Norepinephrine Analogue Research Articles

Cardiac Sympathetic Innervation Imaging with PET Radiotracers.

The present article reviews the pathophysiology of cardiac sympathetic denervation, the principles of positron emission tomography (PET) imaging of the sympathetic innervation of the heart and its potential clinical role, based on current and expected future evidence. Imaging of cardiac sympathetic denervation can be performed with radiolabeled noradrenaline analogues, e.g., 11C-hydroxyephedrine. A greater burden of sympathetic denervation carries prognostic significance, e.g., in patients with ischemic cardiomyopathy and a left ventricular ejection fraction ≤ 35%, who are more likely to experience sudden cardiac death. Abnormalities of sympathetic cardiac innervation have been demonstrated in hypertrophic, dilated, and arrhythmic right ventricular cardiomyopathies, and may be helpful in better phenotyping patients who will benefit from device therapy, e.g., cardiac resynchronization and implantable cardioverter-defibrillator implantation. The results of future trials, e.g., the Prediction of Arrhythmic Events with Positron Emission Tomography (PAREPET) II study, are awaited to inform on the role of PET cardiac sympathetic imaging in the selection of device therapy. PET cardiac sympathetic innervation imaging allows visualization and quantification of autonomic denervation secondary to various cardiac diseases, and has significant potential to influence clinical decision-making, e.g., the titration of pharmacotherapy and more directed selection of candidates for device implantation.

Renal 123I-MIBG Uptake before and after Live-Donor Kidney Transplantation.

Increased sympathetic activity is suggested to be part of the pathogenesis in several diseases. Methods to evaluate sympathetic activity and renal nervous denervation procedural success are lacking. Scintigraphy using the norepinephrine analog Iodine-123 Metaiodobenzylguanidine (123I-MIBG) might provide information on renal sympathetic nervous activity. Renal transplantation induces complete denervation of the kidney and as such represents an ideal model to evaluate the renal 123I-MIBG scintigraphy method. The aim of this study was to evaluate whether renal 123I-MIBG scintigraphy can detect changes in renal sympathetic nervous activity following renal transplantation. Renal 123I-MIBG scintigraphy was performed in eleven renal transplant recipients at 1, 3, and 6 months following transplantation and in their respective living donors prior to their kidney donation. Relative uptake as well as washout was obtained. In transplanted patients, the relative 4 h uptake of 123I-MIBG, as measured by the kidney/background ratio, was 2.7 (0.4) (mean (SD)), 2.7 (0.5), and 2.5 (0.4) at 1, 3, and 6 months post-transplantation, respectively, as compared with the 4.0 (0.4) value in the donor kidney before donor nephrectomy (p < 0.01). There was no significant change in washout-rate between pre-transplantation and any of the follow-up time points. Living donor kidney transplantation was at 6 months post transplantation, associated with an almost 40% reduction in the relative 4 h 123I-MIBG uptake of the kidney. Further studies will help to fully establish its implications as a marker of renal innervation or denervation.

Insight into an Oxidative DNA-Cleaving DNAzyme: Multiple Cofactors, the Catalytic Core Map and a Highly Efficient Variant.

SummaryAn oxidative DNA-cleaving DNAzyme (PL) employs a double-cofactor model “X/Cu2+” for catalysis. Herein, we verified that reduced nicotinamide adenine dinucleotide (NADH), flavin mononucleotide, cysteine, dithiothreitol, catechol, resorcinol, hydroquinone, phloroglucinol, o-phenylenediamine, 3,3′,5,5'-tetramethylbenzidine, and hydroxylamine acted as cofactor X. According to their structural similarities or fluorescence property, we further confirmed that reduced nicotinamide adenine dinucleotide phosphate (NADPH), 2-mercaptoethanol, dopamine, chlorogenic acid, resveratrol, and 5-carboxyfluorescein also functioned as cofactor X. Superoxide anions might be the commonality behind these cofactors. We subsequently determined the conservative change of individual nucleotides in the catalytic core under four different cofactor X. The nucleotides A4 and C5 are highly conserved, whereas the conservative levels of other nucleotides are dependent on the types of cofactor X. Moreover, we observed that the minor change in the PL's secondary structure affects electrophoretic mobility. Finally, we characterized a highly efficient variant T3G and converted its double-cofactor NADH/Cu2+ to sole-cofactor NADH.

