No abstract available

The distribution of dopamine beta-hydroxylase and tyrosine hydroxylase, two key enzymes in the biosynthesis of catecholamines, was investigated by immunocytochemistry in the brain of male and female Japanese quail. Cells or fibers showing dopamine beta-hydroxylase and tyrosine hydroxylase immunoreactivity were considered to be noradrenergic or adrenergic, while all structures showing only tyrosine hydroxylase immunoreactivity were tentatively considered to be dopaminergic. The major dopaminergic and noradrenergic cell groups that have been identified in the brain of mammals could be observed in the Japanese quail, with the exception of a tuberoinfundibular dopaminergic group. The dopamine beta-hydroxylase-immunoreactive cells were found exclusively in the pons (locus ceruleus and nucleus subceruleus ventralis) and in the medulla (area of the nucleus reticularis). The tyrosine hydroxylase-immunoreactive cells had a much wider distribution and extended from the preoptic area to the level of the medulla. They were, however, present in larger numbers in the area ventralis of Tsai and in the nucleus tegmenti pedunculo-pontinus, pars compacta, which respectively correspond to the ventral tegmental area and to the substantia nigra of mammals. A high density of dopamine beta-hydroxylase- and tyrosine hydroxylase-immunoreactive fibers and punctate structures was found in several steroid-sensitive brain regions that are implicated in the control of reproduction. In the preoptic area and in the region of the nucleus accumbens-nucleus stria terminalis, immunonegative perikarya were completely surrounded by immunoreactive fibers forming basket-like structures. Given that some of these cells contain the enzyme aromatase, these structures may represent the morphological substrate for a regulation of aromatase activity by catecholamines. The dopamine beta-hydroxylase-immunoreactive fibers were also present in a larger part of the preoptic area of females than in males. This sex difference in the noradrenergic innervation of the preoptic area presumably reflects the sex difference in norepinephrine content in this region.

Dopamine beta hydroxylase (DBH) deficiency is an extremely rare autosomal recessive disorder with severe orthostatic hypotension, that can be treated with L-threo-3,4-dihydroxyphenylserine (L-DOPS). We aimed to summarize clinical, biochemical, and genetic data of all world-wide reported patients with DBH-deficiency, and to present detailed new data on long-term follow-up of a relatively large Dutch cohort. We retrospectively describe 10 patients from a Dutch cohort and 15 additional patients from the literature. We identified 25 patients (15 females) from 20 families. Ten patients were diagnosed in the Netherlands. Duration of follow-up of Dutch patients ranged from 1 to 21 years (median 13 years). All patients had severe orthostatic hypotension. Severely decreased or absent (nor)epinephrine, and increased dopamine plasma concentrations were found in 24/25 patients. Impaired kidney function and anemia were present in all Dutch patients, hypomagnesaemia in 5 out of 10. Clinically, all patients responded very well to L-DOPS, with marked reduction of orthostatic complaints. However, orthostatic hypotension remained present, and kidney function, anemia, and hypomagnesaemia only partially improved. Plasma norepinephrine increased and became detectable, while epinephrine remained undetectable in most patients. We confirm the core clinical characteristics of DBH-deficiency and the pathognomonic profile of catecholamines in body fluids. Impaired renal function, anemia, and hypomagnesaemia can be part of the clinical presentation. The subjective response to L-DOPS treatment is excellent and sustained, although the neurotransmitter profile in plasma does not normalize completely. Furthermore, orthostatic hypotension as well as renal function, anemia, and hypomagnesaemia improve only partially.

