Reversal of quinpirole inhibition of ventral tegmental area neurons is linked to the phosphatidylinositol system and is induced by agonists linked to G_q

Abstract

Putative dopaminergic (pDAergic) ventral tegmental area neurons play an important role in brain pathways related to addiction. Extended exposure of pDAergic neurons to moderate concentrations of dopamine (DA) results in a time-dependent decrease in sensitivity of pDAergic neurons to DA inhibition, a process called dopamine inhibition reversal (DIR). We have shown that DIR is mediated by phospholipase C and conventional protein kinase C through concurrent stimulation of D2 and D1-like receptors. In the present study, we further characterized this phenomenon by using extracellular recordings in brain slices to examine whether DIR is linked to phosphatidylinositol (PI) or adenylate cyclase (AC) second-messenger pathways. A D1-like dopaminergic agonist associated with PI turnover (SKF83959), but not one linked to AC (SKF83822), promoted reversal of inhibition produced by quinpirole, a dopamine D2-selective agonist. Other neurotransmitter receptors linked to PI turnover include serotonin 5-HT₂, α₁-adrenergic, neurotensin, and group I metabotropic glutamate (mGlu) receptors. Both serotonin and neurotensin produced significant reversal of quinpirole inhibition, but agonists of α₁-adrenergic and group I mGlu receptors failed to significantly reverse quinpirole inhibition. These results indicate that some agonists that stimulate PI turnover can facilitate desensitization of D2 receptors but that there may be other factors in addition to PI that control that interaction.

changes in dopaminergic (DAergic) neurotransmission in the ventral tegmental area (VTA) and its targets have been related to salient and motivational stimuli and are important for the reward and reinforcement of numerous drugs of abuse (Di Chiara and Imperato 1988; Mirenowicz and Schultz 1996; Wise 1996). Among these stimuli, the effects of addictive drugs such as cocaine and ethanol are much more potent and longer lasting than the effects of other stimuli (Tobler et al. 2005). The effect of increased dopamine concentrations in the VTA is not known, but elevated dopamine may affect the excitability of DAergic neurons of the VTA and may produce long-term changes in neurotransmission; for example, elevated dopamine can increase glutamatergic receptor expression in prefrontal cortex (Gao and Wolf 2008; Sun et al. 2008).

Based on structural homology and functional and pharmacological profiles, five classes of dopamine receptors have been identified: two “D1-like” receptors (D1 and D5) and three “D2-like” receptors (D2, D3, and D4) (Ciliax et al. 2000; Khan et al. 2000; Neve et al. 2004; Sibley et al. 1993). Some evidence suggests the existence of possible D6 and D7 receptors, which have physiological and pharmacological actions similar to those of D1 and D2, but such receptors have not been conclusively identified (Murphy 2000). Of the five main classes of dopamine receptors, the DAergic neurons of the VTA possess high densities of D2 (Bouthenet et al. 1991) and D5 receptors (Ciliax et al. 2000; Khan et al. 2000) but low levels of D3 receptors (Bouthenet et al. 1991; Diaz et al. 1995; Gurevich and Joyce 1999). D1 and D4 receptors are quite sparse or are not detectable in the DAergic VTA neurons (Meador-Woodruff et al. 1992; Mengod et al. 1992; Rivera et al. 2008). However, D1 receptors have been reported to be located on presynaptic glutamatergic terminals projecting to the region, not on the DAergic VTA neurons themselves (Caillé et al. 1996).

Putative DAergic (pDAergic) VTA neurons fire action potentials spontaneously in vivo (Bunney et al. 1973) and in vitro (Brodie and Dunwiddie 1987). This spontaneous firing is inhibited by the action of dopamine at D2 autoreceptors on the cell bodies and dendrites of these neurons (Lacey et al. 1987). Inhibition of firing of pDAergic VTA neurons by extracellular dopamine has been reported by many laboratories (Brodie et al. 1990; Lacey et al. 1987). However, we demonstrated that prolonged elevation of dopamine results in a time- and concentration-dependent decrease in the magnitude of dopamine-induced inhibition, a phenomenon that we termed “dopamine inhibition reversal,” or DIR (Nimitvilai and Brodie 2010). This DIR is produced by concurrent stimulation of D2 and D1-like receptors, requires 10–40 min to develop, and persists for up to 90 min (Nimitvilai and Brodie 2010). We also reported that reversal of either dopamine or quinpirole inhibition is mediated by activation of phospholipase C (PLC) and conventional protein kinase C (cPKC), without the involvement of adenylyl cyclase (AC), cyclic AMP, and protein kinase A (Nimitvilai et al. 2011). PLC and PKC can be activated by phosphatidylinositol (PI) via the G_q protein; activation of D2-D1 and D2-D5 heterooligomers produces an increase in PLC and PKC activity through G_q/PI pathway (Lee et al. 2004; Rashid et al. 2007; So et al. 2005). In this study, we used selective agonists to examine whether DIR is the result of concurrent stimulation of D2- and D1-like receptors linked to the G_q/PI pathway or to the AC pathway.

Autoregulation of D2 receptors in VTA neurons can be modulated by many neurotransmitters such as glutamate, GABA, acetylcholine, norepinephrine, dynorphin, neurotensin, and serotonin. Among these neurotransmitters, glutamate, norepinephrine, neurotensin, and serotonin have been linked to G_q, PLC, and PKC pathway. Activation of these receptors may play an important role in modulating D2 receptor sensitivity in VTA neurons. In the present study, therefore, we also examined whether activation of these other G_q-coupled receptors can produce a decrease in sensitivity of D2 receptor to its agonists. Extracellular recordings from pDAergic VTA neurons in rat brain slices were used in all experiments to avoid disrupting the intracellular milieu while monitoring spontaneous firing of these neurons for long continuous time periods.

METHODS

Animals.

Fischer 344 adult rats (4–6 wk old, 90–150 g) used in these studies were obtained from Harlan Sprague Dawley (Indianapolis, IN). All rats were treated in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all experimental methods were approved by the Animal Care Committee of the University of Illinois at Chicago.

Preparation of brain slices.

Brain slices containing the VTA were prepared from the subject animals as previously described (Brodie et al. 1999). Briefly, following brief isoflurane anesthesia and rapid removal of the brain, the tissue was blocked coronally to contain the VTA and substantia nigra; the cerebral cortices and a portion of the dorsal mesencephalon were removed. The tissue block was mounted in the vibratome and submerged in chilled cutting solution to cut coronal sections (400 μm thick). Each slice was placed onto a mesh platform in the recording chamber and was totally submerged in artificial cerebrospinal fluid (aCSF) maintained at a flow rate of 2 ml/min; the temperature in the recording chamber was kept at 35°C. The composition of the aCSF in these experiments was (in mM) 126 NaCl, 2.5 KCl, 1.24 NaH₂PO₄, 2.4 CaCl₂, 1.3 MgSO₄, 26 NaHCO₃, and 11 glucose. The composition of the cutting solution was (in mM) 2.5 KCl, 2.4 CaCl₂, 1.3 MgSO₄, 26 NaHCO₃, 11 glucose, and 220 sucrose. Both solutions were saturated with 95% O₂-5% CO₂ (pH 7.4). Equilibration time of at least 1 h was allowed after placement of tissue in the recording chamber before electrodes were placed in the tissue.

