Patch and matrix striatonigral neurons differentially regulate locomotion.

The striatonigral neurons are known to promote locomotion1,2. These neurons reside in both the patch (also known as striosome) and matrix compartments of the dorsal striatum3-5. However, the specific contribution of patch and matrix striatonigral neurons to locomotion remain largely unexplored. Using molecular identifier Kringle-Containing Protein Marking the Eye and the Nose (Kremen1) and Calbidin (Calb1)6, we showed in mouse models that patch and matrix striatonigral neurons exert opposite influence on locomotion. While a reduction in neuronal activity in matrix striatonigral neurons precedes the cessation of locomotion, fiber photometry recording during self-paced movement revealed an unexpected increase of patch striatonigral neuron activity, indicating an inhibitory function. Indeed, optogenetic activation of patch striatonigral neurons suppressed locomotion, contrasting with the locomotion-promoting effect of matrix striatonigral neurons. Consistently, patch striatonigral neuron activation markedly inhibited dopamine release, whereas matrix striatonigral neuron activation initially promoted dopamine release. Moreover, the genetic deletion of inhibitory GABA-B receptor Gabbr1 in Aldehyde dehydrogenase 1A1-positive (ALDH1A1+) nigrostriatal dopaminergic neurons (DANs) completely abolished the locomotion-suppressing effect caused by activating patch striatonigral neurons. Together, our findings unravel a compartment-specific mechanism governing locomotion in the dorsal striatum, where patch striatonigral neurons suppress locomotion by inhibiting the activity of ALDH1A1+ nigrostriatal DANs.

Advances in the Study of Mirror Neurons and Their Impact on Neuroscience: An Editorial.

The mirror neurons are complex neuronal circuits in the brain, and they respond to the actions that we observe in others. The mirror neurons constitute a revolutionary discovery in the field of neuroscience that has not only reshaped our understanding of social cognition and empathetic behavior but also bridged gaps in our comprehension of the human brain's intricate workings. This article aims to distill the crux of these groundbreaking discoveries and their transformative ramifications regarding our perception of human interactions and the advancement of neurorehabilitation techniques. The integration of non-invasive and patient-centric therapies into clinical practice underscores the immense potential that research on mirror neurons holds in enhancing patient outcomes and quality care. Research in mirror neurons will contribute significantly to the field of neuroscience, specifically neurorehabilitation.

Single-nucleus multi-omics of Parkinson's disease reveals a glutamatergic neuronal subtype susceptible to gene dysregulation via alteration of transcriptional networks.

The genetic architecture of Parkinson's disease (PD) is complex and multiple brain cell subtypes are involved in the neuropathological progression of the disease. Here we aimed to advance our understanding of PD genetic complexity at a cell subtype precision level. Using parallel single-nucleus (sn)RNA-seq and snATAC-seq analyses we simultaneously profiled the transcriptomic and chromatin accessibility landscapes in temporal cortex tissues from 12 PD compared to 12 control subjects at a granular single cell resolution. An integrative bioinformatic pipeline was developed and applied for the analyses of these snMulti-omics datasets. The results identified a subpopulation of cortical glutamatergic excitatory neurons with remarkably altered gene expression in PD, including differentially-expressed genes within PD risk loci identified in genome-wide association studies (GWAS). This was the only neuronal subtype showing significant and robust overexpression of SNCA. Further characterization of this neuronal-subpopulation showed upregulation of specific pathways related to axon guidance, neurite outgrowth and post-synaptic structure, and downregulated pathways involved in presynaptic organization and calcium response. Additionally, we characterized the roles of three molecular mechanisms in governing PD-associated cell subtype-specific dysregulation of gene expression: (1) changes in cis-regulatory element accessibility to transcriptional machinery; (2) changes in the abundance of master transcriptional regulators, including YY1, SP3, and KLF16; (3) candidate regulatory variants in high linkage disequilibrium with PD-GWAS genomic variants impacting transcription factor binding affinities. To our knowledge, this study is the first and the most comprehensive interrogation of the multi-omics landscape of PD at a cell-subtype resolution. Our findings provide new insights into a precise glutamatergic neuronal cell subtype, causal genes, and non-coding regulatory variants underlying the neuropathological progression of PD, paving the way for the development of cell- and gene-targeted therapeutics to halt disease progression as well as genetic biomarkers for early preclinical diagnosis.

