Circadian Behaviour in Neuroglobin Deficient Mice

Neuroglobin (Ngb), a neuron-specific oxygen-binding globin with an unknown function, has been proposed to play a key role in neuronal survival. We have previously shown Ngb to be highly expressed in the rat suprachiasmatic nucleus (SCN). The present study addresses the effect of Ngb deficiency on circadian behavior. Ngb-deficient and wild-type (wt) mice were placed in running wheels and their activity rhythms, endogenous period and response to light stimuli were investigated. The effect of Ngb deficiency on the expression of Period1 (Per1) and the immediate early gene Fos was determined after light stimulation at night and the neurochemical phenotype of Ngb expressing neurons in wt mice was characterized. Loss of Ngb function had no effect on overall circadian entrainment, but resulted in a significantly larger phase delay of circadian rhythm upon light stimulation at early night. A light-induced increase in Per1, but not Fos, gene expression was observed in Ngb-deficient mice. Ngb expressing neurons which co-stored Gastrin Releasing Peptide (GRP) and were innervated from the eye and the geniculo-hypothalamic tract expressed FOS after light stimulation. No PER1 expression was observed in Ngb-positive neurons. The present study demonstrates for the first time that the genetic elimination of Ngb does not affect core clock function but evokes an increased behavioural response to light concomitant with increased Per1 gene expression in the SCN at early night

Handheld FTIR outperforms total organic carbon swab in pharmaceutical cleaning validation

Cleaning Validation is a crucial process in pharmaceutical manufacturing, as it verifies that cross-contamination levels are below acceptable limits. The use of Mid-Infrared (IR) spectroscopy has long been proposed as a rapid method enabling real-time release, but only with the recent emergence of a handheld Fourier-Transform IR (FTIR) does a feasible solution exist for practical implementation in pharmaceutical manufacturing plants. This paper address the model development challenges for multi-product plants without complete traceability of production equipment and produced product. This is done by developing a partial least squares discriminant analysis model determining if the sample is clean or not clean compared to a residual acceptance limit, based on total organic carbon (TOC) measurements. The model is built and tested on artificial samples printed with a chemical printer for multiple products with different spectral peaks based on 91 samples in the calibration set and tested on 30 samples in the validation set. Furthermore, the model also incorporates spectra from surfaces with different surface roughness. The evaluation of the model is based on sensitivity, specificity and class error. The model outperforms or performs equally well as TOC swab with sampling error, dependent on the sampling error distribution for the TOC swab

Light induced cFOS and PER1 in Ngb expressing neurons of the mouse SCN.

<p>The first column shows sections of mid SCN from a mouse euthanized at ZT 1730 without light stimulation. Ngb-IR (light blue) <b>A</b>, PER1 (green) <b>D</b> and cFOS (red) <b>G</b> are shown. Expression of Ngb-IR and PER1, but not cFOS-IR can been observed. Merged images <b>J–M</b> shows PER1/cFOS and PER1/cFOS/Ngb-IR, respectively. There was no co-expression between PER1 and Ngb. Similarly, in the mid column SCN from a mouse euthanized 90 min after light stimuli is shown. Note strong expression of cFOS (<b>H</b>) and a high degree of co-localization (yellow) with PER1-IR (<b>K</b>). No co-expression could be observed between Ngb and PER1-IR, but a subpopulation of Ngb-IR cells in the core was found to be cFOS positive (<b>N</b>). In the last column SCN from a mouse euthanized at ZT 20 after having received a 30 min light stimulus at ZT 16 is shown. Note cFOS-IR has disappeared (<b>I</b>) and substantially less PER1-IR (<b>L</b>) is expressed in both the nuclei and cytoplasm. No co-expression between Ngb-IR and PER1-IR could be seen (<b>O</b>). Oc; optic chiasm; 3 V, 3^rd ventricle. Scale bar 100 µm.</p

Re-entrainment after eight h phase shift of the LD cycle (jetlag) in Ngb deficient mice.

<p><b>A</b>. Representative actogram of Ngb wild type (red) and Ngb deficient mice (blue) entrained to a 12:12 h LD cycled followed by a eight h shift (delay) of the LD cycle. Bars in top of each actogram represent the LD cycle before the shift, the bars below the LD cycle after the shift. <b>B</b>. Quantitative analysis of the eight h phase delay of the LD cycle using the onset as phase marker (n = 7 of each genotype). Note that both groups re-entrain within three cycles. <b>C</b>. Quantitative analysis of the eight h phase delay of the LD cycle using the offset as phase marker (n = 7 of each genotype). Note both groups re-entrain within seven cycles.</p

Summary of activity data.

<p>Summary of activity data.</p

Light induced expression of <i>Per1</i> (A and C) and <i>cFos</i> (B and D) mRNA in Ngb deficient mice during early night.

<p>Left panels (A–B) show the results of quantitative analysis using RT-PCR on SCN tissue ((mRNA/ß2MG mRNA (arbitrary units), see material and methods) and the results in the right panel (C and D) are obtained by semi-quantitative in situ hybridization (optical density, see material and methods). <b>A</b>–<b>B</b>. A 30 min light pulse induced <i>Per1</i> expression in Ngb deficient mice compared to wild type controls which is significant determined by RT-PCR on SCN tissue. Light stimulation significantly induces <i>cFos</i> expression in both genotypes but no difference was found in between the two genotypes (<b>B</b> and <b>D</b>). Error bars = S.E.M., Mann Whitney-U test.</p

Antibodies.

<p>Antibodies.</p

Innervation and co-expression of Ngb in the mouse SCN.

<p>In <b>A–D</b> Ngb-IR (green) can be seen in both the neuronal cell body and processes throughout the mid and ventral part of the SCN. Highest expression of Ngb-IR was observed in the mid/core part of SCN from rostral (<b>A</b>), mid (<b>B–C</b>) and in the ventral-lateral part in the caudal SCN (<b>D</b>). In (<b>E</b>) visual input from the RHT is depicted with cholera toxin subunit B (Ctb) (red). A high degree of innervation of Ngb-IR cells (green) was observed in the ventral and mid part of the SCN shown with white colour (arrows). Likewise Ngb-IR cells were also innervated by NPY-IR fibres (red) originating from the GHT (arrows) (<b>F</b>). In colchicine treated mice no co-expression of Ngb-IR and VIP-IR could be seen (<b>G</b>). Most GRP-IR cells were seen to co-express Ngb-IR (arrows) in colchicine treated mice (<b>H</b>). Oc; optic chiasm; 3 V, 3^rd ventricle. Scale bars 50 µm.</p

Running wheel activity in Ngb deficient mice.

<p><b>A</b>. Double plot actogram representing running wheel activity of Ngb wild type (<b>A</b>) and Ngb deficient mice (<b>B</b>) during a period of 12:12 h LD cycle followed by constant darkness (DD), re-entrainment to a new LD cycle followed by a period of constant light (LL). Both groups behave similarly and have a TAU not significantly different between the two genotypes. <b>C</b> and <b>D</b>. Light stimulation at ZT16 and ZT22 result in phase delays and phase advance, respectively which is illustrated in wild type animals (<b>C</b>) and in Ngb deficient mice (<b>D</b>). Ngb deficient mice display a significantly larger phase delay at ZT16 as shown in E. Yellow arrows indicates the time of the light pulse. Error bars = S.E.M., Mann Whitney-U test.</p