Prefrontal GABAA(δ)R Promotes Fear Extinction through Enabling the Plastic Regulation of Neuronal Intrinsic Excitability.

Extinguishing the previously acquired fear is critical for the adaptation of an organism to the ever-changing environment, a process requiring the engagement of GABAA receptors (GABAARs). GABAARs consist of tens of structurally, pharmacologically, and functionally heterogeneous subtypes. However, the specific roles of these subtypes in fear extinction remain largely unexplored. Here, we observed that in the medial prefrontal cortex (mPFC), a core region for mood regulation, the extrasynaptically situated, δ-subunit-containing GABAARs [GABAA(δ)Rs], had a permissive role in tuning fear extinction in male mice, an effect sharply contrasting to the established but suppressive role by the whole GABAAR family. First, the fear extinction in individual mice was positively correlated with the level of GABAA(δ)R expression and function in their mPFC. Second, knockdown of GABAA(δ)R in mPFC, specifically in its infralimbic (IL) subregion, sufficed to impair the fear extinction in mice. Third, GABAA(δ)R-deficient mice also showed fear extinction deficits, and re-expressing GABAA(δ)Rs in the IL of these mice rescued the impaired extinction. Further mechanistic studies demonstrated that the permissive effect of GABAA(δ)R was associated with its role in enabling the extinction-evoked plastic regulation of neuronal excitability in IL projection neurons. By contrast, GABAA(δ)R had little influence on the extinction-evoked plasticity of glutamatergic transmission in these cells. Altogether, our findings revealed an unconventional and permissive role of extrasynaptic GABAA receptors in fear extinction through a route relying on nonsynaptic plasticity.SIGNIFICANCE STATEMENT The medial prefrontal cortex (mPFC) is one of the kernel brain regions engaged in fear extinction. Previous studies have repetitively shown that the GABAA receptor (GABAAR) family in this region act to suppress fear extinction. However, the roles of specific GABAAR subtypes in mPFC are largely unknown. We observed that the GABAAR-containing δ-subunit [GABAA(δ)R], a subtype of GABAARs exclusively situated in the extrasynaptic membrane and mediating the tonic neuronal inhibition, works oppositely to the whole GABAAR family and promotes (but does not suppress) fear extinction. More interestingly, in striking contrast to the synaptic GABAARs that suppress fear extinction by breaking the extinction-evoked plasticity of glutamatergic transmission, the GABAA(δ)R promotes fear extinction through enabling the plastic regulation of neuronal excitability in the infralimbic subregion of mPFC. Our findings thus reveal an unconventional role of GABAA(δ)R in promoting fear extinction through a route relying on nonsynaptic plasticity.

Chronic microaspiration of bile acids induces lung fibrosis through multiple mechanisms in rats.

Gastroesophageal reflux (GER) and microaspiration of duodenogastric refluxate have been recognized as a risk factor for pulmonary fibrosis. Recent evidence suggests that bile acid microaspiration may contribute to the development of lung fibrosis. However, the molecular evidence is scarce and the underlying mechanisms remain to be elucidated. We have recently demonstrated that bile acids induce activation of alveolar epithelial cells (AECs) and lung fibroblasts in vitro In the present study, a rat model of bile acid microaspiration was established by weekly intratracheal instillation of three major bile acids including chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), and lithocholic acid (LCA). Repeated microaspiration of CDCA, DCA, and LCA caused fibrotic changes, including alveolar wall thickening and extensive collagen deposition, in rat lungs. Bile acid microaspiration also induced alveolar epithelial-mesenchymal transition (EMT), as indicated by up-regulation of mesenchymal markers α-smooth muscle actin (α-SMA) and vimentin, as well as down-regulaton of epithelial markers E-cadherin and cytokeratin in alveolar epithelium of rat lungs. The expression of fibrogenic mediators, including transforming growth factor-ß1 (TGF-ß1), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and periostin, was significantly elevated in rat lungs exposed to microaspiration of bile acids. Furthermore, microaspiration of bile acids also induced p-Smad3 and farnesoid X receptor (FXR) expression in rat lungs. Our findings suggest that microaspiration of bile acids could promote the development of pulmonary fibrosis in vivo, possibly via stimulating fibrogenic mediator expression and activating TGF-ß1/Smad3 signaling and FXR.