The role of Foxo3a in neuron-mediated cognitive impairment.

Cognitive impairment (COI) is a prevalent complication across a spectrum of brain disorders, underpinned by intricate mechanisms yet to be fully elucidated. Neurons, the principal cell population of the nervous system, orchestrate cognitive processes and govern cognitive balance. Extensive inquiry has spotlighted the involvement of Foxo3a in COI. The regulatory cascade of Foxo3a transactivation implicates multiple downstream signaling pathways encompassing mitochondrial function, oxidative stress, autophagy, and apoptosis, collectively affecting neuronal activity. Notably, the expression and activity profile of neuronal Foxo3a are subject to modulation via various modalities, including methylation of promoter, phosphorylation and acetylation of protein. Furthermore, upstream pathways such as PI3K/AKT, the SIRT family, and diverse micro-RNAs intricately interface with Foxo3a, engendering alterations in neuronal function. Through several downstream routes, Foxo3a regulates neuronal dynamics, thereby modulating the onset or amelioration of COI in Alzheimer's disease, stroke, ischemic brain injury, Parkinson's disease, and traumatic brain injury. Foxo3a is a potential therapeutic cognitive target, and clinical drugs or multiple small molecules have been preliminarily shown to have cognitive-enhancing effects that indirectly affect Foxo3a. Particularly noteworthy are multiple randomized, controlled, placebo clinical trials illustrating the significant cognitive enhancement achievable through autophagy modulation. Here, we discussed the role of Foxo3a in neuron-mediated COI and common cognitively impaired diseases.

ATF2/BAP1 Axis Mediates Neuronal Apoptosis After Subarachnoid Hemorrhage via P53 Pathway.

BACKGROUND: Neuronal apoptosis plays an essential role in the pathogenesis of brain injury after subarachnoid hemorrhage (SAH). BAP1 (BRCA1-associated protein 1) is considered to exert pro-apoptotic effects in multiple diseases. However, evidence supporting the effect of BAP1 on the apoptotic response to SAH is lacking. Therefore, we aimed to confirm the role of BAP1 in SAH-induced apoptosis. METHODS: Enzyme-linked immunosorbent assay (ELISA) was used to detect BAP1 expression in the cerebrospinal fluid. Endovascular perforation was performed in mice to induce SAH. Lentiviral short hairpin RNA targeting BAP1 mRNA was transduced into the ipsilateral cortex of mice with SAH to investigate the role of BAP1 in neuronal damage. Luciferase and coimmunoprecipitation assays were performed to investigate the mechanism through which BAP1 participates in hemin-induced SAH. RESULTS: First, BAP1 expression was upregulated in the cerebrospinal fluid of patients with SAH and positively associated with unfavorable outcomes. ATF2 (activating transcription factor-2) then regulated BAP1 expression by binding to the BAP1 promoter. In addition, BAP1 overexpression enhanced P53 activity and stability by reducing P53 proteasome-mediated degradation. Subsequently, elevated P53 promoted neuronal apoptosis via the P53 pathway. Inhibition of the neuronal BAP1/P53 axis significantly reduced neurological deficits and neuronal apoptosis and improved neurological dysfunction in mice after SAH. CONCLUSIONS: Our results suggest that the neuronal ATF2/BAP1 axis exerts a brain-damaging effect by modulating P53 activity and stability and may be a novel therapeutic target for SAH.

The role of the Ventral Nucleus of the Trapezoid Body in the auditory prepulse inhibition of the acoustic startle reflex.

