Psychostimulant-induced reinstatement of place preferences have been used to investigate underlying physiological mechanisms mediating drug-seeking behavior in adolescent and adult rodents; however, it is still unclear how psychostimulant exposure during adolescence affects neuronal communication in the mesolimbic dopamine (DA) pathway and whether these changes would elicit enhanced drug-seeking behavior later in adulthood. The aim of the present study was to investigate the effects of intra-ventral tegmental area (VTA) or intra-nucleus accumbens septi (NAcc) DA D2 receptor antagonist infusions on cocaine-induced reinstatement of cocaine place conditioning in high and low responders for cocaine reward. Adolescent rats were exposed to cocaine place conditioning [postnatal day (PND 28-39)] and divided into high and low responders for cocaine reward based on their place preference expression score. Place preferences were extinguished and guide cannula were implanted into either the VTA or NAcc followed by one of the following: 1) intra-VTA or intra-NAcc infusion of the DA D2 receptor antagonist sulpiride (100 µM) during a cocaine-primed reinstatement test (10 mg/kg/ip cocaine) or 2) measurement of NAcc DA levels during intra-VTA or intra-NAcc infusion of sulpiride (100 µM), a cocaine prime (10 mg/kg cocaine) and re-exposure to the cocaine paired chamber. Infusion of sulpiride into the VTA but not the NAcc blocked reinstatement of cocaine place conditioning in rats exposed to cocaine during adolescence. Furthermore, re-exposure to cocaine-associated cues and simultaneous local infusion of sulpiride into either the VTA or NAcc attenuated cocaine-induced increases in accumbal DA levels for rats pretreated with cocaine during adolescence, regardless of phenotype. These data suggest intrinsic compensatory mechanisms in the mesolimbic DA pathway mediate adolescent behavioral responsivity to cocaine prime-induced reinstatement of cocaine place conditioning later on in adulthood.

In humans, adolescent exposure to illicit drugs predicts the onset of adult drug abuse and suggests that early drug use potentiates adolescent vulnerability to drug addiction. In experiment 1, it was hypothesized that adolescent rats would show a CPP for a low cocaine dose if in fact adolescents are more vulnerable to cocaine's rewarding effects. Place preferences were measured in early adolescent [postnatal day (PND) 35], late adolescent (PND 45) and young adult (PND 60) rats by injecting either 0, 5 or 20 mg/kg cocaine and conditioning them to environmental cues in a 2-chamber place conditioning apparatus. Significant cocaine preferences were found for all ages at the high dose. Interestingly, PND 35's were the only age group to have a CPP at the low dose suggesting that PND 35 rats are more sensitive than late adolescent and young adult rats to cocaine's rewarding effects. In Experiment 2, it was hypothesized that age-related differences in cocaine CPP may be mediated by differences in the mesolimbic dopaminergic (DA) system throughout development. Extracellular DA levels in the nucleus accumbens septi (NAcc) of early adolescent, late adolescent and adult rats were measured via quantitative microdialysis. PND 35, PND 45 and PND 60 rats were injected daily with either 5 mg/kg/ip or saline for 4 days, surgically implanted with a microdialysis probe aimed at the NAcc. Rats were perfused with either 0, 1, 10 or 40 nM DA and the extracellular DA concentration was measured. Our results show that adolescents differ from adults in basal DA with PND 35 rats having low basal DA (0.4 nM), PND 45 rats having high basal DA (1.8 nM) and PND 60 rats having intermediate basal DA (1.3 nM). PND 45 cocaine treated rats showed a 58% decrease in basal DA. All cocaine treated rats, regardless of age, showed a significant increase in DA over baseline in response to a cocaine challenge. Additionally, there were age-related differences in the extraction fraction (Ed), an indirect measure of DA reuptake, with PND 45 and PND 60's showing a decrease in basal Ed, an effect absent in PND 35's. Together these findings suggest that there are substantial ontogenetic differences in extracellular DA and DA reuptake and that these differences may provide an explanation for adolescent vulnerability to addiction. Future research should investigate DA supply and degradation processes in naïve and cocaine treated adolescent rats and vulnerability to addiction.

Various small molecules act as neurotransmitters and orchestrate neural communication. Growing evidence suggests that not only classical neurotransmitters but also several small molecules, including amino acid derivatives, modulate synaptic transmission. As conditions of acute and chronic pain alter neuronal excitability in the nucleus accumbens, we hypothesized that small molecules released in the nucleus accumbens might play important roles in modulating the pain sensation. However, it is not easy to identify possible pain modulators owing to the absence of a method for comprehensively measuring extracellular small molecules in the brain. In this study, through the use of an emerging metabolomics technique, namely ion chromatography coupled with high-resolution mass spectrometry, we simultaneously analyzed the dynamics of more than 60 small molecules in brain fluids collected by microdialysis, under both the application of pain stimuli and the administration of analgesics. We identified N-acetylaspartylglutamate as a potential pain modulator that is endogenously released in the nucleus accumbens. Infusion of N-acetylaspartylglutamate into the nucleus accumbens significantly attenuated the pain induced by the activation of sensory nerves through optical stimulation. These findings suggest that N-acetylaspartylglutamate released in the nucleus accumbens could modulate pain sensation.

