Neuromodulatory co-expression in cardiac vagal motor neurons of the dorsal motor nucleus of the vagus.

Vagal innervation is well known to be crucial to the maintenance of cardiac health, and to protect and recover the heart from injury. Only recently has this role been shown to depend on the activity of the underappreciated dorsal motor nucleus of the vagus (DMV). By combining neural tracing, transcriptomics, and anatomical mapping in male and female Sprague-Dawley rats, we characterize cardiac-specific neuronal phenotypes in the DMV. We find that the DMV cardiac-projecting neurons differentially express pituitary adenylate cyclase-activating polypeptide (PACAP), cocaine- and amphetamine-regulated transcript (CART), and synucleins, as well as evidence that they participate in neuromodulatory co-expression involving catecholamines. The significance of these findings is enhanced by previous knowledge of the role of PACAP at the heart and of the other neuromodulators in peripheral vagal targets.

Autism-associated CHD8 controls reactive gliosis and neuroinflammation via remodeling chromatin in astrocytes.

Reactive changes of glial cells during neuroinflammation impact brain disorders and disease progression. Elucidating the mechanisms that control reactive gliosis may help us to understand brain pathophysiology and improve outcomes. Here, we report that adult ablation of autism spectrum disorder (ASD)-associated CHD8 in astrocytes attenuates reactive gliosis via remodeling chromatin accessibility, changing gene expression. Conditional Chd8 deletion in astrocytes, but not microglia, suppresses reactive gliosis by impeding astrocyte proliferation and morphological elaboration. Astrocyte Chd8 ablation alleviates lipopolysaccharide-induced neuroinflammation and septic-associated hypothermia in mice. Astrocytic CHD8 plays an important role in neuroinflammation by altering the chromatin landscape, regulating metabolic and lipid-associated pathways, and astrocyte-microglia crosstalk. Moreover, we show that reactive gliosis can be directly mitigated in vivo using an adeno-associated virus (AAV)-mediated Chd8 gene editing strategy. These findings uncover a role of ASD-associated CHD8 in the adult brain, which may warrant future exploration of targeting chromatin remodelers in reactive gliosis and neuroinflammation in injury and neurological diseases.

Iron-sulfur cluster loss in mitochondrial CISD1 mediates PINK1 loss-of-function phenotypes.

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra of the midbrain. Familial cases of PD are often caused by mutations of PTEN-induced kinase 1 (PINK1) and the ubiquitin ligase Parkin, both pivotal in maintaining mitochondrial quality control. CISD1, a homodimeric mitochondrial iron-sulfur-binding protein, is a major target of Parkin-mediated ubiquitination. We here discovered a heightened propensity of CISD1 to form dimers in Pink1 mutant flies and in dopaminergic neurons from PINK1 mutation patients. The dimer consists of two monomers that are covalently linked by a disulfide bridge. In this conformation CISD1 cannot coordinate the iron-sulfur cofactor. Overexpressing Cisd, the Drosophila orthologue of CISD1, and a mutant Cisd incapable of binding the iron-sulfur cluster in Drosophila reduced climbing ability and lifespan. This was more pronounced with mutant Cisd and aggravated in Pink1 mutant flies. Complete loss of Cisd, in contrast, rescued all detrimental effects of Pink1 mutation on climbing ability, wing posture, dopamine levels, lifespan, and mitochondrial ultrastructure. Our results suggest that Cisd, probably iron-depleted Cisd, operates downstream of Pink1 shedding light on PD pathophysiology and implicating CISD1 as a potential therapeutic target.

Semaphorin7A patterns neural circuitry in the lateral line of the zebrafish.