Cholinergic signaling is essential to mediate the auditory prepulse inhibition (PPI), an operational measure of sensorimotor gating, that refers to the reduction of the acoustic startle reflex (ASR) when a low-intensity, non-startling acoustic stimulus (the prepulse) is presented just before the onset of the acoustic startle stimulus. The cochlear root neurons (CRNs) are the first cells of the ASR circuit to receive cholinergic inputs from non-olivocochlear neurons of the ventral nucleus of the trapezoid body (VNTB) and subsequently decrease their neuronal activity in response to auditory prepulses. Yet, the contribution of the VNTB-CRNs pathway to the mediation of PPI has not been fully elucidated. In this study, we used the immunotoxin anti-choline acetyltransferase (ChAT)-saporin as well as electrolytic lesions of the medial olivocochlear bundle to selectively eliminate cholinergic VNTB neurons, and then assessed the ASR and PPI paradigms. Retrograde track-tracing experiments were conducted to precisely determine the site of lesioning VNTB neurons projecting to the CRNs. Additionally, the effects of VNTB lesions and the integrity of the auditory pathway were evaluated via auditory brain responses tests, ChAT- and FOS-immunohistochemistry. Consequently, we established three experimental groups: 1) intact control rats (non-lesioned), 2) rats with bilateral lesions of the olivocochlear bundle (OCB-lesioned), and 3) rats with bilateral immunolesions affecting both the olivocochlear bundle and the VNTB (OCB/VNTB-lesioned). All experimental groups underwent ASR and PPI tests at several interstimulus intervals before the lesion and 7, 14, and 21 days after it. Our results show that the ASR amplitude remained unaffected both before and after the lesion across all experimental groups, suggesting that the VNTB does not contribute to the ASR. The%PPI increased across the time points of evaluation in the control and OCB-lesioned groups but not in the OCB/VNTB-lesioned group. At the ISI of 50 ms, the OCB-lesioned group exhibited a significant increase in%PPI (p < 0.01), which did not occur in the OCB/VNTB-lesioned group. Therefore, the ablation of cholinergic non-olivocochlear neurons in the OCB/VNTB-lesioned group suggests that these neurons contribute to the mediation of auditory PPI at the 50 ms ISI through their cholinergic projections to CRNs. Our study strongly reinforces the notion that auditory PPI encompasses a complex mechanism of top-down cholinergic modulation, effectively attenuating the ASR across different interstimulus intervals within multiple pathways.

Modeling mechanisms of chemotherapy-induced peripheral neuropathy and chemotherapy transport using induced pluripotent stem cell-derived sensory neurons.

BACKGROUND: and Purpose: Chemotherapy-induced peripheral neuropathy (CIPN) constitutes a significant health problem due to the increasing prevalence and lack of therapies for treatment and prevention. While pivotal for routine cancer treatment, paclitaxel and vincristine frequently cause CIPN and impact the quality of life among cancer patients and survivors. Here, we investigate molecular mechanisms and drug transport in CIPN. EXPERIMENTAL APPROACH: Human sensory neurons were derived from induced pluripotent stem cells (iPSC-SNs), which were characterized using flow cytometry and immunolabeling. These iPSC-SNs were exposed to different concentrations of the two microtubule-targeting agents, paclitaxel and vincristine, with and without pre-exposure to inhibitors and inducers of efflux transporters. Neuronal networks were quantified via fluorescent staining against sensory neuron markers. Transcriptional effects of the chemotherapeutics were examined using quantitative polymerase chain reactions (qPCR). KEY RESULTS: Paclitaxel exposure resulted in axonal retraction and thickening, while vincristine caused fragmentation and abolishment of axons. Both agents increased the mRNA expression of the pain receptor, transient receptor potential vanilloid (TRPV1), and highly induced neuronal damage, as measured by activating transcription factor 3 (ATF3) mRNA. iPSC-SNs express the efflux transporters, P-glycoprotein (P-gp, encoded by ABCB1) and multidrug resistance-associated protein 1 (MPR1, encoded by ABCC1). Modulation of efflux transporters indicate that P-gp and MRP1 play a role in modulating neuronal accumulation and neurotoxicity in preliminary experiments. CONCLUSION: and Implications: iPSC-SNs are a valuable and robust model to study the role of efflux transporters and other mechanistic targets in CIPN. Efflux transporters may play a role in CIPN pathogenesis as they regulate the disposition of chemotherapy to the peripheral nervous system, and they may present potential therapeutic targets for CIPN.

