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Purpose: Comparison of the parasympathetic and sympathetic neurons, including the dopaminergic neural system, in dry eye (DE)-induced pathophysiology has not been elucidated well. This study investigated the presence of dopamine receptors (DRs) and their functional roles in the lacrimal glands (LGs) of DE-induced mice., Methods: After DE was induced in B6 mice for 2 weeks, the expression of tyrosine hydroxylase (TH), dopamine, and DRs (DR1, DR2, etc.) in the LGs and corneas were measured by quantitative RT-PCR, immunoblot, and ELISA. Using flow cytometry and ELISA, immune cell infiltration and inflammatory cytokine expression were determined in DE-induced LGs with or without DR blockers, SCH-23390 (DR1i), or melperone (DR2i). Corneal erosion scores were also investigated., Results: The mRNA and protein levels of TH significantly increased in DE-induced LGs. The dopamine concentration of LGs was 9.51 pmol in DE (versus naive: 1.39 pmol; P < 0.001). Both DR1 and DR2 mRNA expression were significantly enhanced in desiccating stress compared with those in naive (3.7- and 2.1-fold, P < 0.001). Interestingly, DR1 and DR2 immunostaining patterns stained independently in DE-induced LGs. CD3+ and CD19+ cell infiltration was significantly increased by DR2i (P < 0.001) but not by DR1i. Furthermore, IFN-γ, IL-17, and TNF-α were significantly upregulated by DR2i compared with the blow-only condition. The severity of corneal erosion and inflammation was also aggravated by DR2i., Conclusions: Upregulation of DR1 and DR2 was observed in DE-induced mouse LGs. As the inflammatory conditions are aggravated by the inhibition of DRs, especially DR2, their activity may be an important factor preserving ocular surface homeostasis.

Methamphetamine (Meth) abuse is a common HIV comorbidity. Males and females differ in their patterns of Meth use, associated behaviors, and responses, but the underlying mechanisms and impact of HIV infection are unclear. Transgenic mice with inducible HIV-1 Tat protein in the brain (iTat) replicate many neurological aspects of HIV infection in humans. We previously showed that Tat induction enhances the Meth sensitization response associated with perturbation of the dopaminergic system, in male iTat mice. Here, we used the iTat mouse model to investigate sex differences in individual and interactive effects of Tat and Meth challenge on locomotor sensitization, brain expression of dopamine receptors (DRDs) and regulatory adenosine receptors (ADORAs). Because Meth administration increases the production of reactive oxygen species (ROS), we also determined whether the effects of Meth could be rescued by concomitant treatment with the ROS scavenger N-acetyl cysteine (NAC). After Meth sensitization and a 7-day abstinence period, groups of Tat+ and Tat-male and female mice were challenged with Meth in combination with NAC. We confirmed that Tat expression and Meth challenge suppressed DRD mRNA and protein in males and females' brains, and showed that females were particularly susceptible to the effects of Meth on D1-like and D2-like DRD subtypes and ADORAs. The expression of these markers differed strikingly between males and females, and between females in different phases of the estrous cycle, in a Tat -dependent manner. NAC attenuated Meth-induced locomotor sensitization and preserved DRD expression in all groups except for Tat + females. These data identify complex interactions between sex, Meth use, and HIV infection on addiction responses, with potential implications for the treatment of male and female Meth users in the context of HIV, especially those with cognitive disorders., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The neurocircuitry underlying hunger, satiety, motivation to eat and food reward is complex, however a lot of mechanisms are still unknown. Two main cerebral areas are responsible for controlling feeding through hunger and food reward: the hypothalamus (HPT) and the ventral tegmental area (VTA), respectively. The dopaminergic system modulates both these areas and is essential to control food ingestion. Therefore, we aim to evaluate the effects of restrictive and hyperlipidic diets during pregnancy, lactation and during adult life of the offspring, on the expression of dopaminergic system genes in VTA and HPT of mice dams and their adult male offspring. We also measured diets' effect in locomotor activity in the open field (OF) test. Female mice were divided into control (CONT), restriction (RD) and hyperlipidic (HD) dietary groups, and mated with isogenic male mice. On the 9th postpartum day (PPD), dams were tested in the OF, and on the 22nd PPD cerebral areas were collected. After weaning, the offspring also were divided into one of three diet groups, independently of the diets provided to their dams. In the 80th PPD, the offspring was tested in the OF, and at 100th PPD, VTA and HPT were collected. Gene expression was analyzed by quantitative reverse transcription real-time polymerase chain reaction. The correlation between gene expression and locomotor activity was also assessed. In dams' VTA, both diets upregulated the expression of Th, Slc6a3/Dat1, Drd1 and Drd2 genes. In opposition, in the offspring the maternal diet was associated with a reduction in Th and Ddc gene expression. In the HPT, mice dams that received restriction or hyperlipidic diets had increased Th mRNA levels, but reduced the expression of Drd4 gene. The offspring diet had no effect on the expression of the studied genes in their adult lives. Both diets increased mice dam's locomotion in the OF, however none of them altered the offspring locomotor activity. We detected a positive correlation between the duration of total locomotion in the OF and Slc6a3/Dat1 gene expression in VTA of mice dams. In the HPT, a negative correlation of locomotion and Drd4 mRNA levels, and a positive correlation with Th gene expression was observed. Our results show that restriction and hyperlipidic diets alter mice dams' locomotor activity in the OF and modify the expression of dopaminergic system genes in VTA and HPT of mice dams and in VTA of the offspring., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In preclinical studies serotonin stimulates and dopamine inhibits tumour growth and angiogenesis. Information regarding serotonin and dopamine receptor (5-HTR and DRD) expression in human cancers is limited. Therefore, we screened a large tumour set for receptor mRNA overexpression using functional genomic mRNA (FGmRNA) profiling, and we analysed protein expression and location of 5-HTR1B, 5-HTR2B, DRD1, and DRD2 with immunohistochemistry in different tumour types. With FGmRNA profiling 11,756 samples representing 43 tumour types were compared to 3,520 normal tissue samples to analyse receptor overexpression. 5-HTR2B overexpression was present in many tumour types, most frequently in uveal melanomas (56%). Receptor overexpression in rare cancers included 5-HTR1B in nasopharyngeal carcinoma (17%), DRD1 in ependymoma (30%) and synovial sarcoma (21%), and DRD2 in astrocytoma (13%). Immunohistochemistry demonstrated high 5-HTR2B protein expression on melanoma and gastro-intestinal stromal tumour cells and endothelial cells of colon, ovarian, breast, renal and pancreatic tumours. 5-HTR1B expression was predominantly low. High DRD2 protein expression on tumour cells was observed in 48% of pheochromocytomas, and DRD1 expression ranged from 14% in melanoma to 57% in renal cell carcinoma. In conclusion, 5-HTR1B, 5-HTR2B, DRD1, and DRD2 show mRNA overexpression in a broad spectrum of common and rare cancers. 5-HTR2B protein is frequently highly expressed in human cancers, especially on endothelial cells. These findings support further investigation of especially 5HTR2B as a potential treatment target.

Disturbances in the function of the mesostriatal dopamine system may contribute to the development and maintenance of chronic pain, including its sensory and emotional/cognitive aspects. In the present study, we assessed the influence of chronic constriction injury (CCI) of the sciatic nerve on the expression of genes coding for dopamine and opioid receptors as well as opioid propeptides in the mouse mesostriatal system, particularly in the nucleus accumbens. We demonstrated bilateral increases in mRNA levels of the dopamine D1 and D2 receptors (the latter accompanied by elevated protein level), opioid propeptides proenkephalin and prodynorphin, as well as delta and kappa (but not mu) opioid receptors in the nucleus accumbens at 7 to 14 days after CCI. These results show that CCI-induced neuropathic pain is accompanied by a major transcriptional dysregulation of molecules involved in dopaminergic and opioidergic signaling in the striatum/nucleus accumbens. Possible functional consequences of these changes include opposite effects of upregulated enkephalin/delta opioid receptor signaling vs. dynorphin/kappa opioid receptor signaling, with the former most likely having an analgesic effect and the latter exacerbating pain and contributing to pain-related negative emotional states.

