show that dopamine D2-like receptors inhibit complex EPSCs and burst discharges in the SNR by acting within the STN to suppress transmission in the subthalamonigral pathway. Dopamine receptor-mediated inhibition of polysynaptic connections in the STN might be beneficial in the treatment of Parkinson's disease.

Key points

  • Symptoms of Parkinson's disease are associated with increased bursting activity in the subthalamic nucleus and substantia nigra zona reticulata (SNR).

  • In slices of rat brain, a single electrical stimulus to the STN evokes a complex EPSC and bursts of action potentials in SNR neurons.

  • We show that dopamine acts at D2-like receptors to cause marked and reversible inhibition of the complex EPSC.

  • Dopamine also inhibited stimulus-evoked bursts of action potentials more than reducing spontaneous firing.

  • Inhibition of synaptically generated burst firing by D2 receptor agonists may be a clinically important mechanism in the treatment of Parkinson's disease.

Introduction

The substantia nigra zona reticulata (SNR) and globus pallidus interna (GPI) comprise the major output nuclei of the basal ganglia. As such, these nuclei greatly influence both voluntary and involuntary movements. Both the SNR and GPI receive excitatory inputs from glutamate-containing neurons in the subthalamic nucleus (STN), which serves as a main driver of basal ganglia outflow (Parent & Hazrati, 1995). STN neurons engage in increased burst firing activity as a consequence of dopamine depletion in experimental models of Parkinson's disease (Bergman et al. 1994). Moreover, this increase in burst firing in STN neurons is thought to contribute to classic symptoms of Parkinson's disease, such as bradykinesia, tremor and rigidity (Benedetti et al. 2009). Deep brain stimulation of the STN, which is now widely used as a treatment of Parkinson's disease, is thought to improve symptoms by disrupting abnormal bursting output from the basal ganglia (Vitek, 1997; Maltete et al. 2007). Because dopamine receptor agonists effectively alleviate many symptoms of Parkinson's disease, it is widely assumed that these agents may disrupt bursting activity in basal ganglia outflow nuclei, although experiments in Parkinson's disease subjects have been inconclusive (Levy et al. 2001). Nevertheless, excessive burst firing in STN, SNR and GPI neurons is now widely established as a likely contributor to symptoms of Parkinson's disease (Bergman et al. 1998; Bevan et al. 2002).

Recent patch-clamp experiments in rodent brain slice preparations have shown that electrical stimulation of the STN evokes complex excitatory postsynaptic currents (EPSCs) in SNR and GPI neurons (Shen & Johnson, 2006; Ammari et al. 2010). The complex EPSC is a long-lasting excitatory current that is composed of an initial, monosynaptic EPSC followed by multiple polyphasic EPSCs that are superimposed on a slow inward shift in holding current. Complex EPSCs are evoked in SNR neurons by stimulation of the STN, whereas stimulation of adjacent white matter fails to evoke the complex EPSC. Although local stimulation of the SNR near the recording pipette evokes a simple, monophasic EPSC, complex EPSCs cannot be evoked by local SNR stimulation. Complex EPSCs can be blocked by local application of ionotropic glutamate antagonists to the STN, which supports the hypothesis that complex EPSCs are generated by activation of polysynaptic glutamate-containing recurrent collaterals within the STN (Shen & Johnson, 2006). Because complex EPSCs generate long-lasting bursts of action potentials when recorded under current-clamp conditions, it is possible that these synaptically mediated burst discharges might contribute to Parkinson's disease symptoms.

Recently, Ammari et al. (2011) showed that acute dopamine depletion augmented the complex EPSC charge that was evoked in murine GPI neurons by STN stimulation. This finding suggests that dopamine might normally exert a tonic inhibitory influence on STN output pathways. Because complex EPSPs underlie synaptically mediated bursts of action potentials, this action of dopamine might be an important mechanism for reducing symptoms of Parkinson's disease. The purpose of the present experiments was to characterize the dopamine receptor pharmacology that regulates synaptic transmission in the subthalamonigral pathway. Using patch pipettes to record currents in SNR neurons in slices of rat brain, we show that dopamine acts via D2-like receptors to inhibit complex EPSCs evoked in SNR neurons by STN stimulation. We also show that dopamine selectively inhibits stimulus-evoked burst firing in SNR neurons, leaving spontaneous firing less affected. Inhibition of burst discharges in the subthalamonigral pathway might represent an important action of D2-like receptor stimulation that reduces symptoms of Parkinson's disease.

