Introduction Norepinephrine-containing neurons clustered within the locus coeruleus (LC) provide most of the norepinephrine present within the central nervous system. These cells have tonic pacemaker activity and this activity is regulated by a variety of neurotransmitter inputs. The focus of this review is primarily on classical, non-neuropeptide, neurotransmitter input to the LC and the reciprocal projections of noradrenergic neurons to those classical neurotransmitter systems. Input to the LC from serotonin-, dopamine-, γ-aminobutyric acid (GABA)-, glutamate-, and acetylcholine-containing neurons are described. In addition, input from the neuropeptide, substance P, receives attention because of the interest in this neuropeptide in psychiatric disease. Special attention is given to reciprocal projections from the LC to the monoamine neurotransmitters dopamine and serotonin. See Chapter 1 for a detailed description of the anatomy of the LC. Noradrenergic circuitry: input to the LC Early tract-tracing studies suggested that the LC received widespread input from many sites in the brain. A combination of techniques, however, including discrete injections of a more sensitive tract-tracing compound, anterograde labeling studies, and single-pulse stimulation studies forced a reconsideration of brain areas with direct input to the LC. The major afferents to the LC are rostral medullary in origin, with cell bodies located in the nucleus paragigantocellularis (PGi, using excitatory amino acid neurotransmitters) and nucleus prepositus hypoglossi (PrH, using GABA). However, dense projections from many brain regions terminate in the pericoerulear area, an area heavily invested with dendrites from LC neurons, and in the PGi and PrH.

Glutamate is a major stress-sensitive excitatory input to the noradrenergic locus coeruleus (LC) and pathology of both glutamatergic and noradrenergic systems is strongly implicated in major depressive disorder (MDD). Glutamatergic innervation of the LC originates in the frontal cortex and in local brainstem nuclei. Stress increases the release of glutamate in the LC which results in activation of noradrenergic LC neurons. Previous research has demonstrated abnormalities in the levels of glutamate receptor proteins and gene expressions in the postmortem LC of subjects with MDD as compared to normal control subjects providing further evidence for disruption of glutamate-norepinephrine communication in MDD. A principal mechanism for the termination of the action of neuronally-released glutamate is by reuptake via excitatory amino acid transporters (EAAT) located on glia, cells which express both EAAT1 and EAAT2. Here, the potential contribution of glia to glutamate pathology in the LC in MDD was studied by measuring gene expression of EAAT1 and EAAT2 in astrocytes captured from the immediate region of the postmortem LC from 6 pairs of subjects with MDD and psychiatrically normal control subjects. MDD and control subjects were carefully matched for age, RIN value (RNA integrity number), gender, cigarette smoking or non-smoking, and brain tissue pH. Laser capture microdissection was used to capture astrocytes from tissue sections labeled with a modified rapid GFAP-immunostaining, and gene expression levels were analyzed by quantitative PCR. Three reference genes were used as internal controls and the quality of the capture of astrocytes was confirmed by examining the gene expression of cell-type specific markers, including markers for noradrenergic neurons and oligodendrocytes. The gene expression of EAATs was significantly lower (EAAT1, -60%; p<0.001; EAAT2, -25%, P<0.01) in LC astrocytes from MDD subjects as compared to normal control subjects. To determine the regional specificity of these findings, gene expression levels of EAAT1 and EAAT2 were measured in homogenates of both gray and white matter from Brodmann’s area 10 of the cortex. No differences in EAAT gene expression in these cortical tissues were observed comparing MDD to control subjects. These findings indicate that disrupted glial transport of glutamate may contribute to altered glutamatergic transmission in the noradrenergic LC in MDD.

miRNAs 29b and 181a down-regulate expression of the norepinephrine transporter in PC12 cells. M.X. Deng, G. A. Ordway and M.-Y. Zhu. Dept. of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University, Johnson City, TN, USA MicroRNAs are short non-coding RNAs that provide global regulation of gene expression at the post-transcriptional level. Such regulation has been found to play a role in stress-induced epigenetic responses in the brain. The noradrenaline transporter (NET) is a noradrenergic marker and regulates neurotransmitter signaling by rapidly clearing released norepinephrine from synapses. Our previous studies demonstrated that rat NET mRNA and protein levels are regulated by chronic stress and by administration of corticosterone. Whether miRNAs are intermediaries in the regulation of NET expression remains to be elucidated. The present study was undertaken to determine possible regulatory effects of miRNAs on NET expression in PC12 cells, a cell model for noradrenergic neurons. Using computational target prediction, we identified several miRNAs potentially related to regulation of NET expression. Mimics of these miRNAs were transfected into PC12 cells. NET protein expression was assayed by Western blotting 48 hours after transfection. miR29b- and miR181a-transfected cells showed significantly reduced NET protein levels. To identify the exact target loci, the 3’-UTR of NET mRNA was amplified by PCR from PC12 genomic DNA and cloned downstream of the red firefly gene of the pmirGlo vector. The NET 3’-UTR-bearing pmirGlo and miR29b or miR181a were co-transfected into PC12 cells and luciferase signals were measured 48 hours after transfection. Consistent with Western blots, co-transfection of these miRNAs with rat NET3’-UTR-containing plasmids resulted in reduced levels of luciferase activity in PC12 cells. We conclude that miR29b and miR181a can function as negative regulators of NET translation in vitro. Further studies to determine whether these miRNAs contribute to the regulation of NET expression induced by antidepressants are under way.