Agonistic behaviour is common in an encounter between two crustaceans. It often causes limb disability and consumes a lot of energy, which is harmful for the growth and survival of commercially important crustaceans. In the present study, we mainly focused on the agonistic behaviour of the Chinese mitten crab, Eriocheir sinensis, which is an important species of the aquaculture industry in China. We recorded agnostic behaviour with a high-definition camera and preliminarily evaluated the role of serotonin (5-HT) or dopamine (DA)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway and eyestalk in the behaviour. The results showed that agonistic behaviour in E. sinensis consisted of three stages: approach, contact and fight. We found that the number of fights and cumulative time of fight were significantly higher in the male vs. male group than in the female vs. female and female vs. male groups (P < 0.05). After 1 h of agonistic behaviour, 5-HT concentration showed a significant increase and DA concentration showed a significant decrease when compared with the control group (no encounter; P < 0.05). 5-HT1B and 5-HT2B mRNA levels showed a significant increase in the eyestalk (P < 0.05). 5-HT7 mRNA levels showed significant downregulation in the thoracic ganglia and DA1A mRNA levels showed upregulation in the intestine (P < 0.05). DA2 mRNA levels showed a significant decrease in the eyestalk (P < 0.05). These changes were accompanied by a significant increase in cAMP level and significant decrease in PKA level in the haemolymph (P < 0.05). In addition, a significant decrease in glucose levels was detected after the agonistic behaviour. Crustacean hyperglycemic hormone (CHH) mRNA levels showed significant upregulation in the eyestalk and significant downregulation in the intestine (P < 0.05). The number of fights and cumulative time of fight in the left eyestalk ablation (L-X vs. L-X) group were more and longer than those in the intact eyestalk (C vs. C), right eyestalk ablation (R-X vs. R-X) and bilateral eyestalk ablation (D-X vs. D-X) groups. In short, E. sinensis shows special agonistic behaviour modulated by 5-HT or DA-cAMP-PKA pathway and eyestalk, especially the left eyestalk., (Copyright © 2019. Published by Elsevier Inc.)

Background: Benign prostatic hyperplasia (BPH) and prostate cancer (PCa) are the most prevalent diseases in male population, implicated with fundamental differences between benign and malignant growth of prostate cells. An imbalance through a network of nervous, endocrine, and immune systems initiate a signal of altered growth from the brain to the prostate gland, leading to adverse effects such as inflammation., Objective: The aim of this study was to evaluate the gene expression of dopamine receptor family, COMT, and IL6 to identify novel correlations in BPH and PCa in both blood and tumor of the patients., Methods: Peripheral blood mononuclear cells from BPH (n= 30) and PCa (n= 30) patients, and prostate tumor tissues (n= 14) along with pathologically normal adjacent tissues (n= 14) were isolated, mRNA was extracted, and cDNA was synthesized, respectively. Quantitative real- time PCR was applied for DRD1- DRD5, COMT, and IL6 genes in all samples., Results: We found, for the first time, that the expression of COMT and IL6 genes were inversely correlated with the expression of DRD1 and DRD2 genes through the extent of differentiation of PCa from BPH condition. In addition, the PSA levels were correlated with the expression of DRD1 in BPH cases and DRD1, DRD4, DRD5, and IL6 in PCa cases., Conclusion: Results implicate a potential cross- talk between the signaling pathways derived by IL6 cytokine and dopamine receptors in PCa. Thus, it seems promising to reassemble the consequent signaling pathways by adequate agonists and antagonists to help increase therapeutic efficacy., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Addiction to drugs, including opioids is the result of an interplay between environmental and genetic factors. It has been shown that the progeny of addict people is at higher risk for drug addiction. However, the mechanisms of such trans-generational effects of drugs are not so clear. Here we have evaluated the effects of parental morphine consumption on anxiety, morphine preference, and mRNA expression of dopamine receptors in F1 and F2 male offspring. Morphine was chronically administered to adult male and female Wistar rats followed by 14-day abstinence before mating. Morphine preference and anxiety-like behavior in the offspring were measured by two-bottle-choice paradigm and elevated-plus maze, respectively. Real-time PCR was used to measure the mRNA expression level of dopamine receptors in the striatum, nucleus accumbens, prefrontal cortex, and hippocampus of F1 animals. The results indicated that F1 but not the F2 male progeny of morphine-exposed parents had a greater preference for morphine, and more anxiety-like behavior compared to the offspring of saline-treated parents. In F1 male progeny of morphine-treated parents, D1 and D5 dopamine receptors were significantly increased in the prefrontal cortex and nucleus accumbens. D5 and D2 receptors were decreased in the hippocampus. D4 dopamine receptor was augmented in striatum and hippocampus and decreased in the prefrontal cortex. Adulthood exposure to chronic morphine in male and female rats before conception leads to higher morphine preference and increased anxiety in F1 but not F2 male progeny. Alterations of dopamine receptor expression in the reward system may be one mechanism responsible for observed changes in F1 offspring., (Copyright © 2018 ISDN. Published by Elsevier Ltd. All rights reserved.)

The pro-opiomelanocortin (POMC)-expressing neurons of the hypothalamic arcuate nucleus (ARC) are involved in the control of food intake and metabolic processes. It is assumed that, in addition to leptin, the activity of these neurons is regulated by serotonin and dopamine, but only subtype 2C serotonin receptors (5-HT 2C R) was identified earlier on the POMC-neurons. The aim of this work was a comparative study of the localization and number of leptin receptors (LepR), types 1 and 2 dopamine receptors (D 1 R, D 2 R), 5-HT 1B R and 5-HT 2C R on the POMC-neurons and the expression of the genes encoding them in the ARC of the normal and diet-induced obese (DIO) rodents and the agouti mice (A y /a) with the melanocortin obesity. As shown by immunohistochemistry (IHC), all the studied receptors were located on the POMC-immunopositive neurons, and their IHC-content was in agreement with the expression of their genes. In DIO rats the number of D 1 R and D 2 R in the POMC-neurons and their expression in the ARC were reduced. In DIO mice the number of D 1 R and D 2 R did not change, while the number of LepR and 5-HT 2C R was increased, although to a small extent. In the POMC-neurons of agouti mice the number of LepR, D 2 R, 5-HT 1B R and 5-HT 2C R was increased, and the D 1 R number was reduced. Thus, our data demonstrates for the first time the localization of different types of the serotonin and dopamine receptors on the POMC-neurons and a specific pattern of the changes of their number and expression in the DIO and melanocortin obesity.

Patients with advanced breast cancer often fail to respond to treatment, creating a need to develop novel biomarkers and effective therapeutics. Dopamine (DA) is a catecholamine that binds to five G protein-coupled receptors. We discovered expression of DA type-1 receptors (D1Rs) in breast cancer, thereby identifying these receptors as novel therapeutic targets in this disease. Strong to moderate immunoreactive D1R expression was found in 30% of 751 primary breast carcinomas, and was associated with larger tumors, higher tumor grades, node metastasis and shorter patient survival. DA and D1R agonists, signaling through the cGMP/protein kinase G (PKG) pathway, suppressed cell viability, inhibited invasion and induced apoptosis in multiple breast cancer cell lines. Fenoldopam, a peripheral D1R agonist that does not penetrate the brain, dramatically suppressed tumor growth in two mouse models with D1R-expressing xenografts by increasing both necrosis and apoptosis. D1R-expressing primary tumors and metastases in mice were detected by fluorescence imaging. In conclusion, D1R overexpression is associated with advanced breast cancer and poor prognosis. Activation of the D1R/cGMP/PKG pathway induces apoptosis in vitro and causes tumor shrinkage in vivo. Fenoldopam, which is FDA (Food and Drug Administration) approved to treat renal hypertension, could be repurposed as a novel therapeutic agent for patients with D1R-expressing tumors.

Steroids profoundly influence behavioral responses to alcohol by activating canonical nuclear hormone receptors and exerting allosteric effects on ion channels. Accumulating evidence has demonstrated that steroids can also trigger biological effects by directly binding G-protein-coupled receptors (GPCRs), yet physiological roles of such unconventional steroid signaling in controlling alcohol-induced behaviors remain unclear. The dopamine/ecdysteroid receptor (DopEcR) is a GPCR that mediates nongenomic actions of ecdysteroids, the major steroid hormones in insects. Here, we report that Drosophila DopEcR plays a critical role in ethanol-induced sedation.DopEcR mutants took longer than control flies to become sedated during exposure to ethanol, despite having normal ethanol absorption or metabolism. RNAi-mediated knockdown of DopEcR expression revealed that this receptor is necessary after eclosion, and is required in particular neuronal subsets, including cholinergic and peptidergic neurons, to mediate this behavior. Additionally, flies ubiquitously overexpressing DopEcR cDNA had a tendency to become sedated quickly upon ethanol exposure. These results indicate that neuronal subset-specific expression of DopEcR in adults is required for normal sedation upon exposure to ethanol. We also obtained evidence indicating that DopEcR may promote ethanol sedation by suppressing epidermal growth factor receptor/extracellular signal-regulated kinase signaling. Last, genetic and pharmacological analyses suggested that in adult flies ecdysone may serve as an inverse agonist of DopEcR and suppress the sedation-promoting activity of DopEcR in the context of ethanol exposure. Our findings provide the first evidence for the involvement of nongenomic G-protein-coupled steroid receptors in the response to alcohol, and shed new light on the potential roles of steroids in alcohol-use disorders., Significance Statement: Alcohol abuse is an alarming personal and societal burden. The improvement of prevention and treatment strategies for alcohol-use disorders requires a better understanding of their biological basis. Steroid hormones profoundly affect alcohol-induced behaviors, but the contribution of their unconventional, nongenomic actions during these responses has not yet been elucidated. We found that Drosophila DopEcR, a unique G-protein-coupled receptor (GPCR) with dual specificity for dopamine and steroids, mediates noncanonical steroid actions to promote ethanol-induced sedation. Because steroid signaling and the behavioral response to alcohol are evolutionarily well conserved, our findings suggest that analogous mammalian receptors likely play important roles in alcohol-use disorders. Our work provides a foundation for further characterizing the function and mechanisms of action of nonclassical steroid GPCR signaling., (Copyright © 2016 the authors 0270-6474/16/364647-11$15.00/0.)