The biosynthesis and secretion of dopamine beta-hydroxylase were investigated by radiolabeling rat pheochromocytoma (PC12) cells in culture. Intracellular dopamine beta-hydroxylase from a crude chromaffin vesicle fraction and secreted dopamine beta-hydroxylase from culture medium were immunoprecipitated using antiserum made against purified bovine soluble dopamine beta-hydroxylase. Analysis of the immunoprecipitated enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that: 1) the membrane-bound form of the hydroxylase from crude secretory vesicle membrane extracts contained two nonidentical subunits in approximately stoichiometric amounts (Mr = 77,000 and 73,000); 2) the soluble hydroxylase from the lysate of these secretory vesicles was composed predominantly of a single subunit (Mr = 73,000); and 3) the hydroxylase secreted into the medium under resting conditions was also composed of a single subunit (approximate Mr = 73,000). All subunits of the multiple forms of hydroxylase were glycoproteins. Under resting conditions, the rate of secretion of hydroxylase was approximately 6% of total cellular enzyme/15 min. The secreted form of the hydroxylase incorporated [35S]sulfate, whereas no significant [35S]sulfate was incorporated into the cellular forms of enzyme. We propose that in addition to the dopamine beta-hydroxylase which is found in catecholamine storage vesicles and released during stimulus-coupled exocytosis, PC12 cells also have a constitutive secretory pathway for dopamine beta-hydroxylase and that the enzyme released by this second pathway is sulfated.

Disulfiram has been claimed to be useful in cocaine addiction therapy, its efficacy being attributed to dopamine-beta-hydroxylase (DBH) inhibition. Our previous results indicate that disulfiram and the selective DBH inhibitor nepicastat increase extracellular dopamine (DA) in the rat medial prefrontal cortex (mPFC), and markedly potentiated cocaine-induced increase. Concomitantly, in rats with cocaine self-administration history, cocaine-seeking behavior induced by drug priming was prevented, probably through overstimulation of D1 receptors due to the DA increase. The present research was aimed at studying the neurochemical mechanisms originating the enhanced DA release.

Galanin is a 29-amino acid neuropeptide found in rat spinal cord, autonomic ganglia and gastrointestinal tract, as well as in other areas of the nervous system in rats and other species. As part of an overall objective to determine if peptides contribute to target-specific control of visceral function, this study was designed to determine the percentages of populations of renal and splenic postganglionic neurons that contain galanin, and to determine if these neurons were likely to be adrenergic. Retrogradely transported fluorescent dyes were placed on renal and splenic nerves in male Wistar rats anaesthetized with sodium pento-barbital. Four days post-operatively, rats were perfused transcardially with fixative, and T12-L1 thoracolumbar chain ganglia, splanchnic ganglia and the solar plexus were removed. Immunocytochemical methods were then used to determine the proportions of the retrogradely labelled renal and splenic neurons containing galanin-like immunoreactivity and dopamine beta hydroxylase-like immunoreactivity. In seven rats, 24 +/- 3% of 2838 renal neurons were found to contain galanin-like immunoreactivity; in six rats, 32 +/- 5% of 5102 splenic neurons were found to contain galanin-like immunoreactivity. These proportions of the two populations were not significantly different from one another. In three rats, 94 +/- 2% of 684 renal neurons were found to contain dopamine beta hydroxylase-like immunoreactivity, and 95 +/- 2% of 2597 splenic neurons in three rats also showed dopamine beta hydroxylase-like immunoreactivity. These experiments indicate that subpopulations of both renal and splenic postganglionic sympathetic neurons contain the neuropeptide galanin and that these neurons are likely to be adrenergic in function. These findings suggest a role for galanin in control of the kidney and the spleen by the sympathetic nervous system.

Norepinephrine (NE), and specific adrenoceptors, have been reported to influence distinct aspects of adult hippocampal neurogenesis, including latent stem cell activation, progenitor proliferation, and differentiation. These findings are predominantly based on the use of pharmacological approaches in both in vitro and in vivo systems. Here, we sought to assess the consequences of genetic ablation of NE on adult hippocampal neurogenesis, by examining dopamine β hydroxylase knockout (Dbh -/-) mice, which lack NE from birth. We find that Dbh -/- mice exhibit no difference in adult hippocampal progenitor proliferation and survival. Further, the number of immature newborn neurons, labeled using stage-specific developmental markers within the hippocampal neurogenic niche, was also unaltered in Dbh -/- mice. In contrast, the noradrenergic neurotoxin DSP-4, which had previously been shown to reduce adult hippocampal neurogenesis in rats, also resulted in a decline in hippocampal progenitor proliferation in C57/Bl6N mice. These findings indicate that pharmacological lesioning of noradrenergic afferents in adulthood, but not the complete genetic loss of NE from birth, impairs adult hippocampal neurogenesis in mice.

