Neuroscience Bulletin

Summary paragraphAcupuncture, a traditional Chinese medical treatment that has been practiced for over 2,000 years, is widely used around the world [1]. However, its efficacy and distinction from random stimulation are still being questioned [2, 3]. Over the years, many studies have reported either favorable, neutral or even skeptical outcomes regarding the treatment effect of acupuncture on diverse ailments [4-7]. The major question behind this controversy is whether acupuncture is different from random needle insertion and whether its efficacy can be attributed to the placebo effect [8, 9]. Here, we use micro-positron emission tomography (microPET) imaging in a randomized controlled animal study to show that acupuncture facilitates faster recuperation in comparison to sham acupuncture and blank control. Based on the microPET imaging of subjects undergoing daily acupuncture over two weeks duration, we dynamically monitored the metabolic activity levels in different brain regions and found that both acupoint and non-acupoint stimulation could improve ischemic stroke recovery. This finding is consistent with previous reports that both acupuncture and sham needling show a positive effect in the treatment of diseases [4, 5, 8]. More importantly, we further found that rats receiving acupuncture at Baihui (GV20) and Shuigou (GV26), two commonly used acupoints for stroke rehabilitation based on the concept of the meridian system, showed earlier recovery effects than rats receiving sham needling treatment. This difference mainly appeared in regions involved in motor control and was validated by a balance beam walking test. Our findings, in conjunction with a recent electroacupuncture study that revealed a neuroanatomical pathway to mediate the vagal-adrenal anti-inflammatory axis [10], provide quantitative evidence supporting the specificity of acupoints in acupuncture therapy.

As a pathological hallmark in Parkinsons disease (PD), -synucleinopathy causes multiple cellular damages, including calcium overload, mitochondrial and autophagic dysfunction, and eventually dopamine neuron death. However, the hierarchy of these detrimental events is unclear. In Drosophila, we confirmed that overexpression of -synuclein could induce all these cytotoxic events. To determine the specific cytotoxic events induced by calcium overload, we established a calcium overload model in Drosophila and performed genetic screens. We found that calcium overload caused mitochondrial damage and autophagy failure and cell death, and these cytotoxic processes could be strongly rescued by loss of Tousled-like kinase (TLK). Interestingly, loss of TLK also rescued defects induced by -synuclein overexpression in Drosophila. This suggests that calcium overload acts as the crucial event upstream of mitochondrial and autophagy dysfunction. For TLK regulation of autophagy, our data indicated that a transcriptional factor REPTOR, which regulated the expression of several lysosomal genes, functioned downstream of TLK. In mammalian cells and mice, TLK2 (the homolog of Drosophila TLK) was phosphorylated under calcium overload. Upon phosphorylation, TLK2 increased its kinase activity. In addition, TLK2 could phosphorylate CREBRF (the human homolog of REPTOR) to cause its loss of transcription on the lysosomal genes. Moreover, TLK2 knockout mice rescued multi-aspect cytotoxicity induced by calcium overload and -synuclein overexpression. Our research demonstrates that TLK2 acts as a key regulator to mediate cell death and dysfunctions of mitochondria and autophagy downstream of calcium overload.

Restless legs syndrome (RLS) is a common neurological disorder associated with iron deficiency and dopaminergic (DAergic) neuronal dysfunction. BTBD9 is a genetic risk factor for RLS. However, its molecular function remains largely unknown. Here, we report the interaction between BTBD9, manganese (Mn) and insulin/insulin-like growth factor (IGF) signaling in Caenorhabditis elegans, mouse Neuro2a cells and humans. We found that elevated Mn downregulated BTBD9 mRNA levels; in turn, BTBD9 expression attenuated Mn-induced cellular stress and dopaminergic neurodegeneration. As Mn is a known co-factor for insulin receptor and IGF-1 receptor, which activates IGF signaling, we posited that BTBD9 negatively regulates IGF signaling. Our results showed that the protective effects of BTBD9 against Mn toxicity were dependent on the forkhead box O (FOXO) protein. Furthermore, BTBD9 overexpression significantly elevated FOXO level and decreased PKB level, while phosphoinositide-dependent kinase-1 (PDK1) level remained unchanged. We conclude that BTBD9 acts as a key component in the IGF signaling pathway. Meanwhile, the roles of Mn in DAergic neurotoxicity and regulating BTBD9 shed new light on the etiology of RLS.

