Neuroscience Bulletin

In vivo single-cell capture methods based on micropipettes are essential for correlating morphological characteristics with transcriptomic profiling under physiological conditions. However, they often suffer from significant contamination of off-target cells. Consequently, we developed Hip-seq, which significantly reduces heavily contaminated cells, decreases contamination levels, improves cell reproducibility, and enhances data analysis accuracy compared to patch-seq. Using Hip-seq, we found that axon regeneration failure could be due to abnormal activation of translation and the circadian clock. Axon-regenerable central neurons reactivated axon development-related genes during regeneration. And one-to-one associated analysis helped identify pro-regenerative genes.

In acute aminoglycoside ototoxicity to unilateral inner ear, physical abnormalities, such as nystagmus and postural alteration, are relieved within a few days by neural compensation. To examine exploratory behavior over an extended period, freely moving behavior of a mouse after unilateral kanamycin injection was recorded in a home-cage environment. A tail was excluded from deep learning-mediated object detection because of its delayed movement relative to the body. All detection results were confirmed by convolutional neural network classification model. In kanamycininjected mice, total distance moved in 15 minutes increased at 3 days after surgery. Moreover, the injured mouse turned frequently toward healthy side up to 17 days after surgery. Tail suspension and twist toward healthy side induced fast rotation of trunk around longitudinal axis with dorsal bending after 14 days. Our analysis strategy employing deep learning is useful to evaluate neuronal compensatory process and screen a drug candidate with therapeutic potency.

In Drosophila melanogaster, olfactory projection neurons (PNs) convey odor information from the peripheral olfactory center to higher brain regions. The anatomical and physiological properties of PNs have been well characterized at the cellular and circuit level. The ultrastructural features of PNs remain unknown however, particularly with respect to presynaptic active zones (PAZs) and dense core vesicles (DCVs). In the current study, membrane-labeled electron microscopy was used to volume-reconstruct 89 PN axonal boutons and identify the internal PAZs and DCVs. Based on ultrastructural parameters, these PN boutons could be classified into three morphological distinct subtypes. Interestingly, the distributions of PAZs and DCVs were distinct within these three subtypes. DCVs were enriched in membrane labeled GH146-positive boutons, suggesting that GH146-positive PNs release both neurotransmitters and neuromodulators. The study identified the detailed distributions of PAZs and DCVs in PN boutons and indicates that neuromodulators mediated by DCVs may play an important role in PNs for olfactory processing.

Golgi defects including Golgi fragmentation are pathological features of Alzheimer disease (AD). As a pathogenic factor of AD, amyloid precursor protein (APP) induces Golgi fragmentation in soma. However, how APP regulates Golgi outposts (GOs) in dendrites remains unclear. Given that APP resided and affected GOs movements, especially reversed the distribution of multi-compartment GOs (mcGOs), we investigated the regulatory mechanism of mcGOs movements in Drosophila larvae. Knockdown experiments showed the bidirectional mcGOs movements were cooperatively controlled by dynein heavy chain (Dhc) and kinesin heavy chain subunits. Notably, only Dhc mediated APPs regulation on mcGOs movements. Further, by loss-of-function screening, the adaptor protein Sunday driver (Syd) was identified to mediate APP-induced alteration of the direction of mcGOs movements, and dendritic defects. Collectively, by elucidating a model of bidirectional mcGOs movements, we revealed the mechanism of APPs regulation on the direction of mcGOs movements. It provides new insights into AD pathogenesis.

The loss of motor function in patients with spinal cord injury (SCI) is primarily due to the severing of the corticospinal tract (CST). Spinal motor neurons are located in the anterior horn of the spinal cord, and as the lower neurons of the CST, they control voluntary movement. Furthermore, its intrinsic axonal growth ability is significantly stronger than that of cerebral cortex pyramid neurons, which are the upper CST neurons. Therefore, we established an axonal regeneration model of spinal motor neurons to investigate the feasibility of repairing SCI by promoting axonal regeneration of spinal motor neurons. We demonstrated that conditionally knocking out pten in mature spinal motor neurons drastically enhanced axonal regeneration in vivo, and the regenerating axons of the spinal motor neurons re-established synapses with other cells in the damaged spinal cord. Thus, this strategy may serve as a novel and effective treatment method for SCI.

