Predictable enriched environment prevents development of hyper-emotionality in the VPA rat model of autism.

Understanding the effects of environmental stimulation in autism can improve therapeutic interventions against debilitating sensory overload, social withdrawal, fear and anxiety. Here, we evaluate the role of environmental predictability on behavior and protein expression, and inter-individual differences, in the valproic acid (VPA) model of autism. Male rats embryonically exposed (E11.5) either to VPA, a known autism risk factor in humans, or to saline, were housed from weaning into adulthood in a standard laboratory environment, an unpredictably enriched environment, or a predictably enriched environment. Animals were tested for sociability, nociception, stereotypy, fear conditioning and anxiety, and for tissue content of glutamate signaling proteins in the primary somatosensory cortex, hippocampus and amygdala, and of corticosterone in plasma, amygdala and hippocampus. Standard group analyses on separate measures were complemented with a composite emotionality score, using Cronbach’s Alpha analysis, and with multivariate profiling of individual animals, using Hierarchical Cluster Analysis. We found that predictable environmental enrichment prevented the development of hyper-emotionality in the VPA-exposed group, while unpredictable enrichment did not. Individual variation in the severity of the autistic-like symptoms (fear, anxiety, social withdrawal and sensory abnormalities) correlated with neurochemical profiles, and predicted their responsiveness to predictability in the environment. In saline-exposed animals, the association between socio-affective behaviors, neurochemical profiles and environmental predictability was negligible. This study suggests that rearing in a predictable environment prevents the development of hyper-emotional features in animals exposed to an autism risk factor, and demonstrates that unpredictable environments can lead to negative outcomes, even in the presence of environmental enrichment

Fronto-limbic neural variability as a transdiagnostic correlate of emotion dysregulation

Emotion dysregulation is central to the development and maintenance of psychopathology, and is common across many psychiatric disorders. Neurobiological models of emotion dysregulation involve the fronto-limbic brain network, including in particular the amygdala and prefrontal cortex (PFC). Neural variability has recently been suggested as an index of cognitive flexibility. We hypothesized that within-subject neural variability in the fronto-limbic network would be related to inter-individual variation in emotion dysregulation in the context of low affective control. In a multi-site cohort (N = 166, 93 females) of healthy individuals and individuals with emotional dysregulation (attention deficit/hyperactivity disorder (ADHD), bipolar disorder (BD), and borderline personality disorder (BPD)), we applied partial least squares (PLS), a multivariate data-driven technique, to derive latent components yielding maximal covariance between blood-oxygen level-dependent (BOLD) signal variability at rest and emotion dysregulation, as expressed by affective lability, depression and mania scores. PLS revealed one significant latent component (r = 0.62, p = 0.044), whereby greater emotion dysregulation was associated with increased neural variability in the amygdala, hippocampus, ventromedial, dorsomedial and dorsolateral PFC, insula and motor cortex, and decreased neural variability in occipital regions. This spatial pattern bears a striking resemblance to the fronto-limbic network, which is thought to subserve emotion regulation, and is impaired in individuals with ADHD, BD, and BPD. Our work supports emotion dysregulation as a transdiagnostic dimension with neurobiological underpinnings that transcend diagnostic boundaries, and adds evidence to neural variability being a relevant proxy of neural efficiency

Dynamics of amygdala connectivity in bipolar disorders: a longitudinal study across mood states

Alterations in activity and connectivity of brain circuits implicated in emotion processing and emotion regulation have been observed during resting state for different clinical phases of bipolar disorders (BD), but longitudinal investigations across different mood states in the same patients are still rare. Furthermore, measuring dynamics of functional connectivity patterns offers a powerful method to explore changes in the brain's intrinsic functional organization across mood states. We used a novel co- activation pattern (CAP) analysis to explore the dynamics of amygdala connectivity at rest in a cohort of 20 BD patients prospectively followed-up and scanned across distinct mood states: euthymia (20 patients; 39 sessions), depression (12 patients; 18 sessions), or mania/hypomania (14 patients; 18 sessions). We compared them to 41 healthy controls scanned once or twice (55 sessions). We characterized temporal aspects of dynamic fluctuations in amygdala connectivity over the whole brain as a function of current mood. We identified six distinct networks describing amygdala connectivity, among which an interoceptive- sensorimotor CAP exhibited more frequent occurrences during hypomania compared to other mood states, and predicted more severe symptoms of irritability and motor agitation. In contrast, a default-mode CAP exhibited more frequent occurrences during depression compared to other mood states and compared to controls, with a positive association with depression severity. Our results reveal distinctive interactions between amygdala and distributed brain networks in different mood states, and foster research on interoception and default-mode systems especially during the manic and depressive phase, respectively. Our study also demonstrates the benefits of assessing brain dynamics in BD

nLDL but not oxLDL particles are converted in lipid droplets in INS-1E cells.

<p>(A) Oil Red O staining of INS-1E cells cultured for 24 h in presence or absence (Vh for Vehicule) of 2 mM nLDL or oxLDL particles (black bar = 10 µm). Arrows point to lipid droplets. (B) Quantitative assessment of Plin1 (black bars) and ACC1 (white bars) over L27 mRNA expression in INS-1E cells cultured for 48 h in presence of 2 mM nLDL or oxLDL particles. *P<0.05, **P<0.01 vs. Vh condition. ^$P<0.05, vs. nLDL condition.</p

Cx36 overexpression protects INS-1E cells against oxLDL-induced apoptosis.

