Neuronal Markers Research Articles

AxonFinder: Automated segmentation of tumor innervating neuronal fibers.

Neurosignaling is increasingly recognized as a critical factor in cancer progression, where neuronal innervation of primary tumors contributes to the disease's advancement. This study focuses on segmenting individual axons within the prostate tumor microenvironment, which have been challenging to detect and analyze due to their irregular morphologies. We present a novel deep learning-based approach for the automated segmentation of axons, AxonFinder, leveraging a U-Net model with a ResNet-101 encoder, based on a multiplexed imaging approach. Utilizing a dataset of whole-slide images from low-, intermediate-, and high-risk prostate cancer patients, we manually annotated axons to train our model, achieving significant accuracy in detecting axonal structures that were previously hard to segment. Our analysis includes a comprehensive assessment of axon density and morphological features across different CAPRA-S prostate cancer risk categories, providing insights into the correlation between tumor innervation and cancer progression. Our paper suggests the potential utility of neuronal markers in the prognostic assessment of prostate cancer in aiding the pathologist's assessment of tumor sections and advancing our understanding of neurosignaling in the tumor microenvironment.

Preexisting Skin-Resident CD8 and γδ T-cell Circuits Mediate Immune Response in Merkel Cell Carcinoma and Predict Immunotherapy Efficacy.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer with a ∼50% response rate to immune checkpoint blockade (ICB) therapy. To identify predictive biomarkers, we integrated bulk and single-cell RNA sequencing (RNA-seq) with spatial transcriptomics from a cohort of 186 samples from 116 patients, including bulk RNA-seq from 14 matched pairs pre- and post-ICB. In nonresponders, tumors show evidence of increased tumor proliferation, neuronal stem cell markers, and IL1. Responders have increased type I/II interferons and preexisting tissue resident (Trm) CD8 or Vδ1 γδ T cells that functionally converge with overlapping antigen-specific transcriptional programs and clonal expansion of public T-cell receptors. Spatial transcriptomics demonstrated colocalization of T cells with B and dendritic cells, which supply chemokines and costimulation. Lastly, ICB significantly increased clonal expansion or recruitment of Trm and Vδ1 cells in tumors specifically in responders, underscoring their therapeutic importance. These data identify potential clinically actionable biomarkers and therapeutic targets for MCC. Significance: MCC serves as a model of ICB response. We utilized the largest-to-date, multimodal MCC dataset (n = 116 patients) to uncover unique tumor-intrinsic properties and immune circuits that predict response. We identified CD8 Trm and Vδ1 T cells as clinically actionable mediators of ICB response in major histocompatibility complex-high and -low MCCs, respectively.

A novel super-resolution microscopy platform for cutaneous alpha-synuclein detection in Parkinson's disease.

Alpha-synuclein (aSyn) aggregates in the central nervous system are the main pathological hallmark of Parkinson's disease (PD). ASyn aggregates have also been detected in many peripheral tissues, including the skin, thus providing a novel and accessible target tissue for the detection of PD pathology. Still, a well-established validated quantitative biomarker for early diagnosis of PD that also allows for tracking of disease progression remains lacking. The main goal of this research was to characterize aSyn aggregates in skin biopsies as a comparative and quantitative measure for PD pathology. Using direct stochastic optical reconstruction microscopy (dSTORM) and computational tools, we imaged total and phosphorylated-aSyn at the single molecule level in sweat glands and nerve bundles of skin biopsies from healthy controls (HCs) and PD patients. We developed a user-friendly analysis platform that offers a comprehensive toolkit for researchers that combines analysis algorithms and applies a series of cluster analysis algorithms (i.e., DBSCAN and FOCAL) onto dSTORM images. Using this platform, we found a significant decrease in the ratio of the numbers of neuronal marker molecules to phosphorylated-aSyn molecules, suggesting the existence of damaged nerve cells in fibers highly enriched with phosphorylated-aSyn molecules. Furthermore, our analysis found a higher number of aSyn aggregates in PD subjects than in HC subjects, with differences in aggregate size, density, and number of molecules per aggregate. On average, aSyn aggregate radii ranged between 40 and 200 nm and presented an average density of 0.001-0.1 molecules/nm2. Our dSTORM analysis thus highlights the potential of our platform for identifying quantitative characteristics of aSyn distribution in skin biopsies not previously described for PD patients while offering valuable insight into PD pathology by elucidating patient aSyn aggregation status.