Nerve injury in the somatosensory pathway may induce maladaptive changes at the transcriptional or protein level, contributing to the development and maintenance of neuropathic pain. In contrast to the retrosplenial cortex (RSC), which processes nociceptive information and exhibits structural and molecular changes after nerve injury, detailed transcriptional changes in the RSC are not yet known. Here we confirm the involvement of the RSC in regulating pain sensation and observe that the same peripheral stimulation activates more retrosplenial neurons after nerve injury; reducing the activities of CaMKII+ splenial cells relieves peripheral pain hypersensitivity after nerve injury. Using a single-cell RNA sequencing (scRNA-seq) approach, we identified cell-type-specific gene expression changes after nerve injury, and the gene set enrichment analysis results revealed suppressed ion homeostasis in CaMKII+ neurons. Furthermore, examination of the expression of genes encoding ligand-gated ion channels showed a decrease in Gabar1a but an increase in Gria1 in CaMKII+ neurons; consistently, we confirmed the unbalanced excitatory/inhibitory synaptic transmission by using the electrophysiological recording approach. Moreover, micro-infusion of 1-Naphthyl acetyl spermine in the RSC to reduce excitatory synaptic transmission alleviated peripheral pain hypersensitivity. Our data confirm the involvement of the RSC in pain regulation and provide information on cell type-dependent transcriptomic changes after nerve injury, which will contribute to the understanding of the mechanisms mediating neuropathic pain.

Voltage signals in neurons are highly compartmentalized, which can influence their specific functions within neuronal circuits. Targeting of a genetically encoded voltage indicator (GEVI) to specific subcellular compartments can enhance the signal-to-noise ratio and provide more precise information about the location and timing of synaptic firing across different neuronal regions, reducing spatiotemporal signal convolution. To achieve subcellular targeting of the GEVI, ArcLight, we utilized five different postsynaptic targeting sequences (Shaker K+ channel C-terminus, stargazin C-terminus, rat Neuroligin-1 C-terminus, and anti-homer1 nanobodies HC20 & HC87) to direct ArcLight expression to the excitatory postsynaptic density. Additionally, we assessed a presynaptic-targeting tag (rat Neurexin-1{beta} C-terminus) and a somatodendritic targeting tag (Kv2.1-Lk-Tlcn C-terminus). Patch clamp experiments in HEK293 cells showed that the targeting tags used in this study did not significantly alter ArcLights voltage sensitivity compared to controls. AAV infection in the mouse olfactory bulb demonstrated that the subcellular targeting sequences effectively localized GEVI expression to specific compartments of mitral/tufted cells, including postsynaptic densities, presynaptic terminals, and somatodendritic regions. Furthermore, in vivo voltage imaging in mice expressing targeting-enhanced ArcLight variants revealed odorant-evoked responses similar to those observed with the original ArcLight. This indicates that subcellular targeting did not significantly impact the voltage sensing capability of ArcLight in mitral/tufted cells.

Drosophila melanogaster is a suitable model for investigating how neuropeptides influence animal behaviours and physiology. We previously reported that two behavioural paradigms control mating duration of male Drosophila, called Longer-Mating-Duration (LMD) and Shorter-Mating-Duration (SMD) that are induced through socio-sexual environment prior to copulation. Understanding the molecular and cellular mechanisms by which males exhibit plasticity to different social cues remains poorly understood. Here, we show that SIFa modulates the neural circuitry for both LMD and SMD. Neuropeptide-to-neuropeptide communication, so called neuropeptide relay plays a key role to mediate this control. We identified that 7 neuropeptides expressed in SIFa Receptor-positive cells are functionally important to regulate either LMD and/or SMD. The modulation of two independent mating duration behaviour by the different SIFa-mediated neuropeptide relay will help to further investigate how the neuropeptidergic modulation can control complex behaviours.

