The ion channel TRPA1 is a modulator of the cocaine reward circuit in the nucleus accumbens.

Drug addiction therapies commonly fail because continued drug use promotes the release of excessive and pleasurable dopamine levels. Because the connection between pleasure and drug use becomes hard-wired in the nucleus accumbens (NAc), which interfaces motivation, effective therapies need to modulate this mesolimbic reward system. Here, we report that mice with knockdown of the cation channel TRPA1 (transient receptor potential ankyrin 1) were resistant to the drug-seeking behavior and reward effects of cocaine compared to their wildtype litter mates. In our study, we demonstrate that TRPA1 inhibition in the NAc reduces cocaine activity and dopamine release, and conversely, that TRPA1 is critical for cocaine-induced synaptic strength in dopamine receptor 1-expressing medium spiny neurons. Taken together, our data support that cocaine-induced reward-related behavior and synaptic release of dopamine in the NAc are controlled by TRPA1 and suggest that TRPA1 has therapeutic potential as a target for drug misuse therapies.

Enrichment of infection-associated bacteria in the low biomass brain bacteriota of Alzheimer's disease patients.

Alzheimer's disease (AD) is a neurodegenerative disease accompanied by neuroimmune inflammation in the frontal cortex and hippocampus. Recently, the presence of bacteria in AD-affected brains has been documented, prompting speculation about their potential role in AD-associated neuroinflammation. However, the characterization of bacteriota in human brains affected by AD remains inconclusive. This study aimed to investigate potential associations between specific bacteria and AD pathology by examining brain tissues from AD-associated neurodegenerative regions (frontal cortex and hippocampus) and the non-AD-associated hypothalamus. Employing 16S rRNA gene sequencing, 30 postmortem brain tissue samples from four individuals with normal brain histology (N) and four AD patients were analyzed, along with three blank controls. A remarkably low biomass characterized the brain bacteriota, with their overall structures delineated primarily by brain regions rather than the presence of AD. While most analyzed parameters exhibited no significant distinction in the brain bacteriota between the N and AD groups, the unique detection of Cloacibacterium normanense in the AD-associated neurodegenerative regions stood out. Additionally, infection-associated bacteria, as opposed to periodontal pathogens, were notably enriched in AD brains. This study's findings provide valuable insights into potential link between bacterial infection and neuroinflammation in AD.

Depletion of SMN protein in mesenchymal progenitors impairs the development of bone and neuromuscular junction in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neuromuscular disorder characterized by the deficiency of the survival motor neuron (SMN) protein, which leads to motor neuron dysfunction and muscle atrophy. In addition to the requirement for SMN in motor neurons, recent studies suggest that SMN deficiency in peripheral tissues plays a key role in the pathogenesis of SMA. Using limb mesenchymal progenitor cell (MPC)-specific SMN-depleted mouse models, we reveal that SMN reduction in limb MPCs causes defects in the development of bone and neuromuscular junction (NMJ). Specifically, these mice exhibited impaired growth plate homeostasis and reduced insulin-like growth factor (IGF) signaling from chondrocytes, rather than from the liver. Furthermore, the reduction of SMN in fibro-adipogenic progenitors (FAPs) resulted in abnormal NMJ maturation, altered release of neurotransmitters, and NMJ morphological defects. Transplantation of healthy FAPs rescued the morphological deterioration. Our findings highlight the significance of mesenchymal SMN in neuromusculoskeletal pathogenesis of SMA and provide insights into potential therapeutic strategies targeting mesenchymal cells for the treatment of SMA.

Persistent enhancement of basolateral amygdala-dorsomedial striatum synapses causes compulsive-like behaviors in mice.

Compulsive behaviors are observed in a range of psychiatric disorders, however the neural substrates underlying the behaviors are not clearly defined. Here we show that the basolateral amygdala-dorsomedial striatum (BLA-DMS) circuit activation leads to the manifestation of compulsive-like behaviors. We revealed that the BLA neurons projecting to the DMS, mainly onto dopamine D1 receptor-expressing neurons, largely overlap with the neuronal population that responds to aversive predator stress, a widely used anxiogenic stressor. Specific optogenetic activation of the BLA-DMS circuit induced a strong anxiety response followed by compulsive grooming. Furthermore, we developed a mouse model for compulsivity displaying a wide spectrum of compulsive-like behaviors by chronically activating the BLA-DMS circuit. In these mice, persistent molecular changes at the BLA-DMS synapses observed were causally related to the compulsive-like phenotypes. Together, our study demonstrates the involvement of the BLA-DMS circuit in the emergence of enduring compulsive-like behaviors via its persistent synaptic changes.