Cell identification.

The VTA was clearly visible in the fresh tissue as a gray area medial to the darker substantia nigra and separated from the nigra by white matter. Recording electrodes were placed in the VTA under visual control. pDAergic neurons have been shown to have distinctive electrophysiological characteristics (Grace and Bunney 1984; Lacey et al. 1989). Only those neurons that were anatomically located within the VTA and that conformed to the criteria for pDAergic neurons established in the literature and in this laboratory (Chieng et al. 2011; Lacey et al. 1989; Mueller and Brodie 1989) were studied. These criteria include broad action potentials (2.5 ms or greater, measured as the width of the bi- or triphasic waveform at the baseline), slow spontaneous firing rate (0.5–5 Hz), and a regular interspike interval. Cells were not tested with opiate agonists as has been done by other groups to further characterize and categorize VTA neurons (Chieng et al. 2011; Margolis et al. 2006).

Additional characterization, such as determining the projection target of our cells of study (Margolis et al. 2008), would have been difficult because we used extracellular recording to ensure high-quality, long-duration recordings. The long-duration, low-frequency action potentials that characterized the cells from which we recorded are associated with dopamine-sensitive, dopamine-containing neurons projecting to the nucleus accumbens, and dopamine sensitivity also is associated with DAergic VTA neurons projecting to prefrontal cortex (Margolis et al. 2008). One consequence of differential initial sensitivity to dopamine inhibition among groups of neurons projecting to different brain areas (Lammel et al. 2008; Margolis et al. 2008) would be different amounts of DIR (Nimitvilai and Brodie 2010), resulting in a greater relative change in neurons more sensitive to dopamine inhibition.

Drug administration.

In most experiments, drugs were added to the aCSF by means of a calibrated infusion pump from stock solutions 100 to 1,000 times the desired final concentrations. The addition of drug solutions to the aCSF was performed in such a way as to permit the drug solution to mix completely with aCSF before this mixture reached the recording chamber. Final concentrations were calculated from aCSF flow rate, pump infusion rate, and concentration of drug stock solution. The small volume chamber (about 300 μl) used in these studies permitted the rapid application and washout of drug solutions. Typically drugs reach equilibrium in the tissue after 2–3 min of application.

In some experiments, drugs were added to the microelectrode filling solution (0.9% NaCl) at a concentration about 10 times greater than that which would have been used in the extracellular medium. To allow time for the drug to diffuse from the pipette to the cell, the effects of bath-applied drugs were tested no less than 20 min after the recording was initiated; this pipette application method has produced results comparable to the administration of drugs through the extracellular medium in the cases in which both methods were tested (data not shown), with the advantage of more localized application and reduced expense. Such local delivery of drugs through recording pipettes has been used by our laboratory and others (Nimitvilai et al. 2011; Pesavento et al. 2000; Sokolov and Kleschevnikov 1995). One disadvantage of this method is that the exact concentration of drug received by the neurons from which we recorded is unknown.

Quinpirole, neurotensin, serotonin, ketanserin, phenylephrine, and most of the salts used to prepare the extracellular media were purchased from Sigma (St. Louis, MO). SKF83822 hydrobromide [6-chloro-2,3,4,5-tetrahydro-1-(3-methyl phenyl)-3-(2-propenyl)-1H-3-benzazepine-7,8-diol hydrobromide], SKF83959 hydrobromide [6-chloro-2,3,4,5-tetrahydro-3-methyl-1-(3-methylphenyl)-1H-3-benzazepine-7,8-diol], SCH39166 hydrobromide [(6aS-trans)-11-chloro-6,6a,7,8,9,13b-hexahydro-7-methyl-5H-benzo[d]naphth[2,1-b]azepin-12-ol hydrobromide], Gö6976 (5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile), SR142948 (2-[[[5-(2,6-dimethoxyphenyl)-1-[4-[[[3-(dimethylamino)propyl]methylamino]carbonyl]-2-(1-methylethyl)phenyl]-1H-pyrazol-3-yl]carbonyl]amino]-tricyclo[3.3.1.13,7]decane-2-carboxylic acid), and DHPG [(RS)-3,5-dihydroxy phenylglycine] were purchased from Tocris (Ellisville, MO).

Extracellular recording.

Extracellular recording was chosen for these studies because this method permits the recordings to be of long duration and allows us to assess the effects of extended exposure (>60 min) to drugs. The limitation of only measuring spontaneous action potential frequency (rather than membrane potential or other electrophysiological parameters) is counterbalanced by the advantage of being able to determine the time course of drug actions and interactions. Extracellular recording electrodes were made from 1.5-mm-diameter glass tubing with filament and were filled with 0.9% NaCl. Tip resistance of the microelectrodes ranged from 2 to 5 MΩ. A Fintronics amplifier was used in conjunction with an IBM personal computer-based data acquisition system (ADInstruments, Colorado Springs, CO). Off-line analysis was used to calculate, display, and store the frequency of firing in 1-min intervals. Additional software was used to calculate the firing rate over 5-s intervals. Firing rate was determined before and during drug application. Firing rate was calculated over 1-min intervals before administration of drugs and during the drug effect; peak drug-induced changes in firing rate are expressed as the percent change from the control firing rate according to the formula [(FRD − FRC)/FRC] × 100, where FRD is the firing rate during the peak drug effect and FRC is the control firing rate. The change in firing rate thus is expressed as a percentage of the initial firing rate, which controls for small changes in firing rate that may occur over time. This formula was used to calculate both excitatory and inhibitory drug effects. Peak excitation produced by the drug (e.g., dopamine) was defined as the peak increase in firing rate over predrug baseline. Inhibition was defined as the lowest firing rate below the predrug baseline. Inhibition reversal was identified as a statistically significant reduction in the inhibition during the period of drug application.

Data collection.

For comparison of the time course of effects on firing rate, the data were normalized and averaged. Firing rates over 1-min intervals were calculated and normalized to the 1-min interval immediately preceding dopamine administration. These normalized data were averaged by synchronizing the data to the dopamine administration period, and graphs of the averaged data were made.

Statistical analysis.

Averaged numerical values are means ± SE. Mean response graphs are shown as relative change in firing rate normalized to the inhibition observed in the first 5-min interval; in these cases, the mean percentage of inhibition as a function of baseline firing rate is indicated in the text. Statistical significance of data from different drug conditions and among firing rates during the long drug administration intervals in these studies were assessed with a Student's t-test or one-way repeated-measures ANOVA as appropriate; degrees of freedom and statistical error terms are shown as subscripts to df or F, respectively, in the text (Kenakin 1987). Statistical analyses were performed with OriginPro 8.5 (OriginLab, Northampton, MA.).

RESULTS

VTA neuron characteristics.