Dopamine (DA) is a crucial transmitter in the neuroimmune network, where it contributes to the nervous system-immune system interplay as well as in the communication among immune cells. DA acts through five different dopaminergic receptors (DR) grouped into two families: the D1-like (D1 and D5) and the D2-like (D2, D3 and D4). By use of 5-color flow cytometric analysis, we examined the expression of DR on human CD4+ naive T lymphocytes (CD3+CD4+CD45RA+CCR7+), central memory (TCM, CD3+CD4+CD45RA-CCR7+) and effector memory T cells (TEM, CD3+CD4+CD45RA-CCR7-). In addition, in cultured CD4+ T cells we investigated the changes in DR expression induced by stimulation with antiCD3/antiCD28 antibodies. Results showed that CD4+ T cells always expressed all the five DR: D1-like DR were identified on average on 11.6-13.1 % and D2-like DR on 3.1-8.1 % of the cells. DR on CD4+ naive T cells, TCM, and TEM had distinct expression patterns: naive T cells expressed more D1-like than D2-like DR, which on the contrary were increased in TCM and TEM cells. In cultured CD4+ T cells stimulation with anti-CD3/anti-CD28 antibodies increased the expression of D1-like DR by 71-84 % and of D2-like DR by 55-97 %. The frequency of DR was higher in apoptotic cells in comparison to viable cells, however stimulation increased all DR on viable cells, without affecting their expression on apoptotic cells. The present results contribute to unravel the complexity of dopaminergic pathways in human CD4+ T lymphocytes, suggesting their involvement in memory functions as well as in apoptotic processes. In view of the role of CD4+ memory T cells in neuroinflammation and neurodegeneration during Parkinson's disease, the relevance of these findings must be assessed in the clinical setting.

Sleep fragmentation, particularly reduced and interrupted night sleep, impairs the quality of life of older people. Strikingly similar declines in sleep quality are seen during ageing in laboratory animals, including the fruit fly Drosophila. We investigated whether reduced activity of the nutrient- and stress-sensing insulin/insulin-like growth factor (IIS)/TOR signalling network, which ameliorates ageing in diverse organisms, could rescue the sleep fragmentation of ageing Drosophila. Lowered IIS/TOR network activity improved sleep quality, with increased night sleep and day activity and reduced sleep fragmentation. Reduced TOR activity, even when started for the first time late in life, improved sleep quality. The effects of reduced IIS/TOR network activity on day and night phenotypes were mediated through distinct mechanisms: Day activity was induced by adipokinetic hormone, dFOXO, and enhanced octopaminergic signalling. In contrast, night sleep duration and consolidation were dependent on reduced S6K and dopaminergic signalling. Our findings highlight the importance of different IIS/TOR components as potential therapeutic targets for pharmacological treatment of age-related sleep fragmentation in humans., Competing Interests: The authors have declared that no competing interests exist.

Perturbations in the levels of serotonin expression have a significant impact on behavior and have been implicated in the pathogenesis of several neuropsychiatric disorders including anxiety, mood and appetite. Fetal programming is a risk factor for the development of metabolic diseases during adulthood. Moreover, previous studies have shown that serotonin (5‑HT), dopamine and leptin are important in energy balance. In the present study, the impact of maternal malnutrition‑induced prenatal undernutrition (UN) was investigated in mice and the expression of 5‑HT1A, dopamine (D)1, D2 and Ob‑Rb receptors was analyzed in the hypothalamus during adulthood. The UN group showed a low birth weight compared with the control group. With regard to receptor expression, 5‑HT1A in the UN group was increased in the hypothalamus and D1 was reduced, whereas D2 showed an increase from postnatal day (P)14 in the arcuate nucleus. Ob‑Rb receptor expression was increased in the hypothalamus at P14 and P90. These observations indicated that maternal caloric restriction programs a postnatal body weight gain in offspring with an increased food intake in early postnatal life which continues into adulthood. In addition, UN in mice was found to be affected by Ob‑Rb, 5‑HT1A and D1/2 receptor expression, indicating that these observations may be associated with hyperphagia and obesity.

Obesity has reached global epidemic proportions and creating an urgent need to understand mechanisms underlying excessive and uncontrolled food intake. Ghrelin, the only known circulating orexigenic hormone, potently increases food reward behavior. The neurochemical circuitry that links ghrelin to the mesolimbic reward system and to the increased food reward behavior remains unclear. Here we examine whether VTA-NAc dopaminergic signaling is required for the effects of ghrelin on food reward and intake. In addition, we examine the possibility of endogenous ghrelin acting on the VTA-NAc dopamine neurons. A D1-like or a D2 receptor antagonist was injected into the NAc in combination with ghrelin microinjection into the VTA to investigate whether this blockade attenuates ghrelin-induced food reward behavior. VTA injections of ghrelin produced a significant increase in food motivation/reward behavior, as measured by sucrose-induced progressive ratio operant conditioning, and chow intake. Pretreatment with either a D1-like or D2 receptor antagonist into the NAc, completely blocked the reward effect of ghrelin, leaving chow intake intact. We also found that this circuit is potentially relevant for the effects of endogenously released ghrelin as both antagonists reduced fasting (a state of high circulating levels of ghrelin) elevated sucrose-motivated behavior but not chow hyperphagia. Taken together our data identify the VTA to NAc dopaminergic projections, along with D1-like and D2 receptors in the NAc, as essential elements of the ghrelin responsive circuits controlling food reward behavior. Interestingly results also suggest that food reward behavior and simple intake of chow are controlled by divergent circuitry, where NAc dopamine plays an important role in food reward but not in food intake., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Parkinson's disease (PD) patients have increased susceptibility to impulse control disorders. Recent studies have suggested that alterations in dopamine receptors in the midbrain underlie impulsive behaviors and that more impulsive individuals, including patients with PD, exhibit increased occupancy of their midbrain dopamine receptors. The cellular location of dopamine receptor subtypes and transporters within the human midbrain may therefore have important implications for the development of impulse control disorders in PD. The localization of the dopamine receptors (D1-D5) and dopamine transporter proteins in the upper brain stems of elderly adult humans (n = 8) was assessed using single immunoperoxidase and double immunofluorescence (with tyrosine hydroxylase to identify dopamine neurons). The relative amount of protein expressed in dopamine neurons from different regions was assessed by comparing their relative immunofluorescent intensities. The midbrain dopamine regions associated with impulsivity (medial nigra and ventral tegmental area [VTA]) expressed less dopamine transporter on their neurons than other midbrain dopamine regions. Medial nigral dopamine neurons expressed significantly greater amounts of D1 and D2 receptors and vesicular monoamine transporter than VTA dopamine neurons. The heterogeneous pattern of dopamine receptors and transporters in the human midbrain suggests that the effects of dopamine and dopamine agonists are likely to be nonuniform. The expression of excitatory D1 receptors on nigral dopamine neurons in midbrain regions associated with impulsivity, and their variable loss as seen in PD, may be of particular interest for impulse control., (© 2013 International Parkinson and Movement Disorder Society.)

