Correlation between serum BDNF levels and scores on the <i>Perceived Stress Scale</i> (PSS) by insomnia severity groups according to the <i>Insomnia Severity Index</i> (ISI).

<p>Analyses showed a significant correlation (partial correlation controlled for smoking) between BDNF and stress only in subjects with no insomnia (<i>r_p</i> = −0.511, p = 0.013) compared to subjects with sub threshold (<i>r_p</i> = 0.069, <i>p</i> = 0.814) or clinical insomnia (<i>r_p</i> = 0.199, <i>p</i> = 0.608). White squares represent subjects with no insomnia, black circles represent subjects with sub threshold and black triangles represent subjects with clinical insomnia. (Inset) Mean serum BDNF levels of the insomnia severity groups. Plotted means and standard errors estimated by ANCOVA with serum BDNF as dependent variable, insomnia severity group as independent variable and smoking as covariate. For all three insomnia severity groups the overall effect on serum BDNF was not significant (<i>F</i>(2) = 2.67; <i>p</i> = 0.080). Contrasts showed that serum BDNF levels in the group with no insomnia were significantly higher compared to the groups reporting sub threshold and clinical insomnia (<i>F</i>(1) = 5.33; <i>p</i> = 0.026); (no insomnia n = 24; sub threshold insomnia n = 16, clinical insomnia n = 10). * Denotes statistical significance at <i>p</i><0.05</p

Application of PDF blocks outward K⁺ and inward Na⁺ current components.

<p>In whole-cell patch clamp recordings AMe neurons in primary cell cultures were kept at a holding potential of −60 mV. Voltage-dependent currents were activated by series of depolarizing voltage steps from −140 mV to +80 mV with 10 mV increments. <b>A₁–A₂</b>. Current traces before and after application of 500 nM PDF (2 min) to the extracellular solution indicate that PDF inhibits part of a delayed rectifier type potassium (K⁺) outward current. <b>B</b>. The I-V plot of the same recording at the position indicated by the arrows in <b>A₁ and A₂</b> shows the decline of outward currents while sustained, small inward currents, which counteract outward K⁺ currents at hyperpolarized potentials, are not affected. <b>C</b>. In another recording PDF blocks Ca²⁺-dependent outward K⁺ currents, which cause the characteristic downward bend of the outward currents, while apparently not affecting delayed rectifier type K⁺ currents, or small sustained inward Ca²⁺ currents which counteract K⁺ outward currents at hyperpolarizing potentials. <b>D</b>. In another AMe neuron PDF inhibits voltage-gated fast Na⁺ inward currents that activate around −40 mV. Washing in of PDF-free saline containing the Na⁺ channel antagonist tetrodotoxin (TTX) almost competely blocks the residual inward current.</p

Descriptive characteristics of study participants.

<p>A total sample size of N = 50 was included in the analysis. Descriptive data are presented in means (M) and standard deviations (SD). Absolute numbers of participants are given (N) and expressed as percentage (%).</p><p><b>Abbreviations:</b> means (<i>M</i>); standard deviation (<i>SD</i>), brain-derived neurotrophic factor (BDNF); Insomnia Severity Index (ISI); Perceived Stress Scale (PSS); restless legs syndrome (RLS), periodic limb movement (PLM).</p

Stress experience in subjects suffering from current insomnia.

<p>Correlation between insomnia severity score (indicated by the <i>Insomnia Severity Index</i> (ISI)) and stress perception (indicated by the <i>Perceived Stress Scale</i> (PSS)). Analysis showed a significant correlation between scores on the ISI and the PSS across the whole sample (<i>r_p</i> = 0.548, <i>p</i><0.001). * Denotes statistical significance at <i>p</i><0.05.</p

PDF responses of type 1 pacemakers are mediated by cAMP-dependent pathways.

<p>In addition, PDF effects appear to depend on intracellular Ca²⁺ baseline levels and thus, most likely on the membrane potential. <b>A</b>. Adenylyl cyclase (AC) inhibitor SQ22536 (20 µM) blocks forskolin (AC activator, 10 µM) -dependent rise of the Ca²⁺ baseline and Ca²⁺ activity in this continuous recording of type 1 AMe cells. <b>B</b>. Also, SQ22536 (20 µM) reversibly blocks the PDF-induced increase of the Ca²⁺ baseline and Ca²⁺ activity in type 1 cells. <b>C</b>. Application of 8-bromo cAMP (10 µM) produces rapid and large increases in Ca²⁺ levels, which were not blocked by SQ22536 (20 µM). <b>D</b>. Incubation of the Ca²⁺ channel antagonist mibefradil (10 µM) inhibits spontaneous activity and decreases Ca²⁺ baseline levels apparently causing membrane potential hyperpolarizations. Mibefradil-dependent effects prevent PDF (100 µM) responses in type 1 AMe neurons.</p

The spontaneous, regular activity of type 1 neurons depends on low voltage-activated (LVA) Ca²⁺ channels and on hyperpolarization-activated cyclic nucleotide-gated (HCN) pacemaker currents.