A total of 112 VTA neurons were examined. Their firing rate in normal extracellular medium ranged from 0.74 to 4.58 Hz, with a mean of 2.36 ± 0.1 Hz. All neurons had regular firing rates and were inhibited by dopamine agonists. Quinpirole (25–200 nM) was administered for 5 min, and the concentration was increased if inhibition >50% was not achieved. The concentrations of agonist were adjusted for each neuron so that inhibition exceeded 50%, because inhibition that was <50% was not reliably reversed (Nimitvilai and Brodie 2010). This method of adjusting the concentration of DAergic agonist controlled for differences in sensitivity between neurons but also sometimes resulted in the mean concentrations of quinpirole slightly differing between groups. The mean concentration of quinpirole administered to each group and the mean resultant inhibition are shown in Table 1. Overall, for pDAergic VTA neurons from adult rats that were administered quinpirole in a stepwise manner, the concentration of quinpirole used was 72.73 ± 5.79 nM, which produced a mean change in firing rate of −67.3 ± 1.81%. There were no significant differences in the concentration of DAergic agonists or in the percentage inhibition among the groups (1-way ANOVA, P > 0.05). In some experiments, a single dose of quinpirole (based on stepwise administration experiments) was used to examine whether stepwise increasing quinpirole concentration had an effect on the development of reversal of quinpirole inhibition by DHPG or serotonin (Table 1). Cells that did not return to at least 70% of their pre-DA firing rate during this washout were not used. One benefit of the extracellular recording method used in these studies is that long-duration recordings can be made reliably; the average recording duration was 93.98 ± 0.83 min, with a range of 90 to 105 min.

Table 1.

Concentration of quinpirole administered to each group and resultant change in firing rate

Condition	Quinpirole, nM	Change in Firing Rate at 10 min, %
Stepwise increase of quinpirole concentration
Quinpirole	45 ± 5.75	−79.08 ± 8.0
Quinpirole + SKF83822	44.64 ± 9.7	−75.24 ± 5.3
Quinpirole + SKF83959	46.07 ± 9.48	−68.59 ± 5.6
Quinpirole + SKF83959 + SCH39166	50.83 ± 10.83	−67.67 ± 6.5
Quinpirole + SKF83959 + Gö6976	65 ± 15.8	−70.45 ± 6.4
Quinpirole + serotonin	112.5 ± 22.16	−69.55 ± 6.9
Quinpirole + serotonin + ketanserin	78.57 ± 13.83	−74.25 ± 10.2
Quinpirole + ketanserin	56.25 ± 15.7	−82.37 ± 5.4
Quinpirole + neurotensin	51.88 ± 8.96	−57.56 ± 5.9
Quinpirole + neurotensin + SR142948	49.17 ± 10.9	−84.43 ± 6.7
Quinpirole + SR142948	78 ± 14.28	−81.4 ± 9.9
Quinpirole + phenylephrine	76.67 ± 8.33	−76.83 ± 5.9
Quinpirole + DHPG	91.67 ± 15.37	−63.43 ± 7.6
Single concentration of quinpirole
Quinpirole	45	−62.31 ± 9.5
Quinpirole + DHGP	91	−49.77 ± 8.1
Quinpirole + serotonin	112.5	−68.21 ± 6.9

Values are means ± SE. See text for definitions.

In the course of performing the experiments described below, we used a number of pharmacological agents, delivered via either the extracellular medium or the recording pipette, and these agents were applied for at least 15–35 min before the 40-min administration of quinpirole.

D1/D5 agonist linked to PI, but not to AC, pathway reverses quinpirole inhibition of firing.

Classical activities of dopamine D2 and D1/D5 receptors are to inhibit and stimulate, respectively, the AC/cAMP pathway (Missale et al. 1998; Stoof and Kebabian 1981). However, there is evidence of the action of DAergic agonists on dopamine receptors linked to the PI and PLC systems (Felder et al. 1989; Undie et al. 1994). In addition, concurrent stimulation or heterooligomerization of D2 and D1-like receptors can lead to an activation of PI/PLC pathway (Lee et al. 2004; Rashid et al. 2007; So et al. 2005). We examined the effects of two D1/D5 agonists, SKF83822 and SKF83959, on prolonged application of quinpirole (Fig. 1). Activation of D1/D5 receptors by SKF83822 stimulates AC/cAMP without affecting the PI pathway (O'Sullivan et al. 2004), whereas activation of D1/D5 receptors by SKF83959 stimulates only the PI pathway (Jin et al. 2003; Panchalingman and Undie 2001). Either SKF83822 (100 μM) or SKF83959 (100 μM) was dissolved in saline. The saline alone or saline containing drug was used to fill the recording electrodes. These electrodes were used to make the extracellular recordings of single pDAergic VTA neurons. After the recording of pDAergic neurons was obtained and the drug in the pipette was allowed to act locally for at least 20 min, concentrations of quinpirole were applied in the superfusate in a stepwise fashion, in which each concentration was added for 5 min and then increased until inhibition of 50% or greater was achieved. Quinpirole alone (45 ± 5.75 nM, n = 8) produced a persistent inhibition in firing rate that did not significantly reverse over the time course [1-way repeated-measures ANOVA, F_(7,49) = 1.00, P > 0.05]. When SKF83822 (100 μM) was included in the recording electrode 20 min before quinpirole administration, quinpirole (44.64 ± 9.67 nM; n = 7) produced an inhibition of 70.06 ± 7.2% at the 5-min time point, and this inhibition was sustained over the duration of drug application [1-way repeated-measures ANOVA, F_(7,42) = 1.55, P > 0.05]. With SKF83959 (100 μM) in the recording pipette, however, quinpirole (46.07 ± 9.48 nM; n = 14) produced an inhibition of 65.16 ± 4.04% at the 5-min time point, and this inhibition significantly reversed with time so that the inhibitory effect of quinpirole at the last time point was significantly different from the inhibitory effect at the first three time points [1-way repeated-measures ANOVA, F_(7,91) = 4.44, P < 0.05] (Fig. 1). No significant change in firing rate was observed with SKF83822 alone [1-way repeated-measures ANOVA, F_(11,22) = 0.28, P > 0.05] or SKF83959 alone [1-way repeated-measures ANOVA, F_(11,22) = 0.52, P > 0.05] over 60 min (data not shown).

Fig. 1.

Reversal of quinpirole inhibition required coadministration of quinpirole and D1/D5 agonist linked to the phosphatidylinositol (PI) pathway, but not D1/D5 agonist linked to the adenylyl cyclase (AC) pathway. Relative change in firing rate (mean ± SE) in response to long-duration quinpirole application in the absence or presence of either SKF83822 or SKF83959 is plotted as a function of time. Effect of quinpirole at each time point was normalized by subtracting the change in firing rate (%) at the 5-min time point. A concentration of quinpirole that produced inhibition of 50% or greater was applied for 40 min. Quinpirole alone (■, [Q] = 45 ± 5.75 nM, n = 8) produced inhibition of the firing rate that did not significantly reverse over the 40-min duration of quinpirole application [1-way repeated-measures ANOVA, F_(7,49) = 1.00, P > 0.05]. In the presence of 100 μM SKF83822 in the pipette (●, [Q] = 44.64 ± 9.67 nM, n = 7), quinpirole produced an inhibition in firing rate, and this inhibition was not significantly changed for the duration of quinpirole application [1-way repeated-measures ANOVA, F_(7,42) = 1.55, P > 0.05]. In the presence of 100 μM SKF83959 in the pipette (▼, [Q] = 46.07 ± 9.48 nM, n = 14), there was a significant reduction in quinpirole inhibition over time, with the last time point significantly different from the first 3 time points [1-way repeated-measures ANOVA, F_(7,91) = 4.44, P < 0.05]. When 100 μM SKF83959 and 100 μM SCH39166 were included in the pipette (▽, [Q] = 50.83 ± 10.83 nM, n = 6), quinpirole significantly inhibited the firing rate, with no reversal over time [1-way repeated-measures ANOVA, F_(7,35) = 4.32, P < 0.05].

