Agustin Zapata

Modulation of the Behavioral and Neurochemical Effects of Psychostimulants by κ‐Opioid Receptor Systems

Abstract: The repeated, intermittent use of cocaine and other drugs of abuse produces profound and often long‐lasting alterations in behavior and brain chemistry. It has been suggested that these consequences of drug use play a critical role in drug craving and relapse to addiction. This article reviews the effects of psychostimulant administration on dopaminergic and excitatory amino acid neurotransmission in brain regions comprising the brains motive circuit and provides evidence that the activation of endogenous κ‐opioid receptor systems in these regions opposes the behavioral and neurochemical consequences of repeated drug use. The role of this opioid system in mediating alterations in mood and affect that occur during abstinence from repeated psychostimulant use are also discussed.

Overview of Brain Microdialysis

The technique of microdialysis enables sampling and collecting of small‐molecular‐weight substances from the interstitial space. It is a widely used method in neuroscience and is one of the few techniques available that permits quantification of neurotransmitters, peptides, and hormones in the behaving animal. More recently, it has been used in tissue preparations for quantification of neurotransmitter release. This unit provides a brief review of the history of microdialysis and its general application in the neurosciences. The authors review the theoretical principles underlying the microdialysis process, methods available for estimating extracellular concentration from dialysis samples (i.e., relative recovery), the various factors that affect the estimate of in vivo relative recovery, and the importance of determining in vivo relative recovery to data interpretation. Several areas of special note, including impact of tissue trauma on the interpretation of microdialysis results, are discussed. Step‐by‐step instructions for the planning and execution of conventional and quantitative microdialysis experiments are provided. Curr. Protoc. Neurosci. 47:7.1.1‐7.1.28.

Repeated Ethanol Intoxication Induces Behavioral Sensitization in the Absence of a Sensitized Accumbens Dopamine Response in C57BL/6J and DBA/2J Mice

Repeated exposure to drugs of abuse results in an increased sensitivity to their behavioral effects, a phenomena referred to as behavioral sensitization. It has been suggested that the same neuroadaptations underlying behavioral sensitization contribute to the maintenance and reinstatement of addiction. Dysregulation of dopamine (DA) neurotransmission in the mesoaccumbens system is one neuroadaptation that is thought to lead to the compulsive drug-seeking that characterizes addiction. Evidence that sensitization to psychostimulants and opiates is associated with an enhancement of drug-evoked DA levels in the nucleus accumbens has also been obtained. Like other drugs of abuse, the acute administration of ethanol (ETOH) stimulates DA release in this brain region. Moreover, repeated ETOH experience results in an enhanced behavioral response to a subsequent ethanol challenge. Data regarding the influence of repeated ethanol intoxication and withdrawal upon mesoaccumbal DA neurotransmission is limited. Studies examining ETOH-evoked alterations in mesoaccumbal DA neurotransmission as a function of withdrawal duration are lacking. The present experiments quantified basal and ethanol-evoked DA levels 14 days and 24 h following the cessation of a repeated ETOH intoxication protocol, which results in sensitization to the locomotor activating effects of ethanol. Locomotor activity was assessed in parallel groups of animals. Studies were conducted in two mouse strains, C57BL/6J and DBA/2J, which differ in their behavioral responses to ETOH. The results indicate the development of transient tolerance to both ETOH-induced behavioral activation and evoked accumbens DA release at early withdrawal. Moreover, no enhanced DA response to a subsequent ETOH challenge could be demonstrated in ETOH experienced animals 2 weeks after withdrawal, in spite of the observation of clear behavioral sensitization at this time point. These results suggest that, at least in the case of ethanol, sensitization of the DA mesolimbic system may not be necessary for the development of behavioral sensitization.

Selective D3 receptor agonist effects of (+)-PD 128907 on dialysate dopamine at low doses.

An involvement of the D3 dopamine receptor in the modulation of extracellular dopamine concentrations is suggested by pharmacological studies. However, recent studies using D3 receptor knock out mice indicated that several functions previously attributed to the D3 receptor are mediated by other receptor types. In the present study, we used the no-net flux microdialysis technique to characterize: (i) basal dopamine dynamics in the ventral striatum of D3 knock out and wild type mice and (ii) the effects of the putative D3-receptor selective agonist (+)-PD 128907. Neither the extracellular dopamine concentration nor the in vivo extraction fraction, an indirect measure of basal dopamine uptake, differed between D3 knock out and wild type mice. Moreover, no differences in potassium (60 mM) or cocaine (5 or 20 mg/kg i.p.) evoked dopamine concentrations were detected between the two genotypes. However, intra-striatal or systemic administration of doses of (+)-PD 128907 that failed to modify dopamine concentrations in knock out mice significantly decreased dialysate dopamine concentrations in the wild type. Comparison of the concentration-response curve for (+)-PD 128907 revealed IC(25) values of 61 and 1327 nM in wild type and knock out mice, respectively, after intra-striatal infusions. Similar differences were obtained after systemic administration of the D3 preferring agonist (IC(25) 0.05 and 0.44 mg/kg i.p. in wild type and knock out mice, respectively). We conclude that the activation of the D3 receptor decreases extracellular dopamine levels and that, at sufficiently low doses, the effects of (+)-PD 128907 on extracellular dopamine are selectively mediated by the D3 receptor.