Norepinephrine transporter-derived homing peptides enable rapid endocytosis of drug delivery nanovehicles into neuroblastoma cells

BackgroundCurrently, the diagnosis and treatment of neuroblastomas—the most frequent solid tumors in children—exploit the norepinephrine transporter (hNET) via radiolabeled norepinephrine analogs. We aim to develop a nanomedicine-based strategy towards precision therapy by targeting hNET cell-surface protein with hNET-derived homing peptides.ResultsThe peptides (seq. GASNGINAYL and SLWERLAYGI) were shown to bind high-resolution homology models of hNET in silico. In particular, one unique binding site has marked the sequence and structural similarities of both peptides, while most of the contribution to the interaction was attributed to the electrostatic energy of Asn and Arg (< − 228 kJ/mol). The peptides were comprehensively characterized by computational and spectroscopic methods showing ~ 21% β-sheets/aggregation for GASNGINAYL and ~ 27% α-helix for SLWERLAYGI. After decorating 12-nm ferritin-based nanovehicles with cysteinated peptides, both peptides exhibited high potential for use in actively targeted neuroblastoma nanotherapy with exceptional in vitro biocompatibility and stability, showing minor yet distinct influences of the peptides on the global expression profiles. Upon binding to hNET with fast binding kinetics, GASNGINAYLC peptides enabled rapid endocytosis of ferritins into neuroblastoma cells, leading to apoptosis due to increased selective cytotoxicity of transported payload ellipticine. Peptide-coated nanovehicles significantly showed higher levels of early apoptosis after 6 h than non-coated nanovehicles (11% and 7.3%, respectively). Furthermore, targeting with the GASNGINAYLC peptide led to significantly higher degree of late apoptosis compared to the SLWERLAYGIC peptide (9.3% and 4.4%, respectively). These findings were supported by increased formation of reactive oxygen species, down-regulation of survivin and Bcl-2 and up-regulated p53.ConclusionThis novel homing nanovehicle employing GASNGINAYLC peptide was shown to induce rapid endocytosis of ellipticine-loaded ferritins into neuroblastoma cells in selective fashion and with successful payload. Future homing peptide development via lead optimization and functional analysis can pave the way towards efficient peptide-based active delivery of nanomedicines to neuroblastoma cells.

Noradrenergic uptake in the liver on 123 I-mIBG imaging: Influence of heart failure and diabetes.

Imaging noradrenergic uptake in the liver with norepinephrine analog 123 I-meta-iodobenzylguanidine (mIBG) was explored in normal controls and patients with heart failure (HF). A total of 961 HF (343 with diabetes mellitus [DM]) and 94 control subjects underwent anterior planar mIBG images including upper abdomen at 15min (early) and 3h 50min (late) post-injection. Decay-corrected liver activity normalized to injected activity and body surface area (counts/pixel [cpp]/MBq/m2 ) was compared in three groups: HF with DM; HF without DM; and controls. Associations with plasma norepinephrine, liver function tests, and level of cardiac innervation were explored. In controls, liver mIBG activity decreased over time (early: 2.78 vs late: 2.43cpp/MBq/m2 , P<0.0001); in HF subjects, activity increased during this interval (HF without DM: 2.85 vs 2.93 [P=0.005]; HF with DM: 2.37 vs 2.43 [P=0.054]). Early liver activity was lower in HF with DM subjects than in the other groups (P<0.001); late liver activity was higher in HF without DM than in the other two groups (P<0.01). Subjects with elevated plasma norepinephrine (>520pg/mL) or ≥1 abnormal liver function test had lower early and late liver activity. In subjects with preserved cardiac mIBG uptake, HF subjects had higher and control subjects lower liver activity than comparable subjects with decreased cardiac innervation. In HF subjects, liver mIBG activity increased over time, reversing the normal washout pattern, suggesting a compensatory change in sympathetic nerve function. DM, abnormal liver function tests, and decreased cardiac innervation were associated with decreased liver mIBG uptake in HF.

Molecular imaging of norepinephrine transporter-expressing tumors: current status and future prospects.