Gossypetin Prevents the Progression of Nonalcoholic Steatohepatitis by Regulating Oxidative Stress and AMP-Activated Protein Kinase.

Nonalcoholic steatohepatitis (NASH) is a severe liver metabolic disorder, however, there are still no effective and safe drugs for its treatment. Previous clinical trials used various therapeutic approaches to target individual pathologic mechanisms, but these approaches were unsuccessful because of the complex pathologic causes of NASH. Combinatory therapy in which two or more drugs are administered simultaneously to patients with NASH, however, carries the risk of side effects associated with each individual drug. To solve this problem, we identified gossypetin as an effective dual-targeting agent that activates AMP-activated protein kinase (AMPK) and decreases oxidative stress. Administration of gossypetin decreased hepatic steatosis, lobular inflammation and liver fibrosis in the liver tissue of mice with choline-deficient high-fat diet and methionine-choline deficient diet (MCD) diet-induced NASH. Gossypetin functioned directly as an antioxidant agent, decreasing hydrogen peroxide and palmitate-induced oxidative stress in the AML12 cells and liver tissue of MCD diet-fed mice without regulating the antioxidant response factors. In addition, gossypetin acted as a novel AMPK activator by binding to the allosteric drug and metabolite site, which stabilizes the activated structure of AMPK. Our findings demonstrate that gossypetin has the potential to serve as a novel therapeutic agent for nonalcoholic fatty liver disease /NASH. SIGNIFICANCE STATEMENT: This study demonstrates that gossypetin has preventive effect to progression of nonalcoholic steatohepatitis (NASH) as a novel AMP-activated protein kinase (AMPK) activator and antioxidants. Our findings indicate that simultaneous activation of AMPK and oxidative stress using gossypetin has the potential to serve as a novel therapeutic approach for nonalcoholic fatty liver disease /NASH patients.

Cortical astrocytes modulate dominance behavior in male mice by regulating synaptic excitatory and inhibitory balance.

Social hierarchy is established as an outcome of individual social behaviors, such as dominance behavior during long-term interactions with others. Astrocytes are implicated in optimizing the balance between excitatory and inhibitory (E/I) neuronal activity, which may influence social behavior. However, the contribution of astrocytes in the prefrontal cortex to dominance behavior is unclear. Here we show that dorsomedial prefrontal cortical (dmPFC) astrocytes modulate E/I balance and dominance behavior in adult male mice using in vivo fiber photometry and two-photon microscopy. Optogenetic and chemogenetic activation or inhibition of dmPFC astrocytes show that astrocytes bidirectionally control male mouse dominance behavior, affecting social rank. Dominant and subordinate male mice present distinct prefrontal synaptic E/I balance, regulated by astrocyte activity. Mechanistically, we show that dmPFC astrocytes control cortical E/I balance by simultaneously enhancing presynaptic-excitatory and reducing postsynaptic-inhibitory transmission via astrocyte-derived glutamate and ATP release, respectively. Our findings show how dmPFC astrocyte-neuron communication can be involved in the establishment of social hierarchy in adult male mice.

Major depression-related factor NEGR1 controls salivary secretion in mouse submandibular glands.

Salivary gland cells, which secrete water in response to neuronal stimulation, are closely connected to other neurons. Transcriptomic studies show that salivary glands also express some proteins responsible for neuronal function. However, the physiological functions of these common neuro-exocrine factors in salivary glands are largely unknown. Here, we studied the function of Neuronal growth regulator 1 (NEGR1) in the salivary gland cells. NEGR1 was also expressed in mouse and human salivary glands. The structure of salivary glands of Negr1 knockout (KO) mice was normal. Negr1 KO mice showed tempered carbachol- or thapsigargin-induced intracellular Ca2+ increases and store-operated Ca2+ entry. Of interest, the activity of the large-conductance Ca2+-activated K+ channel (BK channel) was increased, whereas Ca2+-activated Cl- channel ANO1 channel activity was not altered in Negr1 KO mice. Pilocarpine- and carbachol-induced salivation was decreased in Negr1 KO mice. These results suggest that NEGR1 influence salivary secretion though the muscarinic Ca2+ signaling.