Reversal of electrophysiological and behavioral deficits mediated by 5-HT7 receptor upregulation following LP-211 treatment in an autistic-like rat model induced by prenatal valproic acid exposure.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by alterations and imbalances in multiple brain neurochemical systems, particularly the serotonergic neurotransmission. This includes changes in serotonin (5-HT) levels, aberrations in 5-HT transporter activity, and decreased synthesis and expression of 5-HT receptors (5-HT7Rs). The exact role of the brain 5-HT system in the development of ASD remains unclear, with conflicting evidence on its involvement. Recently, we have reported research has shown a significant decrease in serotonergic neurons originating from the raphe nuclei and projecting to the CA1 region of the dorsal hippocampus in autistic-like rats. Additionally, we have shown that chronic activation of 5-HT7Rs reverses the effects of autism induction on synaptic plasticity. However, the functional significance of 5-HT7Rs at the cellular level is still not fully understood. This study presents new evidence indicating an upregulation of 5-HT7R in the CA1 subregion of the hippocampus following the induction of autism. The present account also demonstrates that activation of 5-HT7R with its agonist LP-211 can reverse electrophysiological abnormalities in hippocampal pyramidal neurons in a rat model of autism induced by prenatal exposure to VPA. Additionally, in vivo administration of LP-211 resulted in improvements in motor coordination, novel object recognition, and a reduction in stereotypic behaviors in autistic-like offspring. The findings suggest that dysregulated expression of 5-HT7Rs may play a role in the pathophysiology of ASD, and that agonists like LP-211 could potentially be explored as a pharmacological treatment for autism spectrum disorder.

Variation and convergence in the morpho-functional properties of the mammalian neocortex.

Man's natural inclination to classify and hierarchize the living world has prompted neurophysiologists to explore possible differences in brain organisation between mammals, with the aim of understanding the diversity of their behavioural repertoires. But what really distinguishes the human brain from that of a platypus, an opossum or a rodent? In this review, we compare the structural and electrical properties of neocortical neurons in the main mammalian radiations and examine their impact on the functioning of the networks they form. We discuss variations in overall brain size, number of neurons, length of their dendritic trees and density of spines, acknowledging their increase in humans as in most large-brained species. Our comparative analysis also highlights a remarkable consistency, particularly pronounced in marsupial and placental mammals, in the cell typology, intrinsic and synaptic electrical properties of pyramidal neuron subtypes, and in their organisation into functional circuits. These shared cellular and network characteristics contribute to the emergence of strikingly similar large-scale physiological and pathological brain dynamics across a wide range of species. These findings support the existence of a core set of neural principles and processes conserved throughout mammalian evolution, from which a number of species-specific adaptations appear, likely allowing distinct functional needs to be met in a variety of environmental contexts.

Aberrant evoked calcium signaling and nAChR cluster morphology in a SOD1 D90A hiPSC-derived neuromuscular model.

Familial amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disorder that is due to mutations in one of several target genes, including SOD1. So far, clinical records, rodent studies, and in vitro models have yielded arguments for either a primary motor neuron disease, or a pleiotropic pathogenesis of ALS. While mouse models lack the human origin, in vitro models using human induced pluripotent stem cells (hiPSC) have been recently developed for addressing ALS pathogenesis. In spite of improvements regarding the generation of muscle cells from hiPSC, the degree of maturation of muscle cells resulting from these protocols has remained limited. To fill these shortcomings, we here present a new protocol for an enhanced myotube differentiation from hiPSC with the option of further maturation upon coculture with hiPSC-derived motor neurons. The described model is the first to yield a combination of key myogenic maturation features that are consistent sarcomeric organization in association with complex nAChR clusters in myotubes derived from control hiPSC. In this model, myotubes derived from hiPSC carrying the SOD1 D90A mutation had reduced expression of myogenic markers, lack of sarcomeres, morphologically different nAChR clusters, and an altered nAChR-dependent Ca2+ response compared to control myotubes. Notably, trophic support provided by control hiPSC-derived motor neurons reduced nAChR cluster differences between control and SOD1 D90A myotubes. In summary, a novel hiPSC-derived neuromuscular model yields evidence for both muscle-intrinsic and nerve-dependent aspects of neuromuscular dysfunction in SOD1-based ALS.