Bile acids induce activation of alveolar epithelial cells and lung fibroblasts through farnesoid X receptor-dependent and independent pathways.

BACKGROUND AND OBJECTIVE: The roles of bile acid microaspiration and bile acid-activated farnesoid X receptor (FXR) in the pathogenesis of idiopathic pulmonary fibrosis (IPF) remain unclear. We hypothesized that bile acids activate alveolar epithelial cells (AECs) and lung fibroblasts, which may be regulated by FXR activation. METHODS: Human AECs and normal or IPF-derived lung fibroblast cells were incubated with the three major bile acids: lithocholic acid (LCA), deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA). The AECs injury indices, epithelial-mesenchymal transition (EMT) and lung fibroblast activation were evaluated. FXR expression in IPF lungs and the roles of FXR and FXR-independent pathways in bile acid-induced profibrotic effects were also investigated. RESULTS: LCA, DCA and CDCA reduced cell viability and increased intracellular reactive oxygen species (ROS) production in A549 cells. They all induced EMT, as shown by enhanced α-SMA and vimentin and decreased E-cadherin levels. LCA directly induced differentiation of lung fibroblasts to myofibroblasts. All three bile acids promoted cellular migration but not proliferation of lung fibroblasts. FXR expression was upregulated in IPF lungs, and inhibition of FXR restrained the bile acid-induced EMT and lung fibroblast activation. Differentiation and proliferation were enhanced in lung fibroblasts exposed to conditioned medium from bile acid-stimulated A549 cells, which contained increased levels of profibrotic factors. TGF-ß/Smad3 signaling was also involved in the bile acid-induced EMT and lung fibroblast differentiation. CONCLUSION: Bile acid microaspiration may promote the development of pulmonary fibrosis by inducing activation of AECs and lung fibroblasts via FXR-dependent and independent pathways.

GABAA(δ) receptor hypofunction in the amygdala-hippocampal circuit underlies stress-induced anxiety.

Dysregulated GABAergic inhibition in the amygdala has long been implicated in stress-related neuropsychiatric disorders. However, the molecular and circuit mechanisms underlying the dysregulation remain elusive. Here, by using a mouse model of chronic social defeat stress (CSDS), we observed that the dysregulation varied drastically across individual projection neurons (PNs) in the basolateral amygdala (BLA), one of the kernel amygdala subregions critical for stress coping. While persistently reducing the extrasynaptic GABAA receptor (GABAAR)-mediated tonic current in the BLA PNs projecting to the ventral hippocampus (BLA â†’ vHPC PNs), CSDS increased the current in those projecting to the anterodorsal bed nucleus of stria terminalis (BLA â†’ adBNST PNs), suggesting projection-based dysregulation of tonic inhibition in BLA PNs by CSDS. Transcriptional and electrophysiological analysis revealed that the opposite CSDS influences were mediated by loss- and gain-of-function of δ-containing GABAARs (GABAA(δ)Rs) in BLA â†’ vHPC and BLA â†’ adBNST PNs, respectively. Importantly, it was the lost inhibition in the former population but not the augmentation in the latter population that correlated with the increased anxiety-like behavior in CSDS mice. Virally mediated maintenance of GABAA(δ)R currents in BLA â†’ vHPC PNs occluded CSDS-induced anxiety-like behavior. These findings clarify the molecular substrate for the dysregulated GABAergic inhibition in amygdala circuits for stress-associated psychopathology.

Identification of a prefrontal cortex-to-amygdala pathway for chronic stress-induced anxiety.