Biochemical and functional characterization of skeletal muscle cells differentiated from tonsil-derived mesenchymal stem cells.

INTRODUCTION/AIMS: Human tonsils are a readily accessible source of stem cells for the potential treatment of skeletal muscle disorders. We reported previously that tonsil-derived mesenchymal stem cells (TMSCs) can differentiate into skeletal muscle cells (SKMCs), which renders TMSCs promising candidates for cell therapy for skeletal muscle disorders. However, the functional properties of the myocytes differentiated from mesenchymal stem cells have not been clearly evaluated. In this study we investigated whether myocytes differentiated from TMSCs (skeletal muscle cells derived from tonsil mesenchymal stem cells [TMSC-SKMCs]) exhibit the functional characteristics of SKMCs. METHODS: To test the insulin reactivity of TMSC-SKMCs, the expression of glucose transporter 4 (GLUT4) and phosphatidylinositol 3-kinase/Akt was analyzed after the cells were treated for 30 minutes with 100 nmol/L insulin in normal or high-glucose medium. We also examined whether these cells formed a neuromuscular junction (NMJ) when cocultured with motor neurons, and whether they were stimulated by electrical signals using whole-cell patch clamping. RESULTS: Skeletal muscle cells derived from tonsil mesenchymal stem cells expressed SKMC markers, such as MYOD, MYH3, MYH8, TNNI1, and TTN, at high levels, and exhibited a multinucleated cell morphology and a myotube-like shape. The expression of the acetylcholine receptor and GLUT4 was confirmed in TMSC-SKMCs. In addition, these cells exhibited insulin-mediated glucose uptake, NMJ formation, and transient changes in cell membrane action potential, all of which are representative functions of human SKMCs. DISCUSSION: Tonsil-derived mesenchymal stem cells can be functionally differentiated into SKMCs and may have potential for clinical application for the treatment of skeletal muscle disorders.

Synthesizing a Gel Polymer Electrolyte for Supercapacitors, Assembling a Supercapacitor Using a Coin Cell, and Measuring Gel Electrolyte Performance.

Supercapacitors (SC) have attracted attention as energy storage devices due to their high density and long cycle performance. SCs used in devices operating in stretchable systems require stretchable electrolytes. Gel polymer electrolytes (GPEs) are an ideal replacement for liquid electrolytes. Polyvinyl alcohol (PVA) and polyvinylidene fluoride-co-hexafluoropropylene (PVDF-HFP) have been widely applied as a polymer-matrix-based electrolytes for supercapacitors because of their low cost, chemically stable, wide operating temperature range, and high ionic conductivities. Herein, we describe the procedures for (1) synthesizing a gel polymer electrolyte with PVA and PVDF-HFP, (2) measuring the electrochemical stability of the gel polymer electrolytes by cyclic voltammetry (CV), (3) measuring the ionic conductivity of the gel polymer electrolytes by electrochemical impedance spectroscopy (EIS), (4) assembling symmetric coin cells using activated carbon (AC) electrodes with the PVA- and PVDF-HFP-based gel polymer electrolytes, and (5) evaluating the electrochemical performance using galvanostatic charge-discharge analysis (GCD) and CV at 25 °C. Additionally, we describe the challenges and insights gained from these experiments.

Production of human spinal-cord organoids recapitulating neural-tube morphogenesis.

Human spinal-cord-like tissues induced from human pluripotent stem cells are typically insufficiently mature and do not mimic the morphological features of neurulation. Here, we report a three-dimensional culture system and protocol for the production of human spinal-cord-like organoids (hSCOs) recapitulating the neurulation-like tube-forming morphogenesis of the early spinal cord. The hSCOs exhibited neurulation-like tube-forming morphogenesis, cellular differentiation into the major types of spinal-cord neurons as well as glial cells, and mature synaptic functional activities, among other features of the development of the spinal cord. We used the hSCOs to screen for antiepileptic drugs that can cause neural-tube defects. hSCOs may also facilitate the study of the development of the human spinal cord and the modelling of diseases associated with neural-tube defects.

Evaluating the Electrochemical Properties of Supercapacitors using the Three-Electrode System.