Methods

Ethical approval

All experimental procedures were performed in accordance with the ethical guidelines established and approved by the Portland Veterans Affairs Medical Centre Institutional Animal Care and Use Committee. Every precaution was taken to minimize animal stress and the number of animals used.

Tissue preparation

Horizontal slices containing diencephalon and rostral midbrain were prepared from young Sprague–Dawley rats (postnatal day 8–21; Harlan, Indianapolis, IN, USA). Rats were anaesthetized with isoflurane and killed by severing major thoracic vessels. Brains were rapidly removed and slices (400 μm) were cut with a vibrating blade microtome in an ice-cold sucrose buffer solution of the following composition (in mm): sucrose, 196; KCl, 2.5; MgCl2, 3.5; CaCl2, 0.5; NaH2PO4, 1.2; glucose, 20; and NaHCO3, 26; equilibrated with 95% O2–5% CO2. A slice containing the STN and SNR was then placed on a supporting net and submerged in a continuously flowing solution (2 ml min−1) of the following composition (mm): NaCl, 126; KCl, 2.5; CaCl2, 2.4; MgCl2, 1.2; NaH2PO4, 1.2; NaHCO3, 19; glucose, 11; gassed with 95% O2–5% CO2 (pH 7.4) at 36°C. Using a dissection microscope for visual guidance, the SNR was identified 1–2 mm lateral to the substantia nigra zona compacta, whereas the STN was located as grey matter approximately 2.7 mm lateral to the midline and 2 mm rostral to the centre of the SNR.

Electrophysiological recordings

Whole-cell recordings were made with borosilicate pipettes with an initial resistance of 4–5 MΩ. Unless stated otherwise, internal pipette solution contained (in mm): caesium gluconate, 138; MgCl2, 2; CaCl2, 1; EGTA, 11; Hepes, 10; ATP, 1.5; GTP, 0.3 (pH 7.3). Membrane currents were recorded under voltage clamp at −70 mV holding potential and amplified with an Axopatch-1D amplifier (5 kHz low-pass filter). Membrane potentials have been corrected for the liquid junction potential (10 mV). In some experiments, the ‘loose-patch’ method was used to record extracellular currents and potentials (Nunemaker et al. 2003). Patch pipettes for loose-patch recordings were filled with standard extracellular solution; seal resistance was typically 5–10 times the starting value of electrode resistance (3–8 MΩ). Data were acquired using a personal computer with a Digidata analog/digital interface and analysed using pCLAMP software (Molecular Devices, Sunnyvale, CA, USA).

Synaptic currents

As we described previously (Shen & Johnson, 2006), complex EPSCs were evoked in SNR neurons using a bipolar stimulation electrode (2–4 MΩ impedance at 1000 Hz, 10 nA; Frederick Haer & Co., Bowdoin, ME, USA) that was placed directly within the STN. Complex EPSCs were evoked with one or two rectangular pulses (0.1 ms duration, 5 ms interval) of constant current (50–400 μA) every 10–60 s in the presence of picrotoxin (100 μm). Complex EPSCs were quantified as charge by measuring their integrated area using pCLAMP 9 software (Molecular Devices). Simple, monophasic EPSCs were evoked in SNR neurons by electrical stimulation of the SNR within 300 μm of the recording pipette. Simple EPSCs were quantified by measuring their amplitude.

