Search

Hypoxia induces respiratory responses in mammals and serotonergic neurons in the medulla oblongata participate in respiratory control. However, the morphological changes in serotonergic neurons induced by hypoxia have not yet been examined and respiratory controls of serotonergic neurons have not been clarified. We herein investigated the distribution of immunoreactivity for serotonin (5-hydroxytryptamine; 5-HT) in the medulla oblongata of control rats and rats exposed to 1-6h of hypoxia (10% O 2 ). We also examined the medulla oblongata by multiple immunofluorescence labeling for 5-HT, neurokinin 1 receptors (NK1R), a marker for some respiratory neurons in the pre-Bötzinger complex (PBC), and dopamine β-hydroxylase (DBH), a marker for catecholaminergic neurons. The number of 5-HT-immunoreactive nerve cell bodies in the raphe nuclei was higher in rats exposed to hypoxia than in control rats. The number of 5-HT-immunoreactive nerve fibers significantly increased in the rostral ventrolateral medulla of rats exposed to 1-6h of hypoxia, caudal ventrolateral medulla of rats exposed to 2-6h of hypoxia, and lateral part of the nucleus of the solitary tract and dorsal motor nucleus of the vagus nerve of rats exposed to 1-2h of hypoxia. Multiple immunofluorescence labeling showed that 5-HT-immunoreactive nerve fibers were close to NK1R-immunoreactive neurons in ventrolateral medulla and to DBH-immunoreactive neurons in the medulla. These results suggest that serotonergic neurons partly regulate respiratory control under hypoxic conditions by modulating the activity of NK1R-expressing and catecholaminergic neurons., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Rett syndrome (RTT) is a neurodevelopmental disorder affecting multiple functions, including the norepinephrine (NE) system. In the CNS, NE is produced mostly by neurons in the locus coeruleus (LC), where defects in intrinsic neuronal properties, NE biosynthetic enzymes, neuronal CO2 sensitivity, and synaptic currents have been reported in mouse models of RTT. LC neurons in methyl-CpG-binding protein 2 gene (Mecp2) null mice show a high rate of spontaneous firing, although whether such hyperexcitability might increase or decrease the NE release from synapses is unknown. To activate the NEergic axonal terminals selectively, we generated an optogenetic mouse model of RTT in which NEergic neuronal excitability can be manipulated with light. Using commercially available mouse breeders, we produced a new strain of double-transgenic mice with Mecp2 knockout and channelrhodopsin (ChR) knockin in catecholaminergic neurons. Several RTT-like phenotypes were found in the tyrosine hydroxylase (TH)-ChR-Mecp2(-/Y) mice, including hypoactivity, low body weight, hindlimb clasping, and breathing disorders. In brain slices, optostimulation produced depolarization and an increase in the firing rate of LC neurons from TH-ChR control mice. In TH-ChR control mice, optostimulation of presynaptic NEergic neurons augmented the firing rate of hypoglossal neurons (HNs), which was blocked by the α-adrenoceptor antagonist phentolamine. Such optostimulation of NEergic terminals had almost no effect on HNs from two or three TH-ChR-Mecp2(-/Y) mice, indicating that excessive excitation of presynaptic neurons does not benefit NEergic modulation in mice with Mecp2 disruption. These results also demonstrate the feasibility of generating double-transgenic mice for studies of RTT with commercially available mice, which are inexpensive, labor/time efficient, and promising for cell-specific stimulation. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Stressful events can generate emotional memories linked to the traumatic incident, but they also can impair the formation of nonemotional memories. Although the impact of stress on emotional memories is well studied, much less is known about the influence of the emotional state on the formation of nonemotional memories. We used the novel object-recognition task as a model of nonemotional memory in mice to investigate the underlying mechanism of the deleterious effect of stress on memory consolidation. Systemic, hippocampal, and peripheral blockade of cannabinoid type-1 (CB1) receptors abolished the stress-induced memory impairment. Genetic deletion and rescue of CB1 receptors in specific cell types revealed that the CB1 receptor population specifically in dopamine β-hydroxylase (DBH)-expressing cells is both necessary and sufficient for stress-induced impairment of memory consolidation, but CB1 receptors present in other neuronal populations are not involved. Strikingly, pharmacological manipulations in mice expressing CB1 receptors exclusively in DBH(+) cells revealed that both hippocampal and peripheral receptors mediate the impact of stress on memory consolidation. Thus, CB1 receptors on adrenergic and noradrenergic cells provide previously unrecognized cross-talk between central and peripheral mechanisms in the stress-dependent regulation of nonemotional memory consolidation, suggesting new potential avenues for the treatment of cognitive aspects on stress-related disorders., Competing Interests: The authors declare no conflict of interest.

The central nervous system controls food consumption to maintain metabolic homoeostasis. In response to a meal, visceral signals from the gut activate neurons in the nucleus of the solitary tract (NTS) via the vagus nerve. These NTS neurons then excite brain regions known to mediate feeding behaviour, such as the lateral parabrachial nucleus (PBN). We previously described a neural circuit for appetite suppression involving calcitonin gene-related protein (CGRP)-expressing PBN (CGRP(PBN)) neurons; however, the molecular identity of the inputs to these neurons was not established. Here we identify cholecystokinin (CCK) and noradrenergic, dopamine β-hydroxylase (DBH)-expressing NTS neurons as two separate populations that directly excite CGRP(PBN) neurons. When these NTS neurons are activated using optogenetic or chemogenetic methods, food intake decreases and with chronic stimulation mice lose body weight. Our optogenetic results reveal that CCK and DBH neurons in the NTS directly engage CGRP(PBN) neurons to promote anorexia.

Adolescence is a period of major behavioral and brain reorganization. As diagnoses and treatment of disorders like attention deficit hyperactivity disorder (ADHD) often occur during adolescence, it is important to understand how the prefrontal cortices change and how these changes may influence the response to drugs during development. The current study uses an adolescent rat model to study the effect of standard ADHD treatments, atomoxetine and methylphenidate on attentional set shifting and reversal learning. While both of these drugs act as norepinephrine reuptake inhibitors, higher doses of atomoxetine and all doses of methylphenidate also block dopamine transporters (DAT). Low doses of atomoxetine, were effective at remediating cognitive rigidity found in adolescents. In contrast, methylphenidate improved performance in rats unable to form an attentional set due to distractibility but was without effect in normal subjects. We also assessed the effects of GBR 12909, a selective DAT inhibitor, but found no effect of any dose on behavior. A second study in adolescent rats investigated changes in norepinephrine transporter (NET) and dopamine beta hydroxylase (DBH) density in five functionally distinct sub-regions of the prefrontal cortex: infralimbic, prelimbic, anterior cingulate, medial and lateral orbitofrontal cortices. These regions are implicated in impulsivity and distractibility. We found that NET, but not DBH, changed across adolescence in a regionally selective manner. The prelimbic cortex, which is critical to cognitive rigidity, and the lateral orbitofrontal cortex, critical to reversal learning and some forms of response inhibition, showed higher levels of NET at early than mid- to late adolescence. This article is part of a Special Issue entitled SI: Noradrenergic System., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Previous studies documented a cross-talk between purinergic P2X (P2XR) and nicotinic acetylcholine receptors (nAChR) in heterologous expression systems and peripheral preparations. We now investigated if this occurred in native brain preparations and probed its physiological function. We found that P2XR and nAChR were enriched in hippocampal terminals, where both P2X1-3R and α3, but not α4, nAChR subunits were located in the active zone and in dopamine-β-hydroxylase-positive hippocampal terminals. Notably, P2XR ligands displaced nAChR binding and nAChR ligands displaced P2XR binding to hippocampal synaptosomes. In addition, a negative P2XR/nAChR cross-talk was observed in the control of the evoked release of noradrenaline from rat hippocampal synaptosomes, characterized by a less-than-additive facilitatory effect upon co-activation of both receptors. This activity-dependent cross-inhibition was confirmed in Xenopus oocytes transfected with P2X1-3Rs and α3β2 (but not α4β2) nAChR. Besides, P2X2 co-immunoprecipitated α3β2 (but not α4β2) nAChR, both in HEK cells and rat hippocampal membranes indicating that this functional interaction is supported by a physical association between P2XR and nAChR. Moreover, eliminating extracellular ATP with apyrase in hippocampal slices promoted the inhibitory effect of the nAChR antagonist tubocurarine on noradrenaline release induced by high- but not low-frequency stimulation. Overall, these results provide integrated biochemical, pharmacological and functional evidence showing that P2X1-3R and α3β2 nAChR are physically and functionally interconnected at the presynaptic level to control excessive noradrenergic terminal activation upon intense synaptic firing in the hippocampus., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

BACKGROUND The purpose of this study was to explore the association between 2 single-nucleotide polymorphisms (SNPs) in the dopamine β-hydroxylase (DBH) gene (rs1611115 and rs732833) and the susceptibility to Parkinson's disease (PD). MATERIAL AND METHODS Polymerase chain reaction direct sequencing (PCR-DS) was used to test the genotypes of DBH polymorphisms in 95 PD patients and 100 healthy examinees frequency-matched with the former by age and sex. The genotype and allele distribution differences between the case and control groups were analyzed by chi-square test, and the relative risk of PD in southern Chinese populations was expressed by odds ratio (OR) and 95% confidence interval (CI). Hardy-Weinberg equilibrium (HWE) was also checked by chi-square test. RESULTS The genotype and allele distribution frequencies in rs1611115 were obviously different between PD patients and the healthy control group (P<0.05). The TT genotype may lead to a 2.95 times higher risk of PD occurrence compared with the common genotype CC (OR=2.95, 95%CI=1.02-8.51), and the C allele increased risk of onset of PD (OR=1.81, 95%CI=1.17-2.82). Cognition of the PD patients was different between CC and CT+TT genotypes of rs1611115 (P=0.047). CONCLUSIONS DBH rs1611115 polymorphism was likely to be associated with the susceptibility to PD, but we did not find that rs732833 is a susceptibility marker for PD.