Schizophrenia patients are behaviorally supersensitive to dopamine-like drugs such as amphetamine or methylphenidate, meaning that patients respond to such drugs with increased psychotic symptoms, as compared to control subjects. A basis of such supersensitivity may be an increased pre-synaptic release of dopamine or a post-synaptic elevation of D2 receptors or of D2High receptors in active stages of schizophrenia. While the pre-synaptic release of dopamine is normal in stable patients with schizophrenia, brain imaging studies find that D2 receptors are increased by an average of 5.8% in antipsychotic-free schizophrenia patients. It is possible that the behavioral supersensitivity may stem from more D2High receptors in schizophrenia. Although the antipsychotic/dopamine D2 receptor can exist in vitro in a state of high affinity for dopamine (as D2High), or in a state of low affinity for dopamine (as D2Low), there is no clear evidence that D2High states can be selectively labeled or stably exist in vivo. Nevertheless, two studies revealed an 80% increase in apparent D2High receptors in schizophrenia patients after reducing endogenous dopamine. The elevation in apparent D2High receptors in vivo in schizophrenia matches the elevation in D2High receptors in vitro in animal models of psychosis, including dopamine-supersensitive animals pretreated with amphetamine, marijuana, or phencyclidine, or animals with gene knockouts in various neurotransmitter pathways, including those for glutamate receptors. The elevation of D2High receptors in vitro and the increased apparent D2High receptors in vivo is consistent with behavioral dopamine supersensitivity in schizophrenia patients., (© 2013 Elsevier B.V. and ECNP. All rights reserved.)

Rearing young rodents in socially isolated or environmentally enriched conditions has been shown to affect numerous components of the dopamine system as well as behavior. Methylphenidate (MPH), a commonly used dopaminergic agent, may affect animals differently based on rearing environment. Here we examined the interaction between environment and chronic MPH treatment at clinically relevant doses, administered via osmotic minipump. Young Sprague Dawley rats (PND 21) were assigned to environmentally enriched, pair-housed, or socially isolated rearing conditions, and treated with either 0, 2, 4, or 8 mg/kg/day MPH for 3 weeks. At the end of the treatment period, animals were tested for locomotor activity and anxiety-like behavior. The densities of D1-like and D2-like receptors were measured in the striatum using in vitro receptor autoradiography. Locomotor activity and anxiety-like behavior were increased in isolated animals compared to pair-housed and enriched animals. The density of D1-like receptors was greater in isolated animals, but there were no differences between groups in D2-like receptor density. Finally, there were no effects of MPH administration on any reported measure. This study provides evidence for an effect of early rearing environment on the dopamine system and behavior, and also suggests that MPH administration may not have long-term consequences., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Nicotine, the major addictive substance in tobacco, increases the activity of the central amygdala (CeA). Amygdala is directly implicated in anxiety modulation and sends projections to the vicinity of the midbrain dopamine neurons, including the ventral tegmental area (VTA) which is a key area that controls nicotine dependence processes. In this study, the role of dopamine D(1) and D(2)/(3) receptors of the VTA on anxiogenic-like behavior induced with intra-CeA injection of nicotine has been investigated. Male Wistar rats with cannula aimed to the left CeA and the left VTA were submitted to the elevated plus-maze (EPM). The nicotine injection (1 μg/rat) into the CeA decreased the percentage of open arm time and open arm entries, but not locomotor activity, indicating an anxiogenic-like response. Intra-VTA injection of a dopamine D1 receptor antagonist, SCH23390 (0.25 μg/rat), and a dopamine D2/3 receptor antagonist, sulpiride (0.7 μg/rat), inhibited the anxiogenic-like response caused by intra-CeA injection of nicotine. These results suggest that the relationship between the VTA and the CeA may be involved in nicotine-induced anxiogenic-like behavior via dopamine D(1) and D(2)/(3) receptors. An understanding of these cellular processes will be crucial for the development of new intervention to combat nicotine effect., (Copyright © 2012. Published by Elsevier Inc.)

The aim of the present study was to evaluate the behavioral patterns associated with autism and the prevalence of these behaviors in males and females, to verify whether our model of lipopolysaccharide (LPS) administration represents an experimental model of autism. For this, we prenatally exposed Wistar rats to LPS (100 μg/kg, intraperitoneally, on gestational day 9.5), which mimics infection by gram-negative bacteria. Furthermore, because the exact mechanisms by which autism develops are still unknown, we investigated the neurological mechanisms that might underlie the behavioral alterations that were observed. Because we previously had demonstrated that prenatal LPS decreases striatal dopamine (DA) and metabolite levels, the striatal dopaminergic system (tyrosine hydroxylase [TH] and DA receptors D1a and D2) and glial cells (astrocytes and microglia) were analyzed by using immunohistochemistry, immunoblotting, and real-time PCR. Our results show that prenatal LPS exposure impaired communication (ultrasonic vocalizations) in male pups and learning and memory (T-maze spontaneous alternation) in male adults, as well as inducing repetitive/restricted behavior, but did not change social interactions in either infancy (play behavior) or adulthood in females. Moreover, although the expression of DA receptors was unchanged, the experimental animals exhibited reduced striatal TH levels, indicating that reduced DA synthesis impaired the striatal dopaminergic system. The expression of glial cell markers was not increased, which suggests that prenatal LPS did not induce permanent neuroinflammation in the striatum. Together with our previous finding of social impairments in males, the present findings demonstrate that prenatal LPS induced autism-like effects and also a hypoactivation of the dopaminergic system., (Copyright © 2012 Wiley Periodicals, Inc.)

Background and Purpose: Delayed treatment of patients with stroke with levodopa/benserazide contributes to enhanced functional recovery, but the mechanisms involved are poorly understood. The present study was designed to investigate if levodopa/benserazide treatment improves recovery of lost neurological function and contributes to tissue reorganization in the rat brain after stroke., Methods: Male Wistar rats were subjected to transient occlusion of the middle cerebral artery (120 minutes) and treated with levodopa (1, 5, and 20 mg/kg)/benserazide (15 mg/kg) or saline for 12 consecutive days starting on Day 2 after transient occlusion of the middle cerebral artery. Infarct volume was determined and sensorimotor function was assessed using the rotating pole test, a 28-point neuroscore, and a cylinder test on Days 2, 7, and 14 after transient occlusion of the middle cerebral artery. The spatiotemporal expression pattern of dopamine-1 and dopamine-2 receptors and the dopamine- and cAMP-regulated neuronal phosphoprotein in reactive astrocytes were analyzed in the ischemic hemisphere as well as in cultured astrocytes., Results: Treatment with levodopa/benserazide significantly improved the recovery of sensorimotor function after transient occlusion of the middle cerebral artery without affecting the infarct volume. In addition, we found that different subpopulations of glial fibrillary acidic protein-positive astrocytes in the peri-infarct area express dopamine-1 receptors and dopamine-2 receptors as well as dopamine- and cAMP-regulated neuronal phosphoprotein., Conclusions: Our results strongly corroborate the concept of recovery enhancing actions of levodopa treatment after stroke. Also, astrocytes in the peri-infarct area may contribute to the dopamine enhanced recovery mechanisms.

The timeline of dopamine (DA) system maturation and the signaling properties of DA receptors (DRs) during rat brain development are not fully characterized. We used in situ hybridization and quantitative PCR to map DR mRNA transcripts in the medial frontal cortex (mFC) and striatum (STR) of the rat from embryonic day (E) 15 to E21. The developmental trajectory of DR mRNAs revealed distinct patterns of DA receptors 1 and 2 (DRD1, DRD2) in these brain regions. Whereas the mFC had a steeper increase in DRD1 mRNA, the STR had a steeper increase in DRD2 mRNA. Both DR mRNAs were expressed at a higher level in the STR compared with the mFC. To identify the functional properties of DRs during embryonic development, the phosphorylation states of cyclic AMP response element binding protein, extracellular signal-regulated kinase 1/2, and glycogen synthase kinase 3 beta were examined after DR stimulation in primary neuronal cultures obtained from E15 and E18 embryos and cultured for 3 days to ensure a stable baseline level. DR-mediated signaling cascades were functional in E15 cultures in both brain regions. Because DA fibers do not reach the mFC by E15, and DA was not present in cultures, these data indicate that DRs can become functional in the absence of DA innervation. Because activation of DR signal transduction pathways can affect network organization of the developing brain, maternal exposure to drugs that affect DR activity may be liable to interfere with fetal brain development., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Activation of D1 dopamine receptors expressed in the kidneys promotes the excretion of sodium and regulates sodium levels during increases in dietary sodium intake. A decrease in the expression or function of D1 receptors results in increased sodium retention which can potentially lead to the development of hypertension. Studies have shown that in the absence of functional D1 receptors, in null mice, the systolic, diastolic, and mean arterial pressures are higher. Previous studies have shown that the expression and function of D1 receptors in the kidneys are decreased in animal models of diabetes. The mechanisms that down-regulate the expression of renal D1 receptor gene in diabetes are not well understood. Using primary renal cells and acutely isolated kidneys from the streptozotocin-induced rat diabetic model, we demonstrate that the renal D1 receptor expression is down-regulated by the extracellular cAMP-adenosine pathway in vitro and in vivo. In cultures of primary renal cells, a 3 mm, 60-h cAMP treatment down-regulated the expression of D1 receptors. In vivo, we determined that the plasma and urine cAMP levels as well as the expression of 5'-ectonucleotidase, tissue-nonspecific alkaline phosphatase, and adenosine A2a receptors are significantly increased in diabetic rats. Inhibitors of 5'-ectonucleotidase and tissue-nonspecific alkaline phosphatase, α,β-methyleneadenosine 5'-diphosphate, and levamisole, respectively, blocked the down-regulation of D1 receptors in the primary renal cells and in the kidney of diabetic animals. The results suggest that inhibitors of the extracellular cAMP-adenosine pathway reverse the down-regulation of renal D1 receptor in diabetes.