Drugs

All drugs except picrotoxin were dissolved into aqueous or dimethyl sulfoxide stock solutions. Stock solutions of drugs were diluted at least 1:1000 to the desired concentration in superfusate immediately prior to use. Dimethyl sulfoxide, diluted 1:1000 in artificial spinal fluid, had no effect on either holding current or synaptic currents. Approximately 30 s were required for the drug solution to enter the recording chamber; this delay was due to passage of the perfusate through a heat exchanger. In some experiments, dopamine was applied locally by fast flow through a microapplicator (tip diameter 500 μm). The microapplicator was positioned immediately above the brain slice in the region of interest. In order to minimize unwanted spread of dopamine to the recording pipette, we arranged the brain slice in the tissue chamber such that flow from the microapplicator was always parallel to the direction of bath flow. N-(Methyl-4-(2-cyanophenyl)piperazinyl-3-methylbenzamide maleate (PD-168,077), 3-(4-[4-chlo-rophenyl]piperazin-1-yl)-methyl-1H-pyrrolo[2,3-b]pyridine trihydrochloride (L-745,870), 3-[4-(4- chlorophenyl)-4-hydroxypiperidin-l-yl]methyl-1H-indole (L-741,626), (±)-7-hydroxy-2-dipropylaminote-tralin hydrobromide (7-OH-DPAT), N-(trans-4-[2-(6-cyano-3,4-dihydroisoquinolin-2(1H)-yl)ethyl]cyclohexyl) quinoline-4-carboxamide (SB-277,011A), sulpiride, quinpirole, (±)-2-amino-5-phosphonopentanoic acid (AP5) and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX), and picrotoxin were obtained from Tocris Cookson Inc. (Ellisville, MO, USA). Dopamine and SKF-38393 were obtained from Sigma-Aldrich Co. (St Louis, MO, USA).

Data analysis

Numerical data in the text and error bars in figures are expressed as means ± SEM. Analysis of variance with repeated measures (ANOVA with repeated measures (RM)) and Student's paired or un-paired two-tailed t test were used to test for significant differences (SigmaStat software, Jandel Scientific, San Rafael, CA, USA). A significant difference was accepted when P < 0.05. Concentration–response data were fitted to the Hill–Langmuir equation: y = ax/(x + b), where y is the magnitude of effect, a is maximum effect, x is the drug concentration, and b is the concentration that inhibits the effect by 50% (IC50).

Results

Dopamine inhibits complex EPSCs

As shown in Fig. 1A, focal electrical stimulation of the STN evoked a long-lasting complex EPSC in SNR neurons (Shen & Johnson, 2006). This complex EPSC consists of an initial EPSC (early component) followed by a series of notches and inflections (late component) that are superimposed on a slow inward current lasting 200–500 ms. Also shown in Fig. 1A, dopamine (30 μm) reversibly inhibits the late component of the complex EPSC while leaving the initial EPSC unaffected. When recording with internal pipette solutions that contained potassium instead of caesium gluconate, dopamine (30 μm) also evoked a small, reversible outward current of 10 ± 6 pA (n = 8). Averaged data for dopamine-induced inhibition of complex EPSCs show that the dopamine effect begins within 1 min of perfusion and reaches its peak at 5 min (Fig. 1B). Averaged data in Fig. 1C show that dopamine produces a concentration-dependent reduction in complex EPSCs, with an estimated IC50 of 6.4 μm. Dopamine at 30 μm reduced complex EPSCs by 64 ± 3% (n = 31), whereas 100 μm dopamine inhibited complex EPSCs by 71 ± 3% (n = 11). Although both concentrations of dopamine caused significant inhibition (P < 0.001), inhibition caused by 100 μm dopamine was not significantly greater than that caused by 30 μm dopamine (P = 0.2). Thus, 30 μm dopamine produced a near-maximum effect for inhibition of the complex EPSC. In contrast, the dopamine D1/5 receptor agonist SKF-38393 (10 μm) had no consistent effect on complex EPSCs (105 ± 3% of control; n = 8). Thus, future work focused on dopamine D2, D3 and D4 receptor-mediated actions on complex EPSCs.

Figure 1. STN stimulation evokes a complex EPSCs that is inhibited reversibly by dopamine in SNR neurons.

Figure 1

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A, current traces showing that dopamine (30 μm) reversibly inhibits the complex EPSC. Insets show initial components of complex EPSCs displayed at an expanded time scale. Note that dopamine did not reduce the amplitude of the initial EPSC. B, time course showing the reversible inhibition of the complex EPSCs by dopamine (30 μm). Data are from the same neuron shown in A. Numbers indicate those complex EPSCs that are shown in A. C, dopamine concentration–response curve for inhibition of complex EPSCs. Asterisks indicate significant reductions from control (*P < 0.05; **P < 0.001; paired t tests). Parentheses indicate number of observations for each concentration.

