Toni S. Shippenberg

Opposing tonically active endogenous opioid systems modulate the mesolimbic dopaminergic pathway

The mesolimbic dopaminergic system has been implicated in mediating the motivational effects of opioids and other drugs of abuse. The site of action of opioids within this system and the role of endogenous opioid peptides in modulating dopamine activity therein remain unknown. Employing the technique of in vivo microdialysis and the administration of highly selective opioid ligands, the present study demonstrates the existence of tonically active and functionally opposing mu and kappa opioid systems that regulate dopamine release in the nucleus accumbens, the major terminal area of A10 dopaminergic neurons. Thus, stimulation of mu-type receptors in the ventral tegmental area, the site of origin of A10 dopaminergic neurons, increases dopamine release whereas the selective blockade of this opioid receptor type results in a significant decrease in basal dopamine release. In contrast, stimulation of kappa-type receptors within the nucleus accumbens decreases dopamine release whereas their selective blockade markedly increases basal dopamine release. These data show that tonic activation of mu and kappa receptors is required for the maintenance of basal dopamine release in the nucleus accumbens. In view of the postulated role of the mesolimbic system in the mediation of drug-induced alterations in mood and affect, such findings may have implications for the treatment of opiate dependence and affective disorders.

The Effects of Opioid Peptides on Dopamine Release in the Nucleus Accumbens: An In Vivo Microdialysis Study

Abstract: An involvement of the mesolimbic dopamine (DA) system in mediating the motivational effects of opioids has been suggested. Accordingly, the present study employed the technique of in vivo microdialysis to examine the effects of selective μ‐, δ‐, and k‐ opioids on DA release in the nucleus accumbens (NAC) of anesthetized rats. Microdialysis probes were inserted into the NAC and perfusates were analyzed for DA and its metabolites, dihydroxyphenylacetic acid (DO‐PAC) and homovanillic acid (HVA), using a reverse‐phase HPLC system with electrochemical detection for separation and quantification. Intracerebroventricular (i.c.v.) administration of selective μ‐opioid [D‐Ala2, N‐methyl‐Phe4, Gly5‐ol]‐enkephalin (DAMGO) or δ‐opioid [d‐Pen2, d‐Pen5]‐en‐kephalin (DPDPE) agonists, at doses that function as positive reinforcers in rats, resulted in an immediate and significant increase in extracellular DA. DOPAC and HVA levels were also significantly increased. The effects of DAMGO were blocked by the selective μ‐antagonist d‐Pen‐Cys‐Tyr‐d‐Trp‐Orn‐Thr‐Pen‐Thr‐NH2 (CTOP) whereas those of DPDPE were blocked by the δ‐antagonist allyl2‐Tyr‐Aib‐Aib‐Phe‐Leu‐OH (ICI 174,864). In contrast to μ‐ and δ‐agonists, the k‐agonist N‐CH3‐Tyr‐Gly‐Gly‐Phe‐Leu‐Arg‐N‐CH3‐Arg‐d‐Leu‐NHC2H5 (E‐2078). a dynorphin analog that produces aversive states, decreased DA release in a biphasic manner. Norbin‐altorphimine, a selective k‐antagonist, could block this effect. These results demonstrate that μ‐, δ‐, and k‐opioid agonists differentially affect DA release in the NAC and this action is centrally mediated.

Neural systems underlying opiate addiction

Opiate addiction is a chronically relapsing disorder that is characterized by compulsive drug taking, an inability to limit intake, and bouts of intense drug craving that can be precipitated by the mere presence of people, places, or objects previously associated with drug use. Although knowledge of

Mitogen-Activated Protein Kinase Regulates Dopamine Transporter Surface Expression and Dopamine Transport Capacity