Alteration of mature neuronal marker of β-III tubulin expression in differentiated SH-SY5Y cells by refinement of foetal bovine serum concentration

BackgroundThe process of differentiating neuroblastoma cells (SH-SY5Y) is crucial for obtaining mature neuronal markers. However, there is variability in the concentrations of foetal bovine serum (FBS) used in the differentiation media, ranging from 1 to 10%. This inconsistency in FBS concentrations may contribute to the inconsistent differentiation of cells. To improve the utility of the SH-SY5Y cell line as a model for neuronal cell culture, we investigated the impact of FBS concentrations in the differentiation media using Dulbecco's modified eagle medium and 10 μM all-trans-retinoic acid (ATRA). The aim of this study was to optimise the concentrations of FBS in the differentiation media of SH-SY5Y cells. Our study focused on assessing the length of dendrites in neuronal cells and the expression of β-III tubulin, a marker indicative of mature neurons. SH-SY5Y cells were differentiated with 10 µM of ATRA for 7 days. Four treatment groups with different FBS concentrations (1%, 3%, 5%, and 10%) were examined to assess the alteration of cellular morphology and dendritic length. The expression of the mature neuron marker β-III tubulin was evaluated using immunocytochemistry technique.ResultsSH-SY5Y cells' dendrite length was significantly longer (p < 0.05) when there was a higher concentration of FBS in the differentiation medium. The result was confirmed with the significant increase of β-III tubulin expression (p < 0.001) of the differentiated SH-SY5Y cells that have been incubated with higher concentrations of FBS in the differentiation medium.ConclusionsWe concluded that optimised concentrations of FBS in the differentiation media display longer length of the dendrites and express higher production of β-III tubulin in the differentiated SH-SY5Y cells. The consistency of serum concentration used in the differentiation media is important to produce a sustainable in vitro neuronal model of SH-SY5Y cells for neurodegenerative studies.

Therapeutic potential of Canna edulis RS3-resistant starch in alleviating neuroinflammation and apoptosis in a Parkinson's disease rat model

This study aimed to investigate the effects of Miao medicinal Canna edulis RS3-resistant starch on behavioral performance and substantia nigra neuron apoptosis-related indicators in a rat model of Parkinson's disease (PD). Among the experimental groups, except for the control group, we induced PD rat models by subcutaneous injection of rotenone in the neck and back. After model induction, a 28-day drug intervention was conducted. Various techniques have been employed, including behavioral analysis, Real-time Polymerase Chain Reaction (RT-PCR), western blotting, enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and terminal deoxynucleotidyltransferase-mediated UTP nick-ends. labeling (TUNEL) and Nissl staining to investigate the effect of Canna edulis RS3-resistant starch on the substantia nigra and neuronal apoptosis-related markers in the brains of PD model rats. Our study revealed that Canna edulis RS3, a resistant starch, significantly reduced the climbing time of PD model rats, prolonged their hanging time, lowered the expression levels of the inflammatory factors IL-1β, IL-6, and TNF-α, increased the number of TH-positive neurons in the substantia nigra, and decreased the levels of IL-1β, IL-6, and TNF-α. Furthermore, Canna edulis RS3 elevated the protein expression levels of tyrosine hydroxylase (TH) and Bcl-2 while reducing those of Bax, TLR4, NLRP3,and p-P65, and mitigated apoptosis and morphological changes in dopaminergic neurons in the substantia nigra region. Our results suggest that Canna edulis RS3-resistant starch may offer therapeutic benefits for PD patients with PD by potentially influencing inflammation and apoptosis in the dopaminergic system.