<p>(A–C) INS-1E cells were transfected with a control siRNA (siCtrl) or two different Cx36 siRNA (siCx36#1 and #2). (A) <i>Upper panel</i>: representative Western blot of Cx36 over tubulin expression. <i>Lower panel:</i> data are means ± SEM of 4 independent experiments. (B) Prevalence of apoptosis was evaluated by HO-PI staining after 48 h of exposure to 2 mM nLDL or oxLDL particles. (C) ROS/RNS production was evaluated in live cells after 48 h of exposure to 2 mM nLDL or oxLDL particles. (B–C) Data are means ± SEM of five experiments. *P<0.05, **P<0.01, ***P<0.001 vs. Vh condition.^#P<0.05 ^##P<0.01 vs. siCtrl-transfected condition. (D) Islets from WT (+/+) or Cx36 KO (−/−) mice were exposed for 72 h to 2 mM nLDL or oxLDL particles. Islets viability was evaluated by HO-PI staining. Data are aligned dot plots of individual islet viability values from five animals per group (+/+ or −/−) and two separated LDL preparations. Horizontal bars show mean value ± SEM. n.s. (non-significant), *P<0.05 vs respective WT values (χ2 tests; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055198#pone.0055198.s002" target="_blank">table S1</a>). (E) INS-1E cells were infected or not (NI) with a control adenovirus (Ad-GFP) or a rat Cx36 adenovirus (Ad-Cx36) at various multiplicity of infection (MOI) as indicated. Cells were then exposed or not (Vh for Vehicule) for 48 h to 2 mM native (nLDL) or oxidized LDL (oxLDL). <i>Upper panel:</i> Representative WB of Cx36 over tubulin expression after infection. <i>Lower panel</i>: prevalence of apoptosis was evaluated by HO-PI staining. Data are mean ± SEM of at least 4 independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs. respective Vh and nLDL conditions. ^#P<0.05, ^##P<0.01 vs. respective non-infected and Ad-GFP-infected conditions.</p

Prolonged exposure to oxidized LDL particles decreases Cx36 levels in β-cells.

<p>A) Langerhans islets were isolated from adult male WT or ApoE−/− C57Bl6 mice and immediatly sampled for mRNA extractions. Cx36 and ICER-1γ mRNA levels were analyzed by quantitative RT-PCR, and normalized to the expression of the housekeeping gene L27. Results are means ± SEM of six animals in each group. B) INS-1E cells, MIN6-B1 cells, or primary rat islets were cultured for 72 h in presence or absence (Vh for Vehicule) of 2 mM native (nLDL) or oxidized LDL (oxLDL). Quantitative RT-PCR of Cx36 mRNA levels normalized to the levels of the housekeeping gene L27. Data represent mean ± SEM of four to six independent experiments. *P<0.05; **P<0.01 vs. control. C) Time course analysis of Cx36 mRNA levels in INS-1E cells cultured in absence (Vh for Vehicule) or presence of 2 mM native (nLDL) or oxidized LDL (oxLDL). Data are mean ± SEM of four independent experiments. *P<0.05; **P<0.01 vs control condition (vehicle treated). D–E)Western blot analyses of Cx36 levels in INS-1E cells (D) or primary isolated rat islets (E) cultured for 48 h in presence or absence (Vh for Vehicule) of 2 mM native (nLDL) or oxidized LDL (oxLDL). (D) Data are mean ± SEM of five independent experiments. (E) Blot shows three independent experiments.</p

ICER-1 overexpression mediates the effect of oxLDL on Cx36 expression.

<p>A) INS-1E cells were cotransfected with a plasmid expressing the reporter gene luciferase under the control of a 1 kb fragment of the <i>CX36</i> promoter, or a fragment containing a mutated CRE, together with an empty vector (pCDNA3; black bars) or an antisense ICER plasmid (ICER AS; white bars). 24 h post transfection, cells were cultured for 48 h in presence or absence (Vh for Vehicule) of 2 mM nLDL or oxidized LDL oxLDL. Cx36 promoter activity was evaluated by luciferase assay. Data are mean ± SEM of five to six experiments. **P<0.01 vs. vehicle-treated cells. B) Western Blot analyses of Cx36 over tubulin levels in INS-1E cells non transfected (NT), transfected with an empty vector (pCDNA3) or the antisense ICER plasmid (ICER AS) and treated with native LDL (nLDL) or two different preparations of oxidized LDL (oxLDL1 or 2). Data are representative of three independent experiments.</p

oxLDL overexpress ICER-1 and downregulate Cx36 in a PKA-dependent manner.

<p>A)Western Blot analyses of Cx36 over tubulin levels in INS-1E cells cultured for 48 h in presence or absence (Vh for Vehicule) of 2 mM native (nLDL) or oxidized LDL (oxLDL), together or not with the PKA inhibitor H89 (10 µM). <i>Upper panel:</i> representative Western blot; <i>lower panel</i>: means ± SEM of three independent Western blots. ***P<0.01 vs. INS-1E control. #P<0.05 vs. oxLDL condition in absence of H89. B) Quantitative RT-PCR of ICER-1 over L27 mRNA expression in INS-1E cells, MIN6-B1 cells, or primary rat islets cultured for 72 h in presence or absence (Vh for Vehicule) of 2 mM native (nLDL) or oxidized LDL (oxLDL). Data represent mean ± SEM of four to six independent experiments. C) Time course analysis of ICER-1 mRNA levels in INS-1E cells cultured in presence of oxLDL. *P<0.05; **P<0.01 vs control condition (vehicle treated). D) Western Blot analyses of ICER-1 and ICER-1γ levels in INS-1E cells cultured for 48 h in presence or absence (Vh for Vehicule) of 2 mM native (nLDL) or oxidized LDL (oxLDL), together or not with the PKA inhibitor H89. <i>Upper panel:</i> representative Western blot; <i>lower panel</i>: Results are means ± SEM of three independent Western blots. ***P<0.01 vs. INS-1E control.</p