Dopamine β-hydroxylase (DBH) converts dopamine (DA) to norepinephrine (NE) in noradrenergic/adrenergic cells. DBH deficiency prevents NE production and causes sympathetic failure, hypotension and ptosis in humans and mice; DBH knockout (Dbh -/-) mice reveal other NE deficiency phenotypes including embryonic lethality, delayed growth, and behavioral defects. Furthermore, a single nucleotide polymorphism (SNP) in the human DBH gene promoter (-970C>T; rs1611115) is associated with variation in serum DBH activity and with several neurological- and neuropsychiatric-related disorders, although its impact on DBH expression is controversial. Phenotypes associated with DBH deficiency are typically treated with L-3,4-dihydroxyphenylserine (DOPS), which can be converted to NE by aromatic acid decarboxylase (AADC) in the absence of DBH. In this study, we generated transgenic mice carrying a human bacterial artificial chromosome (BAC) encompassing the DBH coding locus as well as ~45 kb of upstream and ~107 kb of downstream sequence to address two issues. First, we characterized the neuroanatomical, neurochemical, physiological, and behavioral transgenic rescue of DBH deficiency by crossing the BAC onto a Dbh -/- background. Second, we compared human DBH mRNA abundance between transgenic lines carrying either a "C" or a "T" at position -970. The BAC transgene drove human DBH mRNA expression in a pattern indistinguishable from the endogenous gene, restored normal catecholamine levels to the peripheral organs and brain of Dbh -/- mice, and fully rescued embryonic lethality, delayed growth, ptosis, reduced exploratory activity, and seizure susceptibility. In some cases, transgenic rescue was superior to DOPS. However, allelic variation at the rs1611115 SNP had no impact on mRNA levels in any tissue. These results indicate that the human BAC contains all of the genetic information required for tissue-specific, functional expression of DBH and can rescue all measured Dbh deficiency phenotypes, but did not reveal an impact of the rs11115 variant on DBH expression in mice.

The norepinephrine pathway is believed to modulate behavioral and physiological processes, such as mood, overall arousal, and attention. Furthermore, abnormalities in the pathway have been linked to numerous diseases, for example hypertension, depression, anxiety, Parkinson's disease, schizophrenia, Alzheimer's disease, attention deficit hyperactivity disorder, and cocaine dependence. We report the crystal structure of human dopamine β-hydroxylase, which is the enzyme converting dopamine to norepinephrine. The structure of the DOMON (dopamine β-monooxygenase N-terminal) domain, also found in >1600 other proteins, reveals a possible metal-binding site and a ligand-binding pocket. The catalytic core structure shows two different conformations: an open active site, as also seen in another member of this enzyme family [the peptidylglycine α-hydroxylating (and α-amidating) monooxygenase], and a closed active site structure, in which the two copper-binding sites are only 4 to 5 Å apart, in what might be a coupled binuclear copper site. The dimerization domain adopts a conformation that bears no resemblance to any other known protein structure. The structure provides new molecular insights into the numerous devastating disorders of both physiological and neurological origins associated with the dopamine system.

Background: The monoaminergic transmitters dopamine (DA), noradrenaline (NE) and serotonin (5-HT) modulate cerebral functions via their extensive effects in the brain. Investigating their roles has led to the creation of vesicular monoaminergic transporter-2 (VMAT2) knockout (KO) mice. While this mutation results in postnatal death, VMAT2-heterozygous (HET) mice are viable and show a complex behavioural phenotype. However, the simultaneous alteration of the 3 systems prevents investigations into their individual functions., Methods: To assess the specific role of NE, 5-HT and DA, we genetically disrupted their neurotransmission by creating conditional VMAT2-KO mice with targeted recombination. These specific recombinations were obtained by breeding VMAT2(lox/lox) mice with DBHcre, SERTcre and DATcre mice, respectively. We conducted a complete neurochemical and behavioural characterization of VMAT2-HET animals in each system., Results: Conditional VMAT2-KO mice revealed an absence of VMAT2 expression, and a specific decrease in the whole brain levels of each monoamine. Although NE- and 5-HT-depleted mice are viable into adulthood, DA depletion results in postnatal death before weaning. Interestingly, alteration of the DA transmission fully accounted for the increased amphetamine response formerly observed in the VMAT2-HET mice, whereas alteration of the 5-HT system was solely responsible for the increase in cocaine response., Limitations: We used VMAT2-HET mice that displayed a mild phenotype. Because the VMAT2-KO in DA neurons is lethal, it precluded a straightforward comparison of the full KOs in the 3 systems., Conclusion: Given the intermingled functions of NE, 5-HT and DA in regulating cognitive and affective functions, this model will enhance understanding of their respective roles in the pathophysiology of psychiatric disorders.

The experiment was conducted to investigate the effect of high dietary copper on catecholamine concentration and dopamine-β-hydroxylase (DβH) activity in hypothalami and midbrains of growing pigs. Forty-five crossbred weanling pigs with an average body weight of 7.5 kg were randomly assigned to three groups of 15 each to receive a control diet containing 10 mg/kg Cu (diet A) and diets containing 125 (diet B) or 250 (diet C) mg Cu/kg DM for 45 days. Compared to the control, Cu supplementation at both 125 and 250 mg Cu/kg DM increased average daily gain (ADG), average daily feed intake (ADFI), and feed efficiency. High dietary copper increased midbrain and hypothalami dopamine (DA) and norepinephrine (NE) concentrations and midbrain dopamine-β-hydroxylase activity. However, increasing dietary Cu had no effect on hypothalami dopamine-β-hydroxylase activity.

Aims: Transgenic TGR(ASrAOGEN)680 (TGR) rats with specific downregulation of glial angiotensinogen (AOGEN) synthesis develop cardiovascular deficits, anxiety, altered response to stress, and depression. Here, we evaluated whether these deficits are associated with alteration of the integrity of the noradrenergic system originating from locus coeruleus (LC) neurons., Methods: Adult TGR rats were compared to control Sprague Dawley rats in terms of the following: tissue levels of transcripts encoding noradrenergic markers, tissue tyrosine hydroxylase (TH) protein level, in vivo TH activity, density of TH-containing fibers, behavioral response to novelty, locomotor activity, and polysomnography., Results: TH expression was increased in the LC of TGR rats compared to controls. In LC terminal fields, there was an increase in density of TH-containing fibers in TGR rats that was associated with an elevation of in vivo TH activity. TGR rats also displayed locomotor hyperactivity in response to novelty. Moreover, polysomnographic studies indicated that daily paradoxical sleep duration was increased in TGR rats and that the paradoxical sleep rebound triggered by total sleep deprivation was blunted in these rats., Conclusions: Altogether, these results suggest that disruption of astroglial AOGEN synthesis leads to cardiovascular, cognitive, behavioral, and sleep disorders that might be partly due to LC dysfunction., (© 2016 John Wiley & Sons Ltd.)