Integrative analysis of long isoform sequencing and functional data identifies distinct cortical layer neuronal subtypes derived from human iPSCs.

Generation of human induced pluripotent stem cells (iPSCs) through reprogramming was a transformational change in the field of regenerative medicine that led to new possibilities for drug discovery and cell replacement therapy. Several protocols have been established to differentiate hiPSCs into neuronal lineages. However, low differentiation efficiency is one of the major drawbacks of these approaches. Here, we compared the efficiency of two methods of neuronal differentiation from iPSCs cultured in two different culture media, StemFlex Medium (SFM) and Essential 8 Medium (E8M). The results indicated that iPSCs cultured in E8M efficiently generated different types of neurons in a shorter time and without the growth of undifferentiated nonneuronal cells in the culture as compared with those generated from iPSCs in SFM. Furthermore, these neurons were validated as functional units immunocytochemically by confirming the expression of mature neuronal markers (i.e., NeuN, β tubulin, and Synapsin I) and whole cell patch-clamp recordings. Long-read single-cell RNA sequencing confirms the presence of upper and deep layer cortical layer excitatory and inhibitory neuronal subtypes in addition to small populations of GABAergic neurons in day 30 neuronal cultures. Pathway analysis indicated that our protocol triggers the signaling transcriptional networks important for the process of neuronal differentiation in vivo.NEW & NOTEWORTHY Low differentiation efficiency is one of the major drawbacks of the existing protocols to differentiate iPSCs into neuronal lineages. Here, we present time-efficient and robust approach of neuronal differentiation leading to the generation of functional brain units, cortical layer neurons. We found iPSCs cultured in Essential 8 media (E8M) resulted in neuronal differentiation without the signs of growth of spontaneously differentiated cells in culture at any point in 35 days compared with Stemflex media (SFM).

The effects of Atoh8 on postnatal murine neurogenesis.

Introduction Basic helix-loop-helix (bHLH) transcription factors are expressed in various organs and are involved in diverse developmental processes. The mouse atonal homolog 8 (Atoh8), a bHLH transcription factor plays a crucial role in various developmental processes, especially as a regulator of neurogenesis in the retina. Besides, Atoh8 expression has been observed in the central nervous system. The function of Atoh8 during the postnatal neurogenesis is still unclear. Methods This study focuses on elucidating the impact of Atoh8 on postnatal neurogenesis in the brain, particularly in selected regions; the subventricular zone (SVZ), rostral migration stream (RMS) and olfactory bulb (OB), across different life stages, using male homozygous Atoh8-knockout (M6KO) mice. Our morphometric analysis is based on immunohistochemically labeled markers for neuroblasts (Doublecortin) and proliferation (Phospho-Histone H3, PHH3) as well as pan neuronal markers. Results In Atoh8-/- mice alteration in the postnatal neurogenesis can be observed. Immunohistochemical analysis revealed a significant reduction in doublecortin-positive neuroblasts within the SVZ of neonatal M6KO mice compared to wildtype (WT) mice. Interestingly, no differences in cell number and distribution was observed in the subsequent migration of neuroblasts through the rostral migration stream (RMS) to the olfactory bulb (OB). Proliferating PHH3-positive neuronal progenitor cells were significantly diminished in proliferation rate in both the SVZ and RMS of neonatal and young M6KO mice. Furthermore, in the glomerular layer of the OB significantly fewer neurons were detected in the neonatal stage. Conclusion In conclusion, Atoh8 emerges as a positive regulator of postnatal neurogenesis in the brain. Its role encompasses the promotion of neuroblast formation, modulation of proliferation rates, differentiation, and maintenance of mature neurons. Understanding the intricacies of Atoh8 function provides valuable insights into the complex regulatory mechanisms governing neurogenesis.

Distinct In Vitro Differentiation Protocols Differentially Affect Cytotoxicity Induced by Heavy Metals in Human Neuroblastoma SH-SY5Y Cells.

