An enriched environment reduces chronic stress-induced visceral pain through modulating microglial activity in the central nucleus of the amygdala.

Cognitive behavioral therapy (CBT) improves the quality of life for patients with brain-gut disorders; however, the underlying mechanisms of CBT remain to be explored. Previously, we showed that environmental enrichment (EE), an experimental paradigm that mirrors positive behavioral intervention, ameliorates chronic stress-induced visceral hypersensitivity in a rodent model via mechanisms involving altered activity in the central nucleus of amygdala (CeA). In the present study, we investigated whether microglia-mediated synaptic plasticity in the CeA is a potential mechanism underlying the protective effects of EE against stress-induced visceral hypersensitivity. We stereotaxically implanted corticosterone (CORT) micropellets onto the dorsal margin of the CeA shown previously to induce colonic hypersensitivity. Animals were housed in EE cages or standard cages for 14 days after CORT implantation. Visceral sensitivity was assessed via visceromotor behavioral response to colorectal distension. Microglial morphology, microglia-mediated synaptic engulfment, and the expression of synaptic pruning-related signals complement component 1q (C1q), complement component 3 (C3), and C3 receptor (C3R) were measured using immunofluorescence and RNAscope assay. We found that housing CORT implanted rats in EE cages for 14 days attenuated visceral hypersensitivity in both male and female rats as compared with control rats maintained in standard housing. EE reduced CORT-induced microglial remodeling and microglia-mediated synaptic pruning with reduced C1q and CR3, but not C3, expression. Our data suggest that exposure to EE is sufficient to ameliorate stress-induced visceral pain via reducing amygdala microglia-modulated neuronal plasticity.NEW & NOTEWORTHY Clinical studies show that cognitive behavioral therapy (CBT) is effective in ameliorating visceral pain in patient with irritable bowel syndrome (IBS), yet the underlying mechanisms remain unexplored. By using environmental enrichment (EE), an experimental paradigm that mirrors positive behavioral intervention, we demonstrated that microglia-mediated synaptic plasticity in the CeA explains, plays a role, at least in part, in the positive effects of EE to reduce visceral hypersensitivity.

Stress-induced visceral pain in female rats is associated with epigenetic remodeling in the central nucleus of the amygdala.

Stress and anxiety contribute to the pathophysiology of irritable bowel syndrome (IBS), a female-predominant disorder of the gut-brain axis, characterized by abdominal pain due to heightened visceral sensitivity. In the current study, we aimed to evaluate in female rats whether epigenetic remodeling in the limbic brain, specifically in the central nucleus of the amygdala (CeA), is a contributing factor in stress-induced visceral hypersensitivity. Our results showed that 1 h exposure to water avoidance stress (WAS) for 7 consecutive days decreased histone acetylation at the GR promoter and increased histone acetylation at the CRH promoter in the CeA. Changes in histone acetylation were mediated by the histone deacetylase (HDAC) SIRT-6 and the histone acetyltransferase CBP, respectively. Administration of the HDAC inhibitor trichostatin A (TSA) into the CeA prevented stress-induced visceral hypersensitivity through blockade of SIRT-6 mediated histone acetylation at the GR promoter. In addition, HDAC inhibition within the CeA prevented stress-induced histone acetylation of the CRH promoter. Our results suggest that, in females, epigenetic modifications in the limbic brain regulating GR and CRH expression contribute to stress-induced visceral hypersensitivity and offer a potential explanation of how stress can trigger symptoms in IBS patients.

W246G Mutant ELOVL4 Impairs Synaptic Plasticity in Parallel and Climbing Fibers and Causes Motor Defects in a Rat Model of SCA34.

Spinocerebellar ataxia (SCA) is a neurodegenerative disorder characterized by ataxia and cerebellar atrophy. A number of different mutations gives rise to different types of SCA with characteristic ages of onset, symptomatology, and rates of progression. SCA type 34 (SCA34) is caused by mutations in ELOVL4 (ELOngation of Very Long-chain fatty acids 4), a fatty acid elongase essential for biosynthesis of Very Long Chain Saturated and Polyunsaturated Fatty Acids (VLC-SFA and VLC-PUFA, resp., ≥28 carbons), which have important functions in the brain, skin, retina, Meibomian glands, testes, and sperm. We generated a rat model of SCA34 by knock-in of the SCA34-causing 736T>G (p.W246G) ELOVL4 mutation. Rats carrying the mutation developed impaired motor deficits by 2 months of age. To understand the mechanism of these motor deficits, we performed electrophysiological studies using cerebellar slices from rats homozygous for W246G mutant ELOVL4 and found marked reduction of long-term potentiation at parallel fiber synapses and long-term depression at climbing fiber synapses onto Purkinje cells. Neuroanatomical analysis of the cerebellum showed normal cytoarchitectural organization with no evidence of degeneration out to 6 months of age. These results point to ELOVL4 as essential for motor function and cerebellar synaptic plasticity. The results further suggest that ataxia in SCA34 patients may arise from a primary impairment of synaptic plasticity and cerebellar network desynchronization before onset of neurodegeneration and progression of the disease at a later age.

