Emerging strategies for the genetic dissection of gene functions, cell types, and neural circuits in the mammalian brain.

The mammalian brain is composed of a large number of highly diverse cell types with different molecular, anatomical, and functional features. Distinct cellular identities are generated during development under the regulation of intricate genetic programs and manifested through unique combinations of gene expression. Recent advancements in our understanding of the molecular and cellular mechanisms underlying the assembly, function, and pathology of the brain circuitry depend on the invention and application of genetic strategies that engage intrinsic gene regulatory mechanisms. Here we review the strategies for gene regulation on DNA, RNA, and protein levels and their applications in cell type targeting and neural circuit dissection. We highlight newly emerged strategies and emphasize the importance of combinatorial approaches. We also discuss the potential caveats and pitfalls in current methods and suggest future prospects to improve their comprehensiveness and versatility.

Cell-Type-Specific Gene Inactivation and In Situ Restoration via Recombinase-Based Flipping of Targeted Genomic Region.

Conditional gene inactivation and restoration are powerful tools for studying gene functions in the nervous system and for modeling neuropsychiatric diseases. The combination of the two is necessary to interrogate specific cell types within defined developmental stages. However, very few methods and animal models have been developed for such purpose. Here we present a versatile method for conditional gene inactivation and in situ restoration through reversibly inverting a critical part of its endogenous genomic sequence by Cre- and Flp-mediated recombinations. Using this method, we generated a mouse model to manipulate Mecp2, an X-linked dosage-sensitive gene whose mutations cause Rett syndrome. Combined with multiple Cre- and Flp-expressing drivers and viral tools, we achieved efficient and reliable Mecp2 inactivation and restoration in the germline and several neuronal cell types, and demonstrated phenotypic reversal and prevention on cellular and behavioral levels in male mice. This study not only provides valuable tools and critical insights for Mecp2 and Rett syndrome, but also offers a generally applicable strategy to decipher other neurologic disorders.SIGNIFICANCE STATEMENT Studying neurodevelopment and modeling neurologic disorders rely on genetic tools, such as conditional gene regulation. We developed a new method to combine conditional gene inactivation and restoration on a single allele without disturbing endogenous expression pattern or dosage. We applied it to manipulate Mecp2, a gene residing on X chromosome whose malfunction leads to neurologic disease, including Rett syndrome. Our results demonstrated the efficiency, specificity, and versatility of this new method, provided valuable tools and critical insights for Mecp2 function and Rett syndrome research, and offered a generally applicable strategy to investigate other genes and genetic disorders.

microRNA Deficiency in VIP+ Interneurons Leads to Cortical Circuit Dysfunction.

Genetically distinct GABAergic interneuron subtypes play diverse roles in cortical circuits. Previous studies revealed that microRNAs (miRNAs) are differentially expressed in cortical interneuron subtypes, and are essential for the normal migration, maturation, and survival of medial ganglionic eminence-derived interneuron subtypes. How miRNAs function in vasoactive intestinal peptide expressing (VIP+) interneurons derived from the caudal ganglionic eminence remains elusive. Here, we conditionally removed Dicer in postmitotic VIP+ interneurons to block miRNA biogenesis. We found that the intrinsic and synaptic properties of VIP+ interneurons and pyramidal neurons were concordantly affected prior to a progressive loss of VIP+ interneurons. In vivo recording further revealed elevated cortical local field potential power. Mutant mice had a shorter life span but exhibited better spatial working memory and motor coordination. Our results demonstrate that miRNAs are indispensable for the function and survival of VIP+ interneurons, and highlight a key role of VIP+ interneurons in cortical circuits.

[Cortical 5-hydroxytryptamine receptor 3A (Htr3a) positive inhibitory neurons: diversity in type and function].