The human norepinephrine transporter (hNET) is a transmembrane protein responsible for reuptake of norepinephrine in presynaptic sympathetic nerve terminals and adrenal chromaffin cells. Neural crest tumors, such as neuroblastoma, paraganglioma and pheochromocytoma often show high hNET expression. Molecular imaging of these tumors can be done using radiolabeled norepinephrine analogs that target hNET. Currently, the most commonly used radiopharmaceutical for hNET imaging is meta-[123I]iodobenzylguanidine ([123I]MIBG) and this has been the case since its development several decades ago. The γ-emitter, iodine-123 only allows for planar scintigraphy and single photon emission computed tomography imaging. These modalities typically have a poorer spatial resolution and lower sensitivity than positron emission tomography (PET). Additional practical disadvantages include the fact that a two-day imaging protocol is required and the need for thyroid blockade. Therefore, several PET alternatives for hNET imaging are actively being explored. This review gives an in-depth overview of the current status and recent developments in clinical trials leading to the next generation of clinical PET ligands for imaging of hNET-expressing tumors.

Bisoprolol therapy does not reduce right ventricular sympathetic activity in pulmonary arterial hypertension patients.

Right ventricular (RV) function and autonomic dysfunction are important determinants of morbidity and mortality in patients with pulmonary arterial hypertension (PAH). Although successful in animal studies, effects of beta-blocker therapy on RV function in clinical trials were disappointing. To understand this discrepancy, we studied whether beta-blocker therapy changes RV sympathetic activity. Idiopathic PAH (IPAH) patients received beta-blocker therapy (uptitrated to a maximal tolerated dose) and underwent cardiac magnetic resonance imaging, right heart catheterization, and a [11C]-hydroxyephedrine positron emission tomography ([11C]HED PET) scan at baseline to determine, respectively, RV ejection fraction (RVEF), RV pressures, and sympathetic activity. [11C]HED, a norepinephrine analogue, allows determination of sympathetic innervation of the RV. [11C]HED retention index reflects norepinephrine transporter activity. As a consequence of excessive catecholamine levels in the synaptic cleft, this transporter may be downregulated. Therefore, low [11C]HED retention index indicates high sympathetic activity. 13 IPAH patients underwent [11C]HED PET scans at baseline and after bisoprolol treatment. Although heart rate was reduced, systemic modulation of autonomic activity by bisoprolol did not affect local RV sympathetic nerve activity, RV function, or RV wall tension. In PAH patients, RV [11C]HED retention index was lower compared to LV tracer uptake (p<0.01) and was related to systolic wall tension (R2 = 0.4731, p<0.01) and RV function (R2 = 0.44, p = 0.01). In RV failure, the tolerated dosage of bisoprolol did not result in an improvement of RV function nor in a reduction in RV sympathetic activity.

123 I-MIBG scintigraphy in the assessment of heart failure prognosis and effectiveness of cardiac resynchronization therapy

Cardiac resynchronization therapy (CRT) is one of the methods of treating patients with chronic heart failure, which can reduce the mortality rate of this group. Scintigraphic assessment of sympathetic myocardial innervation allows us to evaluate the heart failure prognosis and the effectiveness of interventional treatment. The method is based on use of the radiopharmaceutical 123 I-methiodiobenzylguanidine (123 I-MIBG), which is a structural analogue of norepinephrine and is able to selectively accumulate in the sympathetic nerve endings. This review includes a brief description of norepinephrine metabolism and pharmacokinetics of 123 I-MIBG in the sympathetic nerve ending, a brief description of the study methodology and the clinical significance of this method in patients with heart failure. Particular attention is paid to the possibilities of using this method in patients with severe chronic heart failure before and after CRT.

Characterization of Drosophila octopamine receptor neuronal expression using MiMIC-converted Gal4 lines.

Octopamine, the invertebrate analog of norepinephrine, is known to modulate a large variety of behaviors in Drosophila including feeding initiation, locomotion, aggression, and courtship, among many others. Significantly less is known about the identity of the neurons that receive octopamine input and how they mediate octopamine-regulated behaviors. Here, we characterize adult neuronal expression of MiMIC-converted Trojan-Gal4 lines for each of the five Drosophila octopamine receptors. Broad neuronal expression was observed for all five octopamine receptors, yet distinct differences among them were also apparent. Use of immunostaining for the octopamine neurotransmitter synthesis enzyme Tdc2, along with a novel genome-edited conditional Tdc2-LexA driver, revealed all five octopamine receptors express in Tdc2/octopamine neurons to varying degrees. This suggests autoreception may be an important circuit mechanism by which octopamine modulates behavior.

The many faces of thyroxine.