Effects of dopamine receptor antagonists

We proceeded to test the abilities of the D2/3 receptor antagonist sulpiride and the selective D4 receptor antagonist L-745,870 to block the action of dopamine on complex EPSCs. As shown in the current traces in Fig. 2A and B, dopamine-induced inhibition of the complex EPSC could not be completely prevented by either sulpiride or L-745,870. However, Fig. 2C shows that concurrent treatment with both antagonists nearly completely blocked the action of dopamine. A summary of these data is shown in Fig. 2D. In the presence of sulpiride (3–10 μm) or L-745,870 (10 μm), dopamine (30 μm) reduced complex EPSCs by 31 ± 5% (n = 7) and 31 ± 2% (n = 17), respectively. These values were significantly smaller than the 64 ± 3% inhibition of complex EPSCs produced by dopamine-alone (P < 0.001). Moreover, superfusing slices with sulpiride plus L-745,870 caused dopamine to inhibit complex EPSCs by only 9 ± 2% (n = 8), which was significantly smaller than that produced by dopamine in the presence of either antagonist alone (P < 0.001). These results suggest that dopamine acts on D2, D3 and D4 receptors to inhibit complex EPSCs.

Figure 2. Dopamine-induced inhibition of the complex EPSC is reduced by D2, D3 and D4 receptor antagonists.

Figure 2

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A and B, current traces showing that dopamine (30 μm) produces some inhibition of the complex EPSC in the presence of either 10 μm sulpiride (A) or 10 μm L-745,870 (B). C, superfusing the slice with both sulpiride (10 μm) and L-745,870 (10 μm) nearly completely blocked the action of dopamine. D, bar graph summarizing data indicating that sulpiride- or L-745,870-alone partially reduces dopamine-induced inhibition of complex EPSCs, whereas the combined application of these two antagonists nearly completely blocked the action of dopamine. Significant differences: *P < 0.001 compared to control dopamine effect; #P < 0.001 compared to sulpiride or L-745,870 alone (t tests).

Effects of dopamine receptor agonists

To further investigate the relative roles of D2versus D3, we tested the effects of the selective D2 receptor agonist quinpirole and the D3 receptor agonist 7-OH-DPAT. These effects were compared to that produced by the selective D4 receptor agonist PD-168,077. As shown in Fig. 3A–C, quinpirole, 7-OH-DPAT, and PD-168,077 each produced significant inhibition of complex EPSCs, and these effects were effectively blocked by the D2 antagonist L-741,626, the mixed D2/3 receptor antagonist sulpiride, and by the D4 receptor antagonist L-745,870, respectively. Summaries of these data are shown in Fig. 3D. The D2 agonist quinpirole (10 μm) inhibited complex EPSCs by 30 ± 2% (n = 10; P < 0.001), and this effect was reduced to 1 ± 1% inhibition (n = 9) by the D2 receptor antagonist L-741,626 (10 μm). Similarly, the D3 agonist 7-OH-DPAT (3 μm) inhibited complex EPSCs by 31 ± 3% (n = 18; P < 0.001), and this effect was reduced to 3 ± 2% inhibition (n = 7) by the mixed D2/3 receptor antagonist sulpiride (10 μm). Inhibition by 7-OH-DPAT was also nearly completely blocked by the selective D3 antagonist SB-277,011A (10 μm; n = 10). Finally, the D4 agonist PD-168,077 (1 μm) inhibited complex EPSCs by 63 ± 4% (n = 13; P < 0.001), and this effect was reduced to 18 ± 5% inhibition (n = 6) by the D4 receptor antagonist L-745,870 (10 μm). Although PD-168,077 produced relatively more inhibition of complex EPSCs than did other agonists, the lack of complete concentration–response data preclude drawing conclusions about relative efficacies of these ligands. However, these results support the conclusion that D2, D3 and D4 receptors participate in regulating synaptic transmission in the subthalamonigral pathway.

Figure 3. Dopamine-induced inhibition of the complex EPSC is mimicked by D2, D3 and D4 receptor agonists.