Dysregulated prefrontal control over amygdala is engaged in the pathogenesis of psychiatric diseases including depression and anxiety disorders. Here we show that, in a rodent anxiety model induced by chronic restraint stress (CRS), the dysregulation occurs in basolateral amygdala projection neurons receiving mono-directional inputs from dorsomedial prefrontal cortex (dmPFC→BLA PNs) rather than those reciprocally connected with dmPFC (dmPFC↔BLA PNs). Specifically, CRS shifts the dmPFC-driven excitatory-inhibitory balance towards excitation in the former, but not latter population. Such specificity is preferential to connections made by dmPFC, caused by enhanced presynaptic glutamate release, and highly correlated with the increased anxiety-like behavior in stressed mice. Importantly, low-frequency optogenetic stimulation of dmPFC afferents in BLA normalizes the enhanced prefrontal glutamate release onto dmPFC→BLA PNs and lastingly attenuates CRS-induced increase of anxiety-like behavior. Our findings thus reveal a target cell-based dysregulation of mPFC-to-amygdala transmission for stress-induced anxiety.

Gender-dependent regulation of anxiety-like behavior by δ subunit-containing GABAA receptor during postnatal development.

The δ subunit-containing GABAA receptor [GABAA(δ)R], which is exclusively situated in the extrasynaptic space, has considerable influence on emotion and behavior. Although the expression of this receptor experiences dramatic fluctuation during postnatal development, it remains unknown whether it regulates emotion in a development-dependent manner. Here, by using mice with genetic deletion of GABAA(δ)R (knockout) and their wild-type littermates, we examined the role of GABAA(δ)R in regulating anxiety-like behavior, as measured with open field test (OFT) and elevated plus maze during the transition from puberty to adulthood. We observed that for female mice, the knockout ones at puberty but not adulthood showed increased anxiety-like behavior in the OFT relative to their wild-type littermates. However, such increase was not observed in elevated plus maze. For male mice, no between-genotype differences were observed in both tests at the above two developmental stages. Our results suggest that GABAA(δ)R preferentially affects the anxiety-like behavior in OFT in a development-dependent manner, but only in female mice.

Chronic Stress Causes Projection-Specific Adaptation of Amygdala Neurons via Small-Conductance Calcium-Activated Potassium Channel Downregulation.

BACKGROUND: The role of the amygdala in mediating stress coping has been long appreciated. However, basolateral amygdala (BLA) projection neurons (PNs) are organized into discrete output circuits, and it remains unclear whether stress differentially impacts these circuits. METHODS: Mice were exposed to acute restraint stress or chronic restraint stress (CRS), and c-fos expression was measured as a proxy for neuronal activation in Retrobead retrogradely labeled dorsomedial prefrontal cortex-targeting PNs (BLA→dmPFC) and non-dmPFC-targeting PNs (BLA↛dmPFC). Next, the effects of CRS on neuronal firing and membrane potassium channel current were examined via ex vivo electrophysiology in these neuronal populations and correlated with anxiety-like behavior, as measured in the elevated plus maze and novel open field tests. Lastly, the ability of virus-mediated overexpression of subtype 2 of small-conductance, calcium-activated potassium (SK2) channel in BLA↛dmPFC PNs to negate the anxiety-related effects of CRS was assessed. RESULTS: BLA→dmPFC PNs were transiently activated after CRS, whereas BLA↛dmPFC showed sustained c-fos expression and augmented firing to external input. CRS led to a loss of SK2 channel-mediated currents in BLA↛dmPFC PNs, which correlated with heightened anxiety-like behavior. Virus-mediated maintenance of SK2 channel currents in BLA↛dmPFC PNs prevented CRS-induced anxiety-like behavior. Finally, CRS produced persistent activation of BLA PNs targeting the ventral hippocampus, and virally overexpressing SK2 channels in this projection population were sufficient to prevent CRS-induced anxiety-like behavior. CONCLUSIONS: The current data reveal that chronic stress produces projection-specific functional adaptations in BLA PNs. These findings offer new insight into the neural circuits that contribute to stress-induced psychopathology.

Harmine enhances GABAergic transmission onto basoamygdala projection neurons in mice.