Artesunate improves learning and memory impairment in rats with vascular cognitive impairment by down-regulating the level of autophagy in cerebral cortex neurons.

Background: Vascular cognitive impairment (VCI) is the second leading cause of dementia. Cognitive impairment is a common consequence of VCI. However, there is no effective treatment for VCI and the underlying mechanism of its pathogenesis remains unclear. This study to investigate whether artesunate (ART) can improve the learning and memory function in rats with VCI by down-regulating he level of autophagy in cerebral cortex neurons. Methods: The models for VCI were the rat bilateral common carotid artery occlusion (BACCO), which were randomized into three groups including the sham operation group (Sham), model + vehicle group (Model) and model + ART group (ART). Then the animal behaviors were recorded, as well as staining the results of cortical neurons. Western blot was performed to determine the protein expressions of LC3BⅡ/Ⅰ, p-AMPK, p-mTOR, and Beclin-1. Results: Behavioral outcomes and the protein expressions in Model group were supposedly affected by the induction of autophagy in cerebral cortex neurons. Compared to the Model group, ART improved memory impairment in VCI rats. And the expression of LC3BⅡ/Ⅰ, p-AMPK/AMPK, Beclin-1 is significant decreased in the ART group, while significant increases of p-mTOR/mTOR were showed. These results suggest that ART improved learning and memory impairment in VCI rats by down-regulating the level of autophagy in cerebral cortex neurons. Conclusion: The results suggest that autophagy occurs in cerebral cortex neurons in rats with VCI. It is speculated that ART can improve learning and memory impairment in VCI rats by down-regulating the level of autophagy in cerebral cortex neurons.

Hyperexcitation of the glutamatergic neurons in lateral hypothalamus induced by chronic pain contributes to depression-like behavior and learning and memory impairment in male mice.

Chronic pain can induce mood disorders and cognitive dysfunctions, such as anxiety, depression, and learning and memory impairment in humans. However, the specific neural network involved in anxiety- and depression-like behaviors and learning and memory impairment caused by chronic pain remains poorly understood. In this study, behavioral test results showed that chronic pain induced anxiety- and depression-like behaviors, and learning and memory impairment in male mice. c-Fos immunofluorescence and fiber photometry recording showed that glutamatergic neurons in the LH of mice with chronic pain were selectively activated. Next, the glutamatergic neurons of LH in normal mice were activated using optogenetic and chemogenetic methods, which recapitulates some of the depressive-like behaviors, as well as memory impairment, but not anxiety-like behavior. Finally, inhibition of glutamatergic neurons in the LH of mice with chronic pain, effectively relieved anxiety- and depression-like behaviors and learning and memory impairment. Taken together, our findings suggest that hyperexcitation of glutamatergic neurons in the LH is involved in depression-like behavior and learning and memory impairment induced by chronic pain.

Excitatory Projections of Wide Field Collicular Neurons to the Nucleus of the Optic Tract in the Rat.