The dopamine transporter (DAT) regulates the clearance of dopamine (DA) released into the extracellular space and is an important site on which psychostimulants act to produce their effects. Here, we show that mitogen-activated protein kinase (MAPK) regulates the transport capacity and intracellular trafficking of DAT. Incubation of striatal synaptosomes or epitope-tagged human DAT (hDAT) human embryonic kidney (HEK) 293 cells with the MAPK kinase (MEK) inhibitors 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto) butadiene and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one decreased DA uptake in a concentration- and time-dependent manner. Kinetic studies revealed a decrease in the capacity of transport (Vmax) but no change in Km. Immunoblotting confirmed labeling of p42 and p44 MAPK in untreated striatal synaptosomes and HEK 293 cells, consistent with constitutive MAPK activation, and the inhibitors used decreased MAPK phosphorylation. Biotinylation and confocal imaging studies showed that MAPK inhibition promoted the clathrin-associated redistribution of hDAT from the plasma membrane to the cytosol. In contrast, transient transfection of hDAT-expressing cells with constitutively active MEK increased the Vmax of DA transport without altering Km. However, only a small increase in hDAT cell surface expression was seen. These data demonstrate an involvement of the MAPK cascade in regulating DAT transport capacity in striatum and that inhibition of this cascade decreases DAT cell surface expression in HEK 293 cells. Furthermore, they highlight the potential role of MAPK as a presynaptic mechanism that regulates DA signaling.

Evidence that the aversive effects of opioid antagonists and κ-agonists are centrally mediated

The role of central versus peripheral opioid receptors in mediating the aversive effects of opioids was examined by use of an unbiased place preference conditioning procedure in rats. The non-selective opioid antagonist naloxone (NLX) produced conditioned aversions for the drug-associated place after subcutaneous (SC) as well as intracerebroventricular (ICV) administration. Place aversions were also observed in response to the ICV administration of the selective μ-antagonist CTOP. In contrast, the selective δ-antagonist ICI 174,864 and the selective κ-antagonist norbinaltorphimine (nor-BNI) (ICV) were without effect. Place aversions were also produced by central applications of the selective κ-agonist U50,488H and the dynorphin derivative E-2078. For those opioid ligands tested, the doses required to produce place aversions were substantially lower following ICV as compared to SC administration. These data confirm that κ-agonists and opioid antagonists produce aversive states in the drug-naive animal and demonstrate that this effect is centrally mediated. Furthermore, the ability of NLX and CTOP, in contrast to both ICI 174,864 and nor-BNI, to produce place aversions suggests that the aversive effects of opioid antagonists result from the blockade of μ-receptors.

Place preference conditioning reveals the involvement of D1-dopamine receptors in the motivational properties of μ- and κ-opioid agonists

The role of D1 dopamine receptors in mediating the motivational properties of opioid agonists was investigated by use of place preference conditioning. Administration of the D1 receptor antagonist SCH 23390 (0.001-0.5 mg/kg) or the kappa-opioid receptor agonist U-69593 (0.16 mg/kg) produced conditioned place aversions. In contrast, the mu-opioid agonist, morphine (3.0 mg/kg) was appetitively reinforcing. Chronic infusion of SCH 23390 (1.0 mg/kg/day) during conditioning abolished the effects of both opioid agonists. These data demonstrate the specific involvement of D1 receptors in the motivational properties of mu- and kappa-opioid agonists and suggest that dopaminergic systems are crucial for the expression of both reinforcing and aversive motivational states.

Sensitization to the conditioned rewarding effects of morphine: pharmacology and temporal characteristics.

An unbiased place preference conditioning procedure was used to determine whether the repeated administration of morphine results in sensitization to its conditioned rewarding effects. Rats received once daily injections of saline or morphine (5.0 mg/kg; i.p.) for 5 days in a room distinct from that where conditioning would occur. Place preference conditioning commenced 72 h later. A minimum of three drug conditioning sessions was necessary for the establishment of morphine-induced conditioned place preferences in saline-pretreated rats. The minimum dose producing this effect was 5.0 mg/kg. In animals pre-exposed to morphine, significant place preferences occurred after only two drug conditioning sessions and in response to doses of 3.0 mg/kg and greater. The augmented response to morphine was apparent when conditioning commenced 3, 10 or 21 days after the cessation of morphine pretreatment. It was not apparent when conditioning commenced 1 day after treatment cessation. An enhanced response to morphine was also observed in rats which had previously received either fentanyl (0.016 mg/kg/day) or nicotine (0.4 mg/kg/day) for 5 days. Animals which received morphine or fentanyl in combination with naloxone (0.5 mg/kg; s.c.) for 5 days failed to exhibit a conditioned response to morphine. When, however, naloxone was administered in combination with nicotine, significant morphine-induced place preferences were still seen. These data demonstrate that both sensitization and cross-sensitization develop to the conditioned rewarding effects of morphine. Furthermore, they indicate that the sensitization induced by morphine and fentanyl, but not nicotine, is opioid-receptor mediated.

