Pain experience reduces social avoidance to others in pain: a c-Fos-based functional connectivity network study in mice.

Pain experience increases individuals' perception and contagion of others' pain, but whether pain experience affects individuals' affiliative or antagonistic responses to others' pain is largely unknown. Additionally, the neural mechanisms underlying how pain experience modulates individuals' responses to others' pain remain unclear. In this study, we explored the effects of pain experience on individuals' responses to others' pain and the underlying neural mechanisms. By comparing locomotion, social, exploration, stereotyped, and anxiety-like behaviors of mice without any pain experience (naïve observers) and mice with a similar pain experience (experienced observers) when they observed the pain-free demonstrator with intraperitoneal injection of normal saline and the painful demonstrator with intraperitoneal injection of acetic acid, we found that pain experience of the observers led to decreased social avoidance to the painful demonstrator. Through whole-brain c-Fos quantification, we discovered that pain experience altered neuronal activity and enhanced functional connectivity in the mouse brain. The analysis of complex network and graph theory exhibited that functional connectivity networks and activated hub regions were altered by pain experience. Together, these findings reveal that neuronal activity and functional connectivity networks are involved in the modulation of individuals' responses to others' pain by pain experience.

Sexually dimorphic control of affective state processing and empathic behaviors.

Recognizing the affective states of social counterparts and responding appropriately fosters successful social interactions. However, little is known about how the affective states are expressed and perceived and how they influence social decisions. Here, we show that male and female mice emit distinct olfactory cues after experiencing distress. These cues activate distinct neural circuits in the piriform cortex (PiC) and evoke sexually dimorphic empathic behaviors in observers. Specifically, the PiC → PrL pathway is activated in female observers, inducing a social preference for the distressed counterpart. Conversely, the PiC → MeA pathway is activated in male observers, evoking excessive self-grooming behaviors. These pathways originate from non-overlapping PiC neuron populations with distinct gene expression signatures regulated by transcription factors and sex hormones. Our study unveils how internal states of social counterparts are processed through sexually dimorphic mechanisms at the molecular, cellular, and circuit levels and offers insights into the neural mechanisms underpinning sex differences in higher brain functions.

An insular cortical circuit required for itch sensation and aversion.

Itch encompasses both sensory and emotional dimensions, with the two dimensions reciprocally exacerbating each other. However, whether a shared neural circuit mechanism governs both dimensions remains elusive. Here, we report that the anterior insular cortex (AIC) is activated by both histamine-dependent and -independent itch stimuli. The activation of AIC elicits aversive emotion and exacerbates pruritogen-induced itch sensation and aversion. Mechanistically, AIC excitatory neurons project to the GABAergic neurons in the dorsal bed nucleus of the stria terminalis (dBNST). Manipulating the activity of the AIC → dBNST pathway affects both itch sensation and itch-induced aversion. Our study discovers the shared neural circuit (AIC â†’ dBNST pathway) underlying the itch sensation and aversion, highlights the critical role of the AIC as a central hub for the itch processing, and provides a framework to understand the neural mechanisms underlying the sensation and emotion interaction.

Synaptic homeostasis transiently leverages Hebbian mechanisms for a multiphasic response to inactivity.

Homeostatic regulation of synapses is vital for nervous system function and key to understanding a range of neurological conditions. Synaptic homeostasis is proposed to operate over hours to counteract the destabilizing influence of long-term potentiation (LTP) and long-term depression (LTD). The prevailing view holds that synaptic scaling is a slow first-order process that regulates postsynaptic glutamate receptors and fundamentally differs from LTP or LTD. Surprisingly, we find that the dynamics of scaling induced by neuronal inactivity are not exponential or monotonic, and the mechanism requires calcineurin and CaMKII, molecules dominant in LTD and LTP. Our quantitative model of these enzymes reconstructs the unexpected dynamics of homeostatic scaling and reveals how synapses can efficiently safeguard future capacity for synaptic plasticity. This mechanism of synaptic adaptation supports a broader set of homeostatic changes, including action potential autoregulation, and invites further inquiry into how such a mechanism varies in health and disease.

Clearance of ß-amyloid and synapses by the optogenetic depolarization of microglia is complement selective.

