Treating activated regulatory T cells with pramipexole protects human dopaminergic neurons from 6-OHDA-induced degeneration.

BACKGROUND: Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra, which promotes a sustained inflammatory environment in the central nervous system. Regulatory T cells (Tregs) play an important role in the control of inflammation and might play a neuroprotective role. Indeed, a decrease in Treg number and function has been reported in PD. In this context, pramipexole, a dopaminergic receptor agonist used to treat PD symptoms, has been shown to increase peripheral levels of Treg cells and improve their suppressive function. The aim of this work was to determine the effect of pramipexole on immunoregulatory Treg cells and its possible neuroprotective effect on human dopaminergic neurons differentiated from human embryonic stem cells. METHODS: Treg cells were sorted from white blood cells of healthy human donors. Assays were performed with CD3/CD28-activated and non-activated Treg cells treated with pramipexole at concentrations of 2 or 200 ng/mL. These regulatory cells were co-cultured with in vitro-differentiated human dopaminergic neurons in a cytotoxicity assay with 6-hydroxydopamine (6-OHDA). The role of interleukin-10 (IL-10) was investigated by co-culturing activated IL-10-producing Treg cells with neurons. To further investigate the effect of treatment on Tregs, gene expression in pramipexole-treated, CD3/CD28-activated Treg cells was determined by Fluidigm analysis. RESULTS: Pramipexole-treated CD3/CD28-activated Treg cells showed significant protective effects on dopaminergic neurons when challenged with 6-OHDA. Pramipexole-treated activated Treg cells showed neuroprotective capacity through mechanisms involving IL-10 release and the activation of genes associated with regulation and neuroprotection. CONCLUSION: Anti-CD3/CD28-activated Treg cells protect dopaminergic neurons against 6-OHDA-induced damage. In addition, activated, IL-10-producing, pramipexole-treated Tregs also induced a neuroprotective effect, and the supernatants of these co-cultures promoted axonal growth. Pramipexole-treated, activated Tregs altered their gene expression in a concentration-dependent manner, and enhanced TGFß-related dopamine receptor regulation and immune-related pathways. These findings open new perspectives for the development of immunomodulatory therapies for the treatment of PD.

Assessing Neuronal Mitophagy During Development in the Nematode Caenorhabditis elegans.

Preserving mitochondrial homeostasis is vital, particularly for the energetically demanding and metabolically active nerve cells. Mitophagy, the selective autophagic removal of mitochondria, stands out as a prominent mechanism for efficient mitochondrial turnover, which is crucial for proper neuronal development and function. Dysfunctional mitochondria and disrupted mitophagy pathways have been linked to a diverse array of neurological disorders. The nematode Caenorhabditis elegans, with its well-defined nervous system, serves as an excellent model to unravel the intricate involvement of mitophagy in developing neurons. This chapter describes the use of Rosella biosensor in C. elegans to monitor neuronal mitophagy, providing a user-friendly platform for screening genes and drugs affecting mitophagic pathways under physiological conditions or in the context of neurodevelopmental pathologies.

GABAA receptors and neuroligin 2 synergize to promote synaptic adhesion and inhibitory synaptogenesis.

GABAA receptors (γ-aminobutyric acid-gated receptors type A; GABAARs), the major structural and functional postsynaptic components of inhibitory synapses in the mammalian brain, belong to a family of GABA-gated Cl-/HCO3 - ion channels. They are assembled as heteropentamers from a family of subunits including: α (1-6), ß(1-3), γ(1-3), δ, ε, π, θ and ρ(1-3). GABAARs together with the postsynaptic adhesion protein Neuroligin 2 (NL2) and many other pre- and post-synaptic proteins guide the initiation and functional maturation of inhibitory GABAergic synapses. This study examined how GABAARs and NL2 interact with each other to initiate the formation of synapses. Two functionally distinct GABAAR subtypes, the synaptic type α2ß2γ2-GABAARs versus extrasynaptic type α4ß3δ-GABAARs were expressed in HEK293 cells alone or together with NL2 and co-cultured with striatal GABAergic medium spiny neurons to enable innervation of HEK293 cells by GABAergic axons. When expressed alone, only the synaptic α2ß2γ2-GABAARs induced innervation of HEK293 cells. However, when GABAARs were co-expressed with NL2, the effect on synapse formation exceeded the individual effects of these proteins indicating a synergistic interaction, with α2ß2γ2-GABAAR/NL2 showing a significantly greater synaptogenic activity than α4ß3δ-GABAAR/NL2 or NL2 alone. To investigate the molecular basis of this interaction, different combinations of GABAAR subunits and NL2 were co-expressed, and the degree of innervation and synaptic activity assessed, revealing a key role of the γ2 subunit. In biochemical assays, the interaction between NL2 and α2ß2γ2-GABAAR was established and mapped to the large intracellular domain of the γ2 subunit.