The SH-SY5Y cell line is widely used in neurotoxicity studies. However, the effects of inducing cell differentiation on the cytotoxic effects of heavy metals are unclear. Therefore, we investigated the effects of mercuric chloride (HgCl2), cadmium chloride (CdCl2), arsenic trioxide (As2O3), and methylmercury (MeHg) on SH-SY5Y cells differentiated in the presence of insulin-like growth factor-I (IGF-I) or all-trans retinoic acid (ATRA). Neurite outgrowth with distinct changes in neuronal marker expression, phenotype, and cell cycle was induced in SH-SY5Y cells by IGF-I treatment for 1day or ATRA treatment for up to 7days. The cytotoxic effects of HgCl2 decreased at lower concentrations and increased at higher concentrations in both IGF-I- and ATRA-differentiated cells compared with those in undifferentiated cells. Differentiation with IGF-I, but not with ATRA, increased the cytotoxic effects of CdCl2. Decreased cytotoxic effects of As2O3 and MeHg were observed at lower concentrations in IGF-I-differentiated cells, whereas increased cytotoxic effects of As2O3 and MeHg were observed at higher concentrations in ATRA-differentiated cells. Changes in the cytotoxic effects of heavy metals were observed even after 1day of ATRA exposure in SH-SY5Y cells. Our results demonstrate that the differentiation of SH-SY5Y cells by IGF-I and ATRA induces different cellular characteristics, resulting in diverse changes in sensitivity to heavy metals, which depend not only on the differentiation agents and treatment time but also on the heavy metal species and concentration.

TUMOR-INFILTRATING NOCICEPTOR NEURONS PROMOTE IMMUNOSUPPRESSION.

Nociceptor neurons impact tumor immunity. Removing nociceptor neurons reduced myeloid-derived suppressor cell (MDSCs) tumor infiltration in mouse models of head and neck carcinoma and melanoma. Carcinoma-released small extracellular vesicles (sEVs) attract nociceptive nerves to tumors. sEV-deficient tumors fail to develop in mice lacking nociceptor neurons. Exposure of dorsal root ganglia (DRG) neurons to cancer sEVs elevated expression of Substance P, IL-6 and injury-related neuronal markers while treatment with cancer sEVs and cytotoxic CD8 T-cells induced an immunosuppressive state (increased exhaustion ligands and cytokines). Cancer patient sEVs enhanced DRG responses to capsaicin, indicating increased nociceptor sensitivity. Conditioned media from DRG and cancer cell co-cultures promoted expression of MDSC markers in primary bone marrow cells while DRG conditioned media together with cancer sEVs induced checkpoint expression on T-cells. Our findings indicate that nociceptor neurons facilitate CD8+ T cell exhaustion and enhance MDSC infiltration. Targeting nociceptor-released IL-6 emerges as a novel strategy to disrupt harmful neuro-immune interactions in cancer and enhance anti-tumor immunity.

Neurological recovery in rats with portocaval anastomosis-induced hepatic encephalopathy treated with leuprolide acetate, a GnRH agonist.

Hepatic encephalopathy (HE) is a neuropsychiatric complication of acute liver failure or chronic liver injury. Liver dysfunction impairs ammonia detoxification, allowing it to cross the blood-brain barrier (BBB) and disrupt brain function. The hippocampus becomes a crucial target during elevated ammonia levels, causing spatial memory impairment and decreased learning ability. Leuprolide acetate (LA), a GnRH agonist, has been implicated in neuroprotection and neuroregeneration in several regions of the central nervous system (CNS) including hippocampus. In this study, we aim to evaluate the effects of LA treatment on hippocampus of rats with HE induced by portocaval anastomosis (PCA) trough cognitive tests, histology analysis and expression of neuronal recovery marker proteins, such as neurofilament (NF200) and neurabin II, and astrocyte marker glial fibrillary acidic protein (GFAP). Rats were divided into three groups: SHAM, portocaval anastomosis with saline solution (PCA + SS) and portocaval anastomosis treated with LA (PCA + LA). To evaluate learning and spatial memory elevated T-maze (ETM) and Y-maze test (YMT) were respectively used. Results indicated that LA-treated rats performed significantly better in ETM and YMT than untreated rats. Histological analysis of hippocampus showed increased neuron density, nuclear area, and layer thickness in dentate gyrus of PCA + LA group compared to PCA + SS. Additionally, neurabin II and NF200 expression were higher in LA-treated rats, while GFAP expression was elevated in the PCA + SS group compared to control and PCA + LA groups. In conclusion, LA enhances hippocampal neuron recovery and reduces astrogliosis, suggesting its potential as a therapeutic intervention for attenuating hippocampal damage during HE.