Purpose: Increased adrenergic activity in response to chronic stress is known to promote tumor growth by stimulating the tumor microenvironment. The focus of the current study was to determine whether dopamine, an inhibitory catecholamine, could block the effects of chronic stress on tumor growth., Experimental Design: Expression of dopamine receptors (DR1-DR5) was analyzed by reverse transcriptase-PCR and by Western blotting. In vitro effects of dopamine on cell viability, apoptosis, and migration were examined. For in vivo therapy, murine and human DR2-siRNAs were incorporated into chitosan nanoparticles (CH-NP)., Results: In this model of chronic stress, tumoral norepinephrine levels remained elevated whereas dopamine levels were significantly decreased compared with nonstressed animals. Daily restraint stress resulted in significantly increased tumor growth in both immunodeficient (SKOV3ip1 and HeyA8) and immunocompetent (ID8) ovarian cancer models. This increase was completely blocked with daily dopamine treatment. Dopamine treatment also blocked the stress-induced increase in angiogenesis. Endothelial and ovarian cancer cells expressed all dopamine receptors except for the lack of DR3 expression in ovarian cancer cells. DR2 was responsible for the inhibitory effects of dopamine on tumor growth and microvessel density as well as the stimulatory effect on apoptosis, as the DR2 antagonist eticlopride reversed these effects. Dopamine significantly inhibited cell viability and stimulated apoptosis in vitro. Moreover, dopamine reduced cyclic AMP levels and inhibited norepinephrine and vascular permeability factor/VEGF-induced Src kinase activation., Conclusions: Dopamine depletion under chronic stress conditions creates a permissive microenvironment for tumor growth that can be reversed by dopamine replacement., (©2011 AACR.)

Introduction: It has been reported in some series that gsp+ somatotropinomas are more sensitive to somatostatin analogues (SA) and dopamine's actions which may be related to their somatostatin receptor (SSTR) and dopamine receptor (DR) profile. No previous studies have been undertaken to evaluate the SSTR and DR profile related with the gsp status in somatotropinomas., Objectives: To determine if (1) gsp status is correlated with response to octreotide LAR (LAR) and tumor expression patterns of SSTR1-5 and DR1-5 and (2) cAMP level can directly modulate SSTR and DR mRNA levels., Methods: Response to SA was evaluated by GH and IGF-I percent reduction after 3 and 6 months of treatment with LAR. Conventional PCR and sequencing were used to identify gsp+ tumors. Quantitative real-time PCR was used to determine SSTR and DR tumor expression. Primary pituitary cell cultures of primates were used to study whether SSTR and DR expression is regulated by forskolin., Results: The response to LAR did not significantly differ between patients with gsp+ and gsp- tumors; however, gsp+ tumors expressed higher levels of SSTR1, SSTR2, DR2 and a lower level of SSTR3. Forskolin increased SSTR1, SSTR2, DR1 and DR2 expression in cell cultures., Conclusion: Elevated SSTR1, SSTR2, and DR2 tumor expression may help improve responsiveness to SA and DA therapy; however, this study may not have been appropriately powered to observe significant effects in the clinical response. Elevated cAMP levels could be directly responsible for the upregulation in SSTR1, SSTR2 and DR2 mRNA levels observed in gsp+ patients., (Copyright © 2010 S. Karger AG, Basel.)

Among the best-replicated and most heritable endophenotypes of attention-deficit/hyperactivity disorder (ADHD) are deficits in attention, inhibitory response control and larger intra-individual variability in response latencies. Here, we explored the presence of these heritable ADHD endophenotypes in two commonly used inbred mouse strains, C57BL/6J and DBA/2J, and investigated whether treatment with the stimulant amphetamine affected these phenotypes. Both in an operant Go/No-Go task and 5-choice serial reaction time (5-CSRT) task, DBA/2J mice showed reduced inhibitory response control compared with C57BL/6J mice. Mean correct response latencies of DBA/2J mice were slower in both tasks. Analysis of the distribution of correct response latencies suggested similar processing speed, but DBA/2J mice displayed larger intra-individual variability. Amphetamine did not affect inhibition in the Go/No-Go task but increased omission errors. In contrast, in the 5-CSRT task, amphetamine did not affect omission errors but impaired inhibitory response control, specifically in C57BL/6J mice. The dopamine uptake inhibitor, GBR 12909, mimicked this effect and decreased accurate choice, specifically in C57BL/6J mice, indicating that dopamine modulates inhibitory response control and attention in the murine 5-CSRT task. Amphetamine did not affect response distributions in either task. Furthermore, we extended previous reports on differences in the brain dopamine system of DBA/2J and C57BL/6J mice, by showing differential gene expression levels of three dopamine receptors (Drd1, Drd4 and Drd5) in the mPFC. In conclusion, genetic differences between DBA/2J and C57BL/6J mice translate into multiple ADHD-related phenotypes, indicating that these strains are valuable resources to understand genetic mechanisms underlying ADHD-relevant phenotypes., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Dopamine produced by renal proximal tubules increases sodium excretion via a decrease in renal sodium reabsorption. Dopamine natriuresis is impaired in obese Zucker rats; however, the mechanism is not fully understood. To test the hypothesis that renal expression of one or more of the subtypes are altered in these rats, we measured whole kidney protein levels by immunoblotting of D1-like (D1R and D5R) and D2-like (D2R, D3R, and D4R) dopamine receptors in both male and female obese and lean Zucker rats. In obese males on 1% NaCl diet, D1R, D2R, D4R, and D5R were decreased, while D3R was increased, relative to lean rats. Under a 4% NaCl diet, D2R and D3R levels in obese rats were restored to lean levels. 4% NaCl diet reduced D5R in both body types, relative to 1% NaCl diet. Female rats had higher expression of D1R and D3R than did male; however, the sex difference for D1R was markedly blunted in obese rats. In obese rats, dietary candesartan (angiotensin II type 1 receptor blocker) normalized downregulated D1R and D2R, but either decreased (D3R), did not affect (D4R), or further downregulated (D5R) the other subtypes. Candesartan also decreased D4R in lean rats. In summary, reduced renal protein levels of D1R, D2R, D4R, and D5R in obese Zucker rats could induce salt sensitivity and elevate blood pressure. Increased angiotensin II type 1 receptor activity may be mechanistically involved in the decreased expression of D1R and D2R in obese rats. Finally, reduced D1R and D3R in male rats may contribute to sex differences in blood pressure.

The inducible gene Homer1a has been considered a candidate gene for schizophrenia. Drugs efficacious in schizophrenia and acting as dopamine receptor antagonists induce Homer1a expression, although the specific role of the different dopamine receptors in its induction is not completely known. In this study, we explored Homer1a expression induced by selective antagonists at dopamine receptors (SCH-23390, D(1) receptor selective antagonist, 0.5 mg/kg; L-741,626, D(2) receptor selective antagonist, 2 mg/kg; U-99194, D(3) receptor selective antagonist, 5 mg/kg; L-745,870, D(4) receptor selective antagonist, 3 mg/kg), haloperidol (0.8 mg/kg), and terguride (0.5 mg/kg), a partial agonist at D(2) receptors. Moreover, we evaluated the expression of two Homer1a-related genes which play essential roles in synaptic plasticity: mGluR5 and Homer1b. Gene expression was analyzed in brain regions relevant for schizophrenia pathophysiology and therapy, namely the striatum, the cortex, and the hippocampus. In striatum, Homer1a was induced by D(2) receptor antagonists and, with a different distribution, by SCH-23390. In the cortex, Homer1a was differentially induced by D(1), D(2), and D(3) receptors antagonists, while haloperidol and terguride did not affect or reduced its expression. Homer1b expression was reduced by L-741,626, L-745,870, terguride, and haloperidol in the ventral caudate-putamen, in the nucleus accumbens and in the cortex, while SCH-23390 increased the expression in the core of the accumbens. mGluR5 expression was increased by SCH-23390 in the dorsomedial putamen, the core of the accumbens, and in some hippocampal subregions. A reduction of gene expression by terguride and an increase by L-745,870 was observed in the dorsomedial putamen. The changes in expression suggest that these gene transcripts are differentially regulated by antagonism at different dopamine receptors.