Dihydromyricetin protects sevoflurane-induced mitochondrial dysfunction in HT22 hippocampal cells.

Sevoflurane (Sev) is a commonly used inhalation anaesthetic that has been shown to cause hippocampus dysfunction through multiple underlying molecular processes, including mitochondrial malfunction, oxidative stress and inflammation. Dihydromyricetin (DHM) is a 2,3-dihydroflavonoid with various biological properties, such as anti-inflammation and anti-oxidative stress. The purpose of this study was to investigate the effect of DHM on Sev-induced neuronal dysfunction. HT22 cells were incubated with 10, 20 and 30 µM of DHM for 24 h, and then stimulated with 4% Sev for 6 h. The effects and mechanism of DHM on inflammation, oxidative stress and mitochondrial dysfunction were explored in Sev-induced HT22 cells by Cell Counting Kit-8, flow cytometry, enzyme-linked immunosorbent assay, reverse transcription-quantitative polymerase chain reaction, colorimetric detections, detection of the level of reactive oxygen species (ROS), mitochondrial ROS and mitochondrial membrane potential (MMP), immunofluorescence and western blotting. Our results showed that DHM increased Sev-induced cell viability of HT22 cells. Pretreatment with DHM attenuated apoptosis, inflammation, oxidative stress and mitochondrial dysfunction in Sev-elicited HT22 cells by remedying the abnormality of the indicators involved in these progresses, including apoptosis rate, the cleaved-caspase 3 expression, as well as the level of tumour necrosis factor α, interleukin (IL)-1ß, IL-6, malondialdehyde, superoxide dismutase, catalase, ROS, mitochondrial ROS and MMP. Mechanically, pretreatment with DHM restored the Sev-induced the expression of SIRT1/FOXO3a pathway in HT22 cells. Blocking of SIRT1 counteracted the mitigatory effect of DHM on apoptosis, inflammation, oxidative stress and mitochondrial dysfunction in Sev-elicited HT22 cells. Collectively, pretreatment with DHM improved inflammation, oxidative stress and mitochondrial dysfunction via SIRT1/FOXO3a pathway in Sev-induced HT22 cells.

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.

Involvement of posterior hypothalamic CaMKII-positive neurons in ADHD-like behaviors in mice.

This study explores the behavioral effects of modulating CaMKII-positive (CaMKII+) neurons in the posterior hypothalamus (PH). Utilizing a chemogenetic approach in mice, we discovered that the activation of CaMKII + neurons within the PH is associated with heightened locomotor activity, reduced social interaction, and impulsive behavior unrelated to anxiety or avoidance. These observed behaviors share a significant resemblance with characteristics commonly found in attention deficit and hyperactivity disorder (ADHD). Notably, treatment with clonidine, which is frequently prescribed for ADHD, effectively reduced impulsive behaviors in our mouse model. Our findings uncover the role of the PH that has not been previously explored and suggest a possible involvement of the PH in the manifestation of ADHD-like behaviors.

Drosophila: a Tale of regeneration with MYC.