Cortical GABAergic inhibitory neurons are composed of three major classes, each expressing parvalbumin (PV), somatostatin (SOM) and 5-hydroxytryptamine receptor 3A (Htr3a), respectively. Htr3a+ inhibitory neurons are mainly derived from the caudal ganglionic eminence (CGE). This highly heterogeneous group of inhibitory neurons are comprised of many different subtypes with distinct molecular signatures, morphological and electrophysiological properties and connectivity patterns. In this review, we summarized recent research progress regarding cortical Htr3a+ inhibitory neurons, focusing on their molecular, morphological and electrophysiological diversity, and introduced some genetic mouse tools that were used to study Htr3a+ inhibitory neurons.

Integrating serum pharmacochemistry and network pharmacology to reveal the mechanism of chickpea in improving insulin resistance.

Although chickpea have great potential in the treatment of obesity and diabetes, the bioactive components and therapeutic targets of chickpea to prevent insulin resistance (IR) are still unclear. The purpose of this study was to investigate the chemical and pharmacological characteristics of chickpea on IR through serum pharmacochemistry and network pharmacology. The results revealed that compared with other polar fractions, the ethyl acetate extract of chickpea (CE) had the definitive performance on enhancing the capacities of glucose consumption and glycogen synthesis. In addition, we analyzed the components of CE in vivo and in vitro based on UPLC-Q-Orbitrap HRMS technology. There were 28 kinds of in vitro chemical components, among which the isoflavones included biochanin A, formononetin, ononin, sissotrin, and astragalin, etc. Concerningly, the chief prototype components of CE absorbed into the blood were biochanin A, formononetin, loliolide, and lenticin, etc. Furthermore, a total of 209 common targets between IR and active components of CE were screened out by network pharmacology, among which the key targets involved PI3K p85, NF-κB p65 and estrogen receptor 1, etc. Specifically, KEGG pathway analysis indicated that PI3K-AKT signaling pathway, HIF-1 signaling pathway, and AGE-RAGE signaling pathway may play critical roles in the IR remission by CE. Finally, the in vitro validation experiments disclosed that CE significantly balanced the oxidative stress state of IR-HepG2 cells and inhibited expressions of inflammatory cytokines. In conclusion, the present study will be an important reference for clarifying the pharmacodynamic substance basis and underlying mechanism of chickpea to alleviate IR.

Rapidly enrichment and detection of per-and polyfluoroalkyl substances in foods using a novel bifunctional covalent organic framework.

Per- and polyfluoroalkyl substances (PFASs) are extensively found in foods, posing potential toxicity to humans. Therefore, rapid analysis and monitoring of PFASs in foods are crucial for public health and also a challenge. To detect trace PFASs in foods, construction of sorbents with multiple interactions could be an effective approach. Herein, a cationic-fluorinated covalent organic framework (CF-COF) was prepared by post-modification and used as a magnetic solid-phase extraction adsorbent for adsorption of PFASs. By combining magnetic solid-phase extraction based on CF-COF with liquid chromatography-tandem mass spectrometry (LC - MS/MS), a novel method was developed for determination of eight long-chain PFASs in foods. Under optimized conditions, the method exhibited low detection limits (0.003-0.019 ng/g) and satisfactory recovery rates (73.5-118%) for PFASs. This study introduces a novel idea for the development of adsorbents targeting PFASs, along with a new analytical method for monitoring of PFASs in foods.

Cortical VIP+ Interneurons in the Upper and Deeper Layers Are Transcriptionally Distinct.

Different interneuron classes have distinct laminar distribution patterns which contribute to the layer-specific organization of cortical microcircuits. However, laminar differences within the same interneuron classes are not well recognized. Despite systematic efforts towards neuron cell-type taxonomy in the neocortex by single-cell transcriptomics, less attention has been driven towards laminar differences in interneurons compared to projection neurons. VIP+ interneurons are the major interneuron class that mostly populate superficial layers and mediate disinhibition. A few reports noted the morphological and electrophysiological differences between VIP+ interneurons residing in layers I-III (upper layer) and layers IV-VI (deeper layer), but little is known about their molecular differences. Here, we delineated the laminar difference in their transcriptome employing single-cell RNA sequencing (scRNAseq) data from public databases. Analysis of 1175 high-quality VIP+ interneurons in the primary visual cortex (VISp) showed that the upper layer and deeper layer VIP+ interneurons are transcriptionally distinct distinguished by genes implicated in synapse organization and regulation of membrane potential. Similar differences are also observed in the anterior lateral motor cortex (ALM) and primary motor cortex (MOp). Cross-comparing between the top 10 differentially expressed genes (DEGs) with Allen Mouse Brain in situ hybridization database, we identified Tac2 and CxCl14 as potential marker genes of upper layer VIP+ interneurons across most cortical regions. Importantly, such expression patterns are conserved in the human brain. Together, we revealed significant laminar differences in transcriptomic profiles within VIP+ interneurons, which provided new insight into their molecular heterogeneity that may contribute to their functional diversity.

