Localisation of NMU1R and NMU2R in human and rat central nervous system and effects of neuromedin-U following central administration in rats

Rationale: Neuromedin-U (NmU) is an agonist at NMU1R and NMU2R. The brain distribution of NmU and its receptors, in particular NMU2R, suggests widespread central roles for NmU. In agreement, centrally administered NmU affects feeding behaviour, energy expenditure and pituitary output. Further central nervous system (CNS) roles for NmU warrant investigation. Objectives: To investigate the CNS role of NmU by mapping NMU1R and NMU2R mRNA and measuring the behavioural, endocrine, neurochemical and c-fos response to intracerebroventricular (i.c.v.) NmU. Methods: Binding affinity and functional potency of rat NmU was determined at human NMU1R and NMU2R. Expression of NMU1R and NMU2R mRNA in rat and human tissue was determined using semi-quantitative reverse-transcription polymerase chain reaction. In in-vivo studies, NmU was administered i.c.v. to male Sprague-Dawley rats, and changes in grooming, motor activity and pre-pulse inhibition (PPI) were assessed. In further studies, plasma endocrine hormones, [DOPAC + HVA]/[dopamine] and [5-HIAA]/[5-HT] ratios and levels of Fos-like immunoreactivity (FLI) were measured 20 min post-NmU (i.c.v.). Results: NmU bound to NMU1R (KI, 0.11±0.02 nM) and NMU2R (KI, 0.21±0.05 nM) with equal affinity and was equally active at NMU1R (EC50, 1.25±0.05 nM) and NMU2R (EC50, 1.10±0.20 nM) in a functional assay. NMU2R mRNA expression was found at the highest levels in the CNS regions of both rat and human tissues. NMU1R mRNA expression was restricted to the periphery of both species with the exception of the rat amygdala. NmU caused a marked increase in grooming and motor activity but did not affect PPI. Further, NmU decreased plasma prolactin but did not affect levels of corticosterone, luteinising hormone or thyroid stimulating hormone. NmU elevated levels of 5-HT in the frontal cortex and hypothalamus, with decreased levels of its metabolites in the hippocampus and hypothalamus, but did not affect dopamine function. NmU markedly increased FLI in the nucleus accumbens, frontal cortex and central amygdala. Conclusions: These data provide further evidence for widespread roles for NmU and its receptors in the brain

Positive deviance control-case life history: a method to develop grounded hypotheses about successful long-term avoidance of infection

<p>Abstract</p> <p>Background</p> <p>Prevalence rates for long-term injection drug users in some localities surpass 60% for HIV and 80% for HCV. We describe methods for developing grounded hypotheses about how some injectors avoid infection with either virus.</p> <p>Methods</p> <p>Subjects: 25 drug injectors who have injected drugs 8 – 15 years in New York City. 17 remain without antibody to either HIV or HCV; 3 are double-positives; and 5 are positive for HCV but not HIV. "Staying Safe" methodology compares serostatus groups using detailed biographical timelines and narratives; and information about how subjects maintain access to physical resources and social support; their strategies and tactics to remain safe; how they handle problems of addiction and demands by drug dealers and other drug users; and how their behaviors and strategies do or do not become socially-embedded practices. Grounded theory and life-history analysis techniques compare and contrast doubly-uninfected with those infected with both viruses or only with HCV.</p> <p>Results</p> <p>Themes and initial hypotheses emerging from analyses included two master hypotheses that, if confirmed, should help shape preventive interventions: 1) Staying uninfected is not simply a question of social structure or social position. It involves agency by drug injectors, including sustained hard work and adaptation to changing circumstances. 2) Multiple intentionalities contribute to remaining uninfected. These conscious goals include balancing one's need for drugs and one's income; developing ways to avoid drug withdrawal sickness; avoiding situations where other drug users importune you to share drugs; and avoiding HIV (and perhaps HCV) infection. Thus, focusing on a single goal in prevention might be sub-optimal.</p> <p>Other hypotheses specify mechanisms of enacting these intentionalities. One example is finding ways to avoid extreme social ostracism.</p> <p>Conclusion</p> <p>We have identified strategies and tactics that some doubly-uninfected IDUs have developed to stay safe. Staying Safe methodology develops grounded hypotheses. These can be tested through cohort studies of incidence and prevention trials of hypothesis-based programs to help drug injectors make their injection and sexual careers safer for themselves and others. This positive deviance control-case life history method might be used to study avoiding other infections like genital herpes among sex workers.</p