Genes involved in serotonergic and dopaminergic neurotransmission have been hypothesized to affect different aspects of personality, but findings from genetic association studies did not provide conclusive results so far. In previous studies, however, only one or a few polymorphisms within single genes were investigated neglecting the possibility that the genetic associations might be more complex comprising several genes or gene regions. To overcome this limitation, we performed an extended genetic association study analyzing 17 serotonergic (SLC6A4, HTR1A, HTR1B, HTR2A, HTR2C, HTR3A, HTR6, MAOA, TPH1, TPH2) and dopaminergic genes (SLC6A3, DRD2, DRD3, DRD4, COMT, MAOA, TH, DBH), which have been previously reported to be implicated with personality traits. One hundred and ninety-five single nucleotide polymorphisms (SNPs) within these genes were genotyped with the Illumina BeadChip technology (HumanHap300, Human-1) in a sample of 366 mentally healthy Caucasians. Additionally, we tried to replicate our results in an independent sample of further 335 Caucasians. Personality traits in both samples were assessed with the German version of Cloninger's Tridimensional Personality Questionnaire. From 30 SNPs showing associations at a nominal level of significance, two intronic SNPs, rs2770296 and rs927544, both located in the HTR2A gene, withstood correction for multiple testing. These SNPs were associated with the personality trait novelty seeking. The effect of rs927544 could be replicated for the novelty seeking subscale extravagance, and the same SNP was also associated with extravagance in the combined samples. Our results show that HTR2A polymorphisms modulate facets of novelty seeking behaviour in healthy adults suggesting that serotonergic neurotransmission is involved in this phenotype.

In search for biomarkers of neurodegeneration, increasing attention has been focussed on peripheral blood mononuclear cells (PBMC). In particular, PBMC from patients with Alzheimer disease (AD) have been suggested to carry apoptotic changes and alterations of neurotransmitter receptor expression, which may resemble those occurring in central nervous system neurons. We investigated the expression of apoptosis-related proteins Bcl-2, Bax, and caspase-3 and the levels of dopaminergic receptors (DR) D3 and D5 mRNA in PBMC from 17 AD patients and 11 age-matched healthy subjects. Apoptosis-related proteins were assayed by standard Western blotting analysis and DR mRNA by real-time polymerase chain reaction techniques. PBMC from healthy subjects and from AD patients expressed Bcl-2, Bax, and caspase-3 to about the same extent, and the Bcl-2/Bax ratio did not differ in the 2 groups. Levels of mRNA for DRD3 and DRD5 were similar in cells from healthy subjects and from AD patients. In conclusion, we found no evidence that PBMC from AD patients may express apoptosis-related proteins or DR mRNA to any different extent in comparison to cells from healthy subjects. These findings do not necessarily imply that immune cells cannot be exploited as biomarkers in AD (and in other central nervous system disorders). Future studies, however, should take into account the inherent complexity of the immune network.

The abuse of anabolic androgenic steroids (AASs) at supratherapeutic doses is a problem not only in the world of sports, but also among non-athletes using AASs to improve physical appearance and to become more bold and courageous. Investigations of the possible neurochemical effects of AAS have focused partially on the monoaminergic systems, which are involved in aggressive behaviours and the development of drug dependence. In the present study, we administered nandrolone decanoate (3 or 15 mg/kg/day for 14 days) and measured mRNA expression of dopaminergic and serotonergic receptors, transporters and enzymes in the male rat brain using quantitative real-time polymerase chain reaction. Expression of the dopamine D1-receptor transcript was elevated in the amygdala and decreased in the hippocampus while the transcript level of the dopamine D4-receptor was increased in the nucleus accumbens. No changes in transcriptional levels were detected among the serotonin-related genes examined in this study. The altered mRNA expression of the dopamine receptors may contribute to some of the behavioural changes often reported in AAS abusers of increased impulsivity, aggression and drug-seeking.

The extent of catecholaminergic receptor and respective behavioral alterations associated with prenatal cocaine exposure varies according to exogenous factors such as the amount, frequency, and route of maternal exposure, as well as endogenous factors such as specific brain regions under consideration and sex of the species. The goal of the current study was to use autoradiography to delineate possible moderators of dopaminergic and adrenergic receptor expression in adult rat offspring exposed to cocaine in utero. The current study demonstrated sex-dependent D1 receptor, alpha2, and noradrenergic transporter binding alterations in prelimbic, hippocampus, and anterior cingulate regions of adult rat brains exposed to cocaine during gestational days 8-21. Of further interest was the lack of alterations in the nucleus accumbens for nearly all receptors/transporters investigated, as well as the lack of alterations in D3 receptor binding in nearly all of the regions investigated (nucleus accumbens, prelimbic region, hippocampus, and cingulate gyrus). Thus, the current investigation demonstrated persistent receptor and transporter alterations that extend well into adulthood as a result of cocaine exposure in utero. Furthermore, the demonstration that sex played a mediating role in prenatal cocaine-induced, aberrant receptor/transporter expression is of primary importance for future studies that seek to control for sex in either design or analysis.

In the present study, we analyzed the localization of D1a receptors within the rat substantia nigra pars reticulata (SNr) using specific D1a immunochemistry at the ultrastructural level and RT-PCR. At the electron microscopic level, D1a receptors were strongly associated with axons and axonal endings in the SNr, but also with numerous glutamic acid decarboxylase-positive dendrites and neuronal cell bodies. This neuronal expression of D1a receptors was confirmed using RT-PCR. G(alphaolf) protein-specific immunostaining displayed a similar distribution in dendrites and cell bodies to that of D1a receptors. The localization of D1a receptors in both GABAergic cell bodies and terminals is in accordance with the well known complex action of dopamine in the SNr. Moreover, the intracytoplasmic localization of D1a receptors in cell bodies and dendrites that we observed suggests that these receptors are only effective in specific conditions, or are transported to different nigral targets where they may have a presynaptic function.

A combination of PCR-Select cDNA subtraction and gene array hybridization was used to identify differentially expressed genomic markers in brains of rats fed for 3 weeks in utero and 2 weeks after birth on an n-3 polyunsaturated fatty acid (PUFA)-deficient diet supplied to dams. Total RNA was isolated, switch mechanism at 5'-end of the RNA transcripts (SMART) applied and used for PCR-Select subtraction of PUFA-deficient and adequately-fed control preparations. Subtracted and amplified ds-cDNA end-products were fragmented, terminally labeled with biotin-ddUTP and hybridized with a RN-U34A gene array. A 10-fold increase in potential genes with log2(Tester/Driver) = 1.4 was found compared with traditional gene array technology when the same chip was tested using non-subtracted targets. Reverse transcription-real-time relative PCR confirmed 30% of the transcripts. Among the validated transcripts, D1 and D2 receptors for dopamine (DA), were most prominent among a number of over-expressed neurotransmitter receptors and retinoic acid receptor (RXR alpha-2 and alpha-1). Immunohistochemical staining of brain sections from 2-week-old pups revealed a substantial enrichment of the D2 receptor in discrete regions of the mesolimbic and mesocortical pathways as well as in a large number of brain areas from the n-3 PUFA-deficient pups. Punches of the same areas run on western blots showed similar results. The overwhelming expression of D1 and D2 receptors may be attributed to a behavioral hypersensitivity caused by the possible impairment of DA production during brain development, which may have implications in certain disorders of the nervous system.

The use-dependent modification of synapses is strongly influenced by dopamine, a transmitter that participates in both the physiology and pathophysiology of animal behavior. In the hippocampus, dopaminergic signaling is thought to play a key role in protein synthesis-dependent forms of synaptic plasticity. The molecular mechanisms by which dopamine influences synaptic function, however, are not well understood. Using a GFP-based reporter, as well as a small-molecule reporter of endogenous protein synthesis, we show that dopamine D1/D5 receptor activation stimulates local protein synthesis in the dendrites of hippocampal neurons. We also identify the GluR1 subunit of AMPA receptors as one protein upregulated by dopamine receptor activation, with increased incorporation of surface GluR1 at synaptic sites. The insertion of new GluRs is accompanied by an increase in the frequency of miniature synaptic events. Together, these data suggest a local protein synthesis-dependent activation of previously silent synapses as a result of dopamine receptor stimulation.