Serotoninergic nerves are known to modulate sensitization of dopamine receptors (DA-R) in a rodent model of Parkinson’s disease (PD). However, serotoninergic nerves are not known to have a prominent role on DA exocytosis in intact rats. The current study was undertaken to explore the possible influence of serotoninergic nerves on DA exocytosis in Parkinsonian rats. Rat pups were treated at 3 days after birth with the neurotoxin 6-hydroxydopamine (6-OHDA; 134 μg icv, half into each lateral ventricle; desipramine, 1 h pretreatment), in order to produce marked long-lasting destruction of neostriatal dopaminergic innervation, as evidenced by the 90–95% depletion of DA (p < 0.001) [HPLC/ED] into adulthood. Controls received vehicle/desipramine in place of 6-OHDA. Other groups received the serotoninergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT; 25 μg base, icv, half in each lateral ventricle; desipramine, 1 h; 75 mg/kg pargyline HCl, 30 min) at 3 days post-birth; or both 6-OHDA+5,7-DHT treatments. In adulthood, an in vivo microdialysis study was undertaken to ascertain that p-chloroamphetamine (PCA, 1 mM in the microdialysate)-evoked DA release in the neostriatum was reduced approximately 50% in the 6-OHDA group, while PCA-evoked DA release in the 6-OHDA+5,7-DHT group was substantially increased, to a level equivalent to that of the vehicle control. The baseline neostriatal microdialysate level of 3,4-dihydroxyphenylacetic acid (DOPAC) was also higher in the 6-OHDA+5,7-DHT group vs 6-OHDA group; also, during the 2nd hour of PCA infusion. PCA-enhanced DA exocytosis occurred in the absence of changes in hydroxyl radical (HO·) in the microdialysate (i.e., assay of 2,3- and 2,5-dihydroxybenzoic acid, 2,3-DHBA; 2,5-DHBA). The overall findings demonstrate that an adulthood serotoninergic nerve lesion enhanced PCA-evoked DA exocytosis in a rodent model of severe PD, while susceptibility to oxidative stress was unchanged. The implication is that serotoninergic nerves may normally suppress the release of DA and/or act as an uptake site and storage sink for accumulated DA in parkinsonian-like neostriatum. Potentially, serotoninergic agonists or antagonists, targeting subtype-selective serotonin receptors, may be viable therapeutic adjuncts in PD.

D-amino acid oxidase (DAO) is a flavin-dependent enzyme that is expressed in the mammalian brain. It is the metabolising enzyme of several D-amino acids, including D serine, which is an endogenous agonist at the glycine co-agonist site of the glutamatergic NMDA receptor. As such, regulation of D serine levels in the brain by DAO may indirectly modulate the activity of NMDA receptors. The expression and activity of DAO have been reported to be increased in schizophrenia. It has been identified as a putative susceptibility gene for the disorder, and as a potential therapeutic target. This thesis explored three aspects of the interface between DAO and the DA system. First, the expression of DA was investigated in the ventral tegmental area (VTA), the source of the dopaminergic mesocortical pathway. Traditionally, DAO was considered to be an enzyme confined to the hindbrain and to glia, but more recent studies have reported its expression in additional brain regions, and also in neurons. DAO mRNA and protein was found to be expressed in the VTA, and was present in both neurons and glia in this region, whereas in the cerebellum, DAO expression appeared solely glial. DA output from the VTA is regulated by NMDA receptors, and hence expression of DAO in the VTA suggests that it may serve a role in modulating cortical DA via regulation of D serine levels and NMDA receptor function. The second part of this thesis investigated the effects of DAO inhibition and D serine administration on DA levels in the prefrontal cortex (PFC) using in vivo microdialysis. Systemic DAO inhibition and D serine administration resulted in increases in extracellular levels of DA metabolites in the PFC, despite no detectable change in DA. Similarly, DA metabolites in the PFC increased after local application of D serine to the VTA, but no change was detected in DA. However, local DAO inhibition in the VTA resulted in increased levels of both DA and its metabolites, and DAO inhibition combined with D serine administration also produced increases in DA. This suggested that DAO and its regulation of D-serine levels may serve to indirectly modulate mesocortical DA function, and this may be mediated via the VTA. This notion was supported in the final section of this thesis, in which the expression of three DA genes was measured in the PFC of a novel line of DAO knockout mice. In this pilot study, there was evidence for an increase in Comt and Drd2 mRNAs in the knockout mice. As such, constitutive abolition of DAO activity may also alter mesocortical DA function. These studies provide new insights into the presence and role of DAO beyond the hindbrain, and point to a potentially important physiological function in modulating the activity of the mesocortical DA system via the VTA. This could be therapeutically relevant in the context of elevating cortical DA in the treatment of schizophrenia, and may provide supporting evidence for the clinical use of DAO inhibitors.