Pharmacogenetic Modulation of Orexin Neurons Alters Sleep/Wakefulness States in Mice

Hypothalamic neurons expressing neuropeptide orexins are critically involved in the control of sleep and wakefulness. Although the activity of orexin neurons is thought to be influenced by various neuronal input as well as humoral factors, the direct consequences of changes in the activity of these neurons in an intact animal are largely unknown. We therefore examined the effects of orexin neuron-specific pharmacogenetic modulation in vivo by a new method called the Designer Receptors Exclusively Activated by Designer Drugs approach (DREADD). Using this system, we successfully activated and suppressed orexin neurons as measured by Fos staining. EEG and EMG recordings suggested that excitation of orexin neurons significantly increased the amount of time spent in wakefulness and decreased both non-rapid eye movement (NREM) and rapid eye movement (REM) sleep times. Inhibition of orexin neurons decreased wakefulness time and increased NREM sleep time. These findings clearly show that changes in the activity of orexin neurons can alter the behavioral state of animals and also validate this novel approach for manipulating neuronal activity in awake, freely-moving animals

Safety and acceptability of an organic light-emitting diode sleep mask as a potential therapy for retinal disease

Purpose The purpose of the study was to study the effect of an organic light-emitting diode sleep mask on daytime alertness, wellbeing, and retinal structure/function in healthy volunteers and in diabetic macular oedema (DMO). Patients and methods Healthy volunteers in two groups, 18–30 yrs (A), 50–70 yrs (B) and people with DMO (C) wore masks (504 nm wavelength; 80 cd/m2 luminance; ≤8 h) nightly for 3 months followed by a 1-month recovery period. Changes from baseline were measured for (means): psychomotor vigilance task (PVT) (number of lapses (NL), response time (RT)), sleep, depression, psychological wellbeing (PW), visual acuity, contrast sensitivity, colour, electrophysiology, microperimetry, and retinal thickness on OCT. Results Of 60 participants, 16 (27%) withdrew, 8 (13%) before month 1, due to sleep disturbances and mask intolerance. About 36/55 (65%) who continued beyond month 1 reported ≥1 adverse event. At month 3 mean PVT worsened in Group A (RT (7.65%, P<0.001), NL (43.3%, P=0.005)) and mean PW worsened in all groups (A 28.0%, P=0.01, B 21.2%, P=0.03, C 12.8%, P<0.05). No other clinically significant safety signal was detected. Cysts reduced/resolved in the OCT subfield of maximal pathology in 67% Group C eyes. Thinning was greater at 3 and 4 months for greater baseline thickness (central subfield P<0.001, maximal P<0.05). Conclusion Sleep masks showed no major safety signal apart from a small impairment of daytime alertness and a moderate effect on wellbeing. Masks were acceptable apart from in some healthy participants. Preliminary data suggest a beneficial effect on retinal thickness in DMO. This novel therapeutic approach is ready for large clinical trials

Use of humanised rat basophilic leukaemia cell line RS-ATL8 for the assessment of allergenicity of Schistosoma mansoni proteins.

BACKGROUND Parasite-specific IgE is thought to correlate with protection against Schistosoma mansoni infection or re-infection. Only a few molecular targets of the IgE response in S. mansoni infection have been characterised. A better insight into the basic mechanisms of anti-parasite immunity could be gained from a genome-wide characterisation of such S. mansoni allergens. This would have repercussions on our understanding of allergy and the development of safe and efficacious vaccinations against helminthic parasites. METHODOLOGY/PRINCIPAL FINDINGS A complete medium- to high-throughput amenable workflow, including important quality controls, is described, which enables the rapid translation of S. mansoni proteins using wheat germ lysate and subsequent assessment of potential allergenicity with a humanised Rat Basophilic Leukemia (RBL) reporter cell line. Cell-free translation is completed within 90 minutes, generating sufficient amounts of parasitic protein for rapid screening of allergenicity without any need for purification. Antigenic integrity is demonstrated using Western Blotting. After overnight incubation with infected individuals' serum, the RS-ATL8 reporter cell line is challenged with the complete wheat germ translation mixture and Luciferase activity measured, reporting cellular activation by the suspected allergen. The suitability of this system for characterization of novel S. mansoni allergens is demonstrated using well characterised plant and parasitic allergens such as Par j 2, SmTAL-1 and the IgE binding factor IPSE/alpha-1, expressed in wheat germ lysates and/or E. coli. SmTAL-1, but not SmTAL2 (used as a negative control), was able to activate the basophil reporter cell line. CONCLUSION/SIGNIFICANCE This method offers an accessible way for assessment of potential allergenicity of anti-helminthic vaccine candidates and is suitable for medium- to high-throughput studies using infected individual sera. It is also suitable for the study of the basis of allergenicity of helminthic proteins