Microglia actively monitor the neighboring brain microenvironments and constantly contact synapses with their unique ramified processes. In neurodegenerative diseases, including Alzheimer's disease (AD), microglia undergo morphological and functional alterations. Whether the direct manipulation of microglia can selectively or concurrently modulate synaptic function and the response to disease-associated factors remains elusive. Here, we employ optogenetic methods to stimulate microglia in vitro and in vivo. Membrane depolarization rapidly changes microglia morphology and leads to enhanced phagocytosis. We found that the optogenetic stimulation of microglia can efficiently promote ß-amyloid (Aß) clearance in the brain parenchyma, but it can also enhance synapse elimination. Importantly, the inhibition of C1q selectively prevents synapse loss induced by microglia depolarization but does not affect Aß clearance. Our data reveal independent microglia-mediated phagocytosis pathways toward Aß and synapses. Our results also shed light on a synergistic strategy of depolarizing microglia and inhibiting complement functions for the clearance of Aß while sparing synapses.

Differences in the neural basis and transcriptomic patterns in acute and persistent pain-related anxiety-like behaviors.

Background: Both acute and persistent pain is associated with anxiety in clinical observations, but whether the underlying neural mechanisms differ is poorly understood. Methods: We used formalin or complete Freund's adjuvant (CFA) to induce acute or persistent pain. Behavioral performance was assessed by the paw withdrawal threshold (PWT), open field (OF), and elevated plus maze (EPM) tests. C-Fos staining was used to identify the activated brain regions. Chemogenetic inhibition was further performed to examine the necessity of brain regions in behaviors. RNA sequencing (RNA-seq) was used to identify the transcriptomic changes. Results: Both acute and persistent pain could lead to anxiety-like behavior in mice. The c-Fos expression indicates that the bed nucleus of the stria terminalis (BNST) is activated only in acute pain, whereas the medial prefrontal cortex (mPFC) is activated only in persistent pain. Chemogenetic manipulation reveals that the activation of the BNST excitatory neurons is required for acute pain-induced anxiety-like behaviors. In contrast, the activation of the prelimbic mPFC excitatory neurons is essential for persistent pain-induced anxiety-like behaviors. RNA-seq reveals that acute and persistent pain induces differential gene expression changes and protein-protein interaction networks in the BNST and prelimbic mPFC. The genes relevant to neuronal functions might underline the differential activation of the BNST and prelimbic mPFC in different pain models, and be involved in acute and persistent pain-related anxiety-like behaviors. Conclusion: Distinct brain regions and gene expression patterns are involved in acute and persistent pain-related anxiety-like behaviors.

[Comparative analysis of different fecal DNA extraction methods].

The community structure and diversity of the gut microbiota are associated with human diseases. However, the analysis of different community structure might be influenced by experimental approaches such as the quality of DNA extraction. Therefore, evaluating the efficiency of different DNA extraction methods for specific intestinal species is a guideline for obtaining a comprehensive human gut microbial profile, which may assist the in-depth investigation into the structure of the gut microbial community. The aim of this study was to perform a comparative analysis of five different DNA extraction methods. With the aid of qPCR, the efficiency of five DNA extraction kits was evaluated in terms of the purity of the extracted DNA, the DNA concentration, and the abundance of genomic DNA extracted from specific intestinal species. The results showed that the kit Q gave the best extraction results, especially for Gram-positive bacteria such as Lactobacillus and Bifidobacterium. The average DNA concentration of the N kit was lower than that of the Q kit, but there was no significant difference between the two in terms of the purity. Compared to the other three commercial kits (M, PSP, TG), the efficiency of the N kit in extracting the genomic DNA of the specified microorganisms were the least different from those of the Q kit. In contrast, the DNA extracted by the M kit was of higher quality but of lower concentration, and was not very efficient for Gram-positive bacteria. The DNA extracted by the TG and PSP kits was inferior to the other validated kits in terms of the concentration, quality and bacterial abundance. These results provide a basis for the selection of genomic DNA extraction methods in microecological research experiments.

BDNF Alleviates Microglial Inhibition and Stereotypic Behaviors in a Mouse Model of Obsessive-Compulsive Disorder.

