Morphological Tracing and Functional Identification of Monosynaptic Connections in the Brain: A Comprehensive Guide.

Behavioral studies play a crucial role in unraveling the mechanisms underlying brain function. Recent advances in optogenetics, neuronal typing and labeling, and circuit tracing have facilitated the dissection of the neural circuitry involved in various important behaviors. The identification of monosynaptic connections, both upstream and downstream of specific neurons, serves as the foundation for understanding complex neural circuits and studying behavioral mechanisms. However, the practical implementation and mechanistic understanding of monosynaptic connection tracing techniques and functional identification remain challenging, particularly for inexperienced researchers. Improper application of these methods and misinterpretation of results can impede experimental progress and lead to erroneous conclusions. In this paper, we present a comprehensive description of the principles, specific operational details, and key steps involved in tracing anterograde and retrograde monosynaptic connections. We outline the process of functionally identifying monosynaptic connections through the integration of optogenetics and electrophysiological techniques, providing practical guidance for researchers.

Potential mitigation of titanium dioxide nanoparticles against 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis through inhibiting the canonical NF-κB pathway.

Titanium dioxide nanoparticles (TiO2 NPs) have been widely employed in various industry fields, which makes consumers concerned about their health impact. Our previous work displayed that TiO2 NPs participate in the mitigation of TNBS-induced colitis, but the mechanism is still unknown. This work aimed to explore the role of oxidative stress and NF-κB pathway in the effect of TiO2 NPs on TNBS-induced colitis. The results showed that TiO2 NPs administration reduced the DAI score of colitis mice after TNBS enema. TiO2 NPs did not alter oxidative stress states (GSH/GSSG), but repaired the gut dysbacteriosis and inhibited the canonical NF-κB pathway activation in TNBS-induced colitis mice, manifested as a decrease in pathogenic bacteria and an increase in beneficial bacteria, as well as down-regulation of Toll-like receptors (TLRs), IKKα, IKKß, p65 and pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α and IFN-γ) in mRNA level, and the increased transcription of anti-inflammatory cytokines (IL-10, TGF-ß, and IL-12), along with the declined protein level of TNF-α in TiO2 NPs treated colitis mice. The present study suggested that oral TiO2 NPs administration inhibited the canonical NF-κB pathway activation by repairing gut dysbacteriosis, which made a predominant role in alleviating colitis. These findings provided a new perspective for exploring the safety of TiO2 NPs.

The basal forebrain to lateral habenula circuitry mediates social behavioral maladaptation.

Elucidating the neural basis of fear allows for more effective treatments for maladaptive fear often observed in psychiatric disorders. Although the basal forebrain (BF) has an essential role in fear learning, its function in fear expression and the underlying neuronal and circuit substrates are much less understood. Here we report that BF glutamatergic neurons are robustly activated by social stimulus following social fear conditioning in male mice. And cell-type-specific inhibition of those excitatory neurons largely reduces social fear expression. At the circuit level, BF glutamatergic neurons make functional contacts with the lateral habenula (LHb) neurons and these connections are potentiated in conditioned mice. Moreover, optogenetic inhibition of BF-LHb glutamatergic pathway significantly reduces social fear responses. These data unravel an important function of the BF in fear expression via its glutamatergic projection onto the LHb, and suggest that selective targeting BF-LHb excitatory circuitry could alleviate maladaptive fear in relevant disorders.

The development of Tibetan children's racial bias in empathy: The mediating role of ethnic identity and wrongfulness of ethnic intergroup bias.