Objectives: The vagus nerve has gained a role in the treatment of certain diseases by the use of vagus nerve stimulation (VNS). This study provides detailed morphological information regarding the human cervical vagus nerve at the level of electrode implant., Results: Eleven pairs of cervical vagus nerves and four pairs of intracranial vagus nerves were analysed by the use of computer software. It was found that the right cervical vagus nerve has an 1.5 times larger effective surface area on average than the left nerve [1,089,492 ± 98,337 vs 753,915 ± 102,490 μm(2), respectively, (P < 0.05)] and that there is broad spreading within the individual nerves. At the right side, the mean effective surface area at the cervical level (1,089,492 ± 98,337 μm(2)) is larger than at the level inside the skull base (630,921 ± 105,422) (P < 0.05). This could imply that the vagus nerve receives anastomosing and 'hitchhiking' branches from areas other than the brainstem. Furthermore, abundant tyrosine hydroxylase (TH)- and dopamine ß-hydroxylase (DBH)-positive staining nerve fibres could be identified, indicating catecholaminergic neurotransmission. In two of the 22 cervical nerves, ganglion cells were found that also stained positive for TH and DBH. Stimulating the vagus nerve may therefore induce the release of dopamine and noradrenaline. A sympathetic activation could therefore be part of mechanism of action of VNS. Furthermore, it was shown that the right cervical vagus nerve contains on average two times more TH-positive nerve fibres than the left nerve (P < 0.05), a fact that could be of interest upon choosing stimulation side. We also suggest that the amount of epineurial tissue could be an important variable for determining individual effectiveness of VNS, because the absolute amount of epineurial tissue is widely spread between the individual nerves (ranging from 2,090,000 to 11,683,000 μm(2))., Conclusions: We conclude by stating that one has to look at the vagus nerve as a morphological entity of the peripheral autonomic nervous system, a composite of different fibres and (anastomosing and hitchhiking) branches of different origin with different neurotransmitters, which can act both parasympathetic and sympathetic. Electrically stimulating the vagus nerve therefore is not the same as elevating the 'physiological parasympathetic tone', but may also implement catecholaminergic (sympathetic) effects., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The purpose of the present study was to determine the response of the porcine coeliac-superior mesenteric ganglion complex (CSMG) neurons projecting to the prepyloric area of the porcine stomach to peripheral neuronal damage following partial stomach resection. To identify the sympathetic neurons innervating the studied area of stomach, the neuronal retrograde tracer Fast Blue (FB) was applied to control and partial stomach resection (RES) groups. On the 22nd day after FB injection, following laparotomy, the partial resection of the previously FB-injected stomach prepyloric area was performed in animals of RES group. On the 28th day, all animals were re-anaesthetized and euthanized. The CSMG complex was then collected and processed for double-labeling immunofluorescence. In control animals, retrograde-labelled perikarya were immunoreactive to tyrosine hydroxylase (TH), dopamine β-hydroxylase (DβH), neuropeptide Y (NPY) and galanin (GAL). Partial stomach resection decreased the numbers of FB-positive neurons immunopositive for TH and DβH. However, the strong increase of NPY and GAL expression, as well as de novo-synthesis of neuronal nitric oxide synthase (nNOS) and leu5-Enkephalin (LENK) was noted in studied neurons. Furthermore, FB-positive neurons in all pigs were surrounded by a network of cocaine- and amphetamine-regulated transcript peptide (CART)-, calcitonin gene-related peptide (CGRP)-, and substance P (SP)-, vasoactive intestinal peptide (VIP)-, LENK- and nNOS- immunoreactive nerve fibers. This may suggest neuroprotective contribution of these neurotransmitters in traumatic responses of sympathetic neurons to peripheral axonal damage., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Neuroblastoma is an embryonal pediatric tumor that originates from the developing sympathetic nervous system and shows a broad range of clinical behavior, ranging from fatal progression to differentiation into benign ganglioneuroma. In experimental neuroblastoma systems, retinoic acid (RA) effectively induces neuronal differentiation, and RA treatment has been therefore integrated in current therapies. However, the molecular mechanisms underlying differentiation are still poorly understood. We here investigated the role of transcription factor activating protein 2 beta (TFAP2B), a key factor in sympathetic nervous system development, in neuroblastoma pathogenesis and differentiation. Microarray analyses of primary neuroblastomas (n = 649) demonstrated that low TFAP2B expression was significantly associated with unfavorable prognostic markers as well as adverse patient outcome. We also found that low TFAP2B expression was strongly associated with CpG methylation of the TFAP2B locus in primary neuroblastomas (n = 105) and demethylation with 5-aza-2'-deoxycytidine resulted in induction of TFAP2B expression in vitro, suggesting that TFAP2B is silenced by genomic methylation. Tetracycline inducible re-expression of TFAP2B in IMR-32 and SH-EP neuroblastoma cells significantly impaired proliferation and cell cycle progression. In IMR-32 cells, TFAP2B induced neuronal differentiation, which was accompanied by up-regulation of the catecholamine biosynthesizing enzyme genes DBH and TH, and down-regulation of MYCN and REST, a master repressor of neuronal genes. By contrast, knockdown of TFAP2B by lentiviral transduction of shRNAs abrogated RA-induced neuronal differentiation of SH-SY5Y and SK-N-BE(2)c neuroblastoma cells almost completely. Taken together, our results suggest that TFAP2B is playing a vital role in retaining RA responsiveness and mediating noradrenergic neuronal differentiation in neuroblastoma., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Sympathetic and parasympathetic control of the heart is a classic example of norepinephrine (NE) and acetylcholine (ACh) triggering opposing actions. Sympathetic NE increases heart rate and contractility through activation of β receptors, whereas parasympathetic ACh slows the heart through muscarinic receptors. Sympathetic neurons can undergo a developmental transition from production of NE to ACh and we provide evidence that mouse cardiac sympathetic nerves transiently produce ACh after myocardial infarction (MI). ACh levels increased in viable heart tissue 10-14 d after MI, returning to control levels at 21 d, whereas NE levels were stable. At the same time, the genes required for ACh synthesis increased in stellate ganglia, which contain most of the sympathetic neurons projecting to the heart. Immunohistochemistry 14 d after MI revealed choline acetyltransferase (ChAT) in stellate sympathetic neurons and vesicular ACh transporter immunoreactivity in tyrosine hydroxylase-positive cardiac sympathetic fibers. Finally, selective deletion of the ChAT gene from adult sympathetic neurons prevented the infarction-induced increase in cardiac ACh. Deletion of the gp130 cytokine receptor from sympathetic neurons prevented the induction of cholinergic genes after MI, suggesting that inflammatory cytokines induce the transient acquisition of a cholinergic phenotype in cardiac sympathetic neurons. Ex vivo experiments examining the effect of NE and ACh on rabbit cardiac action potential duration revealed that ACh blunted both the NE-stimulated decrease in cardiac action potential duration and increase in myocyte calcium transients. This raises the possibility that sympathetic co-release of ACh and NE may impair adaptation to high heart rates and increase arrhythmia susceptibility., Significance Statement: Sympathetic neurons normally make norepinephrine (NE), which increases heart rate and the contractility of cardiac myocytes. We found that, after myocardial infarction, the sympathetic neurons innervating the heart begin to make acetylcholine (ACh), which slows heart rate and decreases contractility. Several lines of evidence confirmed that the source of ACh was sympathetic nerves rather than parasympathetic nerves that are the normal source of ACh in the heart. Global application of NE with or without ACh to ex vivo hearts showed that ACh partially reversed the NE-stimulated decrease in cardiac action potential duration and increase in myocyte calcium transients. That suggests that sympathetic co-release of ACh and NE may impair adaptation to high heart rates and increase arrhythmia susceptibility., (Copyright © 2016 the authors 0270-6474/16/360479-10$15.00/0.)

Tyrosine hydroxylase (TH, the rate limiting-enzyme in catecholamine synthesis) is regulated acutely via phosphorylation of 3 serine residues--Ser19, 31 and 40, and chronically via changes in TH protein levels. In this study, we aimed to investigate how TH is regulated in the brain, gut and adrenal gland as well as changes in mature brain-derived neurotrophic factor (mBDNF) and proBDNF levels in a low-dose (2 mg/kg, 5 days/week for 4 weeks) rotenone model of Parkinson's disease (PD). Rearing behaviour decreased by week 3 in the rotenone group (p<0.01), with further decreases in rearing by week 4 (p<0.001); however, TH remained unchanged in the substantia nigra (SN) and striatum; TH levels were also unaltered in other catecholaminergic cell groups of the brainstem such as A1C1 neurons or locus coeruleus. In the olfactory bulb, TH protein decreased (2.5-fold, p<0.01) while Ser31 phosphorylation increased (1.4-fold, p<0.05) in the rotenone group. In contrast, TH protein was increased in the adrenal gland (2-fold, p<0.05) and colon (5-fold, p<0.05) of rotenone rats. mBDNF levels were not changed in the SN but were significantly reduced in plasma and significantly increased in the colon (2-fold, p<0.01) of rotenone-treated rats. This is the first study to assess TH and BDNF in the brain and periphery in the rotenone model before SN/striatum degeneration is evident. Together these results suggest that low-dose rotenone may have some potential to model the early stages of PD., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Dysregulation of the central noradrenergic system is a core feature of post-traumatic stress disorder (PTSD). Here, we examined molecular changes in locus coeruleus (LC) triggered by single-prolonged stress (SPS) PTSD model at a time when behavioral symptoms are manifested, and the effect of early intervention with intranasal neuropeptide Y (NPY). Immediately following SPS stressors, male SD rats were administered intranasal NPY (SPS/NPY) or vehicle (SPS/V). Seven days later, TH protein, but not mRNA, was elevated in LC only of the SPS/V group. Although 90% of TH positive cells expressed GR, its levels were unaltered. Compared to unstressed controls, LC of SPS/V, but not SPS/NPY, expressed less Y2 receptor mRNA with more CRHR1 mRNA in subset of animals, and elevated corticotropin-releasing hormone (CRH) in central nucleus of amygdala. Following testing for anxiety on elevated plus maze (EPM), there were significantly increased TH, DBH and NPY mRNAs in LC of SPS-treated, but not previously unstressed animals. Their levels highly correlated with each other but not with behavioral features on EPM. Thus, SPS triggers long-term noradrenergic activation and higher sensitivity to mild stressors, perhaps mediated by the up-regulation influence of amygdalar CRH input and down-regulation of Y2R presynaptic inhibition in LC. Results also demonstrate the therapeutic potential of early intervention with intranasal NPY for traumatic stress-elicited noradrenergic impairments. Single-prolonged stress (SPS)-triggered long-term changes in the locus coeruleus/norepinephrine (LC/NE) system with increased tyrosine hydroxylase (TH) protein and CRH receptor 1(CRHR1) mRNA and lower neuropeptide Y receptor 2 (Y2R) mRNA levels as well as elevated corticotropin-releasing hormone (CRH) in the central nucleus of amygdala (CeA) that were prevented by early intervention with intranasal neuropeptide Y (NPY). SPS treatment led to increased sensitivity of LC to mild stress of elevated plus maze (EPM), with elevated mRNA for NE biosynthetic enzymes in subset of animals., (© 2015 International Society for Neurochemistry.)

