The generation of knock-in mice expressing fluorescently tagged galanin receptors 1 and 2

The neuropeptide galanin has diverse roles in the central and peripheral nervous systems, by activating the G protein-coupled receptors Gal(1), Gal(2) and the less studied Gal(3) (GalR1–3 gene products). There is a wealth of data on expression of Gal(1–3) at the mRNA level, but not at the protein level due to the lack of specificity of currently available antibodies. Here we report the generation of knock-in mice expressing Gal(1) or Gal(2) receptor fluorescently tagged at the C-terminus with, respectively, mCherry or hrGFP (humanized Renilla green fluorescent protein). In dorsal root ganglia (DRG) neurons expressing the highest levels of Gal(1)-mCherry, localization to the somatic cell membrane was detected by live-cell fluorescence and immunohistochemistry, and that fluorescence decreased upon addition of galanin. In spinal cord, abundant Gal(1)-mCherry immunoreactive processes were detected in the superficial layers of the dorsal horn, and highly expressing intrinsic neurons of the lamina III/IV border showed both somatic cell membrane localization and outward transport of receptor from the cell body, detected as puncta within cell processes. In brain, high levels of Gal(1)-mCherry immunofluorescence were detected within thalamus, hypothalamus and amygdala, with a high density of nerve endings in the external zone of the median eminence, and regions with lesser immunoreactivity included the dorsal raphe nucleus. Gal(2)-hrGFP mRNA was detected in DRG, but live-cell fluorescence was at the limits of detection, drawing attention to both the much lower mRNA expression than to Gal(1) in mice and the previously unrecognized potential for translational control by upstream open reading frames (uORFs)

Melanocortin 4 Receptors Reciprocally Regulate Sympathetic and Parasympathetic Preganglionic Neurons

Melanocortin 4 receptors (MC4Rs) in the central nervous system are key regulators of energy and glucose homeostasis. Notably, obese patients with MC4R mutations are hyperinsulinemic and resistant to obesity-induced hypertension. Although these effects are likely dependent upon the activity of the autonomic nervous system, the cellular effects of MC4Rs on parasympathetic and sympathetic neurons remain undefined. Here, we show that MC4R agonists inhibit parasympathetic preganglionic neurons in the brainstem. In contrast, MC4R agonists activate sympathetic preganglionic neurons in the spinal cord. Deletion of MC4Rs in cholinergic neurons resulted in elevated levels of insulin. Furthermore, re-expression of MC4Rs specifically in cholinergic neurons (including sympathetic preganglionic neurons) restores obesity-associated hypertension in MC4R null mice. These findings provide a cellular correlate of the autonomic side effects associated with MC4R agonists and demonstrate a role for MC4Rs expressed in cholinergic neurons in the regulation of insulin levels and in the development of obesity-induced hypertension

Genome-wide association study of height-adjusted BMI in childhood identifies functional variant in ADCY3

Objective: Genome-wide association studies (GWAS) of BMI are mostly undertaken under the assumption that "kg/m2" is an index of weight fully adjusted for height, but in general this is not true. The aim here was to assess the contribution of common genetic variation to a adjusted version of that phenotype which appropriately accounts for covariation in height in children. Methods: A GWAS of height-adjusted BMI (BMI[x]=weight/heightx), calculated to be uncorrelated with height, in 5809 participants (mean age 9.9 years) from the Avon Longitudinal Study of Parents and Children (ALSPAC) was performed. Results: GWAS based on BMI[x] yielded marked differences in genomewide results profile. SNPs in ADCY3 (adenylate cyclase 3) were associated at genome-wide significance level (rs11676272 (0.28 kg/m3.1 change per allele G (0.19, 0.38), P=6 × 10-9). In contrast, they showed marginal evidence of association with conventional BMI [rs11676272 (0.25 kg/m2 (0.15, 0.35), P=6 × 10-7)]. Results were replicated in an independent sample, the Generation R study. Conclusions: Analysis of BMI[x] showed differences to that of conventional BMI. The association signal at ADCY3 appeared to be driven by a missense variant and it was strongly correlated with expression of this gene. Our work highlights the importance of well understood phenotype use (and the danger of convention) in characterising genetic contributions to complex traits

Cholinergic Interneurons Mediate Fast VGluT3-Dependent Glutamatergic Transmission in the Striatum