OBJECTIVES: Individuals often automatically have more empathy for same-race members. However, there are no studies on racial bias in empathy (RBE) among Tibetan school-aged children. The present study aimed to examine the development of RBEs, including racial bias in cognitive empathy, affective empathy, and behavioral empathy, in Tibetan school-aged children. METHOD: In Experiment 1 (N = 108, aged 7-12), ethnic identity was primed using Tibetan and Han names. Then negative and neutral events were applied to measure the RBEs of Tibetan children. In Experiment 2 (N = 148, aged 7-12), negative events were replaced by pain events. In Experiment 3 (N = 60, aged 7-12), Tibetan children's ethnic identity and the awareness of the wrongfulness of ethnic intergroup bias were added to examine the underlying mechanism. RESULT: Results found that RBEs increased among Tibetan children aged 7-10 and decreased among those aged 11-12, Moreover, we analyzed age as a continuous variable and found that 10 years old was the inflection point in the development of RBEs in Tibetan children. Importantly, children aged 11-12 years old realized more wrongfulness of ethnic intergroup bias than children aged 7-10. The ethnic identity of Tibetan children aged 7-10 mediated the relation between age group and RBEs. And the wrongfulness of ethnic intergroup bias mediated the link between age group and RBEs in Tibetan children aged 9-12. CONCLUSION: Our study sheds light on the development of RBEs in Tibetan school-aged children and highlights the importance of identifying the appropriate timing for intervening in prejudice. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Control of defensive behavior by the nucleus of Darkschewitsch GABAergic neurons.

The nucleus of Darkschewitsch (ND), mainly composed of GABAergic neurons, is widely recognized as a component of the eye-movement controlling system. However, the functional contribution of ND GABAergic neurons (NDGABA) in animal behavior is largely unknown. Here, we show that NDGABA neurons were selectively activated by different types of fear stimuli, such as predator odor and foot shock. Optogenetic and chemogenetic manipulations revealed that NDGABA neurons mediate freezing behavior. Moreover, using circuit-based optogenetic and neuroanatomical tracing methods, we identified an excitatory pathway from the lateral periaqueductal gray (lPAG) to the ND that induces freezing by exciting ND inhibitory outputs to the motor-related gigantocellular reticular nucleus, ventral part (GiV). Together, these findings indicate the NDGABA population as a novel hub for controlling defensive response by relaying fearful information from the lPAG to GiV, a mechanism critical for understanding how the freezing behavior is encoded in the mammalian brain.

Phasic/tonic glial GABA differentially transduce for olfactory adaptation and neuronal aging.

Phasic (fast) and tonic (sustained) inhibition of γ-aminobutyric acid (GABA) are fundamental for regulating day-to-day activities, neuronal excitability, and plasticity. However, the mechanisms and physiological functions of glial GABA transductions remain poorly understood. Here, we report that the AMsh glia in Caenorhabditis elegans exhibit both phasic and tonic GABAergic signaling, which distinctively regulate olfactory adaptation and neuronal aging. Through genetic screening, we find that GABA permeates through bestrophin-9/-13/-14 anion channels from AMsh glia, which primarily activate the metabolic GABAB receptor GBB-1 in the neighboring ASH sensory neurons. This tonic action of glial GABA regulates the age-associated changes of ASH neurons and olfactory responses via a conserved signaling pathway, inducing neuroprotection. In addition, the calcium-evoked, vesicular glial GABA release acts upon the ionotropic GABAA receptor LGC-38 in ASH neurons to regulate olfactory adaptation. These findings underscore the fundamental significance of glial GABA in maintaining healthy aging and neuronal stability.

How well do neural signatures of resting-state EEG detect consciousness? A large-scale clinical study.

The assessment of consciousness states, especially distinguishing minimally conscious states (MCS) from unresponsive wakefulness states (UWS), constitutes a pivotal role in clinical therapies. Despite that numerous neural signatures of consciousness have been proposed, the effectiveness and reliability of such signatures for clinical consciousness assessment still remains an intense debate. Through a comprehensive review of the literature, inconsistent findings are observed about the effectiveness of diverse neural signatures. Notably, the majority of existing studies have evaluated neural signatures on a limited number of subjects (usually below 30), which may result in uncertain conclusions due to small data bias. This study presents a systematic evaluation of neural signatures with large-scale clinical resting-state electroencephalography (EEG) signals containing 99 UWS, 129 MCS, 36 emergence from the minimally conscious state, and 32 healthy subjects (296 total) collected over 3 years. A total of 380 EEG-based metrics for consciousness detection, including spectrum features, nonlinear measures, functional connectivity, and graph-based measures, are summarized and evaluated. To further mitigate the effect of data bias, the evaluation is performed with bootstrap sampling so that reliable measures can be obtained. The results of this study suggest that relative power in alpha and delta serve as dependable indicators of consciousness. With the MCS group, there is a notable increase in the phase lag index-related connectivity measures and enhanced functional connectivity between brain regions in comparison to the UWS group. A combination of features enables the development of an automatic detector of conscious states.