Obsessive-compulsive disorder (OCD) is a severe mental illness characterized by obsessions and compulsions. However, its underlying mechanisms remain to be elucidated. Recent studies have suggested that neuroimmune dysregulation is involved in the pathogenesis of OCD. To investigate the role of microglia in this disorder, we established a pharmacological mouse model by using the serotonin (5-HT) 1A/1B receptor agonist RU24969 to mimic monoamine dysregulation in OCD, and we examined the morphological and functional alterations of microglia in this model. We found that RU24969 treatment led to compulsive circling behavior in mice. Strikingly, we found that the density and mobility of microglia in the prelimbic cortex were much lower in RU24969-treated mice than in control mice. Moreover, the expression of cytokines and chemokines, including BDNF, IL-1ß, IL-6, TNFα, CD80, CD86, MHC-I, and MHC-II, also decreased in RU24969-treated mice. Importantly, we found that injection of BDNF or induction of BDNF expression by trehalose completely reversed microglial dysfunction and reduced stereotypic behavior. These results indicate that microglial dysfunction is closely related to stereotypic behaviors in our mouse model of OCD and that BDNF could be an effective treatment for stereotypic behaviors.

FACS-Based Neuronal Cell Type-Specific RNA Isolation and Alternative Splicing Analysis.

Alternative splicing of pre-mRNAs expands the coding abilities of genomes by generating distinct transcription variants from individual genes. It contributes to the marvelous complexity of the transcriptome in neurons. Given the differential expression of alternative splicing regulators and diversity in alternative splicing programs in neuronal subpopulations, it is urgent and necessary to develop methods to efficiently isolate diverse subgroups of neurons and analyze their transcriptomic diversity. Here, we describe a protocol to isolate RNA from specific neuronal types using a fluorescence-activated cell sorting (FACS)-based method to analyze alternative splicing events in a cell type-specific manner. The method is universally applicable to analyze alternative splicing in fluorescent protein-labeled neuronal types. It was optimized to preserve the transcription state and improve efficiency in cell suspension purification. With our protocol, fluorescent protein-labeled neurons could be efficiently purified. The transcription states suitable for gene expression and alternative splicing analysis could be well-preserved.

Gut Mycobiota Dysbiosis in Pulmonary Tuberculosis Patients Undergoing Anti-Tuberculosis Treatment.

Patients with pulmonary tuberculosis (TB) undergoing anti-tuberculosis (anti-TB) treatment were previously reported to present gut bacterial microbiota dysbiosis, but the role of the mycobiota has not been reported. Here, we conducted a follow-up study of 29 naive TB patients who received first-line anti-TB drug treatment; we collected their fecal samples at different time points, as well as 22 fecal samples from healthy subjects. Fungal ITS2 and bacterial 16S rRNA amplicon sequencing were used to analyze the effects of active TB and anti-TB treatment on the gut microbiota. We found that naive TB patients had bacterial and fungal dysbiosis with altered community composition and a decreased density of the transkingdom correlation network. Anti-TB drug treatment significantly decreased the diversity of bacteria and fungi with altered composition. Notably, we observed that the abundance of Purpureocillium lilacinum tended to decrease and Nakaseomyces spp. tended to increase in the anti-TB treatment, and all of them had increased proportions in the three TB groups compared with healthy subjects. We found that the fungal-bacterial transkingdom network was severely altered in TB patients after 2 months of treatment, and new fungal-enriched connections that were not observed in other groups after 6 months of treatment. This study provides the first detailed analysis of dysbiosis of the gut mycobiota due to active TB and anti-TB treatment. The results suggest that fungi play an important role in the balance of the gut microbiota and may be associated with the progression of TB, influencing the microbiota and immunity homeostasis in those receiving anti-TB treatment. IMPORTANCE Numerous studies have shown that the gut bacterial microbiota is altered in active TB patients and that anti-TB drugs have profound and long-term impacts. However, as an integral part of the microbiota, fungi have rarely been studied. The need to investigate both the bacterial and fungal microbiota, as well as the relationship between them is apparent. The significance of our study is in our examination of the changes in the bacterial and fungal microbiota simultaneously in both active TB and patients receiving anti-TB treatment. We found that fungi play an important role in the bacterial-fungal transkingdom network, especially during the anti-TB therapy. These findings underscore the importance of fungi in gut microbiota dysbiosis during active TB and anti-TB treatment processes. In addition, our findings suggest it is meaningful to research potential adjunctive therapies that reduce fungal expansion and increase commensal bacterial abundance after anti-TB treatment, which would help the recovery of TB patients.