Tet1-mediated 5hmC regulates hippocampal neuroinflammation via wnt signaling as a novel mechanism in obstructive sleep apnoea leads to cognitive deficit

BackgroundObstructive sleep apnoea (OSA) is a sleep-disordered breathing characterized by intermittent hypoxia (IH) that may cause cognitive dysfunction. However, the impact of IH on molecular processes involved in cognitive function remains unclear.MethodsC57BL / 6 J mice were exposed to either normoxia (control) or IH for 6 weeks. DNA hydroxymethylation was quantified by hydroxymethylated DNA immunoprecipitation (hMeDIP) sequencing. ten-eleven translocation 1 (Tet1) was knocked down by lentivirus. Specifically, cognitive function was assessed by behavioral experiments, pathological features were assessed by HE staining, the hippocampal DNA hydroxymethylation was examined by DNA dot blot and immunohistochemical staining, while the Wnt signaling pathway and its downstream effects were studied using qRT-PCR, immunofluorescence staining, and Luminex liquid suspension chip analysis.ResultsIH mice showed pathological changes and cognitive dysfunction in the hippocampus. Compared with the control group, IH mice exhibited global DNA hydroxylmethylation in the hippocampus, and the expression of three hydroxylmethylases increased significantly. The Wnt signaling pathway was activated, and the mRNA and 5hmC levels of Wnt3a, Ccnd2, and Prickle2 were significantly up-regulated. Further caused downstream neurogenesis abnormalities and neuroinflammatory activation, manifested as increased expression of IBA1 (a marker of microglia), GFAP (a marker of astrocytes), and DCX (a marker of immature neurons), as well as a range of inflammatory cytokines (e.g. TNFa, IL3, IL9, and IL17A). After Tet1 knocked down, the above indicators return to normal.ConclusionActivation of Wnt signaling pathway by hippocampal Tet1 is associated with cognitive dysfunction induced by IH.Graphical

Effects on Synaptic Plasticity Markers in Fetal Mice and HT22 Neurons upon F-53B Exposure: The Role of PKA Cytoplasmic Retention

Effects on Synaptic Plasticity Markers in Fetal Mice and HT22 Neurons upon F-53B Exposure: The Role of PKA Cytoplasmic Retention

Sex-Specific changes in stress circuitry of the nucleus tractus solitarius following food deprivation in two rat strains

Food deprivation is used in many experimental models and is becoming increasingly prevalent in human diets. The impact of food deprivation on specific brain regions, including the nucleus of the tractus solitarius (NTS), a region that is involved in hunger and satiety sensing, remains to be determined. The NTS is a heterogeneous nucleus that includes corticotropin releasing factor receptor 1 (CRF1) neurons. CRF1 is implicated in both stress and appetite regulation, but the effects of food deprivation on CRF1 NTS neurons are unclear. We used immunofluorescence to examine the effects of 24-hour food deprivation on NTS activity in male and female Sprague-Dawley (SD) rats and CRF1-cre rats using cFos, an immediate early gene and neuronal marker of activation. NTS activity was increased in food deprived male but not female SD rats. In food deprived CRF1-cre rats, males had an increased proportion of active CRF1 + neurons with no change in females. In CRF1-cre rats, increased global NTS activity was observed in food deprived and refed males. Activation of CRF1 + neurons was also increased after deprivation but was reduced by refeeding. In females, food deprivation decreased global NTS activity that was then increased by refeeding, while CRF1 activity was unchanged.Collectively, these data suggest the NTS is differentially activated after food deprivation in a sex-specific manner, whereby males are more sensitive than females. These results provide insight into the role of brainstem stress circuitry in changes associated with conditions including intermittent fasting and eating disorders like anorexia.

