Cell Proliferation, Movement and Differentiation during Maintenance of the Adult Mouse Adrenal Cortex

Appropriate maintenance and regeneration of adult endocrine organs is important in both normal physiology and disease. We investigated cell proliferation, movement and differentiation in the adult mouse adrenal cortex, using different 5-bromo-2'-deoxyuridine (BrdU) labelling regimens and immunostaining for phenotypic steroidogenic cell markers. Pulse-labelling showed that cell division was largely confined to the outer cortex, with most cells moving inwards towards the medulla at around 13-20 µm per day, though a distinct labelled cell population remained in the outer 10% of the cortex. Pulse-chase-labelling coupled with phenotypic immunostaining showed that, unlike cells in the inner cortex, most BrdU-positive outer cortical cells did not express steroidogenic markers, while co-staining for BrdU and Ki67 revealed that some outer cortical BrdU-positive cells were induced to proliferate following acute adrenocorticotropic hormone (ACTH) treatment. Extended pulse-chase-labelling identified cells in the outer cortex which retained BrdU label for up to 18-23 weeks. Together, these observations are consistent with the location of both slow-cycling stem/progenitor and transiently amplifying cell populations in the outer cortex. Understanding the relationships between these distinct adrenocortical cell populations will be crucial to clarify mechanisms underpinning adrenocortical maintenance and long-term adaptation to pathophysiological states

Isolation of Mutations Affecting Neural Circuitry Required for Grooming Behavior in Drosophila Melanogaster

We have developed a screen for the isolation of mutations that produce neural defects in adult Drosophila melanogaster. In this screen, we identify mutants as flies unable to remove a light coating of applied dust in a 2-hr period. We have recovered and characterized six mutations and have found that they produce coordination defects and some have reduced levels of reflex responsiveness to the stimulation of single tactile sensory bristles. The grooming defects produced by all six of the mutations are recessive, and each of the mutations has been genetically mapped. We have also used our assay to test the grooming ability of stocks containing mutations that produce known neural defects

Subdiaphragmatic Vagotomy Prevents Drinking-Induced Reduction in Plasma Corticosterone in Water-Restricted Rats

Dehydrated rats exhibit a rapid inhibition of the hypothalamic-pituitary-adrenal axis after rehydration. Drinking activates vagal afferents that project to neurons in the nucleus tractus solitarius (NTS). We hypothesized that when dehydrated rats drink, vagal afferents stimulate NTS neurons initiating inhibition of hypothalamic-pituitary-adrenal activity. Experiments assessed NTS activity by measuring Fos expression. Rats were water restricted for 1 or 6 d, limiting access to water to 30 min/d in the morning. Drinking after single or repeated restriction increased Fos, demonstrating increased NTS activity. We next examined the contribution of the vagus by comparing hormonal responses after total subdiaphragmatic vagotomy or sham surgery. Water restriction for 6 d increased plasma arginine vasopressin (AVP), ACTH, and adrenal and plasma corticosterone in both groups. In sham rats, drinking reduced plasma AVP, ACTH, adrenal and plasma corticosterone by 7.5 min. In total subdiaphragmatic vagotomy rats, whereas drinking reduced plasma AVP, ACTH, and adrenal corticosterone, drinking did not reduce plasma corticosterone. To identify the source of vagal activity, hormonal responses to restriction-induced drinking were measured after common hepatic branch vagotomy (HBV). Although pituitary hormonal responses were not affected by HBV, the adrenal and plasma corticosterone responses to water restriction were reduced; in addition, drinking in HBV rats decreased adrenal corticosterone without changing plasma corticosterone. These data indicate that an intact vagus is necessary to reduce plasma corticosterone when water-restricted rats drink and that the common hepatic vagal branch contributes to the response. These findings implicate the vagus in augmenting rapid removal of circulating corticosterone during relief from stress

Forced desynchrony reveals independent contributions of suprachiasmatic oscillators to the daily plasma corticosterone rhythm in male rats.