Neuronal mitochondrial dysfunction is associated with cognitive decline in neurodegenerative disorders such as Alzheimers Disease (AD). In this study, multiple pieces of evidence proved that phosphorylated tau (p-Tau) caused mitochondrial swelling and dysfunction in neurons. In a novel in vitro newborn neurons culture system, we discovered mitochondrial swelling and dysfunction were associated with increased p-Tau, leading to necroptosis activation, which was induced by Complement C3 (C3) produced from activated astrocytes. In the in vivo tauopathy mouse models, the effects of astrocytic C3 on tau-associated mitochondrial dysfunction and necroptosis were also discovered in hippocampal newborn neurons, and we directly showed that p-Tau aggregation was associated with mitochondria swelling in the hippocampal neurons by electron microscopy analysis. In addition, we proved the ability of compound anserine, which can block Tak1-Ikk dependent NF-{kappa}B activation, to further down-regulate astrocytic C3 production and alleviate neuronal mitochondrial dysfunction in vitro and in vivo, respectively. Down-regulation of astrocyte C3-production by anserine could also rescue mortality as well as cognitive and motor functions. Our findings first reported the contribution of p-Tau on neuronal mitochondrial dysfunction and proposed the therapies that down-regulate astrocytic C3 production have a potential role in alleviating this neurotoxic effect.

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.

Reaching to grasp movement is thought to rely upon two independent brain pathways. The dorsomedial one is involved in reaching while the dorsolateral one is dealing with grasping. However, some recent evidences suggested that the dorsomedial pathway might participate in grasp movement. Therefore, it is important to investigate whether PMd is involved in grasp planning, and if participating, what kind of role PMd played in grasp planning. In this study, two macaques monkeys were trained to grasp same object by instructing or freely choosing one of two grips, power grip or hook grip. A 96-channel microelectrode array was implanted to collect the population activity of PMd in each subject. Both single unit activity and population activity were analyzed. We found that nearly 21.0% and 26.8% units in PMd of two monkeys displayed grip selectivity during gesture planning in both instructing or freely choosing conditions. These units exhibit selectivity for different gestures when facing the identical visual stimuli (freely choosing condition). At the same time, similar activity patterns are displayed for the same gesture when faced with different selection strategies (freely choosing condition vs. instructing condition). These results show that some neurons of PMd are mainly involved in the hand shape preparation and have no obvious relationship with external visual stimuli and selection strategies.

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.

Methamphetamine (METH) is frequently abused drug and produces cognitive deficits. METH could induce hyper-glutamatergic state in the brain, which could partially explain METH-related cognitive deficits, but the synaptic etiology remains incompletely understood. To address this issue, we explored the role of dCA1 tripartite synapses and the potential therapeutic effects of electro-acupuncture (EA) in the development of METH withdrawal-induced spatial memory deficits in mice. We found that METH withdrawal weakened astrocytic capacity of glutamate (Glu) uptake, but failed to change Glu release from dCA3, which lead to hyper-glutamatergic excitotoxicity at dCA1 tripartite synapses. By restoring the astrocytic capacity of Glu uptake, EA treatments suppressed the hyper-glutamatergic state and normalized the excitability of postsynaptic neuron in dCA1, finally alleviated spatial memory deficits in METH withdrawal mice. These findings indicate that astrocyte at tripartite synapses might be a key target for developing therapeutic interventions against METH-associated cognitive disorders, and EA represent a promising non-invasive therapeutic strategy for the management of drugs-caused neurotoxicity.

Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death in refractory epilepsy patients. Despite previous accumulating evidence has shown that seizure-induced respiratory arrest (S-IRA) may play the main contributor to SUDEP as an initiating event preeminent cause of mortality, the specific underlying mechanism of action remains unclear. Based on our previous work, serotonin (5-HT) signaling in the dorsal raphe nucleus (DRN) is strongly implicated in S-IRA in animal models, including the DBA/1 mice, on the meanwhile, norepinephrine (NE) neurons of the locus coeruleus (LC) also plays a vital role in regulating respiratory function on its own. Superficially, monoaminergic neuron, as important neurotransmitters in the central nervous system, have similar modes of action in the maintenance of nervous system balance, and each of them has a regulatory effect on SUDEP. However, it remains to be investigated whether monoaminergic neuron family (NE and 5-HT) are related in the mechanism of regulating SUDEP, what is even more curious is whether the two are intrinsically linked. Thus, we hypothesize neural mechanism of central noradrenergic and serotonergic circuits in modulating SUDEP in a synergistic-dependent manner, this endeavor will culminate in a significant breakthrough in elucidating the precise mechanism of action underlying SUDEP. In our study, we will use chemogenetics, optogenetics, calcium signal recording, and bidirectional tracing to explore the internal mechanism of DR-LC regulating the occurrence of SUDEP, and by specifically injecting 5-HT2AR antagonist Ketanserin (KET) and/or NE-1R antagonist Prazosin into the pre-Botzinger complex (PBC), it was finally elucidate that the DR-LC-PBC network can effectively reduce the incidence of SIRA. We firstly proposed a powerful target for exploring the reduction of the incidence of SUDEP, which has great clinical translation potential.

Astrocytes are intricately involved in the activity of neural circuits, however, their basic physiology of interacting with neurons remains controversial. Using dual-indicator two-photon imaging of neurons and astrocytes during stimulations of hippocampal CA3 - CA1 Schaffer collateral (Scc) excitatory synapses, we report that under physiological conditions, the increased glutamate released from the higher frequency stimulation of neurons can accelerate local astrocytic Ca2+ levels. As consequences of extracellular glutamate clearance and maintaining of astrocytic intracellular Na+ homeostasis, the increase of astrocytic membrane Ca2+ permeability via Na+/Ca2+ exchanger (NCX) reverse mode is the primary reason of eliciting astrocytic intracellular Ca2+ elevation upon neuronal stimulation. This Ca2+-induced Ca2+ release is dependent on inositol triphosphate receptor type 2 (IP3R2). In addition, ATP released from Scc excitatory synapses can contribute to this molecular mechanism of Ca2+-induced Ca2+ release in astrocytes.

Neuroanatomical tracing technology is fundamental for unraveling the complex network of brain connectome. Tracing tools that could spread between neurons are urgently needed, especially the rigorous trans-monosynaptic anterograde tracer is still lacking. HSV1 strain H129 was proved to be an anterograde tracer and has been used to trace neuronal networks in several reports. However, H129 has a serious defect that it was demonstrated to infect neurons via axon terminals. Thus, when using H129 to dissect output neural circuit, its terminal take up capacity should be carefully considered. Here, we report a recombinant H129 that carrying the anti-Her2 scFv in glycoprotein D to target genetically defined neurons. With the usage of helper virus complementarily expressing Her2 and gD, we can realize the elucidation of direct projection regions of either a given brain nucleus or a specific neuron type. The retargeted H129 system complements the current neural circuit tracer arsenal, which provides a rigorous and practical anterograde trans-monosynaptic tool.