We studied the effect of testosterone overdose on the number, distribution and chemical coding of ovarian neurons in the sympathetic chain ganglia (SChGs) in pigs. On day 3 of the estrous cycle the ovaries of both the control and experimental gilts were injected with retrograde neuronal tracer Fast Blue to identify the neurons innervating gonads. From the following day to the expected day 20 of the second studied cycle the experimental pigs were injected with testosterone, while the control pigs received oil, and subsequently the SChG Th16-S2 were collected. Testosterone injections increased testosterone (∼3.5 fold) and estradiol-17β (∼1.6 fold) levels in the peripheral blood, and reduced the following in the SChGs: the total number of Fast Blue-positive neurons, the numbers of perikarya in the L3-L5 ganglia, the numbers of perikarya in the ventral, dorsal and central regions of the SChGs, and the numbers of DβH(+)/NPY(+), DβH(+)/GAL(+), DβH(+)/NPY(-), DβH(+)/SOM(-) and DβH(+)/GAL(-) perikarya. In the testosterone-affected SChGs, the perikarya DβH(-)/SOM(+), DβH(-)/GAL(+) and DβH(-)/NPY(-) were absent. In these ganglia, the population of androgen receptor-positive perikarya was increased, while the population of estrogen receptor-expressing perikarya was lowered. Our data indicate that in the pig SChGs elevated androgen levels occurring during pathological states may affect the morphology and chemical coding of ovarian neurons., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Trans-plasma membrane electron transfer is achieved by b-type cytochromes of different families, and plays a fundamental role in diverse cellular processes involving two interacting redox couples that are physically separated by a phospholipid bilayer, such as iron uptake and redox signaling. Despite their importance, no direct recordings of trans-plasma membrane electron currents have been described in plants. In this work, we provide robust electrophysiological evidence of trans-plasma membrane electron flow mediated by a soybean (Glycine max) cytochrome b561 associated with a dopamine β-monooxygenase redox domain (CYBDOM), which localizes to the plasma membrane in transgenic Arabidopsis (Arabidopsis thaliana) plants and CYBDOM complementary RNA-injected Xenopus laevis oocytes. In oocytes, two-electrode voltage clamp experiments showed that CYBDOM-mediated currents were activated by extracellular electron acceptors in a concentration- and type-specific manner. Current amplitudes were voltage dependent, strongly potentiated in oocytes preinjected with ascorbate (the canonical electron donor for cytochrome b561), and abolished by mutating a highly conserved His residue (H292L) predicted to coordinate the cytoplasmic heme b group. We believe that this unique approach opens new perspectives in plant transmembrane electron transport and beyond., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Introduction: 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., Methods: Noradrenergic system ablation was attained by intracerebroventricular (i.c.v.) administration of the neurotoxin anti-DBH-saporin (aDBH-sap). DA, noradrenaline (NA), and DOPAC were assessed by HPLC after ex vivo tissue extraction or in vivo microdialysis. Control and denervated rats were subjected to microdialysis in the mPFC and caudate nucleus to evaluate the effect of nepicastat-cocaine combination on extracellular DA levels and their regulation by α2-adrenoceptors., Results: Fifteen days after neurotoxin or its vehicle administration, tissue and extracellular NA were reduced to less than 2% the control value, while extracellular DA was increased by approximately 100%. In control rats, nepicastat given alone and in combination with cocaine increased extracellular DA by about 250% and 1100%, respectively. In denervated rats, nepicastat slightly affected extracellular DA, while in combination with cocaine increased extracellular DA by 250%. No differences were found in the caudate nucleus. Clonidine almost totally reversed the extracellular DA elevation produced by nepicastat-cocaine combination, while it was ineffective in denervated rats., Conclusions: This research shows that the increase of extracellular DA produced by nepicastat alone or in combination with cocaine was prevented by noradrenergic denervation. The results indicate that nepicastat enhances DA release from noradrenergic terminals supposedly by removing NA from α2-autoreceptors. In addition to the inhibition of DA uptake, the latter mechanism may explain the synergistic effect of cocaine on nepicastat-induced DA release.

Background: Cuff and spared nerve injury (SNI) in the sciatic territory are widely used to model neuropathic pain. Because nociceptive information is first detected in skin, it is important to understand how alterations in peripheral innervation contribute to pain in each model. Over 16 weeks in male rats, changes in sensory and autonomic innervation of the skin were described after cuff and SNI using immunohistochemistry to label myelinated (neurofilament 200 positive-NF200+) and peptidergic (calcitonin gene-related peptide positive-CGRP+) primary afferents and sympathetic fibres (dopamine β-hydroxylase positive-DBH+), Results: Cuff and SNI caused an early loss and later reinnervation of NF200 and CGRP fibres in the plantar hind paw skin. In both models, DBH+ fibres sprouted into the upper dermis of the plantar skin 4 and 6 weeks after injury. Despite these similarities, behavioural pain measures were significantly different in each model. Sympathectomy using guanethidine significantly alleviated mechanical allodynia 6 weeks after cuff, when peak sympathetic sprouting was observed, having no effect at 2 weeks, when fibres were absent. In SNI animals, mechanical allodynia in the lateral paw was significantly improved by guanethidine at 2 and 6 weeks, and the development of cold hyperalgesia, which roughly paralleled the appearance of ectopic sympathetic fibres, was alleviated by guanethidine at 6 weeks. Sympathetic fibres did not sprout into the dorsal root ganglia at 2 or 6 weeks, indicating their unimportance to pain behaviour in these two models., Conclusions: Alterations in sympathetic innervation in the skin represents an important mechanism that contributes to pain in cuff and SNI models of neuropathic pain.

Pharmacological studies in mammals suggest that norepinephrine (NE) plays an important role in promoting arousal. However, the role of endogenous NE is unclear, with contradicting reports concerning the sleep phenotypes of mice lacking NE due to mutation of dopamine β-hydroxylase (dbh). To investigate NE function in an alternative vertebrate model, we generated dbh mutant zebrafish. In contrast to mice, these animals exhibit dramatically increased sleep. Surprisingly, despite an increase in sleep, dbh mutant zebrafish have a reduced arousal threshold. These phenotypes are also observed in zebrafish treated with small molecules that inhibit NE signaling, suggesting that they are caused by the lack of NE. Using genetic overexpression of hypocretin (Hcrt) and optogenetic activation of hcrt-expressing neurons, we also find that NE is important for Hcrt-induced arousal. These results establish a role for endogenous NE in promoting arousal and indicate that NE is a critical downstream effector of Hcrt neurons.

Endocannabinoids (eCBs) are involved in a myriad of physiological processes that are mediated through the activation of cannabinoid receptors, which are ubiquitously distributed within the nervous system. One neurochemical target at which cannabinoids interact to have global effects on behavior is brain noradrenergic circuitry. We, and others, have previously shown that CB type 1 receptors (CB1r) are positioned to pre-synaptically modulate norepinephrine (NE) release in the rat frontal cortex (FC). Diacylglycerol lipase (DGL) is a key enzyme in the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). While DGL-α is expressed in the FC in the rat brain, it is not known whether noradrenergic afferents target neurons expressing synthesizing enzymes for the endocannabinoid, 2-AG. In the present study, we employed high-resolution neuroanatomical approaches to better define cellular sites for interactions between noradrenergic afferents and FC neurons expressing DGL-α. Immunofluorescence microscopy showed close appositions between processes containing the norepinephrine transporter (NET) or dopamine-β-hydroxylase (DβH) and cortical neurons expressing DGL-α-immunoreactivity. Ultrastructural analysis using immunogold-silver labeling for DGL-α and immunoperoxidase labeling for NET or DβH confirmed that NET-labeled axon terminals were directly apposed to FC somata and dendritic processes that exhibited DGL-α-immunoreactivity. Finally, tissue sections were processed for immunohistochemical detection of DGL-α, CB1r and DβH. Triple label immunofluorescence revealed that CB1r and DβH were co-localized in common cellular profiles and these were in close association with DGL-α. Taken together, these data provide anatomical evidence for direct synaptic associations between noradrenergic afferents and cortical neurons exhibiting endocannabinoid synthesizing machinery., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

This study addressed the in vitro construction and biological activity of tissue engineered intervertebral discs with exogenous human dopamine beta-hydroxylase (DBH) nucleus pulposus cells. pSNAV2.0-DBH expression plasmids were utilized to enhance the survival rates of intervertebral disc tissue cells. Various concentrations of transfected nucleus pulposus cells were injected into the discs, and DBH mRNA expression was determined using polymerase chain reaction amplification. Polysaccharide content and total collagen protein content in the engineered disc nucleus pulposus tissue were determined. The visible fluorescence intensities of the 1 x 10(5) and 1 x 10(6) groups vs the 1 x 10(4) group were significantly increased (P < 0.05); no significant difference was observed between the 1 x 10(5) and 1 x 10(6) groups (P > 0.05) at 7 days after injection. DBH mRNA expression could be detected in the all but the EGFP control group at 14 days culture. No significant difference was observed in the protein content between the 1 x 10(4) and the control groups at various times, while the protein content was significantly higher in the 1 x 10(5) vs the control and the 1 x 10(4) groups at 7-, 14-, and 21-day cultures. These results demonstrate that a tissue engineered intervertebral disc with high biological activity can be constructed by utilizing allogeneic intervertebral discs stored in liquid nitrogen and a 1 x 10(5) transfected nucleus pulposus cell complex with in vitro culture for 14 days. This model can be used in animal experiments to study the biological activity of the engineered discs.