The suprachiasmatic nucleus (SCN) is required for the daily rhythm of plasma glucocorticoids; however, the independent contributions from oscillators within the different subregions of the SCN to the glucocorticoid rhythm remain unclear. Here, we use genetically and neurologically intact, forced desynchronized rats to test the hypothesis that the daily rhythm of the glucocorticoid, corticosterone, is regulated by both light responsive and light-dissociated circadian oscillators in the ventrolateral (vl-) and dorsomedial (dm-) SCN, respectively. We show that when the vlSCN and dmSCN are in maximum phase misalignment, the peak of the plasma corticosterone rhythm is shifted and the amplitude reduced; whereas, the peak of the plasma adrenocorticotropic hormone (ACTH) rhythm is also reduced, the phase is dissociated from that of the corticosterone rhythm. These data support previous studies suggesting an ACTH-independent pathway contributes to the corticosterone rhythm. To determine if either SCN subregion independently regulates corticosterone through the sympathetic nervous system, we compared unilateral adrenalectomized, desynchronized rats that had undergone either transection of the thoracic splanchnic nerve or sham transection to the remaining adrenal. Splanchnicectomy reduced and phase advanced the peak of both the corticosterone and ACTH rhythms. These data suggest that both the vlSCN and dmSCN contribute to the corticosterone rhythm by both reducing plasma ACTH and differentially regulating plasma corticosterone through an ACTH- and sympathetic nervous system-independent pathway

The plasma corticosterone rhythm is disrupted on days of misalignment.

<p>Plasma corticosterone (A), ACTH (B) and corticosterone/log ACTH (C) on aligned (<i>black</i>) and misaligned (<i>grey</i>) days. Black horizontal bars denote times when lights are off. Illustrations (<i>top</i>), as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068793#pone-0068793-g001" target="_blank">Figure 1E</a>, show predicted <i>Per1</i> expression patterns in the subregions of the SCN at the midpoint of the lights-on and lights-off on days of alignment and misalignment. Data are means ± SEM and fitted with a sine wave function (see materials and methods). n = 5; the same animals make up both aligned and misaligned groups.</p

Model of output from SCN subregions to the anterior pituitary and adrenal glands.

<p>During the light phase, the dmSCN and vlSCN inhibit ACTH release from the pituitary, while the dmSCN slowly increases plasma corticosterone through either decreasing inhibitory, or increasing stimulatory, output to the adrenal. At the same time, the vlSCN supplies inhibitory output to the adrenals over the light period, keeping plasma corticosterone low until the onset of the dark phase, at which time the pituitary is released from inhibition, resulting in a heightened plasma corticosterone response to ACTH.</p

Plasma corticosterone and ACTH rhythms are disrupted in Sham-operated forced desynchronized rats.

<p>Plasma corticosterone (A) and ACTH (B) in Sham treated rats on aligned (<i>black</i>) and misaligned (<i>grey</i>) days. Black horizontal bars denote times when lights are off. Illustrations (<i>top</i>) show predicted <i>Per1</i> expression patterns, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068793#pone-0068793-g002" target="_blank">Figure 2</a>. Data are means ± SEM and fitted with a sine wave function (see materials and methods). n= 6 rats/group.</p

Forced desynchrony disrupts the plasma corticosterone rhythm.

<p>A) Double-plotted locomotor activity of a representative forced desynchronized rat. Grey bars represent locomotor activity; double diagonal bands indicate the time of lights-on (<i>for box “C”</i>) or time of lights-off (<i>for box “D”</i>); dashed diagonal lines indicate the onset of dmSCN-associated locomotor activity bout with the angle set to match the LD-dissociated period indicated in the periodogram (B); black horizontal boxes indicate the 24-h periods during which blood samples were taken every two hours during misaligned (<i>top, “C”</i>) or aligned (<i>bottom, “D”</i>) phases. B) The X² periodogram analysis of locomotor activity revealing two statistically significant rhythmic components (22 h and 25 h) in the animal shown in (A). C and D) The plasma corticosterone profiles (raw data) determined for the one animal shown in (A) during the days of misalignment and alignment, respectively. The dotted line in (C) represents predicted values between initial (t = 10: 00) and final (t = 8: 00) sampling times. Grey shading indicates time of lights off. E) Schematic diagram of predicted <i>Per1</i> expression patterns in the subregions of the SCN at 5.5 h after lights-on or lights-off on days of alignment and misalignment. Predicted <i>Per1</i> expression patterns (grey and white for high and low expression, respectively) are based on previous studies [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068793#B20" target="_blank">20</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068793#B22" target="_blank">22</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068793#B28" target="_blank">28</a>].</p