Hönes et al. have recently shown that in vivo interference with the apparatus of the nuclear receptor-mediated, gene-driven mechanism of triiodothyronine (T3) actions fails to eliminate all actions of T3. However, the investigators conducting that study provided little information regarding the mechanisms that might be responsible for conferring those implied gene-independent effects. Dratman has long ago suggested a system wherein such gene-free mechanisms might operate. Therefore, since news of that discovery was originally published in 1974, it seems appropriate to describe the progress made since then. We propose that thyroxine and triiodothyronine have many different structural properties that may confer a series of different capabilities on their functions. These conform with our proposal that a series of catecholamine analogs and their conversion to iodothyronamines, allows them to perform many of the functions that previously were attributed to nuclear receptors regulating gene expression. The actions of deiodinases and the differential distribution of iodine substituents are among the critical factors that allow catecholamine analogs to change their effects into ones that either activate their targets or block them. They do this by using two different deiodinases to vary the position of an iodide ion on the diphenylether backbones of thyroxine metabolites. A panoply of these structural features imparts major unique functional properties on the behavior of vertebrates in general and possibly on Homo sapiens in particular.

Comparative antiradical activity and molecular Docking/Dynamics analysis of octopamine and norepinephrine: the role of OH groups

Octopamine is a neurotransmitter in invertebrates and a phenol analog of norepinephrine. The crystallographic and spectral (UV–visUV, and NMR) characteristics of octopamine were investigated experimentally and theoretically by applying appropriate level of theory, B3LYP-D3BJ/6-311++G(d,p), which reproduced well the experimental bond lengths and angles. The intramolecular interactions governing the stability of conformers were described by NBO and QTAIM analyses. The antiradical potencies of octopamine and norepinephrine towards DPPH and ABTS+ were examined with special emphasis on the preferred mechanism and effect of catechol moiety. Several techniques were used to distinguish Hydrogen Atom Transfer (HAT) and Proton Coupled Electron Transfer (PCET) mechanisms for reaction with DPPH. The calculated rate constants of the reactions with both radicals showed that Sequential Proton Loss Electron Transfer (SPLET) mechanism was dominant both thermodynamically and kinetically, with values of thermodynamic functions and rate constants clearly proving the importance of the second hydroxyl group in structure. The Molecular Docking and afterward Molecular Dynamics calculations of formed complexes between octopamine/norepinephrine with β1- and β2- adrenergic receptors examined in details the interactions that lead to the formation of stable complexes. The number of strong interactions of amino acids with norepinephrine was higher, but the absence of hydroxyl group in octopamine did not lead to a significant change in the type of interactions and stability. The formed complexes showed higher flexibility of amino acids, similar compactness of structure as proteins and increased interatomic distances of the backbone when compared to pure proteins.

MIBG in neuroblastoma diagnosis and treatment

Neuroblastoma is a heterogenous disease, with solid tumors arising in the adrenal gland or paraspinal regions in young children. Neuroblastoma is unique, with varied presentation and prognosis based on primary location and tumor stage. Tumor behavior and response to treatment ranges from spontaneous regression to disseminated, lethal disease depending on the individual biology of a patient’s tumor. Stratification of the disease has changed, with patients now placed in low, intermediate, and high-risk categories depending on age, stage, and tumor biology. Long-term survival for the high-risk subset of patients with metastatic disease is <40% despite aggressive multimodal therapy. Derived from sympathoadrenal cells of the adrenal medulla and sympathetic nervous system, both malignant neuroblastoma and differentiated tumors have specialized norepinephrine transporter (NET) receptors which are naturally occurring in the sympathetic nervous system throughout the body. Metaiodobenzylguanidine (MIBG) is a norepinephrine analog that undergoes active uptake by NET receptors resulting in accumulation in neuroblastoma as well as tissues normally expressing the NET receptor. When radioiodine labeled, MIBG can be used for both diagnosis and treatment. This article describes the history of MIBG use in neuroblastoma, including its utility as an imaging modality for diagnosis as well as the varied ways in which is it included in the multimodal treatment algorithm.

DARK Classics in Chemical Neuroscience: NBOMes.