Motivational properties of opioids: evidence that an activation of σ-receptors mediates reinforcement processes

The role of central delta-opioid receptors in the mediation of opioid reinforcement and endogenous reward processes was examined using a non-biased place-preference conditioning procedure. Intracerebroventricular (i.c.v.) administration of the selective delta-receptor agonist, [D-Pen2, D-Pen5]-enkephalin (DPDPE, 10.0-25.0 micrograms) produced a significant preference for the drug-associated place and a similar effect was observed following i.c.v. injections of morphine (10.0 micrograms). Administration of the delta-receptor antagonist, ICI 174,864, at doses (1.0-5.0 micrograms, i.c.v.) which had no aversive effects when tested alone, abolished the reinforcing properties of DPDPE. Such treatment did not, however, modify the effect of morphine. These findings demonstrate the involvement delta- as well as mu-receptors in the motivational properties of opioids and suggest that the activation of either receptor type is sufficient for the elicitation of appetitively reinforcing effects.

PI 3-kinase regulation of dopamine uptake

The magnitude and duration of dopamine (DA) signaling is defined by the amount of vesicular release, DA receptor sensitivity, and the efficiency of DA clearance, which is largely determined by the DA transporter (DAT). DAT uptake capacity is determined by the number of functional transporters on the cell surface as well as by their turnover rate. Here we show that inhibition of phosphatidylinositol (PI) 3‐kinase with LY294002 induces internalization of the human DAT (hDAT), thereby reducing transport capacity. Acute treatment with LY294002 reduced the maximal rate of [3H]DA uptake in rat striatal synaptosomes and in human embryonic kidney (HEK) 293 cells stably expressing the hDAT (hDAT cells). In addition, LY294002 caused a significant redistribution of the hDAT from the plasma membrane to the cytosol. Conversely, insulin, which activates PI 3‐kinase, increased [3H]DA uptake and blocked the amphetamine‐induced hDAT intracellular accumulation, as did transient expression of constitutively active PI 3‐kinase. The LY294002‐induced reduction in [3H]DA uptake and hDAT cell surface expression was inhibited by expression of a dominant negative mutant of dynamin I, indicating that dynamin‐dependent trafficking can modulate transport capacity. These data implicate DAT trafficking in the hormonal regulation of dopaminergic signaling, and suggest that a state of chronic hypoinsulinemia, such as in diabetes, may alter synaptic DA signaling by reducing the available cell surface DATs.

Shift from Goal-Directed to Habitual Cocaine Seeking after Prolonged Experience in Rats

The development of drug-seeking habits is implicated in the transition from recreational drug use to addiction. Using a drug seeking/taking chained schedule of intravenous cocaine self-administration and reward devaluation methods in rats, the present studies examined whether drug seeking that is initially goal-directed becomes habitual after prolonged drug seeking and taking. Devaluation of the outcome of the drug seeking link (i.e., the drug taking link of the chained schedule) by extinction significantly decreased drug seeking indicating that behavior is goal-directed rather than habitual. With, however, more prolonged drug experience, animals transitioned to habitual cocaine seeking. Thus, in these animals, cocaine seeking was insensitive to outcome devaluation. Moreover, when the dorsolateral striatum, an area implicated in habit learning, was transiently inactivated, outcome devaluation was effective in decreasing drug seeking indicating that responding was no longer habitual but had reverted to control by the goal-directed system. These studies provide direct evidence that cocaine seeking becomes habitual with prolonged drug experience and describe a rodent model with which to study the neural mechanisms underlying the transition from goal-directed to habitual drug seeking.