Emerging evidence indicates that loss of inhibitory tone in amygdala with its subsequent overactivation contributes to the development of multiple mental disorders such as anxiety disorders and post-traumatic stress disorder (PTSD). Harmine is a member of natural ß-carboline alkaloids which can readily cross the blood brain barrier and displays significant antidepressant and anxiolytic effects in rodents. However, the underlying neurobiological mechanisms are largely unknown. Here, by using whole-cell patch clamp recordings in in vitro amygdala slices, we examined the effect of harmine on glutamatergic and GABAergic transmission onto basal amygdala (BA) projection neurons (PNs). Our results showed that harmine affected neither the amplitude nor the frequency of spontaneous and miniature excitatory postsynaptic currents (sEPSCs/mEPSCs) of PNs. By contrast, it markedly increased both the amplitude and frequency of the spontaneous inhibitory postsynaptic currents (sIPSCs). For mIPSCs, only an increase of their frequency but not amplitude was observed following harmine perfusion, suggesting that harmine might act through presynaptic mechanism. In parallel, a reduction of paired-pulse ratio of evoked IPSCs emerged in the presence of harmine. Furthermore, the intrinsic excitability of PNs was dramatically decreased upon harmine treatment. Together, our study suggests that harmine selectively potentiates the inhibitory but not excitatory transmission onto BA PNs, which may contribute to its antidepressant and anxiolytic influence.

Overexpression of farnesoid X receptor in small airways contributes to epithelial to mesenchymal transition and COX-2 expression in chronic obstructive pulmonary disease.

BACKGROUND: Epithelial-mesenchymal transition (EMT) and cyclooxygenase-2 (COX-2) contribute to airway remodelling and inflammation in chronic obstructive pulmonary disease (COPD). Recent data suggest that the farnesoid X receptor (FXR), a nuclear receptor traditionally considered as bile acid-activated receptor, is also expressed in non-classical bile acids target tissues with novel functions beyond regulating bile acid homeostasis. This study aimed to investigate the potential role of FXR in the development of COPD, as well as factors that affect FXR expression. METHODS: Expression of FXR, EMT biomarkers and COX-2 was examined by immunohistochemistry in lung tissues from non-smokers, smokers, and smokers with COPD. The role of FXR in TGF-ß1-induced EMT and COX-2 expression in human bronchial epithelial (HBE) cells was evaluated in vitro. Factors regulating FXR expression were assessed in cultured HBE cells and a cigarette smoke-induced rat model of COPD. RESULTS: Expression of FXR, EMT markers and COX-2 was significantly elevated in small airway epithelium of COPD patients compared with controls. The staining scores of FXR in small airway epithelium were negatively related with FEV1% of predicted of smokers without and with COPD. FXR agonist GW4064 remarkably enhanced and FXR antagonist Z-Guggulsterone significantly inhibited EMT changes in TGF-ß1-treated HBE cells. Both chenodeoxycholic acid (CDCA) and GW4064 increased COX-2 expression in HBE cells, whereas Z-Guggulsterone dramatically restrained CDCA-induced COX-2 expression. Finally, FXR expression is induced by IL-4 and IL-13 in HBE cells, as well as by cigarette smoke exposure in a rat model of COPD. CONCLUSIONS: Overexpression of FXR in small airway may contribute to airway remodelling and inflammation in COPD by regulating EMT and COX-2 expression.

Application of a stand-alone anchored spacer in noncontiguous anterior cervical arthrodesis with radiologic analysis of the intermediate segment.

The purpose of this study was to describe the clinical features of noncontiguous cervical degenerative disc disease (cDDD), investigate the efficacy and complications of a stand-alone anchored spacer (SAAS) for patients with noncontiguous cDDD, and present radiologic analysis of the intermediate segment (IS) after skip-level fusion. Nineteen consecutive patients with noncontiguous cDDD who underwent skip-level anterior cervical discectomy and fusion (ACDF) with SAAS from January 2010 to December 2012 were enrolled in this study. Clinical outcomes were assessed preoperatively and at 24 months postoperatively using the Japanese Orthopaedic Association score, Neck Disability Index, and Visual Analog Scale. Overall cervical alignment (OCA) of the cervical spine, and the range of motion (ROM), intervertebral disc height (IDH), disc signal intensity and disc protrusion of IS were measured and compared before and after surgery. Clinical outcomes significantly improved compared to preoperative scores. The OCA was corrected and maintained at 24 months postoperatively compared with preoperative values (p<0.05). There were no significant differences in the ROM and IDH of the IS at each follow-up (p>0.05). However, decreased signal intensity on T2-weighted MRI was evidenced in three mobile IS at final follow-up (20.0%). Skip-level ACDF with SAAS may be an efficacious option for the treatment of noncontiguous cDDD.