We also examined whether inhibition of D1/D5 receptors by D1/D5 antagonist SCH39166 suppressed SKF83959 reversal of quinpirole-induced inhibition. Saline containing SKF83959 (100 μM) and SCH39166 (100 μM) was used to fill the recording pipettes, and these pipettes were used to record changes in firing rate of pDAergic neurons. After 20-min administration of SKF83959 and SCH39166, concentrations of quinpirole were added in the superfusate in a stepwise fashion, in which each concentration was applied for 5 min until inhibition of 50% or greater was achieved, and these concentrations were sustained for 40 min. In the presence of the combination of SCH39166 and SKF83959 in the recording pipettes, quinpirole (50.83 ± 10.83 nM, n = 6) produced a significant inhibition in firing rate with no reversal [1-way repeated-measures ANOVA, F_(7,35) = 4.32, P < 0.05]. These results suggest that D1/D5-linked PI/PLC, but not AC/cAMP, pathway mediates D2 receptor desensitization that can be suppressed by inhibition of D1/D5 receptors.

Inhibition of cPKC suppressed the reversal of quinpirole inhibition produced by SKF83959.

We reported previously that inhibition of either PLC or cPKC suppressed DIR and that activation of PKC produced the reversal of quinpirole inhibition (Nimitvilai et al. 2011). In the present study, we examined whether the reversal of quinpirole inhibition produced by D1/D5-linked PI pathway SKF83959 was blocked when the cPKC inhibitor Gö6976 was applied (Fig. 2). Saline containing SKF83959 (100 μM) and Gö6976 (1 μM) was used to fill the recording pipettes. The 1 μM Gö6976 included in the recording pipettes produced a comparable result to the 100 nM Gö6976 applied in the superfusate (data not shown), and this concentration inhibits cPKC but not novel and atypical PKCs (Gschwendt et al. 1996; Martiny-Boron et al. 1993). After 20-min administration of Gö6976 and SKF83959, concentrations of quinpirole were applied in the superfusate in a stepwise fashion, in which each concentration was added for 5 min until inhibition of 50% or greater was achieved, and these concentrations were sustained for 40 min. In the presence of Gö6976, the firing rate significantly decreased over time, with no reversal of quinpirole-induced inhibition despite the presence of SKF83959 (quinpirole concentration [Q] = 65 ± 15.8 nM, n = 7) [1-way repeated-measures ANOVA, F_(7,42) = 4.12, P < 0.05]. This indicates that desensitization of D2 receptors requires stimulation of D2 and D1/D5 receptors and activation of the PI/cPKC pathway.

Fig. 2.

Reversal of quinpirole inhibition by D1/D5 agonist linked to the PI pathway was blocked by a conventional protein kinase C (cPKC) inhibitor. Relative change in firing rate (mean ± SE) in response to long-duration quinpirole application is plotted as a function of time. Effect of quinpirole at each time point was normalized by subtracting the change in firing rate (%) at the 5-min time point. The response to a concentration of quinpirole that produced inhibition (crossed open circles and dashed line) and the response to SKF83959 that reversed quinpirole inhibition (▽) (both from Fig. 1) are shown for reference. Saline containing 100 μM SKF83959 and 1 μM Gö6976 was used to fill the recording pipette. After 20-min administration of Gö6976 and SKF83959, a concentration of quinpirole that produced inhibition of 50% or greater was applied for 40 min. In the presence of Gö6976, there was no reversal of quinpirole inhibition produced by SKF83959, and the firing rate was significantly inhibited (●, [Q] = 65 ± 15.8 nM, n = 7) [1-way repeated-measures ANOVA, F_(7,42) = 4.12, P < 0.05].

Activation of 5-HT₂ receptors reversed quinpirole-induced inhibition.

Autoregulation of D2 receptors in the VTA neurons can be modulated by many neurotransmitters, including serotonin (5-hydroxytryptamine, 5-HT) (Olijslagers et al. 2006). Within the VTA there are serotonin-containing nerve fibers (Fuxe 1965), and the VTA receives a prominent 5-HT innervation originating in the raphe nuclei of the brain stem (Azmitia and Segal 1978; Hervé et al. 1987; Steinbusch 1981; Van Bockstaele et al. 1994). High densities of 5-HT_1B, 5-HT_2A, and 5-HT_2C receptors have been demonstrated on VTA neurons; however, low or undetectable densities of 5-HT_2B and 5-HT₃ receptors are present (Bubar and Cunningham 2007; Clemet et al. 2000; Doherty and Pickel 2000; Ikemoto et al. 2000; Kilpatrick et al. 1996; Mengod et al. 1990a, 1990b; Morilak et al. 1993). Among these 5-HT receptors, 5-HT₂ subtypes are coupled to G_q/11 and activate the downstream effector PLC, resulting in stimulation of diacylglycerol (DAG) and inositol trisphosphate (IP₃) receptors. Activation of 5-HT₂ receptors modulates the function of DAergic VTA neurons by increasing the acute inhibitory potency of D2 agonists (Brodie and Bunney 1996), increases the activity of G protein-coupled inwardly rectifying potassium channel (GIRK) (Olijslagers et al. 2006), reduces the activity of hyperpolarization-activated cation channels (I_h) through PKC (Liu et al. 2003), and potentiates the effect of ethanol-induced excitation of VTA neurons (Brodie et al. 1995). In this study, we tested whether stimulation of 5-HT₂ receptors, which are coupled to G_q and activate PLC and PKC, causes the reversal of quinpirole-induced inhibition (Fig. 3).

Fig. 3.

Coadministration of quinpirole and serotonin produced the reversal of quinpirole inhibition, which was suppressed by the 5-HT₂ receptor antagonist ketanserin. Relative change in firing rate (mean ± SE) in response to long-duration quinpirole application is plotted as a function of time. Effect of quinpirole at each time point was normalized by subtracting the change in firing rate (%) at the 5-min time point. The effect of quinpirole alone (from Fig. 1) is shown for comparison (crossed open circles and dashed line). A concentration of quinpirole that produced 50% inhibition or greater was applied for 40 min. In the presence of 50 μM serotonin in the superfusate (▼, [Q] = 112.5 ± 22.16 nM, n = 8), there was a significant reduction in quinpirole inhibition over time, with the last 3 time points significantly different from the 5-min time point [1-way repeated-measures ANOVA, F_(7,1) = 7.22, P < 0.05]. In the presence of 50 μM serotonin in the superfusate and 50 μM ketanserin in the recording pipettes (●, [Q] = 78.57 ± 13.83 nM, n = 6), quinpirole produced a significant inhibition in firing rate, with no reversal over time [1-way repeated-measures ANOVA, F_(7,1) = 3.17, P < 0.05]. No reversal of quinpirole inhibition was observed when 50 μM ketanserin was present in the recording pipettes (▽, [Q] = 56.25 ± 15.7 nM, n = 4) [1-way repeated-measures ANOVA, F_(7,1) = 4.51, P < 0.05].

