Chronic morphine induces visible changes in the morphology of mesolimbic dopamine neurons.

The mesolimbic dopamine system, which arises in the ventral tegmental area (VTA), is an important neural substrate for opiate reinforcement and addiction. Chronic exposure to opiates is known to produce biochemical adaptations in this brain region. We now show that these adaptations are associated with structural changes in VTA dopamine neurons. Individual VTA neurons in paraformaldehyde-fixed brain sections from control or morphine-treated rats were injected with the fluorescent dye Lucifer yellow. The identity of the injected cells as dopaminergic or nondopaminergic was determined by immunohistochemical labeling of the sections for tyrosine hydroxylase. Chronic morphine treatment resulted in a mean approximately 25% reduction in the area and perimeter of VTA dopamine neurons. This reduction in cell size was prevented by concomitant treatment of rats with naltrexone, an opioid receptor antagonist, as well as by intra-VTA infusion of brain-derived neurotrophic factor. In contrast, chronic morphine treatment did not alter the size of nondopaminergic neurons in the VTA, nor did it affect the total number of dopaminergic neurons in this brain region. The results of these studies provide direct evidence for structural alterations in VTA dopamine neurons as a consequence of chronic opiate exposure, which could contribute to changes in mesolimbic dopamine function associated with addiction.

Increased dopamine turnover in the prefrontal cortex impairs spatial working memory performance in rats and monkeys.

The selective activation of the prefrontal cortical dopamine system by mild stress can be mimicked by anxiogenic beta-carbolines such as FG7142. To investigate the functional relevance of elevated levels of dopamine turnover in the prefrontal cortex, the current study examined the effects of FG7142 on the performance of spatial working memory tasks in the rat and monkey. FG7142 selectively increased prefrontal cortical dopamine turnover in rats and significantly impaired performance on spatial working memory tasks in both rats and monkeys. Spatial discrimination, a task with similar motor and motivational demands (rats), or delayed response performance following zero-second delays (monkeys) was unaffected by FG7142. Further, biochemical analysis in rats revealed a significant positive correlation between dopamine turnover in the prefrontal cortex and cognitive impairment on the delayed alternation task. The cognitive deficits in both rats and monkeys were prevented by pretreatment with the benzodiazepine receptor antagonist, RO15-1788, which blocked the increase in dopamine turnover and by the dopamine receptor antagonists, haloperidol, clozapine, and SCH23390. These findings indicate that excessive dopamine activity in the prefrontal cortex is detrimental to cognitive functions mediated by the prefrontal cortex.

Most central nervous system D2 dopamine receptors are coupled to their effectors by Go

We reported previously that Go-deficient mice develop severe neurological defects that include hyperalgesia, a generalized tremor, lack of coordination, and a turning syndrome somewhat reminiscent of unilateral lesions of the dopaminergic nigro-striatal pathway. By using frozen coronal sections of serially sectioned brains of normal and Go-deficient mice, we studied the ability of several G protein coupled receptors to promote binding of GTPγS to G proteins and the ability of GTP to promote a shift in the affinity of D2 dopamine receptor for its physiologic agonist dopamine. We found a generalized, but not abolished reduction in agonist-stimulated binding of GTPγS to frozen brain sections, with no significant left–right differences. Unexpectedly, the ability of GTP to regulate the binding affinity of dopamine to D2 receptors (as seen in in situ [35S]sulpiride displacement curves) that was robust in control mice, was absent in Go-deficient mice. The data suggest that most of the effects of the Gi/Go-coupled D2 receptors in the central nervous system are mediated by Go instead of Gi1, Gi2, or Gi3. In agreement with this, the effect of GTP on dopamine binding to D2 receptors in double Gi1 plus Gi2- and Gi1 plus Gi3-deficient mice was essentially unaffected.

A novel nonneuronal catecholaminergic system: exocrine pancreas synthesizes and releases dopamine.