Regeneration is vital for many organisms, enabling them to repair injuries and adapt to environmental changes. The mechanisms underlying regeneration are complex and involve coordinated events at the cellular and molecular levels. Moreover, while some species exhibit remarkable regenerative capabilities, others, like mammals, have limited regenerative potential. Central to this process is the regulation of gene expression, and among the numerous genes involved, MYC emerges as a regulator of relevant processes during regeneration with roles conserved in several species, including Drosophila. This mini-review aims to provide valuable insights into the regeneration process in flies, focusing on significant organs where the role of MYC has been identified: from the imaginal discs, where MYC regulates cell growth, structure, and proliferation, to the gut, where it maintains the balance between renewal and differentiation of stem cells, and the central nervous system, where it influences the activities of neural stem cells and the interaction between glia and neuronal cells. By emphasizing the molecular mechanisms regulated by MYC, its significance in controlling regeneration mechanisms, and its conserved role in flies, we aim to offer valuable insights into the utility of Drosophila as a model for studying regeneration. Moreover, unraveling MYC's function in Drosophila during regeneration may help translate findings into the mechanisms underlying human tissue repair.

Anatomical topology of extrahippocampal projections from dorsoventral CA pyramidal neurons in mice.

The hippocampus primarily functions through a canonical trisynaptic circuit, comprised of dentate granule cells and CA1-CA3 pyramidal neurons (PNs), which exhibit significant heterogeneity along the dorsoventral axis. Among these, CA PNs are known to project beyond the hippocampus into various limbic areas, critically influencing cognitive and affective behaviors. Despite accumulating evidence of these extrahippocampal projections, the specific topological patterns-particularly variations among CA PN types and between their dorsal and ventral subpopulations within each type-remain to be fully elucidated. In this study, we utilized cell type-specific Cre mice injected with fluorescent protein-expressing AAVs to label each CA PN type distinctly. This method further enabled the dual-fluorescence labeling of dorsal and ventral subpopulations using EGFP and tdTomato, respectively, allowing a comprehensive comparison of their axonal projections in an animal. Our findings demonstrate that CA1 PNs predominantly form unilateral projections to the frontal cortex (PFC), amygdala (Amy), nucleus accumbens (NAc), and lateral septum (LS), unlike CA2 and CA3 PNs making bilateral innervation to the LS only. Moreover, the innervation patterns especially within LS subfields differ according to the CA PN type and their location along the dorsoventral axis of the hippocampus. This detailed topographical mapping provides the neuroanatomical basis of the underlying functional distinctions among CA PN types.

A linearized modeling framework for the frequency selectivity in neurons postsynaptic to vibration receptors.

Vibration is an indispensable part of the tactile perception, which is encoded to oscillatory synaptic currents by receptors and transferred to neurons in the brain. The A2 and B1 neurons in the drosophila brain postsynaptic to the vibration receptors exhibit selective preferences for oscillatory synaptic currents with different frequencies, which is caused by the specific voltage-gated Na+ and K+ currents that both oppose the variations in membrane potential. To understand the peculiar role of the Na+ and K+ currents in shaping the filtering property of A2 and B1 neurons, we develop a linearized modeling framework that allows to systematically change the activation properties of these ionic channels. A data-driven conductance-based biophysical model is used to reproduce the frequency filtering of oscillatory synaptic inputs. Then, this data-driven model is linearized at the resting potential and its frequency response is calculated based on the transfer function, which is described by the magnitude-frequency curve. When we regulate the activation properties of the Na+ and K+ channels by changing the biophysical parameters, the dominant pole of the transfer function is found to be highly correlated with the fluctuation of the active current, which represents the strength of suppression of slow voltage variation. Meanwhile, the dominant pole also shapes the magnitude-frequency curve and further qualitatively determines the filtering property of the model. The transfer function provides a parsimonious description of how the biophysical parameters in Na+ and K+ channels change the inhibition of slow variations in membrane potential by Na+ and K+ currents, and further illustrates the relationship between the filtering properties and the activation properties of Na+ and K+ channels. This computational framework with the data-driven conductance-based biophysical model and its linearized model contributes to understanding the transmission and filtering of vibration stimulus in the tactile system.

Inhibitory fear memory engram in the mouse central lateral amygdala.

Engrams, which are cellular substrates of memory traces, have been identified in various brain areas, including the amygdala. While most identified engrams are composed of excitatory, glutamatergic neurons, GABAergic inhibitory engrams have been relatively overlooked. Here, we report the identification of an inhibitory engram in the central lateral amygdala (CeL), a key area for auditory fear conditioning. This engram is primarily composed of GABAergic somatostatin-expressing (SST(+)) and, to a lesser extent, protein kinase C-δ-expressing (PKC-δ(+)) neurons. Fear memory is accompanied by a preferential enhancement of synaptic inhibition onto PKC-δ(+) neurons. Silencing this CeL GABAergic engram disinhibits the activity of targeted extra-amygdaloid areas, selectively increasing the expression of fear. Our findings define the behavioral function of an engram formed exclusively by GABAergic inhibitory neurons in the mammalian brain.