Dopamine beta-hydroxylase (DβH) plays a key role in the synthesis of catecholamines (CAs) in the neuroendocrine regulatory network. The DβH gene was identified from the razor clam Sinonovacula constricta and referred to as ScDβH. The ScDβH gene is a copper type II ascorbate-dependent monooxygenase with a DOMON domain and two Cu2_monooxygen domains. ScDβH transcript expression was abundant in liver and hemolymph. During early development, ScDβH expression significantly increased at the umbo larval stage. Furthermore, the inhibitors and siRNA of DβH were screened. After challenge with DβH inhibitor, the larval metamorphosis and survival rates, and juvenile growth were obviously decreased. Under the siRNA stress, the larval metamorphosis and survival rates were also significantly decreased. Therefore, ScDβH may play an important regulating role in larval metamorphosis and juvenile growth.

Locus coeruleus (LC) neurons in the brainstem have long been associated with attention and arousal. Optogenetic stimulation of LC-NE neurons induces immediate sleep-to-wake transitions. However, LC neurons also secrete other neurotransmitters in addition to NE. To interrogate the role of NE derived from the LC in regulating wakefulness, we applied in vivo cell type-specific CRISPR/Cas9 technology to disrupt the dopamine beta hydroxylase (dbh) gene selectively in adult LC-NE neurons. Unilateral dbh gene disruption abolished immediate arousal following optogenetic stimulation of LC. Bilateral LC-specific dbh disruption significantly reduced NE concentration in LC projection areas and reduced wake length even in the presence of salient stimuli. These results suggest that NE may be crucial for the awakening effect of LC stimulation and serve as proof-of-principle that CRISPR gene editing in adult neurons can be used to interrogate gene function within genetically-defined neuronal circuitry associated with complex behaviors.

By balancing the ratios of dopamine and norepinephrine, dopamine beta hydroxylase (DBH) plays an important role in brain reward circuit that is involved with behavioral effects of heroin addiction. DBH -1021C/T (rs1611115) is a functional variant with strong correlation with plasma DBH activity and several nerval and psychic disorders. In the present study, we have collected 333 male cases with heroin addiction and 200 male healthy controls to explore the role of -1021C/T in heroin addiction. There is no evidence of association between -1021C/T and heroin addiction on both genotype and allele levels (P>0.05). In the injection subgroup of cases, -1021TT carriers have longer heroin addiction time (P<0.001) and higher dosage of self-administered heroin (P=0.045) than carriers with -1021CC or -1021CT, suggesting that patients with TT genotype are likely to have more progressive style of heroin users with injection route. In conclusion, our results support -1021TT genotype may be implicated with a more progressive nature of heroin addiction, although DBH -1021C/T is unlikely to be involved in the risk of heroin addiction.

Electroacupuncture (EA) is a modified form of acupuncture that utilizes electrical stimulation. We previously showed that EA stimulated rats were divided into responders that were sensitive to EA and non-responders that were insensitive to EA based on the tail flick latency (TFL) test. The dopamine beta-hydroxylase (DBH) gene was more abundantly expressed in the hypothalamus of responder rats than non-responder rats. To determine whether overexpression of DBH gene expression in the hypothalamus modulate EA analgesia, we constructed a DBH encoding adenovirus and which was then injected into the hypothalamus of SD rats. Microinjection of DBH or control GFP virus into the hypothalamus had no changes on the basal pain threshold measured by a TFL test without EA treatment. However, the analgesic effect of EA was significantly enhanced from seven days after microinjection of the DBH virus, but not after injection of the control GFP virus. DBH expression was significantly higher in the hypothalamus of DBH virus injected rat than control GFP virus or PBS injected rats. Moreover, expression of the DBH gene did not affect the body core temperature, body weight, motor function or learning and memory ability. Although the functional role of DBH in the hypothalamus in the analgesic effect of EA remains unclear, our findings suggest that expression of the DBH gene in the hypothalamus promotes EA analgesia without obvious side-effects.