<p><b>A</b>. Type 1 AMe neurons express regular Ca²⁺ transients, which can be blocked by the voltage-dependent Ca²⁺ channel antagonist mibefradil (10 µM), the HCN-channel antagonist DK-AH 269 (10 µM; which also reduces the baseline), and the Na⁺ channel antagonist TTX (100 nM). Consecutive recordings during constant perfusion of the same pacemaker neuron reveal that TEA-dependent block of K⁺ channels does not mimic all PDF effects in type 1 cells. <b>B</b>. Type 1 pacemakers increase baseline Ca²⁺ levels, amplitude, and frequency of Ca²⁺ transients with increasing concentrations (1–20 mM) of the K⁺ channel blocker TEA. Coapplication of the Na⁺ channel antagonist TTX (50 nM) increases the speed of TEA effects while truncating their durations, favoring burst-activity. <b>C</b>. Increasing the concentration of TTX (10–100 nM) finally blocks spontaneous activity and shortens TEA responses to a brief burst.</p

Application of PDF to accessory medulla (AMe) pacemakers in primary cell culture allows to distinguish 4 different response types 1–4 in Ca²⁺ imaging experiments.

<p><b>A</b>. Type 1 AMe neurons express regular Ca²⁺ transients. PDF increases the frequency of spontaneous Ca²⁺ transients and the Ca²⁺ baseline dose-dependently and reversibly. <b>B</b>. Type 2 AMe neurons are not spontaneously active and have low baseline Ca²⁺ levels. PDF slowly increases Ca²⁺ baseline levels. <b>C</b>. Type 3 cells are less regularly spontaneously active than type 1 cells. PDF application slightly increases baseline Ca²⁺ levels while suppressing high amplitude Ca²⁺ transients. <b>D</b>. Type 4 AMe neurons have a high Ca²⁺ baseline level which is decreased by PDF application.</p

Hypothetical model of PDF signaling in spontaneously active type 1 circadian pacemaker neurons.

<p><b>A</b>. We suggest that PDF signals via adenylyl cylcase activity in type 1 cells. The cAMP-dependent block of K⁺ channels depolarizes the cell and thereby opens voltage-gated Ca²⁺ channels. The resulting rise in intracellular Ca²⁺ together with the rise in cAMP concentrations then feeds back to the molecular clockwork (TTFL) via activation of PKA and PKC, thereby phase-advancing circadian rhythms of circadian pacemaker neurons. <b>B</b>. In contrast, PDF-dependent block of Na⁺ channels hyperpolarizes and thereby phase-delays pacemaker neurons only via PKA, but not concomitant PKC-dependent feedback to the TTFL. <b>C</b>. When PDF blocks both K⁺ and Na⁺ channels it promotes rhythmic membrane potential oscillations and causes bursting. <b>D</b>. Finally, PDF promotes fast synchronization between two pacemakers, which are coupled via their common PDF-sensitivity. If the PDF releasing pacemaker is also PDF-sensitive, because of autoreceptor expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108757#pone.0108757-Choi2" target="_blank">[48]</a> it will synchronize with the postsynaptic PDF-sensitive pacemaker neuron. PDF-dependent rhythmic bursting is suggested to promote fast synchronization.</p

Aβ42 binds to and impairs ABAD activity, while HA (human amylin) does not.

<p>(A) Treatment of SH-SY5Y human neuroblastoma cells with Aβ42 causes decreased levels of estradiol, indicative of an impairment of ABAD activity, while HA does not. Results are means ± SE, (n = 5 to 6 per group), <b>**</b>, <i>P</i><0.01 (B) Pull-down of ABAD from SH-SY5Y cells shows that different from HA, Aβ42 can bind to ABAD <i>in vitro</i>. (C) Structure of the ABAD inhibitor, AG18051 (adapted from Kissinger et al., JMB 2004).</p

The ABAD inhibitor AG18051 partially prevents the toxicity of HA, but not its metabolic impairment.

<p>(A) Co-incubation of HA with AG18051 partially maintains levels of LDH release in SH-SY5Y cells suggesting that the toxicity of HA is partially mediated by ABAD. (B) Treatment with 0.5 µM HA significantly decreases metabolic activity as shown with the MTT assay, which is not prevented with co-incubation with AG18051. <b>*</b>, <i>P</i><0.05; <b>**</b>, <i>P</i><0.01.</p