Mechanical and cold polymodality coexist in tactile peripheral afferents, and it's not mediated by TRPM8.

In the mammalian somatosensory system, polymodality is defined as the competence of some neurons to respond to multiple forms of energy (e.g., mechanical and thermal). This ability is thought to be an exclusive property of nociceptive neurons (polymodal C-fiber nociceptors) and one of the pillars of nociceptive peripheral plasticity. The current study uncovered a completely different neuronal sub-population with polymodal capabilities on the opposite mechanical modality spectrum (tactile). We have observed that several tactile afferents (1/5) can respond to cold in non-nociceptive ranges. These cells' mechanical thresholds and electrical properties are similar to any low-threshold mechano-receptors (LT), conducting in a broad range of velocities (Aδ to Aß), lacking CGRP and TRPM8 receptors. Due to its density, cold-response range, speed, and response to injury (or lack thereof), we speculate on its role in controlling reflexive behaviors (wound liking and rubbing) and modulation of nociceptive spinal cord integration. Further studies are required to understand the mechanisms behind this neuron's polymodality, central architecture, and impact on pain perception.

Dysembryoplastic Neuroepithelial Tumor: A Case Report of A Benign Intracranial Lesion Masquerading as Seizure Disorder.

The uncommon, benign dysembryoplastic neuroepithelial tumor (DNET, WHO grade 1) is frequently linked to epilepsy. It is a glioneuronal neoplasm in the cerebral cortex of children or young adults defined by the presence of a pathognomonic glioneuronal element that may be linked to glial nodules and activating mutations of fibroblast growth factor receptor 1 (FGFR1) (CNS WHO grade 1 according to WHO classification of CNS and pituitary tumors, 2021 ). The cerebral cortex is primarily affected. The most frequent areas are the temporal lobe, particularly the medial lobe, frontal lobe, and other cortex. This study reports the instance of a 31-year-old male who had a history of seizures for the past 20 years and complained of a sudden headache and vomiting at the hospital. MRI revealed a cortical-based lesion in the left posterior temporo-occipital region. A biopsy sample was sent for histopathological examination. DNETs are usually benign, non-recurring lesions and rarely can be a malignant transformation. Although they are frequently stable tumors, surgical excision seldom results in recurrence.

Coding of self and environment by Pacinian neurons in freely moving animals.

Pacinian corpuscle neurons are specialized low-threshold mechanoreceptors (LTMRs) that are tuned to detect high-frequency vibration (∼50-2,000 Hz); however, it is unclear how Pacinians and other LTMRs encode mechanical forces encountered during naturalistic behavior. Here, we developed methods to record LTMRs in awake, freely moving mice. We find that Pacinians, but not other LTMRs, encode subtle vibrations of surfaces encountered by the animal, including low-amplitude vibrations initiated over 2 m away. Strikingly, Pacinians are also highly active during a wide variety of natural behaviors, including walking, grooming, digging, and climbing. Pacinians in the hindlimb are sensitive enough to be activated by forelimb- or upper-body-dominant behaviors. Finally, we find that Pacinian LTMRs have diverse tuning and sensitivity. Our findings suggest a Pacinian population code for the representation of vibro-tactile features generated by self-initiated movements and low-amplitude environmental vibrations emanating from distant locations.

Alcohol, HMGB1, and Innate Immune Signaling in the Brain.