The hph-1 mice have defective tetrahydrobiopterin biosynthesis and share many neurochemical similarities with l-dopa-responsive dystonia (DRD) in humans. In both, there are deficiencies in GTP cyclohydrolase I and low brain levels of dopamine (DA). Striatal tyrosine hydroxylase (TH) levels are decreased while the number of DA neurones in substantia nigra (SN) appears normal. The hph-1 mouse is therefore a useful model in which to investigate the biochemical mechanisms underlying dystonia in DRD. In the present study, the density of striatal DA terminals and DA receptors and the expression of D-1, D-2, and D-3 receptors, preproenkephalin (PPE-A), preprotachykinin (PPT), and nitric oxide synthase (NOS) mRNAs in the striatum and nucleus accumbens and nigral TH mRNA expression were examined. Striatal DA terminal density as judged by specific [3H]mazindol binding was not altered while the levels of TH mRNA were elevated in the SN of hph-1 mice compared to control (C57BL) mice. Total and subregional analysis of the striatum and nucleus accumbens showed that D-2 receptor ([3H]spiperone) binding density was increased while D-1 receptor ([3H]SCH 23390) and D-3 receptor ([3H]7-OH-DPAT) binding density was not altered. In the striatum and nucleus accumbens, expression of PPT mRNA was elevated but PPE-A mRNA, D-1, D-2 receptor, and nNOS mRNA were not changed in hph-1 mice compared to controls. These findings suggest that an imbalance between the direct strionigral and indirect striopallidal output pathways may be relevant to the genesis of DRD. However, the pattern of changes observed is not that expected as a result of striatal dopamine deficiency and suggests that other effects of GTP cyclohydrolase I deficiency may be involved.

We reported previously a model of polyglutamine repeat disorders with insertion of 146 CAG repeats into the murine hypoxanthine phosphoribosyl transferase locus (Hprt(CAG)146; Ordway et al. [1997] Cell 91:753-763), which does not normally contain polyglutamine repeats. These mice develop an adult-onset neurologic phenotype of incoordination, involuntary limb clasping, seizures, and premature death. Histologic analysis demonstrates widespread ubiquinated neuronal intranuclear inclusions (NIIs). We now report characterization of the age of onset of behavioral abnormalities, correlated with the time course of occurrence of NIIs in several brain regions, and the occurrence of NIIs in non-neuronal tissues. Onset of behavioral abnormalities occurred at approximately 22 weeks of age. There was variable time course of expression of NIIs in several brain regions. Assessment of several non-neuronal tissues revealed nuclear inclusions in hepatocytes and choroid plexus epithelium. Gamma-aminobutyric acid (GABA)/benzodiazepine receptors, dopamine D1-like and D2-like receptors, and type 2 vesicular monoamine transporter (VMAT2) binding sites were assayed before and after the onset of behavioral abnormalities. GABA/benzodiazepine receptors were unchanged either before or after the onset of behavioral abnormalities in any region analyzed, whereas striatal D1-like and D2-like receptors were diminished after but not before the onset of symptoms. Dorsal striatal VMAT2 binding sites were decreased before the onset of behavioral changes. Mitochondrial electron transport chain components were assayed with histochemical methods before and after the onset of behavioral changes. There was no change in behaviorally presymptomatic or symptomatic animals. Hprt(CAG)146 mice did not exhibit increased susceptibility to the mitochondrial toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Hprt(CAG)146 mice are a useful model for studying polyglutamine repeat disorders., (Copyright 2003 Wiley-Liss, Inc.)

Recent advances in molecular genetics have greatly increased the understanding of the pathophysiology of certain neurobehavioral disorders and the core symptoms of these disorders. This paper reviews key concepts important in understanding the genetics of neuropsychiatric disorders, and gives an overview of several different types of genetic disorders, including trinucleotide repeat disorders, and functional polymorphisms of monoamine neurotransmitter systems.

The mesocorticolimbic circuitry has been implicated in the pathophysiology of several neuropsychiatric syndromes like chronic pain and addiction. The aim of this study was to evaluate the effects of dizocilpine (MK-801), a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, on sensorimotor behaviors and the consequent changes in the dopamine, glutamate, and opiate systems in rats. Five groups of rats were subjected to acute tests for nociception (hot plate and paw pressure) before and after MK-801 (0.05, 0.1, 0.2 and 0.4 mg/kg, i.p.) or saline. Another two groups received daily i.p. saline or MK-801 (0.4 mg/kg) for 15 days. The nociceptive tests were performed on days 1, 7, and 14. On day 15 the rats received the last injection and were immediately sacrificed. We measured the mRNA expression, by in situ hybridization (ISH), of various dopamine and glutamate receptors, and enkephalin (Enk), dynorphin (Dyn), and substance P (SP) in the striatum, nucleus accumbens (NAC), piriform and cingulate cortex. Acute MK-801, dose-dependently, resulted in hyperalgesia. The chronic effects of 0.4 mg/kg MK-801 showed an extinction of the acute hyperalgesic effects especially with the hot plate test. The ISH studies revealed a decrease in mRNA expression of Enk and SP in the striatum and NAC. Our results indicate that the reversal of acute MK-801-induced hyperalgesia, with repeated exposure to systemic MK-801, is not directly related to changes in dopamine and glutamate receptors and might involve alteration of the striatal neuropeptide system.

We used in situ hybridization histochemistry to assess expression of dopamine receptors (D1R, D2R and D3R), neurotensin, proenkephalin and glutamate decarboxylase-67 (GAD67) in the prefrontal cortex, striatum, and/or nucleus accumbens in adult rats with neonatal ventral hippocampal (VH) lesions and in control animals after acute and chronic treatment with antipsychotic drugs clozapine and haloperidol. We also acquired these measures in a separate cohort of treatment-naïve sham and neonatally VH-lesioned rats used as an animal model of schizophrenia. Our results indicate that the neonatal VH lesion did not alter expression of D1R, D3R, neurotensin or proenkephalin expression in any brain region examined. However, D2R mRNA expression was down-regulated in the striatum, GAD67 mRNA was down-regulated in the prefrontal cortex and prodynorphin mRNA was up-regulated in the striatum of the VH-lesioned rats as compared with sham controls. Antipsychotic drugs did not alter expression of D1R, D2R or D3R receptor mRNAs but elevated neurotensin and proenkephalin expression in both groups of rats; patterns of changes were dependent on the duration of treatment and brain area examined. GAD67 mRNA was up-regulated by chronic antispychotics in the nucleus accumbens and the striatum and by chronic haloperidol in the prefrontal cortex in both sham and lesioned rats. These results indicate that the developmental VH lesion changed the striatal expression of D2R and prodynorphin and robustly compromised prefrontal GAD67 expression but did not modify drug-induced expression of any genes examined in this study.

The diverse physiological effects of dopamine are mediated by multiple receptor systems. The dDA1 represents one of the Drosophila dopamine receptors that activate the cAMP cascade. To gain insight into the role of dDA1, we generated a polyclonal antibody against the unique sequence in dDA1 and investigated dDA1 distribution in the central nervous system (CNS) of Drosophila melanogaster. In both larval and adult CNS pronounced dDA1 immunoreactivity was present in the neuropil of the mushroom bodies, a brain structure crucial for learning and memory in insects, and four unpaired neurons in each thoracic segment. In addition, the larval abdominal ganglion contained two dDA1 cells in each segment. This expression pattern appeared to be maintained in the condensed adult abdominal ganglion although the precise number and the intensity of staining were somewhat variable. The adult CNS also exhibited intense dDA1 immunoreactivity in the central complex, a structure controlling higher-order motor function, moderate expression in several neurosecretory cells, and weak staining in two unpaired neurons in the mesothoracic neuromere. The dDA1 expression in these areas was only detected in adult, but not in third instar larval CNS.

Although changes in nucleotide sequence affecting the composition and the structure of proteins are well known, functional changes resulting from nucleotide substitutions cannot always be inferred from simple analysis of DNA sequence. Because a strong synonymous codon usage bias in the human DRD2 gene, suggesting selection on synonymous positions, was revealed by the relative independence of the G+C content of the third codon positions from the isochoric G+C frequencies, we chose to investigate functional effects of the six known naturally occurring synonymous changes (C132T, G423A, T765C, C939T, C957T, and G1101A) in the human DRD2. We report here that some synonymous mutations in the human DRD2 have functional effects and suggest a novel genetic mechanism. 957T, rather than being 'silent', altered the predicted mRNA folding, led to a decrease in mRNA stability and translation, and dramatically changed dopamine-induced up-regulation of DRD2 expression. 1101A did not show an effect by itself but annulled the above effects of 957T in the compound clone 957T/1101A, demonstrating that combinations of synonymous mutations can have functional consequences drastically different from those of each isolated mutation. C957T was found to be in linkage disequilibrium in a European-American population with the -141C Ins/Del and TaqI 'A' variants, which have been reported to be associated with schizophrenia and alcoholism, respectively. These results call into question some assumptions made about synonymous variation in molecular population genetics and gene-mapping studies of diseases with complex inheritance, and indicate that synonymous variation can have effects of potential pathophysiological and pharmacogenetic importance.