Serotonin (50 μM) was applied in the superfusate for at least 15 min, and then concentrations of quinpirole were administered in a stepwise fashion until inhibition of 50% was achieved. In the presence of serotonin, quinpirole (112.5 ± 22.16 nM) produced an inhibition in firing rate initially, and this inhibition partially reversed with time (n = 8); there was a significant difference between firing rate at the last three time points compared with the firing rate at the 5-min time point [1-way repeated-measures ANOVA, F_(7,1) = 7.22, P < 0.05]. Serotonin alone did not significantly change the firing rate over time (paired t-test, t = 1.01, df = 7, P > 0.05) (Table 2).

Table 2.

Effect of serotonin, neurotensin, DHPG, and phenylephrine on firing rate

Chemical Name	Chemical Concentration (in delivery tubing), μM	No. of Cells	Mean Firing Rate at Baseline, Hz	Mean Firing Rate at 15 min, Hz	Change in Firing Rate, %	P Value
Serotonin	50	8	2.65 ± 0.21	2.51 ± 0.25	−5.5 ± 4.8	>0.05
Neurotensin	0.01	8	3.09 ± 0.27	4.33 ± 0.39	40.8 ± 8.9	<0.05
DHPG	10	6	2.96 ± 0.47	4.33 ± 0.59	50.5 ± 11.3	<0.05
Phenylephrine	10	6	2.77 ± 0.32	2.93 ± 0.4	6.4 ± 8.6	>0.05

Values are means ± SE.

Because VTA neurons contain several subtypes of 5-HT receptors, we tested whether the reversal of quinpirole inhibition was due to serotonin activating G_q-coupled 5-HT₂ receptors. The selective 5-HT₂ antagonist ketanserin (50 μM) was dissolved in saline, and the saline containing ketanserin was used to fill the recording pipettes. These pipettes were used to record the cell firing rate of DAergic VTA neurons at least 20 min before serotonin and quinpirole were applied, respectively. In the presence of ketanserin in the pipettes (n = 7), serotonin failed to reverse quinpirole inhibition over the time course; there was no significant change in the inhibitory effect of quinpirole over time ([Q] = 78.57 ± 13.83 nM) [1-way repeated-measures ANOVA, F_(7,1) = 0.92, P > 0.05]. With ketanserin alone (n = 4), quinpirole (56.25 ± 15.7 nM) produced a significant reduction in firing rate, with no reversal [1-way repeated-measures ANOVA, F_(7,1) = 4.51, P < 0.05]. These results suggest that serotonin produces the reversal of quinpirole-induced inhibition through the activation of 5-HT₂ receptors.

Activation of neurotensin receptor reversed quinpirole-induced inhibition.

The DAergic VTA neurons express G_q-coupled neurotensin receptors, predominantly the NTS1 subtype (Palacios and Kuhar 1981; Quirion et al. 1985). Activation of neurotensin receptors causes an increase in firing rate of DAergic neurons (Jiang et al. 1994; Lacey 1993; Stowe et al. 1991) through a rise in intracellular calcium concentration via the G_q/PLC pathway, which further activates IP₃ receptors, inducing calcium release from the intracellular store (St-Gelais et al. 2004). In addition, stimulation of neurotensin receptor is able to decrease D2 autoreceptor function through PLC/PKC pathway in the substantia nigra, VTA, human embryonic kidney HEK293 cells, and cultured dopamine neurons (Shi and Bunney 1990; Thibault et al. 2011; Werkman et al. 2000). Neurotensin receptor activation of PKC mediates the phosphorylation of D2 receptors, resulting in the desensitization and internalization of the receptors through β-arrestins in HEK293 cells (Namkung and Sibley 2004; Thibault et al. 2011). In this study, therefore, we tested whether neurotensin can mediate the reversal of quinpirole inhibition in the pDAergic VTA neurons (Fig. 4). Neurotensin (10 nM) was applied in the superfusate for at least 15 min, and then concentrations of quinpirole were applied in a stepwise fashion until inhibition of 50% was achieved. In the presence of neurotensin (n = 8), there was a small but significant reversal of quinpirole inhibition ([Q] = 51.88 ± 8.96 nM) [1-way repeated-measures ANOVA, F_(7,1) = 4.39, P < 0.05]. Neurotensin alone produced a significant increase in baseline firing frequency by 40.84 ± 8.9% (paired t-test, t = −5.33, df = 7, P < 0.05) (Table 2).

Fig. 4.

Coadministration of quinpirole and neurotensin produced the reversal of quinpirole inhibition, which was suppressed by neurotensin receptor antagonist SR142948. Relative change in firing rate (mean ± SE) in response to long-duration quinpirole application is plotted as a function of time. Effect of quinpirole at each time point was normalized by subtracting the change in firing rate (%) at the 5-min time point. The effect of quinpirole alone (from Fig. 1) is shown for comparison (crossed open circles and dashed line). A concentration of quinpirole that produced inhibition of 50% or greater was applied for 40 min. In the presence of 10 nM neurotensin in the superfusate (■, [Q] = 51.88 ± 8.96 nM, n = 8), there was a small but significant reduction in quinpirole inhibition over time, with the last 3 time points significantly different from the 5-min time point [1-way repeated-measures ANOVA, F_(7,1) = 4.39, P < 0.05]. In the presence of 10 nM neurotensin in the superfusate and 1 μM SR142948 in the recording pipettes (●, [Q] = 49.17 ± 10.9 nM, n = 6), quinpirole produced a significant inhibition in firing rate, with no reversal over time [1-way repeated-measures ANOVA, F_(7,1) = 18.98, P < 0.05]. No reversal of quinpirole inhibition was observed when 1 μM SR142948 was present in the recording pipettes (▼, [Q] = 78 ± 14.28 nM, n = 5) [1-way repeated-measures ANOVA, F_(7,1) = 3.56, P < 0.05].

The effect of neurotensin receptor activation on quinpirole inhibition reversal was confirmed by application of neurotensin receptor antagonist SR142948. The SR142948 (1 μM) was dissolved in saline, and the saline containing SR142948 was used to fill the recording electrodes. These electrodes were used to make the extracellular recordings of single pDAergic VTA neurons. After the recording of pDAergic neurons was obtained and the drug in the pipette was allowed to act locally for at least 20 min, neurotensin (10 nM) was applied for 15 min and then concentrations of quinpirole were applied in the superfusate in a stepwise fashion, in which each concentration was added for 5 min and increased until inhibition of 50% or greater was achieved. In the presence of SR142948 in the pipettes (n = 6), neurotensin failed to reverse quinpirole-induced inhibition ([Q] = 49.17 ± 10.9 nM); there was a significant inhibition in firing rate produced by quinpirole over the time course [1-way repeated-measures ANOVA, F_(7,1) = 18.98, P < 0.05]. Likewise, treatment with SR142948 alone was unable to induce the reversal of quinpirole inhibition; quinpirole produced a significant inhibition in firing rate over time (n = 5, [Q] = 78 ± 14.28 nM) [1-way repeated-measures ANOVA, F_(7,1) = 3.56, P < 0.05].