Glial reactivity is linked to synaptic dysfunction across the aging and Alzheimer's disease spectrum.

Previous studies have shown that glial and neuronal changes may trigger synaptic dysfunction in Alzheimer's disease(AD). However, the link between glial and neuronal markers and synaptic abnormalities in the living brain is poorly understood. Here, we investigated the association between biomarkers of astrocyte and microglial reactivity and synaptic dysfunction in 478 individuals across the aging and AD spectrum from two cohorts with available CSF measures of amyloid-β(Aβ), phosphorylated tau(pTau181), astrocyte reactivity(GFAP), microglial activation(sTREM2), and synaptic biomarkers(GAP43 and neurogranin). Elevated CSF GFAP levels were linked to presynaptic and postsynaptic dysfunction, regardless of cognitive status or Aβ presence. CSF sTREM2 levels were associated with presynaptic biomarkers in cognitively unimpaired and impaired Aβ + individuals and postsynaptic biomarkers in cognitively impaired Aβ + individuals. Notably, CSF pTau181 levels mediated all associations between GFAP or sTREM2 levels and synaptic dysfunction biomarkers. These results suggest that neuronal-related synaptic biomarkers could be used in clinical trials targeting glial reactivity in AD.

Neuronal repair after spinal cord injury by in vivo astrocyte reprogramming mediated by the overexpression of NeuroD1 and Neurogenin-2

BackgroundAs a common disabling disease, irreversible neuronal death due to spinal cord injury (SCI) is the root cause of functional impairment; however, the capacity for neuronal regeneration in the developing spinal cord tissue is limited. Therefore, there is an urgent need to investigate how defective neurons can be replenished and functionally integrated by neural regeneration; the reprogramming of intrinsic cells into functional neurons may represent an ideal solution.MethodsA mouse model of transection SCI was prepared by forceps clamping, and an adeno-associated virus (AAV) carrying the transcription factors NeuroD1 and Neurogenin-2(Ngn2) was injected in situ into the spinal cord to specifically overexpress these transcription factors in astrocytes close to the injury site. 5-bromo-2´-deoxyuridine (BrdU) was subsequently injected intraperitoneally to continuously track cell regeneration, neuroblasts and immature neurons marker expression, neuronal regeneration, and glial scar regeneration. In addition, immunoprotein blotting was used to measure the levels of transforming growth factor-β (TGF-β) pathway-related protein expression. We also evaluated motor function, sensory function, and the integrity of the blood-spinal cord barrier(BSCB).ResultsThe in situ overexpression of NeuroD1 and Ngn2 in the spinal cord was achieved by specific AAV vectors. This intervention led to a significant increase in cell regeneration and the proportion of cells with neuroblasts and immature neurons cell properties at the injury site(p < 0.0001). Immunofluorescence staining identified astrocytes with neuroblasts and immature neurons cell properties at the site of injury while neuronal marker-specific staining revealed an increased number of mature astrocytes at the injury site. Behavioral assessments showed that the intervention did not improve The BMS (Basso mouse scale) score (p = 0.0726) and gait (p > 0.05), although the treated mice had more sensory sensitivity and greater voluntary motor ability in open field than the non-intervention mice. We observed significant repair of the BSCB at the center of the injury site (p < 0.0001) and a significant improvement in glial scar proliferation. Electrophysiological assessments revealed a significant improvement in spinal nerve conduction (p < 0.0001) while immunostaining revealed that the levels of TGF-β protein at the site of injury in the intervention group were lower than control group (p = 0.0034); in addition, P70 s6 and PP2A related to the TGF-β pathway showed ascending trend (p = 0.0036, p = 0.0152 respectively).ConclusionsThe in situ overexpression of NeuroD1 and Ngn2 in the spinal cord after spinal cord injury can reprogram astrocytes into neurons and significantly enhance cell regeneration at the injury site. The reprogramming of astrocytes can lead to tissue repair, thus improving the reduced threshold and increasing voluntary movements. This strategy can also improve the integrity of the blood-spinal cord barrier and enhance nerve conduction function. However, the simple reprogramming of astrocytes cannot lead to significant improvements in the striding function of the lower limbs.