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Objective: To evaluate pressor sensitivity to catecholamines in dogs after induction of iatrogenic hyperadrenocorticism (I-HAC) by serial arterial blood pressure measurements during infusions of increasing dose rates of norepinephrine. Animals: Eight dogs with I-HAC induced by administration of oral hydrocortisone at a mean dose of 3.3 mg/kg PO TID for 42-49 days and 8 control dogs which received empty gelatin capsules PO TID for 42-49 days. Procedure: Systolic, diastolic, mean blood pressure and heart rate measurements were recorded after sequential administration of increasing dose rates of norepinephrine (0.1, 0.125, 0.2, 0.3, 0.4, 0.6 and 0.8 mg/kg/min) for 10 minutes. The changes in systolic, diastolic, mean blood pressure and heart rate were compared between control dogs and dogs with I-HAC. Results: Dogs in the I-HAC group had a more pronounced pressor response to norepinephrine infusions than control dogs. The infusions were not completed in 7 of the 8 dogs in the I-HAC group versus 3 dogs in the control group due to severe elevations in systolic blood pressure. The mean change in systolic blood pressure was consistently higher in dogs in the I-HAC group. The difference was statistically significant at the 0.2 mg/kg/min norepinephrine dose rate. The mean change in heart rate was consistently lower in the I-HAC group, a difference that was significant at the 0.2 mg/kg/min norepinephrine dose rate. Conclusions and clinical relevance: Increased pressor sensitivity or decreased baroreceptor response to norepinephrine was seen in dogs with I-HAC suggesting that this mechanism is involved in the development of hypertension in canine hyperadrenocorticism. / Master of Science

A better understanding of the neural mechanisms associated with Attention Deficit Hyperactivity Disorder (ADHD) and related cognitive deficits can potentially clarify the neural circuits involved in ADHD symptoms, help define neurobiologically informed subtypes and aid in developing more refined treatments. Two neurotransmitter (NT) systems have been implicated in ADHD: Dopamine (DA), and Norepinephrine (NE), and the primary cognitive deficits associated with ADHD are in working memory, response inhibition, reaction time variability, and reward processing. Frank et al. (2007a) proposes, based on computational models, that DA is associated with deficits in reward-based learning and updating of working memory, while NE is associated with deficits in response inhibition and greater response variability. Therefore, it might be possible to learn more about the NT systems' specific roles in ADHD by studying the associated cognitive deficits. The primary goal of this study was to assess performance in adolescents with and without ADHD on a number of cognitive tasks. We expected that the Attention Deficit Hyperactivity Disorder - Inattentive Subtype (ADHD-I) group would perform the worst on NE tasks and that the Attention Deficit Hyperactivity Disorder - Combined Subtype (ADHD-C) group would perform the worst on DA tasks, and that both groups would perform worse than controls on all tasks. Instead, we found that the ADHD-I group performed the most poorly on updating of working memory, while the ADHD-C group performed the best on this variable. However, the ADHD-C group performed worst on overall working memory. Dimensional analyses revealed that hyperactivity/impulsivity is positively correlated with updating of working memory, while inattention is negatively correlated with updating of working memory. In addition, hyperactivity/impulsivity was positively correlated with reaction time variability. In conclusion, it is likely that the roles of these NT systems are not as mutually exclusive as initially expected. It is also possible that our ADHD group was performing more like control groups in other studies, which might be due to a more 'pure' ADHD sample with less comorbid Oppositional Defiant Disorder (ODD) and Conduct disorder (CD), or could be due to a less symptomatic ADHD group.

Effects of norepinephrine (NE) on macrophage cytokine release are complex because the cells have both α2 and β2 adrenergic receptors, which mediate opposing actions. Furthermore, β2-adrenergic agonists are reported to have both stimulatory and inhibitory effects on interleukin-6 (IL-6). This study was designed to clarify the autocrine role of macrophage-derived NE on IL-6 production in activated peritoneal macrophages. Effects of NE on IL-6 production in the RAW264.7 macrophage cell-line also were investigated. Treatment of activated peritoneal macrophages with endotoxin, the α2-adrenergic antagonists yohimbine or RS79948 revealed that the α2-adrenergic receptor mediates a stimulatory autocrine action of catecholamines on IL-6 production. When peritoneal macrophages were treated with the β2 antagonist ICI-118,551 (ICI), there was both inhibition and stimulation of IL-6. Treatment of RAW264.7 macrophages with high and low concentrations of NE and various concentrations of ICI provided evidence that the concentration of NE determines whether the β2-adrenergic receptor mediates stimulation or inhibition of IL-6 production.