It is generally believed that humans are the only species that values life. However, it is not well understood whether animals have a nature that values life. In this study, we attempted to determine whether male Wistar rats have this nature. Normally-reared rats did not show lifesaving-like actions towards anesthesia-induced comatose rats, although they seemed to distinguish life from death. Considering the possibility that different rearing conditions may foster a life-valuing nature, male Wistar rat pups were reared in several ways: normal rearing, loving rearing (LR; rats reared as if they were cute pets), rearing in an enriched environment, reared with gentle stroking of the back, and normal rearing of offspring of rats raised under LR conditions. When placed in an anxiety-producing environment, only LR rats escaped into the hands of the person who reared them, indicating attachment. Only the LR rats displayed lifesaving-like actions towards unknown comatose rats or drowning pups. LR rats also stopped attacks by biting ICR mice that were attacking C57BL/6 mice. Thus, rearing in an affectionate environment may foster a life-valuing nature, even in rats, suggesting that the valuing of life may be neither innate nor human-specific.

The prosperity of electronic nicotine delivery systems (ENDS) or e-cigarette use has been regarded to lead an increasing risk of nicotine addiction, especially among youth. Understanding and evaluating the behaviors induced by ENDS are fundamental to the study of neuropsychiatric effects of e-cigarettes. However, little is known regarding the behavioral features during e-cigarette exposure in mice. Current behavioral assessments for nicotine addiction are based on nicotine withdrawal-induced anxiety which can be only performed after ENDS exposures. Here we developed MiceVAPORDot, a novel high-throughput tool for automated in situ behavioral characterization during e-cigarette exposure. The integration of a deep learning-based animal pose tracking method by MiceVAPORDot allows precise characterization on behavioral phenotypes of e-vapor exposed mice, which were unable revealed by traditional evaluation methodology such as conditioned place preference and elevated plus maze tests. The behavioral fingerprints recognized by MiceVAPORDot can be used for high-throughput screening on incentive nature of e-cigarette flavors as well as medications for smoking cessation.

Alzheimers disease (AD) is the most common neurodegenerative disease. To date, its cause is unclear and there are no effective treatments or preventive measures. Despite there are accumulating evidences for the existence of AD pathological hallmarks in the brain of aging rhesus monkeys, it remains a mainstream notion that monkeys do not develop AD naturally. This is an important issue because it will determine how we use monkeys in AD studies. To settle down this issue, a group (n=10) of aged rhesus monkeys 26 years old or above went through a systematic AD screening procedure in this study. Three of these monkeys showed severe memory impairments (SMI) after evaluated with a classic working memory test. Further behavioral testing revealed that the SMI monkeys also exhibited apathy-like behavior, which is another core AD clinical symptom. In addition to the cognitive deficits, two of the three SMI monkeys developed all of the three AD pathological hallmarks, including neurofibrillary tangles, senile plaques and neuronal loss. According to the diagnostic criteria of human AD, the two SMI monkeys were clearly naturally occurring AD monkeys. These results suggest that AD is not a uniquely human disease and monkeys have great potential for the development of much needed etiological AD models, which are vital for better understanding of developmental process of AD and the base of identification of early diagnostic biomarkers and effective therapeutic targets of AD.

Neuronal intranuclear inclusion disease (NIID) is a polyglycine disease that primarily affects the neuronal and neuromuscular systems. Here, we developed a novel transgenic mouse model that faithfully recapitulates the multisystemic impairments associated with polyG intranuclear inclusions. Our findings demonstrate that polyG expression induces neurodegeneration, behavioral deficits, and age-dependent accumulation of uN2CpolyG aggregates across multiple tissues.

Neurotropic virus tracers, particularly those with low toxicity and high efficient tracing, are powerful tools for structural and functional dissections of neural circuits. The retrograde trans-mono-synaptic technology based on rabies virus CVS-N2c strain has reduced cytotoxicity and enhanced efficiency, attains long-term gene manipulation for functional studies, but suffers from difficult preparation and low yield. To overcome these shortcomings, an improved production system was established for rapid rescue and preparation of CVS-N2c-{Delta}G virus, CVS-N2c-{Delta}G with the same titer as SAD-B19-{Delta}G can be prepared within a short time. Meanwhile, we found that N2cG coated CVS-N2c-{Delta}G allows efficient retrograde access to projection neurons, and further expand its application in VTA/SNc to DLS pathway that unaddressed by rAAV9-Retro, and the efficiency is 6 folds higher than that of rAAV9-Retro. Then the trans-synaptic efficiency of CVS-N2c-{Delta}G virus was evaluated. Results showed that the trans-mono-synaptic efficiency of oG-mediated CVS-N2c-{Delta}G was 2-3 folds higher than that of oG-mediated SAD-B19-{Delta}G, but there was no difference between oG-mediated and N2cG-mediated CVS-N2c-{Delta}G system. In addition, codon modified N2cG (optiG) did not increase the efficiency of CVS-N2c-{Delta}G tracing. Finally, we found that the CVS-N2c-{Delta}G produced by the improved method can be used for monitoring neural activity of projection neurons, and the time window can be maintained for 3 weeks, and it can also express sufficient recombinases for efficient transgene recombination. That is, the virus produced by the improved production system does not affect its own function, paving the way for its further optimization, popularization and application in structural and functional studies of neural circuits.