N-Benzylphenethylamines, commonly known as NBOMes, are synthetic psychedelic compounds derived from the phenethylamine class of psychedelics (2C-X compounds), which originally have been derived from the naturally occurring alkaloid mescaline. Analogously to their parent compounds and other classical psychedelics, such as psilocybin and lysergic acid diethylamide (LSD), NBOMes are believed to exert their main pharmacological effects through activation of serotonin 2A (5-HT2A) receptors. Since their introduction as New Psychoactive Substances (NPSs) in 2010, NBOMes have been widely used for recreational purposes; this has resulted in numerous cases of acute toxicity, sometimes with lethal outcomes, leading to the classification of several NBOMes as Schedule I substances in 2013. However, in addition to their recreational use, the NBOMe class has yielded several important biochemical tools, including [11C]Cimbi-36, which is now being used in positron emission tomography (PET) studies of the 5-HT2A and 5-HT2C receptors in the mammalian brain, and 25CN-NBOH, one of the most selective 5-HT2A receptor agonists developed to date. In this Review, the history, chemistry, structure-activity relationships, ADME (absorption, distribution, metabolism, and excretion) properties, and safety profiles of NBOMes will be outlined and discussed.

The flight response impairs cytoprotective mechanisms by activating the insulin pathway.

An animal’s stress response requires different adaptive strategies depending on the nature and duration of the stressor. While acute stressors, like predation, induce a rapid and energy-demanding fight or flight response, long-term environmental stressors induce the gradual and long-lasting activation of highly conserved cytoprotective processes1–3. In animals across the evolutionary spectrum the continued activation of the fight-or-flight response weakens the animal’s resistance to environmental challenges4,5. However, the molecular and cellular mechanisms that regulate the trade-off between flight response and long-term stressors are poorly understood. Here we show that repeated induction of the C. elegans flight response shortens lifespan and inhibits conserved cytoprotective mechanisms. The flight response activates neurons that release tyramine, the invertebrate analog of adrenaline/noradrenaline. Tyramine stimulates the DAF-2/Insulin/IGF-1 pathway and precludes the induction of stress response genes by activating an adrenergic-like receptor in the intestine. In contrast, long-term environmental stressors, such as heat or oxidative stress, reduce tyramine release allowing the induction of cytoprotective genes. These findings demonstrate that a neural stress-hormone supplies a state-dependent neural switch between acute flight and long-term environmental stress responses and provides mechanistic insights into how the flight response impairs cellular defense systems and accelerates aging.

Fast enzymatic synthesis of n.c.a. 6-[18 F]fluorodopamine (FDA) from n.c.a. 6-[18 F]FDOPA and the fate of 6-FDOPA and 6-FDA in neuroblastoma and Caki-1 cells after their uptake.

The catecholamine analogue [123 I]mIBG has been used for scintigraphic imaging of neuroblastoma since 1984. It is taken up by the noradrenaline transporter (NAT), which is present in most neuroblastoma cells. An alternative imaging method could be PET with 6-[18 F]fluorodopamine, which is also taken up by NAT, but-in contrast to mIBG-also by dopamine transporter (DAT), present in neuroblastoma cells (NAT>DAT). An enzymatic method was established allowing a rapid, quantitative transformation of FDOPA to FDA by DOPA decarboxylase within 25minutes. This strategy was applied to [18 F]FDOPA, which was produced via nucleophilic synthesis (RCY 15%, 10GBq, 50GBq/μmol) and subsequently converted to [18 F]FDA (RCY 35%-50%, n=5). Uptake and metabolism of FDOPA and FDA were analyzed in human Kelly and SK-N-SH neuroblastoma cell lines and in human Caki-1 kidney cells that can take up catecholamines and mIBG via an organic cation transporter (OCT). FDOPA and FDA were taken up by all three cells, but FDOPA could only be converted to FDA in neuroblastoma cells. As today, [18 F]FDOPA is well available in high yields, efficient enzymatic conversion to [18 F]FDA to be used for NAT/DAT PET imaging in neuroendocrine tumors is an attractive, alternative synthesis route.

Development of covalent antagonists for β1- and β2-adrenergic receptors.

The selective covalent tethering of ligands to a specific GPCR binding site has attracted considerable interest in structural biology, molecular pharmacology and drug design. We recently reported on a covalently binding noradrenaline analog (FAUC37) facilitating crystallization of the β2-adrenergic receptor (β2ARH2.64C) in an active state. We herein present the stereospecific synthesis of covalently binding disulfide ligands based on the pharmacophores of adrenergic β1- and β2 receptor antagonists. Radioligand depletion experiments revealed that the disulfide-functionalized ligands were able to rapidly form a covalent bond with a specific cysteine residue of the receptor mutants β1ARI2.64C and β2ARH2.64C. The propranolol derivative (S)-1a induced nearly complete irreversible blockage of the β2ARH2.64C within 30 min incubation. The CGP20712A-based ligand (S)-4 showed efficient covalent blocking of the β2ARH2.64C at very low concentrations. The analog (S)-5a revealed extraordinary covalent cross-linking at the β1ARI2.64C and β2ARH2.64C mutant while retaining a 41-fold selectivity for the β1AR wild type over β2AR. These compounds may serve as valuable molecular tools for studying β1/β2 subtype selectivity or investigations on GPCR trafficking and dimerization.