Biphasic release of betamethasone from an injectable HA hydrogel implant for alleviating lumbar disc herniation induced sciatica.

Epidural steroid injection (ESI) is a common therapeutic approach for managing sciatica caused by lumbar disc herniation (LDH). However, the short duration of therapeutic efficacy and the need for repeated injections pose challenges in LDH treatment. The development of a controlled delivery system capable of prolonging the effectiveness of ESI and reducing the frequency of injections, is highly significant in LDH clinical practice. In this study, we utilized a thiol-ene click chemistry to create a series of injectable hyaluronic acid (HA) based release systems loaded with diphasic betamethasone, including betamethasone dipropionate (BD) and betamethasone 21-phosphate disodium (BP) (BD/BP@HA). BD/BP@HA hydrogel implants demonstrated biocompatibility and biodegradability to matched neuronal tissues, avoiding artificial compression following injection. The sustained release of betamethasone from BD/BP@HA hydrogels effectively inhibited both acute and chronic neuroinflammation by suppressing the nuclear factor kappa-B (NF-κB) pathway. In a mouse model of LDH, the epidural administration of BD/BP@HA efficiently alleviated LDH-induced sciatica for at least 10 days by inhibiting the activation of macrophages and microglia in dorsal root ganglion and spinal dorsal horn, respectively. The newly developed HA hydrogels represent a valuable platform for achieving sustained drug release. Additionally, we provide a simple paradigm for fabricating BD/BP@HA for epidural injection, demonstrating greater and sustained efficiency in alleviating LDH-induced sciatica compared to traditional ESI and displaying potentials for clinical translation. This system has the potential to revolutionize drug delivery for co-delivery of both soluble and insoluble drugs, thereby making a significant impact in the pharmaceutical industry. STATEMENT OF SIGNIFICANCE: Lumbar disc herniation (LDH) is a common degenerative disorder leading to sciatica and spine surgery. Although epidural steroid injection (ESI) is routinely used to alleviate sciatica, the efficacy is short and repeated injections are required. There remains challenging to prolong the efficacy of ESI. Herein, an injectable hyaluronic acid (HA) hydrogel implant by crosslinking acrylated-modified HA (HA-A) with thiol-modified HA (HA-SH) was designed to achieve a biphasic release of betamethasone. The hydrogel showed biocompatibility and biodegradability to match neuronal tissues. Notably, compared to traditional ESI, the hydrogel better alleviated sciatica in vivo by synergistically inhibiting the neuroinflammation in central and peripheral nervous systems. We anticipate the injectable HA hydrogel implant has the potential for clinical translation in treating LDH-induced sciatica.

How Society Anxiety Influences Attention Control in College Students: The Moderated Mediation Effect of Cognitive Flexibility and Resting-state Electroencephalography Activity.

Social anxiety is a prevalent issue among college students, adversely affecting their overall well-being. Drawing from the cognitive model of social anxiety and attention control theory, heightened levels of social anxiety may correspond to poorer attention control ability. However, little is known about the underlying cognitive mechanisms of the relationship between social anxiety and attention control. To address this research gap, the current study recruited a sample of 156 college students (56 women) who underwent self-report measures of social anxiety, cognitive flexibility, and attention control, followed by a resting-state EEG recording. The results revealed a significant negative predictive effect of social anxiety on attention control, with cognitive flexibility partially mediating this relationship. Furthermore, resting-state theta power emerged as a significant moderator, accentuating the negative impact of social anxiety on cognitive flexibility among individuals with lower theta power. In addition, frontal alpha asymmetry (FAA) demonstrated a moderating effect, with lower FAA intensifying the predictive influence of cognitive flexibility on attention control. Taken together, these results suggested that social anxiety can predict attention control either directly or indirectly via the mediating role of cognitive flexibility, and lower theta power and FAA has a risk amplification effect, which provide novel insights into the treatment and prevention of social anxiety and its negative impact on college students.