In a developing nervous system, axonal arbors often undergo complex rearrangements before neural circuits attain their final innervation topology. In the lateral line sensory system of the zebrafish, developing sensory axons reorganize their terminal arborization patterns to establish precise neural microcircuits around the mechanosensory hair cells. However, a quantitative understanding of the changes in the sensory arbor morphology and the regulators behind the microcircuit assembly remain enigmatic. Here, we report that Semaphorin7A (Sema7A) acts as an important mediator of these processes. Utilizing a semi-automated three-dimensional neurite tracing methodology and computational techniques, we have identified and quantitatively analyzed distinct topological features that shape the network in wild-type and Sema7A loss-of-function mutants. In contrast to those of wild-type animals, the sensory axons in Sema7A mutants display aberrant arborizations with disorganized network topology and diminished contacts to hair cells. Moreover, ectopic expression of a secreted form of Sema7A by non-hair cells induces chemotropic guidance of sensory axons. Our findings propose that Sema7A likely functions both as a juxtracrine and as a secreted cue to pattern neural circuitry during sensory organ development.

Balanced anesthesia in pigeons (Columba livia): a protocol that ensures stable vital parameters and feasibility during long surgeries in cognitive neuroscience.

In neuroscience, numerous experimental procedures in animal models require surgical interventions, such as the implantation of recording electrodes or cannulas before main experiments. These surgeries can take several hours and should rely on principles that are common in the field of research and medicine. Considering the characteristics of the avian respiratory physiology, the development of a safe and replicable protocol for birds is necessary to minimize side effects of anesthetic agents, circumvent technical limitations due to the insufficient availability of patient monitoring, and to maintain stable intraoperative anesthesia. Through the consistent and responsible implementation of the three R principle of animal welfare in science ("Replace, Reduce, Refine"), we aimed to optimize experimental methods to minimize the burden on pigeons (Columba livia) during surgical procedures. Here, surgeries were conducted under balanced anesthesia and perioperative monitoring of heart rate, oxygen saturation, body temperature, and the reflex state. The protocol we developed is based on the combination of injectable and inhalative anesthetic drugs [ketamine, xylazine, and isoflurane, supported by the application of an opiate for analgesia (e.g., butorphanol, buprenorphine)]. The combination of ketamine and xylazine with a pain killer is established in veterinary medicine across a vast variety of species. Practicability was verified by survival of the animals, fast and smooth recovery quantified by clinical examination, sufficiency, and stability of anesthesia. Independent of painful stimuli like incision or drilling, or duration of surgery, vital parameters were within known physiological ranges for pigeons. Our approach provides a safe and conservative protocol for surgeries of extended duration for scientific applications as well as for veterinary medicine in pigeons which can be adapted to other bird species.

Systemic gene therapy corrects neurological phenotype in a mouse model of NGLY1 deficiency.

The cytoplasmic peptide:N-glycanase (NGLY1) is ubiquitously expressed and functions as a de-N-glycosylating enzyme that degrades misfolded N-glycosylated proteins. NGLY1 deficiency due to biallelic loss-of-function NGLY1 variants is an ultrarare autosomal recessive deglycosylation disorder with multisystemic involvement; the neurological manifestations represent the major disease burden. Currently, there is no treatment for this disease. To develop a gene therapy, we first characterized a tamoxifen-inducible Ngly1 knock-out (iNgly1) C57BL/6J mouse model, which exhibited symptoms recapitulating human disease, including elevation of the biomarker GlcNAc-Asn (GNA), motor deficits, kyphosis, Purkinje cell loss, and gait abnormalities. We packaged a codon-optimized human NGLY1 transgene cassette into two adeno-associated virus (AAV) capsids, AAV9 and AAV.PHPeB. Systemic administration of the AAV.PHPeB vector to symptomatic iNgly1 mice corrected multiple disease features at eight weeks post-treatment. Furthermore, another cohort of AAV.PHPeB-treated iNgly1 mice were monitored over a year, and showed near-complete normalization of the neurological aspects of the disease phenotype, demonstrating the durability of gene therapy. Our data suggested that brain-directed NGLY1 gene replacement via systemic delivery is a promising therapeutic strategy for NGLY1 deficiency. Although the superior CNS tropism of AAV.PHPeB vector does not translate to primate, emerging AAV capsids with enhanced primate CNS tropism will enable future translational studies.

A human fetal cerebellar map of the late second trimester reveals developmental molecular characteristics and abnormality in trisomy 21.