Amygdala microglia modify neuronal plasticity via complement C1q/C3-CR3 signaling and contribute to visceral pain in a rat model.

Stress can trigger symptoms in patients with irritable bowel syndrome (IBS). Previously we demonstrated that chronic psychological stress induced microglial remodeling in the central nucleus of amygdala (CeA) and contributed to the development of visceral hypersensitivity via synaptic engulfment. However, the specific signaling mechanisms that microglia depend upon to recognize target neurons to facilitate visceral pain remain unknown. Here, we test the hypothesis that the microglia in the CeA contribute to chronic stress-induced visceral hypersensitivity via complement C1q/C3-CR3 signaling-mediated synaptic remodeling. In male and female Fischer-344 rats, micropellets of corticosterone (CORT) or cholesterol (control) were stereotaxically implanted bilaterally onto the CeA. After 7 days, microglial C1q, complement receptor 3 (CR3) expression, and microglia-mediated synaptic engulfment were assessed via RNAscope, quantitative PCR, and immunofluorescence. The microglial inhibitor minocycline, CR3 antagonist neutrophil inhibitory factor (NIF), or vehicle were daily infused into the CeA following CORT implantations. Visceral sensitivity was assessed via a visceromotor response (VMR) to graded pressures of isobaric colorectal distension (CRD). Our results suggest that chronic exposure to elevated CORT in the CeA induced visceral hypersensitivity and amygdala microglial morphological remodeling. CORT increased microglial C1q and CR3 expression and increased microglia-mediated synaptic engulfment. Both groups of animals with minocycline or NIF infusions reversed microglia-mediated synaptic remodeling and attenuated CORT-induced visceral hypersensitivity. Our findings demonstrate that C1q/C3-CR3 signaling is critical for microglia-mediated synaptic remodeling in the CeA and contributes to CORT-induced visceral hypersensitivity.NEW & NOTEWORTHY Patients with irritable bowel syndrome (IBS) show altered amygdala activity. We showed previously that stress induces visceral hypersensitivity partially through microglia-modulated synaptic plasticity in the central nucleus of the amygdala (CeA). Our current data suggest that the C1q/C3-CR3 cascade initiates microglia-mediated synaptic remodeling in the CeA. Blocking C3-CR3 interaction attenuates stress-induced visceral hypersensitivity. These findings uncover a role of microglia-synapse signaling in the brain-gut regulation and support a future therapeutic target to treat visceral pain.

Inhibition of Microglial Activation in the Amygdala Reverses Stress-Induced Abdominal Pain in the Male Rat.

BACKGROUND & AIMS: Psychological stress is a trigger for the development of irritable bowel syndrome and associated symptoms including abdominal pain. Although irritable bowel syndrome patients show increased activation in the limbic brain, including the amygdala, the underlying molecular and cellular mechanisms regulating visceral nociception in the central nervous system are incompletely understood. In a rodent model of chronic stress, we explored the role of microglia in the central nucleus of the amygdala (CeA) in controlling visceral sensitivity. Microglia are activated by environmental challenges such as stress, and are able to modify neuronal activity via synaptic remodeling and inflammatory cytokine release. Inflammatory gene expression and microglial activity are regulated negatively by nuclear glucocorticoid receptors (GR), which are suppressed by the stress-activated pain mediator p38 mitogen-activated protein kinases (MAPK). METHODS: Fisher-344 male rats were exposed to water avoidance stress (WAS) for 1 hour per day for 7 days. Microglia morphology and the expression of phospho-p38 MAPK and GR were analyzed via immunofluorescence. Microglia-mediated synaptic remodeling was investigated by quantifying the number of postsynaptic density protein 95-positive puncta. Cytokine expression levels in the CeA were assessed via quantitative polymerase chain reaction and a Luminex assay (Bio-Rad, Hercules, CA). Stereotaxic infusion into the CeA of minocycline to inhibit, or fractalkine to activate, microglia was followed by colonic sensitivity measurement via a visceromotor behavioral response to isobaric graded pressures of tonic colorectal distension. RESULTS: WAS induced microglial deramification in the CeA. Moreover, WAS induced a 3-fold increase in the expression of phospho-p38 and decreased the ratio of nuclear GR in the microglia. The number of microglia-engulfed postsynaptic density protein 95-positive puncta in the CeA was increased 3-fold by WAS, while cytokine levels were unchanged. WAS-induced changes in microglial morphology, microglia-mediated synaptic engulfment in the CeA, and visceral hypersensitivity were reversed by minocycline whereas in stress-naïve rats, fractalkine induced microglial deramification and visceral hypersensitivity. CONCLUSIONS: Our data show that chronic stress induces visceral hypersensitivity in male rats and is associated with microglial p38 MAPK activation, GR dysfunction, and neuronal remodeling in the CeA.