Overactivity of the sympathetic nervous system has an important role in the development and progression of arterial hypertension. Catheter-based renal nerve ablation for the treatment of drug-resistant hypertension has recently been developed. An alternative strategy for the modulation of sympathetic nerve function is to reduce the biosynthesis of noradrenaline (NA) by inhibiting dopamine β-hydroxylase (DβH), the enzyme that catalyzes the conversion of dopamine (DA) to NA in the sympathetic nerves. Renal denervation (RDN) surgery was performed in spontaneously hypertensive rats (SHR) to evaluate the effect of RDN on the DA and NA levels and on blood pressure over a 28-day period. The selective peripheral DβH inhibitor etamicastat (30 mg kg (-1)day(-1)) was administered to another cohort of SHR. RDN and etamicastat treatment had no effect on the renal function, as assessed by measuring the water balance response, renal function and urinary electrolyte levels. RDN significantly decreased the systolic blood pressure (SBP) and the diastolic blood pressure (DBP). A gradual return of the SBP and the DBP to the high baseline levels was observed over time. Conversely, treatment with etamicastat resulted in a significant decrease in the SBP and the DBP at all time points. On the last day of the assessment, NA levels in renal tissue were significantly decreased in both RDN and etamicastat-treated groups. In contrast, the NA levels in the left ventricle were decreased only in the etamicastat-treated group. Thus, RDN produces transitory decreases in blood pressure, whereas prolonged downregulation of sympathetic drive with the DβH inhibitor etamicastat results in a sustained decrease in the SBP and the DBP.

In recent years, non-motor symptoms have been recognised as of vital importance in Parkinson's disease (PD); among these, cardiovascular dysfunctions are commonly seen in PD patients before their motor signs. The role of cardiovascular dysfunction in the progression of PD pathology, and its underlying mechanisms, are largely unknown. In the present study, in rotenone-induced PD rats, there was a gradual reduction in the number of nigral tyrosine hydroxylase-immunoreactive (TH-ir) neurons after 7, 14 and 21 days treatment. With the 56% reduction in striatal dopamine content and 52% loss of TH-ir neurons on the 14th day, the rats showed motor dysfunctions. However, from ECG power spectra, reductions in normalised low-frequency power and in the low-frequency power : high-frequency power ratio, as well as in mean blood pressure, were observed as early as the 3rd day. Plasma norepinephrine (NE) and epinephrine (E) levels were decreased by 39% and 26% respectively at the same time. Pearson's correlation analysis showed that both plasma NE and plasma E levels were positively correlated with MBP. Our results also showed that the loss of catecholaminergic neurons in the rostral ventrolateral medulla (RVLM), but not in the caudal ventrolateral medulla or the nucleus tractus solitarii, emerged earlier than the loss of nigral dopaminergic neurons. This suggests that dysfunction of catecholaminergic neurons in the RVLM might account for the reduced sympathetic activity, MBP and plasma catecholamine levels in the early stages of PD., (© 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

It has been shown that vagus nerve stimulation (VNS) has an antidepressant-like effect in the forced swim test. The mechanism of action underlying this effect is incompletely understood, but there is evidence suggesting that the locus coeruleus (LC) may play an important role. In this study, noradrenergic LC neurons were selectively lesioned to test their involvement in the antidepressant-like effect of VNS in the forced swim test. Forced swim test behavior was assessed in rats that were subjected to VNS or sham treatment. In half of the VNS-treated animals, the noradrenergic neurons from the LC were lesioned using the selective neurotoxin DSP-4 [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride], yielding three experimental arms: sham, VNS and DSP-4-VNS (n = 8 per group). Furthermore, the open field test was performed to evaluate locomotor activity. A dopamine-β-hydroxylase immunostaining was performed to confirm lesioning of noradrenergic LC neurons. VNS significantly reduced the percentage of immobility time in the forced swim test compared to sham treatment (median: 56%, interquartile range: 41% vs. median: 75%, interquartile range: 12%). This antidepressant-like effect of VNS could not be demonstrated in the DSP-4-VNS group (median: 79%, interquartile range: 33%). Locomotor activity in the open field test was not different between the three treatment arms. The absence of hippocampal dopamine-β-hydroxylase immunostaining in the DSP-4-treated rats confirmed the lesioning of noradrenergic neurons originating from the brainstem LC. The results of this study demonstrate that the noradrenergic neurons from the LC play an important role in the antidepressant-like effect of VNS., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Respiratory responses to hypoxia and/or hypercapnia, and their relationship to neural activity in the ventrolateral medulla (VLM), which includes the respiratory center, have not yet been elucidated in detail. We herein examined respiratory responses during exposure of 10% O2 (hypoxia), 10% CO2 (hypercapnia), and 10% O2-10% CO2 (hypercapnic hypoxia) using plethysmography. In addition to recording respiration, Fos expressions were examined in the VLM of the rat exposed to each gas to analyze neural activity. Respiratory frequency was increased in rats exposed to hypoxia, and Fos-positive neurons were observed in the caudal VLM (cVLM) and medial VLM (mVLM). Tidal volume was increased in rats exposed to hypercapnia, and Fos-positive neurons were observed in the rostral VLM (rVLM) includes the retrotrapezoid nucleus (RTN) and mVLM. Tidal volume was enhanced in rats exposed to hypercapnic hypoxia, similar to that in hypercapnia-exposed rats, and Fos-positive neurons were observed in the entire region of the VLM. In the mVLM and cVLM, double immunofluorescence showed Fos-immunoreactive nerve cells were also immunoreactive to dopamine β-hydroxylase, the marker for A1/C1 catecholaminergic neuron. These results suggested that hypoxia and hypercapnia modulated rhythmogenic microcircuits in the mVLM via A1/C1 neurons and the RTN, respectively., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Both lateral hypothalamic orexinergic neurons and hindbrain catecholaminergic neurons contribute to control of feeding behavior. Orexin fibers and terminals are present in close proximity to hindbrain catecholaminergic neurons, and fourth ventricular (4V) orexin injections that increase food intake also increase c-Fos expression in hindbrain catecholamine neurons, suggesting that orexin neurons may stimulate feeding by activating catecholamine neurons. Here we examine that hypothesis in more detail. We found that 4V injection of orexin-A (0.5 nmol/rat) produced widespread activation of c-Fos in hindbrain catecholamine cell groups. In the A1 and C1 cell groups in the ventrolateral medulla, where most c-Fos-positive neurons were also dopamine β hydroxylase (DBH) positive, direct injections of a lower dose (67 pmol/200 nl) of orexin-A also increased food intake in intact rats. Then, with the use of the retrogradely transported immunotoxin, anti-DBH conjugated to saporin (DSAP), which targets and destroys DBH-expressing catecholamine neurons, we examined the hypothesis that catecholamine neurons are required for orexin-induced feeding. Rats given paraventricular hypothalamic injections of DSAP, or unconjugated saporin (SAP) as control, were implanted with 4V or lateral ventricular (LV) cannulas and tested for feeding in response to ventricular injection of orexin-A (0.5 nmol/rat). Both LV and 4V orexin-A stimulated feeding in SAP controls, but DSAP abolished these responses. These results reveal for the first time that catecholamine neurons are required for feeding induced by injection of orexin-A into either LV or 4V., (Copyright © 2015 the American Physiological Society.)