Rett syndrome is a neurodevelopmental disorder caused by Mecp2 gene mutations. In RTT patients and Mecp2-null (Mecp2(-/Y)) mice, norepinephrine (NE) content drops significantly, which may play a role in breathing arrhythmia, sleep disorders and sudden death. However, the underlying mechanisms for the NE defect are not fully understood. The NE defect may result from decreased NE biosynthesis, loss of catecholaminergic neurons or both. Although deficiency in tyrosine hydroxylase (TH) has been demonstrated, it is possible that dopamine beta-hydroxylase (DBH), the critical enzyme converting dopamine to NE, is also affected. To test these possibilities, we studied DBH expressions in pontine catecholaminergic neurons of Mecp2(-/Y) mice identified with breathing abnormalities. In comparison to the wild type, Mecp2(-/Y) mice at 2months of age showed approximately 50% decrease in the expressions of DBH and TH, at both protein and mRNA levels in the locus coeruleus (LC) region. Consistently, DBH and TH immunoreactivity was markedly decreased in LC neurons of Mecp2(-/Y) mice. No evidence was found for selective deficiency in TH- or DBH-containing neurons in Mecp2(-/Y) mice, as almost all TH-positive cells expressed DBH. By counting TH-immunoreactive cells in the LC, we found that the Mecp2(-/Y) mice lost only approximately 5% of the catecholaminergic neurons as compared to wild-type, although their LC volume shrank by approximately 15%. These results strongly suggest that the NE defect in Mecp2(-/Y) mice is likely to result from deficient expression of not only TH but also DBH without significant loss of catecholaminergic neurons in the LC.

Dopamine-beta-hydroxylase (D beta H) catalyzes the conversion of dopamine to norepinephrine and is released from sympathetic neurons into the circulation. Plasma-D beta H activity varies widely between individuals, and a subgroup of the population has very low activity levels. Mounting evidence suggests that the DBH structural gene is itself the major quantitative-trait locus (QTL) for plasma-D beta H activity, and a single unidentified polymorphism may account for a majority of the variation in activity levels. Through use of both sequencing-based mutational analysis of extreme phenotypes and genotype/phenotype correlations in samples from African American, European American (EA), and Japanese populations, we have identified a novel polymorphism (--1021C-->T), in the 5' flanking region of the DBH gene, that accounts for 35%--52% of the variation in plasma-D beta H activity in these populations. In EAs, homozygosity at the T allele predicted the very low D beta H-activity trait, and activity values in heterozygotes formed an intermediate distribution, indicating codominant inheritance. Our findings demonstrate that --1021C-->T is a major genetic marker for plasma-D beta H activity and provide new tools for investigation of the role of both D beta H and the DBH gene in human disease.

CRISPR/Cas9 mediated DNA double strand cutting is emerging as a powerful approach to increase rates of homologous recombination of large targeting vectors, but the optimization of parameters, equipment and expertise required remain barriers to successful mouse generation by single-step zygote injection. Here, we sought to apply CRISPR/Cas9 methods to traditional embryonic stem (ES) cell targeting followed by blastocyst injection to overcome the common issues of difficult vector construction and low targeting efficiency. To facilitate the study of noradrenergic function, which is implicated in myriad behavioral and physiological processes, we generated two different mouse lines that express FLPo recombinase under control of the noradrenergic-specific Dopamine-Beta-Hydroxylase (DBH) gene. We found that by co-electroporating a circular vector expressing Cas9 and a locus-specific sgRNA, we could target FLPo to the DBH locus in ES cells with shortened 1 kb homology arms. Two different sites in the DBH gene were targeted; the translational start codon with 6-8% targeting efficiency, and the translational stop codon with 75% targeting efficiency. Using this approach, we established two mouse lines with DBH-specific expression of FLPo in brainstem catecholaminergic populations that are publically available on MMRRC (MMRRC_041575-UCD and MMRRC_041577-UCD). Altogether, this study supports simplified, high-efficiency Cas9/CRISPR-mediated targeting in embryonic stem cells for production of knock-in mouse lines in a wider variety of contexts than zygote injection alone.