Neuropathic pain triggered by chemotherapy poses a significant clinical challenge. Investigating cell type-specific alterations through single-cell transcriptome analysis holds promise in understanding symptom development and pathogenesis. In this study, we performed single nuclei RNA (snRNA) sequencing of dorsal root ganglions (DRG) to explore the molecular mechanism underlying paclitaxel-induced neuropathic pain. Mouse exposed to repeated paclitaxel doses developed persistent pain hypersensitivity lasting at least 21 days. The snRNA sequencing unveiled seven major cell types within DRGs, with neurons further subdivided into 12 distinct subclusters using known markers. Notably, type C low-threshold mechanoreceptors (C_LTMR) exhibited the most pronounced transcriptomic changes post-paclitaxel administration. Differential gene expression and Gene Ontology (GO) analysis highlighted suppressed potassium-related currents, microtubule transport, and mitochondrial functions in C_LTMR following paclitaxel treatment. Meanwhile, Gene Set Enrichment Analysis (GSEA) suggested increased Interleukin 17 production in C_LTMR after paclitaxel exposure. Pseudo-time analysis uncovered nine distinct states (state 1 to 9) of C_LTMR. State 1 exhibits higher prevalence in paclitaxel-treated mice and altered neurotransmission properties, likely contributing to paclitaxel-induced pain hypersensitivity. This comprehensive exploration sheds light on the molecular mechanisms driving paclitaxel-induced neuropathic pain, offering potential avenues for therapeutic intervention.

Perturbed neuronal migration and abnormal axonogenesis have been shown to be implicated in the pathogenesis of autism spectrum disorder (ASD). However, the molecular mechanism remains unknown. Here we demonstrate that dendritic cell factor 1(DCF1) is involved in neuronal migration and axonogenesis. The deletion of dcf1 in mice delays the localization of callosal projection neurons, while dcf1 overexpression restores normal migration. Delayed neurons appear as axon swelling and axonal boutons loss, resulting in a permanent deficit in the callosal projections. Western blot analysis indicates that absence of dcf1 leads to the abnormal activation of ERK signal. Differential protein expression assay shows that PEBP1, a negative regulator of the ERK signal, is significant downregulation in dcf1 KO mice. Direct interaction between DCF1 and PEBP1 is confirmed by Co-immunoprecipitation test, thus indicating that DCF1 regulates the ERK signal in a PEBP1-dependent pattern. As a result of the neurodevelopmental migration disorder, dcf1 deletion results in ASD-like behaviors in mice. This finding identifies a link between abnormal activated ERK signaling, delayed neuronal migration and autistic-like behaviors in humans.

One of the central questions in neural development is how individual neurons assemble functional networks. To address this, it is essential to elucidate how coordinated activity emerges during development. However, tracking the functional maturation of neuronal populations over time remains challenging, as it requires long-term, non-invasive monitoring of membrane potential dynamics. Here, we developed a voltage imaging approach for zebrafish embryos using genetically encoded voltage indicators (GEVIs), enabling fast, direct and cell-type-specific measurements of membrane potentials from defined neuronal populations in a non-invasive manner. Using this approach, we detected coordinated voltage changes in spinal motor neurons with high spatiotemporal resolution. Depolarization and hyperpolarization events were observed at the population, single-cell, and subcellular levels. Notably, long-term voltage imaging revealed the early emergence and progressive maturation of membrane potential dynamics, characterized by increased firing rate, coupling strength and axonal outgrowth. This optical approach constitutes a significant advancement in the study of neural development, providing a powerful tool for investigating the spatiotemporal dynamics of neuronal populations in vivo.