The superficial layers of the mammalian superior colliculus (SC) contain neurons that are generally responsive to visual stimuli but can differ considerably in morphology and response properties. To elucidate the structure and function of these neurons, we combined extracellular recording and juxtacellular labeling, detailed anatomical reconstruction, and ultrastructural analysis of the synaptic contacts of labeled neurons, using transmission electron microscopy. Our labeled neurons project to different brainstem nuclei. Of particular importance are neurons that fit the morphological criteria of the wide field (WF) neurons and whose dendrites are horizontally oriented. They display a rather characteristic axonal projection pattern to the nucleus of optic tract (NOT); thus, we call them superior collicular WF projecting to the NOT (SCWFNOT) neurons. We corroborated the morphological characterization of this neuronal type as a distinct neuronal class with the help of unsupervised hierarchical cluster analysis. Our ultrastructural data demonstrate that SCWFNOT neurons establish excitatory connections with their targets in the NOT. Although, in rodents, the literature about the WF neurons has focused on their extensive projection to the lateral posterior nucleus of the thalamus, as a conduit for information to reach the visual association areas of the cortex, our data suggest that this subclass of WF neurons may participate in the optokinetic nystagmus.

A spatial-temporal map of glutamatergic neurogenesis in the murine embryonic cerebellar nuclei uncovers a high degree of cellular heterogeneity.

The nuclei are the main output structures of the cerebellum. Each and every cerebellar cortical computation reaches several areas of the brain by means of cerebellar nuclei processing and integration. Nevertheless, our knowledge of these structures is still limited compared to the cerebellar cortex. Here, we present a mouse genetic inducible fate-mapping study characterizing rhombic lip-derived glutamatergic neurons of the nuclei, the most conspicuous family of long-range cerebellar efferent neurons. Glutamatergic neurons mainly occupy dorsal and lateral territories of the lateral and interposed nuclei, as well as the entire medial nucleus. In mice, they are born starting from about embryonic day 9.5, with a peak between 10.5 and 12.5, and invade the nuclei with a lateral-to-medial progression. While some markers label a heterogeneous population of neurons sharing a common location (BRN2), others appear to be lineage specific (TBR1, LMX1a, and MEIS2). A comparative analysis of TBR1 and LMX1a distributions reveals an incomplete overlap in their expression domains, in keeping with the existence of separate efferent subpopulations. Finally, some tagged glutamatergic progenitors are not labeled by any of the markers used in this study, disclosing further complexity. Taken together, our results obtained in late embryonic nuclei shed light on the heterogeneity of the excitatory neuron pool, underlying the diversity in connectivity and functions of this largely unexplored cerebellar territory. Our findings contribute to laying the groundwork for a comprehensive functional analysis of nuclear neuron subpopulations.

Lhx4 surpasses its paralog Lhx3 in promoting the differentiation of spinal V2a interneurons.

Paralog factors are considered to ensure the robustness of biological processes by providing redundant activity in cells where they are co-expressed. However, the specific contribution of each factor is frequently underestimated. In the developing spinal cord, multiple families of transcription factors successively contribute to differentiate an initially homogenous population of neural progenitors into a myriad of neuronal subsets with distinct molecular, morphological, and functional characteristics. The LIM-homeodomain transcription factors Lhx3, Lhx4, Isl1 and Isl2 promote the segregation and differentiation of spinal motor neurons and V2 interneurons. Based on their high sequence identity and their similar distribution, the Lhx3 and Lhx4 paralogs are considered to contribute similarly to these processes. However, the specific contribution of Lhx4 has never been studied. Here, we provide evidence that Lhx3 and Lhx4 are present in the same cell populations during spinal cord development. Similarly to Lhx3, Lhx4 can form multiproteic complexes with Isl1 or Isl2 and the nuclear LIM interactor NLI. Lhx4 can stimulate a V2-specific enhancer more efficiently than Lhx3 and surpasses Lhx3 in promoting the differentiation of V2a interneurons in chicken embryo electroporation experiments. Finally, Lhx4 inactivation in mice results in alterations of differentiation of the V2a subpopulation, but not of motor neuron production, suggesting that Lhx4 plays unique roles in V2a differentiation that are not compensated by the presence of Lhx3. Thus, Lhx4 could be the major LIM-HD factor involved in V2a interneuron differentiation during spinal cord development and should be considered for in vitro differentiation of spinal neuronal populations.

Magnitude and angle dynamics in training single ReLU neurons.