Environmental enrichment prevents chronic stress-induced brain-gut axis dysfunction through a GR-mediated mechanism in the central nucleus of the amygdala.

BACKGROUND: Cognitive behavioral therapy (CBT) improves quality of life of patients with irritable bowel syndrome (IBS), a disorder characterized by chronic visceral pain and abnormal bowel habits. Whether CBT can actually improve visceral pain in IBS patients is still unknown. The aim of this study is to evaluate whether environment enrichment (EE), the animal analog of CBT, can prevent stress-induced viscero-somatic hypersensitivity through changes in glucocorticoid receptor (GR) signaling within the central nucleus of the amygdala (CeA). METHODS: Rats were housed in either standard housing (SH) or EE for 7 days before and during daily water avoidance stress (WAS) exposure (1-h/d for 7 days). In the first cohort, visceral and somatic sensitivity were assessed via visceromotor response to colorectal distention and von Frey Anesthesiometer 24 hous and 21 days after WAS. In another cohort, the CeA was isolated for GR mRNA quantification. KEY RESULTS: Environment enrichment for 7 days before and during the 7 days of WAS persistently attenuated visceral and somatic hypersensitivity when compared to rats placed in SH. Environment enrichment exposure also prevented the WAS-induced decrease in GR expression in the CeA. CONCLUSION & INFERENCES: Pre-exposure to short-term EE prevents the stress-induced downregulation of GR, and inhibits visceral and somatic hypersensitivity induced by chronic stress. These results suggest that a positive environment can ameliorate stress-induced pathology and provide a non-pharmacological therapeutic option for disorders such as IBS.

Importance of Non-pharmacological Approaches for Treating Irritable Bowel Syndrome: Mechanisms and Clinical Relevance.

Chronic visceral pain represents a major unmet clinical need with the severity of pain ranging from mild to so severe as to prevent individuals from participating in day-to-day activities and detrimentally affecting their quality of life. Although chronic visceral pain can be multifactorial with many different biological and psychological systems contributing to the onset and severity of symptoms, one of the major triggers for visceral pain is the exposure to emotional and physical stress. Chronic visceral pain that is worsened by stress is a hallmark feature of functional gastrointestinal disorders such as irritable bowel syndrome (IBS). Current pharmacological interventions for patients with chronic visceral pain generally lack efficacy and many are fraught with unwanted side effects. Cognitive behavioral therapy (CBT) has emerged as a psychotherapy that shows efficacy at ameliorating stress-induced chronic visceral pain; however, the molecular mechanisms underlying CBT remain incompletely understood. Preclinical studies in experimental models of stress-induced visceral pain employing environmental enrichment (EE) as an animal model surrogate for CBT are unraveling the mechanism by which environmental signals can lead to long-lasting changes in gene expression and behavior. Evidence suggests that EE signaling interacts with stress and nociceptive signaling. This review will (1) critically evaluate the behavioral and molecular changes that lead to chronic pain in IBS, (2) summarize the pharmacological and non-pharmacological approaches used to treat IBS patients, and (3) provide experimental evidence supporting the potential mechanisms by which CBT ameliorates stress-induced visceral pain.

Age-Related Cognitive Impairment: Role of Reduced Synaptobrevin-2 Levels in Deficits of Memory and Synaptic Plasticity.