Males, but not females, overproduce dopamine receptors in the striatum of rats across the periadolescent period followed by their elimination during young adulthood. In order to investigate the role that gonadal hormones play in this pubertal process, rats were castrated or ovariectomized at postnatal day (P) 28 when estrogen and testosterone levels are beginning to surge. Dopamine D1 and D2 striatal receptor density was then determined with autoradiography at P40 (adolescence) and P80 (young adulthood) to determine if either testosterone stimulates the overproduction of receptors in males or if estrogen inhibits this process in females. Neither castration nor ovariectomy altered dopamine receptor density, although enhanced testosterone levels increased D1 receptor binding 4.2% and 19.5% in males and females, respectively. The results of this study suggest that the endogenous rise in gonadal steroid hormones during puberty is not responsible for the overproduction of receptors in males or the lack of overproduction in females.

The age-associated changes in dopamine subtype receptors were examined in Aplysia californica. The density of the subtype receptors D1, D2, D3 and D4 was examined in the ganglia from 4.5-, 6-, 8-, 9- and 12-month animals. Receptor analysis was performed by examining the binding of radiolabeled ligands to the individual subtypes. [3H]SCH23390 and [3H]Clozapine were used to analyze D1 and D4 specific binding. [3H]Quinpirole was used for determining D2 and D3 specific binding. Specific binding was found to be present for all four receptor subtypes. All receptor subtypes showed an increase in density from 4.5 to 6 months. From 6 to 8 months D2 and D3 decreased, while D1 and D4 increased. D4 showed the strongest increase. All four subtypes examined showed decreases from 8 to 12 months. ANOVA results indicated age was a significant factor in the subtype receptor density for all receptor types.

The relatively few dopaminergic (DA) neurons in the mammalian brain regulate many important neural functions, including motor integration, neuroendocrine hormone release, cognition, emotive behaviors and reward. A number of laboratories, including ours, have contributed to unravel the mechanisms of DA phenotype induction and maturation and elucidated the role of epigenetic factors involved in specification, development and maintenance of midbrain dopaminergic functions. DA progenitors are first "committed" to give rise to DA neurons by the action of two secreted factors, Sonic hedgehog and fibroblast growth factor 8 (FGF8). Subsequently, the function of selectively activated transcription factors, Nurr1 and Ptx3, is required for the DA final determination. Further development of DA neurotransmission requires specific interactions with the developing target striatal cells, which modulate key DA functions, namely synthesis and uptake of the neurotransmitter. Committed and determined DA neurons express the key genes involved in DA neurotransmission at different times in development. In rodents, synthesis and intracellular accumulation of DA is achieved shortly after expression of Nurr1, while the onset of high affinity uptake, responsible for ending the neurotransmission, takes place after a few days. Cell contacts between the presynaptic DA neurons and target striatal neurons are apparently necessary for the fine modulation of DA function, in vivo and in vitro. Strikingly, the in situ maturation and phenotypic specialization of DA neurons grafted into the adult striatum/caudate-putamen parallels the normal development of committed fetal dopamine neurons during neurogenesis. The correct matching between the right presynaptic and postsynaptic neurons is required also for grafted DA cells.

1. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to investigate the effects of haloperidol treatment (1 mg/kg/day for 2, 7 or 21 consecutive days) on the expression of D1B and D3 dopamine receptor mRNAs in the rat lymphocytes. 2. The expression of D1B receptor mRNA was significantly decreased after 2 days of treatment and progressively returned toward basal values at the end of treatment. Conversely, haloperidol failed to modify the expression of lymphocyte D3 receptor mRNA. 3. These results indicate short-lasting dynamic changes of expression of lymphocyte D1B dopamine receptor mRNA by haloperidol and suggest that the effects of dopamine and dopaminergic drugs on the immune system might be mediated, at least in part, by direct interaction of these substances with dopamine receptors on lymphocyte membrane.

Fluorescence calcium imaging with fura-2 and whole cell, patch-clamp electrophysiology was applied to cultured Kenyon cells (interneurons) isolated from the mushroom bodies of adult crickets (Acheta domesticus) to demonstrate the presence of functional neurotransmitter receptors. In all cells investigated, 5 microM acetylcholine (ACh, n = 52) evoked an increase in intracellular free calcium ([Ca2+]i). Similar effects were observed in response to 10 microM nicotine. The ACh response was insensitive to atropine (50 microM) but was reduced by mecamylamine (50 microM) and alpha-bungarotoxin (alpha-bgt, 10 microM). ACh-induced inward ion currents (n = 28, EACh approximately 0 mV) were also blocked by 1 microM mecamylamine and by 1 microM alpha-bgt. Nicotine-induced inward currents desensitized more rapidly than ACh responses. Thus functional alpha-bgt-sensitive nicotinic ACh receptors are abundant on all Kenyon cells tested, and their activation leads to an increase in [Ca2+]i. gamma-Aminobutyric acid (GABA, 100 microM) triggered a sustained decrease in [Ca2+]i. Similar responses were seen with a GABAA agonist, muscimol (100 microM), and a GABAB agonist, 3-APPA (1 mM), suggesting that more than one type of GABA receptor can affect [Ca2+]i. This action of GABA was not observed when the extracellular KCl concentration was lowered. All cells tested (n = 26) with patch-clamp electrophysiology showed picrotoxinin (PTX)-sensitive, GABA-induced (30-100 microM) currents with a chloride-sensitive reversal potential. Thus, an ionotropic PTX-sensitive GABA receptor was found on all Kenyon cells tested. Most (61%) of the 54 cells studied responded to -glutamate (100 microM) application either with a biphasic increase in [Ca2+]i or with a single, delayed, sustained [Ca2+]i increase. Nearly all cells tested (95%, n = 19) responded to (100 microM) -glutamate with rapidly desensitizing, inward currents that reversed at approximately -30 mV. Dopamine (100 microM) elicited either a rapid or a delayed increase in [Ca2+]i in 63% of the 26 cells tested. The time course of these responses varied greatly among cells. Dopamine failed to elicit currents in patch-clamped cells (n = 4). A brief decrease in [Ca2+]i was induced by octopamine (100 microM) in approximately 54% of the cells tested (n = 35). However, when extracellular CaCl2 was lowered, octopamine triggered a substantial increase in [Ca2+]i in 35% of the cells tested (n = 26). No octopamine-elicited currents were detected in patched-clamped cells (n = 10).

Loss of neurotransmitter receptors, especially glutamate and dopamine receptors, is one of the pathologic hallmarks of brains of patients with Huntington disease (HD). Transgenic mice that express exon 1 of an abnormal human HD gene (line R6/2) develop neurologic symptoms at 9-11 weeks of age through an unknown mechanism. Analysis of glutamate receptors (GluRs) in symptomatic 12-week-old R6/2 mice revealed decreases compared with age-matched littermate controls in the type 1 metabotropic GluR (mGluR1), mGluR2, mGluR3, but not the mGluR5 subtype of G protein-linked mGluR, as determined by [3H]glutamate receptor binding, protein immunoblotting, and in situ hybridization. Ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate receptors were also decreased, while N-methyl-D-aspartic acid receptors were not different compared with controls. Other neurotransmitter receptors known to be affected in HD were also decreased in R6/2 mice, including dopamine and muscarinic cholinergic, but not gamma-aminobutyric acid receptors. D1-like and D2-like dopamine receptor binding was drastically reduced to one-third of control in the brains of 8- and 12-week-old R6/2 mice. In situ hybridization indicated that mGluR and D1 dopamine receptor mRNA were altered as early as 4 weeks of age, long prior to the onset of clinical symptoms. Thus, altered expression of neurotransmitter receptors precedes clinical symptoms in R6/2 mice and may contribute to subsequent pathology.

The presence of mRNAs for dopamine receptor subtypes and dopamine transporter in rat peripheral sensory and sympathetic ganglia was investigated using polymerase chain reaction (PCR) and DNA sequencing. Dopamine D1, D2, D3, D5 receptor subtype mRNAs and dopamine transporter mRNA were detected in both superior cervical sympathetic ganglia (SCG) and dorsal root ganglia (DRG) in the rat; the expression of D4 mRNA was only detected in DRG. While two alternatively spliced isoforms of D2 were detected in both ganglia, the alternative splicing transcripts for D3 and D4 were only found in the DRG. These results are useful in further studying the roles of dopamine and the effects of dopaminergic agents in the peripheral nervous system., (Copyright 1998 Elsevier Science B.V.)