Temporal-spatial Generation of Astrocytes in the Developing Diencephalon.

Astrocytes are the largest glial population in the mammalian brain. However, we have a minimal understanding of astrocyte development, especially fate specification in different regions of the brain. Through lineage tracing of the progenitors of the third ventricle (3V) wall via in-utero electroporation in the embryonic mouse brain, we show the fate specification and migration pattern of astrocytes derived from radial glia along the 3V wall. Unexpectedly, radial glia located in different regions along the 3V wall of the diencephalon produce distinct cell types: radial glia in the upper region produce astrocytes and those in the lower region produce neurons in the diencephalon. With genetic fate mapping analysis, we reveal that the first population of astrocytes appears along the zona incerta in the diencephalon. Astrogenesis occurs at an early time point in the dorsal region relative to that in the ventral region of the developing diencephalon. With transcriptomic analysis of the region-specific 3V wall and lateral ventricle (LV) wall, we identified cohorts of differentially-expressed genes in the dorsal 3V wall compared to the ventral 3V wall and LV wall that may regulate astrogenesis in the dorsal diencephalon. Together, these results demonstrate that the generation of astrocytes shows a spatiotemporal pattern in the developing mouse diencephalon.

Single-Cell Transcriptomes and Immune Repertoires Reveal the Cell State and Molecular Changes in Pemphigus Vulgaris.

The etiology and pathogenesis of pemphigus vulgaris (PV) entail intricate interactions between immune cells and epithelial cells. However, the specific subtypes of immune cells involved in PV, along with their respective roles, remain elusive. Likewise, the precise functions and mechanisms by which glucocorticoids affect cell types within the disease context require further elucidation. To address these knowledge gaps, we performed 5' single-cell RNA sequencing, combined with V(D)J enrichment on buccal mucosal lesions and peripheral blood samples from treatment-naive patients with PV, in conjunction with post-treatment peripheral blood samples obtained after oral prednisone treatment. Our findings suggest that the IL-1α signaling pathway, myeloid APCs, inflammatory CD8+ resident memory T cells, and dysfunctional CD4+ regulatory T cells are involved in the pathogenesis of PV. Part of these findings were validated by immunohistochemical assays and multiplex immunofluorescence assays. Furthermore, our results highlight the significant impact of prednisone treatment on monocytes and mucosal-associated invariant T cells while revealing a limited effect on CD4+ regulatory T cells. Additionally, we present the CDR3 amino acid sequence of BCR related to PV disease and investigate the characteristics of TCR/BCR clonotypes. In conclusion, our study provides a comprehensive understanding of PV, particularly focusing on the mucosal-dominant type, and sheds light on the effects of glucocorticoids within the PV context. These insights hold promise for the development of new therapeutic strategies in this autoimmune disorder.

Neural adaption in midbrain GABAergic cells contributes to high-fat diet-induced obesity.

Overeating disorders largely contribute to worldwide incidences of obesity. Available treatments are limited. Here, we discovered that long-term chemogenetic activation of ventrolateral periaqueductal gray (vlPAG) GABAergic cells rescue obesity of high-fat diet-induced obesity (DIO) mice. This was associated with the recovery of enhanced mIPSCs, decreased food intake, increased energy expenditure, and inguinal white adipose tissue (iWAT) browning. In vivo calcium imaging confirmed vlPAG GABAergic suppression for DIO mice, with corresponding reduction in intrinsic excitability. Single-nucleus RNA sequencing identified transcriptional expression changes in GABAergic cell subtypes in DIO mice, highlighting Cacna2d1 as of potential importance. Overexpressing CACNA2D1 in vlPAG GABAergic cells of DIO mice rescued enhanced mIPSCs and calcium response, reversed obesity, and therefore presented here as a potential target for obesity treatment.

Sustained antidepressant effect of ketamine through NMDAR trapping in the LHb.