Cognitive impairment in the aging population is quickly becoming a health care priority, for which currently no disease-modifying treatment is available. Multiple domains of cognition decline with age even in the absence of neurodegenerative diseases. The cellular and molecular changes leading to cognitive decline with age remain elusive. Synaptobrevin-2 (Syb2), the major vesicular SNAP receptor protein, highly expressed in the cerebral cortex and hippocampus, is essential for synaptic transmission. We have analyzed Syb2 protein levels in mice and found a decrease with age. To investigate the functional consequences of lower Syb2 expression, we have used adult Syb2 heterozygous mice (Syb2+/-) with reduced Syb2 levels. This allowed us to mimic the age-related decrease of Syb2 in the brain in order to selectively test its effects on learning and memory. Our results show that Syb2+/- animals have impaired learning and memory skills and they perform worse with age in the radial arm water maze assay. Syb2+/- hippocampal neurons have reduced synaptic plasticity with reduced release probability and impaired long-term potentiation in the CA1 region. Syb2+/- neurons also have lower vesicular release rates when compared to WT controls. These results indicate that reduced Syb2 expression with age is sufficient to cause cognitive impairment.

Dysregulation of the SNARE-binding protein Munc18-1 impairs BDNF secretion and synaptic neurotransmission: a novel interventional target to protect the aging brain.

Brain-derived neurotrophic factor (BDNF) has a central role in maintaining and strengthening neuronal connections and to stimulate neurogenesis in the adult brain. Decreased levels of BDNF in the aging brain are thought to usher cognitive impairment. BDNF is stored in dense core vesicles and released through exocytosis from the neurites. The exact mechanism for the regulation of BDNF secretion is not well understood. Munc18-1 (STXBP1) was found to be essential for the exocytosis of synaptic vesicles, but its involvement in BDNF secretion is not known. Interestingly, neurons lacking munc18-1 undergo severe degeneration in knock-out mice. Here, we report the effects of BDNF treatment on the presynaptic terminal using munc18-1-deficient neurons. Reduced expression of munc18-1 in heterozygous (+/-) neurons diminishes synaptic transmitter release, as tested here on individual synaptic connections with FM1-43 fluorescence imaging. Transduction of cultured neurons with BDNF markedly increased BDNF secretion in wild-type but was less effective in munc18-1 +/- cells. In turn, BDNF enhanced synaptic functions and restored the severe synaptic dysfunction induced by munc18-1 deficiency. The role of munc18-1 in the synaptic effect of BDNF is highlighted by the finding that BDNF upregulated the expression of munc18-1 in neurons, consistent with enhanced synaptic functions. Accordingly, this is the first evidence showing the functional effect of BDNF in munc18-1 deficient synapses and about the direct role of munc18-1 in the regulation of BDNF secretion. We propose a molecular model of BDNF secretion and discuss its potential as therapeutic target to prevent cognitive decline in the elderly.

Munc18-1 haploinsufficiency impairs learning and memory by reduced synaptic vesicular release in a model of Ohtahara syndrome.

Ohtahara syndrome, also known as type 4 of Early Infantile Epileptic Encephalopathy with suppression bursts (EIEE-4) is currently an untreatable disorder that presents with seizures and impaired cognition. EIEE-4 patients have mutations most frequently in the STXBP1 gene encoding a Sec protein, munc18-1. The exact molecular mechanism of how these munc18-1 mutations cause impaired cognition, remains elusive. The leading haploinsufficiency hypothesis posits that mutations in munc18-1 render the protein unstable leading to its degradation. Expression driven by the healthy allele is not sufficient to maintain the physiological function resulting in haploinsufficiency. The aim of this study has been to understand how munc18-1 haploinsufficiency causes cognitive impairment seen in EIEE-4. Here we present results from behavioral to cellular effects from a mouse model of munc18-1 haploinsufficiency. Munc18-1 heterozygous knock-out mice showed impaired spatial learning and memory in behavior tests as well as reduced synaptic plasticity in hippocampal CA1 long-term potentiation. Cultured munc18-1 heterozygous hippocampal neurons had significantly slower rate of synaptic vesicle release and decreased readily releasable vesicle pool compared to wild-type control neurons in fluorescent FM dye assays. These results demonstrate that reduced munc18-1 levels are sufficient to impair learning and memory by reducing neurotransmitter release. Therefore, our study implicates munc18-1 haploinsufficiency as a primary cause of cognitive impairment seen in EIEE-4 patients.

Hypertension-induced synapse loss and impairment in synaptic plasticity in the mouse hippocampus mimics the aging phenotype: implications for the pathogenesis of vascular cognitive impairment.