Behavioural sensitization and enhanced dopamine response in the nucleus accumbens after intravenous cocaine self-administration in mice

The behavioural effects of cocaine are enhanced in animals with a prior history of repeated cocaine administration. This phenomenon, referred to as sensitization, is also associated with an increase in cocaine‐evoked extracellular dopamine levels in the nucleus accumbens. Behavioural and neurochemical sensitization has been demonstrated in rats with a prior history of cocaine self‐administration and in those that had received experimenter‐administered cocaine. Although it is clear that the repeated non‐contingent administration also results in behavioural sensitization in the mouse, the issue of whether behavioural and neurochemical sensitization also occur in this species following intravenous cocaine self‐administration has not been assessed. The present study used the technique of in vivo microdialysis in conjunction with operant self‐administration to characterize cocaine‐evoked locomotor activity and dopamine levels in the nucleus accumbens in mice with a prior history of intravenous cocaine self‐administration or those that had received yoked infusions of cocaine. Mice that had received contingent or non‐contingent infusions of cocaine exhibited an enhanced behavioural response to cocaine and increased cocaine‐evoked dopamine levels in the nucleus accumbens. There was no difference between groups in the magnitude of this effect. Prior exposure to cocaine did not modify baseline dopamine levels in the nucleus accumbens. These data demonstrate that mice with previous cocaine self‐administration experience show an enhanced behavioural and dopamine response to cocaine in the nucleus accumbens. Furthermore, control over cocaine infusion does not significantly alter the magnitude of the sensitized behavioural and presynaptic dopamine responses observed in response to a challenge dose of cocaine.

Mu opioid receptor modulation of somatodendritic dopamine overflow: GABAergic and glutamatergic mechanisms

Mu opioid receptor (MOR) regulation of somatodendritic dopamine neurotransmission in the ventral tegmental area (VTA) was investigated using conventional microdialysis in freely moving rats and mice. Reverse dialysis of the MOR agonist DAMGO (50 and 100 μm) into the VTA of rats produced a concentration‐dependent increase in dialysate dopamine concentrations. Basal dopamine overflow in the VTA was unaltered in mice lacking the MOR gene. However, basal γ‐aminobutyric acid (GABA) overflow in these animals was significantly increased, whereas glutamate overflow was decreased. Intra‐VTA perfusion of DAMGO into wild‐type (WT) mice increased dopamine overflow. GABA concentrations were decreased, whereas glutamate concentrations in the VTA were unaltered. Consistent with the loss of MOR, no effect of DAMGO was observed in MOR knockout (KO) mice. These data provide the first direct demonstration of tonically active MOR systems in the VTA that regulate basal glutamatergic and GABAergic neurotransmission in this region. We hypothesize that increased GABAergic neurotransmission following constitutive deletion of MOR is due to the elimination of a tonic inhibitory influence of MOR on GABAergic neurons in the VTA, whereas decreased glutamatergic neurotransmission in MOR KO mice is a consequence of intensified GABA tone on glutamatergic neurons and/or terminals. As a consequence, somatodendritic dopamine release is unaltered. Furthermore, MOR KO mice do not exhibit the positive correlation between basal dopamine levels and the glutamate/GABA ratio observed in WT mice. Together, our findings indicate a critical role of VTA MOR in maintaining an intricate balance between excitatory and inhibitory inputs to dopaminergic neurons.

Quantitative no-net-flux microdialysis permits detection of increases and decreases in dopamine uptake in mouse nucleus accumbens

A number of investigators are using the quantitative no-net-flux microdialysis technique to monitor basal neurotransmitter dynamics in discrete brain regions of behaving animals. The predictive validity of the probe extraction fraction (Ed) for quantifying decreases in the rate of dopamine (DA) clearance from the extracellular space is well documented. It was recently suggested, however, that Ed may be insensitive to increases in DA clearance. Here we report that the Ed for DA in the nucleus accumbens (NAc) of the behaving mouse is increased following pharmacological inactivation of kappa-opioid receptors, a treatment previously shown to augment DA uptake. The Ed obtained in control mice and those that received the long-acting kappa-opioid receptor antagonist, nor-binaltorphimine (nor-BNI), satisfied the requirement that the mean values of each were lower than the mean value in vitro for the same probes immersed in well-stirred artificial cerebrospinal fluid. The Ed was increased in the NAc of nor-BNI-treated mice as compared to saline-treated control animals. The corresponding increase in the DA uptake rate was quantified by using the Ed values to calculate a change in the apparent clearance rate constant. Nor-BNI treatment did not alter the apparent extracellular dopamine concentration represented by the point of no-net-flux indicating that the rates of DA uptake and release were both increased.