A group of social animals, including humans, distributes work and rewards and establishes inter-individual relationships through shared experiences of working together. Understanding the neural mechanisms underlying group work is essential for elucidating how social relationships are formed. Thus, a behavioral paradigm for studying group work in laboratory rodents is required. Here, we developed a Tsunahiki task, a novel group-based operant task for mice. In this task, three mice jointly pulled three ropes, and once all ropes were pulled out, all members gained access to a reward area, regardless of who performed the work. The mice acquired the task within a few days. Importantly, repeated experience with the Tsunahiki task led to a shift in workload toward subordinate individuals and induced rank consolidation. These findings suggest that group work induces consolidation of intra-group disparities based on the dominance hierarchy. The Tsunahiki task provides a useful framework for investigating the neurobiological mechanisms underlying collaborative work, group formation, and social inequality in rodents. TeaserA group-based operant task for mice was newly developed, in which hierarchy affected work and reward distributions.

Identifying genetic variants associated with nicotine-motivated behavioral traits is an important strategy to understand the fundamental mechanisms underpinning smoking and tobacco abuse. For suitable emulation of behavioral phenotype with the full advantage of this invertebrate model, we newly established a worm model of nicotine seeking by Conditioned Cue Preference (CCP). We demonstrated that C. elegans also exhibited pivotal features of nicotine-motivated behaviors as in mammals. First, we identified the nicotine-elicited cue preference is mediated by nicotinic acetylcholine receptors in worms. Additionally, we exhibited dopamine is also required for the development of CCP. Subsequently, we identified the nAChRs subunits associated with the facilitation of nicotine preference. Accordingly, we validated human GWAS candidates associated with nicotine dependence involved in the role of those nAChR subunits. we addressed the cross-species functional validation to determine the GWAS candidate genes have authentic roles in nicotine seeking associated with tobacco abuse. The loss of function strain of CACNA2D3 orthologue, calcium voltage-gated channel auxiliary subunit alpha2delta 3, was tested for CCP. We also tested the knock-out (KO) strain of the CACNA2D2 orthologue, calcium voltage-gated channel auxiliary subunit alpha2delta 2, which is closely related to CACNA2D3 in the same family and shared the human smoking phenotypes. Our orthogonal test suggests the functional conservation of the 2{delta} subunit of calcium channel in nicotine motivated behavior.

Accumulating evidence indicates that the molecular circadian clock underlies the mating behavior of Drosophila melanogaster. However, information about which gene affects circadian mating behavior is poorly understood in animals. The present study found that feeding Myo-inositol enhanced the close-proximity (CP) rhythm of D. melanogaster mating behavior and lengthened the period of the CP rhythm. Then, to understand a role for inositol synthesis to fly mating behavior, we established the Inos (Myo-inositol 1-phosphate synthase) gene knock down fly strains with RNAi. Interestingly, the CP behavior of this three-different driver knock down strains was arrhythmic, but the locomotor rhythm was rhythmic. The data of three-different Inos knock down strains suggests that Inos gene expression of upper LNd, l-LNV, 5ths-LNv in brain is necessary for proper CP rhythm generation in D. melanogaster. The data indicated that the Inos gene is involved in the role for the circadian rhythm of D. melanogaster mating behavior.

The cyclic-AMP dependent protein kinase A (Protein kinase A, PKA) regulates dopaminergic function in the substantia nigra pars compacta (SNc). However, whether PKA is involved in the pathogenesis of Parkinsons disease (PD) is unknown. Here, by collecting and analyzing the current worldwide SNc transcriptomic datasets of PD patients, we found a decline of PKA-RII{beta} subunit level in the SNc of PD patients. The decreased PKA-RII{beta} subunit level was positively correlated with decreased dopamine synthesis and increased oxidative stress in the SNc of PD patients. PKA-RII{beta} subunit is expressed in the striatum and the SNc. PKA-RII{beta} gene knockout mice (RII{beta}-KO) showed a age-related parkinsonism at 12 months of age. Using Cre-LoxP system, we observed that RII{beta} re-expression in the SNc dopaminergic neurons rescued parkinsonism of RII{beta}-KO mice. RII{beta} re-expression in striatal neurons did not affect parkinsonism of RII{beta}-KO mice. The spontaneous parkinsonism could be developed in 12-month-old SNc dopaminergic neuron-specific RII{beta}-deficient mice. Single-nucleus RNA sequencing revealed decreased PKA activity, reduced dopamine synthesis and raised oxidative stress in the SNc dopaminergic neurons of RII{beta}-KO mice. Adeno-associated virus (AAV)-mediated gene therapy targeting PKA-RII{beta} in the SNc dopaminergic neurons rescued parkinsonism in PD mouse model. Taken together, these findings indicate that PKA-RII{beta} may be a key factor of human genetic etiologies of PD. The therapy targeting PKA-RII{beta} in the SNc dopaminergic neurons may be promising for PD treatment.