Activation of α₁-adrenergic or metabotropic glutamate receptors fails to reverse quinpirole-induced inhibition.

Other G_q-coupled receptors such as α₁-adrenergic and metabotropic glutamate (mGlu) receptors have been reported to play a role in modulating dopamine D2 receptor function. α-Adrenergic receptor consists of α₁ and α₂ subtypes, both of which are present in the VTA neurons. In addition, the VTA neurons receive 50% of their adrenergic inputs from locus coeruleus and other pontine structures (Guiard et al. 2008). The α₁-adrenergic receptor is coupled to G_q, and activation of this receptor results in an increase in dopamine-mediated locomotion in rats and mice through PKC pathway. Blocking α₁-adrenergic receptor also decreases locomotor effects and attenuates behavioral sensitization to both cocaine and amphetamine (Drouin et al. 2002a; Jiménez-Rivera et al. 2006). In this study, we tested whether prolonged activation of α₁-adrenergic receptor causes a decrease in the inhibitory effect of quinpirole (Fig. 5A). The α₁-adrenergic receptor agonist phenylephrine (10 μM) was administered in the superfusate for at least 15 min, and this concentration was continued for the rest of the experiment. Concentrations of quinpirole were applied in a stepwise fashion until inhibition of 50% or greater was achieved, and this concentration was sustained for 40 min. In the presence of phenylephrine (n = 6), quinpirole (76.67 ± 8.33 nM) produced a significant inhibition in firing rate, with no reversal [1-way repeated-measures ANOVA, F_(7,1) = 6.43, P < 0.05]. Phenylephrine alone did not significantly change the baseline firing frequency (paired t-test, t = −0.75, df = 5, P > 0.05) (Table 2). Therefore, stimulation of α₁-adrenergic receptors did not produce desensitization of D2 receptors in the VTA neurons.

Fig. 5.

Coadministration of quinpirole and agonist of either α₁-adrenergic or group I metabotropic glutamate (mGlu) receptors did not produce the reversal of quinpirole inhibition. Relative change in firing rate (mean ± SE) in response to long-duration quinpirole application is plotted as a function of time. Effect of quinpirole at each time point was normalized by subtracting the change in firing rate (%) at the 5-min time point. The effect of quinpirole alone (from Fig. 1) is shown for comparison (crossed open circles and dashed line). A and B: a concentration of quinpirole that produced inhibition of 50% or greater was applied for 40 min. In the presence of the α₁-adrenergic receptor antagonist phenylephrine (10 μM; A), quinpirole produced a significant reduction in firing rate, with no reversal over time (■, [Q] = 76.67 ± 8.33 nM, n = 6) [1-way repeated-measures ANOVA, F_(7,1) = 6.43, P < 0.05]. In the presence of the group I mGlu receptor antagonist DHPG (10 μM; B), no significant reversal of quinpirole-induced inhibition was observed (■, [Q] = 91.67 ± 15.37 nM, n = 6) [1-way repeated-measures ANOVA, F_(7,1) = 0.81, P > 0.05]. C: a single concentration of quinpirole was applied for 40 min alone or in the presence of either DHPG or serotonin. Quinpirole alone (■, 45 nM, n = 7) produced a significant inhibition in firing rate, with no reversal over time [1-way repeated-measures ANOVA, F_(7,42) = 8.85, P < 0.05]. In the presence of 10 μM DHPG, quinpirole (■, 90 nM, n = 8) significantly inhibited the firing rate, and this inhibition persisted for the duration of drug application [1-way repeated-measures ANOVA, F_(7,49) = 4.32, P < 0.05]. In the presence of 50 μM serotonin, a single dose of quinpirole (▼, 112.5 nM, n = 5) produced an initial inhibition in firing rate, and this inhibition partially reversed with time [1-way repeated measures ANOVA, F_(7,28) = 7.22, P < 0.05].

There is evidence that group I mGlu receptors (mGluR1 and mGluR5) are prominently expressed in midbrain dopamine neurons (Martin et al. 1992; Testa et al. 1994) and can cause a sustained increase in spontaneous firing rate and depolarization of DAergic mesencephalic neurons (Mercuri et al. 1993). Because group I mGluRs cause activation of PKC (Riberio et al. 2010), it is possible that activation of group I mGluRs in the VTA neurons also may modulate the sensitivity of D2 receptors. In this study, we tested whether the selective group I mGlu agonist DHPG causes the reversal of quinpirole-induced inhibition (Fig. 5B). DHPG (10 μM) was applied in the superfusate for at least 15 min. Concentrations of quinpirole were added in a stepwise fashion, in which each concentration was applied for 5 min until inhibition of 50% or greater was achieved, and this concentration was sustained for 40 min. In the presence of DHPG (n = 6), no significant reversal of quinpirole-induced inhibition was observed ([Q] = 91.67 ± 15.37 nM) [1-way repeated-measures ANOVA, F_(7,1) = 0.81, P > 0.05]. DHPG alone significantly increased baseline firing rate by 50.48 ± 11.3% (paired t-test, t = −4.74, df = 5, P < 0.05) (Table 2).

Quinpirole inhibition in the presence of DHPG differed from that of quinpirole inhibition alone or in the presence of either SKF83822 or phenylephrine; with DHPG, the inhibition produced by quinpirole did not increase during the first 15 min. It is possible that DHPG produced a more rapid desensitization of D2 receptors as a result of the stepwise application of quinpirole so that it was essentially complete at the 5-min time point of the final concentration of quinpirole. Therefore, we examined whether DHPG produced reversal of quinpirole inhibition when a single dose of quinpirole was applied. The 90 nM quinpirole concentration was used because it was the mean concentration used in Fig. 5B (also see Table 1). After 15-min application of DHPG (10 μM), quinpirole (90 nM) was added in the superfusate and both DHPG and quinpirole were sustained for 40 min. As shown in Fig. 5C, in the presence of DHPG (n = 8), quinpirole produced a significant inhibition in firing rate, with the last four time points significantly different from the 5-min time point [1-way repeated-measures ANOVA, F_(7,49) = 4.32, P < 0.05]. In these experiments, 90 nM quinpirole produced a mean change in firing rate of −49.8 ± 8.1% at the 10-min time point. This result suggests that activation of group I mGluRs does not induce desensitization of D2 receptors in the VTA neurons.

For comparison with the DHPG results with a single quinpirole concentration (Fig. 5C), we also examined whether application of a single dose of quinpirole alone (45 nM, a mean concentration of quinpirole in the control experiment, Fig. 1) or a single dose of quinpirole in the presence of 50 μM serotonin ([Q] = 112.5 nM, a mean concentration of quinpirole used in the presence of serotonin, Fig. 3) produced results similar to a stepwise increase in quinpirole concentrations. As shown in Fig. 5C, quinpirole alone (45 nM, n = 7) significantly inhibited firing rate, and this inhibition did not reverse for the duration of quinpirole application [1-way repeated-measures ANOVA, F_(7,42) = 8.85, P < 0.05]. In these experiments, 45 nM quinpirole produced a mean change in firing rate of −62.3 ± 9.5% at the 10-min time point. In the presence of 50 μM serotonin (n = 5), quinpirole (112.5 nM) produced an inhibition in firing rate with maximum inhibition of 68.21 ± 6.9% at 10 min, and this inhibition partially reversed over time, with the last three time points significantly different from the 10-min time point [1-way repeated-measures ANOVA, F_(7,28) = 7.22, P < 0.05]. These results indicate that both a single and a stepwise application of quinpirole concentrations produce comparable results.