Our understanding of human fetal cerebellum development during the late second trimester, a critical period for the generation of astrocytes, oligodendrocytes, and unipolar brush cells (UBCs), remains limited. Here, we performed single-cell RNA sequencing (scRNA-seq) in human fetal cerebellum samples from gestational weeks (GWs) 18-25. We find that proliferating UBC progenitors distribute in the subventricular zone of the rhombic lip (RLSVZ) near white matter (WM), forming a layer structure. We also delineate two trajectories from astrogenic radial glia (ARGs) to Bergmann glial progenitors (BGPs) and recognize oligodendrogenic radial glia (ORGs) as one source of primitive oligodendrocyte progenitor cells (PriOPCs). Additionally, our scRNA-seq analysis of the trisomy 21 fetal cerebellum at this stage reveals abnormal upregulated genes in pathways such as the cell adhesion pathway and focal adhesion pathway, which potentially promote neuronal differentiation. Overall, our research provides valuable insights into normal and abnormal development of the human fetal cerebellum.

Enkephalin-mediated modulation of basal somatic sensitivity by regulatory T cells in mice.

CD4+CD25+Foxp3+ regulatory T cells (Treg) have been implicated in pain modulation in various inflammatory conditions. However, whether Treg cells hamper pain at steady state and by which mechanism is still unclear. From a meta-analysis of the transcriptomes of murine Treg and conventional T cells (Tconv), we observe that the proenkephalin gene (Penk), encoding the precursor of analgesic opioid peptides, ranks among the top 25 genes most enriched in Treg cells. We then present various evidence suggesting that Penk is regulated in part by members of the Tumor Necrosis Factor Receptor (TNFR) family and the transcription factor Basic leucine zipper transcription faatf-like (BATF). Using mice in which the promoter activity of Penk can be tracked with a fluorescent reporter, we also show that Penk expression is mostly detected in Treg and activated Tconv in non-inflammatory conditions in the colon and skin. Functionally, Treg cells proficient or deficient for Penk suppress equally well the proliferation of effector T cells in vitro and autoimmune colitis in vivo. In contrast, inducible ablation of Penk in Treg leads to heat hyperalgesia in both male and female mice. Overall, our results indicate that Treg might play a key role at modulating basal somatic sensitivity in mice through the production of analgesic opioid peptides.

NF1-dependent disruption of the blood-nerve-barrier is improved by blockade of P2RY14.

The blood-nerve-barrier (BNB) that regulates peripheral nerve homeostasis is formed by endoneurial capillaries and perineurial cells surrounding the Schwann cell (SC)-rich endoneurium. Barrier dysfunction is common in human tumorigenesis, including in some nerve tumors. We identify barrier disruption in human NF1 deficient neurofibromas, which were characterized by reduced perineurial cell glucose transporter 1 (GLUT1) expression and increased endoneurial fibrin(ogen) deposition. Conditional Nf1 loss in murine SCs recapitulated these alterations and revealed decreased tight junctions and decreased caveolin-1 (Cav1) expression in mutant nerves and in tumors, implicating reduced Cav1-mediated transcytosis in barrier disruption and tumorigenesis. Additionally, elevated receptor tyrosine kinase activity and genetic deletion of Cav1 increased endoneurial fibrin(ogen), and promoted SC tumor formation. Finally, when SC lacked Nf1, genetic loss or pharmacological inhibition of P2RY14 rescued Cav1 expression and barrier function. Thus, loss of Nf1 in SC causes dysfunction of the BNB via P2RY14-mediated G-protein coupled receptor (GPCR) signaling.

FTO variant is associated with changes in BMI, ghrelin and brain function following bariatric surgery.

BACKGROUND: A polymorphism in the fat mass and obesity-associated gene (FTO) is linked to enhanced neural sensitivity to food-cues and attenuated ghrelin suppression. Risk allele carriers regain more weight than non-carriers after bariatric surgery. It remains unclear how FTO variation affects brain function and ghrelin following surgery. METHODS: Resting-state functional magnetic resonance imaging (RS-fMRI) and cue-reactivity fMRI with high-/low-caloric food-cues were performed at pre-surgery and 1-, 6-, and 12-months post-surgery to examine brain function in 16 carriers with one copy of the rs9939609 A allele (AT) and 26 non-carriers (TT). Behavioral assessments up to five years post-surgery were also conducted. RESULTS: AT relative to TT group had smaller BMI-loss at 12 to 60 months post-surgery and lower resting-state activity in posterior cingulate cortex following LSG (group-by-time interaction effects). Meanwhile, AT relative to TT group showed greater food-cue responses in dorsolateral prefrontal cortex (DLPFC), dorsomedial prefrontal cortex (DMPFC) and insula (group effects). There were negative associations of weight-loss with ghrelin and greater activation in DLPFC, DMPFC and insula in AT but not TT group. CONCLUSION: These findings indicate that FTO variation is associated with the evolution of ghrelin signaling and brain function after bariatric surgery, which might hinder weight-loss.