Enriched Environment Prevents Surgery-Induced Persistent Neural Inhibition and Cognitive Dysfunction.

Perioperative neurocognitive disorders (PND) encompass short-term delirium and long-term cognitive dysfunction. Aging increases the susceptibility to PND, yet the neural mechanism is not known. In this study, we monitored the dynamic changes of neuronal activity in the prelimbic cortex before and after surgery. We found that anesthesia combined with surgery, but not anesthesia alone, induced a prolonged decrease in neuronal activity during the post-operation period in the aged mice, but not in the adult mice. The prolonged decrease in neuronal activity was accompanied by surgery-induced microglial activation and proinflammatory cytokines expression. Importantly, we found that the enriched environment (EE) completely prevented both the prolonged neural inhibition and neuroinflammation, and improved cognitive function in the aged mice. These results indicate that the prolonged neural inhibition correlated to PND and that EE before the surgery could effectively alleviate the surgery- induced cognitive dysfunction.

Distinct behavioral traits and associated brain regions in mouse models for obsessive-compulsive disorder.

BACKGROUND: Obsessive-compulsive disorder (OCD) is a mental disease with heterogeneous behavioral phenotypes, including repetitive behaviors, anxiety, and impairments in cognitive functions. The brain regions related to the behavioral heterogeneity, however, are unknown. METHODS: We systematically examined the behavioral phenotypes of three OCD mouse models induced by pharmacological reagents [RU24969, 8-hydroxy-DPAT hydrobromide (8-OH-DPAT), and 1-(3-chlorophenyl) piperazine hydrochloride-99% (MCPP)], and compared the activated brain regions in each model, respectively. RESULTS: We found that the mouse models presented distinct OCD-like behavioral traits. RU24969-treated mice exhibited repetitive circling, anxiety, and impairments in recognition memory. 8-OH-DPAT-treated mice exhibited excessive spray-induced grooming as well as impairments in recognition memory. MCPP-treated mice showed only excessive self-grooming. To determine the brain regions related to these distinct behavioral traits, we examined c-fos expression to indicate the neuronal activation in the brain. Our results showed that RU24969-treated mice exhibited increased c-fos expression in the orbitofrontal cortex (OFC), anterior cingulate cortex (ACC), prelimbic cortex (PrL), infralimbic cortex (IL), nucleus accumbens (NAc), hypothalamus, bed nucleus of the stria terminalis, lateral division, intermediate part (BSTLD), and interstitial nucleus of the posterior limb of the anterior commissure, lateral part (IPACL), whereas in 8-OH-DPAT-treated mice showed increased c-fos expression in the ACC, PrL, IL, OFC, NAc shell, and hypothalamus. By contrast, MCPP did not induce higher c-fos expression in the cortex than control groups. CONCLUSION: Our results indicate that different OCD mouse models exhibited distinct behavioral traits, which may be mediated by the activation of different brain regions.

Intracellular recording of cardiomyocyte action potentials by nanobranched microelectrode array.

Electrophysiological study that records the action potential of cardiomyocyte served as excellent tool to explore cardiology and neuroscience, disease investigation and pharmacological screening. Advances of micro/nanotechnologies promote the development of three-dimensional (3D) nanodevices to record high-quality intracellular recordings by various perforation approaches of cells, however, the complicated fabrication processes limited their large-scale manufacture. In this work, a unique nanobranched microelectrode array (NBMEA) platform is developed to achieve high-quality intracellular recording of cultured cardiomyocytes in a minimally invasive manner. The NBMEA is consisting of high aspect ratio conductive nanobranches fabricated on patterned microelectrodes combining hydrothermal growth and standard microfabrication. The 3D structure of nanobranches enables the electrode to form tight coupling with cardiomyocytes to achieve the low voltage cell electroporation and high-quality intracellular recording. The recorded intracellular action potentials of cardiomyocytes by NBMEA exhibited significant enhancement on amplitude (~5 mV), signal-to-noise ratio (SNR) (~67.47 dB), recording duration (up to 105 min), and recording yield (69.5 ± 17.8%). This NBMEA platform is a promising and powerful tool for electrophysiology that opens up new opportunities for high-quality and stable intracellular recording of cardiomyocytes.