The three-electrode system is a basic and general analytical platform for investigating the electrochemical performance and characteristics of energy storage systems at the material level. Supercapacitors are one of the most important emergent energy storage systems developed in the past decade. Here, the electrochemical performance of a supercapacitor was evaluated using a three-electrode system with a potentiostat device. The three-electrode system consisted of a working electrode (WE), reference electrode (RE), and counter electrode (CE). The WE is the electrode where the potential is controlled and the current is measured, and it is the target of research. The RE acts as a reference for measuring and controlling the potential of the system, and the CE is used to complete the closed circuit to enable electrochemical measurements. This system provides accurate analytical results for evaluating electrochemical parameters such as the specific capacitance, stability, and impedance through cyclic voltammetry (CV), galvanostatic charge-discharge (GCD), and electrochemical impedance spectroscopy (EIS). Several experimental design protocols are proposed by controlling the parameter values of the sequence when using a three-electrode system with a potentiostat device to evaluate the electrochemical performance of supercapacitors. Through these protocols, the researcher can set up a three-electrode system to obtain reasonable electrochemical results for assessing the performance of supercapacitors.

Elaborate Control of Inkjet Printer for Fabrication of Chip-based Supercapacitors.

There are tremendous efforts in various fields to apply the inkjet printing method for the fabrication of wearable devices, displays, and energy storage devices. To get high-quality products, however, sophisticated operation skills are required depending on the physical properties of the ink materials. In this regard, optimizing the inkjet printing parameters is as important as developing the physical properties of the ink materials. In this study, optimization of the inkjet printing software parameters is presented for fabricating a supercapacitor. Supercapacitors are attractive energy storage systems because of their high power density, long lifespan, and various applications as power sources. Supercapacitors can be used in the Internet of Things (IoT), smartphones, wearable devices, electrical vehicles (EVs), large energy storage systems, etc. The wide range of applications demands a new method that can fabricate devices in various scales. The inkjet printing method can break through the conventional fixed-size fabrication method.

SALM4 negatively regulates NMDA receptor function and fear memory consolidation.

Many synaptic adhesion molecules positively regulate synapse development and function, but relatively little is known about negative regulation. SALM4/Lrfn3 (synaptic adhesion-like molecule 4/leucine rich repeat and fibronectin type III domain containing 3) inhibits synapse development by suppressing other SALM family proteins, but whether SALM4 also inhibits synaptic function and specific behaviors remains unclear. Here we show that SALM4-knockout (Lrfn3-/-) male mice display enhanced contextual fear memory consolidation (7-day post-training) but not acquisition or 1-day retention, and exhibit normal cued fear, spatial, and object-recognition memory. The Lrfn3-/- hippocampus show increased currents of GluN2B-containing N-methyl-D-aspartate (NMDA) receptors (GluN2B-NMDARs), but not α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors (AMPARs), which requires the presynaptic receptor tyrosine phosphatase PTPσ. Chronic treatment of Lrfn3-/- mice with fluoxetine, a selective serotonin reuptake inhibitor used to treat excessive fear memory that directly inhibits GluN2B-NMDARs, normalizes NMDAR function and contextual fear memory consolidation in Lrfn3-/- mice, although the GluN2B-specific NMDAR antagonist ifenprodil was not sufficient to reverse the enhanced fear memory consolidation. These results suggest that SALM4 suppresses excessive GluN2B-NMDAR (not AMPAR) function and fear memory consolidation (not acquisition).

Muscarinic Receptors and BK Channels Are Affected by Lipid Raft Disruption of Salivary Gland Cells.

Activity-dependent fluid secretion is the most important physiological function of salivary glands and is regulated via muscarinic receptor signaling. Lipid rafts are important for G-protein coupled receptor (GPCR) signaling and ion channels in plasma membranes. However, it is not well understood whether lipid raft disruption affects all membrane events or only specific functions in muscarinic receptor-mediated water secretion in salivary gland cells. We investigated the effects of lipid raft disruption on the major membrane events of muscarinic transcellular water movement in human salivary gland (HSG) cells. We found that incubation with methyl-ß-cyclodextrin (MßCD), which depletes lipid rafts, inhibited muscarinic receptor-mediated Ca2+ signaling in HSG cells and isolated mouse submandibular acinar cells. However, MßCD did not inhibit a Ca2+ increase induced by thapsigargin, which activates store-operated Ca2+ entry (SOCE). Interestingly, MßCD increased the activity of the large-conductance Ca2+-activated K+ channel (BK channel). Finally, we found that MßCD did not directly affect the translocation of aquaporin-5 (AQP5) into the plasma membrane. Our results suggest that lipid rafts maintain muscarinic Ca2+ signaling at the receptor level without directly affecting the activation of SOCE induced by intracellular Ca2+ pool depletion or the translocation of AQP5 into the plasma membrane.