The neurotransmitter glutamate is released by excitatory projection neurons throughout the brain. However, non-glutamatergic cells, including cholinergic and monoaminergic neurons, express markers that suggest that they are also capable of vesicular glutamate release. Striatal cholinergic interneurons (CINs) express the Type-3 vesicular glutamate transporter (VGluT3), although whether they form functional glutamatergic synapses is unclear. To examine this possibility, we utilized mice expressing Cre-recombinase under control of the endogenous choline acetyltransferase locus and conditionally expressed light-activated Channelrhodopsin2 in CINs. Optical stimulation evoked action potentials in CINs and produced postsynaptic responses in medium spiny neurons that were blocked by glutamate receptor antagonists. CIN-mediated glutamatergic responses exhibited a large contribution of NMDA-type glutamate receptors, distinguishing them from corticostriatal inputs. CIN-mediated glutamatergic responses were insensitive to antagonists of acetylcholine receptors and were not seen in mice lacking VGluT3. Our results indicate that CINs are capable of mediating fast glutamatergic transmission, suggesting a new role for these cells in regulating striatal activity

An ultra-wide bandwidth (704 to 4 032 MHz) receiver for the Parkes radio telescope

We describe an ultra-wide-bandwidth, low-frequency receiver recently installed on the Parkes radio telescope. The receiver system provides continuous frequency coverage from 704 to 4032 MHz. For much of the band ( ${∼}60$ ), the system temperature is approximately 22 K and the receiver system remains in a linear regime even in the presence of strong mobile phone transmissions. We discuss the scientific and technical aspects of the new receiver, including its astronomical objectives, as well as the feed, receiver, digitiser, and signal processor design. We describe the pipeline routines that form the archive-ready data products and how those data files can be accessed from the archives. The system performance is quantified, including the system noise and linearity, beam shape, antenna efficiency, polarisation calibration, and timing stability

Transgenic studies of growth hormone secretagogues physiology

SIGLEAvailable from British Library Document Supply Centre- DSC:DXN055656 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Transgenic studies of growth hormone secretagogue physiology

The synthetic growth hormone secretagogues (GHS) exert their growth hormone (GH) releasing effects by activating their receptor (GHS-R) in hypothalamic neurones and pituitary GH cells. The aim of this study was to investigate hypothalamic and pituitary GHS physiology by in vitro studies and transgenesis, over-expressing the GHS-R in hypothalamic GH releasing hormone (GHRH) neurones or pituitary GH cells. Initially, to analyse transgene expression driven by two targeting cosmids - a rat GHRH or a human GH cosmid - transgenic mice expressing enhanced green fluorescent protein (eGFP) were generated. In rGHRH-eGFP transgenic mice eGFP protein was visible in brightly fluorescing GHRH neurone cell bodies and median eminence nerve terminals. hGH-eGFP transgenic mice showed high levels of eGFP fluorescence limited to GH expressing pituitary cells. These mice provide useful new tools to study GHRH and GH cell physiology. In vitro studies with a stably GHS-R transfected GH cell line showed that processing of the GHS-R cDNA resulted in a functionally active receptor. In transgenic mice overexpression of the GHS-R in GHRH neurones caused alteration of the GH axis with increased pituitary GH content and body weight. GH responses to a GHS injection seemed enhanced. However, prolonged GHS exposure did not cause enhanced body weight gain compared to WT littermates. These studies suggested that enhancement of GHS-R abundance in GHRH neurones caused alteration of the GH and GHS axis, implicating GHRH neurones as an important GHS target in GH release. In contrast, transgenic mice with 50-fold GHS-R over-expression in GH cells showed reduced pituitary GH content, body weight and GH responses to GHRH. These data suggest that somatotroph function might be compromised by high levels of GHS-R over-expression. Crossing the hGHS-R over-expressing mice with their eGFP counterpart mice will facilitate the further investigation of their physiological phenotype by enabling in vivo analysis of GHS effects directly in identified cells

Sexual dimorphism in offspring glucose-sensitive hypothalamic gene expression and physiological responses to maternal high-fat diet feeding

A wealth of animal and human studies demonstrate that early life environment significantly influences adult metabolic balance, however the etiology for offspring metabolic misprogramming remains incompletely understood. Here, we determine the effect of maternal diet per se on offspring sex-specific outcomes in metabolic health and hypothalamic transcriptome regulation in mice. Furthermore, to define developmental periods of maternal diet misprogramming aspects of offspring metabolic balance, we investigated offspring physiological and transcriptomic consequences of maternal high-fat/high-sugar diet feeding during pregnancy and/or lactation. We demonstrate that female offspring of high-fat/high-sugar diet-fed dams are particularly vulnerable to metabolic perturbation with body weight increases due to postnatal processes, whereas in utero effects of the diet ultimately lead to glucose homeostasis dysregulation. Furthermore, glucose- and maternal-diet sensitive gene expression modulation in the paraventricular hypothalamus is strikingly sexually dimorphic. In summary, we uncover female-specific, maternal diet-mediated in utero misprogramming of offspring glucose homeostasis and a striking sexual dimorphism in glucose- and maternal diet-sensitive paraventricular hypothalamus gene expression adjustment. Notably, female offspring metabolic vulnerability to maternal high-fat/high-sugar diet propagates a vicious cycle of obesity and type 2 diabetes in subsequent generations