DISCUSSION

We have previously reported that extended exposure of pDAergic VTA neurons to moderate concentrations of dopamine results in a time- and concentration-dependent decrease in dopamine-induced inhibition, which persists for up to 90 min (Nimitvilai and Brodie 2010). This decrease in dopamine sensitivity requires concurrent stimulation of D2 and D1/D5 receptors so that it is neither homologous nor heterologous desensitization, and we termed this phenomenon “dopamine inhibition reversal,” or DIR. Even though a high quinpirole concentration (3 μM) is able to induce desensitization of D2 receptors in the pDAergic VTA neurons, as described previously (Bartlett et al. 2005), this high quinpirole fails to mediate D2 desensitization in the presence of D1/D5 antagonist (Nimitvilai and Brodie 2010). We have also demonstrated that dopamine inhibition reversal is mediated by PLC and phorbol ester-sensitive PKC and is dependent on extracellular calcium concentration and intracellular calcium release; this profile suggests an involvement of cPKC, rather than novel or atypical PKC (Nimitvilai et al. 2011). In the present study, we demonstrated that the D1/D5-linked PI pathway (SKF83959) could mediate the reversal of quinpirole inhibition, whereas D1/D5-linked AC pathway (SKF83822) had no effect on quinpirole inhibition. In addition, the effect of SKF83959 reversal of quinpirole inhibition was blocked by the cPKC inhibitor Gö6976, suggesting that stimulation of D2 and D1/D5 receptors mediates D2 desensitization through the PI/PLC/cPKC pathway. The present study supports the idea that activation of D2 and D1/D5 receptors mediates D2 desensitization through the G_q/PLC/cPKC pathway, without an involvement of AC/cAMP/PKA. This study also indicates that activation of some, but not all, G_q-coupled receptors in VTA neurons produces desensitization of D2 receptors. Desensitization has been studied in many G protein-coupled receptors (GPCRs), such as the adrenergic β-receptor. The mechanism is generally related to phosphorylation of the GPCR at its COOH terminus or within the third intracellular loop under conditions of agonist occupancy, which results in decoupling of the receptor and the G protein, decreasing the function of the receptor (Cho et al. 2006; Namkung and Sibley 2004). A role of PKC in desensitization of D2 receptors has been reported in many systems such as HEK293 cells and striatal and hippocampal neurons (Bofill-Cardona et al. 2000; Namkung and Sibley 2004; Rogue et al. 1990; Thibault et al. 2011). In site-directed mutagenesis experiments, two PKC phosphorylation sites within the third intracellular loop of the D2 receptor have been identified (Namkung and Sibley 2004). Activation of PKC mediates the phosphorylation of D2 receptors, resulting in the desensitization and internalization of the receptors through β-arrestins (Namkung and Sibley 2004; Thibault et al. 2011). Phosphorylation of D2 receptors may be a mechanism underlying the reversal of dopamine inhibition in the pDAergic VTA neurons. Whether PKC phosphorylates D2 receptors in pDAergic VTA neurons and whether D2 receptor phosphorylation results in receptor internalization needs to be characterized in future studies.

Interaction between PKC and D2 receptors has been observed previously. Calcium influx induced by D2 agonist was reduced in Ltk⁻ cells expressing the short form of the D2 receptor (D_2S) but not the long form (D_2L) when treated with a phorbol ester activator of PKC (Liu et al. 1992). Desensitization of the D_2S receptor by PKC was observed in Ltk⁻ cells and was linked to a PKC pseudosubstrate site that is found on the D_2L receptor but not the D_2S receptor (Morris et al. 2007). In contrast, neurotensin has been shown to induce internalization of both D_2S and D_2L forms of the D2 receptor, although the effect on the D_2S receptor was larger in magnitude and demonstrated faster kinetics (Thibault 2011). Because the short form of the D2 receptor is the predominating form of the D2 receptor in DAergic VTA neurons (Khan et al. 1998), our data are consistent with the idea of PKC control of desensitization via DIR in DAergic VTA neurons.

Although it is clear that coactivation of D2 and D1-like receptors is necessary to produce DIR (Nimitvilai and Brodie 2010), it is not certain that both D2 and D1-like receptors reside on the same neurons. Because all of our experiments were conducted in 400-μm-thick brain slices, it is possible that there were polyneuronal interactions within the slice, including GABAergic interneurons and glutamatergic terminals. Although the most parsimonious model for the interaction suggests colocalization of D2 and D1-like receptors on pDAergic VTA neurons, additional experiments are necessary to determine the exact locations of the D1-like and D2 receptors, and to examine whether polyneuronal interactions are involved in the reversal of dopamine-induced inhibition.

A persistent increase in dopamine concentration in the VTA as the result of exposure to drugs of abuse causes an increase in DAergic transmission in the reward system in a sustained manner (Di Chiara et al. 2004), a process that may be related to the craving and seeking for drugs even in the presence of negative consequences. We have previously demonstrated that DIR can occur at much lower concentrations of dopamine when cocaine is present (Nimitvilai et al. 2011). Cocaine may increase the likelihood that dopamine in the VTA can activate PKC and subsequently decrease D2 receptor sensitivity. In addition, cocaine has been reported to cause an enhancement of NMDA/AMPA glutamate transmission and subsequent increases in postsynaptic calcium concentration that ultimately lead to an induction of long-term potentiation (LTP) in the VTA (Borgland et al. 2004; Ungless et al. 2001). Therefore, the enhancement of glutamate neurotransmission in the VTA by drugs of abuse may contribute to the desensitization of D2 receptors, since activation of this system increases intracellular calcium concentrations and PKC activity (Luu and Malenka 2008; Skeberdis et al. 2001). In addition to a potentiation of glutamatergic transmission induced by abused drugs, NMDA/AMPA receptor-mediated excitatory postsynaptic currents can be enhanced by activation of D1/D5 receptors in hippocampal neurons (Yang 2000). Injection of the D1/D5 antagonist SCH23390 into the VTA has been reported to block the effect of cocaine-induced increases in extracellular dopamine in the nucleus accumbens (NAc), but not the development of behavioral sensitization (Steketee 1998). On the other hand, intra-VTA administration of the D1/D5 agonist SKF38393 potentiates both the cocaine-induced increase in dopamine concentrations in the NAc and the behavioral response to cocaine (Pierce et al. 1996). Thus stimulation of D1/D5 receptors in the pDAergic VTA neurons might lead to increased sensitivity of glutamatergic receptors, resulting in a rise in intracellular calcium and activation of PKC that further phosphorylates and desensitizes D2 receptors. Glutamate receptors not only activate PKC but also can be phosphorylated by PKC, resulting in LTP. Glutamate neurotransmission may interact with intracellular processes involved in the reversal of dopamine-induced inhibition in the VTA, and there may be a synergistic interaction of glutamate excitation with D2 dopamine receptor desensitization.