Ketamine, an N-methyl-D-aspartate receptor (NMDAR) antagonist1, has revolutionized the treatment of depression because of its potent, rapid and sustained antidepressant effects2-4. Although the elimination half-life of ketamine is only 13 min in mice5, its antidepressant activities can last for at least 24 h6-9. This large discrepancy poses an interesting basic biological question and has strong clinical implications. Here we demonstrate that after a single systemic injection, ketamine continues to suppress burst firing and block NMDARs in the lateral habenula (LHb) for up to 24 h. This long inhibition of NMDARs is not due to endocytosis but depends on the use-dependent trapping of ketamine in NMDARs. The rate of untrapping is regulated by neural activity. Harnessing the dynamic equilibrium of ketamine-NMDAR interactions by activating the LHb and opening local NMDARs at different plasma ketamine concentrations, we were able to either shorten or prolong the antidepressant effects of ketamine in vivo. These results provide new insights into the causal mechanisms of the sustained antidepressant effects of ketamine. The ability to modulate the duration of ketamine action based on the biophysical properties of ketamine-NMDAR interactions opens up new opportunities for the therapeutic use of ketamine.

Dopamine release and negative valence gated by inhibitory neurons in the laterodorsal tegmental nucleus.

GABAergic neurons in the laterodorsal tegmental nucleus (LDTGABA) encode aversion by directly inhibiting mesolimbic dopamine (DA). Yet, the detailed cellular and circuit mechanisms by which these cells relay unpleasant stimuli to DA neurons and regulate behavioral output remain largely unclear. Here, we show that LDTGABA neurons bidirectionally respond to rewarding and aversive stimuli in mice. Activation of LDTGABA neurons promotes aversion and reduces DA release in the lateral nucleus accumbens. Furthermore, we identified two molecularly distinct LDTGABA cell populations. Somatostatin-expressing (Sst+) LDTGABA neurons indirectly regulate the mesolimbic DA system by disinhibiting excitatory hypothalamic neurons. In contrast, Reelin-expressing LDTGABA neurons directly inhibit downstream DA neurons. The identification of separate GABAergic subpopulations in a single brainstem nucleus that relay unpleasant stimuli to the mesolimbic DA system through direct and indirect projections is critical for establishing a circuit-level understanding of how negative valence is encoded in the mammalian brain.

Oral intake of titanium dioxide nanoparticles affect the course and prognosis of ulcerative colitis in mice: involvement of the ROS-TXNIP-NLRP3 inflammasome pathway.

BACKGROUND: Titanium dioxide (TiO2), no matter in nanoscale or micron sizes, has been widely used in food industry as additives for decades. Given the potential impact of TiO2 on the gastrointestinal epithelial and parenchymal cells, including goblet cells, the public consumers may suffer the risk of diseases caused by its widespread dissemination in food products. We therefore set out to investigate the impact of TiO2 NPs on the course and prognosis of ulcerative colitis by oral gavaging TiO2 NPs at the doses levels of 0, 30, 100, and 300 mg/kg during the induction (7 days, from day 1 to day 7) and recovery (10 days, from day 8 to day 17) phases of colitis in mice. RESULTS: The ulcerative colitis (UC) disease model was established by administrating of 2.5% dextran sulfate sodium (DSS) solution. Our results show that TiO2 NPs significantly enhanced the severity of DSS-induced colitis, decreased the body weight, increased the disease activity index (DAI) and colonic mucosa damage index (CMDI) scores, shortened the colonic length, increased the inflammatory infiltration in the colon. The most significant changes occurred in the low dose (30 mg/kg) group of TiO2 NPs exposure during the development phase of UC and the high dose (300 mg/kg) group of TiO2 NPs during UC self-healing phase. Increased reactive oxygen species (ROS) level and upregulation of anti-oxidant enzymes including total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-PX) and catalase (CAT), demonstrate that the TiO2 NP exposure has triggered oxidative stress in mice. Moreover, the upregulation of caspase-1 mRNA and increased expression of thioredoxin interacting protein (TXNIP) further demonstrate the involvement of the ROS-TXNIP-NLR family pyrin domain containing 3 (NLRP3) inflammasome pathway in aggravating the development of UC. CONCLUSION: Oral intake of TiO2 NPs could affect the course of acute colitis in exacerbating the development of UC, prolonging the UC course and inhibiting UC recovery.