A norepinephrine (NE) deficiency has been observed in aged rats and in patients with Alzheimer's disease and is thought to cause cognitive disorder. Which endogenous factor induces NE depletion, however, is largely unknown. In this study, we investigated the effects of aging-associated formaldehyde (FA) on the inactivation of NE in vitro and in vivo, and on memory behaviors in rodents. The results showed that age-related DNA demethylation led to hippocampal FA accumulation, and when this occurred, the hippocampal NE content was reduced in healthy male rats of different ages. Furthermore, biochemical analysis revealed that FA rapidly inactivated NE in vitro and that an intrahippocampal injection of FA markedly reduced hippocampal NE levels in healthy adult rats. Unexpectedly, an injection of FA (at a pathological level) or 6-hydroxydopamine (6-OHDA, a NE depletor) can mimic age-related NE deficiency, long-term potentiation (LTP) impairments, and spatial memory deficits in healthy adult rats. Conversely, an injection of NE reversed age-related deficits in both LTP and memory in aged rats. In agreement with the above results, the senescence-accelerated prone 8 (SAMP8) mice also exhibited a severe deficit in LTP and memory associated with a more severe NE deficiency and FA accumulation, when compared with the age-matched, senescence-resistant 1 (SAMR1) mice. Injection of resveratrol (a natural FA scavenger) or NE into SAMP8 mice reversed FA accumulation and NE deficiency and restored the magnitude of LTP and memory. Collectively, these findings suggest that accumulated FA is a critical endogenous factor for aging-associated NE depletion and cognitive decline., (© 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Brainstem catecholaminergic neurons significantly participate in the regulation of neuroendocrine system activity, particularly during stressful conditions. However, so far the precise quantitative characterisation of basal and stress-induced changes in gene expression and protein levels of catecholaminergic biosynthetic enzymes in these neurons has been missing. Using a quantitative reverse transcription-polymerase chain reaction method, we investigated gene expression of catecholamine biosynthetic enzymes in brainstem noradrenergic and adrenergic cell groups in rats under resting conditions as well as in acutely and repeatedly stressed animals. For the first time, we described quantitative differences in basal levels of catecholamine biosynthetic enzyme mRNA in brainstem catecholaminergic ascending and descending projecting cell groups. Moreover, we found and defined some differences among catecholaminergic cell groups in the time-course of mRNA levels of catecholaminergic enzymes following a single and especially repeated immobilisation stress. The data obtained support the assumption that brainstem catecholaminergic cell groups represent a functionally differentiated system, which is highly (but specifically) activated in rats exposed to stress. Therefore, potential interventions for the treatment of stress-related diseases need to affect the activity of brainstem catecholaminergic neurons not uniformly but with some degree of selectivity., (© 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Psychological stress has long been reported to be linked with the disease activity of patients with inflammatory bowel disease (IBD). However, the mechanisms of psychological stress involved in pathogenesis of IBD are still to be elucidated. We have previously shown that catecholamine participates in progression of acute colitis through a2-adrenoreceptors. The study aimed to explore the pivotal role of catecholamine in psychological stress-induced colitis. The expression of dopamine β-hydroxylase (DBH), the rate-limiting enzyme in regulation of catecholamine synthesis, was induced in colon tissues of mice with restraint stress, indicating the association of catecholamine synthesis with psychological stress. Notably, pretreatment with RX821002, an a2-adrenoceptor antagonist, attenuated inflammatory responses of psychological stress-induced colitis. Intriguingly, DBH levels were elevated in colon tissues of patients with active IBD. The study suggests that a2-adrenoreceptors/catecholamine play pivotal role in psychological stress-induced colitis and might contribute to the development of human IBD.

Background: The descending antinociceptive system (DAS) is thought to play crucial roles in the antinociceptive effect of spinal cord stimulation (SCS), especially through its serotonergic pathway. The nucleus raphe magnus (NRM) in the rostral ventromedial medulla is a major source of serotonin [5-hydroxytryptamine (5-HT)] to the DAS, but the role of the dorsal raphe nucleus (DRN) in the ventral periaqueductal gray matter is still unclear. Moreover, the influence of the noradrenergic pathway is largely unknown. In this study, we evaluated the involvement of these serotonergic and noradrenergic pathways in SCS-induced antinociception by behavioral analysis of spinal nerve-ligated (SNL) rats. We also investigated immunohistochemical changes in the DRN and locus coeruleus (LC), regarded as the adrenergic center of the DAS, and expression changes of synthetic enzymes of 5-HT [tryptophan hydroxylase (TPH)] and norepinephrine [dopamine β-hydroxylase (DβH)] in the spinal dorsal horn., Results: Intrathecally administered methysergide, a 5-HT1- and 5-HT2-receptor antagonist, and idazoxan, an α2-adrenergic receptor antagonist, equally abolished the antinociceptive effect of SCS. The numbers of TPH-positive serotonergic and phosphorylated cyclic AMP response element binding protein (pCREB)-positive neurons and percentage of pCREB-positive serotonergic neurons in the DRN significantly increased after 3-h SCS. Further, the ipsilateral-to-contralateral immunoreactivity ratio of DβH increased in the LC of SNL rats and reached the level seen in naïve rats, even though the number of pCREB-positive neurons in the LC was unchanged by SNL and SCS. Moreover, 3-h SCS did not increase the expression levels of TPH and DβH in the spinal dorsal horn., Conclusions: The serotonergic and noradrenergic pathways of the DAS are involved in the antinociceptive effect of SCS, but activation of the DRN might primarily be responsible for this effect, and the LC may have a smaller contribution. SCS does not potentiate the synthetic enzymes of 5HT and norepinephrine in the neuropathic spinal cord.

In the present study, we tested the hypothesis that the potent and selective dopamine-β-hydroxylase (DβH) inhibitor nepicastat would have minimal effects on cardiovascular and pharmacokinetic parameters associated with cocaine administration and would reduce the positive subjective effects produced by cocaine. We conducted a double-blind, placebo-controlled, inpatient study of oral nepicastat (0, 80 and 160mg) concurrent with intravenous (IV) cocaine (0, 10, 20 and 40mg) in non-treatment seeking participants who metcriteria for cocaine use disorder. Safety analyses revealed that nepicastat was well-tolerated and there were no differences in adverse events observed after nepicastat plus cocaine vs. cocaine alone. In addition, the pharmacokinetic properties of cocaine administration were not altered by nepicastat treatment. Cocaine-induced cardiovascular and subjective effects were evaluated for completers in the cohort randomized to nepicastat (n=13) using a within-subjects statistical analysis strategy. Specifically, the cardiovascular and subjective effects of cocaine were assessed in the presence of placebo (0mg), 80mg of nepicastat or 160mg of nepicastat on study Days 4, 8 and 12, respectively. Analyses revealed a main effect of nepicastat to reduce several cocaine-induced positive subjective effects. Taken together, these data indicate that nepicastat is safe when co-administered with cocaine and may suppress its positive subjective effects, and may be viable as a pharmacotherapy for treatment of cocaine use disorder., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The interaction of etamicastat, a novel peripherally acting dopamine-β-hydroxylase (DBH) inhibitor, with the enzyme was studied using a classical kinetic approach and the pharmacodynamics effect of the compound upon administration to rats was also evaluated. SK-N-SH cell homogenates convert tyramine into octopamine with a Km value of 9 mM, and a Vmax of 1747 nmol/mg protein/h. The K(m) value for ascorbate was 3 mM. The inhibition of DBH by etamicastat and nepicastat, a known centrally acting DBH inhibitor, with IC50 values of 107 and 40 nM, respectively, was fully reversed by dilution. Non-linear fitting of the velocities, determined at various concentrations of substrate (tyramine) and co-substrate (ascorbic acid), and of etamicastat and nepicastat, indicated that the inhibition of DBH by both compounds follows a mixed-model inhibition mechanism, approaching competitive behavior with regards to the substrate tyramine, with K(i) values of 34 and 11 nM, respectively. Relatively to ascorbate, both compounds followed a mixed-model inhibition mechanism, approaching uncompetitive behavior. Oral administration of both compounds (at 30 mg/kg) inhibited adrenal DBH activity over time and significantly decreased noradrenaline levels in the heart. Nepicastat also decreased noradrenaline levels in the parietal cortex, but not etamicastat. Both compounds significantly decreased systolic and diastolic blood pressure in spontaneously hypertensive rats. In conclusion, etamicastat and nepicastat behave as multisubstrate DBH inhibitors, binding reversibly and preferentially to the reduced form of the enzyme, and simultaneously at the substrate and oxygen binding sites. Etamicastat, in contrast to nepicastat, offers the advantage of peripheral selectivity without central effects., (Copyright © 2015 Elsevier B.V. All rights reserved.)

As development proceeds from the embryonic to fetal stages, cardiac energy demands increase substantially, and oxidative phosphorylation of ADP to ATP in mitochondria becomes vital. Relatively little, however, is known about the signaling mechanisms regulating the transition from anaerobic to aerobic metabolism that occurs during the embryonic period. The main objective of this study was to test the hypothesis that adrenergic hormones provide critical stimulation of energy metabolism during embryonic/fetal development. We examined ATP and ADP concentrations in mouse embryos lacking adrenergic hormones due to targeted disruption of the essential dopamine β-hydroxylase (Dbh) gene. Embryonic ATP concentrations decreased dramatically, whereas ADP concentrations rose such that the ATP/ADP ratio in the adrenergic-deficient group was nearly 50-fold less than that found in littermate controls by embryonic day 11.5. We also found that cardiac extracellular acidification and oxygen consumption rates were significantly decreased, and mitochondria were significantly larger and more branched in adrenergic-deficient hearts. Notably, however, the mitochondria were intact with well-formed cristae, and there was no significant difference observed in mitochondrial membrane potential. Maternal administration of the adrenergic receptor agonists isoproterenol or l-phenylephrine significantly ameliorated the decreases in ATP observed in Dbh-/- embryos, suggesting that α- and β-adrenergic receptors were effective modulators of ATP concentrations in mouse embryos in vivo. These data demonstrate that adrenergic hormones stimulate cardiac energy metabolism during a critical period of embryonic development., (Copyright © 2015 the American Physiological Society.)

Critical periods are developmental windows during which the stimuli an animal encounters can reshape response properties in the affected system to a profound degree. Despite this window's importance, the neural mechanisms that regulate it are not completely understood. Pioneering studies in visual cortex initially indicated that norepinephrine (NE) permits ocular dominance column plasticity during the critical period, but later research has suggested otherwise. More recent work implicating NE in experience-dependent plasticity in the adult auditory cortex led us to re-examine the role of NE in critical period plasticity. Here, we exposed dopamine β-hydroxylase knock-out (Dbh(-/-)) mice, which lack NE completely from birth, to a biased acoustic environment during the auditory cortical critical period. This manipulation led to a redistribution of best frequencies (BFs) across auditory cortex in our control mice, consistent with prior work. By contrast, Dbh(-/-) mice failed to exhibit the expected redistribution of BFs, even though NE-deficient and NE-competent mice showed comparable auditory cortical organization when reared in a quiet colony environment. These data suggest that while intrinsic tonotopic patterning of auditory cortical circuitry occurs independently from NE, NE is required for critical period plasticity in auditory cortex., (Copyright © 2015 the authors 0270-6474/15/352432-06$15.00/0.)