To obtain insight into the noradrenergic system of amphibians, the distribution of noradrenaline was studied immunohistochemically with antibodies against noradrenaline (NA) and dopamine-beta-hydroxylase (DBH) in the brain of the South African clawed frog Xenopus laevis. Noradrenaline-containing cell bodies are found in the hypothalamic periventricular organ, the isthmic region, and in an area ventral and medial to the solitary tract. Noradrenaline-immunoreactive (NAi) fibers are widely, but not uniformly, distributed throughout the brain and spinal cord. In the telencephalon, dense plexuses of NAi fibers are present dorsomedial to the nucleus accumbens, in the nucleus of the diagonal band, the dorsolateral part of the striatum, the medial amygdala, and in an area that encompasses the lateral forebrain bundle. In the diencephalon, dense plexuses are found ventrolateral to the periventricular organ, in the posterior tubercle, and in the intermediate lobe of the hypophysis. Compared to the forebrain, the brainstem and spinal cord are less densely innervated by NAi fibers. The distribution of DBHi cell bodies and fibers resembles the pattern revealed with the NA antibodies. An exception is formed by the liquor contacting cells of the hypothalamic periventricular organ, which are immunonegative for the DBH antiserum. It is suggested that these cells accumulate rather than metabolize catecholamines. The present study combined with the results of a previous report in Xenopus on the distribution of dopamine (González, Tuinhof, Smeets, '93, Anat. Embryol. 187:193-201) offers the opportunity to differentiate between the two catecholamines. For example, it is now shown that both dopaminergic and noradrenergic fibers innervate the intermediate lobe of the hypophysis and that, therefore, both catecholamines are likely involved in background adaptation.

To study the innervation of the major sublingual gland by means of immunohistochemistry.

Treatment with nerve growth factor was found to influence the subunit forms of dopamine beta-hydroxylase in PC12 pheochromocytoma cells. In untreated cells, near equal amounts of two subunit forms were observed (apparent Mr = 77,000 and 73,000) by labeling with [35S]methionine. Upon treatment of PC12 cells with nerve growth factor for several days, the Mr = 73,000 subunit form of dopamine beta-hydroxylase was almost exclusively observed. The shift in subunit forms became apparent only after a day of treatment and was maximal with 3 days or more of exposure to nerve growth factor. The dose-response curve was similar to most other nerve growth factor-induced responses in PC12 cells. Neurite outgrowth, however, was not essential for the shift in predominance of the Mr = 73,000 subunit form. This effect of nerve growth factor also occurred in suspension cultures or in the presence of low concentrations of inhibitors of transcription sufficient to prevent neurite outgrowth. Pulse-chase experiments with nerve growth factor-treated cells indicated that the Mr = 77,000 form is initially synthesized (5 min) and is then converted to the Mr = 73,000 form by 30-60 min. Insulin (100 ng/ml) and epidermal growth factor (1 ng/ml) had no effect on the subunit forms of dopamine beta-hydroxylase. However, treatment of PC12 cells for several days with dexamethasone (10(-5)M) or dibutyryl cyclic AMP (1 mM) leads to predominance of the Mr = 73,000 form of the enzyme. These experiments suggest that the proportions of the subunit forms of dopamine beta-hydroxylase can be regulated in cells by external signals and this may reflect alterations in post-translational processing enzymes and may serve as a potential mechanism to regulate catecholamine metabolism.