Morpho-Anatomical Degeneration of Dopaminergic Neurons in Adult Zebrafish Brain after Exposure to Nickel

Chronic exposure to heavy metals has been widely demonstrated to induce pathological features in different tissues and, in particular, in the central nervous system. Specific neurons, including dopaminergic neurons, were observed to be more susceptible to toxic agents. Several previous studies performed on zebrafish (Danio rerio) models observed that exposure to nickel (one of the most abundant heavy metals) induces impairment of memory and anxious-like behaviors. Nevertheless, this phenotypical evidence has not been associated with dopaminergic system damage, and no reports showing the effects of nickel on dopaminergic neurons are available. In this study, we aim to analyze the precise distribution and variation in dopaminergic neurons in adult zebrafish after chronic (96 h) exposure to nickel ions dissolved in water at different sub-lethal doses (0.4 mg L−1; 2 mg L−1 and 4 mg L−1). The effects of treatment on dopaminergic neurons were evaluated by measuring transcript and protein levels of tyrosine hydroxylase (TH), described as a dopaminergic neuron marker. As shown, the expression of the th1 and th2 genes was reduced in the entire brain of zebrafish treated with nickel. Immunostaining analysis allowed us to localize TH-expressing neurons mainly in the posterior tuberculum, where they were observed to be reduced after nickel treatment in a dose-dependent fashion. Consistently, the TUNEL assay revealed a significant increase in apoptosis of TH-expressing cells after treatment with 2 mg L−1 and 4 mg L−1 of nickel. Our findings represent the first evidence of the effect of nickel on the dopaminergic system.

Standardization of a Novel Semi-Automatic Software for Neurite Outgrowth Measurement.

Effective live-imaging techniques are crucial to assess neuronal morphology in order to measure neurite outgrowth in real time. The proper measurement of neurite outgrowth has been a long-standing challenge over the years in the neuroscience research field. This parameter serves as a cornerstone in numerous in vitro experimental setups, ranging from dissociated cultures and organotypic cultures to cell lines. By quantifying the neurite length, it is possible to determine if a specific treatment worked or if axonal regeneration is enhanced in different experimental groups. In this study, the aim is to demonstrate the robustness and accuracy of the Incucyte Neurotrack neurite outgrowth analysis software. This semi-automatic software is available in a time-lapse microscopy system which offers several advantages over commonly used methodologies in the quantification of the neurite length in phase contrast images. The algorithm masks and quantifies several parameters in each image and returns neuronal cell metrics, including neurite length, branch points, cell-body clusters, and cell-body cluster areas. Firstly, we validated the robustness and accuracy of the software by correlating its values with those of the manual NeuronJ, a Fiji plug-in. Secondly, we used the algorithm which is able to work both on phase contrast images as well as on immunocytochemistry images. Using specific neuronal markers, we validated the feasibility of the fluorescence-based neurite outgrowth analysis on sensory neurons in vitro cultures. Additionally, this software can measure neurite length across various seeding conditions, ranging from individual cells to complex neuronal nets. In conclusion, the software provides an innovative and time-effective platform for neurite outgrowth assays, paving the way for faster and more reliable quantifications.

Hippocampal neurogenesis modulated by Quinic acid: A therapeutic strategy for the neurodegenerative disorders.