Humidity sensing ability is crucial to terrestrial animals for fitting the environment. Researchers made great progress in recent study about humidity sensing mechanisms of terrestrial animals. However, it is poorly understood whether humidity sensing exists in aquatic animals. Here, we demonstrate that the aquatic planarians, one of the primitive forerunners of later animals, has the ability of humidity sensing and is capable of using the ability to perceive the water beneath itself from a drought place to seek survival. The behavior we discovered is described as diving because the worms twist its body to break away from the mucus that make them adhere to the drought place and drop into the water. The behavior is triggered by rapidly increasing humidity. This finding suggests that humidity sensing ability exists in the lower aquatic animals, and the ability might be used to seek for water when aquatic animals are facing desiccation. The finding also suggests that survival-seeking and decision-making behavior have appeared in the primitive planarian worms.

AbstractEarly and economical diagnosis of acute ischemic stroke (AIS) is pivotal for therapeutic efficacy, particularly for the settings where medical imaging resource is deficient. We have obtained evidence supporting our hypothesis that collective properties of the green autofluorescence (AF) of the fingernails and certain skins positions may be a novel diagnostic biomarker for AIS: Both the green AF intensity and AF asymmetry of the AIS patients in their Index Fingernails and most examined skins positions were significantly higher than that of the healthy subjects and the Non-AIS subjects. ROC analyses and machine learning-based analyses on the AF properties showed that AUC was 0.93 and 0.87, respectively, for differentiating the AIS patients from the healthy subjects and for differentiating the AIS patients from the Non-AIS subjects. The AIS patients had significantly higher AF intensity and AF asymmetry at several examined positions, compared to those of the patients of Parkinsons disease, pulmonary infection and transient ischemic attack. The AUC was 0.79 - 0.88 for differentiating AIS patients from each of these diseases. There was evidence suggesting that the AF originates from keratins. Collectively, our study has indicated that the characteristic AISs Pattern of AF is a novel diagnostic biomarker for the disease. The Pattern of AF Technology holds excellent potential to become a new non-invasive, label-free and economical diagnostic approach for AIS, which is particularly valuable when MRI or CT imaging resource is deficient.

Gut microbiota influence neurodevelopment of brain and programing of behaviors. However, the mechanism underlining the relationship between shoals behaviors and intestinal microbiota remain controversial and the roles of neurotransmitters are still unclear. Here we show that, shoaling behavior affected the innate color preference of shoals, indicating that shoals tended to choose a favorable color environment that benefits social contact. Meanwhile, administration of D1-R antagonist disrupted the social interaction which led to the deficits of color preference. More importantly, the altered microbiota caused by an antibiotic OTC decreased the sociability and weakened shoals color preference. When given a supplement of LGG after OTC exposure, fish exhibited an unexpectedly recovery capability in social cohesion and color preference. Our findings show that dopamine level of brain could mediate both social recognition and color preference, and highlight the pathway of microbial metabolites through the microbiota-gut-brain axis that coordinate the production of dopamine.

The color discrimination can confer survival advantages by helping animals to find nutritious food and shelter and to avoid predator. Zebrafish as a social species, data on innate color preference in shoals remain controversial and there are limited data for this organism. Here we showed that, when given a choice among two color combinations (R-Y, R-G, Y-G, B-G, B-R, B-Y), shoals of zebrafish exhibited a complex pattern of color preference and the order of RYGB preference was R>Y>G, B>G. By contrast, the individual zebrafish showed marked changes, completely losing their preference for all the tested color combinations. To investigate the role of shoaling behavior in color preference, we selected a D1-receptor antagonist (SCH23390), which could disrupt social preference and decrease social interaction in zebrafish. Interestingly, the shoals that were treated by SCH23390 showed no color preference for all color combinations. Our findings indicate that social interaction is involved in color-driven behavior in zebrafish, and reveal the possible mechanisms that the dopaminergic system may contribute to innate color preference in shoals of zebrafish. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY Graphical Abstract C_FIG_DISPLAY