Understanding the training dynamics of deep ReLU networks is a significant area of interest in deep learning. However, there remains a lack of complete elucidation regarding the weight vector dynamics, even for single ReLU neurons. To bridge this gap, our study delves into the training dynamics of the gradient flow w(t) for single ReLU neurons under the square loss, dissecting it into its magnitude ‖w(t)‖ and angle φ(t) components. Through this decomposition, we establish upper and lower bounds on these components to elucidate the convergence dynamics. Furthermore, we demonstrate the empirical extension of our findings to general two-layer multi-neuron networks. All theoretical results are generalized to the gradient descent method and rigorously verified through experiments.

Inhibition of Mast Cell Degranulation by Novel Small Molecule MRGPRX2 Antagonists.

BACKGROUND: Mas-related G-protein coupled receptor X2 (MRGPRX2) is a promiscuous receptor on mast cells that mediates IgE-independent degranulation and has been implicated in multiple mast cell-mediated disorders, including chronic urticaria, atopic dermatitis, and pain disorders. Although it is a promising therapeutic target, few potent, selective, small molecule antagonists have been identified, and functional effects of human MRGPRX2 inhibition have not been evaluated in vivo. OBJECTIVE: We identified and characterized novel, potent, and selective orally active small molecule MRGPRX2 antagonists for potential treatment of mast cell-mediated disease. METHODS: Antagonists were identified using multiple functional assays in cell lines overexpressing human MRGPRX2, LAD2 mast cells, human peripheral stem cell-derived mast cells, and isolated skin mast cells. Skin mast cell degranulation was evaluated in Mrgprb2em(-/-) knockout (KO) and Mrgprb2em(MRGPRX2) transgenic human MRGPRX2 knock-in (KI) mice by assessment of agonist-induced skin vascular permeability. Ex vivo skin mast cell degranulation and associated histamine release was evaluated by microdialysis of human skin tissue samples. RESULTS: MRGPRX2 antagonists potently inhibited agonist-induced MRGPRX2 activation and mast cell degranulation in all mast cell types tested, in an IgE-independent manner. Orally administered MRGPRX2 antagonists also inhibited agonist-induced degranulation and resulting vascular permeability in MRGPRX2 KI mice. In addition, antagonist treatment dose dependently inhibited agonist-induced degranulation in ex vivo human skin. CONCLUSION: MRGPRX2 small molecule antagonists potently inhibited agonist-induced mast cell degranulation in vitro and in vivo as well as ex vivo in human skin, supporting potential therapeutic utility as a novel treatment for multiple human diseases involving clinically relevant mast cell activation.

Immunohistochemical characterization of interstitial cells and their relationship to motor neurons within the mouse esophagus.

Interstitial cells of Cajal (ICC) and PDGFRα+ cells regulate smooth muscle motility in the gastrointestinal (GI) tract. However, their role(s) in esophageal motility are still unclear. The mouse esophagus has traditionally been described as almost entirely skeletal muscle in nature though ICC have been identified along its entire length. The current study evaluated the distribution of skeletal and smooth muscle within the esophagus using a mouse selectively expressing eGFP in smooth muscle cells (SMCs). The relationship of SMCs to ICC and PDGFRα+ cells was also examined. SMCs declined in density in the oral direction however SMCs represented ~ 25% of the area in the distal esophagus suggesting a likeness to the transition zone observed in humans. ANO1+ intramuscular ICC (ICC-IM) were distributed along the length of the esophagus though like SMCs, declined proximally. ICC-IM were closely associated with SMCs but were also found in regions devoid of SMCs. Intramuscular and submucosal PDGFRα+ cells were densely distributed throughout the esophagus though only intramuscular PDGFRα+ cells within the LES and distal esophagus highly expressed SK3. ICC-IM and PDGFRα+ cells were closely associated with nNOS+, VIP+, VAChT+ and TH+ neurons throughout the LES and distal esophagus. GFAP+ cells resembling intramuscular enteric glia were observed within the muscle and were closely associated with ICC-IM and PDGFRα+ cells, occupying a similar location to c. These data suggest that the mouse esophagus is more similar to the human than thought previously and thus set the foundation for future functional and molecular studies using transgenic mice.

