Conditional deletion of hypothalamic Y2 receptors reverts gonadectomy-induced bone loss in adult mice

Reduction in levels of sex hormones at menopause in women is associated with two common, major outcomes, the accumulation of white adipose tissue, and the progressive loss of bone because of excess osteoclastic bone resorption exceeding osteoblastic bone formation. Current antiresorptive therapies can reduce osteoclastic activity but have only limited capacity to stimulate osteoblastic bone formation and restore lost skeletal mass. Likewise, the availability of effective pharmacological weight loss treatments is currently limited. Here we demonstrate that conditional deletion of hypothalamic neuropeptide Y2 receptors can prevent ongoing bone loss in sex hormone-deficient adult male and female mice. This benefit is attributable solely to activation of an anabolic osteoblastic bone formation response that counterbalances persistent elevation of bone resorption, suggesting the Y2-mediated anabolic pathway to be independent of sex hormones. Furthermore, the increase in fat mass that typically occurs after ovariectomy is prevented by germ line deletion of Y2 receptors, whereas in male mice body weight and fat mass were consistently lower than wild-type regardless of sex hormone status. Therefore, this study indicates a role for Y2 receptors in the accumulation of adipose tissue in the hypogonadal state and demonstrates that hypothalamic Y2 receptors constitutively restrain osteoblastic activity even in the absence of sex hormones. The increase in bone formation after release of this tonic inhibition suggests a promising new avenue for osteoporosis treatment

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Differential actions of NPY on seizure modulation via Y1 and Y2 receptors: Evidence from receptor knockout mice

Summary: Purpose: Neuropeptide Y (NPY) has been shown to modulate seizure activities. To provide further understanding of the involvement of two of the most abundantly expressed NPY receptors, Y1 and Y2, we assessed the effect of Y1 and Y2 gene deletion on systemic kainic acid\u96induced seizures. We also examined the effect of rAAV-mediated hippocampal NPY overexpression on seizure susceptibility in these receptor knockout mice. Methods: Recombinant adeno-associated viral vector overexpressing NPY (rAAV-NPY) or an empty vector control (rAAV-Empty) was injected into the hippocampus of adult C57BL/6-129/SvJ wild-type male mice and mice deficient of Y1 or Y2 receptors on the same background. Four weeks after vector injection, mice were subjected to systemic kainic acid-induced seizures, and the seizure behaviors were scored. Results: The rAAV-mediated hippocampal overexpression of NPY led to a twofold reduction in seizures induced by systemic kainic acid in wild-type mice and Y1 receptor knockout mice but not in mice deficient of Y2 receptors. A differential action by the receptors was observed in the seizure-induced mortality rate, with increased fatality in Y2/ mice. In addition, although NPY overexpression did not significantly affect the mortality rate in Y2/ and wild-type mice, it abolished KA-induced mortality in Y1/mice. Conclusions: This study shows for the first time an altered susceptibility to chemically induced seizures in Y1 and Y2 knockout mice and demonstrates a differential seizure modulation mediated by these receptors via a genetic approach

Expression of proopiomelanocortin (POMC), glutamic acid decarboxylase 65 (GAD65) and neuropeptide Y (NPY) mRNA in the arcuate nucleus of the hypothalamus at 30 minutes after i.p. injection of saline or pancreatic polypeptide (PP).

<p>Data are means ± standard error of 5 wild type mice per group, with neuron labeling intensity expressed as a percentage of saline-injected controls. **, P<0.01; ***P<0.001 versus saline-injected controls.</p

Conditional deletion of Y4 receptors in the arcuate nucleus of the hypothalamus (ARC) alters pancreatic polypeptide (PP)-induced c-Fos immunoreactivity and POMC mRNA expression.