Cells of the exocrine pancreas produce digestive enzymes potentially harmful to the intestinal mucosa. Dopamine has been reported to protect against mucosal injury. In looking for the source of dopamine in the small intestine, we found that the duodenal juice contains high levels of dopamine and that the pancreas itself has a high dopamine [and dihydroxyphenylalanine (dopa)] content that does not change significantly after chemical sympathectomy. Furthermore, we were able to demonstrate tyrosine hydroxylase (TH) activity in control pancreas as well as in pancreas from rats after chemical sympathectomy. Immunostaining and in situ hybridization histochemistry confirmed both the presence of TH, dopamine, and the dopamine transporter, and the mRNAs encoding TH and dopamine transporter, and the presence of both types of vesicular monoamine transporters in the exocrine cells of the pancreas. Since there are no catecholaminergic enteric ganglia in the pancreas, the above results indicate that pancreatic cells have all the characteristics of dopamine-producing cells. We suggest that the pancreas is an important source of nonneuronal dopamine in the body, and that this dopamine has a role in protecting the intestinal mucosa and suggests that dopamine D1b receptor agonists might be used to help mucosal healing in the gastrointestinal tract.

Regulation of the phosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa in vivo by dopamine D1, dopamine D2, and adenosine A2A receptors

Dopamine D1, dopamine D2, and adenosine A2A receptors are highly expressed in striatal medium-sized spiny neurons. We have examined, in vivo, the influence of these receptors on the state of phosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32). DARPP-32 is a potent endogenous inhibitor of protein phosphatase-1, which plays an obligatory role in dopaminergic transmission. A dose-dependent increase in the state of phosphorylation of DARPP-32 occurred in mouse striatum after systemic administration of the D2 receptor antagonist eticlopride (0.1–2.0 mg/kg). This effect was abolished in mice in which the gene coding for the adenosine A2A receptor was disrupted by homologous recombination. A reduction was also observed in mice that had been pretreated with the selective A2A receptor antagonist SCH 58261 (10 mg/kg). The eticlopride-induced increase in DARPP-32 phosphorylation was also decreased by pretreatment with the D1 receptor antagonist SCH 23390 (0.125 and 0.25 mg/kg) and completely reversed by combined pretreatment with SCH 23390 (0.25 mg/kg) plus SCH 58261 (10 mg/kg). SCH 23390, but not SCH 58261, abolished the increase in DARPP-32 caused by cocaine (15 mg/kg). The results indicate that, in vivo, the state of phosphorylation of DARPP-32 and, by implication, the activity of protein phosphatase-1 are regulated by tonic activation of D1, D2, and A2A receptors. The results also underscore the fact that the adenosine system plays a role in the generation of responses to dopamine D2 antagonists in vivo.

Dopamine tone regulates D1 receptor trafficking and delivery in striatal neurons in dopamine transporter-deficient mice

In vivo, G protein-coupled receptors (GPCR) for neurotransmitters undergo complex intracellular trafficking that contribute to regulate their abundance at the cell surface. Here, we report a previously undescribed alteration in the subcellular localization of D1 dopamine receptor (D1R) that occurs in vivo in striatal dopaminoceptive neurons in response to chronic and constitutive hyperdopaminergia. Indeed, in mice lacking the dopamine transporter, D1R is in abnormally low abundance at the plasma membrane of cell bodies and dendrites and is largely accumulated in rough endoplasmic reticulum and Golgi apparatus. Decrease of striatal extracellular dopamine concentration with 6-hydroxydopamine (6- OHDA) in heterozygous mice restores delivery of the receptor from the cytoplasm to the plasma membrane in cell bodies. These results demonstrate that, in vivo, in the central nervous system, the storage in cytoplasmic compartments involved in synthesis and the membrane delivery contribute to regulate GPCR availability and abundance at the surface of the neurons under control of the neurotransmitter tone. Such regulation may contribute to modulate receptivity of neurons to their endogenous ligands and related exogenous drugs.

Dopamine β-hydroxylase deficiency impairs cellular immunity

Norepinephrine, released from sympathetic neurons, and epinephrine, released from the adrenal medulla, participate in a number of physiological processes including those that facilitate adaptation to stressful conditions. The thymus, spleen, and lymph nodes are richly innervated by the sympathetic nervous system, and catecholamines are thought to modulate the immune response. However, the importance of this modulatory role in vivo remains uncertain. We addressed this question genetically by using mice that lack dopamine β-hydroxylase (dbh−/− mice). dbh−/− mice cannot produce norepinephrine or epinephrine, but produce dopamine instead. When housed in specific pathogen-free conditions, dbh−/− mice had normal numbers of blood leukocytes, and normal T and B cell development and in vitro function. However, when challenged in vivo by infection with the intracellular pathogens Listeria monocytogenes or Mycobacterium tuberculosis, dbh−/− mice were more susceptible to infection, exhibited extreme thymic involution, and had impaired T cell function, including Th1 cytokine production. When immunized with trinitrophenyl-keyhole limpet hemocyanin, dbh−/− mice produced less Th1 cytokine-dependent-IgG2a antitrinitrophenyl antibody. These results indicate that physiological catecholamine production is not required for normal development of the immune system, but plays an important role in the modulation of T cell-mediated immunity to infection and immunization.