Visualized analysis of hotspots and frontiers in diabetes-associated periodontal disease research: a bibliometric study.

Background: Diabetes-associated periodontal disease is caused by diabetes-enhanced host immune-inflammatory responses to bacterial insult. An increasing number of papers related to diabetes-associated periodontal disease have been published. This study analyzed research on diabetes-associated periodontal disease with bibliometrics methods. The objective of this study was to identify hotspots and frontiers in the diabetes-associated periodontal disease research field. Methods: Publications were extracted from the Web of Science core collection database, and the document types included were limited to articles and reviews. The bibliometric analysis software CiteSpace5 was used to analyze the number of articles, research fields, countries/regions, institutions, authors, keywords, and other information. Outcomes were visualized to analyze the hotspots and research frontiers of diabetes-associated periodontal disease. Results: A total of 3,572 articles were retrieved. Among the research fields, dentistry, oral surgery, and medicine accounted for the highest proportion of publications, and public, environmental, and occupational health had the highest betweenness centrality. The number of publications from the United States ranked first among all the countries, while Columbia University ranked first among all the institutions. Global cooperation was not frequent. Keyword analysis showed that inflammatory pathways were the hotspots. Burst words analysis indicated that early prevention was a research frontier. Conclusions: The bibliometric method helped identify research hotspots and frontiers. Inflammatory pathways were hotspots, and early prevention was a frontier in diabetes-associated periodontal disease.

Ablation of microRNAs in VIP+ interneurons impairs olfactory discrimination and decreases neural activity in the olfactory bulb.

AIM: MicroRNAs (miRNAs) are abundantly expressed in vasoactive intestinal peptide expressing (VIP+ ) interneurons and are indispensable for their functional maintenance and survival. Here, we blocked miRNA biogenesis in postmitotic VIP+ interneurons in mice by selectively ablating Dicer, an enzyme essential for miRNA maturation, to study whether ablation of VIP+ miRNA affects olfactory function and neural activity in olfactory centres such as the olfactory bulb, which contains a large number of VIP+ interneurons. METHODS: A go/no-go odour discrimination task and a food-seeking test were used to assess olfactory discrimination and olfactory detection. In vivo electrophysiological techniques were used to record single units and local field potentials. RESULTS: Olfactory detection and olfactory discrimination behaviours were impaired in VIP+ -specific Dicer-knockout mice. In vivo electrophysiological recordings in awake, head-fixed mice showed that both spontaneous and odour-evoked firing rates were decreased in mitral/tufted cells in knockout mice. The power of ongoing and odour-evoked beta local field potentials response of the olfactory bulb and anterior piriform cortex were dramatically decreased. Furthermore, the coherence of theta oscillations between the olfactory bulb and anterior piriform cortex was decreased. Importantly, Dicer knockout restricted to olfactory bulb VIP+ interneurons recapitulated the behavioural and electrophysiological results of the global knockout. CONCLUSIONS: VIP+ miRNAs are an important factor in sensory processing, affecting olfactory function and olfactory neural activity.

Transcardiac Perfusion of the Mouse for Brain Tissue Dissection and Fixation.

Transcardiac perfusion with saline followed by 4% paraformaldehyde (PFA) is widely used to clear blood and preserve brain for immunostaining or in situ hybridization. PFA breaks into formaldehyde in solution, which cross-link protein and DNA molecules to preserve tissue and cell structure. Here we provide a step by step guide for performing this procedure in mouse.