Exosomal TNF-α mediates voltage-gated Na+ channels 1.6 overexpression and contributes to brain-tumor induced neuronal hyperexcitability.

Patients affected by glioma frequently suffer of epileptic discharges, however the causes of brain tumor-related epilepsy (BTRE) are still not completely understood. We investigated the mechanisms underlying BTRE by analyzing the effects of exosomes released by U87 glioma cells and by patient-derived glioma cells. Rat hippocampal neurons incubated for 24 h with these exosomes exhibited increased spontaneous firing, while their resting membrane potential shifted positively by 10-15 mV. Voltage clamp recordings demonstrated that the activation of the Na+ current shifted towards more hyperpolarized voltages by 10-15 mV. To understand the factors inducing hyperexcitability we focused on exosomal cytokines. Western Blot and ELISA assays show that TNF-α is present inside glioma-derived exosomes. Remarkably, incubation with TNF-α fully mimicked the phenotype induced by exosomes, with neurons firing continuously, while their resting membrane potential shifted positively. RT-PCR revealed that both exosomes and TNF-α induced over-expression of the voltage-gated Na channel Nav1.6, a low-threshold Na+ channel responsible for hyperexcitability. When neurons were preincubated with Infliximab, a specific TNF-α inhibitor, the hyperexcitability induced by exosomes and TNF-α were drastically reduced. We propose that Infliximab, an FDA approved drug to treat rheumatoid arthritis, could ameliorate the conditions of glioma patients suffering of BTRE.

Disrupting glioblastoma networks with tumor treating fields (TTFields) in in vitro models.

PURPOSE: This study investigates the biological effect of Tumor Treating Fields (TTFields) on key drivers of glioblastoma's malignancy-tumor microtube (TM) formation-and on the function and overall integrity of the tumor cell network. METHOD: Using a two-dimensional monoculture GB cell network model (2DTM) of primary glioblastoma cell (GBC) cultures (S24, BG5 or T269), we evaluated the effects of TTFields on cell density, interconnectivity and structural integrity of the tumor network. We also analyzed calcium (Ca2+) transient dynamics and network morphology, validating findings in patient-derived tumoroids and brain tumor organoids. RESULTS: In the 2DTM assay, TTFields reduced cell density by 85-88% and disrupted network interconnectivity, particularly in cells with multiple TMs. A "crooked TM" phenotype emerged in 5-6% of treated cells, rarely seen in controls. Ca2+ transients were significantly compromised, with global Ca2+ activity reduced by 51-83%, active and periodic cells by over 50%, and intercellular co-activity by 52% in S24, and almost completely in BG5 GBCs. The effects were more pronounced at 200 kHz compared to a 50 kHz TTFields. Similar reductions in Ca2+ activity were observed in patient-derived tumoroids. In brain tumor organoids, TTFields significantly reduced tumor cell proliferation and infiltration. CONCLUSION: Our comprehensive study provides new insights into the multiple effects of Inovitro-modeled TTFields on glioma progression, morphology and network dynamics in vitro. Future in vivo studies to verify our in vitro findings may provide the basis for a deeper understanding and optimization of TTFields as a therapeutic modality in the treatment of GB.

IL-6 is associated with poor seizure control in low-grade glioma patients undergoing primary resection.