Strong epidemiological and experimental evidence indicates that hypertension has detrimental effects on the cerebral microcirculation and thereby promotes accelerated brain aging. Hypertension is an independent risk factor for both vascular cognitive impairment (VCI) and Alzheimer's disease (AD). However, the pathophysiological link between hypertension-induced cerebromicrovascular injury (e.g., blood-brain barrier disruption, increased microvascular oxidative stress, and inflammation) and cognitive decline remains elusive. The present study was designed to characterize neuronal functional and morphological alterations induced by chronic hypertension and compare them to those induced by aging. To achieve that goal, we induced hypertension in young C57BL/6 mice by chronic (4 weeks) infusion of angiotensin II. We found that long-term potentiation (LTP) of performant path synapses following high-frequency stimulation of afferent fibers was decreased in hippocampal slices obtained from hypertensive mice, mimicking the aging phenotype. Hypertension and advanced age were associated with comparable decline in synaptic density in the stratum radiatum of the mouse hippocampus. Hypertension, similar to aging, was associated with changes in mRNA expression of several genes involved in regulation of neuronal function, including down-regulation of Bdnf, Homer1, and Dlg4, which may have a role in impaired synaptic plasticity. Collectively, hypertension impairs synaptic plasticity, reduces synaptic density, and promotes dysregulation of genes involved in synaptic function in the mouse hippocampus mimicking the aging phenotype. These hypertension-induced neuronal alterations may impair establishment of memories in the hippocampus and contribute to the pathogenesis and clinical manifestation of both vascular cognitive impairment (VCI) and Alzheimer's disease (AD).

Biomechanical Performances of Networked Polyethylene Glycol Diacrylate: Effect of Photoinitiator Concentration, Temperature, and Incubation Time.

Nutrient conduit networks can be introduced within the Polyethylene Glycol Diacrylate (PEGDA) tissue construct to enable cells to survive in the scaffold. Nutrient conduit networks can be created on PEGDA by macrochannel to nanochannel fabrication techniques. Such networks can influence the mechanical and cell activities of PEGDA scaffold. There is no study conducted to evaluate the effect of nutrient conduit networks on the maximum tensile stress and cell activities of the tissue scaffold. The study aimed to explore the influence of the network architecture on the maximum tensile stress of PEGDA scaffold and compared with the nonnetworked PEGDA scaffold. Our study found that there are 1.78 and 2.23 times decrease of maximum tensile stress due to the introduction of nutrient conduit networks to the PEGDA scaffold at 23°C and 37°C temperature conditions, respectively. This study also found statistically significant effect of network architecture, PI concentration, temperature, and wait time on the maximum failure stress of PEGDA samples (P value < 0.05). Cell viability results demonstrated that networked PEGDA hydrogels possessed increased viability compared to nonnetworked and decreased viability with increased photoinitiator concentrations. The results of this study can be used for the design of PEGDA scaffold with macrosize nutrient conduit network channels.

OKN-007 decreases VEGFR-2 levels in a preclinical GL261 mouse glioma model.

Angiogenesis is essential to tumor progression, and the precise imaging of the angiogenic marker vascular endothelial growth factor receptor 2 (VEGFR-2) may provide an accurate evaluation for angiogenesis during a therapeutic response. With the use of molecular magnetic resonance imaging (mMRI), an in vitro cell assay indicated significantly decreased T1 relaxation values when tumor endothelial cells (TEC), which positively expressed VEGFR-2 (Western blot), were in the presence of the VEGFR-2 probe compared to TEC alone (P < 0.001). For in vivo mMRI evaluations, we assessed VEGFR-2 levels in untreated and OKN-007-treated GL261 mouse gliomas. Regarding treatment response, OKN-007 was also able to significantly decrease tumor volumes (P < 0.01) and increase survival (P < 0.001) in treated animals. Regarding in vivo detection of VEGFR-2, OKN-007 was found to significantly decrease the amount of VEGFR-2 probe (P < 0.05) compared to an untreated control group. Fluorescence imaging for the VEGFR-2 probe indicated that there was colocalization with the endothelial marker CD31 in an untreated tumor bearing mouse and decreased levels for an OKN-007-treated animal. Immuno-fluorescence imaging for VEGFR-2 indicated that OKN-007 treatment significantly decreased VEGFR-2 levels (P < 0.0001) when compared to untreated tumors. Immuno-electron microscopy was used with gold-labeled anti-biotin to detect the anti-VEGFR-2 probe within the plasma membrane of GL261 tumor endothelial cells. This is the first attempt at detecting in vivo levels of VEGFR-2 in a mouse GL261 glioma model and assessing the anti-angiogenic capability of an anticancer nitrone. The results indicate that OKN-007 treatment substantially decreased VEGFR-2 levels in a GL261 glioma model, and can be considered as an anti-angiogenic therapy in human gliomas.