PURPOSE: Binge drinking (i.e., consuming enough alcohol to achieve a blood ethanol concentration of 80 mg/dL, approximately 4-5 drinks within 2 hours), particularly in early adolescence, can promote progressive increases in alcohol drinking and alcohol-related problems that develop into compulsive use in the chronic relapsing disease, alcohol use disorder (AUD). Over the past decade, neuroimmune signaling has been discovered to contribute to alcohol-induced changes in drinking, mood, and neurodegeneration. This review presents a mechanistic hypothesis supporting high mobility group box protein 1 (HMGB1) and Toll-like receptor (TLR) signaling as key elements of alcohol-induced neuroimmune signaling across glia and neurons, which shifts gene transcription and synapses, altering neuronal networks that contribute to the development of AUD. This hypothesis may help guide further research on prevention and treatment. SEARCH METHODS: The authors used the search terms "HMGB1 protein," "alcohol," and "brain" across PubMed, Scopus, and Embase to find articles published between 1991 and 2023. SEARCH RESULTS: The database search found 54 references in PubMed, 47 in Scopus, and 105 in Embase. A total of about 100 articles were included. DISCUSSION AND CONCLUSIONS: In the brain, immune signaling molecules play a role in normal development that differs from their functions in inflammation and the immune response, although cellular receptors and signaling are shared. In adults, pro-inflammatory signals have emerged as contributing to brain adaptation in stress, depression, AUD, and neurodegenerative diseases. HMGB1, a cytokine-like signaling protein released from activated cells, including neurons, is hypothesized to activate pro-inflammatory signals through TLRs that contribute to adaptations to binge and chronic heavy drinking. HMGB1 alone and in heteromers with other molecules activates TLRs and other immune receptors that spread signaling across neurons and glia. Both blood and brain levels of HMGB1 increase with ethanol exposure. In rats, an adolescent intermittent ethanol (AIE) binge drinking model persistently increases brain HMGB1 and its receptors; alters microglia, forebrain cholinergic neurons, and neuronal networks; and increases alcohol drinking and anxiety while disrupting cognition. Studies of human postmortem AUD brain have found elevated levels of HMGB1 and TLRs. These signals reduce cholinergic neurons, whereas microglia, the brain's immune cells, are activated by binge drinking. Microglia regulate synapses through complement proteins that can change networks affected by AIE that increase drinking, contributing to risks for AUD. Anti-inflammatory drugs, exercise, cholinesterase inhibitors, and histone deacetylase epigenetic inhibitors prevent and reverse the AIE-induced pathology. Further, HMGB1 antagonists and other anti-inflammatory treatments may provide new therapies for alcohol misuse and AUD. Collectively, these findings suggest that restoring the innate immune signaling balance is central to recovering from alcohol-related pathology.

The activation of D2-like dopamine receptors increases NMDA currents in the dorsal raphe serotonergic neurons.

The dorsal raphe nucleus (DRN) receives dopaminergic inputs from the ventral tegmental area (VTA). Also, the DRN contains a small population of cells that express dopamine (DRNDA neurons). However, the physiological role of dopamine (DA) in the DRN and its interaction with serotonergic (5-HT) neurons is poorly understood. Several works have reported moderate levels of D1, D2, and D3 DA receptors in the DRN. Furthermore, it was found that the activation of D2 receptors increased the firing of putative 5-HT neurons. Other studies have reported that D1 and D2 dopamine receptors can interact with glutamate NMDA receptors, modulating the excitability of different cell types. In the present work, we used immunocytochemical techniques to determine the kind of DA receptors in the DRN. Additionally, we performed electrophysiological experiments in brainstem slices to study the effect of DA agonists on NMDA-elicited currents recorded from identified 5-HT DRN neurons. We found that D2 and D3 but not D1 receptors are present in this nucleus. Also, we demonstrated that the activation of D2-like receptors increases NMDA-elicited currents in 5-HT neurons through a mechanism involving phospholipase C (PLC) and protein kinase C (PKC) enzymes. Possible physiological implications related to the sleep-wake cycle are discussed.

Neurotrophins and Their Receptors: BDNF's Role in GABAergic Neurodevelopment and Disease.

Neurotrophins and their receptors are distinctly expressed during brain development and play crucial roles in the formation, survival, and function of neurons in the nervous system. Among these molecules, brain-derived neurotrophic factor (BDNF) has garnered significant attention due to its involvement in regulating GABAergic system development and function. In this review, we summarize and compare the expression patterns and roles of neurotrophins and their receptors in both the developing and adult brains of rodents, macaques, and humans. Then, we focus on the implications of BDNF in the development and function of GABAergic neurons from the cortex and the striatum, as both the presence of BDNF single nucleotide polymorphisms and disruptions in BDNF levels alter the excitatory/inhibitory balance in the brain. This imbalance has different implications in the pathogenesis of neurodevelopmental diseases like autism spectrum disorder (ASD), Rett syndrome (RTT), and schizophrenia (SCZ). Altogether, evidence shows that neurotrophins, especially BDNF, are essential for the development, maintenance, and function of the brain, and disruptions in their expression or signaling are common mechanisms in the pathophysiology of brain diseases.