Inflammatory bowel diseases (IBD) are associated with altered neuronal regulation of the gastrointestinal (GI) tract and release of norepinephrine (NE). As sympathetic innervation of the GI tract modulates motility, blood flow, and immune function, changes in NE signaling may alter the risk of developing IBD. Dopamine beta-hydroxylase (DβH), the enzyme responsible for NE production, has been suggested to play a critical role in IBD, however the exact mechanism is unknown. We hypothesized that genetic variants of DβH could increase the risk of IBD. We performed genetic analysis on 45 IBD patients and 74 controls. IBD patients were screened by targeted exome sequencing and compared with NeuroX DβH single nucleotide polymorphism (SNP) genotyping data of the controls. Serum DβH protein levels for 15 IBD patients and 13 controls were evaluated using immunoblots and competitive ELISA. Seven SNPs were observed from DβH targeted exome sequencing in the 45 IBD patients. A single non-synonymous SNP, rs6271 (Arg549Cys), had a significant association with IBD patients; the odds ratio was a 5.6 times higher SNP frequency in IBD patients compared to controls (p = 0.002). We also examined the function and availability of the protein in both the IBD and control patients' sera bearing DβH Arg549Cys. Both control and IBD subjects bearing the heterozygote allele had statistically lower DβH protein levels while the intrinsic enzyme activity was higher. This is the first report of a noradrenergic genetic polymorphism (rs6271; Arg549Cys) associated with IBD. This polymorphism is associated with significantly lower levels of circulating DβH.

There is currently a great deal of interest in using linkage disequilibrium (LD) mapping to locate both disease and quantitative-trait loci on a genomewide scale. Recent findings suggest that much of the human genome is organized in discrete "blocks" of low haplotype diversity, but the utility of such blocks in identifying genes influencing complex traits is not yet known and must ultimately be tested empirically through use of real data. We recently identified a putative functional polymorphism (-1021C-->T) in the 5' upstream region of the DBH gene that accounted for 35%-52% of the total phenotypic variance in plasma dopamine beta-hydroxylase (DBH) activity in samples from three distinct populations. In the present study, we genotyped 11 diallelic markers at the DBH locus surrounding -1021C-->T in 386 unrelated individuals of European origin. We identified a single 10-kb block containing -1021C-->T, in which four haplotypes comprised 93% of the observed chromosomes. Only markers within the block were highly associated with phenotype (P< or =2.2 x 10(-10)), with one exception. In general, association with phenotype was strongly correlated with the degree of LD between each marker and -1021C-->T. Of four LD measures assessed, d(2) was the best predictor of this relationship. Had one attempted to map quantitative-trait loci for plasma DBH activity on a genomewide basis without prior knowledge of candidate regions and not included (by chance) markers within this haplotype block, the DBH locus might have been missed entirely. These results provide a direct example of the potential value of constructing a haplotype map of the human genome prior to embarking on large-scale association studies.

Preganglionic neurones in the lumbosacral spinal cord give rise to nerves providing the parasympathetic and sympathetic innervation of pelvic organs. These neurones are modulated by neurotransmitters released both from descending supra-spinal pathways and spinal interneurones. Though serotonin has been identified as exerting a significant influence on these neurones, few studies have investigated the circuitry through which it achieves this particularly in relation to sympathetic preganglionic neurones. Using a combination of neuronal tracing and multiple immunolabeling procedures, the current study has shown that pelvic preganglionic neurones receive a sparse, and probably non-synaptic, axosomatic/proximal dendritic input from serotonin-immunoreactive terminals. This was in marked contrast to dopamine beta hydroxylase-immunoreactive terminals, which made multiple contacts. However, the demonstration of both serotonin, and dopamine beta hydroxylase immunoreactive terminals on both parasympathetic and sympathetic preganglionic neurones provides evidence for direct modulation of these cells by both serotonin and norepinephrine. Serotonin-containing terminals displaying conventional synaptic morphology were often seen to contact unlabeled somata and dendritic processes in regions surrounding the labeled preganglionic cells. It is possible that these unlabeled structures represent interneurones that might allow the serotonin containing axons to exert an indirect influence on pelvic preganglionic neurones. Since many spinal interneurones employ GABA as a primary fast acting neurotransmitter we examined the relationship between terminals that were immunoreactive for serotonin or GABA and labeled pelvic preganglionic neurones. These studies were unable to demonstrate any direct connections between serotonin and GABA terminals within the intermediolateral or sacral parasympathetic nuclei. Colocalization of serotonin and GABA was very rare but terminals immunoreactive for each were occasionally seen to contact the same unlabeled processes in close proximity. These results suggest that in the rat, the serotonin modulation of pelvic preganglionic neurones may primarily involve indirect connections via local interneurones.