<p>(A) Schematic representation of primer positions used for PCR verification of Y4 receptor gene knockout.The un-deleted gene is 3.4 kilobases, and after Cre-mediated Y4 receptor gene deletion a PCR product of 290 base pairs is produced. (B) Confirmation of Y4 receptor deletion (indicated by production of the 290 base pair PCR product) from DNA isolated from the hypothalamus (Hypo) but not from the cerebellum (Cer) of Y4 receptor conditional knockout (Y4F) mice injected with AAV-Cre recombinase into the ARC, or from mice not injected with AAV-Cre(Y4F Hypo). (C) Photomicrograph showing c-Fos immunoreactivity and (D+E) emulsion-dipped autoradiograph of POMC mRNA expression in the brain of a conditional Y4 receptor knockout mouse, 30 minutes after i.p. injection of PP. Mice received unilateral injection of (D) AAV-Cre or (E) AAV-empty 28 days prior to PP injection in order to induce local deletion of Y4 receptors by virally-delivered Cre-recombinase, the contra-lateral side (at left in C, D and E) was used as control. Scale bar  = 100 µm in C and 25 µm in D and E. 3V, third cerebral ventricle.</p

Pancreatic polypeptide (PP) injection induces c-Fos immunoreactivity in neurons positive for alpha melanocyte stimulating hormone (α-MSH) and glutamic acid decarboxylase 65 (GAD65) in the arcuate nucleus of the hypothalamus (ARC).

<p>(A) c-Fos immunoreactivity, (B) α-MSH immunoreactivity, and (C) overlay of c-Fos and α-MSH immunoreactivity in neurons as indicated by white arrows at 30 minutes after i.p. injection of PP. Sale bar for A–C  = 25 µm. (D) Brightfield micrograph showing c-Fos and GAD65 immunoreactivity at 30 minutes after i.p. injection of PP. Scale bar  = 200 µm. (E) Higher magnification of the boxed area from D. Black arrows indicate neurons positive for c-Fos immunoreactivity only. These neurons are darkly stained. Black arrowheads indicate neurons positive for GAD65 immunoreactivity only. These neurons are lightly stained. Red arrows indicate neurons double-labeled for c-Fos and GAD65. The double staining on these neurons makes these neurons appear larger than the neurons positive only for c-Fos or CAG65 immunoreactvity. Scale bar  = 5 µm. 3V, third cerebral ventricle.</p

Number of c-Fos-like immunoreactive neurons in the brainstem and hypothalamus of wild type and Y4 receptor knockout (<i>Y4</i>^−/−) mice at 30 or 90 minutes after i.p. injection of saline or pancreatic polypeptide (PP).

<p>Data are means ± SEM of 4–6 mice per groups.* P<0.05 versus saline-injected wild type mice; # P<0.05 versus PP-injected wild type mice (90 minutes). NTS: nucleus tractus solitarus; AP: area postrema; ARC: arcuate nucleus of the hypothalamus; PVN: paraventricular nucleus of the hypothalamus; VMHDM: dorsomedial part of the ventromedial nucleus of the hypothalamus; LHA: lateral hypothalamic area.</p

Immunoreactivity for phosphorylated extracellular signal-regulated kinases 1 and 2 (P-Erk1/2) in the hypothalamus and co-expression of c-Fos and tyrosine hydroxylase (TH) immunoreactivity in the brainstem after PP injection.

<p>(A) Brightfield micrograph showing P-Erk1/2 immunoreactivity in the arcuate nucleus of the hypothalamus (ARC) at 30 minutes after i.p. injection of PP in wild type mice. Scale bar = 200 µm. (B) Higher magnification of the boxed area from A. Scale bar  = 5 µm. (C, E) Fluorescence micrographs of the nucleus tractus solitarius (NTS) and area postrema (AP) respectively, 30 minutes after i.p. injection of PP. Scale bar  = 40 µm. (D, F) Higher magnifications of the NTS and AP, respectively, from the boxed areas in C and E. Scale bar  = 25 µm. White arrows indicate neurons positive for c-Fos immunoreactivity only (red fluorescence); white arrowheads indicate neurons positive for TH immunoreactivity only (green fluorescence); blue arrows indicate neurons double-labeled for c-Fos and TH. 4V, fourth cerebral ventricle; cc, central canal.</p

Effects of pancreatic polypeptide (PP) on the expression of proopiomelanocortin (POMC) and GAD65 mRNA in the arcuate nucleus of the hypothalamus (ARC).

<p>Emulsion-dipped autoradiographs showing POMC mRNA at 30 minutes after i.p. injection of (A) saline or (C) PP, orGAD65 mRNA at 30 minutes after i.p. injection of (B) saline or (D) PP. Scale bar for all panels  = 25 µm. 3V, third cerebral ventricle.</p