Hypoxia-induced chemosensory activity in the carotid body (CB) may be enhanced by the sympathetic regulation of vascular tone in the CB. In the present study, we recorded cervical sympathetic nerve activity in rats exposed to hypoxia, and examined noradrenaline (NA)- and serotonin (5-HT)-induced intracellular Ca(2+) ([Ca(2+)]i) responses in smooth muscle cells and pericytes in isolated blood vessels from the CB. Multifiber electrical activity recorded from the cervical sympathetic trunk was increased during the inhalation of hypoxic gas. NA induced [Ca(2+)]i increases in smooth muscle cells in arteriole specimens, whereas 5-HT did not cause any [Ca(2+)]i responses. However, NA did not induce [Ca(2+)]i increases in pericytes in capillaries, whereas 5-HT did and this response was inhibited by the 5-HT2 receptor antagonist, ketanserin. In conclusion, cervical sympathetic nerves enhanced by hypoxia may reduce blood flow in the CB in order to increase chemosensitivity. Thus, hypoxic chemosensitivity in the CB may involve a positive feedback mechanism via sympathetic nerves., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Aims: We investigated the norepinephrine pathway changes from the locus coeruleus (LC) to the preoptic area of the hypothalamus (POAH) in the brain of ovariectomized rats under low estrogen levels and explored the therapeutic effects of estradiol valerate (E2) and Remifemin (ICR) on these changes., Methods: 40 female Sprague-Dawley rats were randomly divided into the following groups: surgery with vehicle (SHAM), ovariectomy surgery with vehicle (OVX), ovariectomy with E2 treatment (OVX + E2), and ovariectomy with Remifemin (OVX + ICR). After 4 weeks of treatment, we observed the changes by immunohistochemistry., Results: (1) The average optical density of DBH-ir fibers and the number of α1-adrenoreceptor- and estrogen receptor (ER)α-positive neurons in the main nuclei of POAH were all reduced in OVX rats compared with the SHAM group. The above changes were normalized in all nuclei of the POAH in the E2 group, while they were normalized in some nuclei in the ICR group. Coexpression of ERα and α1-adrenoreceptor was observed in the POAH. (2) The number of DBH- and ERα-positive neurons in the LC decreased in the OVX group compared with the SHAM group and increased after treatment with E2 and ICR. Coexpression of ERα and DBH was observed in the LC., Conclusion: Low estrogen (OVX) altered norepinephrine synthesis in the LC, the projection of norepinephrine fibers and α1-adrenoreceptor expression in the POAH. Both E2 and ICR normalized the norepinephrine pathway, but E2 achieved greater effects than ICR. ICR had different effects in different nuclei in the POAH and its therapeutic effect was better in the LC., (© 2015 S. Karger AG, Basel.)

1. This study explores the impact of permeability and P-glycoprotein (P-gp) efflux, upon brain exposure to etamicastat, a new dopamine-β-hydroxylase (DBH) inhibitor and consequently brain levels of catecholamines. 2. Brain exposure to etamicastat (10 mg/kg), following intravenous administration to mice, was residual and upon oral administration of the same dose no compound was detected, concurring with the absence of effects upon brain catecholamines. The intravenous co-administration of elacridar (1.0 mg/kg), a known P-gp/BCRP dual modulator, significantly increased brain etamicastat exposure, but the levels attained were very low when compared to those of nepicastat, a centrally active DBH inhibitor. 3. In vitro permeability studies from apical-to-basal direction conducted in Caco-2 cells and MDCK-II cells showed that etamicastat apparent permeability was 1.2 × 10(-5) and 1.1 × 10(-6 )cm/s, respectively, 5- and 50-fold lower as compared to nepicastat. The secretory efflux ratio in MDCK-II cells overexpressing human P-gp showed an efflux ratio greater than 2, for both compounds, which was significantly decreased by elacridar. Despite its lower bioavailability and higher clearance, as compared to nepicastat, etamicastat showed preferential distribution to peripheral tissues and high plasma free fraction (15.5%), which may explain its effects upon peripheral DBH and catecholamine levels. 4. Though P-gp-mediated efflux may contribute to the limited brain penetration of etamicastat, the low permeability along with the pharmacokinetic properties of etamicastat may be perceived as the main contributors for its peripheral selectivity, which is advantageous for a cardiovascular drug candidate.

Background/aim: Increased dopamine production may be a feature of head and neck paraganglioma (HNPGL). 18F-fluorodihydroxyphenylalanine positron emission tomography scintigraphy has a high sensitivity for detecting HNPGLs. These observations strongly suggest that HNPGLs have the capacity for L-3,4-dihydroxyphenylalanine uptake and conversion towards dopamine. Therefore, our aim was to demonstrate the presence of catecholamine-synthesizing enzymes, i.e. tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC) and dopamine β-hydroxylase (DBH) in HNPGL tissue., Methods: A single-center study was performed among patients who underwent surgery for HNPGL at a single university referral center between 1994 and 2012. HNPGL tissue was immunohistochemically stained for TH, AADC and DBH. Data on paraganglioma-associated germline mutations, preoperative biochemical phenotype and imaging studies were retrieved. Catecholamine excess was defined as preoperative plasma and/or urinary levels of metanephrine, normetanephrine or 3-methoxytyramine above the upper reference limit., Results: Nineteen HNPGLs from 18 patients were evaluated. All tumor tissues (100%) stained positive for AADC, 6 (32%) for TH and 2 (11%) for DBH. Of 3 HNPGLs staining positive for DBH, 2 were also positive for AADC and TH. Catecholamine excess was only present in 1 patient (5%). The HNPGLs of this single patient only showed positive staining for AADC., Conclusions: Catecholamine-synthesizing enzymes, in particular AADC, are expressed in the majority of HNPGL tissues.

Amygdalar norepinephrine (NE) plays a key role in regulating neural responses to emotionally arousing stimuli and is involved in memory consolidation of emotionally charged events. Corticotropin-releasing factor (CRF) and dynorphin (DYN), two neuropeptides that mediate the physiological and behavioral responses to stress, are abundant in the central nucleus of the amygdala (CeA), and directly innervate brainstem noradrenergic locus coeruleus (LC) neurons. Whether the CRF- and DYN-containing amygdalar neurons receive direct noradrenergic innervation has not yet been elucidated. The present study sought to define cellular substrates underlying noradrenergic modulation of CRF- and DYN-containing neurons in the CeA using immunohistochemistry and electron microscopy. Ultrastructural analysis revealed that NE-labeled axon terminals form synapses with CRF- and DYN-containing neurons in the CeA. Semi-quantitative analysis showed that approximately 31 % of NET-labeled axon terminals targeted CeA neurons that co-expressed DYN and CRF. As a major source of CRF innervation to the LC, it is also not known whether CRF-containing CeA neurons are directly targeted by noradrenergic afferents. To test this, retrograde tract tracing using FluoroGold from the LC was combined with immunocytochemical detection of CRF and NET in the CeA. Our results revealed a population of LC-projecting CRF-containing CeA neurons that are directly innervated by NE afferents. Analysis showed that approximately 34 % of NET-labeled axon terminals targeted LC-projecting CeA neurons that contain CRF. Taken together, these results indicate significant interactions between NE, CRF and DYN in this critical limbic region and reveal direct synaptic interactions of NE with amygdalar CRF that influence the LC-NE arousal system.

Rationale: Dopamine β-hydroxylase (DBH) catalyzes the conversion of dopamine to norepinephrine in the central nervous system and peripherally. DBH variants are associated with large changes in circulating DBH and implicated in multiple disorders; yet causal relationships and tissue-specific effects remain unresolved., Objective: To characterize regulatory variants in DBH, effect on mRNA expression, and role in modulating sympathetic tone and disease risk., Methods and Results: Analysis of DBH mRNA in human tissues confirmed high expression in the locus coeruleus and adrenal gland, but also in sympathetically innervated organs (liver>lung>heart). Allele-specific mRNA assays revealed pronounced allelic expression differences in the liver (2- to 11-fold) attributable to promoter rs1611115 and exon 2 rs1108580, but only small differences in locus coeruleus and adrenals. These alleles were also associated with significantly reduced mRNA expression in liver and lung. Although DBH protein is expressed in other sympathetically innervated organs, mRNA levels were too low for analysis. In mice, hepatic Dbh mRNA levels correlated with cardiovascular risk phenotypes. The minor alleles of rs1611115 and rs1108580 were associated with sympathetic phenotypes, including angina pectoris. Testing combined effects of these variants suggested protection against myocardial infarction in 3 separate clinical cohorts., Conclusions: We demonstrate profound effects of DBH variants on expression in 2 sympathetically innervated organs, liver and lung, but not in adrenals and brain. Preliminary results demonstrate an association of these variants with clinical phenotypes responsive to peripheral sympathetic tone. We hypothesize that in addition to endocrine effects via circulating DBH and norepinephrine, the variants act in sympathetically innervated target organs., (© 2014 American Heart Association, Inc.)