Topographical organization of mammillary neurogenesis and efferent projections in the mouse brain.

The mammillary body is a hypothalamic nucleus that has important functions in memory and spatial navigation, but its developmental principles remain not well understood. Here, we identify progenitor-specific Fezf2 expression in the developing mammillary body and develop an intersectional fate-mapping approach to demonstrate that Fezf2+ mammillary progenitors generate mammillary neurons in a rostral-dorsal-lateral to caudal-ventral-medial fashion. Axonal tracing from different temporal cohorts of labeled mammillary neurons reveal their topographical organization. Unsupervised hierarchical clustering based on intrinsic properties further identify two distinct neuronal clusters independent of birthdates in the medial nuclei. In addition, we generate Fezf2 knockout mice and observe the smaller mammillary body with largely normal anatomy and mildly affected cellular electrophysiology, in contrast to more severe deficits in neuronal differentiation and projection in many other brain regions. These results indicate that Fezf2 may function differently in the mammillary body. Our results provide important insights for mammillary development and connectivity.

Viral approaches to study the mammalian brain: Lineage tracing, circuit dissection and therapeutic applications.

Viral vectors are widely used to study the development, function and pathology of neural circuits in the mammalian brain. Their flexible payloads with customizable choices of tool genes allow versatile applications ranging from lineage tracing, circuit mapping and functional interrogation, to translational and therapeutic applications. Different applications have distinct technological requirements, therefore, often utilize different types of virus. This review introduces the most commonly used viruses for these applications and some recent advances in improving the resolution and throughput of lineage tracing, the efficacy and selectivity of circuit tracing and the specificity of cell type targeting.

Visualized analysis of trends and hotspots in global oral microbiome research: A bibliometric study.

The oral microbiome contains numerous bacteria, which directly or indirectly participate in various human functions and continuously exchange signals and substances with the human body, significantly affecting human life cycle, health, and disease. This study aimed to conduct bibliometric studies on the scientific outputs of global oral microbiome research by Citespace software. The data were obtained from the Thomson Reuters' Web of Science Core Collection (WoSCC), from the first relevant literature published until December 31st, 2019, and a total of 2225 articles and reviews were identified. The top country and institutions are the United States and Harvard University. Keywords analysis showed that periodontal disease, oral microbes, and dental plaque are research hotspots. The burst word analysis indicates that early childhood caries, squamous cell carcinoma, gut microbiome, Helicobacter pylori, Candida albicans, and dysbiosis are likely to become the research hotspots of the next era. We also recommend the use of knowledge mapping methods to track specific knowledge areas efficiently and objectively regularly, which can accurately identify hotspots and frontiers and provide valuable information for practitioners in the field, including related scientists, students, journals, and editors.

Enormous influence of TNIK knockdown on intracellular signals and cell survival.

Basic cellular activities and coordinated cell actions are governed by intracellular signals, among which the Wnt signaling cascade plays an important role in tissue polarity and cell adhesion or movement through the activation of c-Jun N-terminal kinase (JNK) pathway. As one of the central transcriptional factors, Traf2- and Nck-interacting kinase (TNIK) mediates the transactivation of Wnt target genes and promotes the activity of c-Jun N-terminus kinase (JNK)2 when overexpressed. To further understand the function of TNIK, changes in intracellular signals were detected in colon cancer cell lines using a knockdown strategy. In this study, we found that the short-hairpin RNA-mediated knockdown of TNIK decreased the expressions of CD44, c-MYC and cyclin D1, which was consistent with the results of a TCF-4 reporter assay. Our data showed, for the first time, that the activation of both JNK1 and JNK2 by TNFα could be blocked through TNIK knockdown, which dampened the AP1 luciferase activity accordingly. In addition, adenovirus mediated the downregulation of TNIK-triggered intrinsic apoptosis in SW480 cells by activating caspase-9 and PARP-1. We conclude that TNIK is essential for the activation of both the canonical Wnt pathway and the JNK pathway, and serves as a pro-survival factor.