Figure 3

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A, current traces showing that inhibition of the complex EPSC by the D2 receptor agonist quinpirole (10 μm; upper traces) is antagonized by the selective D2 receptor antagonist L-741,626 (10 μm; lower). B, inhibition of the complex EPSC by the D3 receptor agonist 7-OH-DPAT (3 μm; upper) is blocked by the mixed D2/3 receptor antagonist sulpiride (10 μm; lower). C, inhibition of the complex EPSC by the D4 receptor agonist PD-168,077 (1 μm; upper) is blocked by the selective D4 receptor antagonist L-745,870 (10 μm; lower). D, bar graph summarizing effects of dopamine D2, D3 and D4 receptor agonists and their reversals by antagonists. Each bar represents the mean of 6–18 cells. Asterisks indicate significant reductions in the inhibitory effect of each agonist by their respective antagonist agent (P < 0.001; paired t tests).

Inhibition of complex EPSCs by local application of dopamine

Because previous studies have demonstrated that dopamine can inhibit glutamate synaptic transmission in both the STN and SNR (Shen & Johnson, 2000; Ibañez-Sandoval et al. 2006), inhibition of complex EPSCs could occur by dopamine acting at either site. We used a fast-flow microapplicator to apply dopamine (100 μm) locally to either the STN or SNR to investigate the site of action for inhibition of complex EPSCs. As shown in Fig. 4A, local application of dopamine to the STN produced prominent inhibition of the complex EPSC. In contrast, local application of dopamine to the SNR near the recording pipette produced only a small reduction in complex EPSC (Fig. 4B). As a control experiment, Fig. 4C shows that dopamine application to the internal capsule failed to inhibit the complex EPSC. These data are summarized in Fig. 4D. On average, local application of dopamine to the STN reduced the complex EPSC charge by 55 ± 2%, which was significantly more than the 15 ± 2% reduction produced by dopamine application to the SNR (P < 0.001, n = 8, paired t test). These data suggest that the STN is the more important site of action for dopamine-induced inhibition of complex EPSCs.

Figure 4. Local application of dopamine to STN effectively inhibits complex EPSCs.

Figure 4

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A–C, current traces showing effects of local fast-flow application of dopamine (100 μm) to the STN (A), SNR (B), and internal capsule (C). D, averaged data showing that local application of dopamine to the STN caused significantly greater inhibition compared to application to SNR (*P < 0.001, paired t test). Dopamine had no effect when applied to the internal capsule. Data were obtained from 8 SNR neurons.

Dopamine actions on monophasic EPSCs

To further explore the possible site of dopamine action, we studied the effect of dopamine and dopamine agonists on monophasic, simple EPSCs that were evoked in SNR neurons by local electrical stimulation of the SNR near the recording site. Recorded in picrotoxin (100 μm), these EPSCs were completely blocked by superfusing the slice with the glutamate receptor antagonists AP5 (50 μm) and NBQX (10 μm; data not shown). As shown in Fig. 5A, dopamine (30 μm) caused a relatively small reduction in monophasic EPSC amplitude. Averaged data (Fig. 5B) show that dopamine caused a 20 ± 2% reduction in the amplitude of monophasic EPSCs (n = 14). Although this effect was statistically significant (P < 0.001, paired t test), it was much smaller than the 64% reduction in complex EPSCs that was produced by this same concentration of dopamine (see Fig. 1C). Effects of dopamine agonists are summarized in Fig. 5B. On average, monophasic EPSCs were reduced 9 ± 2% by 10 μm quinpirole (n = 11), 9 ± 1% by 3 μm 7-OH-DPAT (n = 12), and 12 ± 1% by 1 μm PD-168,077 (n = 21). Although each of these reductions in monophasic EPSC amplitude was statistically significant (P < 0.01), each effect was much smaller than the 30–63% reductions in complex EPSCs that were produced by the same concentrations of dopamine agonist (see Fig. 3D). Thus, it appears that an action within the SNR is not likely to account for the strong dopamine-induced inhibition of the complex EPSC.

Figure 5. Effects of dopamine receptor stimulation on monophasic EPCS evoked by local SNR stimulation.

Figure 5

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A, current traces showing that 30 μm dopamine causes a modest reduction in the amplitude of the monophasic EPSC. B, bar graph summarizing effects of dopamine and dopamine agonists on monophasic EPSC amplitudes. Each bar is the average response of 11–21 neurons. Dopamine (30 μm), quinpirole (10 μm), 7-OH-DPAT (3 μm) and PD-168,077 (1 μm) caused small but significant reductions in EPSC amplitude (P < 0.01, paired t tests).