Microglia modulate general anesthesia through P2Y12 receptor.

General anesthesia (GA) is an unconscious state produced by anesthetic drugs, which act on neurons to cause overall suppression of neuronal activity in the brain. Recent studies have revealed that GA also substantially enhances the dynamics of microglia, the primary brain immune cells, with increased process motility and territory surveillance. However, whether microglia are actively involved in GA modulation remains unknown. Here, we report a previously unrecognized role for microglia engaging in multiple GA processes. We found that microglial ablation reduced the sensitivity of mice to anesthetics and substantially shortened duration of loss of righting reflex (LORR) or unconsciousness induced by multiple anesthetics, thereby promoting earlier emergence from GA. Microglial repopulation restored the regular anesthetic recovery, and chemogenetic activation of microglia prolonged the duration of LORR. In addition, anesthesia-accompanying analgesia and hypothermia were also attenuated after microglial depletion. Single-cell RNA sequencing analyses showed that anesthesia prominently affected the transcriptional levels of chemotaxis and migration-related genes in microglia. By pharmacologically targeting different microglial motility pathways, we found that blocking P2Y12 receptor (P2Y12R) reduced the duration of LORR of mice. Moreover, genetic ablation of P2Y12R in microglia also promoted quicker recovery in mice from anesthesia, verifying the importance of microglial P2Y12R in anesthetic regulation. Our work presents the first evidence that microglia actively participate in multiple processes of GA through P2Y12R-mediated signaling and expands the non-immune roles of microglia in the brain.

Single-cell immune profiling reveals immune responses in oral lichen planus.

Introduction: Oral lichen planus (OLP) is a common chronic inflammatory disorder of the oral mucosa with an unclear etiology. Several types of immune cells are involved in the pathogenesis of OLP. Methods: We used single-cell RNA sequencing and immune repertoire sequencing to characterize the mucosal immune microenvironment of OLP. The presence of tissue-resident memory CD8+ T cells are validated by multiplex immunofluorescence. Results: We generated a transcriptome atlas from four OLP biopsy samples and their paired peripheral blood mononuclear cells (PBMCs), and compared them with two healthy tissues and three healthy PBMCs samples. Our analysis revealed activated tissue-resident memory CD8+ T cells in OLP tissues. T cell receptor repertoires displayed apperant clonal expansion and preferrential gene pairing in OLP patients. Additionally, obvious BCR clonal expansion was observed in OLP lesions. Plasmacytoid dendritic cells, a subtype that can promote dendritic cell maturation and enhance lymphocyte cytotoxicity, were identified in OLP. Conventional dendritic cells and macrophages are also found to exhibit pro-inflammatory activity in OLP. Cell-cell communication analysis reveals that fibroblasts might promote the recruitment and extravasation of immune cells into connective tissue. Discussion: Our study provides insights into the immune ecosystem of OLP, serving as a valuable resource for precision diagnosis and therapy of OLP.

A parabrachial to hypothalamic pathway mediates defensive behavior.

Defensive behaviors are critical for animal's survival. Both the paraventricular nucleus of the hypothalamus (PVN) and the parabrachial nucleus (PBN) have been shown to be involved in defensive behaviors. However, whether there are direct connections between them to mediate defensive behaviors remains unclear. Here, by retrograde and anterograde tracing, we uncover that cholecystokinin (CCK)-expressing neurons in the lateral PBN (LPBCCK) directly project to the PVN. By in vivo fiber photometry recording, we find that LPBCCK neurons actively respond to various threat stimuli. Selective photoactivation of LPBCCK neurons promotes aversion and defensive behaviors. Conversely, photoinhibition of LPBCCK neurons attenuates rat or looming stimuli-induced flight responses. Optogenetic activation of LPBCCK axon terminals within the PVN or PVN glutamatergic neurons promotes defensive behaviors. Whereas chemogenetic and pharmacological inhibition of local PVN neurons prevent LPBCCK-PVN pathway activation-driven flight responses. These data suggest that LPBCCK neurons recruit downstream PVN neurons to actively engage in flight responses. Our study identifies a previously unrecognized role for the LPBCCK-PVN pathway in controlling defensive behaviors.