Neural stem cells derived from human midbrain organoids as a stable source for treating Parkinson's disease: Midbrain organoid-NSCs (Og-NSC) as a stable source for PD treatment.

Successful clinical translation of stem cell-based therapy largely relies on the scalable and reproducible preparation of donor cells with potent therapeutic capacities. In this study, midbrain organoids were yielded from human pluripotent stem cells (hPSCs) to prepare cells for Parkinson's disease (PD) therapy. Neural stem/precursor cells (NSCs) isolated from midbrain organoids (Og-NSCs) expanded stably and differentiated into midbrain-type dopamine(mDA) neurons, and an unprecedentedly high proportion expressed midbrain-specific factors, with relatively low cell line and batch-to-batch variations. Single cell transcriptome analysis followed by in vitro assays indicated that the majority of cells in the Og-NSC cultures are ventral midbrain (VM)-patterned with low levels of cellular senescence/aging and mitochondrial stress, compared to those derived from 2D-culture environments. Notably, in contrast to current methods yielding mDA neurons without astrocyte differentiation, mDA neurons that differentiated from Og-NSCs were interspersed with astrocytes as in the physiologic brain environment. Thus, the Og-NSC-derived mDA neurons exhibited improved synaptic maturity, functionality, resistance to toxic insults, and faithful expressions of the midbrain-specific factors, in vitro and in vivo long after transplantation. Consequently, Og-NSC transplantation yielded potent therapeutic outcomes that are reproducible in PD model animals. Collectively, our observations demonstrate that the organoid-based method may satisfy the demands needed in the clinical setting of PD cell therapy.

Enhanced Prefrontal Neuronal Activity and Social Dominance Behavior in Postnatal Forebrain Excitatory Neuron-Specific Cyfip2 Knock-Out Mice.

The cytoplasmic fragile X mental retardation 1 (FMR1)-interacting protein 2 (CYFIP2) gene is associated with epilepsy, intellectual disability (ID), and developmental delay, suggesting its critical role in proper neuronal development and function. CYFIP2 is involved in regulating cellular actin dynamics and also interacts with RNA-binding proteins. However, the adult brain function of CYFIP2 remains unclear because investigations thus far are limited to Cyfip2 heterozygous (Cyfip2+/- ) mice owing to the perinatal lethality of Cyfip2-null mice. Therefore, we generated Cyfip2 conditional knock-out (cKO) mice with reduced CYFIP2 expression in postnatal forebrain excitatory neurons (CaMKIIα-Cre). We found that in the medial prefrontal cortex (mPFC) of adult Cyfip2 cKO mice, CYFIP2 expression was decreased in both layer 2/3 (L2/3) and layer 5 (L5) neurons, unlike the L5-specific CYFIP2 reduction observed in adult Cyfip2+/- mice. Nevertheless, filamentous actin (F-actin) levels were increased only in L5 of Cyfip2 cKO mPFC possibly because of a compensatory increase in CYFIP1, the other member of CYFIP family, in L2/3 neurons. Abnormal dendritic spines on basal, but not on apical, dendrites were consistently observed in L5 neurons of Cyfip2 cKO mPFC. Meanwhile, neuronal excitability and activity were enhanced in both L2/3 and L5 neurons of Cyfip2 cKO mPFC, suggesting that CYFIP2 functions of regulating F-actin and excitability/activity may be mediated through independent mechanisms. Unexpectedly, adult Cyfip2 cKO mice did not display locomotor hyperactivity or reduced anxiety observed in Cyfip2+/- mice. Instead, both exhibited enhanced social dominance accessed by the tube test. Together, these results provide additional insights into the functions of CYFIP2 in the adult brain.

Protocol for Quantitative Analysis of Synaptic Vesicle Clustering in Axons of Cultured Neurons.

Clustering of synaptic vesicles along the neuronal axons is a critical mechanism underpinning proper synaptic transmission. Here, we provide a detailed protocol for analyzing the distribution of synaptic vesicles in presynaptic boutons of cultured neurons. The protocol covers preparation of cultured neurons, expression of synaptic vesicle-enriched proteins, and quantification procedures. Utilizing neurons from postnatal transgenic mice, this method can be applied to investigate the roles of synaptic genes in regulating vesicle dynamics at synaptic sites. For complete details on the use and execution of this protocol, please refer to Han et al. (2020a).