Dopamine inhibits stimulus-evoked burst firing

It is now well-established by our lab and others that complex EPSCs cause bursts of action potentials when SNR neurons are recorded in current-clamp mode (Shen & Johnson, 2006; Ammari et al. 2010). Having established that dopamine suppresses complex EPSCs, we then tested the effect of dopamine on stimulus-evoked burst firing in SNR neurons recorded in loose-patch configuration with pipettes that were filled with normal extracellular solution. As shown in Fig. 6A, dopamine (30 μm) in the superfusate caused a reversible suppression of stimulus-evoked action potentials while also slowing spontaneous action potential discharge. Stimulus-evoked bursts recovered 10–15 min after dopamine washout. Averaged data in Fig. 6B show that 30 μm dopamine reduced the number of spikes measured up to 100 ms after the stimulus from a control value of 8.6 ± 1.3 to 3.1 ± 0.5 spikes (n = 12; P < 0.001, one-way ANOVA with RM). Figure 6B also shows that this concentration of dopamine reduced the spontaneous firing rate from a control value of 6.4 ± 0.8 to 4.2 ± 0.8 Hz (n = 12; P < 0.001, one-way ANOVA with RM). We then normalized these data by expressing the effect of dopamine in terms of percentage of control values. Results in Fig. 6C show that dopamine caused a significantly greater reduction in stimulus-evoked spikes (36 ± 5% of control) compared to reduction in spontaneous firing rate (59 ± 9% of control; P < 0.01, paired t test).

Figure 6. Loose-patch recordings show that dopamine and PD-168,077 inhibit stimulus-evoked firing to a greater extent than inhibition of spontaneous firing.

Figure 6

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Bursts of actions potentials were evoked in SNR neurons by focal STN stimulation. Patch pipettes were filled with normal extracellular solution. A, voltage trace showing that dopamine (30 μm) greatly inhibits the stimulus-evoked burst of action potentials and slows spontaneous firing. B, summarized data showing inhibition of stimulus-evoked spike number and spontaneous firing rate by 30 μm dopamine (n = 12). C, normalized data from B showing that 30 μm dopamine caused a significantly greater reduction in evoked spikes compared to inhibition of tonic firing rate. D, voltage trace showing that PD-168,077 (1 μm) reversibly eliminates the stimulus-evoked burst and decelerates the rate of tonic firing. E, summarized data showing inhibition of stimulus-evoked spike number and tonic firing rate by PD-168,077 (1 μm; n = 5). F, normalized data from E showing that PD-168,077 (1 μm) produced a significantly greater reduction in evoked spikes compared to inhibition of tonic firing rate. Arrow heads in A and D denote stimulus artifacts. Stimulus-evoked spikes were counted during the 100 ms interval immediately following STN stimulation, whereas tonic firing rate was measured in the absence of stimulation. Asterisks indicate significant differences from control (*P < 0.05; **P < 0.01; ***P < 0.001).

Because the D4 agonist PD-168,077 caused relatively strong inhibition of complex EPSCs (Fig. 3D), we also examined the effect of this agonist on stimulus-evoked burst firing. As shown in Fig. 6D, PD-168,077 (1 μm) reversibly suppressed stimulus-evoked action potentials while slowing spontaneous action potential discharge. Averaged data in Fig. 6E show that 1 μm PD-168,077 reduced the number of evoked spikes from a control value of 7.4 ± 1.0 to 4.2 ± 0.7 (n = 5; P < 0.01, one-way ANOVA with RM). Figure 6E also shows that this concentration of PD-168,077 reduced the spontaneous firing rate from a control value of 6.4 ± 0.5 to 5.4 ± 0.7 Hz (n = 5; P < 0.05, one-way ANOVA with RM). We then normalized these data by expressing the effect of PD-168,077 in terms of percentage of control values. Results in Fig. 6F show that PD-168,077 caused a significantly greater reduction in stimulus-evoked spikes (57 ± 5% of control) compared to reduction in spontaneous firing rate (84 ± 7% of control; P < 0.05, paired t test). These data show that dopamine and PD-168,077 cause preferential inhibition of stimulus-evoked bursts compared to inhibition of spontaneous activity.