In this study, 198 patients with low-grade gliomas (LGGs) undergoing primary resection were evaluated for seizure status at 24 months after primary resection with the Engel classification of seizures, and 120 patients had good seizure control (class I) while 78 patients had poor seizure control (class II-IV). Multivariate analysis showed that cortex involvement, subtotal resection, serum IL-6 concentration, and neutrophil to lymphocyte ratio (NLR) were associated with poor seizure control. The area under curve (AUC) of serum IL-6 concentration, NLR and their combination applied in predicting poor seizure control was 0.756, 0.714, and 0.857, respectively. The AUC of combination prediction was significantly higher than those of individual prediction. Therefore, elevated serum IL-6 concentration was associated with poor seizure control in LLG patients undergoing primary resection and could be applied in predicting seizure control, and the predictive value could be elevated through adding other serum indices to IL-6.

Aberrant regulation of serine metabolism drives extracellular vesicle release and cancer progression.

Cancer cells secrete extracellular vesicles (EVs) to regulate cells in the tumor microenvironment to benefit their own growth and survive in the patient's body. Although emerging evidence has demonstrated the molecular mechanisms of EV release, regulating cancer-specific EV secretion remains challenging. In this study, we applied a microRNA library to reveal the universal mechanisms of EV secretion from cancer cells. Here, we identified miR-891b and its direct target gene, phosphoserine aminotransferase 1 (PSAT1), which promotes EV secretion through the serine-ceramide synthesis pathway. Inhibition of PSAT1 affected EV secretion in multiple types of cancer, suggesting that the miR-891b/PSAT1 axis shares a common mechanism of EV secretion from cancer cells. Interestingly, aberrant PSAT1 expression also regulated cancer metastasis via EV secretion. Our data link the PSAT1-controlled EV secretion mechanism and cancer metastasis and show the potential of this mechanism as a therapeutic target in multiple types of cancer.

Protocol for adeno-associated virus-mediated optogenetic activation of olfactory output neurons in neonatal mice.

Optogenetic manipulation has proven a powerful tool for investigating the mechanisms underlying the function of neuronal networks, but implementing the technique on mammals during early development remains challenging. Here, we present a comprehensive workflow to specifically manipulate mitral/tufted cells (M/TCs), the output neurons in the olfactory circuit, mediated by adeno-associated virus (AAV) transduction and light stimulation in neonatal mice and monitor neuronal and network activity with in vivo electrophysiology. This method represents an efficient approach to elucidate functional brain development. For complete details on the use and execution of this protocol, please refer to Chen et al.1,2,3.

An enhanced pattern detection and segmentation of brain tumors in MRI images using deep learning technique.

Neuroscience is a swiftly progressing discipline that aims to unravel the intricate workings of the human brain and mind. Brain tumors, ranging from non-cancerous to malignant forms, pose a significant diagnostic challenge due to the presence of more than 100 distinct types. Effective treatment hinges on the precise detection and segmentation of these tumors early. We introduce a cutting-edge deep-learning approach employing a binary convolutional neural network (BCNN) to address this. This method is employed to segment the 10 most prevalent brain tumor types and is a significant improvement over current models restricted to only segmenting four types. Our methodology begins with acquiring MRI images, followed by a detailed preprocessing stage where images undergo binary conversion using an adaptive thresholding method and morphological operations. This prepares the data for the next step, which is segmentation. The segmentation identifies the tumor type and classifies it according to its grade (Grade I to Grade IV) and differentiates it from healthy brain tissue. We also curated a unique dataset comprising 6,600 brain MRI images specifically for this study. The overall performance achieved by our proposed model is 99.36%. The effectiveness of our model is underscored by its remarkable performance metrics, achieving 99.40% accuracy, 99.32% precision, 99.45% recall, and a 99.28% F-Measure in segmentation tasks.

The enigma of sleep: Implications of sleep neuroscience for the dental clinician and patient.