Imaging cardiac innervation in amyloidosis

Cardiac amyloidosis is a form of restrictive cardiomyopathy resulting in heart failure and potential risk on arrhythmia, due to amyloid infiltration of the nerve conduction system and the myocardial tissue. The prognosis in this progressive disease is poor, probably due the development of cardiac arrhythmias. Early detection of cardiac sympathetic innervation disturbances has become of major clinical interest, because its occurrence and severity limits the choice of treatment. The use of iodine-123 labelled metaiodobenzylguanidine ([I-123]MIBG), a chemical modified analogue of norepinephrine, is well established in patients with heart failure and plays an important role in evaluation of sympathetic innervation in cardiac amyloidosis. [I-123]MIBG is stored in vesicles in the sympathetic nerve terminals and is not catabolized like norepinephrine. Decreased heart-to-mediastinum ratios on late planar images and increased wash-out rates indicate cardiac sympathetic denervation and are associated with poor prognosis. Single photon emission computed tomography provides additional information and has advantages for evaluating abnormalities in regional distribution in the myocardium. [I-123]MIBG is mainly useful in patients with hereditary and wild-type ATTR cardiac amyloidosis, not in AA and AL amyloidosis. The potential role of positron emission tomography for cardiac sympathetic innervation in amyloidosis has not yet been identified.

Assessment of myocardial sympathetic innervation by PET in patients with heart failure: a review of the most recent advances and future perspectives

Life-threatening ventricular arrhythmias (VA) are a major cause of death in patients with congestive heart failure (HF). Among various factors, the sympathetic nervous system may give rise to VA in several pathophysiological pathways due to an impaired function of presynaptic sympathetic nerve terminals. Positron emission tomography (PET) with labeled catecholamine analogues represents a reliable tool to assess the sympathetic innervation activity. This review aims at summarising the most relevant and recent literature findings on the current role of PET in the evaluation of cardiac sympathetic activity in patients with heart failure. A comprehensive literature search strategy using PubMed databases was carried out looking for articles on the role of Positron emission tomography/Computed Tomography (PET/CT) in the assessment of myocardial sympathetic innervation in patients with heart failure. The literature search limited to the last 5 years retrieved 40 papers. Most of the papers dealt with PET studies with 11C-HED. 19 pre-clinical, first-in-human and clinical studies highlighting the current role of PET and future perspectives resulted eligible for inclusion in the present review. The assessment of myocardial sympathetic activity in patients with heart failure with PET will play a pivotal role in clinical practice. Its capability to predict the occurrence of life-threatening VA and the effectiveness of resynchronization therapy makes this technique ideal in the era of personalized medicine.

Synthesis of Deuterium- or Tritium-Labeled Norepinephrine and Evaluation of Its Biological Activity

A labeled norepinephrine analogue was prepared by bromination of norepinephrine with subsequent debromination with tritium or deuterium gas. The yield of tritiated norepinephrine was 10–20% with a molar radioactivity of 27 Ci/mmol. The biological activity of tritiated norepinephrine was evaluated by analyzing its specific capture by noradrenergic neutrons in thick sections of the brainstem and midbrain of Wistar rats, and in a suspension of cells of pheochromocytoma PC12 expressing the membrane transporter of norepinephrine. It is shown that tritium-labeled norepinephrine synthesized by the proposed method does not differ in biological activity from the natural norepinephrine.

A sleep state in Drosophila larvae required for neural stem cell proliferation.

Sleep during development is involved in refining brain circuitry, but a role for sleep in the earliest periods of nervous system elaboration, when neurons are first being born, has not been explored. Here we identify a sleep state in Drosophila larvae that coincides with a major wave of neurogenesis. Mechanisms controlling larval sleep are partially distinct from adult sleep: octopamine, the Drosophila analog of mammalian norepinephrine, is the major arousal neuromodulator in larvae, but dopamine is not required. Using real-time behavioral monitoring in a closed-loop sleep deprivation system, we find that sleep loss in larvae impairs cell division of neural progenitors. This work establishes a system uniquely suited for studying sleep during nascent periods, and demonstrates that sleep in early life regulates neural stem cell proliferation.