Neuronal Inactivity Co-opts LTP Machinery to Drive Potassium Channel Splicing and Homeostatic Spike Widening.

Homeostasis of neural firing properties is important in stabilizing neuronal circuitry, but how such plasticity might depend on alternative splicing is not known. Here we report that chronic inactivity homeostatically increases action potential duration by changing alternative splicing of BK channels; this requires nuclear export of the splicing factor Nova-2. Inactivity and Nova-2 relocation were connected by a novel synapto-nuclear signaling pathway that surprisingly invoked mechanisms akin to Hebbian plasticity: Ca2+-permeable AMPA receptor upregulation, L-type Ca2+ channel activation, enhanced spine Ca2+ transients, nuclear translocation of a CaM shuttle, and nuclear CaMKIV activation. These findings not only uncover commonalities between homeostatic and Hebbian plasticity but also connect homeostatic regulation of synaptic transmission and neuronal excitability. The signaling cascade provides a full-loop mechanism for a classic autoregulatory feedback loop proposed ∼25 years ago. Each element of the loop has been implicated previously in neuropsychiatric disease.

Fatty Liver Disease Caused by High-Alcohol-Producing Klebsiella pneumoniae.

The underlying etiology of nonalcoholic fatty liver disease (NAFLD) is believed to be quite varied. Changes in the gut microbiota have been investigated and are believed to contribute to at least some cases of the disease, though a causal relationship remains unclear. Here, we show that high-alcohol-producing Klebsiella pneumoniae (HiAlc Kpn) is associated with up to 60% of individuals with NAFLD in a Chinese cohort. Transfer of clinical isolates of HiAlc Kpn by oral gavage into mice induced NAFLD. Likewise, fecal microbiota transplant (FMT) into mice using a HiAlc-Kpn-strain-containing microbiota isolated from an individual with NASH induced NAFLD. However, selective elimination of the HiAlc Kpn strain before FMT prevented NAFLD in the recipient mice. These results suggest that at least in some cases of NAFLD an alteration in the gut microbiome drives the condition due to excess endogenous alcohol production.

The Gut Microbiome Signatures Discriminate Healthy From Pulmonary Tuberculosis Patients.

Cross talk occurs between the human gut and the lung through a gut-lung axis involving the gut microbiota. However, the signatures of the human gut microbiota after active Mycobacterium tuberculosis infection have not been fully understood. Here, we investigated changes in the gut microbiota in tuberculosis (TB) patients by shotgun sequencing the gut microbiomes of 31 healthy controls and 46 patients. We observed a dramatic changes in gut microbiota in tuberculosis patients as reflected by significant decreases in species number and microbial diversity. The gut microbiota of TB patients were mostly featured by the striking decrease of short-chain fatty acids (SCFAs)-producingbacteria as well as associated metabolic pathways. A classification model based on the abundance of three species, Haemophilus parainfluenzae, Roseburia inulinivorans, and Roseburia hominis, performed well for discriminating between healthy and diseased patients. Additionally, the healthy and diseased states can be distinguished by SNPs in the species of B. vulgatus. We present a comprehensive profile of changes in the microbiota in clinical TB patients. Our findings will shed light on the design of future diagnoses and treatments for M. tuberculosis infections.

Inulin Can Alleviate Metabolism Disorders in ob/ob Mice by Partially Restoring Leptin-related Pathways Mediated by Gut Microbiota.