Nucleus accumbens-derived glial cell line-derived neurotrophic factor is a retrograde enhancer of dopaminergic tone in the mesocorticolimbic system.

Spontaneous firing of ventral tegmental area (VTA) dopamine (DA) neurons provides ambient levels of DA in target areas such as the nucleus accumbens (NAc) and the prefrontal cortex (PFC). Here we report that the glial cell line-derived neurotrophic factor (GDNF), produced in one target region, the NAc, is retrogradely transported by DA neurons to the VTA where the growth factor positively regulates the spontaneous firing activity of both NAc- and PFC-projecting DA neurons in a mechanism that requires the activation of the mitogen-activated protein kinase (MAPK) pathway. We also show that the consequence of GDNF-mediated activation of the MAPK signaling cascade in the VTA is an increase in DA overflow in the NAc. Together, these results demonstrate that NAc-produced GDNF serves as a retrograde enhancer that upregulates the activity of the mesocorticolimbic DA system.

NAALADase (GCP II) inhibition prevents cocaine-kindled seizures

The prediction that inhibition of NAALADase, an enzyme catalyzing the cleavage of glutamate from N-acetyl-aspartyl-glutamate, would produce antiepileptogenic effects against cocaine was tested. Cocaine kindled seizures were developed in male, Swiss-Webster mice by daily administration of 60 mg/kg cocaine for 5 days. The NAALADase inhibitor 2-(phosphonomethyl)pentanedioic acid (2-PMPA) produced dose-dependent protection (10-100 mg/kg) against both the development of seizure kindling and the occurrence of seizures during the kindling process without observable behavioral side-effects. It is not likely that 2-PMPA produced protection against cocaine kindling by altering the potency of the convulsant stimulus as daily administration of 2-PMPA did not alter the convulsant thresholds for cocaine. Lower daily doses of cocaine (40 mg/kg) did not increase the incidence of seizures but produced kindling, as evidenced by the increase in seizure susceptibility when mice were probed with a higher dose of cocaine. 2-PMPA was also effective in preventing the development of sensitization to this covert kindling process. In contrast to its efficacy against cocaine kindled seizures, 2-PMPA failed to attenuate the convulsions engendered by acute challenges with pentylenetetrazole, bicuculline, N-methyl-D-aspartate, maximal electroshock or cocaine. Similarly, acutely-administered 2-PMPA did not block cocaine seizures in fully-kindled mice. NAALADase inhibition thus provides a novel means of attenuating the development of cocaine seizure kindling.

Selective Deletion of PTEN in Dopamine Neurons Leads to Trophic Effects and Adaptation of Striatal Medium Spiny Projecting Neurons

The widespread distribution of the tumor suppressor PTEN in the nervous system suggests a role in a broad range of brain functions. PTEN negatively regulates the signaling pathways initiated by protein kinase B (Akt) thereby regulating signals for growth, proliferation and cell survival. Pten deletion in the mouse brain has revealed its role in controlling cell size and number. In this study, we used Cre-loxP technology to specifically inactivate Pten in dopamine (DA) neurons (Pten KO mice). The resulting mutant mice showed neuronal hypertrophy, and an increased number of dopaminergic neurons and fibers in the ventral mesencephalon. Interestingly, quantitative microdialysis studies in Pten KO mice revealed no alterations in basal DA extracellular levels or evoked DA release in the dorsal striatum, despite a significant increase in total DA tissue levels. Striatal dopamine receptor D1 (DRD1) and prodynorphin (PDyn) mRNA levels were significantly elevated in KO animals, suggesting an enhancement in neuronal activity associated with the striatonigral projection pathway, while dopamine receptor D2 (DRD2) and preproenkephalin (PPE) mRNA levels remained unchanged. In addition, PTEN inactivation protected DA neurons and significantly enhanced DA-dependent behavioral functions in KO mice after a progressive 6OHDA lesion. These results provide further evidence about the role of PTEN in the brain and suggest that manipulation of the PTEN/Akt signaling pathway during development may alter the basal state of dopaminergic neurotransmission and could provide a therapeutic strategy for the treatment of Parkinsons disease, and other neurodegenerative disorders.