Effect of sex on chronic stress induced alterations in hindbrain catecholaminergic system and autonomic dysfunction resulting in gastrointestinal dysmotility.

It has been reported that the clinical symptoms of functional dyspepsia (FD) exacerbate upon stress while the gender-related factors have been incompletely understood. This study aims to investigate the role of sex in chronic heterotypic stress (CHS)-induced autonomic and gastric motor dysfunction. For CHS, the rats were exposed to the combination of different stressors for 7 consecutive days. Subsequently, electrocardiography was recorded in anesthetized rats to evaluate heart rate variability (HRV) for the determination of autonomic outflow and sympathovagal balance. Solid gastric emptying (GE) was measured in control and CHS-loaded male and female rats. The immunoreactivities of catecholaminergic cell marker tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), corticotropin releasing factor (CRF), and estrogen receptor (ER-α/ß) were evaluated in medullary and pontine brainstem sections by immunohistochemistry. Compared with the controls, CHS significantly delayed GE in males but not in females. There was no significant sex-related difference in parasympathetic indicator HF under either control or CHS conditions. Sympathetic indicator LF was significantly higher in control females compared to the males. The higher sympathetic output in females was found to be attenuated upon CHS; in contrast, the elevated sympathetic output was detected in CHS-loaded males. No sex- or stress-related effect was observed on ChAT immunoreactivity in the dorsal motor nucleus of N.vagus (DMV). In males, greater number of TH-ir cells was observed in the caudal locus coeruleus (LC), while they were more densely detected in the rostral LC of females. Regardless of sex, CHS elevated immunoreactivity of TH throughout the LC. Under basal conditions, greater number of TH-ir cells was detected in the rostral ventrolateral medulla (RVLM) of females. In contrast, CHS remarkably increased the number of TH-ir cells in the RVLM of males which was found to be decreased in females. There was no sex-related alteration in TH immunoreactivity in the nucleus tractus solitarius (NTS) of control rats, while CHS affected both sexes in a similar manner. Compared with females, CRF immunoreactivity was prominently observed in control males, while both of which were stimulated by CHS. ER-α/ß was found to be co-expressed with TH in the NTS and LC which exhibit no alteration related to either sex or stress status. These results indicate a sexual dimorphism in the catecholaminergic and the CRF system in brainstem which might be involved in the CHS-induced autonomic and visceral dysfunction occurred in males.

The recruitment of mechanosensitive enteric neurons in the guinea pig gastric fundus is dependent on ganglionic stretch level.

BACKGROUND: Serving as a reservoir, the gastric fundus can expand significantly, with an initial receptive and a following adaptive relaxation, controlled by extrinsic and intrinsic reflex circuits, respectively. We hypothesize that mechanosensitive enteric neurons (MEN) are involved in the adaptive relaxation, which is initiated when a particular gastric volume and a certain stretch of the stomach wall is reached. To investigate whether the responsiveness of MEN in the gastric fundus is dependent on tissue stretch, we performed mechanical stimulations in stretched versus ganglia "at rest". METHODS: Responses of myenteric neurons in the guinea pig gastric fundus were recorded with membrane potential imaging using Di-8-ANEPPS. MEN were identified by small-volume intraganglionic injection in ganglia stretched to different degrees using a self-constructed stretching tool. Immunohistochemical staining identified the neurochemical phenotype of MEN. Hexamethonium and capsaicin were added to test their effect on recruited MEN. KEY RESULTS: In stretched compared to "at rest" ganglia, significantly more MEN were activated. The change in the ganglionic area correlated significantly with the number of additional recruited MEN. The additional recruitment of MEN was independent from nicotinic transmission and the ratio of active MEN in stretched ganglia shifted towards a nitrergic phenotype. CONCLUSION AND INFERENCES: The higher number of active MEN with increasing stretch of the ganglia and their greater share of nitrergic phenotype might indicate their contribution to the adaptive relaxation. Further experiments are necessary to address the receptors involved in mechanotransduction.