The dramatic response of most motor and some nonmotor symptoms to dopaminergic therapies has contributed to maintaining the long-established identity of Parkinson's disease (PD) as primarily a nigrostriatal dopamine (DA) deficiency syndrome. However, DA neurotransmission may be neither the first nor the major neurotransmitter casualty in the neurodegenerative sequence of PD. Growing evidence supports earlier norepinephrine (NE) deficiency resulting from selective degeneration of neurons of the locus coeruleus and sympathetic ganglia. Dopaminergic replacement therapy therefore would seem to neglect some of the motor, behavioral, cognitive, and autonomic impairments that are directly or indirectly associated with the marked deficiency of NE in the brain and elsewhere. Therapeutic strategies to enhance NE neurotransmission have undergone only limited pharmacological testing. Currently, these approaches include selective NE reuptake inhibition, presynaptic α2 -adrenergic receptor blockade, and an NE prodrug, the artificial amino acid L-threo-3,4-dihydroxyphenylserine. In addition to reducing the consequences of deficient noradrenergic signaling, enhancement strate gies have the potential for augmenting the effects of dopaminergic therapies in PD. Furthermore, early recognition of the various clinical manifestations associated with NE deficiency, which may precede development of motor symptoms, could provide a window of opportunity for neuroprotective interventions., (© 2014 International Parkinson and Movement Disorder Society.)

Mottled-dappled (Mo-dp) is a mouse model of Menkes disease caused by a large, previously uncharacterized deletion in the 5' region of Atp7a, the mouse ortholog of ATP7A. Affected mutants die in utero at embryonic day 17, and show bending and thickening of the ribs and distortion of the pectoral and pelvic girdles and limbs. To characterize this allele, we designed a custom 4x180K microarray on the mouse X chromosome and performed comparative genomic hybridization using extracted DNA from normal and carrier Mo-dp females, and identified an approximately 9 kb deletion. We used PCR to fine-map the breakpoints and amplify a junction fragment of 630 bp. Sequencing of the junction fragment disclosed the exact breakpoint locations and that the Mo-dp deletion is precisely 8990 bp, including approximately 2 kb in the promoter region of Atp7a. Western blot analysis of Mo-dp heterozygous brains showed diminished amounts of Atp7a protein, consistent with reduced expression due to the promoter region deletion on one allele. In heterozygous females, brain copper levels tended to be lower compared to wild type whereas neurochemical analyses revealed higher dihydroxyphenylacetic acid:dihydroxyphenylglycol (DOPAC:DHPG) and dopamine:norepinephrine (DA:NE) ratios compared to normal (P=0.002 and 0.029, respectively), consistent with partial deficiency of dopamine-beta-hydroxylase, a copper-dependent enzyme. Heterozygous females showed no significant differences in body weight compared to wild type females. Our results delineate the molecular details of the Mo-dp mutation for the first time and define novel biochemical findings in heterozygous female carriers of this allele., (Published by Elsevier Inc.)

Background: The medulla is capable of controlling and modulating ingestive behavior and gastrointestinal function. These two functions, which are critical to maintaining homeostasis, are governed by an interconnected group of nuclei dispersed throughout the medulla. As such, in vitro experiments to study the neurophysiologic details of these connections have been limited by spatial constraints of conventional slice preparations., New Method: This study demonstrates a novel method of sectioning the medulla so that sensory, integrative, and motor nuclei that innervate the gastrointestinal tract and the oral cavity remain intact., Results: Immunohistochemical staining against choline-acetyl-transferase and dopamine-β-hydroxylase demonstrated that within a 450 μm block of tissue we are able to capture sensory, integrative and motor nuclei that are critical to oromotor and gastrointestinal function. Within slice tracing shows that axonal projections from the NST to the reticular formation and from the reticular formation to the hypoglossal motor nucleus (mXII) persist. Live-cell calcium imaging of the slice demonstrates that stimulation of either the rostral or caudal NST activates neurons throughout the NST, as well as the reticular formation and mXII., Comparison With Existing Methods: This new method of sectioning captures a majority of the nuclei that are active when ingesting a meal. Tradition planes of section, i.e. coronal, horizontal or sagittal, contain only a limited portion of the substrate., Conclusions: Our results demonstrate that both anatomical and physiologic connections of oral and visceral sensory nuclei that project to integrative and motor nuclei remain intact with this new plane of section., (Published by Elsevier B.V.)

Idiopathic Parkinson's disease (PD) is a late-onset, chronic, and progressive motor dysfunction attributable to loss of nigrostriatal dopamine neurons. Patients with PD experience significant gastrointestinal (GI) issues, including gastroparesis. We aimed to evaluate whether 6-hydroxy-dopamine (6-OHDA)-induced degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) induces gastric dysmotility via dysfunctions of the brain-gut axis. 6-OHDA microinjection into the SNpc induced a >90% decrease in tyrosine hydroxylase-immunoreactivity (IR) on the injection site. The [13C]-octanoic acid breath test showed a delayed gastric emptying 4 wk after the 6-OHDA treatment. In control rats, microinjection of the indirect sympathomimetic, tyramine, in the dorsal vagal complex (DVC) decreased gastric tone and motility; this inhibition was prevented by the fourth ventricular application of either a combination of α1- and α2- or a combination of D1 and D2 receptor antagonists. Conversely, in 6-OHDA-treated rats, whereas DVC microinjection of tyramine had reduced effects on gastric tone or motility, DVC microinjection of thyrotropin-releasing hormone induced a similar increase in motility as in control rats. In 6-OHDA-treated rats, there was a decreased expression of choline acetyl transferase (ChAT)-IR and neuronal nitric oxide synthase (NOS)-IR in DVC neurons but an increase in dopamine-β-hydroxylase-IR in the A2 area. Within the myenteric plexus of the esophagus, stomach, and duodenum, there were no changes in the total number of neurons; however, the percentage of NOS-IR neurons increased, whereas that of ChAT-IR decreased. Our data suggest that the delayed gastric emptying in a 6-OHDA rat model of PD may be caused by neurochemical and neurophysiological alterations in the brain-gut axis., (Copyright © 2014 the American Physiological Society.)

Despite the importance of sympathetic nervous system in pathophysiological mechanisms of cardiac heart failure and essential hypertension, therapy specifically targeting the sympathetic nervous system is currently underutilized. Etamicastat is a novel dopamine-ß-hydroxylase (DBH) inhibitor that is oxidized into BIA 5-965 and deaminated followed by oxidation to BIA 5-998, which represents 13% of total etamicastat and quantified metabolites. However, the primary metabolic pathway of etamicastat in rats was found to be the N-acetylation (BIA 5-961), which represents 44% of total etamicastat and quantified metabolites. Trace amounts of BIA 5-961 de-sulfated and S-glucuronide were also detected. All the main metabolites of etamicastat inhibited DBH with IC50 values of 306 (228, 409), 629 (534, 741), 427 (350, 522) nM for BIA 5-965, BIA 5-998 and BIA 5-961, respectively. However, only etamicastat (IC50 of 107 (94; 121) nM) was able to reduce catecholamine levels in sympathetic nervous system innervated peripheral tissues, without effect upon brain catecholamines. Quantitative whole body autoradiography revealed a limited transfer of etamicastat related radioactivity to brain tissues and the mean recovery of radioactivity was ~90% of the administered radioactive dose, eliminated primarily via renal excretion over 5 days. The absolute oral bioavailability of etamicastat was 64% of the administered dose. In conclusion, etamicastat is a peripheral selective DBH inhibitor mainly N-acetylated in the aminoethyl moiety and excreted in urine. Etamicastat main metabolites inhibit DBH, but only etamicastat demonstrated unequivocal pharmacological effects as a DBH inhibitor with impact upon the activity of the sympathetic nervous system under in vivo conditions., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Satiety signals arising from the gastrointestinal (GI) tract and related digestive organs during food ingestion and digestion are conveyed by vagal sensory afferents to the hindbrain nucleus of the solitary tract (NST). Two intermingled but chemically distinct NST neuronal populations have been implicated in meal size control: noradrenergic (NA) neurons that comprise the A2 cell group, and glucagon-like peptide-1 (GLP-1)-positive neurons. Previous results indicate that A2 neurons are activated in a meal size-dependent manner in rats that have been acclimated/entrained to a feeding schedule in order to increase meal size, whereas feeding under the same conditions does not activate GLP-1 neurons. The present study was designed to test the hypothesis that both A2 and GLP-1 neuronal populations are recruited in non-entrained rats after voluntary first-time intake of an unrestricted, satiating volume of liquid Ensure. DBH-positive A2 neurons within the caudal visceral NST were progressively recruited to express cFos in rats that consumed progressively larger volumes of Ensure. Among these DBH-positive neurons, the prolactin-releasing peptide (PrRP)-positive subset was more sensitive to feeding-induced activation than the PrRP-negative subset. Notably, significant activation of GLP-1-positive neurons occurred only in rats that consumed the largest volumes of Ensure, corresponding to nearly 5% of their BW. We interpret these results as evidence that progressive recruitment of NA neurons within the caudal NST, especially the most caudally-situated PrRP-positive subset, effectively "tracks" the magnitude of GI satiety signals and other meal-related sensory feedback. Conversely, GLP-1 neurons may only be recruited in response to the homeostatic challenge of consuming a very large, unanticipated meal., (Copyright © 2014 Elsevier Inc. All rights reserved.)