Because DIR is mediated by the PI pathway and PKC, we hypothesized that agonists at other receptors that activate PKC in pDAergic VTA neurons could also mediate time-dependent desensitization of D2 receptors. The activity of DAergic VTA neurons can be modulated by many neurotransmitters, including serotonin, norepinephrine, dynorphin, acetylcholine, GABA, glutamate, and neurotensin. Of these neurotransmitters, serotonin, norepinephrine, glutamate, and neurotensin have been reported to activate G_q and the PI/PLC/cPKC pathway. We have shown in this study that not all agents that activate PI and PKC could produce desensitization of D2 receptors; serotonin and neurotensin did, but DHPG and phenylephrine did not.

Two- to threefold higher quinpirole concentrations were needed when quinpirole was coapplied with serotonin ([Q] = 112.5 ± 22.16 nM), DHPG ([Q] = 91.67 ± 15.37 nM), or phenylephrine ([Q] = 76.67 ± 8.33 nM), compared with the quinpirole concentration alone ([Q] = 45 ± 5.75 nM). It is possible that activation of 5-HT₂, type I mGlu, or α₁-adrenergic receptors initially produces a decrease in the affinity of D2 receptor for quinpirole. For example, application of type I mGlu receptor agonist to rat striatal neurons diminishes the affinity of D2 receptor for its agonists and lessens the D2 receptor agonist-mediated motor activation (Ferrè et al. 1999; Popoli et al. 2001). Even though activation of 5-HT₂ receptor by 5–10 μM serotonin in the VTA neurons has been reported to enhance the inhibitory effect of D2 agonists (Brodie and Bunney 1996), in these experiments, higher concentrations of quinpirole were required in the presence of 50 μM serotonin to produce the same amount of inhibition. Numerous factors may contribute to these opposing effects of serotonin; concentrations of serotonin, differential action of serotonin on pDAergic VTA neurons and other neurons in the slice preparation, and methodological differences may cause one effect of serotonin to be observed over the other. Ultimately, it is clear that the sensitivity of autoreceptors of pDAergic VTA neurons is under the control of numerous factors. In this study, we demonstrated that activation of some, but not all, G_q-coupled receptors produced a decrease in the inhibitory effect of quinpirole over time. Neurotensin and DHPG by themselves produced an increase in firing rate of DAergic VTA neurons; however, neurotensin but not DHPG mediated the reversal of quinpirole inhibition. On the other hand, serotonin and phenylephrine by themselves did not significantly change the baseline firing rate of dopamine neurons; however, quinpirole inhibition reversal was observed when serotonin, but not phenylephrine, was coadministered. The lack of correlation between changes in firing rate produced by the receptors themselves and their modulatory effects on D2 receptors indicates that the direct action of these agents on firing rate may be regulated differently and may be a result of other factors, such as specific location of the receptors on the dendrites and cell soma, or the proximity of these receptors to D2 or other receptors.

The spontaneous firing rate of DAergic VTA neurons can be modulated by neurotensin. High-affinity neurotensin receptors NTS1 are found predominantly in dopamine-rich areas such as the VTA and substantia nigra (Jennes et al. 1982; Quirion et al. 1985; Uhl 1982; Uhl et al. 1977). As shown in many laboratories (Jiang et al. 1994; Seutin et al. 1989; Wu et al. 1995) and in the present study, activation of neurotensin receptors causes an increase in firing rate of dopamine neurons. In addition, we report here that neurotensin produced a decrease in the inhibitory effect of quinpirole over 40 min of drug application, an effect that was blocked by a selective neurotensin antagonist. Desensitization of D2 dopamine receptors produced by neurotensin has been reported in many systems, such as the striatal neurons (von Euler et al. 1991), cultured mesencephalic dopamine neurons (Jomphe et al. 2006), HEK293 cells (Thibault et al. 2011), nucleus accumbens, and olfactory tubercle (Li et al. 1995). A decrease in the sensitivity of D2 receptors by neurotensin requires activation of G_q/PLC/PKC pathway and is dependent on calcium. In cultured mesencephalic dopamine neurons, a decrease in the inhibitory effect of quinpirole by neurotensin is dependent on extracellular calcium influx, without an involvement of calcium release from the intracellular stores (Jomphe et al. 2006). We have previously reported that DIR produced by concurrent stimulation of D2 and D1/D5 receptors is dependent on both calcium influx from extracellular medium and calcium release from the intracellular stores (Nimitvilai et al. 2011). It is possible that differences between cultured neurons and mature neurons in brain slices account for this difference in calcium dependence or that neurotensin regulation of D2 receptor desensitization differs from that mediated by D1-like receptors.

Type I mGlu (mGluR1 and mGluR5) and α₁-adrenergic receptors have been reported to play a role in modulating the activity of D2 receptors in other systems (Drouin et al. 2002b; Ferrè et al. 1999; Jiménez-Rivera et al. 2006; Popoli et al. 2001). In the present study, however, we found that neither the mGluR agonist DHPG nor the α₁-adrenergic receptor agonist phenylephrine produced significant reversal of quinpirole-induced inhibition. Interestingly, there was a subtle change in the quinpirole inhibition curve in the presence of DHPG compared with controls (Fig. 5, B and C); in the presence of DHPG, quinpirole did not appear to inhibit as strongly in the first 15 min as under control conditions. Despite this, the sustained level of inhibition from the 15- to 40-min time points does not support a role of DHPG to reverse quinpirole inhibition. Stimulation of α₁-adrenergic or type I mGlu receptors in the VTA might produce an increase in the activity of different PKC isoforms that are not involved in the reversal of D2 agonist inhibition. Our results also suggest more generally that not all G_q-coupled receptors can produce a decrease in sensitivity of D2 receptor to its agonists.

Exposure to drugs of abuse causes an increase in DAergic neurotransmission in the reward system in a sustained manner (Di Chiara et al. 2004), a process that may be related to drug craving and drug seeking even in the presence of negative consequences. The increase in DAergic neurotransmission might be due to the desensitization of D2 autoreceptors in the VTA neurons through enhancement of DIR. The present study further defines the mechanism of that desensitization, with the involvement of the PI/PLC/cPKC pathway. In addition, the action of drugs of abuse on excitatory neurotransmitter systems, like serotonin or neurotensin, may reduce autoreceptor inhibition of DA VTA neurons, resulting in an increase in the firing rate of these important reward/reinforcement neurons. Understanding molecular mechanisms underlying the effects of sustained increases in dopamine concentration in the VTA may contribute to medication discovery for more effective treatment of addiction disorders.

GRANTS

We gratefully acknowledge support from National Institute on Alcohol Abuse and Alcoholism Grant AA05846.

DISCLOSURES

None of the authors have any conflict of interest associated with the content of this report.

AUTHOR CONTRIBUTIONS

Author contributions: S.N. and M.S.B. conception and design of research; S.N., M.A.M., and D.S.A. performed experiments; S.N. and M.A.M. analyzed data; S.N. and M.S.B. interpreted results of experiments; S.N. prepared figures; S.N. drafted manuscript; S.N. and M.S.B. edited and revised manuscript; S.N., M.A.M., D.S.A., and M.S.B. approved final version of manuscript.