Discussion

Our results showed that the strong and reversible inhibition of complex EPSCs by dopamine was mediated by activation of D2, D3 and D4 receptors. Thus, the dopamine-induced inhibition of complex EPSCs could be partially mimicked by the D2 agonist quinpirole, by the D3 agonist 7-OH-DPAT, and the D4 agonist PD-168,077. Moreover, simultaneous application of the mixed D2/3 antagonist sulpiride and the D4 antagonist L-745,870 nearly completely blocked the inhibitory action of dopamine. These results show that the D2 family of dopamine receptors underlies the inhibitory action of dopamine on synaptic transmission in the subthalamonigral pathway.

Site of action of dopamine

In theory, an inhibitory effect of dopamine on subthalamonigral transmission could occur by activation of D2-like receptors that are located in either the STN or the SNR. In support of the STN as the dominant site of action of dopamine, our results showed that fast-flow, local application of dopamine to the STN was more effective for inhibition of complex EPSCs than was application to the SNR. However, we also showed that dopamine produced a small but significant reduction in the monophasic EPSC that was evoked in SNR neurons by local stimulation near the recording pipette. This result agrees with in vivo microdialysis studies showing that local application of quinpirole to the SNR reduced glutamate release evoked in the SNR by STN stimulation (Hatzipetros & Yamamoto, 2006). Furthermore, Ibañez-Sandoval et al. (2006) used paired-pulse studies to show that inhibition of monophasic EPSCs by dopamine was due to presynaptic stimulation of D2-like receptors in the SNR. But our finding that dopamine inhibited the complex EPSC more effectively than inhibition of the monophasic EPSC suggests that D2-like receptors located outside the SNR are more significant for this effect of dopamine. In support of this hypothesis, previous studies showed that the long-duration, multiphasic complex EPSC is mediated by polysynaptic connections in the STN (Shen & Johnson, 2006; Ammari et al. 2010). Histological studies have also shown the existence of a small but significant number of recurrent collateral innervations in the STN (Kita et al. 1983; Sato et al. 2000). Moreover, polysynaptic transmission is clearly more sensitive than monosynaptic transmission to inhibitors of synaptic function (Berry & Pentreath, 1976; Rose & Metherate, 2005). Therefore, D2-like receptors located in the STN are likely to be most significant for inhibition of complex EPSCs by dopamine.

Distribution of D2-like receptors

Regional distributions of D2-like receptors in the basal ganglia should also be considered when assessing possible sites of action for dopamine-induced inhibition of complex EPSCs. Flores et al. (1999) demonstrated that the rat STN expresses significant mRNA and receptor binding for D2 and D3 dopamine receptors. Thus, dopamine could inhibit complex EPSCs by activating D2 and D3 receptors that are located either on STN neurons or on nerve terminals of the subthalamonigral pathway. SNR neurons also reportedly express low levels of mRNA for both D2 and D3 receptors (Bouthenet et al. 1991; Gurevich & Joyce, 1999). However, the fact that most D2-like receptor binding in the rat substantia nigra largely disappears after 6-hydroxydopamine treatment suggests that D2-like receptors are predominantly expressed by substantia nigra dopamine neurons rather than SNR neurons (Bouthenet et al. 1987).

With regard to D4 expression, mRNA for this receptor is reportedly absent in STN and SNR in rat brain (Flores et al. 1999; Noaín et al. 2006), although human STN reportedly expresses D4 message (Matsumoto et al. 1996). Although D4 mRNA is absent in rat tissue, both the STN and SNR contain binding for this receptor (Mrzljak et al. 1996; Flores et al. 1999), which suggests that the D4 receptor resides on nerve terminals that are afferent to the STN and SNR. D4 receptor stimulation reportedly inhibits GABA release in both STN and SNR, which suggests that this receptor may reside on nerve terminals of inputs that arise from GPI or striatum (Rivera et al. 2003; Floran et al. 2004; Acosta-Garcìa et al. 2009). Lack of D4 expression by STN neurons is also consistent with the report that agonists at D2 and D3– but not D4– receptors inhibit calcium-dependent K+ currents in STN neurons from rat brain (Ramanathan et al. 2008). Although D4 agonists have been reported to inhibit glutamate-mediated EPSCs evoked in GPI neurons by STN stimulation, paired-pulse experiments suggested that this was a postsynaptic action on GPI neurons rather than a presynaptic action to reduce glutamate release (Hernández et al. 2006). Another possible source of D4 binding in STN is on nerve terminals from cerebral cortical neurons, which are known to have robust expression of D4 in rat brain (Noaín et al. 2006; Rivera et al. 2008). Thus, it is possible that D4 receptor activation could reduce glutamate release in the corticosubthalamic pathway and thereby inhibit the stimulus-induced complex EPSC in SNR neurons. The location of D4 receptors in SNR is less clear. However, our results showing that the D4 agonist PD-168,077 inhibited monophasic EPSCs in SNR neurons suggest that this receptor has a significant action in the SNR. Although it is possible that dopamine could act within either the STN or the SNR to inhibit complex EPSCs, reports on the distribution of D2-like receptors in rat brain tend to favour the STN as the more important site of action.