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 coronavirus disease of 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), remains a major health issue after nearly 7 millions of death toll in the last four years. As the world is recovering with improving vaccines and antiviral treatments, the alarming rate of long-COVID, or Post-acute Sequelae of COVID-19 (PASC), calls for further investigations. Among a list of symptoms associated with multi-organ dysfunctions, the neurological complications are particularly intriguing, yet the underlying mechanisms remain elusive. With the recently developed mouse adapted SARS-CoV-2 stain, we are now able to model the mild COVID infection in C57BL/6 mice and study the chronic immune responses and subsequent damages in different organs long after the viruses are clearly naturally in the body. More specifically, we found adult C57BL/6J mice developed neurological impairments, including behavior changes related to sensorimotor coordination, depression- and anxiety-like behaviors, and inflammation in multiple organs including lung, liver and brain, which persisted over at least 4 weeks in mice even with mild infection. Therefore, this model can be used to further explopred the mechanisms of PASC, as well as potential intervention or therapeutic approaches.

The elegant functions of the brain are facilitated by sophisticated connections between neurons, the architecture of which is frequently characterized by one nucleus connecting to multiple targets via projection neurons. Delineating the sub-nucleus fine architecture of projection neurons in a certain nucleus could greatly facilitate its circuit, computational, and functional resolution. Here, we developed multi-fluorescent rabies virus to delineate the fine organization of corticothalamic projection neuron subsets in the primary visual cortex (V1). By simultaneously labeling multiple distinct subsets of corticothalamic projection neurons in V1 from their target nuclei in thalamus (dLGN, LP, LD), we observed that V1-dLGN corticothalamic neurons were densely concentrated in layer VI, except for several sparsely scattered neurons in layer V, while V1-LP and V1-LD corticothalamic neurons were localized to both layers V and VI. Meanwhile, we observed a fraction of V1 corticothalamic neurons targeting multiple thalamic nuclei, which was further confirmed by fMOST whole-brain imaging. We further conceptually proposed an upgraded sub-nucleus tracing system with higher throughput (21 subsets) for more complex architectural tracing. The multi-fluorescent RV tracing tool can be extensively applied to resolve architecture of projection neuron subsets, with a strong potential to delineate the computational and functional organization of these nuclei.

Left-right (LR) asymmetry is a conserved characteristic of the brain in various animals and is related to its higher-order functions. The Drosophila brain has an LR asymmetric structure known as an asymmetrical body (AB). LR asymmetric neurite remodeling lateralizes the AB, and ecdysone signaling determines LR specificity. However, the mechanisms underlying LR specificity remain unclear. We found that the Slit/Dreadlocks/Roundabout/p21-activated kinase (Pak) signaling axis determines the LR polarity of the AB downstream of ecdysone signaling in the type II neuroblast lineage before LR asymmetric neurite remodeling. In Drosophila, the intrinsic chirality of cells (cell chirality) defines the LR asymmetry of various non-neuronal organs. We suggested that neurons derived from type II neuroblasts exhibit cell chirality, which is established through Pak and ecdysone signaling and determines the LR polarity of the AB. As cell chirality is broadly observed in eukaryotes, our study reveals a novel mechanism underlying LR asymmetry of the brain.

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.