Cloning of an arylalkylamine N-acetyltransferase (aaNAT1) from Drosophila melanogaster expressed in the nervous system and the gut.

In insects, neurotransmitter catabolism, melatonin precursor formation, and sclerotization involve arylalkylamine N-acetyltransferase (aaNAT, EC 2.3.1.87) activity. It is not known if one or multiple aaNAT enzymes are responsible for these activities. We recently have purified an aaNAT from Drosophila melanogaster. Here, we report the cloning of the corresponding aaNAT cDNA (aaNAT1) that upon COS cell expression acetylates dopamine, tryptamine, and the immediate melatonin precursor serotonin. aaNAT1 represents a novel gene family unrelated to known acetyl-transferases, except in two weakly conserved amino acid motifs. In situ hybridization studies of aaNAT1 mRNA in embryos reveal hybridization signals in the brain, the ventral cord, the gut, and probably in oenocytes, indicating a broad tissue distribution of aaNAT1 transcripts. Moreover, in day/ night studies we demonstrate a diurnal rhythm of melatonin concentration without a clear-cut change in aaNAT1 mRNA levels. The data suggest that tissue-specific regulation of aaNAT1 may be associated with different enzymatic functions and do not exclude the possibility of additional aaNAT genes.

Regulation of striatal D1A dopamine receptor gene transcription by Brn-4.

Brn-4 is a member of the POU transcription factor family and is expressed in the central nervous system. In this study, we addressed whether Brn-4 regulates expression of the D1A dopamine receptor gene. We found a functional Brn-4 responsive element in the intron of this gene by means of cotransfection chloramphenical acetyltransferase assays. This region contains two consensus sequences for binding of POU factors. Gel mobility-shift assays using glutathione S-transferase-Brn-4 fusion protein indicated that Brn-4 binds to these sequences. Both these sites are essential for transactivation by Brn-4 because deletion of either significantly reduced this enhancer activity. In situ hybridization revealed colocalization of Brn-4 and D1A mRNAs at the level of a single neuron in the rat striatum where this dopamine receptor is most abundantly expressed. Gel mobility-supershift assay using rat striatal nuclear extract and Brn-4 antibody confirmed the presence of Brn-4 in this brain region and its ability to bind to its consensus sequences in the D1A gene. These data suggest a functional role for Brn-4 in the expression of the D1A dopamine receptor gene both in vitro and in vivo.

Retrograde axonal transport of glial cell line-derived neurotrophic factor in the adult nigrostriatal system suggests a trophic role in the adult.

The recently cloned, distant member of the transforming growth factor beta (TGF-beta) family, glial cell line-derived neurotrophic factor (GDNF), has potent trophic actions on fetal mesencephalic dopamine neurons. GDNF also has protective and restorative activity on adult mesencephalic dopaminergic neurons and potently protects motoneurons from axotomy-induced cell death. However, evidence for a role for endogenous GDNF as a target-derived trophic factor in adult midbrain dopaminergic circuits requires documentation of specific transport from the sites of synthesis in the target areas to the nerve cell bodies themselves. Here, we demonstrate that GDNF is retrogradely transported by mesencephalic dopamine neurons of the nigrostriatal pathway. The pattern of retrograde transport following intrastriatal injections indicates that there may be subpopulations of neurons that are GDNF responsive. Retrograde axonal transport of biologically active 125I-labeled GDNF was inhibited by an excess of unlabeled GDNF but not by an excess of cytochrome c. Specificity was further documented by demonstrating that another TGF-beta family member, TGF-beta 1, did not appear to affect retrograde transport. Retrograde transport was also demonstrated by immunohistochemistry by using intrastriatal injections of unlabeled GDNF. GDNF immunoreactivity was found specifically in dopamine nerve cell bodies of the substantia nigra pars compacta distributed in granules in the soma and proximal dendrites. Our data implicate a specific receptor-mediated uptake mechanism operating in the adult. Taken together, the present findings suggest that GDNF acts endogenously as a target-derived physiological survival/maintenance factor for dopaminergic neurons.