Dopamine has multiple actions

Previous studies have shown that dopamine exerts a depolarizing, excitatory influence on STN neurons due to a D2 receptor-mediated reduction of a resting K+ conductance (Mintz et al. 1986; Zhu et al. 2002). By regulating intrinsic membrane properties through this mechanism, dopamine exerts an important influence on firing pattern of STN neurons (Beurrier et al. 1999; Baufreton et al. 2005; Wilson & Bevan, 2011). However, results of the present study suggest that dopamine at D2-like receptors has a net inhibitory effect on synaptic transmission in the subthalamonigral pathway. Thus, it would appear that the postsynaptic excitatory effect of dopamine on STN neurons is counterbalanced by an inhibitory action of dopamine to limit transmission through polysynaptic connections in the STN. One might predict that the effect of dopamine would be to limit polysynaptic transmission in the subthalamonigral pathway, while maintaining intrinsic excitability of STN neurons and preserving monosynaptic output from the STN.

Selective inhibition of stimulus-evoked bursts

Because complex EPSCs evoke bursts of action potentials in SNR neurons when recorded under current clamp conditions, it is not surprising that we showed that dopamine also effectively inhibited bursts of spikes evoked by STN stimulation. However, we also showed that dopamine inhibited stimulus-evoked bursts more effectively than it reduced spontaneous firing of action potentials. This selective action of dopamine was observed despite the presence of a small outward current that would be expected to inhibit spontaneous and evoked activity equally. In fact, we may have underestimated the selectivity of this action of dopamine because evoked spikes were counted up to 100 ms after each stimulus, which would have included some spontaneous spikes as well as evoked spikes. This selective action of dopamine, which we have also demonstrated previously for the GABAB agonist baclofen (Shen & Johnson, 2012), is likely to be related to a preferential inhibition of polysynaptic transmission by dopamine in the STN. Because excessive burst firing in the subthalamonigral pathway is linked to symptoms of Parkinson's disease (Maltete et al. 2007; Benedetti et al. 2009), suppression of synaptically induced bursts by dopamine might have therapeutic potential in the treatment of this disease. It is possible that D2-like agonists such as pramipexole and ropinirole that are used in the treatment of Parkinson's disease might owe some of their clinical efficacy to inhibition of synaptically generated burst firing in the subthalamonigral pathway.

Concluding remarks

In summary, our results show that dopamine acts at D2, D3 and D4 receptors to inhibit synaptic transmission in the subthalamonigral pathway. Selective inhibition of stimulus-dependent bursts of action potentials in the SNR is most likely due to suppression of polysynaptic connections in the STN. We suggest that inhibition of synaptically generated burst firing by D2 receptor agonists may be a clinically important mechanism for symptomatic treatment of Parkinson's disease.

Acknowledgments

This work was supported by United States Public Health Service grants NS38715 and NS060662, and by the Portland Veterans Affairs Parkinson's Disease Research, Education, and Clinical Center.

Glossary

Abbreviations

GPI

globus pallidus interna

SNR

substantia nigra zona reticulata

STN

subthalamic nucleus

Author contributions

K-Z.S. performed data collection and analysis and wrote the initial draft of the Methods and Results sections of the manuscript; S.W.J. conceived and designed experiments, wrote the Introduction and Discussion sections, and put the manuscript and figures in final form for publication. All work was done at the Portland VA Medical Center. Both authors approved the final version for publication.

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