Reversal of Prolonged Dopamine Inhibition of Dopaminergic Neurons of the Ventral Tegmental Area

Drug abuse-induced plasticity of putative dopaminergic (pDAergic) ventral tegmental area (VTA) neurons may play an important role in changes in the mesocorticolimbic system that lead to the development of addiction. In the present study, extracellular recordings were used to examine timedependent effects of dopamine (DA) on pDAergic VTA neurons in rat brain slices. Administration of DA (2.5–10 M) for 40 min resulted in inhibition followed by partial or full reversal of that inhibition. The reduced sensitivity to DA inhibition lasted 30 to 90 min after washout of the long-term dopamine administration. The inhibition reversal was not observed with 40-min administration of the D2 agonist quinpirole (25–200 nM), so this phenomenon was not the result of desensitization induced solely by stimulation of D2 DA receptors. Inhibition reversal could be observed with the coapplication of quinpirole and the D1/D5 agonist SKF38393 [( )-1-phenyl2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrobromide], suggesting a D1/D5 mechanism for the reversal. Furthermore, D1/D5 antagonists, given in the presence of prolonged DA exposure, prevented the inhibition reversal. Application of 3 M quinpirole caused desensitization to low quinpirole concentrations that was blocked by a D1/D5 antagonist. These data suggest that coactivation of D1/D5 receptors and D2 receptors in the VTA results in desensitization of autoinhibitory D2 receptors. Prolonged increases in pDAergic tone in the VTA that may occur in vivo with drugs of abuse could reduce the regulation of firing by D2 dopamine receptor activation, producing long-term alteration in information processing related to reward and reinforcement. Putative dopaminergic (pDAergic) neurons of the ventral tegmental area (VTA) are important for the rewarding and reinforcing properties of numerous drugs of abuse (Wise, 1996). Drugs of abuse increase dopaminergic neurotransmission (Imperato and Di Chiara, 1986; Imperato et al., 1986; Di Chiara and Imperato, 1988). Most studies have examined dopamine (DA) release in the terminal target regions of the DA VTA neurons, the nucleus accumbens and the prefrontal cortex, and have found that drugs of abuse increase the DA concentrations in these regions (Di Chiara and Imperato, 1988; Di Chiara et al., 2004). Because there is dendritic DA release in response to increased activity of mesencephalic DA neurons (Cragg et al., 1997), it is likely that the DA concentrations in the VTA are also increased in response to most drugs of abuse. The effect of these elevated dopamine concentrations in the VTA is not known, but it is known that elevated dopamine can 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). There are five classes of DA receptors: two “D1-like” receptors (D1 and D5) and three “D2-like” receptors (D2, D3, and D4). D1-like (D5) receptor immunoreactivity on perikarya of mesencephalic DA neurons has been demonstrated (Ciliax et al., 2000; Khan et al., 2000), and mRNA for D5 receptors has been observed in the substantia nigra by some groups (Choi et al., 1995) but not by all groups (Meador-Woodruff et al., 1992). The D2-like mesolimbic DA receptors are predominantly D2 receptors (Sesack et al., 1994). The D1 receptors located in dopaminergic brain areas seem to be on terminals This study was supported by the National Institutes of Health National Institute on Alcohol Abuse and Alcoholism [Public Health Service Grants AA09125, AA05846]. Some of this work was presented previously: Nimitvilai S and Brodie MS (2008) Biphasic effects of dopamine on the firing rate of dopaminergic ventral tegmental area neurons: involvement of h-current; 2009 Nov 15–19; 2008 Neuroscience Meeting Planner. Program 727.4, Society for Neuroscience, Washington, DC. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.109.163931. ABBREVIATIONS: pDAergic, putative dopaminergic; DA, dopamine; VTA, ventral tegmental area; LTP, long term potentiation; SKF38393, ( )-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrobromide; SCH39166, (6aS-trans)-11-chloro-6,6a,7,8,9,13b-hexahydro7-methyl-5H-benzo[d]naphth[2,1-b]azepin-12-ol hydrobromide; SCH23390, (R)-( )-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5tetrahydro-1H-3-benzazepine hydrochloride; ANOVA, analysis of variance; aCSF, artificial cerebrospinal fluid. 0022-3565/10/3332-555–563$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 333, No. 2 Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 163931/3581379 JPET 333:555–563, 2010 Printed in U.S.A. 555 at A PE T Jornals on Jne 4, 2017 jpet.asjournals.org D ow nladed from projecting to the region, not on the DA neurons themselves (Caillé et al., 1996). Dopamine D5 receptors are present on the cell bodies of dopaminergic VTA neurons (Ciliax et al., 2000). pDAergic VTA neurons fire action potentials spontaneously in vivo (Bunney et al., 1973) and in vitro (Pinnock et al., 1979; Brodie and Dunwiddie, 1987). This spontaneous firing is inhibited by the action of DA at D2 autoreceptors on the cell bodies and dendrites of these neurons (Lacey et al., 1987). Stimulation of D2 autoreceptors activates G proteinlinked potassium channels, which seem to be activated directly by G proteins without the involvement of cAMP or adenylate cyclase (Kim et al., 1995). Drugs of abuse produce increases in dopaminergic neurotransmission, either by increasing firing rate of DA VTA neurons or by blocking reuptake or reversing DA transporter activity in terminal regions (Mueller et al., 2004), and it is likely that the DA concentration in the VTA, released in the somatodendritic area (Cragg et al., 1997; Rice et al., 1997), can remain elevated during drug abuse episodes. For this reason, we tested the effects of sustained administration of exogenous DA on DA inhibition of DA VTA neurons. In vitro electrophysiological experiments using whole-cell patch clamping cannot reliably maintain healthy neuronal recordings for periods longer than 1 h. We used extracellular recording from DA VTA neurons in brain slices, a technique that avoids disrupting the intracellular milieu and that makes it possible to monitor spontaneous firing of these neurons for long continuous time periods (1–4 h). Materials and Methods Animals. Fischer 344 (F344; 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 described previously (Brodie et al., 1999a). In brief, after 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 system and submerged in chilled cutting solution. Coronal sections (400 m thick) were cut, and the slice was placed onto a mesh platform in the recording chamber. The slice was totally submerged in 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 126 mM NaCl, 2.5 mM KCl, 1.24 mM NaH2PO4, 2.4 mM CaCl2, 1.3 mM MgSO4, 26 mM NaHCO3, and 11 mM glucose. In some experiments, a HEPES-aCSF hybrid solution was used; composition of this solution was 106 mM NaCl, 2.5 mM KCl, 1.24 mM NaH2PO4, 2.4 mM CaCl2, 1.3 mM MgSO4, 20 mM HEPES, 26 mM NaHCO3, 11 and mM glucose. The composition of the cutting solution was 2.5 mM KCl, 2.4 mM CaCl2, 1.3 mM MgSO4, 26 mM NaHCO3, 11 mM glucose, and 220 mM 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 (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 biphasic 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 (Margolis et al., 2006). It should be noted that some neurons with the characteristics that we used to identify DA VTA neurons may not, in fact, be DA-containing (Margolis et al., 2006). Drug Administration. Drugs were added to the aCSF by means of a calibrated infusion pump from stock solutions 100 to 1000 times the desired final concentrations. The addition of drug solutions to the aCSF was performed in such a way 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 (approximately 300 l) used in these studies permitted the rapid application and washout of drug solutions. Typically drugs reach equilibrium in the tissue after 2 to 3 min of