Inulin has been used as a prebiotic to alleviate glucose and lipid metabolism disorders in mice and humans by modulating the gut microbiota. However, the mechanism underlying the alleviation of metabolic disorders by inulin through interactions between the gut microbiota and host cells is unclear. We use ob/ob mice as a model to study the effect of inulin on the cecal microbiota by 16S rRNA gene amplicon sequencing and its interaction with host cells by transcriptomics. The inulin-supplemented diet improved glucose and lipid metabolism disorder parameters in ob/ob mice, alleviating fat accumulation and glucose intolerance. The α diversity of gut microbial community of ob/ob mice was reduced after inulin treatment, while the ß diversity tended to return to the level of wild type mice. Interestingly, Prevotellaceae UCG 001 (family Prevotellaceae) was obviously enriched after inulin treatment. A comparative analysis of the gene expression profile showed that the cecal transcriptome was changed in leptin gene deficiency mice, whereas the inulin-supplemented diet partially reversed the changes in leptin gene-related signaling pathways, especially AMPK signaling pathway, where the levels of gene expression became comparable to those in wild type mice. Further analysis indicated that Prevotellaceae UCG 001 was positively correlated with the AMPK signaling pathway, which was negatively correlated with markers of glycolipid metabolism disorders. Our results suggest that the inulin-supplemented diet alleviates glucose and lipid metabolism disorders by partially restoring leptin related pathways mediated by gut microbiota.

Effect of Functional Oligosaccharides and Ordinary Dietary Fiber on Intestinal Microbiota Diversity.

Functional oligosaccharides, known as prebiotics, and ordinary dietary fiber have important roles in modulating the structure of intestinal microbiota. To investigate their effects on the intestinal microecosystem, three kinds of diets containing different prebiotics were used to feed mice for 3 weeks, as follows: GI (galacto-oligosaccharides and inulin), PF (polydextrose and insoluble dietary fiber from bran), and a GI/PF mixture (GI and PF, 1:1), 16S rRNA gene sequencing and metabolic analysis of mice feces were then conducted. Compared to the control group, the different prebiotics diets had varying effects on the structure and diversity of intestinal microbiota. GI and PF supplementation led to significant changes in intestinal microbiota, including an increase of Bacteroides and a decrease of Alloprevotella in the GI-fed, but those changes were opposite in PF fed group. Intriguing, in the GI/PF mixture-fed group, intestinal microbiota had the similar structure as the control groups, and flora diversity was upregulated. Fecal metabolic profiling showed that the diversity of intestinal microbiota was helpful in maintaining the stability of fecal metabolites. Our results showed that a single type of oligosaccharides or dietary fiber caused the reduction of bacteria species, and selectively promoted the growth of Bacteroides or Alloprevotella bacteria, resulting in an increase in diamine oxidase (DAO) and/or trimethylamine N-oxide (TMAO) values which was detrimental to health. However, the flora diversity was improved and the DAO values was significantly decreased when the addition of nutritionally balanced GI/PF mixture. Thus, we suggested that maintaining microbiota diversity and the abundance of dominant bacteria in the intestine is extremely important for the health, and that the addition of a combination of oligosaccharides and dietary fiber helps maintain the health of the intestinal microecosystem.

Community-Metabolome Correlations of Gut Microbiota from Child-Turcotte-Pugh of A and B Patients.

The gut flora are widely involved in the cometabolism with the host and have evident effects on the metabolic phenotype of host. This study performed a metabolome analysis of the intestinal microbiota specific for liver cirrhosis. The study population included patients with Child-Turcotte-Pugh score of A (AP, n = 5) and B (BP, n = 5), and control subjects (NM, n = 3). Metagenomic DNA from fecal microbiota was extracted followed by metagenomic sequencing through Illumina MiSeq high throughput sequencing of 16S rRNA regions. The detection of metabolites from fecal samples was performed using high-performance liquid phase chromatography and gas chromatography coupled with tandem mass spectrometry. Intestinal microbiota community and metabolite analysis both showed separation of cirrhotic patients from control participants, moreover, the microbiota-metabolite correlations changed in cirrhotic patients. Fecal microbiota from cirrhotic patients, with the reduced diversity, contained a decreased abundance of Bacteroidetes and an increased abundance of Firmicutes and Proteobacteria compared with the normal samples. Analysis of metabolome revealed a remarkable change in the metabolic potential of the microbiota in cirrhotic patients, with specific higher concentrations of amine, unsaturated fatty acid, and short-chain fatty acids, and lower concentrations of sugar alcohol and amino acid, suggesting the initial equilibrium of gut microbiota community and co-metabolism with the host were perturbed by cirrhosis. Our study illustrated the relationship between fecal microbiota composition and metabolome in cirrhotic patients, which may improve the clinical prognosis of cirrhosis.