Immune Regulation of Interleukin-27 in Malignant Pleural Effusion.

BACKGROUND: Interleukin (IL)-27 has been reported to have anti-proliferate and anti-angiogenic activities on cancer cells. However, the involvement of IL-27 in malignant pleural effusion (MPE) remains unknown. Thus, in this research, we compared the immune functions of IL-27, interferon (IFN)-γ, and IL-17 on lung cancer cells and revealed the regulatory mechanism of IL-27 in MPE. METHODS: The distribution of IL-27 in both MPE and blood was evaluated by enzyme-linked immunosorbent assay and flow cytometry. The expressions of cytokine receptors and the levels of the phosphorylated signal transducer and activator of transcription (STAT) signalings were detected by flow cytometry. As well as the effects of proliferation, apoptosis, migration, and adherent activity of IL-27, IFN-γ, and IL-17 on lung cancer cells were also explored. RESULTS: The expression of IL-27 was increased in MPE when compared with blood (147.3 ± 25.1 pg/ml vs. 100.3 ± 13.9 pg/ml, P = 0.04). IL-27 was noted to suppress both proliferation (18.33 ± 0.21 vs. 27.77 ± 0.88, P = 0.0005) and migration (1.82 ± 0.44 vs. 3.13 ± 0.07, P = 0.04) of A549 cells, but obviously promoted apoptosis of A549 cells (9.47 ± 1.14 vs. 4.96 ± 0.17, P = 0.02) by activating STAT1 signaling. Interestingly, IL-27 played totally opposite effects on A549 cells by activating STAT3 pathway. Moreover, IL-27 exerted different intercellular adherent activities of A549 cells to pleural mesothelial cell monolayer by activating different STAT signalings. CONCLUSIONS: IL-27 might exert an important immune regulation on lung cancer cells in human pleural malignant environment.

IL-27 and IL-27-producing CD4+ T cells in human tuberculous pleural effusion.

The objective of the present study was to figure out whether human IL-27-producing CD4(+) T cells represent a distinct T cell subset in tuberculosis pleural effusion (TPE). Distribution, phenotypic features of IL-27-producing CD4(+) T cells in TPE were determined. The required transcription factors and signal transductions for IL-27-producing CD4(+) T cell differentiation were explored. The immune regulation of IL-27 on pleural mesothelial cells was observed. We have determined the presence of a subset of human Th cells that infiltrated into tuberculous pleural effusion, which was characterized by the secretion of IL-27, and somehow IFN-γ, but not of IL-4, IL-9, IL-17, or IL-22. These IL-27-producing CD4(+) T cells were effector memory cells and exhibited a transcription profile clearly separated from those of Th2, Th17, Th9, and Th22 cells. The in vitro experiments showed that IL-1ß, IL-2 and IL-12, or their various combinations could promote IL-27(+)CD4(+) T cell differentiation from naive CD4(+) T cells by means of phosphorylation of STAT3, STAT4, or/and STAT5. Transcription factors c-Fos and T-bet were required for IL-27(+)CD4(+) T cell differentiation. By activating STAT3 signaling, IL-27 not only restored a clear epithelial phenotype of pleural mesothelial cells, but also further reversed IFN-γ-induced epithelial-mesenchymal transition of pleural mesothelial cells. These data suggested that human IL-27(+)CD4(+) T cells might represent a distinct human T cell subset with unique expression profiles of transcription factors and proinflammatory cytokines, and these IL-27(+)CD4(+) T cells may play important roles in tuberculosis immunity by affecting pleural mesothelial cells.