BACKGROUND: Sleep disturbances have been shown to result in considerable morbidity and mortality. It is important for dental clinicians to understand the neuroscience behind sleep disorders. TYPES OF STUDIES REVIEWED: The authors conducted a search of the literature published from January 1990 through March 2024 of sleep medicine-related articles, with a focus on neuroscience. The authors prioritized articles about the science of sleep as related to dental medicine. RESULTS: The authors found a proliferation of articles related to sleep neuroscience along with its implications in dental medicine. The authors also found that the intricate neuroscientific principles of sleep medicine are being investigated robustly. The salient features of, and the differences between, central and obstructive sleep apneas have been elucidated. Sleep genes, such as CRY, PER1, PER2, and CLOCK, and their relationship to cancer and neurodegeneration are also additions to this rapidly developing science. CONCLUSIONS AND PRACTICAL IMPLICATIONS: The dental clinician has the potential to be the first to screen patients for possible sleep disorders and make prompt referrals to the appropriate medical professionals. This can be lifesaving as well as minimize potential future morbidity for the patient.

Aberrant hippocampal Ca2+ microwaves following synapsin-dependent adeno-associated viral expression of Ca2+ indicators.

Genetically encoded calcium indicators (GECIs) such as GCaMP are invaluable tools in neuroscience to monitor neuronal activity using optical imaging. The viral transduction of GECIs is commonly used to target expression to specific brain regions, can be conveniently used with any mouse strain of interest without the need for prior crossing with a GECI mouse line, and avoids potential hazards due to the chronic expression of GECIs during development. A key requirement for monitoring neuronal activity with an indicator is that the indicator itself minimally affects activity. Here, using common adeno-associated viral (AAV) transduction procedures, we describe spatially confined aberrant Ca2+ microwaves slowly travelling through the hippocampus following expression of GCaMP6, GCaMP7, or R-CaMP1.07 driven by the synapsin promoter with AAV-dependent gene transfer in a titre-dependent fashion. Ca2+ microwaves developed in hippocampal CA1 and CA3, but not dentate gyrus nor neocortex, were typically first observed at 4 wk after viral transduction, and persisted up to at least 8 wk. The phenomenon was robust and observed across laboratories with various experimenters and setups. Our results indicate that aberrant hippocampal Ca2+ microwaves depend on the promoter and viral titre of the GECI, density of expression, as well as the targeted brain region. We used an alternative viral transduction method of GCaMP which avoids this artefact. The results show that commonly used Ca2+-indicator AAV transduction procedures can produce artefactual Ca2+ responses. Our aim is to raise awareness in the field of these artefactual transduction-induced Ca2+ microwaves, and we provide a potential solution.

Glutamate neurotransmission from leptin receptor cells is required for typical puberty and reproductive function in female mice.

The hypothalamic ventral premammillary nucleus (PMv) is a glutamatergic nucleus essential for the metabolic control of reproduction. However, conditional deletion of leptin receptor long form (LepRb) in vesicular glutamate transporter 2 (Vglut2) expressing neurons results in virtually no reproductive deficits. In this study, we determined the role of glutamatergic neurotransmission from leptin responsive PMv neurons on puberty and fertility. We first assessed if stimulation of PMv neurons induces luteinizing hormone (LH) release in fed adult females. We used the stimulatory form of designer receptor exclusively activated by designer drugs (DREADDs) in LeprCre (LepRb-Cre) mice. We collected blood sequentially before and for 1 hr after intravenous clozapine-N-oxide injection. LH level increased in animals correctly targeted to the PMv, and LH level was correlated to the number of Fos immunoreactive neurons in the PMv. Next, females with deletion of Slc17a6 (Vglut2) in LepRb neurons (LeprΔVGlut2) showed delayed age of puberty, disrupted estrous cycles, increased gonadotropin-releasing hormone (GnRH) concentration in the axon terminals, and disrupted LH secretion, suggesting impaired GnRH release. To assess if glutamate is required for PMv actions in pubertal development, we generated a Cre-induced reexpression of endogenous LepRb (LeprloxTB) with concomitant deletion of Slc17a6 (Vglut2flox) mice. Rescue of Lepr and deletion of Slc17a6 in the PMv was obtained by stereotaxic injection of an adeno-associated virus vector expressing Cre recombinase. Control LeprloxTB mice with PMv LepRb rescue showed vaginal opening, follicle maturation, and became pregnant, while LeprloxTB;Vglut2flox mice showed no pubertal development. Our results indicate that glutamatergic neurotransmission from leptin sensitive neurons regulates the reproductive axis, and that leptin action on pubertal development via PMv neurons requires Vglut2.