Role of the telomeric factor TRF2 in post-hypoxic brain damages.

The neuronal excitotoxicity that follows reoxygenation after a hypoxic period may contribute to epilepsy, Alzheimer's disease, Parkinson's disease and various disorders that are related to inadequate supplement of oxygen in neurons. Therefore, counteracting the deleterious effects of post-hypoxic stress is an interesting strategy to treat a large spectrum of neurodegenerative diseases. Here, we show that the expression of the key telomere protecting protein Trf2 decreases in the brain of mice submitted to a post-hypoxic stress. Moreover, downregulating the expression of Terf2 in hippocampal neural cells of unchallenged mice triggers an excitotoxicity-like phenotype including glutamate overexpression and behavioral alterations while overexpressing Terf2 in hippocampal neural cells of mice subjected to a post-hypoxic treatment prevents brain damages. Moreover, Terf2 overexpression in culture neurons counteracts the oxidative stress triggered by glutamate. Finally, we provide evidence that the effect of Terf2 downregulation on excitotoxicity involves Sirt3 repression leading to mitochondrial dysfunction. We propose that increasing the level of Terf2 expression is a potential strategy to reduce post-hypoxic stress damages.

Visualizing and Measuring Dendrite Arborization in Drosophila Somatosensory Neurons.

Dendrites of neurons receive synaptic or sensory inputs and are important sites of neuronal computation. The morphological features of dendrites not only are hallmarks of the neuronal type but also largely determine a neuron's function. Thus, dendrite morphogenesis has been a subject of intensive study in neuroscience. Quantification of dendritic morphology, which is required for accurate assessment of phenotypes, can often be a challenging task, especially for complex neurons. Because manual tracing of dendritic branches is labor-intensive and time-consuming, automated or semiautomated methods are required for efficient analysis of a large number of samples. A popular in vivo model system for studying the mechanisms of dendrite morphogenesis is dendritic arborization (da) neurons in the Drosophila larval peripheral nervous system. In this chapter, we introduce methods for visualizing and measuring the dendritic arbors of these neurons. We begin with an introduction of da neurons and an overview of the methods that have been used for measuring da neuron dendrites. We then discuss the techniques and detailed steps of neuron visualization and image acquisition. Finally, we provide example steps for dendrite tracing and measurement.

Isolation and Culture of Adult Rat Dorsal Root Ganglion Neurons for the In Vitro Analysis of Peripheral Nerve Degeneration and Regeneration.

Isolation and culture of dorsal root ganglion (DRG) neurons from adult animals is a useful experimental system for evaluating neural plasticity after axonal injury, as well as the neurological dysfunction resulting from aging and various types of disease. In this chapter, we will introduce a detailed method for the culture of mature rat DRG neurons. About 30-40 ganglia are dissected from a rat and mechanically and enzymatically digested. Subsequently, density gradient centrifugation of the digested tissue using 30% Percoll efficiently eliminates myelin debris and non-neuronal cells, to afford neuronal cells with a high yield and purity.

Live Imaging Analysis of Axonal Regeneration in Human iPSC-Derived Motor Neurons Using a Microfluidic System.

Axonal damage is a common feature of traumatic injury and neurodegenerative disease. The capacity for axons to regenerate and to recover functionality after injury is a phenomenon that is seen readily in the peripheral nervous system, especially in rodent models, but human axonal regeneration is limited and does not lead to full functional recovery. Here we describe a system where dynamics of human axonal outgrowth and regeneration can be evaluated via live imaging of human-induced pluripotent stem cell (hiPSC)-derived neurons cultured in microfluidic systems, in which cell bodies are isolated from their axons. This system could aid in studying axonal outgrowth dynamics and could be useful for testing potential drugs that encourage regeneration and repair of the nervous system.