The high affinity, selective dopamine transporter ligand GBR 12909 has served as a template for the design of two novel classes of dopamine transporter ligands. A series of 3-[2- (diarylmethoxyethyidenyl)]-N-substituted tropane derivatives were synthesized and the binding affinities of these compounds were determined at the dopamine (DAT), serotonin (SERT) and norepinephrine (NET) transporters in rat brain tissue preparations. The tropane derivatives were found to exhibit more potent affinity and selectivity for DAT than GBR 12909. From the SAR of the tropane analogues and GBR 12909, a novel series of 3-[2-(diarylmethoxyethylidenyl)]-Nsubstituted azetidine derivatives has been developed.

In an effort to search for potential therapeutic agents for cocaine addiction, a novel class of compounds was synthesized and evaluated for in vitro dopamine and serotonin transporter affinities. These unique 3ƒÀ-aryl-3ƒ¿-arylmethoxytropane analogues incorporated the structure of dopamine selective 2-substituted-3-phenyltropanes and the design of serotonin selective meperidine derivatives. In general, the 3ƒÀ-aryl-3ƒ¿-arylmethoxytropane analogues exhibited greater potency for the serotonin transporter than the dopamine transporter. The most potent compounds of this series were 3ƒÀ-phenyl-3ƒ¿.(3, 4-dichlorophenyl)methoxy-8.azabicyclo [3.2.1]nortropane (Ki = 0.06 nM) and 3ƒÀ-(4Œ-chlorophenyl)-3ƒ¿.(4-chlorophenyl)methoxy-8. azabicyclo[3.2.1]nortropane (Ki = 0.09 nM) at the serotonin transporter and their binding affinities were equipotent with paroxetine and fluoxetine (Prozac). A series of 8-azabicyclo[3.2.1]oct-2-ene derivatives were synthesized from 3-tropinone based on the structure of triple re-uptake inhibitor, DOV 216, 303. The compounds were designed as potential triple re-uptake inhibitors which could exhibit equipotent affinities at the monoamine transporters for dopamine, serotonin and norepinephrine. A short and efficient synthetic methodology was developed for the synthesis of unique compounds which could exhibit potency for both the dopamine and serotonin transporters. The 3ƒÀ-aryl-3ƒ¿-(4Œ, 4-disubstituteddiphenylmethoxy)tropane analogues were designed as hybrid structures of the dopamine transporter selective benztropines and the serotonin transporter selective meperidine derivatives.

While selective serotonin (5-HT) reuptake inhibitors (SSRIs) are utilized as the first-line strategy in treating depression, new approaches are still desired. Using in vivo electrophysiological techniques, the effects of co-administration of bupropion with the SSRI escitalopram on the firing rate of dorsal raphe 5-HT and locus coeruleus norepinephrine (NE) neurons were investigated. Escitalopram significantly decreased the firing of 5-HT and NE neurons at day 2. The 5-HT firing rate, unlike that of NE, recovered after the 14-day escitalopram regimen. Bupropion did not increase 5-HT firing but decreased that of NE after 2 days. Following 14-day bupropion, 5-HT firing was markedly enhanced, and NE firing was back to baseline. Co-administration of escitalopram and bupropion doubled 5-HT firing after 2 and 14 days, whereas NE neurons were inhibited after 2, but partially recovered after 14 days. Although sustained bupropion administration did not alter the sensitivity of 5-HT1A receptors in hippocampus, the tonic activation of postsynaptic 5-HT1A receptors was enhanced in 14-day bupropion-treated rats to a greater extent than in the 2-day and control rats. The function of terminal 5-HT1B autoreceptors was not changed. The inhibitory action of α2-adrenergic receptors on 5-HT terminals was, however, diminished. The function of terminal α2-adrenergic autoreceptors was also attenuated in rats given bupropion for 14 days. Administration of the antidepressant trazodone suppressed the 5-HT firing at day 2, which recovered to baseline following 14 days. Prolonged trazodone-administration enhanced the tonic activation of postsynaptic 5-HT1A receptors in hippocampus, and decreased the function of terminal 5-HT1B autoreceptors. Finally, a novel psychotropic agent asenapine showed potent antagonistic activity at 5-HT2A, D2, and α2-adrenoceptors. Asenapine, however, acted as a partial agonist at 5-HT1A receptors in dorsal raphe and hippocampus. Overall, the therapeutic effects of various antidepressants may be, at least in part, due to the enhancement of 5-HT and/or NE neurotransmission.