Phospholipase C-β4 Is Essential for the Progression of the Normal Sleep Sequence and Ultradian Body Temperature Rhythms in Mice

BACKGROUND: THE SLEEP SEQUENCE: i) non-REM sleep, ii) REM sleep, and iii) wakefulness, is stable and widely preserved in mammals, but the underlying mechanisms are unknown. It has been shown that this sequence is disrupted by sudden REM sleep onset during active wakefulness (i.e., narcolepsy) in orexin-deficient mutant animals. Phospholipase C (PLC) mediates the signaling of numerous metabotropic receptors, including orexin receptors. Among the several PLC subtypes, the beta4 subtype is uniquely localized in the geniculate nucleus of thalamus which is hypothesized to have a critical role in the transition and maintenance of sleep stages. In fact, we have reported irregular theta wave frequency during REM sleep in PLC-beta4-deficient mutant (PLC-beta4-/-) mice. Daily behavioral phenotypes and metabotropic receptors involved have not been analyzed in detail in PLC-beta4-/- mice, however. METHODOLOGY/PRINCIPAL FINDINGS: Therefore, we analyzed 24-h sleep electroencephalogram in PLC-beta4-/- mice. PLC-beta4-/- mice exhibited normal non-REM sleep both during the day and nighttime. PLC-beta4-/- mice, however, exhibited increased REM sleep during the night, their active period. Also, their sleep was fragmented with unusual wake-to-REM sleep transitions, both during the day and nighttime. In addition, PLC-beta4-/- mice reduced ultradian body temperature rhythms and elevated body temperatures during the daytime, but had normal homeothermal response to acute shifts in ambient temperatures (22 degrees C-4 degrees C). Within the most likely brain areas to produce these behavioral phenotypes, we found that, not orexin, but group-1 metabotropic glutamate receptor (mGluR)-mediated Ca(2+) mobilization was significantly reduced in the dorsal lateral geniculate nucleus (LGNd) of PLC-beta4-/- mice. Voltage clamp recordings revealed that group-1 mGluR-mediated currents in LGNd relay neurons (inward in wild-type mice) were outward in PLC-beta4-/- mice. CONCLUSIONS/SIGNIFICANCE: These lines of evidence indicate that impaired LGNd relay, possibly mediated via group-1 mGluR, may underlie irregular sleep sequences and ultradian body temperature rhythms in PLC-beta4-/- mice

Coordinated changes in energy intake and expenditure following hypothalamic administration of neuropeptides involved in energy balance

OBJECTIVE: The hypothalamic control of energy balance is regulated by a complex network of neuropeptide-releasing neurons. Whilst the effect of these neuropeptides on individual aspects of energy homeostasis has been studied, the coordinated response of these effects has not been comprehensively investigated. We have simultaneously monitored a number of metabolic parameters following ICV administration of 1nmol and 3nmol of neuropeptides with established roles in the regulation of feeding, activity and metabolism. Ad libitum fed rats received the orexigenic neuropeptides neuropeptide Y (NPY), agouti-related protein (AgRP), melanin-concentrating hormone (MCH) or orexin-A. Overnight food deprived rats received an ICV injection of the anorectic peptides α-MSH, corticotrophin releasing factor (CRF) or neuromedin U (NMU). RESULTS: Our results reveal the temporal sequence of the effects of these neuropeptides on both energy intake and expenditure, highlighting key differences in their function as mediators of energy balance. NPY and AgRP increased feeding and decreased oxygen consumption, with the effects of AgRP being more prolonged. In contrast, orexin-A increased both feeding and oxygen consumption, consistent with an observed increase in activity. The potent anorexigenic effects of CRF were accompanied by a prolonged increase in activity whilst NMU injection resulted in significant but short-lasting inhibition of food intake, ambulatory activity and oxygen consumption. Alpha-MSH injection resulted in significant increases in both ambulatory activity and oxygen consumption, and reduced food intake following administration of 3nmol of the peptide. CONCLUSION: We have for the first time, simultaneously measured several metabolic parameters following hypothalamic administration of a number of neuropeptides within the same experimental system. This work has demonstrated the interrelated effects of these neuropeotides on activity, energy expenditure and food intake thus facilitating comparison between the different hypothalamic systems