Neural progenitor cells (NPCs) reside in the brain and participate in the mechanism of neurogenesis that permits the brain to generate the building blocks for enhancement of cognitive abilities and acquisition of new skills. The existence of NPCs in brain has opened a novel dimension of research to explore their potential for treatment of various neurodegenerative disorders. The present study provides novel insights into the intracellular mechanisms in neuronal cells proliferation, maturation and differentiation regulated by Quinic acid (QA). Furthermore, this study might help in discovery and development of lead molecule that can overcome the challenges in the treatment of neurodegenerative diseases. The growth supporting effect of QA was studied using MTT assay. For that purpose, hippocampal cell cultures of neonatal rats were treated with different concentrations of QA and incubated for 24, 48 and 72 h. Gene and protein expressions of the selected molecular markers nestin, neuron-specific class III beta-tubulin (Tuj-1), neuronal nuclear protein (NeuN), neuronal differentiation 1 (NeuroD1), glial fibrillary acidic protein (GFAP), neuroligin (NLGN) and vimentin were analyzed. QA-induced cell proliferation and differentiation of hippocampal progenitor cells was also accompanied by significantly increased expression of progenitor and immature neuronal marker, mature neuronal marker and differentiating factor, that is, nestin, Tuj-1, NeuN and NeuroD1, respectively. On the other hand, vimentin downregulation and constant GFAP expression were observed following QA treatment. Additionally, the effects of QA on the recovery of stressed cells was studied using in vitro model of oxygen glucose deprivation (OGD). It was observed that hippocampal cells were able to recover from OGD following the treatment with QA. These findings suggest that QA treatment promotes hippocampal neurogenesis by proliferating and differentiating of NPCs and recovers neurons from stress caused by OGD. Thus, the neurogenic potential of QA can be explored for the treatment of neurodegenerative disorders.

Fasting-induced activity changes in MC3R neurons of the paraventricular nucleus of the thalamus.

The brain controls energy homeostasis by regulating food intake through signaling within the melanocortin system. Whilst we understand the role of the hypothalamus within this system, how extra-hypothalamic brain regions are involved in controlling energy balance remains unclear. Here we show that the melanocortin 3 receptor (MC3R) is expressed in the paraventricular nucleus of the thalamus (PVT). We tested whether fasting would change the activity of MC3R neurons in this region by assessing the levels of c-Fos and pCREB as neuronal activity markers. We determined that overnight fasting causes a significant reduction in pCREB levels within PVT-MC3R neurons. We then questioned whether perturbation of MC3R signaling, during fasting, would result in altered refeeding. Using chemogenetic approaches, we show that modulation of MC3R activity, during the fasting period, does not impact body weight regain or total food intake in the refeeding period. However, we did observe significant differences in the pattern of feeding-related behavior. These findings suggest that the PVT is a region where MC3R neurons respond to energy deprivation and modulate refeeding behavior.

Formation and Long-Term Culture of hiPSC-Derived Sensory Nerve Organoids Using Microfluidic Devices.

Although methods for generating human induced pluripotent stem cell (hiPSC)-derived motor nerve organoids are well established, those for sensory nerve organoids are not. Therefore, this study investigated the feasibility of generating sensory nerve organoids composed of hiPSC-derived sensory neurons using a microfluidic approach. Notably, sensory neuronal axons from neurospheres containing 100,000 cells were unidirectionally elongated to form sensory nerve organoids over 6 mm long axon bundles within 14 days using I-shaped microchannels in microfluidic devices composed of polydimethylsiloxane (PDMS) chips and glass substrates. Additionally, the organoids were successfully cultured for more than 60 days by exchanging the culture medium. The percentage of nuclei located in the distal part of the axon bundles (the region 3-6 mm from the entrance of the microchannel) compared to the total number of cells in the neurosphere was 0.005% for live cells and 0.008% for dead cells. Molecular characterization confirmed the presence of the sensory neuron marker ISL LIM homeobox 1 (ISL1) and the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1). Moreover, capsaicin stimulation activated TRPV1 in organoids, as evidenced by significant calcium ion influx. Conclusively, this study demonstrated the feasibility of long-term organoid culture and the potential applications of sensory nerve organoids in bioengineered nociceptive sensors.