Pharmacological Blockade of Serotonin 5-HT7 Receptor Reverses Working Memory Deficits in Rats by Normalizing Cortical Glutamate Neurotransmission

The role of 5-HT7 receptor has been demonstrated in various animal models of mood disorders; however its function in cognition remains largely speculative. This study evaluates the effects of SB-269970, a selective 5-HT7 antagonist, in a translational model of working memory deficit and investigates whether it modulates cortical glutamate and/or dopamine neurotransmission in rats. The effect of SB-269970 was evaluated in the delayed non-matching to position task alone or in combination with MK-801, a non-competitive NMDA receptor antagonist, and, in separate experiments, with scopolamine, a non-selective muscarinic antagonist. SB-269970 (10 mg/kg) significantly reversed the deficits induced by MK-801 (0.1 mg/kg) but augmented the deficit induced by scopolamine (0.06 mg/kg). The ability of SB-269970 to modulate MK-801-induced glutamate and dopamine extracellular levels was separately evaluated using biosensor technology and microdialysis in the prefrontal cortex of freely moving rats. SB-269970 normalized MK-801 -induced glutamate but not dopamine extracellular levels in the prefrontal cortex. Rat plasma and brain concentrations of MK-801 were not affected by co-administration of SB-269970, arguing for a pharmacodynamic rather than a pharmacokinetic mechanism. These results indicate that 5-HT7 receptor antagonists might reverse cognitive deficits associated with NMDA receptor hypofunction by selectively normalizing glutamatergic neurotransmission

Widespread Regulation of miRNA Biogenesis at the Dicer Step by the Cold-Inducible RNA-Binding Protein, RBM3

MicroRNAs (miRNAs) play critical roles in diverse cellular events through their effects on translation. Emerging data suggest that modulation of miRNA biogenesis at post-transcriptional steps by RNA-binding proteins is a key point of regulatory control over the expression of some miRNAs and the cellular processes they influence. However, the extent and conditions under which the miRNA pathway is amenable to regulation at posttranscriptional steps are poorly understood. Here we show that RBM3, a cold-inducible, developmentally regulated RNA-binding protein and putative protooncogene, is an essential regulator of miRNA biogenesis. Utilizing miRNA array, Northern blot, and PCR methods, we observed that over 60% of miRNAs detectable in a neuronal cell line were significantly downregulated by knockdown of RBM3. Conversely, for select miRNAs assayed by Northern blot, induction of RBM3 by overexpression or mild hypothermia increased their levels. Changes in miRNA expression were accompanied by changes in the levels of their ∼70 nt precursors, whereas primary transcript levels were unaffected. Mechanistic studies revealed that knockdown of RBM3 does not reduce Dicer activity or impede transport of pre-miRNAs into the cytoplasm. Rather, we find that RBM3 binds directly to ∼70 nt pre-miRNA intermediates and promotes / de-represses their ability as larger ribonucleoproteins (pre-miRNPs) to associate with active Dicer complexes. Our findings suggest that the processing of a majority of pre-miRNPs by Dicer is subject to an intrinsic inhibitory influence that is overcome by RBM3 expression. RBM3 may thus orchestrate changes in miRNA expression during hypothermia and other cellular stresses, and in the euthermic contexts of early development, differentiation, and oncogenesis where RBM3 expression is highly elevated. Additionally, our data suggest that temperature-dependent changes in miRNA expression mediated by RBM3 may contribute to the therapeutic effects of hypothermia, and are an important variable to consider in in vitro studies of translation-dependent cellular events